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Feeding of detritivores in freshwater sediments
Vos, J.H.
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Vos, J. H. (2001). Feeding of detritivores in freshwater sediments Amsterdam
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Download date: 15 Jun 2017
Feedingg of Detritivores in Freshwater Sediments
11
IN N
FEEDINGG OF DETRITIVORES IN FRESHWATER SEDIMENTS
Cover: :
Photopgraphh and design by Ronald Gylstra
FEEDINGG OF DETRITIVORES IN FRESHWATER SEDIMENTS
ACADEMISCHH PROEFSCHRIFT
terr verkrijging van de graad van doctor aan de Universiteit van Amsterdam op gezag
vann de Rector Magnificus Prof. Dr. J.J.M. Franse ten overstaan van een door het college
voorr promoties ingestelde commissie in het openbaar te verdedigen in de Aula der
Universiteitt op donderdag 10 mei 2001, te 12.00 uur
doorr José Henriëtte Vos
geborenn te Veendam
PROMOTOR R
Prof.. Dr. W. Admiraal
OVERIGEE LEDEN VAN DE COMMISSIE
Prof.. Dr. M.W. Sabelis
Prof.. Dr. L.R. Mur
Prof.. Dr. G. Van der Velde
Prof.. Dr. M. Scheffer
Dr.. H.A. Verhoef
Dr.. W. Goedkoop
Dr.. M.H.S. Kraak
Faculteitt der Natuurwetenschappen, Wiskunde en Informatica
Universiteitt van Amsterdam
Thiss study was partly financed by RIZA-RWS (Directorate-General for Public Works
andd Watermanagement) and was conducted at the Department of Aquatic Ecology and
Ecotoxicology,, Institute for Biodiversity and Ecosystem Dynamics, University of
Amsterdam,, Kruislaan 320, 1098 SM, Amsterdam, The Netherlands.
ALLESS IS WAT HET IS OMDAT HET ZO GEWORDEN IS
D'ARCC Y THOMPSON 1917
MM
CONTENTS S
Page e
GENERALGENERAL INTRODUCTION
2..
3..
4..
5..
6..
9
INTERACTION BETWEEN FOOD AVAILABILITY AND FOOD QUALITY
DURINGG GROWTH OF EARLY INSTAR CHIRONOMID LARVAE
23
GROWTH RESPONSE OF A BENTHIC DETRITIVORE TO ORGANIC MATTER
COMPOSITIONN OF SEDIMENTS
41
PARTICLE SIZE EFFECT ON PREFERENTIAL SETTLEMENT AND GROWTH
RATEE OF DETRITIVOROUS CHIRONOMID LARVAE AS INFLUENCED
BYY FOOD LEVEL
63 3
NUTRITIONAL VALUE OF SEDIMENTS ASA FACTOR STRUCTURING
MACROFAUNAA COMMUNITIES IN SHALLOW EUTROPHIC WATERS
CONCLUDING REMARKS
REFERENCESS
83
107
H" 7
SUMMARYY
131
SAMENVATTINGG
139
NAWOORDD
141
rr
CHAPTERR 1
GENERALL INTRODUCTION
9 9
ChapterChapter 1
Inn all natural waters particles of non-living organic matter are present, which are
derivedd from autotrophic plants or dying animals. These non-living particles, or detritus,
supportt distinct assemblages of animals which use the particles as a food source. When
thiss food source is suspended it requires filter feeding to be consumed, but when the
detrituss particles are sedimented in aquatic systems, the bottom material serves as both
habitatt and food for detritivores. Detritivory is defined as the intake of nonliving particulatee organic matter together with the microorganisms that are associated with it
(Cumminss 1973). Although the general view of food webs supposes a dominance of
grazerss cropping plant material, these herbivores being eaten by carnivores, in fact as
muchh as 90% of primary plant production in aquatic systems enters the detritus food
webb (Pomeroy 1980). Detritivores form a major link in the foodweb processing
nutritionallyy low quality dead organic matter. Through their activity they stimulate
degradationn of organic matter by microorganisms (Ten Winkel et al. 1982, Harris 1983,
Shepardd & Minshall 1984, van de Bund & Davids 1993, van den Bund et al. 1994,
Goedkoopp 1994). In aquatic systems detritivores in soft sediments are one of the main
foodd sources for predators, like fish and birds, or carnivorous invertebrates such as
mitess and damselfly larvae (Healey 1984, Walde & Davies 1984, Hershey 1985a & b,
Hersheyy & Dodson 1987, Ten Winkel et al. 1989, review Wallace & Webster 1996).
Benthicc detritivores can be partioned into functional feeding groups based on
food-acquiringg mechanisms. Deposit feeders, also termed collector gatherers are bulkfeeders,, processing large volumes of sediment at high rate. Deposit feeders typically
processs at least one body weight of sediment daily. A way to attain a high rate of
sedimentt processing is to sort the sediment but reject most before ingestion as pseudofaeces.. Collector gatherers feed on fine particle detritus (Fine Particular Organic Matter,
FPOM,, < 1 mm) deposited on the substrate surface. Filtering gatherers feed on particles
inn suspension and therefore are also called suspension feeders. Shredders are
detritivorouss species that use plant debris with somewhat bigger particle size (Coarse
Particularr Organic Matter, CPOM, > 1 mm) compared to the gatherers. Shredders only
processs 0.01 to 0.4 times their body weights daily. Quite often taxa can not be assigned
too one particular functional feeding group only. For instance, some species of polychaetess have the ability to switch between suspension feeding and deposit feeding,
dependingg on the availability of suspended particles, while certain species of bivalves
10 0
GeneralGeneral Introduction
simultaneouslyy display both suspension and deposit feeding. Shredders often ingest
FPOMM while feeding on leaf litter (Cummins 1973, Cummins & Klug 1979, review
Lopezz & Levinton 1987).
Thee principal food sources for detritivores are materials that are derived from
algaee and macrophytes, but detritus may also include fragments of dead animals and
terrestriall run-off introducing soil particles and leaf litter. The nutritional value of the
foodd sources of detritivores varies spatially and temporally. Spatial differences depend
onn the contribution of the different sources to detritus. Detritus of large lakes will
containn more algal material formed in the water column, whereas detritus of small
streamss is likely to contain more material from macrophytes, eroded soil particles, and
leaff litter.
Detritivoress are confronted with different quantities of organic matter admixed
withh mineral particles with different nutritional qualities throughout the year, depending
onn a number of factors such as seasonal input of leaf litter, algal blooms, and macrophytess growth, on the depth of the watersystem, and the degradation rate of phytodetriruss (Johannson & Beaver 1983, Johnson 1987, Lopez & Levinton 1987, Moore
1987,, Marsh & Tenore 1990, Hill et al. 1992, Cheng et al. 1993, Ahlgren et al. 1997).
Seasonalityy of algal input to the benthic ecosystem is illustrated by a study of Ahlgren et
al.. (1997). Plankton net samples and sedimenting matter in traps from mesotrophic Lake
Erkenn were analysed for carbon, nitrogen, phosphorus, total lipids and fatty acid content
too follow seasonal changes in food availibility. Organic matter abundance in the pelagic
andd the benthic zone depended on respectively the presence and sedimentation of
phytoplankton.. Biochemical analyses of plankton and sedimenting matter showed that
thee benthic fauna have access to high-quality food only during spring and autumn due
thee dominance of diatoms during these periods. Studies on benthic-pelagic coupling in
thee field reported a rapid response of the macrofauna community to an increase of the
inputt from the pelagic zone, suggesting food limitation during much of the year (Graf et
al.. 1982, Graf 1989, Lopez & Levinton 1987, Goedkoop & Johnson 1996). Field studies
alsoo showed that algal blooms composed of different algal species caused different
responsess of local macrofauna communities (Marsh et al. 1989, Marsh & Tenore 1990,
Chengg et al. 1993) indicating regulation of macroinvertebrates by organic matter
composition.. Noting that food is a primary need for heterotrophic animals, the supply
11 1
ChapterChapter 1
ratee and quality of detritus particles reaching the benthos is a key limiting factor. It is
hypothesizedd that temporal and spatial differences in the availibility of food sources for
detritivoress are main regulating factors for communities of benthic invertebrates. This
regulationn is the main subject of this thesis.
Compositionn of organic matter in sediments
Compositionn of organic matter is dependent of its source and of the degree of
degradation.. During the complex process of decomposition the most labile components
aree degraded first. Conversely, the refractory matter is slowly broken down and tends to
accumulatee in the sediment (Fry 1987, Kemp & Johnston 1979). Therefore, depositfeederss mostly depend on low-quality organic matter as food bulk compared to organismss that consume selectively other food items (Bowen 1987, Ahlgren et al. 1997).
Decayy of the organic matter encompasses fragmentation, cell leaching, bacterial
decomposition,, and chemical oxidation. In the pelagic column zooplankters strip the
sestonn of useful biochemical compounds (Cavaletto & Gardner 1999). After sedimentation,, oxidation and fragmentation of organic matter continues on the lake bottom.
Bioturbationn of surface sediments may prolong the exposure of the sedimented matter to
oxidation-reductionn cycles.
Microorganismss are mostly the first to take advantage of the most labile
componentss that are released from decaying organic matter. Benthic microbial activity
andd biomass are observed to increase within hours after sedimentation of algae (Graf
1992).. In aerobic environments, aquatic hyphomycetes are dominant during the early
stagess of decomposition of plant matter. The fungal hyphae invade the structure of the
plantt and fungal exoenzymes break down the structural cellulose. This weakens the
plantt tissue. Bacteria typically colonize the remaining fragments.
Microbiall conditioning enhances the palatibility of leaf material to detritivores
(Barlocherr & Kendrick 1975, McGrath & Matthews 2000). Being part of detrital aggregatess microorganisms contribute to the potential food sources of detritivores. However,
bacteriaa are often estimated to contribute less than 1% to the weight of fine detrital
particles.. A number of studies showed that microorganisms' biomass consumed by
detritivoress accounts for less than 10% of the detritivore's growth (Bowen et al. 1984,
Findlayy et al. 1984). Other studies, however, stress that microbes are efficiently digested
12 2
GeneralGeneral Introduction
andd provide a nutrient rich diet (Martin et al. 1980, review Bowen 1987, review Graf
1992).. Even though the microorganisms' biomass constitutes a low share of the ingested
food,, small quantities of microorganisms may supply significant quantities of nutrients
suchh as vitamins and amino acids (review Phillips 1984a, Wolf et al. 1997). Thus,
colonizationn by microorganisms may increase overall nutritional value of originally
refractivee organic matter significantly, which is called microbial enrichment. The term
enrichmentt is also used because the microorganisms convert soluble components that
otherwisee would be lost for further consumption by benthic invertebrates into biomass.
Inn summary, degradation of organic matter by microorganisms is based on at least two
mechanisms.. 1) Microorganisms convert refractory material and soluble components
intoo microbial biomass which is more easy to digest and more nutritious compared to
thee degradating organic matter. 2) Microbial degradation breaks organic matter into
subunitss digestible for detritus-feeders.
Duringg the complex process of degradation the originally nutritious organic
materiall (e.g. from recently died algae) is deprived of the most nutritious components
andd only part of the lost biomass is replaced by microbial biomass. Therefore, detritivoress probably have to deal with food sources of low nutritional value which will vary
withh states of degradation and microbial conditioning. The present study aims to investigatee the variety in composition of food exploited by detritivores and thereby to relate
thee nutritional value of the food source for detritivores to its degradation state.
Nutritionall requirements of invertebrates
Assimilationn may follow basically similar patterns among different animals. The
needss of heterotrophic organisms in regard to nutrition can roughly be divided into two
classes.. Firstly, the organism needs energy for activity and internal maintenance. The
needd for energy can be satisfied by a variety of compounds that are oxidized and
thereforee is called a non-specific need. Secondly, heterotrophic organisms need a supply
off specific substances for synthesis of new tissue. Such specific needs can only be satisfiedd by a limited suite of organic compounds: the essential nutrients.
Influencee of food abundance, and hence of available energy, has been examined
inn several studies on a number of invertebrate species. Manipulation of food availability
inn laboratory experiments show that life history responses to food limitation range from
13 3
ChapterChapter 1
retardedd growth and an increase in longevity to lowered fertility and reproduction rate
andd as an extreme consequence, death. Changes in life history parameters due to food
limitationn lower the intrinsic rate of population growth.
Requirementss for some essential nutrients (e.g., polyunsaturated fatty acids and
vitamins)) are fairly consistent among vertebrates and invertebrates, whereas the
requirementss for others vary, depending on the particular taxonomie or physiological
groupp or developmental stage of the animal (Downer 1981, Phillips
1984A).
Polyun-
saturatedd fatty acids (PUFA) are a relatively well studied group of essential components.. PUFA are responsible for regulation of animal cell membrane physiology and
servee as precursors to eicosanoids. Eicosanoids are critical in a wide range of
physiologicall processes in invertebrates. Deficiencies in PUFA can impair functioning
off membranes and membrane-bound enzyme systems and are needed for optimal eggproduction,, egg-laying, spawning, and hatching (Brett & Müller-Navarra 1995).
Animalss can convert one form of PUFA to another through elongation and
desaturation,, but very few species can synthesize PUFA de novo (review Blomquist et
al.. 1991). Polyunsaturated fatty acids are almost exlusively synthesized by plants.
Consequently,, most freshwater organisms exhibit a strong dietary demand for PUFA.
Thee vast body of literature on aquaculture shows that diets rich in PUFA are essential
forr fishes, molluscs, crustaceans, and zooplankton. This literature is reviewed by Brett
&& Müller-Navarra (1995). Prawns are most thoroughly studied because of their
economicc importance as highly valued food for humans. Studies show that PUFA
contentt of artificial and natural diets impacts survival, growth, fecundity, egg
hatchability,, molting and osmotic stress tolerance of shrimps (D'Abramo & Sheen 1993,
Xuu et al. 1993, Rees et al. 1994). Prawns are primarily carnivores, but literature on
pelagicc herbivores also documents a strong dietary demand for PUFA-rich phytoplankton.. All herbivorous insects require PUFA of plant origin (Blomquist et al. 1991).
Addingg emulsions of PUFA to algae cultures can markedly increase the growth rates of
herbivorouss zooplankton (Brett & Müller-Navarra 1995).
Literaturee on PUFA-demands of benthic detritivores is scarce. Although overall
nutritionall demands of organisms may be similar among taxa it should be noted that
adaptationss in specific species may have taken place due to existence of special environmentall conditions or specific modes of behavior. In regard to modes of feeding physio-
14 4
GeneralGeneral Introduction
logicall adaptations may have occurred to be able to digest certain food sources. Hanson
ett al. (1985) presented a study on the fatty acid composition of aquatic insects from
differentt orders and with different modes of feeding. Fatty acid composition differed
predictablyy among orders and functional feeding groups, most noticeably for the
polyunsaturatedd fatty acids. Collector gatherers of the order Diptera contained relatively
lowerr levels of PUFA than filterers, predators, scrapers, and shredders from the same
order,, implying that the collector gatherers use diets poor in PUFA compared to the
foodd sources of the Diptera taxa with other modes of feeding. Collector gatherers from
thee order Trichoptera, however, contained levels of PUFA comparable to PUFA levels
measuredd in Trichoptera specimen with other feeding modes. Thus, the effect of food
sourcess on detritivores cannot be predicted with precision by means of data on other
orderss of insects with similar modes of feeding or data on insects of the same order but
withh different feeding modes.
Studiess on detritivores
NutritionalNutritional demands
Literaturee on the influence of food composition on detritivores is limited and
concentratess on the effect of microbial enrichment (Martin et al. 1980, Bowen 1987),
foodd particle size selection, and ingestion rate as a function of food value (Taghon
1982),, but mostly does not take biochemical composition into consideration. Food
particlee size selection often is the result of the limitation of most detritivores in their
abilityy to handle or ingest certain particle sizes (Jumars et al. 1982, Taghon 1982). Some
studiess on detritivores indicate that particle size selection originates from the aim to
maximizee net rate of energy gain. Two schools of thought have arisen on the effects of
foodd quality on ingestion rate. One group reports that ingestion rate vary inversely with
foodd quality as mechanism to maintain constant intake rate of some food component,
suchh as energy (Calow 1975, Cammen 1980, Phillips 19846). Conversely, others find
ingestionn rate to be positively related to food quality (refs. in Taghon 1981).
DigestionDigestion of refractory food sources
Detritivoress seem to have adapted their digestion system to the low palatibility
off their food. Bjarnov (1972) compared the digestion of saccharides by Chironomus
15 5
ChapterChapter 1
plumosusplumosus and C. antracinus, Gammarus pulex, and various Trichoptera which have
differentt feeding modes and different food sources. Significant differences between
speciess was only found for the degradation of polysaccharides. All a- and fi -glucosides
andd galactosides were degraded by all tested species. Marked species differences existed
inn the degradation of pectin, xylan and chitin. Digestion of pectine and xylan was
clearlyy observed for 5 species of Trichoptera, which were all shredders or suspension
feeders.. The carnivores were better adapted to digest chitin. The results suggest that
mostt invertebrates are not able to digest the long chain cellulose and other structural
polysaccharidess on their own, but also show that aquatic invertebrates have adapted
theirr digestion to their particular food source.
AA number of studies focussed on the digestive systems in stream detritivores,
e.g.. several Gammarus species, Hydropsyche betteni, Tipula caloptera, T. abdominalis,
PteronarcysPteronarcys proteus, and Pycnopsyche luculenta (Martin et al. 1980, Sinsabaugh et al.
1985,, Barlocher & Porter 1986, Chamier & Willoughby 1986, Chamier 1991, McGrath
&& Matthews 2000). The ability to digest major plant polysaccharides was restricted to
thee Gammarus species, Pteronarcys proteus, and Pycnopsyche luculenta, but enzymes
capablee of breaking glycosidic linkages, similar to enzymes that are released during
microbiall breakdown of leaf polysaccharides, were present in the gut fluid of all
animals.. Ingested fungi are partly responsible for the cellulase activity in the gut of
GammarusGammarus species, but production of an endogenous cellulase system by the insects has
alsoo been noted (Sinsabaugh et al. 1985, Chamier & Willoughby 1986, Chamier 1991,
Harriss 1993).
Forr grazers percentages of 10 to 50 % of the plant material that is actually
consumedd are mentioned not to be digested but excreted as faeces or pseudofaeces
(Pomeroyy 1980). Since detritivores cope with more refractory food than grazers the
percentagee of ingested food that is not digested probably will be higher. Furthermore, a
rapidd rate of passage through the gut probably allows little time for most detritivores to
exercisee extensive digestion and therefore will not be able to thoroughly digest cellulose
orr other polysaccharides (Bjarnov 1972).
16 6
GeneralGeneral Introduction
SedimentSediment as habitat
Sedimentt constitutes both the food source and the physical environment of
benthicc detritivores. The physical characteristics of sediments are mainly determined by
particlee size distribution and organic matter. The impact of organic matter content and
grainn size distribution on detritivores in the field is difficult to distinguish, because they
oftenn covary in natural sediments (e.g., Rabeni & Minshall 1977, Pinder 1986, 1995,
Suedell & Rodgers 1994, Maxon et al. 1997, Reinhold-Dudok van Heel & den Besten
1999). .
Severall ways have been proposed in which particle size could affect preference
andd performance of a sediment inhabitant. Particle size distribution of a substrate may
influencee the suitability of a substratum to borrow in as reflected by penetration rate by
thee organisms (Wiley 1981a, Winnell & Jude 1984). Particle size distribution determiness if the organism is able to construct a living tube, because many insects are limited
inn their ability to handle particles of certain sizes (Brennan & McLachlan 1979).
Inhabitingg a tube diminishes the risk of predation by damselflies, stoneflies, mites and
fishfish as is found in several laboratory and field studies (Hershey 1985 & 1987, Ten
Winkell 1987, Macchiusi & Baker 1991, Baker & Ball 1995). Particle size distribution
andd organic matter also influence oxygen concentrations within the substrate, consequentlyy selecting the species that are able to sustain themselves in the substrate
(Verdonschott 1990, Heinis 1993).
Detritivoress may show preference for a distinct type of habitat. Such preference
forr a habitat is often related to the options for the organisms to develop (Wiley 1981ft).
Suitabilityy of a sediment type can be tested through the migration of an organism from a
referencee substrate, although migration towards a suitable substrate has hardly been
observedd over larger distances (Wiley 1981a, Butman 1987, Rosillon 1987). During a
laboratoryy study by Sibley et al. (1998) on preferences of Chironomus tentans larvae the
interactionn of physical characteristics with organic matter content was obviated by using
minerall particle substrates with different particle size ranges and by imposing uniform
oxygenn conditions. C. tentans larvae consistently selected the smaller particle size range
off two substrates when they only needed to travel short distances (cms).
Nott all detritivores will be able to handle the broad range of particle sizes as
chironomidd larvae do. In addition, chironomid larvae are probably more tolerant to low
17 7
ChapterChapter 1
oxygenn levels in substrates than other detritivores. One other possible mode of action of
particlee size distribution on detritivores has still been unexplored. Deposit-feeders ingest
inorganicc particles together with the organic detritus (Rasmussen 1984, Lopez &
Levintonn 1987). I expected that ingestion of inorganic material obstructs food uptake
andd therefore may hamper growth of detritivores.
Biologyy and ecology of the test organism Chironomus riparius
Too study the impact of nutritional value of sediments we choose C riparius as
modell species being a well-known deposit-feeder (Rasmussen 1984). C. riparius
belongss to the dipteran family of Chironomidae which encompasses at least 15,000
differentt species. The family is the most widely distributed group of insects, having
adaptedd to nearly every type of aquatic or semiaquatic environment, ranging from large
lakess to small streams (Armitage et al. 1995, Lindegaard & Brodersen 1995, Batzer &
Wissingerr 1996, Silver Botts 1997). The midges account for most of the macroinvertebratee numbers in freshwater environments. In many aquatic habitats this group
constitutess more than half of the total number of macroinvertebrate species present.
Chironomidss are often the earliest colonizers to arrive in newly formed or disturbed
habitatss (Sheldon 1984, Layton & Voshell 1991, Batzer & Wissinger 1996).
Abundancess of certain species or species groups of Chironomidae are often used to
characterizee types of watersystems (Saether 1975, Resh & Rosenberg 1984, Johnson
1995)) and as biological indicators of water quality.
Deposit-feedingg is the most common feeding mode exhibited by chironomids.
Mostt detritivorous and tube-dwelling chironomids feed by extending the head and
anteriorr part of the body outside the tube while using the posterior prolegs to maintain
contactt with the inner surface of the tube. Therefore, foraging areas are restricted to a
regionn immediately surrounding the tube. Detritivores cope with special feeding conditions.. Firstly, the organisms mostly deal with organic matter of low nutritious value as
wass argued above. The second special condition is the high mineral particle content of
mostt sediments. Mineral particles may be ingested along with organic matter, thereby
potentiallyy reducing the nutritional value of the food intake.
Thee larvae of C. riparius are found in both lentic and lotic environments. The
speciess favours eutrophic conditions or conditions with organic loading (Armitage et al.
18 8
GeneralGeneral
Introduction
1995),, where it can reach densities upto 50,000 individuals per square meter (Köhn &
Frankk 1980, Rasmussen 1984). Larvae of C. riparius are known as "bloodworms"
especiallyy to fishermen due to their red colour. The red colour is caused by haemoglobinee which aids the chironomids to tolerate reduced levels of dissolved oxygen. Low
oxygenn levels are rarely a problem for invertebrates living on stones or submerged
macrophytess but may cause problems for species such as chironomid larvae that live in
softt sediments with a high content of organic matter. Oxygen consumption within mud
cann create sharp gradients of reducing oxygen concentration in the few millimetres or
centimetress above the sediment (Watling 1991, Heinis 1993, Armitage et al. 1995).
Thee ability of chironomids to construct tubes decreases the risk of predation by
vertebratess and invertebrates and may minimize dislodgement by currents. C. riparius is
knownn to build protective tubes from detritus, algae and other sediment particles. Particless are joined together with the larvae's saliva (Edgar & Meadows 1969). Larvae are
capablee of handling only a certain range of particle size for tube construction. Similarly,
penetrationn of a substrate by chironomid larvae is dependent of particle size distribution
(Armitagee et al. 1995, Wiley 1981a). Thus, the suitability of a substrate for chironomids
too settle in depends on the particle size distribution.
Thee life-cycle of chironomids comprises an egg stage, four larval stages, and a
pupall stage, which all live in the aquatic environment. In principal the first instar larvae
aree planktonic, while older individuals inhabit the upper layer of the sediment.
Winteringg of C. riparius occurs in the 3rd or 4th larval stage. The pupal stage lasts a few
Fourthh instar larvae of Chironomus riparius.
19 9
ChapterChapter 1
dayss and takes place in the mud. These stages of aquatic life are followed by a terrestrial
adultt stage which does not feed. Adults often emerge simultaneously and form vast
matingg clouds. After swarming and mating of male and female adults, the females
depositt egg masses at the water surface and attach them to some kind of substrate. Egg
massess may contain upto 600 eggs. At 20 °C hatching occurs within three days after egg
deposition.. In temperate regions C. riparius displays multivoltine life cycles (Groenendijkk et al. 1996) although the number of consecutive generations per year is strongly
relatedd to the water temperature (Mackey 1977).
Lifee cycle of Chironomidae displaying the egg stage, the four larval instars, the pupal
stagee and the terrestrial imago (adopted from Timmermans 1991).
20 0
GeneralGeneral Introduction
Objectivess of this study
Althoughh food is recognized as potentially important factor for macrofauna
communities,, the influence of sedimentary organic matter and its biochemical compositionn on benthic detritivores has been neglected so far. Therefore, this study aims to
clarifyy the influence of the nutritional value of sediments on benthic detritivores. Focal
pointss are the influence of sedimentary organic matter and its biochemical composition
onn the survival and growth of benthic detritivores and the interaction between particle
sizee distribution and nutritional value of sediments.
Duringg the present study efforts were made to answer the following questions:
1)) Which biochemical components of potential food influence growth and survival of
ChironomusChironomus riparius under controlled conditions and which components
influencee growth of this species in natural sediments?
2)) Do physical characteristics of sediments, which serve simultaneously as food and
habitat,, alter behaviour, growth, and survival of C. riparius!
3)) How do differences in nutritional value of sediments lead to differences in multispeciess communities of detritus feeders in the field?
21 1
ChapterChapter 1
Outlinee of this thesis
Thee present study aims to clarify the influence of nutritional value of sediments
onn detritivores. Firstly, the effects of organic matter abundance and composition on
detritivorouss invertebrates were assessed. For this purpose a set of artificial food items
weree analyzed biochemically and offered in concentration series to first instar larvae of
thee model species Chironomus riparius in standardized mineral substrate (Chapter 2).
Growthh after one week at limiting food levels and at excess of food was correlated to
biochemicall composition of the food and
keyy parameters for food limitation and
saturationn of growth were derived. The
resultss of this study were verified for the
fieldfield situation in Chapter 3. In this
chapterr sediments were sampled in the
fieldfield and growth of chironomid larvae on
thesee substrates was determined in the laboratory. The sediments were analyzed for a set
off biochemical variables, water content, and particle size distribution. Correlations were
soughtt between the biochemical variables and larval growth. The generally sub-optimal
growthh of chironomid larvae measured in natural sediments was shown not to be caused
byy physical characteristics of the substrates tested. Yet, results suggested that
indigestiblee sediment particles which were ingested indiscriminately with food reduced
thee growth potential. Therefore, Chapter 4 focused on the effect of particle size distributionn on chironomid larval growth using artificial mineral substrates. Simultaneously,
preferencee of the chironomid larvae for the particle size distribution was examined.
Becausee suitability of substrates may be expressed in preference of organisms, the
activee search strategies of the larvae for sediment substrates with different food
concentrationss was also explored in Chapter 4. In Chapter 5 the observations on the
effectt of nutritional value of sediments on the model species C. riparius is extrapolated
too the field. Biochemical composition of a number of sediments is correlated to the
densitiess of taxa, with reference to modes of feeding of the benthic invertebrates. In
Chapterr 6 the main findings of this thesis are summarized and reviewed.
22 2
CHAPTERR 2
INTERACTIONN BETWEEN FOOD AVAILABILITY AND FOOD QUALITY
DURINGG GROWTH OF EARLY INSTAR CHIRONOMID LARVAE
J.. H. Vos1, M. A. G. Ooijevaar, J. F. Postma2 and W. Admiraal. 2000.
Journall of the North American Benthological Society 19: 158-168.
Departmentt of Aquatic Ecology and Ecotoxicology, University of Amsterdam, Kruislaan
320,, 1098 SM Amsterdam, The Netherlands, 'E-mail: [email protected], 2present
address:: AquaSense, P.O. Box 95125, 1090 HC Amsterdam, The Netherlands
23 3
ChapterChapter 2
Abstract t
Thee nutritional requirements of sediment-feeding invertebrates are poorly
understood.. Therefore, growth experiments with larvae of the midge Chironomus
ripariusriparius (Meigen) were performed using food items of differing composition. Firstinstarr larvae were reared in the presence of different concentrations of each food item,
andd larval length and instar were recorded after 1 wk. Saturation growth curves were
fitted,fitted, and for each food item the slope and the maximum length attained by larvae were
estimated.. Food items were analyzed for organic matter, C, N, P, carbohydrates,
proteins,, and total fat content. Maximum length attained by larvae reared on fish foods
andd on food items of animal origin was higher than maximum length reached on food
itemss of plant origin. In general, slopes of the growth curves for larvae reared on foods
off plant origin were higher than slopes of the growth curves for larvae reared on fish
foodd and food of animal origin. Foods of plant origin had lower N, P, and lipid content
andd higher carbohydrate content than fish food and food of animal origin. Ordination of
foodd composition and the saturation growth-curve parameters indicated that the optimal
foodd composition depended on the amount of food available. For instance, high N, P,
andd lipid contents stimulated growth at high food levels, whereas the amount of
carbohydratee appeared to be important in defining growth at low food levels. We
suggestt that this interaction is caused by limiting energy availability at low food levels
versusversus limiting food quality at high food levels.
24 4
QualityQuality of organic matter as food for chironomids
Introduction n
Thee role of food as a factor regulating population dynamics of sediment-feeding
freshwaterfreshwater invertebrates is widely recognized (e.g., Rasmussen 1985, Pinder 1995).
Bothh quantity (Ristola 1995) and quality of food are principal factors influencing the life
historiess of benthic invertebrates (Beenakkers et al. 1981). Food quality often is
investigatedd in the framework of microbial production or enrichment (e.g., Cummins &
Klugg 1979, Ward & Cummins 1979, Findlay et al. 1984, Phillips 1984, Bowen 1987,
Couchh et al. 1996). Studies in which life-history parameters of benthic invertebrates are
relatedd to the biochemical composition of food are scarce, probably because of problems
withh chemical analyses. These benthic invertebrate studies are performed mostly with
artificiall diets and focus on only 1 component or 1 category of components (Cowey &
Forsterr 1971, Roman 1983, Cargill et al. 1985, D'Abramo & Sheen 1993).
Inn contrast to benthic invertebrates, the role of food composition for zooplankton,, especially cladocerans,
has been studied more extensively. Most of this
researchh has focussed on elemental C, N, P, and on fatty acids (e.g., Brett & MiillerNavarraa 1997, DeMott & Müller-Navarra 1997, Gulati & DeMott 1997), although
additionall components (carbohydrates and protein) also have been studied (Sterner
1993,, Cowie & Hedges 1996, Kilham et al. 1997, Lürling & van Donk 1997). Both P
andd polyunsaturated fatty acids (PUFAs) are positively correlated with food quality for
pelagicc grazers, in terms of the chemical composition of living phytoplankton. These
samee parameters may not be suitable as indicators of food quality in sediments because
processess such as zooplankton grazing, chemical oxidation, and bacterial decomposition
havee taken place before phytoplankton settles on the sediment as detritus (Bowen 1987,
Ahlgrenn et al. 1997). The organic matter in sediments is of lower nutritional value
comparedd to living phytoplankton, as shown by the large differences in the PUFA
contentt of the 2 food resources (Ahlgren et al. 1997). Thus, food composition may be a
limitingg factor in the life history of detritus-feeding invertebrates.
Thee objective of our study was to determine the relative importance of both food
quantityy and composition for benthic invertebrates. We focussed on artificial food
sources,, reasoning that the parameters with the strongest interaction with invertebrate
growthh would be promising factors for future characterization of food quantity and
25 5
ChapterChapter 2
qualityy in freshwater sediments. Early instar larvae of the midge Chironomus riparius
weree used as test organisms because chironomids represent an abundant group of
benthicc insects in freshwater ecosystems.
Methods s
FoodFood items
Tenn different food items were tested: 2 fish foods that often are used in
laboratoryy experiments (Trouvits and TetraMin®), 2 food items of animal origin
{Gammarus{Gammarus and Chaoborus), 4 items of plant origin (Ceratophyllum, Potamogeton,
Utriculaha,Utriculaha, and Populus), 1 algal species {Scenedesmus acuminatus), and 1 yeast
{Saccharomyces{Saccharomyces cerevisiae). Trouvit® and TetraMin® Flaked Staple Food are comm
ciallyy available fishfoods. Gammarus pulex originated from Tetra Gammarus® and
ChaoborusChaoborus partim from Ruto Frozen Fishfood®. Living leaves of Ceratophyllum
demersum,demersum, Potamogeton lucens, and Utriculaha vulgaris were harvested in June 1996
att Lake Maarsseveen, an oligo-mesotrophic lake situated in the center of the Netherlands.. Leaves of Populus tremula were gathered as fresh litter from the ground at
Pampus,, Almere, in August 1996. The leaves were gently washed with destilled water
andd major veins were removed before further treatment. Scenedesmus acuminatus was
grownn in batch culture using Wood's Hole medium (Guillard 1975) without Na2Si02.
Thee algae were grown at 20°C and 200-220 uE-m~2s"'. After 3-4 d the algae were
harvestedd by filtration over a Whatman 0.45 um GFC filter and rinsed with distilled
water.. Yeast was bought at a bakery.
Alll food items were freeze-dried, ground, sieved through a 106-ftm mesh, and
storedd at -18°C before conducting chemical analyses and growth experiments.
GrowthGrowth experiments with series of food concentrations
Growthh experiments were conducted in polyethylene containers (10 x 10 x 6.5
cm)) containing 50 g of prewashed and combusted Litofix® sand (<500 fim, heated at
550°CC for 6 h). Two hundred ml of artificial freshwater (Dutch Standard Water, pH 8.2,
2100 mg CaC0 3 , not sterile) were added per container, and the water was aerated
26 6
QualityQuality of organic matter as food for chironomids
constantly.. The food items were tested in concentration series, and each series was
replicatedd 4 times.
Eachh concentration series consisted of 9 containers in which freeze-dried food
wass added to the overlying water and allowed to settle on the sand. The food amounts
rangedd from 0 to 128 mg ( 1% m/m of sediment per container). For most food items, 0,
2,, 4, 8, 16, 32, 64, 96, and 128 mg of food were used, resulting in 36 containers per food
item.. Different concentrations were used for Scenedesmus, Populus, and yeast to cover
thee complete saturation growth curve, i.e. 25 and 50 mg for Scenedesmus and yeast, and
10,, 25, 160, 200, and 400 mg for Populus. Replicates of each concentration series were
startedd on different days to ensure complete statistical independence. Growth experimentsriments on a concentration series were considered successful if larval survival exceeded
80%% in the 96-mg containers. Preliminary growth experiments showed that survival at
saturatingg food levels was optimal and generally exceeded 80% after 7 d of incubation.
Alll growth periods were restricted to 7 d to avoid possible bacterial or fungal growth or
developmentt of anaerobic conditions in high-food concentrations. Experiments were
conductedd at 20°C
1°C with a light/dark regimen of 16:8 h under incandescent light.
Forr each concentration series, larvae (< 24 h old) hatched from at least 10 egg
massess of laboratory cultured Chironomus riparius (Meigen) were combined, and 20
randomlyy selected individuals were added to each container. An additional group of 20
larvaee was randomly collected to determine the length of the larvae at the start of the
experiment.. The length of all surviving larvae was measured after 7 d, and survival in
eachh experimental unit was reported. Larval length was measured rather than biomass
becausee the biomass of individuals was below the accurate detection limit of the
analyticall balance. The duration of the experiment was restricted to 1 wk to ensure that
larvaee did not reach the 4th instar. Under favourable food conditions the larvae enter the
33rd instar within 1 wk. The 4th instar was avoided because during that stage both growth
andd development towards the pupal and adult stage occur, and the larvae become
sexuallyy dimorphic (Gilbert 1967, Beenakkers et al. 1981). Moreover, last-instar larvae
off several Diptera species have a different chemical composition than earlier instars,
particularlyy a pronounced increase of lipid content (Gilbert 1967, Beenakkers et al.
1981),, which may imply a difference in growth response to food composition compared
too earlier-instar larvae.
27 7
ChapterChapter
2
TetraMin n
Trouvit t
jj
I
I
Gammarus Gammarus
ii
oo
i
i
i
I
i_
Chaoborus Chaoborus
i
ii
i
i
££
11 3
rr
"Si i
22
ff
2
11
Potamogeton Potamogeton
Ceratophyllum Ceratophyllum
OO
jj
I
I
I
I
i_
i
_ ii
i
i
I
I
i _
44
33
rr
22
11
Scenedesmus Scenedesmus
Utricularia Utricularia
OO
jj
I
I
I
i
i
JJ
I
I
I
I
L_
_ii
I
I
I
I
i_
20
30
40
50
60
44
33
_ii
i
yf^—i— yf^—i—
22
11
OO
00
Yeast t
Populus Populus
JJ
10
I
I
I
I
I
20
30
40
50
60
0
10
70
foodd (mg organic matter container 1)
Fig.. 1. Length (
SE) of Chironomus riparius larvae after 1 wk of growth on 10 food items as a
functionn of food concentration and the saturation growth curves.
28 8
QualityQuality of organic matter as food for chironomids
ChemicalChemical analyses
Thee organic matter content (ash-free dry mass, AFDM) of food samples was
determined,, according to the loss-on-ignition technique, by combusting samples at
550°CC for 6 h (Luczak et al. 1997). Total C and N were measured with a Carbo-Erba
Elementt Analyser. Total P was determined according to Murphy and Riley (1962) and
proteinss were determined following Lowry et al. (1951). Carbohydrate analysis was
conductedd using the phenol-sulphuric acid-method of Dubois et al. (1956). Total lipid
contentt was measured gravimetrically after 6 h of soxhlet-extraction using n-hexane (de
Boerr 1988). Energy content was calculated by assuming that 1 g of fat releases 38.9 kJ,
11 g of protein releases 17.3 kJ, and 1 g of carbohydrate yields 16.9 kJ.
StatisticalStatistical analyses
AA saturation growth curve was fitted using the least squares method to estimate
thee slope (k) and the maximum value (lmax) of the saturation growth curves for larvae
rearedd on the individual food items. This model provided easily interpretable parameters
andd a good fit was obtained. The model follows the equation:
.,, food ,
- ( * ** —
// - /
wheree
_ ƒ - U *
_ (1
ll — l max
\l max
1
lmaxx
100
t nun ) * t
= larval length (mm) for a certain food level, achieved after 7 d of growth,
=
maximum larval length (mm), achieved on an individual food item after
77 d of growth,
lminn = minimum larval length (mm), achieved after 7 d of growth with no food
present, ,
kk
= slope of the growth curve (mm mg"1 organic matter), and
foodd = food level (mg organic matter added to the experimental unit).
Bothh maximum larval length and slope of the saturation growth curve for each
concentrationn series were estimated using averages of larval lengths for each container.
Foodd was expressed as the amount of food added per container in terms of mass of
organicc matter (AFDM). Lmax was used as an indicator of optimal growth on the specific
foodd item, k was used as an indicator of the effect of additional food on growth when
foodd level was non-saturating.
29 9
ChapterChapter 2
Multivariatee ANOVA (MANOVA) was used to analyze differences in lmax and k
betweenn food items, using the saturation-curve parameters that were estimated for each
concentrationn series. The chi-square test of heterogeneity of independence (Lozan 1992)
wass used to analyze differences in food composition, using the averages of food
componentt contents per food item, expressed as %s of total freeze-dried matter and of
organicc matter. This rather unusual test was used because it can accommodate a large set
off dependent variables such as the chemical parameters of the food items.
Multivariatee analyses (redundancy analysis [RDA] and detrended correspondence
analysiss [DCA]) were performed using the canonical community ordination program
CANOCO££ 3.11 (ter Braak 1988). Chemical parameters of food, expressed as %s of
organicc matter, were chosen as "species", food items as "samples", and lmax and k,
resultingg from fitting the saturation growth curve, as "explanatory variables". The chemicall parameters were ln(x+l) transformed, centered, and standardized to correct for differencess in the range of measurements between the different parameters. First, the length
off gradient was calculated by DC A to determine the model that was followed by the
relationshipp between the chemical parameters of food items and lmax and k. Because the
lengthh of gradient was < 2.0 (0.7), the linear response model (RDA) was chosen for final
ordinationn (ter Braak 1986). This ordination technique constrained the ordination axes to
Tablee 1. Food composition expressed in %s of organic matter content (
SE). Coefficients of
variationn (CV, %) for each of the food composition variables are given in the last row.
%% organic
Biochemicall analysess (%)
Energyy content
(kJJ g"1)
matter r
carbohydrates s
Trouvit ' '
86.8(0.14) )
20.11 (0.64)
TetraMin® ®
87.66 (0.05)
30.11 (0.99)
6.33 (0.07) 37.66 (2.63)
14.44 (0.50)
Gammarus Gammarus 74.55 (0.09)
12.7(0.59) )
11.66 (0.13) 41.8(0.28) )
14.22 (0.08)
0
lipids s
protein n
17.99 (0.05) 38.22 (2.54)
17.3(0.54) )
Chaoborus Chaoborus 93.33 (0.09)
8.77 (0.40)
14.22 (0.56) 42.11 (0.66)
14.66 (0.25)
Ceratophyllum Ceratophyllum
91.11 (0.34)
53.55 (0.86)
3.99 (0.20) 40.11 (0.60)
17.9(0.21) )
Potamogeton Potamogeton
80.55 (0.05)
45.8(0.91) )
2.44 (0.09) 47.88 (0.60)
17.4(0.21) )
Utricularia Utricularia
85.99 (0.28)
45.77 (0.59)
4.66
37.00 (0.47)
16.3(0.13) )
-
Scenedesmus Scenedesmus
95.8(0.17) )
42.6(1.39) )
9.66 (0.63) 36.88 (0.26)
17.7(0.33) )
Populus Populus
90.2(0.31) )
38.8(0.91) )
3.55 (0.10) 50.8(1.58) )
17.2(0.32) )
Yeast t
93.7(0.13) )
41.11 (1.01)
2.22 (0.04) 42.33 (0.68)
15.5(0.21) )
7.4 4
45.1 1
CV V
30 0
71.5 5
11.3 3
9.2 2
QualityQuality of organic matter as food for chironomids
bee linear combinations of lmax and k. A direct gradient analysis was used because the
experimentall setup aimed to analyze the influence of food composition on growth. The
variancee inflation factors (VIFs) of lmax and k were used to determine the correlation
betweenn parameters. The VIFs of lmax and k were both below 2, implying low collinearity,, and consequently none of the variables was deleted from the dataset. In the
resultingg ordination diagram, positively correlated chemical parameters, food items, and
growth-curvee parameters were placed near each other, and negatively correlated objects
weree placed far apart.
Results s
FoodFood composition
Thee food items had comparable organic matter contents, ranging between 75 and
96%% (Table 1). Between 65% (Chaoborus) and 98% {Ceratophyllum) of the organic
matterr in food items consisted of carbohydrates, lipids and proteins. Consequently,
betweenn 35 and 2% of the organic matter was not analyzed by the biochemical analyses.
Organicc matter, C, protein, and energy content were similar among foods with coefficientss of variation (CV) of < 11 %, calculated across all food items. The other chemical
Tablee 1. Extended
Elementall analyses (%)
CC
Elementall ratios
NN
PP
C/N N
C/P P
N/P P
55.88 (0.36)
9.66 (0.05)
1.2(0.04) )
5.88 (0.03)
47.9(1.57) )
8.22 (0.28)
53.55 (0.08)
9.11 (0.01)
1.2(0.10) )
5.99 (0.00)
46.00 (5.08)
7.88 (0.87)
55.5(0.19) )
10.3(0.04) )
1.3(0.06) )
5.4(0.01) )
41.7(1.86) )
7.77 (0.34)
51.6(0.08) )
11.11 (0.03)
1.2(0.04) )
4.7(0.01) )
40.99 (0.83)
8.8(0.16) )
46.44 (0.46)
4.11 (0.03)
0.3(0.01) ) 11.4(0.07) ) 177.5(9.71) )
15.6(0.81) )
48.7(0.18) )
4.33 (0.03)
0.3(0.01) ) 11.2(0.09) ) 134.11 (3.97)
12.0(0.39) )
47.33 (0.40)
4.22 (0.03)
0.33 (0.02) 11.2(0.09) ) 152.0(7.82) )
13.6(0.79) )
52.99 (0.08)
5.33 (0.05)
0.66 (0.02) 10.0(0.08) )
80.0(1.08) )
8.0(0.12) )
51.7(0.72) )
4.11 (0.06)
0.22 (0.02) 12.66 (0.07) 231.8(30.3) )
18.4(2.37) )
50.44 (0.42)
9.22 (0.07)
1.5(0.04) )
6.2 2
41.3 3
61.8 8
5.55 (0.03)
37.7 7
33.11 (0.92)
71.5 5
6.11 (0.16)
38.3 3
31 1
ChapterChapter 2
parameterss (%N, %P, %carbohydrates, %lipids, C/N, C/P, and N/P) had CVs of > 35%.
Forr example, N content of the food items varied between 4 and 11%. The lowest N
contentss were found in plant items, whereas the highest %s were found in animal foods.
PP content also was lowest in the plant materials (< 0.64%). In contrast, the carbohydrate
content,, which varied between 9 and 53%, was lowest in the animal foods and highest in
thee plant (macrophyte and algae) foods. Last, lipid content was lowest for yeast,
followedd by the plant items, whereas the highest lipid contents were observed in
Trouvit®® and the animal foods. Significant differences in composition between food
itemss are shown in Table 2. In general, food composition was similar among items of
plantt origin, and among items of animal origin and fish foods.
GrowthGrowth and survival
Larvall survival generally exceeded 80%, except in the experimental units with
thee lowest food levels (2 mg organic matter container"1), where survival ranged between
255 and 100%. Maximum larval length was reached at 70 mg food container"1 for all food
itemss {Fig. 1). The x-axis of Fig. 1 is truncated at 70 mg food container"1 to present
differencess in slope clearly.
Averagee length of 1st instar larvae at the start of the growth experiments was 0.95 (
0.02)) mm. After 7 d larval length had reached 1.37 ( 0.02) mm in the experimental
unitss in which no food was added (lmin). Larval length after 7 d of growth increased with
increasingg food concentrations, but the food concentrations at which lmax was reached
Tablee 2. Chi-square test of heterogeneity of independence values (20 df). Significant differences
(P(P < 0.05) between chemical composition of food items are indicated with an *.
Trouvit® ®
TetraMin® ®
TetraMin® ® Gammarus Gammarus
Chaoborus Chaoborus
Ceratophyllum Ceratophyllum
0.440 0
Gammarus Gammarus0.802 2
0.036* *
Chaoborus Chaoborus0.269 9
0.001* *
0.956 6
Ceratophyllum Ceratophyllum
0.000* *
0.000* *
0.000* *
0.000* *
Potamogeton Potamogeton
0.000* *
0.000* *
0.000* *
0.000* *
0.692 2
Utricularia Utricularia 0.000* *
o.ooo* *
0.000* *
0.000* *
0.999 9
Scenedesmus Scenedesmus
0.007* *
0.290 0
0.000* *
0.000* *
0.000* *
Populus Populus
0.000* *
0.000* *
0.000* *
0.000* *
0.251 1
Yeast t
0.006* *
0.794 4
0.000* *
0.000* *
0.000* *
32 2
QualityQuality of organic matter as food for
chironomids
differedd between food items. For example, larvae grown with Ceratophyllum,
Potamogeton,Potamogeton, Utricularia, and Populus reached their lmax at food levels of 10 to 20 mg
organicc matter container"1. In contrast, larvae grown with Scenedesmus reached their lmax
att food concentrations of 70 mg organic matter container"1. Midge larvae fed plant items
reachedd lower lmax values (2.5 to 3.9 mm) than midge larvae fed artificial fish foods and
animall matter (3.7 to 4.8 mm) (Table 3). The slopes of the saturation growth curves for
larvaee fed plant items and Trouvit® were greater than for the other foods. Significant
differencess between saturation growth-curve parameters of the individual food items
occuredd between almost all food items (Table 4).
OrdinationOrdination triplot
Thee ordination explained 55% of the variance in the chemical parameters of the food
items,, unevenly divided over 2 axes (Fig. 2). Most of the variance of the data set was
expressedd on the 1st axis (48%), whereas the 2nd axis explained only 7%. The food items
weree ordinated roughly into 2 groups, consisting of either plant and algal material or
commerciall fish food and animal items. Maximum length was most strongly associated
withh the commercial fish foods and animal items group. In general, these foods were
highh in C, N, P, and lipids, and lmax was positively and significantly correlated with %C,
%N,, %P, and %lipids (Table 5). Slope was most strongly associated with the plant and
algall group (Fig. 2). In general, these foods were high in C/N, C/P, N/P, and carbohydratess and k was positively and significantly correlated with the ratios C/N, C/P, and
Tablee 2. Extended.
PotamogetonPotamogeton
0.945 5
0.006**
Utricularia
Scenedesmus Populus
0.005*
0.1066
0.297
0.000*
0.000**
0.000*
0.007*
0.000*
33 3
ChapterChapter 2
Tablee 3. Saturation growth-curve parameters ( SE). Maximum length (Lax, mm) and slope {k,
mmm mg" organic matter). R2 was calculated for the whole saturation curve as an estimate for
thee goodness of fit.
F? F?
kk
'max x
Trouvif f
3.77 (0.07)
19.99 (4.22)
0.82 2
TetraMin® ®
4.00 (0.20)
9.99 (3.28)
0.63 3
Gammarus Gammarus4.88 (0.18)
8.22 (1.71)
0.82 2
Chaoborus Chaoborus4.11 (0.18)
7.44 (2.06)
0.72 2
Ceratophyllum Ceratophyllum
2.99 (0.10)
16.77 (5.17)
0.60 0
Potamogeton Potamogeton
2.55 (0.11)
18.22 (8.42)
0.43 3
Utricularia Utricularia 2.55 (0.08)
17.55 (5.82)
0.59 9
Scenedesmus Scenedesmus
3.99 (0.22)
4.66 (1.64)
0.64 4
Populus Populus
2.77 (0.06)
28.33 (7.40)
0.69 9
Yeast t
3.55 (0.11)
8.00 (1.93)
0.75 5
Tablee 4. F-values of MANOVA (18 df) used to compare maximum larval length and slope of the
growthh curves of the individual food items. Significant differences (P < 0.05) between growth
curvee parameters of food items are indicated with an *.
Trouvif f
TetraMin® ®
TetraMin® ®
Gammarus Gammarus
114* *
40* *
Chaoborus Chaoborus
51* *
22
Ceratophyllum Ceratophyllum
Gammarus Gammarus
Chaoborus Chaoborus
Ceratophyllum Ceratoph
17* *
29* *
68* *
108* *
415* *
307* *
100* *
109* *
664* *
405* *
165* *
Utricularia Utricularia
97* *
485* *
353* *
342* *
46* *
Scenedesmus Scenedesmus
12 2
22
23* *
30* *
238* *
Populus Populus
65* *
98* *
539* *
55* *
17* *
22
18 8
396* *
11 1
139* *
Potamogeton Potamogeton
Yeast t
34 4
QualityQuality of organic matter as food for chironomids
N/PP (Table 5). Proteins, organic matter, and energy content were situated near the center
off the plot because of their low variance in the dataset, indicating little influence on
larvall growth under the experimental conditions in this study.
Discussion n
Thee high level of variance explained by the ordination suggests that the set of
chemicall parameters measured in this study was appropriate for explaining the growth of
earlyy instar chironomid larvae and consequently also for determining food quality. A
partt of the remaining unexplained variance is most probably attributable to larval growth
resultingg from feeding of the 1st instar larvae on the remains of the egg masses before the
startt of the growth experiment, or from microbial growth during the experiment. Another
partt is probably attributable to variability of growth among individual larvae. However,
thesee factors were of minor importance when compared to the effect of composition and
quantityy of the food items on larval growth reflected in the high level of variance
explainedd by the ordination.
Bothh quantity and biochemical composition of food influenced larval growth of
thee midge as indicated by differences in k and in lmax values of the saturation growth
curves.. In addition, our results demonstrated an interaction between food quantity and
foodd quality on larval growth; i.e., different food components were the limiting factors
forr growth at different food levels. High N, P, and lipid contents stimulated growth at
Tablee 4. Extended
PotamogetonPotamogeton
0.1 1
316**
10**
779**
282*
20*
195*
Utricularia Scenedesmus
126*
28*
Populus
143*
35 5
ChapterChapter 2
highh food levels (lmax), whereas the amount of carbohydrates appeared to be important in
definingg growth at low food levels (k). The interaction of food quality and quantity
mightt be the consequence of 2 coexisting functions of food. Food can be used as an
energyy source (e.g., for maintenance), but it also is used as a source of nutrients essential
forr the production of new tissue. Compounds that are easily respired, or have a high
energyy content (e.g., carbohydrates) are mainly used as an energy source. Other food
componentss such as proteins and lipids can be used as energy source, but often are used
preferentiallyy to deliver the components necessary for growth. Proteins supply essential
aminoo acids (Prosser & Brown 1965, Kimball 1968), and lipids contain fatty acids,
somee of which cannot be synthesized by animals and are needed as building blocks of
animall tissue (e.g., polyunsaturated fatty acids, Turunen 1979, Cook 1996).
Wee found correlations between lmax and certain food components. Because a
surpluss of energy is present at high food levels, the qualitative composition of food is
thee limiting factor for growth. Lmax was correlated with lipids, N, and P, indicating that
thesee factors represent growth restricting assimilative compounds at high food levels.
Duringg low food availability, it is more probable that energy is the limiting factor for
growth.. The addition of high energy compounds under low food availability may lead to
Tablee 5. Spearman coefficients of the correlations of maximum larval length (lmax) and slope (k)
withh chemical parameters of the food items. Significant correlations (P < 0.05) are indicated with
ann *. Chemical parameters and saturation growth-curve parameters were not transformed.
'max x
kk
%% organic matter
++ 0.152
%C C
++ 0.661*
-0.188 8
%% N
++ 0.770*
-- 0.552
%P P
++ 0.697*
-- 0.661*
%% carbohydrates
-- 0.782*
++ 0.285
%% lipids
++ 0.697*
-- 0.273
%% protein
-- 0.261
++ 0.358
Energyy content
++ 0.139
++ 0.406
C/N N
-- 0.782*
++ 0.648*
C/P P
-- 0.658*
++ 0.648*
N/P P
-- 0.638*
++ 0.685*
36 6
-- 0.261
QualityQuality of organic matter as food for chironomids
thee effective use of scarce building blocks for growth. This reasoning is confirmed by
thee RDA plot where the high energy and easily dissimilable carbohydrates and growth at
loww food levels (k) were ordinated on the same side of axis 1. Therefore, it would be
expectedd that lipids also are correlated with k, because lipids also contain higher levels
off energy than the other food components. Nevertheless, lipid content was not correlated
withh k, implying that lipids were not as easily available and respired as carbohydrates
(Prosserr & Brown 1965, Kimball 1968). A similar example of the effective use of scarce
buildingg blocks for growth is the "protein sparing" effect that has been observed in
invertebratess and other organisms (Roman 1983). The protein sparing effect is the result
off high-energy compounds being used for maintenance, allowing more proteins to be
usedd for growth. Interaction of food quality and food quantity also has been found in
daphnidss (Sterner et al. 1993, Sterner & Robinson 1994). In these studies, the effect of
foodd quantity depended on algal food composition, although only C, N, and P were used
too define food quality.
Ourr study illustrates the importance of food quantity and food quality in
regulatingg the growth of early instar chironomid larvae for one week under laboratory
conditions.. It can be expected that both factors also influence later life stages of
chironomids.. Processes such as wing development during the last larval instar, pupation,
succesfull emergence, and egg development are crucial to complete the life cycle, but
alsoo are processes with their own special nutrient demands (Beenakkers et al. 1981,
Stanley-Samuelsonn 1994). Specific fatty acids in the diet are important for normal
growth,, successful emergence, egg production, and survival of a number of
invertebratess species (Gilbert 1967, Beenakkers et al. 1981, D'Abramo & Sheen 1993,
Stanley-Samuelsonn 1994). Other food components such as P and N influence growth,
survival,, and fecundity of zooplankton (Brett & Miiller-Navarra 1997, Gulati & DeMott
1997).. Nutritional growth demands of early instar larvae probably are similar to the
growthh demands during the larval stages that follow. However, during the prepupal
stagee in which the first changes towards the adult imago take place, different nutritional
needss may be expected, reflected in dependencies on food quality and quantity that
differr from those of young larvae.
Bothh food quality and quantity are potential factors in the regulation of
invertebratee life history and field distribution because of their variation in place and
37 7
ChapterChapter 2
Fig.. 2. Redundancy analysis plot showing the direct ordination of saturation growth-curve
parameterss and food composition, with 48% of variance on ais 1 and 7% on axis 2. Open
triangless indicate chemical parameters, black squares food items, and arrows the saturation
growth-curvee parameters. Lmax is an indicator of optimal growth on the specific food item, k is an
indicatorr of the effect of additional food on growth when the food level is non-saturating.
POPULUS S
TROUVIT® ®
rr C
N/PT T
..
pro
c/p*
C/N<<
TETRAMIN**
!. eins
CERATOPHYLLUM M
E-contenff
~-«^
--
r lipids
GAMMARUS S
—» N
^^ ""
>>
** -"
organicc matter
CHAOBORUSS
carbohydrates
UTRICULARIA A
POTAMOGETON N
"f "f
/
max max
SCENEDESMUS S
YEAST T
Axiss 1, eigenvalue 0.48
+10
time.. For example, algal inputs are characterized by strong seasonal fluctuations and are
subjectedd to a certain degree of microbial degradation and chemical oxidation (Bowen
1987,, Canuel & Martens 1993, Goedkoop & Johnson 1996, Ahlgren et al. 1997,
Kreegerr et al. 1997). Moreover, organic matter composition is dependent on the source
off detritus (Cowie and Hedges 1992, Canuel & Martens 1993, Hecky et al. 1993,
Kreegerr et al. 1997). It can be expected that both food quantity and quality are lower in
thee field compared to the present study (e.g., see N/P, C/P, and C/N ratios in Ahlgren et
al.. 1997), which makes it even more probable that these factors are important in
regulatingg growth of sediment-feeding chironomids. Therefore, more attention should be
38 8
QualityQuality of organic matter as food for chironomids
paidd to biochemical composition and quantity of natural food sources to understand
growthh of sediment-feeding chironomids under natural conditions.
Acknowledgements s
Thee study was supported by the Institute for Inland Water Management and
Wastee Water Treatment (RIZA), Lelystad. We thank Ronald Gylstra and Paul J. van den
Brinkk for their help with the canonical and statistical analyses, and Heather A. Leslie for
herr comments on the English grammar. We thank Pamela Silver and the 2 anonymous
refereess for improving the manuscript with their valuable suggestions.
39 9
40 0
CHAPTERR 3
GROWTHH RESPONSE OF A BENTHIC DETRITIVORE TO ORGANIC
MATTERR COMPOSITION OF SEDIMENTS
J.H.. Vos1,, P.J. van den Brink2, F.P. van den Ende, M.A.G. Ooijevaar, A.J.P. Oosthoek,
J.F.. Postma3, and W. Admiraal
Departmentt of Aquatic Ecology and Ecotoxicology, Institute for Biodiversity and
Ecosystemm Dynamics, University of Amsterdam, Kruislaan 320, 1098 SM Amsterdam,
Thee Netherlands, '[email protected],
2
ALTERRA Green World Research,
Departmentt of Water and the Environment, P.O. Box 47, 6700 AA, Wageningen, The
Netherlands,, [email protected], 3present address: AquaSense Consultants, P.O. Box
95125,, 1090 HC Amsterdam, The Netherlands, [email protected]
Submittedd for publication
41 1
ChapterChapter 3
Abstract t
Thee biochemical composition of lake and stream sediments were analyzed and
comparedd to detritivore growth and survival to determine which biochemical parameters
correlatedd most strongly with sediment food quality. Sediments were collected from soft
bottomss of 41 sites and fed to the midge larvae of Chironomus riparius. These
sedimentss were analyzed for organic matter (OM) content, C, N, P, carbohydrates,
protein,, fatty acids, pigments, and grain size distribution. A microbial assay was used as
indicatorr of the fraction easily biodegradable OM. Data were analyzed by means of
univariatee and multivariate analyses. Positive correlations of growth or survival with
polyunsaturatedd fatty acids (PUFAs), pigments, and labile OM were found when
standardizedd on dry weight. When variables were standardized based on mass of OM,
additionall significant positive correlations with P, carbohydrates, proteins, and fatty
acidss of bacterial origin were detected. Similarly, multivariate analyses revealed
strongerr correlations between larval growth and survival and biochemical variables
standardizedd on OM compared to those standardized on dry weight. It was postulated
thatt dilution of OM by mineral particles caused the difference between the
standardizationn methods. Organic matter composition constituted an important factor
influencingg detritivore growth. Labile OM was found to support the highest larval
growth. .
42 2
ChironomidChironomid larvae growth on sediments
Introduction n
Detritivorouss animals feed on particles that vary through time in abundance and
inn state of decomposition. Consequently, food resources available to detritivores differ
inn nutritional quality during the course of the year (e.g. Hill et al. 1992, Canuel and
Martenss 1993, Ahlgren et al. 1997, Cavaletto and Gardner 1999). The strong positive
responsee of deposit feeders to seasonal inputs of newly produced organic matter (OM)
suggestss that food abundance is a limiting factor much of the year (Lopez and Levinton
1987,, Goedkoop and Johnson 1996). Indeed the species composition of algal blooms
hass also been recognized as a factor regulating the population dynamics of benthic
faunaa (Marsh and Tenore 1990, Cheng et al. 1993). Such factors may be entirely
explainedd through the varying quality of OM in sediments. Food quality of the pelagic
inputt to the benthic pool of detritus is dependent on the species that comprise the algae
blooms.. For instance, diatoms and flagellates form a high quality food due to their high
polyunsaturatedd fatty acid (PUFA) content, whereas the low PUFA content of cyanobacteriaa make their quality as food for detritivores poor (Ahlgren et al. 1997, Brett and
Müller-Navarraa 1997, review Napolitano 1999). During sedimentation, the biochemical
contentt of algae changes due to chemical oxidation, bacterial decomposition, cell
leaching,, and stripping by zooplankters prior to settling on to the substrate. The more
labilee components degrade first, leaving less easily digested compounds. This process
continuess after sedimentation and is promoted by bioturbation. As a consequence, sedimentss typically contain less digestible OM compared to other food sources consumed
byy aquatic animals (Bowen 1987, Fry 1987, Meyers and Ishiwatari 1995). Ahlgren et al.
(1997)) found distinct differences in chemical composition between plankton samples
takenn in the photic zone and sedimentation samples collected in the aphotic zone of a
largee mesotrophic lake. C/N ratios of sedimentation samples were mostly higher than
thosee of plankton, but the greatest differences between plankton and sedimentation
sampless were found for PUF As. Thus, ongoing degradation leads to a lower food qualityy to which benthic invertebrates have access compared to that accessible to pelagic
zooplankters. .
Planktonicc algae are not the sole sources of OM in sediments. Macrophytes, OM
off animal origin, and litter from terrestrial vegetation also add to sediment organic
43 3
ChapterChapter 3
material.. The diverse sources of OM and degradation histories are expected to result in
highlyy different food sources for the detritivorous component of benthos inhabiting
substratess of water systems that differ in primary and secondary production, current
regime,, and terrestrial vegetation type. This study explored differences in food
compositionn and nutritional state between individual sediments from various sites as
regulatingg factors of detritivore growth. Field samples of unpolluted sediments of
streamss and lakes in the Netherlands and the Pripyat basin (Republic of Belorussia)
weree taken to the laboratory where growth of the detritivorous Chironomus riparius
Meigenn larvae on the individual substrates was determined. In conjunction, the sedimentss were characterized with respect to a large number of chemical and physical parameterss to identify the main factors regulating growth of the invertebrates. Growth and
survivall of the larvae were correlated to these sediment parameters by means of univariateriate and multivariate analyses.
Methods s
SedimentSediment sampling
AA total of 41 freshwater sediment samples taken from habitats ranging from
smalll streams to large lakes were obtained using an Eckman-Birdge grab which was
adjustedd to sample the upper 4 cm surface layer. All sediments were analyzed by the
analyticall laboratory AlControl, Hoogvliet, the Netherlands, for metals, PAHs, PCBs,
andd pesticides. The sediments contained the sum of PCBs (PCB-28, -52, -101, -118, 138,, -153, and -180) 7 < ug kg"1, sum of pesticides (aldrin, dieldrin, endrin, DDT, endosulfan,, HCHs, heptachlor, and heptachlorepoxide) < 13 ug kg"1, sum of chloride benzeness (di-, tri-, tetra-, penta-, and hexachloride benzenes) < 1 ug kg"1, sum of PAKs
(naftalene,, benzo(a)antracene, benzo(ghi)perylene, benzo(a)pyrene, fenanthene, indeno(1,2,, 3-cd)pyrene, anthacene, benzo(k)fluoranthene, chrysene, fluoranthene) < 0.55 mg
kg*1,, EOX < 0.31 mg kg"1, mineral oil 47 < mg kg"1, and Cd < 0.4 mg kg"1, Hg < 0.05
mgg kg"1, Cu < 5 mg kg"1, Ni < 8 mg kg"1, Pd < 13 mg kg"1, Zn < 16 mg kg"1, Cr < 15 mg
kg*1,, and As < 4 mg kg"1 dry mass. According to the Dutch regulations (Evaluatienota
Waterr 1994) all 41 sediments were clean.
Thee sediment samples were collected between March 1997 and October 1998
44 4
ChironomidChironomid larvae growth on sediments
fromfrom 38 sites throughout the Netherlands and from 3 sites in the Republic of Belorussia
(Pripyatt basin). Of the 41 samples, 20 were taken from large and shallow lakes, 6 from
smalll lakes, 6 from large rivers (width > 5m), and 9 from small streams. Riparian vegetationn ranging from woods to meadows. All sediments were frozen at -20°C within 6 h
afterr sampling. After thawing the sediments were sieved through a 1000 um mesh to
removee larger particles such as pebbles, leaves, and twigs. The sediments were frozen a
secondd time to ensure the removal of all indigenous animals.
GrowthGrowth experiments with chironomid larvae
Growthh experiments were conducted at 20°C
1°C and a 16:8 hour light:dark
regime.. Experiments were carried out in polyethylene containers (10 x 10 x 6.5 cm).
1000 ml of homogenized sediment and 200 ml of artificial freshwater (Dutch Standard
Water,, pH 8.2, 210 mg CaC0 3 l') were added to each container and the water was
continuouslyy gently aerated. The sediments were allowed to settle and stabilize for 24 h,
andd pH and oxygen saturation of the overlying water were measured at the beginning of
thee experiment.
Att the start of each experiment, larvae originating from at least 10 egg masses of
ChironomusChironomus riparius (Meigen), a detritivorous (Rasmussen 1984, 1985) laboratory
culturedd midge, were used. Experiments were started by randomly adding 20 I s instar
larvaee less than 24 h old to each container. An additional group of 20 larvae was
randomlyy collected to determine initial larval length by means of a binocular microscope.. During the experiment, oxygen levels and pH were checked every 2-3 d. In
addition,, ammonium, nitrite, and nitrate concentrations in the overlying water were
measuredd at the start of the experiment using Quantofix® test sticks (SE = 5 mg N 1" for
NH4+)) and TetraTest Nitrite® test kit (SE - 0.05 mg N l"1 for N02" and N03"). After 14
d,, the lengths of surviving larvae were measured with a binocular microscope. Since
larvaee decrease in length during the prepupal stage it was decided to give these larvae
thee greatest length found in the reference containers of growth experiments that were
startedd at the same day. Growth experiments on each sediment sampled in 1997 were
replicatedd 4 times while the growth experiments sampled on sediments sampled in 1998
weree replicated 3 times. To test for food limitation, larval growth on the 1997 sediments
wass also tested 4 times with additions of 100 mg of the fishfood mixture each week.
45 5
ChapterChapter 3
Thee condition of 1st instar larvae used in the growth experiments was monitored by
takingg along 3 control units each day growth experiments were started. Controls consistedd of containers with 50 g of combusted Litofix® sand (<500 urn, heated at 550°C for 6
h),, 200 ml of Dutch Standard Water and 100 mg of a mixture of the commercial
availablee fishfoods Trouvit® and Tetraphyll® (95:5 m:m) each week. Replicates of each
sedimentt were started at different days to ensure complete statistical independence.
ChemicalChemical
analyses
Sedimentt samples for chemical analyses were collected in containers similar to
thosee used for growth experiments, and were treated identically as sediment used for the
growthh experiments. These sediments were frozen directly after starting the growth
experiments.. The resulting 3 or 4 subsamples per sediment were mixed quantitatively,
thann freeze-dried and stored at -20°C. Grain size distribution was determined by sieving
andd the pipet method descibed in ISO 11277 (1998). Water content was determined by
freezee drying a preweighted sediment sample in triplicate.
Thee OM content was determined as loss-on-ignition by combusting the material
att 550°C for 6 h (Luczak et al. 1997) in triplicate. Total C was measured in duplicate
withh a Carbo-Erba Element Analyser. N was measured according to Kjeldahl (ISO
112611 1995).
Totall P was determined according to Murphy and Riley (1962) and protein
accordingg to Rice (1982), with both analyses conducted in duplicate. For analyzing
carbohydrates,, a modified method based on the phenol-sulphuric acid-method of Dubois
ett al. (1956) was used. It was noted that the baseline of the photometric spectra between
4000 and 600 nm of the phenol-sulphuric acid solution of the individual sediments
differedd in intercept. It was decided to correct absorption at 485 nm with additional
spectrophotometricc measurements at 440 and 550 nm according to the following
calculationn for both sediment samples and calibration curve: Abs 485 n m , = Abs 4g5 nm ((Abss 550 nm + ((Abs 440 nm - Abs 550nm)*65/l 10)), in which Abs = absorption and Abs 485
nm'== Abs at 485 nm corrected for intercept of the baseline.
Chlorophyll-^^ and pheaophytin were measured according to Nusch and Palme
(1975)) in duplicate sediment samples. The ethanol solution was centrifuged in closed
test-tubess to avoid optical disturbance by suspended sediment. Chlorophyll-a and
46 6
ChironomidChironomid larvae growth on sediments
pheaophytinn contents were summed because during the analytical procedure chlorophyll-aa was found to partly degrade into pheaophytin indicated by a large standard error
off chlorophyll:pheaophytin ratio among replicates.
Lipidss were extracted with a 1:1:0.9 v/v/v chloroform:methanol:water mixture
followingg the Bligh and Dyer (1959) procedure. Prior to the extraction, preweighed
sedimentt samples of 5 - 10 g together with 8 ml double destilled water were sonificated
forr at least 5 minutes in a -4°C water bath. The resulting collected chloroform was
evaporatedd with nitrogen gas. For analyses of fatty acid composition the lipid samples
weree diluted again in an appropriate volume of hexane containing heneicosaenoic acid
(21:0,, 838.75 mg ml"1) as an internal standard and BHT (2,6-di-tert-butyl-p-cresol, 50
mgg ml"1) as an antioxidant with nitrogen as the overstanding gas and were stored at
-20°CC until transesterification. Fatty acid methyl esters (FAME) were obtained by mild
alcanolicc methanolic transesterification as described in Guckert et al. (1985). The
FAMEE samples were stored at -20°C with overstanding nitrogen gas for no longer than
22 months before Gas Chromatographic analysis took place. GC separation of the FAME
wass performed by injecting a 1 \il aliquot in a very polar 50 m CP-Sil 88 column (ID
0.255 mm, film thickness 0.20 mm) with a splitflow of 1:40. Optimal separation of
FAMEE peaks was obtained with a temperature program that began at injection with an
initiall column temperature of 180°C for 10 minutes followed by a rise of 3°C min"1 to a
finall temperature of 225°C, where it was held for 10 minutes. Fatty acid nomenclature
usedd in this study conforms to the A:BcoC model where A designates the number of
carbonn atoms in the fatty acid methyl ether, B the number of double bonds, and C the
distancee of the closest double bond from the aliphatic (co) end of the molecule. The fatty
acidss (FAs) mentioned in this study and used for further calculation and statistical
analysess were all cw-isomers.
Tentativee identification of FAME peaks was based on co-elution with the 21:0
standardd and by comparison of relative retention times, calculated as the retention time
(RT)) of the peak minus the time at which the solvent peak appeared divided by the
retentionn time of 16:0 or 21:0 minus the RT of the solvent peak. Final identification was
basedd on Mass Spectrometry analyses of 4 sediment FA samples performed by dr. Eric
Boschker,, NIOO, Yerseke, The Netherlands. A PUFA variable was obtained by
summingg up the peak areas of 16:2co4, 16:3co4, 18:2co6, 18:3<o3, and 18:3co6. Peak
47 7
ChapterChapter 3
areass of FAMEs of bacterial origin (i.e. Z14:0, i\5:0, al5:0, 15:1, /16:0, z'16:l, il7:0, and
«17:0;«17:0; Parkes 1987; Napolitano 1999) were added to obtain a bacterial FA variable. A
measuree for total FA was calculated by adding all fatty acid peaks from 12:0 up to the
lastt peak appearing in the chromatogram before the 21:0 internal standard peak
(18:3co3).. Only FAME peaks which appeared before the internal standard in the
chromatogramm were used for further calculations because the peaks appearing later in
thee chromatogram showed irregular retention times.
Energyy content (E-content) was calculated by assuming that 1 g of fat releases
38.99 kj, 1 g of protein releases 17.3 kJ, and 1 g of carbohydrate yields 16.9 kJ.
AssessmentAssessment of the most labile fraction of the organic matter
AA microbial assay was used to obtain a measure of the labile, i.e. easily
degradable,, fraction of the sediment organic matter. Microbial mineralization was
measuredd as CO2 production. Wet sediments were used that had been kept frozen until
analysis.. A bacteria inoculum was prepared from the surface layer of a sediment
containingg a decaying cyanobacterial mat (Oscillatoria sp). Bacteria were detached by
ultrasonicc treatment and particles were removed by centrifugation (5 min 50 g). In a 77
mll gas-tight bottle 4 ml of sediment was suspended in 11 ml of a 55 mM phosphate
bufferr (pH 7.1) and 1 ml of the bacteria inoculum was added. After 30 min of aeration
pHH was measured and the bottles were capped gas-tight. The bottles were placed on a
rotaryy shaker at 20°C in the dark and after allowing one hour equilibration the C0 2
concentrationn in the headspace was measured gaschromatographically. After 48 hours
incubationn the C0 2 concentration was measured again and the pH was measured. The
headspacee contained sufficient oxygen to maintain oxic conditions throughout the
experiments.. Gas in the headspace was assumed to remain at atmospheric pressure and
thee CO2 concentration in equilibrium with the aqueous phase. Total CO2 in carbonic
acid,, bicarbonate and carbonate in the aqueous phase was calculated according to
Stummm and Morgan (1981). Due to the large size of the headspace (61 ml) around half
thee total C0 2 was in the gas-phase thus ensuring accuracy of the measurement. C0 2
productionn was calculated by subtracting the total concentration in water and headspace
att the start from the total concentration after 48 h. Blancs showed that CO2 produced
fromm organic matter in the inoculum was negligible. Controls with yeast extract (2 mg
48 8
ChironomidChironomid larvae growth on
sediments
perr bottle) in stead of sediment were used to check for reproducibility during the
measurementss and to allow comparison with future experiments. None of the sediments
wass rich in inorganic carbonates so no attempt was made to remove these. During the
incubationn the pH declined less that 0.25 units, thus limiting possible interference of
carbonates. .
UnivariateUnivariate statistical analyses
Correlationss between larval growth, survival and chemical variables were
determinedd with Pearson correlation using the average larval survival and growth per
sedimentt and the mean of repeated analyses for the different sediment variables.
Correlationss were performed with variables standardized on both dry weight (DW) and
OM.. Growth and survival on sediments collected in 1997, with and without surplus of
foodd were compared with a two-way ANOVA.
MultivariateMultivariate analyses
Multivariatee analyses were performed using Principal Component Analysis to
obtainn a graphical summary of the data set and an overview of mutual relationships
betweenn sediment variables on the one hand and larval growth and survival as
determinedd in the laboratory tests on the other hand (ter Braak 1995). PCA was chosen
forr final ordination because the length of gradient was < 2.0 as was determined with
Detrendedd Corresponce Analysis (DCA). PCA is an ordination method which uses a
Tablee 1. Summary of variables found in the 41 substrates. Averages are calculated from the
averagee values of each indiviual sediment. CV = coefficient of variance. Min = minimum and
maxx = maximum value as found in the dataset. SWC = sediment water content (% of wet
weight),, OM = organic matter (% of dry weight), %<63 urn = percentage of volume particles <
633 (im, and %<210 urn = percentage of volume particles 0-210 urn.
average e
min n
max x
CV V
SWC C
32.9 9
14.1 1
90.6 6
24 4
OM M
2.3 3
0.2 2
8.8 8
121 1
C/N N
24.2 2
3.0 0
107 7
91 1
%<633 urn
20.8 8
0.7 7
79 9
126 6
%<210um m 60.9 9
5.1 1
96 6
50 0
49 9
ChapterChapter 3
Tablee 2. Overview of variables expressed as portion of dry weight or of organic matter
measuredd in the 41 sediments. Averages are calculated from the average values of the
individuall sediments. CV = coefficient of variance. Minimum (min) and maximum (min) value as
foundd in the dataset.
CO O
CD D
oo
oo
oo
CD D
CO O
ID ID
CM M
CM M
00 0
CD D
00 0
CM M
CM M
CD D
lO O
r*--
**
CM M
CD D
a: :
co o
>>
CD D
CM M
CD D
00 0
mm
CO O
^1--
mm
oo
CD D
IT) )
CM M
oo
1——
oo
00 0
IT) IT)
co
0) )
>
>
CD D
>>
CC
ÜÜ
T3 3
XX
U> >
CO O
NN
FF
UU
01 1 _c c
>
>
TS TS
V) V)
D
m m CD
T_ _
T— —
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CD D
in n
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CO O
CD D
mm
<r>
<r>
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00 0
55 cc
P-- E E
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.555
o
^
OO
linearr model for dimension reduction. In PCA, imaginary, latent explanatory variables
(principall components) are calculated from the data set which best explain the variation
inn sediment parameter composition between sites. The first principle component is
constructedd in such a way that it explains the largest part of the total variance possible,
50 0
ChironomidChironomid larvae growth on sediments
thee second one the largest part of the remaining variance, etc. The first two principle
componentss were used as axes to construct an ordination diagram and the weights of the
parameterss and sites with these variables are plotted in the diagram represented by
points.. After the construction of the diagram, the larval performance parameters (growth
andd survival) were superposed on the diagram, i.e. they were regressed on the axes
usingg the site points. Within PCA, all sediment characteristics were standardized and
centeredd to make them mathematically equally important. This is necessary because all
characteristicss were measured or expressed in different units. Two analyses were performed,, 1 on the sediment data with biochemical variables standardized on DW, and 1
onn data with biochemical variables standardized on OM. Permutation tests were performedd on whether larval growth and survival have a significant relation with the variation
inn sediment characteristics among sites, using the constrained version of PCA, Redundancyy Analysis (ter Braak and Smilauer 1998). All multivariate analyses were performedd with the CANOCO for Windows software package (ter Braak and Smilauer 1998).
Results s
CompositionComposition of the sediments
Grainn size distribution of sediments samples ranged from silty to sandy (Table
1).. Sediment water content ranged from 14.1 to 90.6 % of wet weight, but the coefficientt of variation among samples was relatively low (CV = 24%). The variation in
organicc matter (OM) content was well spread over the samples (CV = 121%). All
biochemicall variables showed a high CV when standardized on dry weight (DW) (Table
2).. Standardization on OM content resulted in lower variation for most of the biochemicall variables. Only XFA, PUF As, and labile OM had slightly higher CVs when standardizedd on OM.
Iff the variables were standardized on DW many significant correlations were
foundd among the biochemical variables (Table 3). Most biochemical variables standardizedd on DW were also found to be significantly correlated with OM content. This may
bee expected, since the biochemical variables are all considered to represent part of the
OMM fraction. Levels of biochemical variables expressed as proportion of DW may
thereforee be expected to follow the level of OM. Only IFA and PUF As were found to
51 1
ChapterChapter 3
Tablee 3. Correlations of sediment parameters with the biochemical variables standardized on
totall fatty acids, PUFAs = polyunsaturated fatty acids, bac. FAs = fatty acids of bacterial origin,
%<633 um = percentage of volume particles smaller than 63 urn, %<210 urn = percentage of
CC
NN
NN
+0.81** *
PP
+0.70** * +0.52" "
PP
carbohydrates protein I F A
PUFAs s bac.. FAs
carbohydrates s + 0 . 7 7 " " +0.83" " +0.37" "
protein n
+0.51** * + 0 . 5 7 " " +0.67" " ++ 0.45"
IFA A
+0.17 7 +0.12 2 +0.26 6
++ 0.10
+0.18 8
PUFAs s
+0.26 6 +0.06 6 +0.55** * ++ 0.05
+0.266
bacteriall FAs
+0.70" " +0.44" " +0.86** * ++ 0.37" +0.54"" +0.20 ++ 0 . 7 1 "
pigments s
+0.51** * +0.35* * +0.44** * +0.33" " +0.34**
E-content t
+0.64** * +0.70" " +0.68" " +0.64" " +0.96"" +0.33* ++ 0.26 ++ 0.57"
C0 22 production + 0 . 5 1 " " +0.38" " +0.64** * +0.33* *
+0.21
+0.06 ++ 0.29* ++ 0.50"
+0.52"" +0.10 ++ 0.43" ++ 0.68"
havee a low number of significant correlations with the other chemical variables if
expressedd as portion of DW. Standardization on OM content decreased the number of
correlationss between biochemical variables, thus indicating a diverse composition of
OMM (Table 4). OM was strongly correlated to particle sizes < 63 um (R2 = 0.92, P <
0.01)) and less strong to particle sizes < 210 um (R2 = 0.31, P < 0.05).
Tablee 4. Correlations of sediment parameters with the biochemical variables standardized on
totall fatty acids, PUFAs = polyunsaturated fatty acids, bac. FAs = fatty acids of bacterial origin,
%<63umm = percentage of volume particles smaller than 63 um, %<210 um = percentage of
CC
NN
NN
+0.23 3
PP
+0.67" " +0.09 9
carbohydrates s -0.00 0
PP
carbohydrates protein I F A
PUFAs s bac.. FAs
+0.10 0 +0.08 8
protein n
+0.11 1 +0.07 7 +0.33* * +0.08 8
IFA A
+0.21 1 -0.02 2 +0.29* * +0.05 5
+0.05 5
PUFAs s
+0.54" " +0.20 0 +0.78** * +0.19 9
+0.34* * +0.20 0
bacteriall FAs
+0.36* * +0.06 6 +0.58** * +0.12 2
+0.28* * +0.09 9 +0.83** *
pigments s
+0.11 1 +0.18 8 +0.21 1
+0.04 4 +0.05 5 +0.55" " +0.49" "
E-content t
+0.18 8 +0.07 7 +0.42** * +0.29* *
C 0 22 production +0.03 3 +0.07 7 +0.16 6
52 2
+0.31* *
+0.17 7
+0.87" " +0.49** *+ 0 . 4 1 " " +0.29* *
+0.24 4 +0.16 6 +0.47" " +0.58" "
ChironomidChironomid larvae growth on
sediments
dryy weight. Pearson coefficient R2 and significance level. * = P < 0.05, ** = P < 0.01. I F A =
E-contentt = energy content, SWC = sediment water content, OM = organic matter,
volumee particles smaller than 210 um.
pigments s E-content t SWC C
+0.35* *
+0.64** *
OM M
C/N N
%<633 um %<2100 um
++ 0.89** ++ 0.83**
-0.20 0 ++ 0.77** +0.26* *
++ 0.63" ++ 0.74**
+0.09 9 ++ 0.81** +0.41** *
++ 0.84** ++ 0.79**
+0.07 7 ++ 0.63** +0.29* *
++ 0.54** ++ 0.57**
-0.12 2 ++ 0.56** +0.34* *
++ 0.14
++ 0.12
+0.04 4 ++ 0.20
+0.07 7
++ 0.25
++ 0.16
+0.11 1 ++ 0.25
+0.49** *
++ 0.63** ++ 0.69**
+0.09 9 ++ 0.73** +0.49** *
++ 0.44** ++ 0.52**
+0.08 8 ++ 0.40** +0.37** *
++ 0.68** ++ 0.69**
-0.07 7 ++ 0.65** +0.36** *
+0.52** * ++ 0.50** ++ 0.53**
-0.08 8 ++ 0.52** +0.56** *
GrowthGrowth and survival of midge larvae
Lengthh of the larvae at the start of the experiments averaged 0.89 mm (SE =
0.02).. After 14 d larvae in the controls averaged 12.5 mm (SE = 0.28) in length and
>70%% were in their 4th instar. After 14 days larval survival exceeded 80% in all controls.
Survivall per experimental unit with sampled sediment ranged between 0 and 100% and
organicc matter. Pearson coefficient R2 and significance level. * = P < 0.05, ** = P < 0.01. I F A =
E-contentt = energy content, SWC = sediment water content, OM = organic matter,
volumee particles smaller than 210 um.
pigmentss
E-content SWC
OM
C/N
%<63 um %<210um
-0.08 8
-0.45** * -0.04 4
-0.11 1
-0.22 2
+0.11 1
-0.14 4
+0.29* *
+0.10 0
+0.02 2
-0.08 8
-0.04 4
+0.31* *
-0.09 9
-0.17 7
-0.16 6
-0.16 6
+0.34* *
-0.25 5
-0.30* *
+0.02 2
-0.25 5
-0.19 9
-0.14 4
-0.30* *
+0.00 0
-0.26 6
+0.23 3
-0.14 4
-0.26 6
-0.07 7
-0.22 2
+0.15 5
-0.25 5
-0.35* *
-0.17 7
-0.37** * +0.15 5
-0.17 7
-0.27* *
-0.14 4
-0.25 5
-0.32* *
-0.43** *
-0.24 4
-0.43** * +0.05 5
+0.06 6
+0.01 1
+0.27* *
53 3
ChapterChapter 3
Fig.. 1. Larval growth (mm) and survival (%) after 14 days on sediments provided with and
withoutt surplus of food. White bars represent sediments supplemented with 100 mg of fish food
perr week, black bars represent sediments without additional food. Errors bars = SE.
M— —
cc
oo
C C 1— —
X. X. CD D CD D CO O
CD D C C o o
JZ JZ
(1) )
CD D C
O O
1_ _
^ ^
m m
n n oo X X
o o
CD D
"O O
<D D
-L L
CD D
CD D
m m CD D
EE
v v
C/) )
CD D
E E
CO O
CD D
m m b b5 5
c c b b _CO O
CD D
OO
OO
growthh between 0.8 and 11.0 mm. Growth was highly significantly correlated with
survivall in sediments not augmented with fish food (R2 = 0.69; P < 0.01).
Larvall growth and survival on sediments that were provided with fish food
(thosee collected in 1997) were similar to growth in the controls (Fig. 1). Survival was
>80%% in all experimental units provided with fish food. Growth and survival were significantlyy lower (P < 0.05) on the sediments not provided with fish food compared to
thee sediments provided with excess of food.
54 4
ChironomidChironomid larvae growth on sediments
Tablee 5. Correlations of growth and survival of C. riparius larvae after 14 days with sediment
parameters.. Pearson coefficient R2 and significance level. * = P < 0.05, ** = P < 0.01. SWC =
sedimentt water content, OM = organic matter, %<63 urn = percentage of volume particles
smallerr than 63 urn, %<210 urn = percentage of volume particles between 0 and 210 urn.
growth h
survival l
survival l
+0.69** *
SWC C
-0.01 1
-0.15 5
OM M
-0.14 4
-0.25 5
C/N N
-0.24 4
-0.33* *
%<633 nm
-0.15 5
-0.19 9
%<2100 urn
+0.51** *
+0.34 4
Duringg the growth experiments, the oxygen saturation level did not fall below
60%% in any of the experimental units of the 41 sediments. All N02" concentrations were
<11 mg l"1, the N t V concentrations <10 mg l"1, and the pH in the overlying water varied
betweenn 7.1 and 8.5 during the experimental period.
CorrelationsCorrelations of larval growth and survival with sediment parameters
Larvall growth and survival in non-augmented sediments were significantly correlatedd (P < 0.01, Table 5). Growth was positivily correlated with the particle size fractionn < 210 urn.
Whenn biochemical variables were expressed on the basis of DW, correlations of
growthh and survival were found with PUF As, pigments, and labile OM (P < 0.05, Table
6).. When sediment variables were standardized on OM content, the strength and number
off correlations increased, with additional significant correlations (P < 0.05) found with
P,, carbohydrates, protein, F As of bacterial origin, and energy-content.
MultivariateMultivariate analyses
Thee first 2 axes of the PC A plot based on biochemical variables expressed on a
DWW basis display 62% of the total variance among sediment variable (Fig. 2, Table 7).
Alll of the sediment characteristics are displayed on the right side of the diagram, indicatingg that they are all, to some extent, positively correlated with each other (see also
Tablee 3). Larval growth and survival correlated most strongly with sediment PUFA
content.. The results of the Monte Carlo permutation tests also show a significant corre-
55 5
ChapterChapter 3
Tablee 6. Correlations of growth and survival of C. riparius larvae after 14 days with the
biochemicall variables expressed per unit of dry weight or organic matter weight. Pearson
coefficientt F? and significance level. * = P < 0.05, ** = P < 0.01. I F A = total fatty acids, PUFAs
== polyunsaturated fatty acids, E-content = energy content.
variabless standardized on
dryy weight
growth h
survival l
cc
-0.10 0
NN
-0.06 6
organicc matter
growth h
survival l
-0.27* *
+0.24 4
+0.02 2
-0.20 0
+0.21 1
+0.12 2
PP
+0.12 2
+0.06 6
+0.45** *
+0.31* *
carbohydrates s
-0.01 1
-0.22 2
+0.41** *
+0.20 0
protein n
+0.21 1
+0.13 3
+0.32* *
+0.29* *
EFA A
-0.05 5
-0.01 1
-0.03 3
+0.08 8
PUFAs s
+0.51" "
+0.39** *
+0.58** *
+0.49** *
bacteriall FAs
+0.25 5
+0.21 1
+0.49** *
+0.50** *
pigments s
+0.39** *
+0.11 1
+0.70** *
+0.51** *
E-content t
+0.15 5
+0.04 4
+0.33* *
+0.32* *
C 0 22 production
+0.56** *
+0.41** *
+0.54** *
+0.59** *
lationn between the larval performance parameters and the sediment characteristics
(Tablee 7).
Thee biplot of the sediment characteristics with biochemical variables expressed
onn an OM basis explained 43% of the total variance, while larval performance explained
20%% of the total variance (Fig. 3, Table 7). Again all sediment characteristics are
displayedd on the right in the diagram, with the exception of water content. The setting of
thee biplot indicates a positive correlation with most of the sediment characteristics, and
especiallyy with CO2 production, pigments, fatty acids of bacterial origin, energycontent,, protein, carbohydrates, and N. A very strong correlation between the larval
performancee parameters and the sediment characteristics is indicated by the Monte
Carloo permutation tests (Table 7).
Strongerr correlation of larval performance and sediment characteristics (lower
P-values)) are found if the biochemical variables are standardized on OM content
comparedd to standardization of the biochemical variables on DW (Table 7). Moreover, a
higherr % of variance used to explain larval growth and survival is displayed in the
biplott with the standardization on OM whereas the total variance expressed of the sedi-
56 6
ChironomidChironomid larvae growth on sediments
mentt characteristics is lower. This means that a lower % of variance is used to explain a
higherr % of variance of larval growth and survival in the data set with the biochemical
variabless standardized on OM content compared to the data set with the biochemical
variabless standardized on DW.
Discussion n
Duringg this study, sediments were sampled in several different water systems
rangingg from large lakes to small streams and from silty to sandy sediments. The origin
off the organic matter (OM) presumably varied between predominantly algal in some
systemss to being largely land-derived in others. In spite of the broad variation in the
substratess used in this study, growth and survival of Chironomus ripctrius larvae were
welll correlated to chemical characteristics of these sediments. Evidently, the nutritional
valuee of ingested matter is highly limiting for larval growth and survival in many
sediments,, since supplementation with high quality fish food stimulated larval growth in
sedimentss from all 41 sites, simultaneously showing that physical characteristics of the
sedimentss did not influence larval growth of C. riparius. This finding supports the
hypothesiss posed by a few field studies that OM limits and regulates population dynamicss of benthic macrofauna (Lopez and Levinton 1987; Goedkoop and Johnson 1996).
Thee degree of food limitation that we demonstrate may partly be affected by the use of
C.C. riparius as a test species. C, riparius is known to abound in organically enriched
waters.. The very low survival and growth rates in the poorest sediments may have been
causedd by relatively high food demands of this species.
Besidess growth limitation caused by food supply, specific biochemical componentss were found to correlate with larval growth and survival. Standardization of these
variabless on the basis of sediment OM content resulted in more and stronger
correlationss with growth and survival of midge larvae than did standardization on basis
off dry weight (DW). The PCA analyses also showed more variance of larval performancee was explained when sediment characteristics were expressed on the basis of OM
content.. OM content of the sediments was strongly correlated with the small particulate
fractionn (< 63 urn). C. riparius larvae are mainly deposit feeders that eat whole sedimentss with a particle size limit determined by the mentum width (for instance, between
57 7
ChapterChapter 3
Fig.. 2. PCA biplot showing characteristics of 41 unpolluted sediments with the (bio)chemical
variabless standardized on dry weight. Open circles represent sediments, black dots sediments
parameters,, and arrows larval performance parameters. For percentages of total variance and
resultss of additional Monte Carlo permutation tests see Table 7.
+1.0 0
OO
carbohydratess
OO
organicc matter
oo
C
cPP
OO
€© ©
c
N
E-content
proteins s
w owo
OO
E/N N totall FA
OO
pigments s
PP
C0 22 production
%>210pm m
survivalsurvival\ \
bacterial FA
O O growthgrowth
OO \
1
PUFA A
-1.0 0
-1.0 0
Axiss 1, eigenvalue 0.49
+1.0 0
422 and 65 urn for 2" instar larvae, Chapter 4). The strong correlation of OM content
withh the small particle fraction of the sediments secures that standardization of variables
onn an OM basis includes the part of the sediments that potentially can be used as food
sourcee by the chironomid larvae. The small particle fraction probably not only containedd OM, but also mineral particles. The midge larvae used in our experiments inevitablybly ingested different quantities of small mineral particles along with the OM during
feedingg trials. Consequently, growth may have been limited to varying extents by dilutionn effects, associated with the portion of small mineral particles ingested (Chapter 4).
Differencess in mineral constituents and thus in the quantity of ingested OM among the
58 8
ChironomidChironomid larvae growth on sediments
Fig.. 3. PCA biplot showing characteristics of 41 sediments with the biochemical variables
standardizedd on organic matter content. Open circles represent sediments, black dots
sedimentss parameters, and arrows larval performance parameters. For displayed percentages
off total variance and results of additional Monte Carlo permutation tests is referred to Table 7.
+1.0 0
oo
OO
oo
oo
swc c
o
OO O
o
%>210um m
carbohydratess C0 2 production
NN
proteins growth
88
^w^w
^ ^^
^
cc
^^
O
E-content
survival
pigments
nn
0) )
O) )
UU
bacteriall
JJ
o
© OO o
p
oo
totall FA u
oo
F ^ ^
O
CC
oo )c)
OO
oo
C/N N
-1.0 0
-1.0 0
Axiss 1, eigenvalue 0.30
+1.0 0
individuall sediments may have disguished the influence of a set of biochemical constituentss of OM present in the sediments on the life history parameters of C. riparius when
thesee biochemical constituents were expressed on a DW basis. However, correlating
larvall growth to biochemical variables standardized based on OM content revealed the
influencee of organic matter composition directly. The effect of organic matter compositionn is independent of organic matter abundance and consequently is not influenced by
overalll food ingestion rate of the chironomid larvae.
Inn most studies, chemical parameters of sediments are expressed as portion of
DW.. If sedimentation traps are used to examine food sources of detritivorous inver-
59 9
ChapterChapter 3
tebrates,, the two different standardization procedures will likely not lead to the differencess found during the present study because organic content of the sedimented matter
iss expected to be high. However, as in the present study, sampled sediments often containn a broad range of mineral contents, potentially obfuscating a number of relationships
iff biochemical variables are standardized based on DW alone.
Growthh and survival of Chironomus riparius larvae did correlate with a few
variabless standardized based on DW, e.g. CO2 production as indicator of labile OM,
PUFF As, and pigments. The strong correlation between larval growth and PUF As may
havee arisen from the inability of animals to synthesize essential fatty acids (especially
co33 and co6 fatty acids; Brett and Miiller-Navarra 1997, Napolitano 1999). Therefore,
PUFAss have to be obtained from the diet, for instance from algae with high PUFA
contents.. Fatty acids in the sediments sampled during this study represented only a
smalll portion of the total amount of OM and, as such, PUFAs were a minor portion of
thee total fatty acids in the substrates. Consequently, it is probable that PUFAs were a
limitingg factor for larval growth in most of the sediments tested during this study. Labile
OMM was assessed with a microbial degradation assay. Microorganisms were chosen in
orderr to obtain a measure of overall digestibility of OM. Enzymatic assays are used to
Tablee 7. Summary of the PCA analyses. Percentage of total variance displayed on the first and
secondd axis and percentage of the total variance explained by larval performance (growth and
survival).. P-values of permutation tests.
biochemicall variables
standardizedd on
dryy weight
organic matter
49
30
%% of total variance
off sediment characteristics displayed on axis 1
off sediment characteristics displayed on axis 2
13
13
off sediment characteristics explaining larval performance
11
20
explainingg larval performance displayed on axis 1
28
76
explainingg larval performance displayed on axis 2
41
8
PP -value growth
0.035
<0.005
PP -value survival
0.050
<0.005
Permutationn test
60 0
ChironomidChironomid larvae growth on sediments
establishh the digestable fraction of specific components such as proteins (Dauwe et al.
I999a,b),I999a,b), but comparable assays that cover all food components are not available.
Judgedd from the good correlation between larval performance and labile OM the microbiall assay was adequate to determine the digestible fraction of OM. Similar to shortchainn PUF As, pigments may be considered as indicators of fresh algal material (Napolitanoo 1999), while CO2 production is an overall assessment of digestibility of OM.
Together,, correlations of PUFAs, pigments, and labile OM with chironomid larvae
growthh suggest that the nutritious and relatively easy digestible OM of algal origin was
ann important factor regulating growth of chironomid larvae in the field.
Organicc matter composition of sediments appeared to constitute a major factor
influencingg the growth and survival of Chironomus riparius larvae, in spite of the
complexx nature of this material. We base this conclusion on the high number of correlationss between the food quality parameters standardized on OM with larval growth and
survival,, and the high percentage of variance explained by ordination. For instance, P
wass found to be positively correlated with growth and survival. In an extensive number
off studies on daphnid crustaceans, both P and PUFAs have been identified as good
predictorss of food quality of living phytoplankton (e.g., Brett and Müller-Navarra 1997,
Gulatii and DeMott 1997). Carbohydrates were also found to be positively correlated
withh larval growth in spite of the diversity of polymeric sugars. The presently used
carbohydratee analysis not only measures simple sugars, but also complex sugars such as
cellulosee which are hardly digestible. At low food levels, carbohydrates are preferentiallyy used for maintenance of the body structures, leaving essential nutrients for
buildingg of new tissue (Roman 1983, Vos et al. 2000). A similar mechanism may have
aidedd the chironomid larvae to survive in the low nutritional conditions of some of the
sedimentss sampled during this study. Proteins are also a heterogeneous group, consistingg of a range of amino acids, some of which are essential and others non-essential
nutrients.. The chironomid larvae may have used proteins as source of essential amino
acidss (Cowey and Forster 1971, Cowie and Hedges 1996), of nitrogen, or of amino N
(Kreegerr et al. 1996).
Inn conclusion, the composition of OM in sediments strongly influences growth
andd survival of chironomid larvae. The presence of newly produced and labile OM,
indicatedd by pigments, PUFAs, and microbial mineralization rate, was strongly
61 1
ChapterChapter 3
correlatedd with larval growth. That population dynamics of benthic invertebrates in lake
sedimentss follow seasonal inputs from the pelagic zone, and that the response to algal
inputss varies with the species composition of algal blooms, has been previously
establishedd (Marsh and Tenore 1990, Goedkoop and Johnson 1996, Goedkoop et al.
1998).. Here, we stress the importance of the variable organic matter composition as a
keyy factor regulating the growth of detritivorous invertebrates. This study gives solid
evidencee that spatial and temporal differences in detritus quality are a major factor
regulatingg the growth dynamics of detritivores in soft bottoms.
Acknowledgements s
Partt of the project was financed by the Institute for Inland Water Management
andd Waste Water Treatment (RIZA), Lelystad, The Netherlands. We thank Ronald
Gylstraa and Edwin Peeters (Wageningen University) for their valuable comments on
thiss paper. Steven Arisz (UvA, Amsterdam, The Netherlands) greatly helped with the
fattyy acid analyses, by improving the GC analysis and with theoretical support. We are
endebtedd to Eric Boschker (NIOO, Yerseke, The Netherlands) who identified fatty acid
peakss with the MS. Joke Westerveld (UvA, Amsterdam, The Netherlands) supported the
CO22 analysis.
62 2
CHAPTERR 4
PARTICLEE SIZE EFFECT ON PREFERENTIAL SETTLEMENT AND
GROWTHH RATE OF DETRITIVOROUS CHIRONOMID LARVAE AS
INFLUENCEDD BY FOOD LEVEL
''
J.H.. Vos', M. Teunissen, J.F. Postma , F.P. van den Ende
Departmentt of Aquatic Ecology and Ecotoxicology, University of Amsterdam,
Kruislaann 320, 1098 SM Amsterdam, the Netherlands. Phone (0031)-20-5257718, fax
(0031)-20-5257716,, '[email protected], 2present address: AquaSense Consultants,
P.O.. Box 95125, 1090 HC Amsterdam, The Netherlands
Submittedd for publication
63 3
ChapterChapter 4
Abstract t
Sedimentt particle size distribution and organic matter content are reported to
determinee the field distribution of chironomid larvae. Therefore, combinations of
minerall particles of different size ranges and food level were tested for the influence on
thee preference and growth of detritivorus chironomid larvae. Mineral particle size had
noo effect on larval growth at saturating food supply. However, at limiting food levels
growthh of third instar larvae was hampered by ingestion of small mineral grains (8-63
urn).. Second, third and fourth instar larvae were able to construct tubes of silty and
sandyy substrates, but tubes that were found in the larger particle size range (550-1200
urn)) were less stable compared to those constructed from small particles.
Larvaee showed a clear preference for the compartment supplied with food when
offeredd a choice between two different particle size substrates with only one particle
sizee substrate supplied with surplus of food, independent of the particle size ranges.
Duringg preference experiments in which larvae were offered two food levels, a thresholdd concentration between 0.075 and 0.10 mg ml"1 was found below which larvae
continuedd crawling and did not settle to construct tubes as they did at higher food levels.
Whenn allowed to choose between two food levels both above the threshold concentration,, larvae preferred the higher food concentration if the difference between food
levelss exceeded 0.75 mg ml" .
Foodd level and substrate particle size determined larval motility and preference.
Thee influence of both factors was governed by food threshold and saturation
concentrations.. It is postulated that organic matter content and particle size distribution
inn the natural environment are interacting factors determining the abundance of detritivoruss chironomid larvae.
64 4
ParticleParticle size and food level effect on C. riparius
Introduction n
Chironomidd larvae are often a quantitatively important component of macrofaunaa communities inhabiting freshwater sediments. Hence, their distribution has been
welll studied and related to sediment composition and other environmental factors (see
Finderr 1986, 1995 for reviews). In general, detritus feeding chironomids are more
numerouss in silty sediments than in sandy sediments (e.g., Thienemann 1954, Maitland
1979,, Pinder 1986, 1995), although chironomid-substratum associations may vary with
morphologicall characteristics of the taxa (Winnell & Jude 1984). The impact of organic
matterr content and grain size distribution on the distribution of chironomids in the field
iss difficult to distinguish, because they often covary in natural sediments (e.g., Rabeni &
Minshalll 1977, Pinder 1986, 1995, Suedel & Rodgers 1994, Maxon et al. 1997,
Reinhold-Dudokk van Heel & den Besten 1999). For Chironomus riparius Meigen, a
detritivorouss species of eutrophic waters (Rasmussen 1984, 1985), a negative correlationn of density with particle size, and a positive correlation with organic matter
contentt have been found (Learner & Edwards 1966, Leppanen et al. 1998). In the River
Dommell (The Netherlands), a lowland river with high organic input from partially
treatedd sewage, C. riparius is the most abundant midge species (Groenendijk et al.
1998).. The sediment consists mainly of sand, with detritus and silt accumulating locally
alongg the banks. It is in this patchy environment that the highest density of larvae is
foundd (Leppanen et al. 1998).
ChironomusChironomus riparius larvae are highly sedentary for most of their lifespan. Egg
massess are laid on substrata just below the water surface and, after emergence, the first
instarr larvae drift away to find a suitable place to settle. Within a few days the larvae
startt to build a living-tube. The larvae keep in physical contact with their tube and
thereforee the foraging area is restricted to the immediate surroundings of the tube
entrancee (personal observations in laboratory cultures). Consequently, the choice of
settlementt site is of critical importance. If larvae leave their burrow they will have to
findfind a new site that meets their demands. Site choice is not restricted to the early instar
larvae,, but also takes place during later life stages. Larval drift in the River Dommel
wass studied by Groenendijk et al. (1998), who found that the majority of the drifting
larvaee consisted of 3rd instars.
65 5
ChapterChapter 4
Severall ways have been proposed in which particle size could affect larval
preferencee and performance, including suitability of the substratum for burrowing
(Wileyy 1981a, Winnell & Jude 1984) and for tube construction (Brennan & McLachlan
1979)) and the risk of physical damage by large particles (Winnell & Jude 1984). Apart
fromm these factors we hypothesize that particle size determines the feeding mode,
therebyy influencing growth opportunities. C. riparius is a deposit feeder ingesting
inorganicc particles together with the organic detritus that constitutes the principal food
(Rasmussenn 1984&). However, if the inorganic particles are too large to be swallowed
thee larvae can only feed on organic aggregates or scrape the organic film from solid
surfaces. .
Inn this study we used an experimental approach to examine if site choice of C.
ripariusriparius larvae results from active selection for food level, particle size or both, and if
preferencess arise from suitability of the substrate as a food source or habitat. Two types
off experiments were performed: (1) to test the effect of combinations of particle size
andd food supply on larval growth. Growth was studied at saturated and non-saturated
foodd levels with mineral particles of different sizes as substrate. During these growth
experimentss larval intestines were checked for the presence of mineral particles. (2) to
determinee the capacity of larvae to differentiate between food levels and particle size
ranges.. Results of growth and preference experiments are discussed in relation to
substratee suitability and foraging behavior of chironomid larvae.
Methods s
OverviewOverview of the experiments and general experimental setup
Twoo growth experiments and two preference experiments were performed. The
firstt growth experiment was carried out with a set of different particle size ranges and
withh a surplus of food to examine the effect of particle size as sole factor influencing
growthh and survival of chironomid larvae. The second growth experiment was
performedd with two particle size ranges and a set of food concentrations to examine the
influencee of mineral particles present in the larval intestines. The small particle size
substratee of 8-63 um and the large particle size substrate of 550-1200 um were chosen,
becausee it was expected that the former could be ingested and the latter could not be
66 6
ParticleParticle size and food level effect on C. riparius
ingestedd by 3rd instar larvae based on mentum width of 3rd instar larvae (mentum widths
aree presented below). Third instars (7 days old) were used, because the intestines of
thesee larvae are easily dissectable. A growth period of 1 week was chosen, because at
thee age of 14 days the first prepupal stages can already be observed. Growth was
measuredd per individual larva as increase of length.
Duringg the first preference experiment (experiment 3) both 1st and 3 r instars
weree allowed to choose between the 2 halves of the experimental unit containing
particless of different size ranges. This experiment was performed first, with excess of
foodd on both halves of the container and second, with an excess of food on just 1 half of
thee container. The aim was to evaluate the relative importance of particle size and food
ass determinants of substrate selection by the chironomid larvae. During the second
preferencee experiment (experiment 4) the ability of 3rd instar larvae to distinguish
betweenn different food levels was tested. During experiments 1 (Larval growth at
saturatingg food level in a range of different particle sizes) and 3 (Larval preference for
particlee sizes in the presence and absence of food), excess food was added at the surface
off the substrate to ensure easily available food. The excess food level chosen was 0.4
mgg cm"2 to allow comparison with Sibley et al. (1998). Different food concentrations
duringg the experiments 2 (Larval growth response to limiting food levels in two
differentt particle size ranges) and 4 (Preference for food level) were obtained by mixing
preweighedd amounts of food through a certain volume of mineral substrate.
Forr all experiments polyethylene containers (10 x 10 x 6.5 cm) were used as
experimentall units; Dutch Standard Water (DSW, NEN 6503, pH 8.2, 210 mg CaC03)
ass artificial freshwater, and a 95%/5% (m/m) mixture of the commercially available
fishfoodss Trouvit® (95%) (Trouw, France) and Tetraphyll® (5%) (TetraWerke, Germany)) as food source were used. For each experimental series at least 10 egg masses
fromm a laboratory culture of C. riparius Meigen were used. Newly hatched 1st instar
larvaee (<24 h old) were obtained directly from egg ropes. Early 3rd instar larvae were
obtainedd by culturing newly hatched larvae for 7 days in an aerated aquarium containing
DSW,, a layer of sand and a surplus of food. Larval instar was determined for all
individualss based on head capsule width, measured using a binocular microscope. Head
capsulee width of 1st, 2nd, 3rd, and 4th instars ranged between 94 and 140, 195 and 246,
3355 and 428, and between 484 and 605 urn respectively. Mentum width was measured
67 7
ChapterChapter 4
forr 20 individuals per instar and ranged between 28 and 42, 42 and 65, 84 and 112 and
betweenn 158 and 167 urn respectively for 1st, 2nd, 3rd, and 4th instars. This resulted in a
mentum/headd capsule ratio ranging between 0.25 and 0.31 for all instars.
Thee quartz particle size ranges used in the experiments are presented in Table 1.
Beforee use in the experiments the substrates were washed and organic matter was
removedd by combustion at 550°C for 6 hours. All particle sizes were checked by laser
diffractionn (Master Sizer X Ver. 1.2a, Malvern).
Alll experiments were conducted at 20°C
1°C. Growth experiments were
performedd under a light/dark regime of 16/8 hours. The preference experiments were
performedd in the dark, since Baker & Ball (1995) have found negative phototactic
behaviorr for C. tentans larvae which had not constructed a tube and similar behavior
wass observed for C. hparius during preliminary studies (unpubl. data). The containers
off both the growth and the preference experiments were not aerated in order to avoid
resuspensionn of food and fine grained material (1.5-20 urn).
ExperimentExperiment 1: Larval growth at saturating food level in a range of different
particleparticle sizes
Thee effect of particle size on larval growth was determined after 7 and 14 days
usingg the 6 different particle size ranges. Growth on each particle size range was tested
55 times for both growth periods. Replicates were started on different days with larvae
originatingg from different egg masses. In each of the containers 400 ml of artificial
freshwaterr was added to 200 ml of substrate with a defined grain size range, resulting
Tablee 1. Mineral particle size ranges ranked after the 5 and 95% percentiles of volume (5-95
vol%),, median particle size and origin of the substrate
grainn size range (urn, 5-95 vol%)
1.5-20 0
8-63 3
50-175 5
105-300 0
275-600 0
550-1200 0
68 8
median particle size (urn)
3.5 5
26 6
95 5
173 3
392 2
757 7
origin
Sibelcoo quartz powder M500
Sibelco®® quartz powder M10
Sibelco®® quartz sand
Sibelco®® quartz sand
Praxis®® playing sand
Praxis®® playing sand
ParticleParticle size and food level effect on C. riparius
inn a substrate layer of 2 cm. One hundred mg of food was added and allowed to settle on
thee substrate after which 20 1st instar larvae were released in the water. The length of a
groupp of 20 randomly chosen larvae was measured to determine initial length. After 7
dayss half of the culture vessels were randomly chosen and analyzed. The remaining
vesselss were provided an additional 100 mg of food and this part of the growth experimentriment was terminated after 14 days. At the end of the experiments larval body length
wass measured and instar stage was identified of each surviving larva, after which gut
contentss were studied through a binocular microscope. During the experiments behavior
off the larvae and the presence and position of the living tubes were noted. One-way
ANOVAA was used to compare growth and survival in the different particle size ranges
(P<0.05). .
ExperimentExperiment 2: Larval growth response to limiting food levels in two different
particleparticle size ranges
Too 200 ml of silty (8-63 um) or coarse (550-1200 urn) substrate 5, 10, 25, 50,
100,, 150, 200, 400 or 800 mg of food was added and substrate and food were
thoroughlyy mixed. This resulted in food concentrations of 0.025, 0.05, 0.125, 0.25, 0.5,
0.75,, 1.0, 2.0 and 4.0 mg food ml"1 mineral substrate respectively. Four hundred ml of
artificiall freshwater was added carefully. At the start of each experiment 20 3 rd instar
larvaee were added to the water and the initial length was measured on a group of 20
randomlyy chosen larvae. After 7 days the length of the surviving larvae was measured.
Eachh experimental unit was replicated twice. The effect of particle size combined with
foodd level was analyzed using a two-way ANOVA (P < 0.05).
ExperimentExperiment 3: Larval preference for particle sizes in the presence and absence
ofof food
Thee containers were filled with 400 ml artificial freshwater and divided
diagonallyy in half by a polyethylene barrier. On both sides of the barrier 100 ml
substratee of a specific particle size range was released and allowed to settle, after which
100 or 20 mg food was added to the water at both sides of the strip, resulting in 0.2 or 0.4
mgg food cm 2 . The barrier was removed after settling of the food. The containers with
0.22 mg food cm"2 were used to test preference of 1st instar larvae and the 0.4 mg cm"2
69 9
ChapterChapter 4
containerss for preference tests with 3 rd instar larvae. Each possible combination of the
particlee size ranges 1.5-20, 8-63, 105-300 and 550-1200 um was replicated 4 times,
includingg pairings with the same particle size substrate serving as controls. Replicates
weree started at different days. The experiments were started by randomly adding 20 1st
orr 20 3 r instar larvae above the interface between the particle sizes. The experiments
weree ended by replacing the barrier after 72 h after which the larvae were counted at
eachh side of the separating barrier.
Additionall experiments were performed, similar in setup, except that food was
addedd to only one side of the container (10 mg for 1st instars, 20 mg for 3rd instars), and
thee other side was left without food. All possible combinations of particle size ranges
weree tested once with food on the first half and once with food on the second half of the
container.. Pairings of the same particle size served as controls.
AA chi-square test was used to compare the number of larvae on each side of the
containerr (P < 0.05), with 50% of the total larvae as the expected number of larvae for
eachh half of the container.
ExperimentExperiment 4: Preference for food level
Containerss were divided in halves by a polyethylene barrier and on each side
1000 ml of 8-63 urn substrate was added together with a specific amount of food (0, 1.0,
2.5,, 5.0, 7.5, 10, 17.5, 25, 37.5, 50,100 or 200 mg). Substrate and food were thoroughly
mixed,, resulting in food concentrations of 0, 0.01, 0.025, 0.05, 0.075, 0.10, 0.175, 0.25,
0.375,, 0.50, 1.0 and 2.0 mg ml"1 respectively. Four hundred ml of artificial freshwater
wass added gently. The barrier was removed and 20 3rd instar larvae were released in the
waterr above the interface. After 72 h the number of larvae on each side of the containers
wass counted. Each combination of food levels was tested once, including combinations
off the same food level that represented the controls.
AA chi-square test was used to compare the number of larvae on each side of the
containerr (P < 0.05), with 50% of the total larvae as expected number of larvae per half
container. .
70 0
ParticleParticle size and food level effect on C. riparius
Results s
ExperimentExperiment 1: Larval growth at saturating food level in a range of different
particleparticle sizes
Averagee length of the 1st instar larvae at the start of the experiments was 0.95
(SEE = 0.02) mm. No significant differences in larval growth and survival after 7 days or
afterr 14 days, were found between any of the particle size ranges tested (F = 0.98, df =
1.5-200
8-63
50-175
105-300 275-600 550-1200
particlee size ranges (jjm)
Fig.. 1. Survival (%) and growth (final - initial length, mm) of larvae after 7 days (grey bars) and
afterr 14 days (black bars) on substrates with different particle size ranges and with saturating
foodd levels of 0.4 mg cm"2. Error bars represent standard errors. Initial length of the 1 s ' instar
larvaee was 0.95 ( 0.02) mm.
71 1
ChapterChapter 4
5,, MS = 0.18) (Fig. 1). After one week an average length of 5.2 mm
rd
0.1) was
nd
reached.. Most of the larvae were in the 3 instar, although a few 2 instar larvae were
foundd (<1%). After two weeks of growth the larvae were mainly 4th instars with an
averagee length of 10.2 mm ( 0.1). Survival in both tests and in all experimental units
exceededd 80%. The 2nd, 3rd, and 4th instars were able to build tubes in all particle size
ranges,, using both mineral and food particles. The tubes were mainly situated horizontally,, on the surface of the substrates. Up to approximately 30% of the tubes in each
particlee size range was built into the substrate, with the exception of the 1.5-20 um and
8-633 urn substrates, where all tubes were situated vertically in the substrate. During the
11 and 2 week growth periods it was noted that the tubes that were constructed in the
largestt particle size range (550-1200 um) were less stable than tubes constructed using
smallerr particles when they were gently crumbled between the fingers. In all particle
sizee ranges most of the tubes were 1.5 to 2 times the length of the inhabiting larvae, but
occasionallyy tubes were found with lengths up to 3-4 times the length of the inhabiting
larvae. .
Dissectionn of the larval gut contents showed that 2nd instar larvae (n = 4) were
ablee to ingest grains with diameters up to approximately 30 um, 3 rd instar larvae (n =
20)) up to approximately 60 um while the guts of 4th instar larvae (n = 20) contained
particless up to 100 um. Thus, grain size diameters found in the guts of the individual
instarss were smaller than the measured minimum mentum width of the corresponding
instar. .
Initiall length of the 3rd instar larvae used in the experiment was 4.0 mm ( 0.05).
Survivall at the end of the experiment exceeded 80% in all experimental units.
ExperimentExperiment 2: Larval growth response to limiting food levels in two different
particleparticle size ranges
Larvall growth on the 8-63 \im substrate was significantly lower than growth on
thee 550-1200 um substrate (F = 0.77, df = 7, P < 0.05 ) (Fig. 2) at limiting food levels.
Maximumm increase in length was similar for both particle size ranges, but was reached
att 2 mg ml"1 on 8-63 urn and at 1 mg ml"1 on 550-1200 um. The larvae that were grown
att the highest food concentrations were still in the growing phase, since the 4th instar
larvaee of C. riparius can reach lengths up to 18 mm.
72 2
ParticleParticle size and food level effect on C. riparius
Fig.. 2. Length (mm) of 3rd instar larvae after one week of growth on two particle size ranges and
differentt amounts of food. Black circles: 8-63 urn substrate, grey squares: 550-1200 urn
substrate.. Error bars represent standard errors. Initial length of the 3rd instar larvae was 4.0 mm
(
0.05) (0.26 mm3)
EE
en n
CD D
Onn average higher larval motility was observed in the 550-1200 urn containers
comparedd to the 8-63 urn containers, although at the highest food levels (1-2 mg ml" ) in
bothh particle size ranges hardly any larval activity was observed. This activity encompassedd mainly crawling over the surface of the substrate, efforts to penetrate the
substratee and occasionally swimming. In containers with the highest food levels of both
particlee size ranges the longest tubes were found with lengths up to 3-4 times of the
larvall length. Observations with a binocular microscope indicated that guts of larvae
grownn in the 8-63 urn substrates were filled with proportions of mineral particles to
foodd volume similar to the proportions observed in the substrates.
ExperimentExperiment 3: Larval preference for particle sizes in the presence and absence
offood offood
Bothh 1st and 3 rd instar larvae were observed to swim freely after being released
inn the water and readily moved between particle sizes. If an excess of food was provided
onn both sides of the container no significant preference for a particle size range was
73 3
ChapterChapter 4
Tablee 2. Preference of 1 s instar larvae for combinations of particle sizes (urn) and presence of
food.. Percentage of the initial 20 larvae found on the section provided with food (0.2 mg cm"2)
(
SE) after 72 hours
sectionn without food
sectionn with food
1.5-20 0
8-63 3
105-300 0
550-1200 0
1.5-20 0
90(5) )
92 2
81 1
92 2
8-63 3
85 5
95(7) )
85 5
93 3
105-300 0
89 9
93 3
92(2) )
85 5
550-1200 0
85 5
100 0
97 7
1000 (0)
foundd for either 1st or 3 rd instars (data not shown). Average of the highest number of
larvaee found at one side was 10.6
0.35) in the experimental units containing different
particlee size substrates on both sides (n = 24).
Duringg the experiments in which only one side of the container was
supplementedd with food, 1st and 3rd instar larvae showed a significant preference for the
foodd side, independent of the particle size (Table 2 and 3). On average 91% ( 1.4) of
thee 1st instar larvae and 94% ( 1.5) of the 3 rd instar larvae were found on the food side
afterr 72 h of incubation. Observations of the 3rd instar larvae indicated that the larvae
eitherr started swimming after release or immediately penetrated the substrate they had
landedd on. If the larvae had penetrated the substrate on the side without food they
mostlyy were found to crawl on the surface or to swim again after a few hours. If the half
containingg food was penetrated the larvae were observed to build tubes within a few
hourss (1.5-2 h) after release.
Tablee 3. Preference of 3r instar larvae for combinations of particle sizes (urn) and presence of
food.. Percentage of the initial 20 larvae found on the section provided with food (20 mg cm"2) (
SE)) after 72 hours
sectionn without food
sectionn with food
1.5-20 0
8-63 3
105-300 0
550-1200 0
1.5-20 0
89(11) )
95 5
85 5
90 0
8-63 3
90 0
100(0) )
95 5
100 0
105-300 0
89 9
100 0
92(6) )
85 5
550-1200 0
100 0
95 5
85 5
91(9) )
74 4
ParticleParticle size and food level effect on C. riparius
ExperimentExperiment 4: Preference for food level
Foodd concentrations were split into 2 groups: low food concentrations of 00.0755 mg ml"' and high food concentrations of 0.1-2 mg ml"1 to illustrate differences in
reactionn of larvae to the food concentration in Figures 3 and 4. This resulted in three
combinationss of food concentration in the experimental containers: low with low food
concentrations,, low with high and high with high food concentrations. If food levels on
bothh sides of the container were low no preference of the 3rd instars for a food level was
observed.. If food concentration differences between both halves were > 0.75 mg ml"1 or
low/highh food concentration < 0.3 almost all larvae were found on the side with the
higherr food level. Interestingly, the percentage of larvae that were found in the highest
foodd concentration hardly ever reached 100% if both sides contained a high food
concentration.. If there was a high food concentration on one side and on the other side a
100 0
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1.5
differencee in food concentration (mg ml"1)
Fig.. 3. Preference of third instar larvae for different food levels. The X-axis represents
differencess in food concentration (mg ml"1) between the two halves of the container. The Y-axis
representss the percentage of larvae at the highest food level. The area between the two
horizontall lines in the graph marks the field where preference was not significant.
Blackk squares: food concentrations on both sides > 0.1 mg ml"'.
Crosses::
lower food concentration < 0.1 mg ml'1, higher food concentration > 0.1 mg ml"1.
Greyy circles:
food concentrations at both sides < 0.1 mg ml"1.
75 5
ChapterChapter 4
loww food concentration and food concentration differences were > 0.75 mg ml" 100%
off the larvae were found on the side with the higher food concentration. No consistent
preferencee was observed when concentration differences were < 0.75 mg ml"1. In fig. 4
foodd concentration differences are expressed as lower food concentration:higher food
concentrationn ratio. The curve shown in the graph follows the equation the curve y =
100/(x+l)) in which y = % of larvae at the higher food side and x = lower/higher food
concentration.. The equation expresses the larval distribution over the 2 halves of the
experimentall unit if this distribution is proportional to the distribution of the food.
Larvall distribution roughly followed this equation but fit was low (R = 0.38).
120 120
cc
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y^ y^
roro 100
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01 1
lower:higherfoodd concentration
Fig.. 4. Preference of third instar larvae for different food levels. The X-axis represents the ratio
lowerr food concentration : higher food concentration. The Y-axis represents the percentage of
larvaee at the highest food level side. The curve represents the distribution of the larvae following
thee curve y = 100 x (x+1)"1,in which y = % of larvae at the higher food side and x = lower/ higher
foodd concentration.
Blackk squares: food concentrations at both sides > 0.1 mg ml' .
Crosses::
lower food concentration < 0.1 mg ml"1, higher food concentration > 0.1 mgml"1.
Openn circles:
food concentrations at both sides < 0.1 mg ml".
76 6
ParticleParticle size and food level effect on C. riparius
Similarr to the experiment on larval preference for combinations of particle size
andd food level, it was observed that the 3rd instar larvae started to swim if the food level
off the half of the container that they had landed on was low, or first penetrated the
substratee and started swimming or crawling again after a few hours. Consequently, in
containerss which had a low food concentration (0-0.075 mg ml"1) on both sides the
larvaee were relatively active and
50% of the larvae were found not to inhabit a tube
afterr 72 h of incubation. This resulted in no observed preference for either of the food
concentrations.. In the containers with both sides having substrates with > 0.10 mg ml" ,
alll larvae inhabited a tube.
Discussion n
Thee data gathered during this study give new insights into the possible role that
minerall particle sizes in combination with different food levels play in the habitat of
detritivores.. Firstly, differences in tube construction and feeding mode of Chironomus
ripariusriparius larvae were found when grown on different particle size ranges. The 2n , 3 r ,
andd 4th instar larvae were observed to be able to construct tubes of all particle size
ranges,, but the tubes that were found in the large particle size ranges seemed to be less
stablee than tubes constructed from smaller particles. A few studies reported that
chironomidd larvae do not discriminate among particles for tube building, but in the few
studiess done only small particles and small ranges of particle sizes were examined
(Edgarr & Meadows 1969, Brennan & McLachlan 1979, Dudgeon 1990). In our study,
foodd and faecal pellets may also have been used as tube building material, and thus
mightt have compensated for a possible lack of smaller particle size substrate for tube
constructionn in the large particle size containers. Nevertheless, at lower, non-saturating
foodd levels, a clear effect of particle size was noted. Larval growth was negatively
affectedd by the small and ingestible particle substrate. This lower growth can be
explainedd by differences in food availability resulting in different modes of feeding. In
thee small particle substrate the larvae had unselectively ingested the food together with
thee substrate. In the large particle substrate the larvae did not ingest mineral particles
andd were able to feed solely on the added fishfood by specifically picking food items
betweenn sand grains, or scraping adhered food from particle surfaces. Consequently, in
77 7
ChapterChapter 4
larvaee feeding on small particle substrates the amount of non-food particles in the gut at
non-saturatingg food levels may have limited food uptake. In contrast, food uptake in
largee particle substrates is not hampered by mineral particles occupying gut space. Food
uptakee diluted with inedible particles is likely to be a common phenomenon in nature.
Inn the field small mineral particles are often associated with fine organic material and
thenn buikfeeding detritivores will be bound to ingest mineral particles together with
organicc matter in order to feed.
Thee C. hparius larvae showed different particle size preference than C. tentans
att high food levels (Sibley et al. 1998). The C. hparius larvae did not show any preferencee for particle size in contrast with C. tentans. A strong selection preference of C.
tentanstentans was found for the smaller particle size range of 2 substrates at surplus food
levelss in both halves of the container. In agreement with our findings, both 1st and 3rd
instarr C. tentans larvae showed a strong response to food availability independent of the
meann particle size of the substrate (Sibley et al. 1998). When offered a choice in our
experimentss between substrates with and without food, the larvae immediately selected
thee side with food and showed no preference for particle size. In an additional
experimentt that is not described in the preceding paragraphs larvae were observed to
quicklyy find a small spot with surplus of food in a sediment otherwise devoid of food.
Thiss indicates that similar to C. tentans (Baker and Ball 1995) olfactory sense is used by
C.C. hparius to find food.
Thee threshold food concentration for 3 rd instar larvae of C. hparius to reside in
duringg the food preference experiment was found between 0.075 and 0.10 mg fishfood
ml"" . The larvae may not have been able to sense food below this threshold concentrationn and therefore may not have been motivated to settle. Moreover, food
concentrationss below the threshold offer only low growth potential, which also might
stimulatee larvae to find better growth conditions elsewhere. If in one compartment a
foodd concentration was below, and in the other compartment above, the threshold
concentrationn all larvae were found in the higher food concentration. The larvae
probablyy ended up at the higher food concentration because below the threshold the
larvaee did not settle, leaving only the higher food concentration for residence. At higher
foodd concentrations (0.1-2 mg ml"1) the C. hparius larvae were observed to penetrate
thee substrate and to build tubes. The higher food concentrations were only distinguished
78 8
ParticleParticle size and food level effect on C. riparius
iff food concentration differences were large (> 0.75 mg ml"1). A possible motivation to
leavee a substrate above the threshold after settling was observed if another high food
concentrationn was present in the vicinity, e.g. the ability to find food through olfaction.
Thee ability to sense differences in food concentration would facilitate choosing a site of
settlementt or might motivate larvae to leave the place of residence. However, it is not
yett known whether the larvae also can sense quantitative differences in available food.
Anotherr possible trigger for chironomid larvae to leave the substrate might be depletion
off food at the place of residence until a threshold concentration is reached. At low food
concentrationss food will locally be depleted faster than in high food concentrations and
consequentlyy larvae will leave low food level substrates faster to find other places to
feed.. Within our experimental setup each time a larva leaves the substrate it has a
chancee to end up at the higher food concentration. Thus, higher food concentrations will
holdd the larvae for a longer time compared to lower food concentrations resulting in
higherr chironomid densities at higher food concentrations.
However,, it remains unclear why a small proportion of larvae was still found in
thee lower of the two food concentrations above the threshold even if food concentration
differencess exceeded 1.5 mg ml"1. Wiley (1981e) examined the influence of chironomid
densityy and habitat suitability on emigration rates of chironomid larvae in artificial
streamm chambers. Emigration rates were positively correlated with population density,
butt if a suitable substrate type was offered emigration rates decreased and higher
populationn densities were sustained. Similar interaction of population density and
preferencee may have occurred during our food level preference experiments. The length
off the 3rd instar larvae during the 72 hours preference experiments ranged between 4.0
andd 8.0 mm. Assuming that a larvae keeps physical contact with the tube entrance and
extendss up to 60% of larva's total body length (Wiley 19816) one larvae is able to cover
22-900 mm2. If the larvae would evenly and ideally disperse over the triangular area of
500 cm2 one half of an experimental container would be able to sustain 40-200 larvae. A
numberr of field studies reported densities of C. riparius up to 19 times the density that
iss reached when all larvae were found at one side of the experimental unit (4,000 ind
m~2).. Under favorable conditions, C. riparius can reach densities between 30,000 and
50,0000 individuals m"2 (Learner & Edwards 1966, Köhn & Frank 1980, Davies &
Hawkess 1981). Groenendijk et al. (1998) even recorded peak densities of 42,797
79 9
ChapterChapter 4
individualss m 2 at a clean site and 75,000 rrf2 at a highly polluted site, although generallyy larval densities did not exceed 40,000 m"2. Taking these densities into considerationn it seems unlikely that overcrowding has occurred during our experiments.
Nevertheless,, no data are available on emigration rates and differences in population
densitiess between substrates with different organic matter contents. Therefore, overcrowdingg as a stimulant to emigrate during our experiments can not be ruled out.
Rasmussenn (1985) found significant negative effects of chironomid larvae density on
growthh rates of C. riparius. Consequently, the lower food concentration could still be
rewardingg to settle in for a low number of larvae, having a bigger area at their disposal
andd less stress coming from competing neighbouring larvae.
Resultss of this study suggest that the higher density of chironomids in silty
sedimentss compared to sandy sediments can partly be explained by the preference of the
larvaee for substrates with high food concentrations even though silty sediments contain
smalll and ingestible mineral grains that may limit food uptake and therefore also larval
growth.. Besides preference for high organic matter contents, higher chironomid
densitiess in silty sediments may have resulted from lower risk of predation compared to
sandyy sediments. Lower risk of predation in silty sediments is expected because in
generall silty sediments contain higher organic matter contents compared to sandy
sediments.. Higher organic matter contents hold the larvae for a longer time at one place
simultaneouslyy reducing the risk of predation by fish that is associated with migration
(Hersheyy 1987, Ten Winkel 1987, Macchiusi & Baker 1991). Risk of predation may
alsoo be influenced by the suitability of sediments for building larval tubes. Silty
sedimentss contain small mineral particles that were observed to be constructed into
strongerr tubes compared to tubes constructed from big particles. The high organic
matterr contents of silty sediments also will facilitate tube construction. Inhabiting a tube
diminishess the risk of predation in damselfiies, stoneflies, mites and fish as is found in
severall laboratory and field studies (Hershey 1985 & 1987, Ten Winkel 1987,
Macchiusii & Baker 1991, Baker & Ball 1995). Overall chironomid densities are
diminishedd by predation, but relative decrease in densities of tube dwelling chironomids
aree typically lower than those of free-living species (Walde & Davies 1984, Hershey
1987,, Ten Winkel 1987).
80 0
ParticleParticle size and food level effect on C. riparius
Thiss study did not point out one single factor regulating chironomid densities in
thee field. Results show that organic matter content and particle size distribution may not
onlyy be correlated in the field, but are interacting master factors determining the
abundancee of C. riparius in situ. Coping with these environmental factors has been
shownn to be ruled by specific threshold values, food saturation values, and migration or
settlingg behaviour. Therefore, we propose quantitative studies on other detritivorous
benthicc species that might provide insight in the distribution of the large diversity of coexistingg detrivorus species in the natural environment.
81 1
82 2
CHAPTERR 5
NUTRITIONALL VALUE OF SEDIMENTS AS A FACTOR
S T R U C T U R I N GG
MACROFAUNA
COMMUNITIES
IN
SHALLOW
EUTROPHICC WATERS
J.H.. Vos1'3, E.T.H.M. Peeters2'4,R. Gylstra2,5, M.H.S. Kraak16, and W. Admiraal1,7
'Departmentt of Aquatic Ecology and Ecotoxicology, Institute for Biodiversity and
Ecosystemm Dynamics, University of Amsterdam, Kruislaan 320, 1098 SM Amsterdam,
Thee Netherlands, 2Aquatic Ecology and Water Quality Management Group, Department
off Environmental Sciences, Wageningen University & Research Center, PO Box 8080,
67000 DD Wageningen, The Netherlands, [email protected], 4edwin.peeters@aqec.
wkao.wau.nl,, 5ronald.gylstra@aqec. wkao.wau.nl, [email protected], 7admiraal@
bio.. uva.nl
Submittedd for publication
83 3
ChapterChapter 5
Abstract t
Thee role of the nutritional quality of organic matter of soft-bottom sediments as
aa factor structuring natural macrofauna communities was studied in shallow eutrophic
waters.. Growth experiments with Chironomus riparius were conducted to obtain a
directt measure of nutritional value of a range of sediments sampled in The Netherlands.
Sedimentss were also analyzed for water content, organic matter content, C, N, P,
carbohydrates,, protein, fatty acids, pigments, C0 2 production, and grain size distribution.. Macrofauna species were enumerated and their occurrence in the sediment
sampless was correlated to the results of the bioassays and to biochemical sediment
compositionn by means of univariate and multivariate analyses. Newly produced organic
matter,, represented by the variables polyunsaturated fatty acids (PUFA), bacterial fatty
acids,, pigments, and mineralization rate, was associated with abundances of a number
off detritivorous taxa and therefore is most likely a key factor for the nutritional value of
sediments.. Chironomid larval growth correlated well with the abundance of Chironomidaee taxa but not with taxa that have other modes of feeding. Therefore, growth of C.
ripariusriparius was found to effectively indicate the nutritional value of sediment bulk feeders.
Thee use of bioassays with midge larvae excludes indirect effects caused by covariation
off organic matter content with other factors determining the habitat for macrofauna
species,, e.g. oxygen regime or stability of the substrate. It is postulated that such nonfoodd parameters select the species that dominate sites. Yet, nutritional value determines
thee overall density of detritivores and therefore is concluded to be a major structuring
factorr for faunal composition.
84 4
OrganicOrganic matter structuring macrofauna
Introduction n
Knowledgee on the role of organic matter in sediments is crucial to fully
understandd the mechanisms influencing population dynamics of macroinvertebrates in
thee field, because the abundance of organic matter and organic matter composition
determiness the nutritional value of a sediment (Prosser and Brown 1965). If a sediment
doess not suffice nutritional demands of macroinvertebrates these organisms tend to
migratee or development and reproduction are inhibited (Johnson 1984, Gresens and
Lowee 1994, Wolf et al. 1997). Thus, the nutritional value is likely to be fundamental for
thee capacity of sediments to sustain a certain number of invertebrate individuals.
Patternss in community structure in relation to temporal changes in organic
matterr input can be derived from a few studies in large waterbodies monitoring
macrofaunaa structure and input of phytodetritus. In general, macrofauna densities
increase,, but diversities of faunal assemblages tend to decrease with a rise in food
supplyy (Marsh and Tenore 1990, Goedkoop and Johnson 1996, Dauwe et al. 1998).
Organicc matter composition has also been shown to influence the life history of benthic
invertebratess in laboratory experiments testing artificial diets (Marsh et al. 1989,
D'Abramoo and Sheen 1993, Vos et al. 2000). Likewise, in the field, algal blooms
composedd of different algal species caused different responses of macrofauna communitiess indicating a significant influence of organic matter composition on seasonal
variationn in macrofauna community composition (Marsh et al. 1989, Marsh and Tenore
1990,, Cheng etal. 1993).
Inn spite of the obvious importance of organic matter abundance and
composition,, most studies on macrofauna communities do not explicitly take into
accountt the nutritional quality of organic matter in terms of biochemical composition.
Thiss ignores the fact that systems with similar organic matter contents may contain
organicc matter of different nutritional quality, because organic input may originate from
differentt sources or differs in degradation state. Differences in nutritional value between
substratess have been examined in Chapter 3 by determining growth of the detritivorous
larvaee of Chironomus hparius. Distinct differences in larval growth were found, which
couldd be attributed to a number of biochemical variables representing freshly produced
organicc matter. The physical characteristics of the sediments did not influence larval
85 5
ChapterChapter 5
growthh of C. riparius. However, the response to food supply may be taxon-specific,
somee taxa being able to take more advantage of organic matter input than others
(Mackeyy 1977, Montagna et al. 1983, Goedkoop and Johnson 1996). Therefore, it is
hypothesizedd that the nutritional quality of organic matter may be a differentiating
factorr for macrofauna community composition.
Thee present study focusses on soft sediments of eutrophic and shallow
freshwaterr systems and their benthic macroinvertebrate fauna. Benthic detritivores
constitutee a substantial part of the benthic community in the majority of eutrophic
watersystemss in The Netherlands (Moller Pillot & Buskens 1990, Armitage et al. 1995).
Ourr aim was to examine the nutritional value of sediments as a factor spatially
structuringg natural macrofauna communities. The nutritional value of sediments
dependss on several (bio)chemical variables which may interact with each other, with
environmentall factors (temperature, light, current), and with biological factors
(predation,, competition). Therefore, the nutritional value of sediments was assessed by
chemicall analyses (water content, organic matter content, C, N, P, carbohydrates,
protein,, fatty acids, pigments, labile organic matter). The overall nutritional value of
unpollutedd sediments can be assessed by laboratory tests with benthic detritivores
(Chengg et al. 1993). To this purpose data were incorporated from a previous study on
growthh of detritivorous Chironomus riparius larvae on the same sediment samples as
usedd during the present study (Chapter 3). To identify the main components responsible
forr changes in macrofauna composition taxa densities were correlated to larval growth
off C. riparius and to biochemical composition of sediments by means of univariate and
multivariatee analyses.
Materialss and Methods
StudyStudy sites and sampling
Sedimentss were sampled from May until October 1998 throughout the
Netherlandss (Fig. 1). Peeters et al. (subm.) showed that the duration of the sampling
periodd had no significant effect on the macrofauna communities. For sediment sampling
ann Ekman-Birdge grab was used that was adjusted to sample the upper 4 cm surface
layerr (400 cm ). The dataset comprised only unpolluted substrates according to Dutch
86 6
OrganicOrganic matter structuring
macrofauna
standardss (Evaluatienota Water, 1994). The sediments originated from large, shallow
lakess (width > 5 m, #17), rivers (width > 5 m, #3), and small lakes (#4), all classified as
beingg eutrophic. Samples were taken in unvegetated or scarcely vegetated zones at < 1.5
mm of depth. Sediments were frozen at -20°C within 6 h after sampling. After thawing,
thee sediments were sieved over a mesh of 1000 um in order to remove the larger
particless such as pebbles, leaves, and twigs. The sediments were frozen a second time to
ensuree the death of indigenous animals.
Besidess sediment samples for biochemical analyses and growth experiments,
macrofaunaa was sampled in triplicate per sampling point. The identification level is
shownn in Table 1 (Appendix I). Information on feeding behavior was extracted from
Verdonschott (1990) and Merrit and Cummins (1996) (Table 1, Appendix I). Shannon-
Fig.. 1. Distribution of the 24 sampling sites in the Netherlands. Several sites are located at short
distancess and therefore are indicated by a single symbol.
87 7
ChapterChapter 5
Weaverr index of diversity (H' log base 10) was calculated according to Shannon and
Weaverr (1949) and Lambshead et al. (1983).
Bioassays Bioassays
AA detailed description of the growth experiments can be found in Chapter 3.
Shortly,, the growth experiments were carried out in polyethylene containers (surface
areaa of 100 cm2). 100 ml of homogenized sediment and 200 ml of artificial freshwater
(Dutchh Standard Water; pH 8.2, 210 mg CaC0 3 1~') was added to each container. The
sedimentss were allowed to settle for 24 h after which the water was gently added.
Oxygenn concentration and pH were measured at the beginning of the experiment.
Controlss consisted of containers with 50 g of combusted Litofix® sand (<500 um,
heatedd at 550°C for 6 h), 200 ml of Dutch Standard Water and 100 mg of a mixture of
thee commercial available fishfoods Trouvit
s
and Tetraphyll £' (95:5 m/m) each week.
Growthh experiments were started by randomly adding 20 1st instar larvae less than 24 h
oldd to each container. An additional group of 20 larvae was collected to determine the
lengthh of the larvae at the start of the experiment. During the growth experiment the
experimentall units were constantly and gently aerated. After 14 d, the length of all
survivingg larvae was measured with a binocular and growth was calculated by
subtractingg the average starting length from larval length after 14 d. Growth
experimentss were replicated 3 times, starting on different days.
SedimentSediment characterization
Chemicall analyses were performed on freeze-dried substrates described in
Chapterr 3. In short, organic matter content was determined in triplicate according to the
loss-on-ignitionn technique by combusting the material at 550°C for 6 h (Luczak et al.
1997).. Total C was measured in duplicate with a Carbo-Erba Element Analyser. N was
measuredd according to Kjeldahl (ISO 11261 1995). Total P was determined according
too Murphy and Riley (1962) in duplicate. The protein analysis was performed according
too Rice (1982) in duplicate. For analysis of carbohydrates, a modified method based on
thee phenol-sulphuric acid-method of Dubois et al. (1956) was used (Chapter 3).
Chlorophyll-aa and phaeophytin were measured according to Nusch en Palme (1975) in
88 8
OrganicOrganic matter structuring macrofauna
duplicate.. Chlorophyll-a and phaeophytin contents were added up to obtain a measure
off pigment content.
Lipidss were extracted with a 1:1:0.9 v/v/v chloroform:methanol:water mixture
followingg the Bligh and Dyer procedure (1959). Fatty acid methyl esters (FAME) were
obtainedd by mild alcanolic methanolic transesterification as described in Guckert et al.
(1985).. Gas chromatographic separation of the FAME was performed by injecting a 1
u.11 aliquot in the very polar 50 m CP-Sil 88 column (ID 0.25 mm, film thickness 0.20
mm)) with a splitflow of 1:40. Optimal separation of FAME peaks was obtained with a
temperaturee program that began at injection with an initial column temperature of
180°CC for 10 minutes followed by a rise of 3°C min"1 to a final temperature of 225°C,
wheree it was held for 10 minutes. A PUF A variable was obtained by summing up the
peakk areas of 16:2u)4, 16:3co4, 18:2(o6, 18:3oo3, and 18:3co6. Peak areas of FAME of
bacteriall origin (i.e. Z14:0, H5:0, al5:0, 15:1, i'16:0, il6:l, /17:0, and
A17:0;
Napolitano
1999)) were added to obtain a bacterial FA variable. A measure for total FA was
calculatedd by adding all fatty acid peaks from 12:0 upto the last peak appearing in the
chromatogramm before the 21:0 internal standard peak (18:3co3). Only FAME peaks
whichh appeared before the internal standard in the chromatogram were used for further
calculationss because the peaks appearing later in the chromatogram showed irregular
retentionn times.
Grainn size distribution was determined by sieving and the pipet method descibed
inn ISO 11277 (1998). Water content was determined by freeze drying a preweighted
sedimentt sample in triplicate.
AssessmentAssessment of the most labile fraction of the organic matter
AA microbial assay was used to obtain a measure of the most labile, i.e. easily
degradable,, fraction of the sediment organic matter. The procedure is described in
Chapterr 3. In short, wet sediments were used that had been kept frozen until analysis. A
bacteriaa inoculum was prepared from a decaying cyanobacterial mat {Oscillatoria sp).
Bacteriaa were detached by ultrasonic treatment and particles were removed by
centrifugationn (5 min 50 g). In a 77 ml gas-tight bottle 4 ml of sediment was suspended
inn 11 ml of a 55 mM phosphate buffer (pH 7.1) and 1 ml of the bacteria inoculum was
added.. After 30 min of aeration the pH was measured and the bottles were capped gas-
89 9
ChapterChapter 5
tight.. The bottles were placed on a rotary shaker at 20°C in the dark and after allowing
onee hour equilibration the CO2 concentration in the headspace was measured gaschromatographically.. After 48 hours incubation the CO2 concentration was measured again
andd the pH was measured.
MultivariateMultivariate analyses
Multivariatee analyses were performed with sediment variables and distribution
off all taxa. Multivariate analyses (redundancy analysis [RDA] and detrended
correspondencee analysis [DCA]) were performed using CANOCO for Windows
softwaree package (Ter Braak and Smilauer 1998). In all analyses species densities were
logg (x+1) transformed and the species were centered. Species densities were not
standardizedd in order to give the species the weight during the ordination directly
dependentt of their presence. Environmental variables included results of bioassays and
sedimentt variables. Two analyses were performed, one with the biochemical variables
standardizedd on dry weight and one with the biochemical variables standardized on
10,000 0
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Fig.. 2. Average densities of taxa (ind m ) for all stations. Error bars represent standard errors.
90 0
OrganicOrganic matter structuring macrofauna
organicc matter content. First, the length of gradient was calculated by DC A to determine
thee model that fitted the relationship between the species densities and sediment
variabless best. The linear response model (RDA) was chosen for final ordination,
becausee the length of gradient was < 2.0 for both datasets (Ter Braak 1995). The Monte
Carloo Permutation test was used to calculate the significance of the influence of the
sedimentt variables on the macrofauna species distribution.
Duringg the ordination of the dataset containing biochemical variables
standardizedd on dry weight the variance inflation factors (VIFs) showed high collinearityy of organic matter, N-, P-, and the particle size fraction < 63 um-content (VIFs >
20).. In the dataset with biochemical variables standardized on organic matter content
thee particle size fraction < 63 urn and C showed high VIFs. The variables with VIFs >
200 were deleted from the datasets for further ordination. In the dataset with the
biochemicall variables standardized on dry weight axis 1 had an eigenvalue of 0.28 and
axiss 2 of 0.11. The 2 axes represent 58% of the species-environment correlation. In the
multivariatee analysis of the dataset containing the biochemical variables standardized on
organicc matter content the eigenvalue of axis 1 was 0.33 and of axis 2 was 0.12. In this
analysiss axes 1 and 2 represent 63% of the species-environment correlation.
UnivariateUnivariate statistical analyses
Correlationss were determined according to the Pearson method using the
averagess of chemical and physical analyses, averages of larval growth, and of species
densitiess per substrate. Numbers of individuals were log(x+l) transformed. Densities of
taxaa were only correlated with results of growth data or biochemical analyses if the taxa
occurredd in 8 or more substrates to avoid correlations based on a low number of
observations. .
Results s
Macrofauna Macrofauna
Thee densities of total number of individuals varied between 40 and 36,100 ind
22
m" with an average density of 8,800 ind m"2 (Fig. 2). Average density of oligochaetes
foundd in the field was ca. 4,000 ind m"2, but the maximum density was 15,300 ind m~2.
91 1
ChapterChapter 5
Oligochaetess made up 0 to 98% of the total macrofauna individuals with an average of
ca.. 54%. The chironomids comprised 1 to 88%» of the individuals and were found in
densitiess of 8 to 30,160 ind m"2 with an average of 4,200 ind m" . One of the most
abundantt groups of chironomid taxa were the Tanytarsini, reaching a maximum density
off 25,730 ind m"2 and comprising 0 to 71%» of the macrofauna specimens. Average
densityy of Tanytarsini was the highest among the subfamilies of Chironomidae (2,450
indd m"2) followed by the Chironomini (1,600 ind rrf2). Malacostraca, Ephemeroptera,
Coleoptera,, and Ceratopogonidae occasionally made out a high percentage of macrofaunaa individuals, but all had on average a presence of < 20%o of the total individuals.
Thee number of sites where a species occurred is shown in Table 1. Oligochaetes,
SphaeridaeSphaeridae spec, Hydracarina spp., Gammarus tigrinus, Ceratopogonidae spp., Stich-
tochironomustochironomus spp., Cryptochironomus spec, Chironomus spp., Glyptotendipes sp
CladotanytarsusCladotanytarsus spec, Procladius s.1., Polypedilum gr. nubeculosum, and Polyped
gr.. bicrenatum, were present in >50% of the sediments. However, most taxa were
presentt in less than 20% of the sediments.
Tablee 2. Overview of variables standardized on dry weight measured in the 24 sediments.
Averagess are calculated from the average values of the individual sediments. Growth and
survivall of Chironomus hparius. CV = coefficient of variance. Minimum (min) and maximum
(min)) value observed in the dataset.
average e
min n
mgg'
11
15 5
mgg"
11
0.8 8
11
unit t
cc
NN
PP
mgg
max x
CV V
2.0 0
51 1
110 0
0.1 1
3.5 5
123 3
0.3 3
0.02 2
1.4 4
113 3
mgg"
11
0.5 5
0.03 3
2.0 0
120 0
protein n
mgg"
11
2.2 2
0.02 2
18.1 1
168 8
totall FA
ugg' 1 1
181 1
2.8 8
535 5
85 5
PUFA A
11
carbohydrates s
1.8 8
00
7.4 4
126 6
bacteriall FA
ugg"
11
4.4 4
00
20.3 3
115 5
pigments s
ugg11
11.0 0
0.3 3
49.0 0
108 8
1
7.3 3
00
^gg
18.2 2
76 6
growthh
mm
4.4
0.7
11.0
60
survivall
%
76
0
100
C 0 22 production
92 2
mmoll g"
108
OrganicOrganic matter structuring
macrofauna
Iff densities of taxa with the same feeding mode were added up, detriti(herbi)voress were found in 23, herbivores in 22, and carnivores in 23 of the 24 sampled
substrates.. Shannon-Weaver index of diversity (H' log base 10) ranged from 0.047 to
0.8922 with a coefficient of variance of 49 % among all 24 sites. The sites with the
lowestt diversities (n = 2, S < 0.1) only comprised oligochaetes, and a number of Chironomidaee species (mostly inhabited by Mollusca, Malacostraca, Ephemeroptera, Ceratopogonidae,, and members of subfamilies of Chironomidae other than Chironomini.
BioassaysBioassays with Chironomus riparius
Lengthh of the 1st instar larvae (< 24 h) at the start of the growth experiments was
0.899 mm ( 0.02). Larvae in the controls reached lengths of 12.5 mm ( 0.28) in 14 d
Tablee 3. P-values of correlations between sediment variables and macrofauna community
compositionn calculated with the Monte Carlo permutation test. Biochemical
variables
standardizedd on dry weight or on organic matter content. SWC = sediment water content, %<63
umm = particle size < 63 urn.
biochemicall variable standardized on
dryy weight
organic matter
CC
0.095 5
0.026 6
NN
0.082 2
0.275 5
PP
0.102 2
0.001 1
carbohydrates s
0.286 6
0.566 6
proteins s
0.304 4
0.625 5
totall FA
0.691 1
0.977 7
PUFA A
0.003 3
0.002 2
bacteriall FA
0.127 7
0.000 0
pigments s
0.775 5
0.016 6
C0 22 production
0.126 6
0.004 4
SWC C
0.034 4
organicc matter
0.070 0
%<633 um
0.004 4
%>2100 um
0.212 2
C/N N
0.185 5
growth h
0.004 4
survival l
0.021 1
93 3
ChapterChapter 5
andd were mostly (70%) in the 41 larval stage. Survival in the controls exceeded 80% in
eachh experimental unit. Growth in the individual substrates ranged between 0.7 and 11.0
mmm with an average of 4.4 mm and a coefficient of variance (CV) of 60% (Table 2).
Survivall in the individual substrates ranged between 0 and 100% per experimental unit
withh an average of 76% and a CV of 108%. Growth and survival were significantly
correlatedd (R2 = 0.71; P < 0.01).
SedimentSediment composition
AA range from silty to sandy sediments was sampled. Particles < 63 urn constitutedd 0.8 - 79.0% DW with an average of 21%. Particles > 210 um ranged between 0.3
andd 81% DW with an average of 24%. Sediment water content ranged from low to high
(20.6-59.1%% of wet weight), with an average of 32.3%. Organic matter content ranged
betweenn 0.3-8.4% of DW among the substrates with an average of 2.0%. C/N-ratio had
ann average of 20.5 and ranged between 5.9 and 55.0.
Biochemicall variables were expressed in 2 ways: standardized on dry weight
andd standardized on organic matter content. The high CVs of the biochemical variables
standardizedd on DW indicate a broad variety of organic matter types present in the
individuall substrates (Table 2). Standardization on dry weight resulted in a high number
off correlations among biochemical variables in contrast to standardization on organic
matterr content, which showed low correlation coefficients in most cases (data not
shown). .
Multi-Multi- and univariate analyses
Multivariatee analyses were used to survey correlations between biochemical
sedimentt composition, bioassay determined nutritional value and macrofauna communityy composition. Univariate analyses were performed to determine the correlations
betweenn individual taxa and sediment variables.
Multivariatee analyses
Multivariatee analyses of the dataset was carried out in two modifications; one
usingg biochemical variables expressed per unit dry weight and one with the variables
normalizedd on organic matter. The two standardization methods affected the interactionss of macrofauna community and individual biochemical variables, as calculated
94 4
OrganicOrganic matter structuring macrofauna
withh the Monte Carlo permutation test (Table 3). Only PUFA-content was significantly
correlatedd to macrofauna community composition when variables were standardized on
dryy weight. A distinct higher number of significant interactions of biochemical variables
withh macrofauna community was found (P < 0.05) when biochemical variables were
standardizedd on organic matter content. In that case C, P, PUF A, bacterial FA,
pigments,, and labile organic fraction measured as bacterial CO2 production were significantlyficantly correlated to species densities in the field. Larval growth and survival, sediment
waterr content (SWC), and particle size fraction < 63 \im were also signicantly correlatedd to species densities. A high collinearity among biochemical variables standardized
onn dry weight and the high number of significant correlations for biochemical variables
standardizedd on organic matter prompted us to further analyse the dataset with the latter
typee of standardization.
Fig.. 3 shows the biplot resulting from the direct ordination (RDA) of the dataset.
Thee parameters for sediment characteristics were plotted as arrows and the ordination of
faunaa composition as individual species or groups of species. The arrows of the food
relatedd parameters 'larval growth' and 'survival' of Chironomus riparius, pigments,
PUFA,, P, bacterial FA, and CO2 production all point to the right. Densities of the
detriti(herbi)vorouss Tanytarsini, Chironomini, Polypedilum gr. nubeculosum, Cladotanytarsusnytarsus spec, Cryptochironomus spec, Stichtochironomus spp., and Einfeldia/Fleuria,
andd the total numbers of individuals (group 6 in Fig. 3) were positioned near these
sedimentt variables, away from the origin of the biplot. Thus, this faunal group shows a
positivee correlation with growth and survival of C. riparius in bioassays and with the
(bio)chemicall parameters PUFA, P, pigments, bacterial FA, and CO2 production.
Thee non-food parameters SWC and the particle size fraction > 210 um together
withh organic matter content were ordinated in the opposite direction of the food
parameters,, in the left panel of Fig. 3. The carnivorous taxa Tanypus punctipennis cf,
andd Micronectra spec, and the detritivorous Chaetocladius piger agg. were associated
withh high organic matter contents. The position of group 3, that consists mostly of
carnivorouss and herbivorous taxa, suggests a positive association of these taxa with
sedimentss particle sizes > 210 um. The setting of the biplot indicates a strong negative
correlationn of organic matter content with Shannon index.
95 5
ChapterChapter 5
Fig.. 3. Redundancy analysis plot (RDA) showing the direct ordination of species densities,
sedimentt variables and results of bioassays with Chironomus riparius, with 33% of variance on
axiss 1 and 12% on axis 2. Arrows represent the sediment variables and growth and survival of
C.C. riparius. EINFFLEU = EinfeldialFleuria, CRICSYLV = Cricotopus sylvestris agg., POPENUBE
== Polypedilum gr. nubeculosum, TANYPUNC = Tanypus punctipennis cf., PARACONC = Paracorixacorixa concinna, MINECTSP = Micronecta spp. larvae, CHAEPIGE = Chaetocladius piger agg.,
PROACOXAPROACOXA = Proasellus coxalis, LIMNOPHI = Limnophila spec, TABANIAE = Tabanidae
spec,, CLTANERV = Clinotanypus nervosus. Group 1 consists of Neomysis integer, Chironomusmus spp., Cricotopus intersectus agg., and Micropsectra spec. Group 2: Erpobdella octoculata,
BithyniaBithynia tentaculata, Gyraulus albus, Gammarus pulex, Sigara falleni/longipalis, Sialis lutaria,
LaccophilusLaccophilus spec larvae, Psychodidae spec, Endochironomus albipennis, Tanypodinae,
ProcladiusProcladius s.l. Group 3: Valvata piscinalis, Zygotera spec juveniles, Haliplus spec, larvae,
Oxyethira,Oxyethira, spec, Ecnomus tenellus, Mystacides spec, Ceratopogonidae spec, Pseudochirono-
+1.0 0
SWI SWI
bacterialbacterial t :A
TANYPUNC C
organicorganic matter
oo
0) )
cc
CD D
g> >
MINECTSP P
CHAEPIGEE PROACOXA
LIMNOPHI I
--
TABAWAE
survivalsurvival
Chironomir
CLTANERV V
%>210fim %>210fim
Tanytarsini i
C0C022 production
Groupp with overlapping
speciess names
-1.0 0
-1.0 0
96 6
Axiss 1, eigenvalue 0.33
+1.0 0
OrganicOrganic matter structuring
macrofauna
musmus spec, Tribelos intextus. Group 4: Helobdella stagnalis, Mollusca, Potamopyrgus antipodarum,darum, Lymnaea spec, juveniles, Planorbis spec, juveniles, Sphaeridae spec, Hydracarina
spp.,, Malacostraca, Gammarus tigrinus, Corophium curvispinum, Caenis horaria, Agraylea
multipunctata,multipunctata, Oecetis ochracea, Cyrnus spec, Lepidoptera spec, larvae, Dicrotendipes gr.
nervosus,nervosus, Mircotendipes chloris agg., Corynoneura scutellata agg., Ablabesmyia
spec,
TanytarsusTanytarsus spec, Paratanytarsus Neureclipsis bimaculata, Athripsodes spec, juveniles, Simuliumlium spec, Polypedilum gr. bicrenatum, Glyptotendipes spp., Demicryptochironomus vulneratus,
HamischiaHamischia spec, Cryptotendipes spec, spec. Group 5: Va/vate cristata, Dreissena polymorpha,
AseHusAseHus aquaticus, Baetis spec, Hydrophilidae spec, larvae, Oulimnius spec larvae, Paroecetis
struckii,struckii, Hydroptila spec, Parachironomus
gr. arcuatus, Psectrocladius
gr. sordidellus/
limbatellus,limbatellus, Orthocladiinae, Cricotopus bicinctus, Prodiamesa olivacea, Orthociadius orthocladius,cladius, Conchapelopia spec, Macropelopia spec, Rheotanytarsus spec, Stempellina spec
Groupp 6: Stichtochironomus spp., Cryptochironomus spec, Cladotanytarsus spec, total number
off individuals.
AA high number of taxa is positioned near the centre of the biplot (group 1 and 2
inn Fig. 3). Most of these taxa were found in a low number of sites (#<6) or have feeding
modess other than of detriti(herbi)vores. The position near the centre of the first axis
implicatess that the densities of the taxa did not correlate well with the sediment variabless analyzed during this study. Protein, carbohydrates, N, and total FA were also
ordinatedd near the centre of the biplot meaning that these variables hardly contributed to
thee variance explained by the sediment variables.
Univariatee analyses
Thee univariate analyses were also done with the two methods of standardizing
biochemicall variables. Densities of macrofauna species and higher taxonomie units
weree correlated with these biochemical variables and results of bioassays. Table 4
presentss the significant correlations using biochemical variables standardized on organic
matter.. Densities of the detritivore groups Chironomini, Tanytarsini, Chironomus spp.,
andd Stichtochironomus spp. as well as total number of individuals correlated positively
withh growth and survival of Chironomus riparius larvae in the bioassays (P < 0.05) and
withh the variables representing fresh organic matter (PUFA, bacterial FA, pigments, and
CO22 production). Densities of individual species that correlated positively with growth
andd survival were Gammarus tigrinus, Cryptochironomus spec, Polypedilum gr. nubeculosum,culosum, Polypedilum gr. bicrenatum, and Cladotanytarsus spec, which are also detriti-
97 7
ChapterChapter 5
(herbi)vores.. Fig. 4 shows that the sediments were highly different in their capacity to
supportt growth of C. riparius. These differences are due to different nutritional value,
sincee amendment with artificial food stimulated uniform rapid growth (Chapter 3).
Faunall diversity was not correlated to larval growth and survival of C. riparius, but a
positivee correlation was found with water content and negative correlations with the
fractionss of organic matter and silt. Carnivores such as Ceratopogonidae spp., Hydracarinacarina spp., and Procladius s.1. do not occur in Table 4, because these animals show no
Tablee 4. Significant correlations of sediment variables with species densities. Biochemical variabless are standardized on organic matter content. Growth and survival of Chironomus riparius.
SWCC = sediment water content, %<16 um = particle size < 16 um. Positive correlations with P <
0.055 = +, with P < 0.01 = ++, and negative correlations with P < 0.05 = -, and with P < 0.01 = - .
Oligochaeta a
++
Malacostraca a
++
Orthocladiinae e
++ +
-
survival l
growth h
-
+ + + + + +
+++
Tanytarsini i
C/N N
%<633 um
organicc matter
+
+++
Chironomini i
SWC C
C022 production
pigments s
PUFA A
bacteriall FA
carbohydrates s
PP
NN
CC
Taxaa densities are log (x+1) transformed.
++
+
+
+
Tanypodinae e
-
-
++
++
++
++
++
SphaeridaeSphaeridae spec.
GammarusGammarus tigrinus
+++
++ spp. +
StictochironomusStictochironomus
+
+
+
+
+
PolypedilumPolypedilum gr. nubeculosum
++
+
+
+
PolypedilumPolypedilum gr. bicrenatum
+ + ++
+
+
CryptochironomusCryptochironomus spec.
++
++
+
+
+
+
++
++
+
+
+
+
-
-
.
+
.
++
++
+
++
++
++
++
++
ChironomusChironomus spp.
++
GlyptotendipesGlyptotendipes spp. +++
CladotanytarsusCladotanytarsus++spec.
## of individuals
Shannon n
98 8
+
++
++
++
-
+
+++ —
++
++
++
+
OrganicOrganic matter structuring macrofauna
significantt correlation with the food related parameters, but carnivore abundance is
significantlyy correlated (P < 0.05) to the total number of herbivores (R2 = 0.61) and
detriti(herbi)voress (R2 = 0.42). The same univariate analysis described above has been
performedd with biochemical variables standardized on dry weight of sediment. Many
correlationss between biochemical variables and taxa abundances were found to be
weakerr or absent (data not shown), which is consistent with the multivariate analyses. In
casee abundances of taxa were significantly correlated to biochemical variables standardizedd on dry weight, similar correlations of these taxa with organic matter content were
found. .
Discussion n
Thee present study identified the influence of organic matter composition and the
nutritionall value of sediments on the distribution of macroinvertebrate species in the
field.. Searching for correlations between the occurrence of macrofauna species and
experimentallyy determined growth and survival of midge larvae circumvented some
problemss of covariation of factors in field observations, i.e. biochemical composition,
organicc matter content, grain size, etc. During the bioassays in the laboratory larval
growthh and survival were determined by the nutritional value of the sediments (cf.
Chapterr 3) and not by other conditions such as suitability for larval settling, oxygen
regimes,, and predation. Although larval growth can be influenced by ingestion of a
variablee fraction of mineral particles, the bioassays gave a good measure of nutritional
valuee of sediments as experienced by the sediment swallowing invertebrates. The
densitiess of a number of invertebrate taxa correlated well with tested larval growth
indicatingg that the nutritional value of sediments determines the capacity to sustain a
certainn density of individuals. Such positive correlations between larval growth and
densitiess of detritivorous animals were conspicuous for detritivores among the closely
relatedd group of the chironomids, while such correlations were non-significant for the
sedimentt swallowing oligochaetes. The latter group, however, also included species
withh other feeding types, such as herbivores that feed on the sediment surface. The
causall relationship of detritivore abundance with measured food quality of detritus in
thee present study is underlined by the opposing observations on carnivores. The
99 9
ChapterChapter 5
occurrencee of these animals was not correlated to any quality of the detritus, but to the
totall number of invertebrates, i.e. the density of prey. Using chironomid larval growth
ass indicator of nutritional value of substrates seems to be effective for sediment bulk
feeders,, but is not appropriate for organisms with different feeding modes or other food
sources.. Not withstanding these restrictions it is concluded that the bioassays using
growthh of Chironomus riparius larvae are a better alternative to quantify the nutritional
valuee of substrates for macroinvertebrates than determination of organic matter content
off sediments.
Thee abundance of detritivorous macrofaunal species also correlated strongly
withh biochemical parameters, especially when these were standardized on organic
matter.. This standardization highlights food composition, whereas the less effective
standardizationn on dry weight puts emphasis on food quantity. When standardized on
dryy weight biochemical variables were strongly correlated among each other and with
organicc matter content showing that standardization on dry weight resulted in variables
whichh in fact represented alternative measures for organic matter content. The positive
correlationss of abundances of detritivorous macrofauna species and organic matter
qualityy in the present study confirms the earlier analysis of such correlations between
measuredd growth and survival of C. riparius and biochemical quality of sediments
(Chapterr 3). Abundance of freshly produced organic matter represented by the variables
polyunsaturatedd fatty acids (PUFA), bacterial fatty acids, pigments, and labile organic
matterr was associated with the abundance of invertebrate species in the field. Insects
needd to obtain PUFA from their diet, because they are not able to synthesize these
essentiall fatty acids themselves. For benthic invertebrates algae are an important source
off PUFA (Napolitano 1999). A few field studies mentioned algal input as stimulating
factorr of macrofauna densities (Marsh and Tenore 1990, Goedkoop and Johnson 1996).
However,, these studies did not determine organic matter composition of the algal input
andd therefore were not able to separate the influence organic matter abundance from
organicc matter composition. PUFA, bacterial fatty acids, pigments, and the most labile
fractionn of organic matter were also found to be the most important factors stimulating
larvall growth and survival of C. riparius (Chapter 3). Thus, freshly produced organic
matterr is likely to promote the nutritional value of sediments thereby increasing the
capacityy of sediments to sustain numbers of detritus feeders. It is concluded that the
100 0
OrganicOrganic matter structuring
macrofauna
Fig.. 4. Scatterplots of measured larval growth of Chironomus riparius with observed densities of
Oligochaetaa (black diamonds, R2 = 0.377, P > 0.05), Chironomini (crosses, R2 = 0.584, P <
0.01),, and Cryptochironomus spec, (open circles, R2 = 0.608, P < 0.01).
100,000 0
10,000 0
XX
« ! **
CM M
EE
xx
v * ** X
1,000
cc
CO O
cc
a> >
XX
XX
100 0
T3 3
10 0
oo
§
o
oo
OO #
oo
oo
o
oo
oo
11 -o—e-
~ii
44
i
6
growthh (mm)
r
8
10 0
12 2
specificc biochemical parameters measured in the present study, like PUF A, bacterial
fattyy acids, and CO2 production are accurate descriptors of the nutritional value of
sedimentt for macrofauna species.
Ass previously observed by Vos et al. (subm.) standardization of biochemical
componentss on organic matter content renders quality parameters independent of organicc matter abundance. This circumvents covariation of organic matter with factors other
thann food. These factors include oxygen levels, grain size and the twoway dependance
betweenn organic matter abundance and densities of macrofauna, e.g. top-down control
byy invertebrates and bottom-up control by organic matter content. Organic matter
contentt may influence densities of macroinvertebrates, but grazing by macroinvertebratess may decrease organic matter contents and thereby the nutritional value of
sediments.. Organic matter is known to influence oxygen levels in sediments through
oxygenn consumption and by increasing the packing of the sediment, generally resulting
inn lowered oxygen concentrations in substrates with high organic matter contents
101 1
ChapterChapter 5
(Watlingg 1991). In sediments with elevated organic matter levels species diversity was
reducedd and mainly oligochaetes and chironomid species (Cryptochironomus spec,
ChironomusChironomus spp., Tanypus punctipennis) were found, indeed species which are resistan
too low oxygen levels (Verdonschot 1990, Heinis 1993). This makes it probable that
organicc matter abundance constituted a factor indirectly structuring the macrofauna
communitiess by influencing oxygen concentrations in the substrate. Certainly, the
presentt series of macrofauna communities are representative for organically rich
sedimentss that are prone to oxygen depletion (Heinis 1993). Organic matter composition
didd not correlate to species diversity suggesting that habitat factors selected the number
off species that were able to inhabit the particular substrates. This contrasts with the role
off organic matter composition proposed here as a factor determining the capacity of a
sedimentt to sustain a certain density of detritivores. The separate mode of actions of
organicc matter composition and physical characteristics to structure macrofauna
communitiess is visualized by the separation of biochemical variables and physical
variabless in the ordination biplot. The diversity of physical characteristics of the
sedimentss within the present dataset was high (e.g. silty to sandy substrates). Therefore,
itt is expected that physical characteristics contributed to the selection of species which
couldd inhabit the substrate. For instance, particle size distribution is known to influence
thee suitability of the substrate for tube construction and penetration which are both
indispensablee for settlement of several invertebrate species (Brennan and McLachlan
1979,, Wiley 1981).
Thee present study showed that the overall nutritional value of sediments
measuredd in the tests with C. riparius determines the number of detritivorous specimens
thatt can maintain themselves in the substrate, indicating a strong bottom-up control of
thiss group of animals. The role of organic matter abundance in terms of food supply can
nott be quantified separately from the multiple other influences of oxygen, grain size,
etc.,, on so many macroinvertebrate species. The high number of non-food parameters
(e.g.. organic matter abundance) may have selected the species that occupy the niche of
thee benthic detritivores. Yet, it can be concluded that the quality of fresh organic matter
iss likely to determine the abundance of detritivores and therefore is a main structuring
factorr in soft-bottom communities.
102 2
OrganicOrganic matter structuring macrofauna
Acknowledgments s
Wee thank E. Bleeker for his comments on the manuscript. M. Ooijevaar, A.J.P.
Oosthoek,, and S. Liicker helped with field sampling and chemical analyses. S. Arisz
(Departmentt of Plant Fysiology, University of Amsterdam) greatly helped with the fatty
acidd analyses. The study was partly supported by the Institute for Inland Water
Managementt and Waste Water Treatment (RIZA), Lelystad.
103 3
ChapterChapter 5
Appendixx I.
Tablee 1. Feeding mode and number of occurences of macrofauna species taxa. # substrates =
numberr out of the total of 24 sampled sites where species or taxa were found.
modee of feeding
Oligochaeta a
Hirudinea a
Gastropoda a
OligochaetaOligochaeta spp.
detritiherbivore e
HelobdellaHelobdella stagnate
carnivore e
ErpobdellaErpobdella octoculata
carnivore e
ValvataValvata piscinalis
herbivore e
ValvataValvata cristata
herbivore e
BithyniaBithynia tentaculata
herbivore e
# substrates
24 4
PotamopyrgusPotamopyrgus antipodarum herbivore e
Bivalvia a
LymnaeaLymnaea spec, juveniles
herbivore e
GyrautusGyrautus albus
herbivore e
PlanorbisPlanorbis spec, juveniles
herbivore e
SphaeridaeSphaeridae spec.
herbivore e
14 4
DreissenaDreissena polymorpha
herbivore e
44
12 2
Hydracarina a
HydracarinaHydracarina spp.
carnivore e
Amphipoda a
GammarusGammarus pulex
detritiherbivore e
11
GammarusGammarus tigrinus
detritiherbivore e
12 2
CorophiumCorophium curvispinum
detritiherbivore e
11
AsellusAsellus aquaticus
detritivore e
11
ProasellusProasellus coxalis
detritivore e
11
Mysidae e
NeomysisNeomysis integer
detritivore e
22
Ephemeroptera a
CaenisCaenis horaria
detritivore e
88
BaetisBaetis spec.
detritiherbivore e
11
22
Isopoda a
Odonata a
ZygopteraZygoptera spec, juveniles
carnivore e
Hemiptera a
SigaraSigara fallenillongipalis
herbivore e
MicronectaMicronecta spp. larvae
omnivore e
ParacorixaParacorixa concinna
carnivore e
Megaloptera a
SialisSialis lutaria
carnivore e
Coleoptera a
HaliplusHaliplus spec, larvae
herbivore e
LaccophilusLaccophilus spec, larvae
carnivore e
HydrophilidaeHydrophilidae spec, larvae
carnivore e
OulimniusOulimnius spec, larvae
herbivore e
AgrayleaAgraylea multipunctata
herbivore e
OxyethiraOxyethira spec.
herbivore e
EcnomusEcnomus tenellus
carnivore e
Trichoptera a
104 4
OrganicOrganic matter structuring
Lepidoptera a
MystacidesMystacides spec.
detritii herbivore
ParoecetisParoecetis struckii
detritiherbivore e
OecetisOecetis ochracea
omnivore e
HydroptilaHydroptila spec.
herbivore e
NeureclipsisNeureclipsis bimaculata
herbivore e
AthripsodesAthripsodes spec, juveniles
detritiherbivore e
CyrnusCyrnus spec.
carnivore e
LepidopteraLepidoptera spec, larvae
herbivore e
macrofauna
Diptera a
Ceratopogonidae e CeratopogonidaeCeratopogonidae spp.
carnivore e
14 4
LimnophilaLimnophila spec.
detritiherbivore e
11
Psychodidae e
PsychodidaePsychodidae spec.
detritivore e
11
Tabanidae e
TabanidaeTabanidae spec.
carnivore e
11
Simuliidae e
SimuliumSimulium spec.
detritiherbivore e
11
Tipulidae e
Chironomidaee
Chironomini:
detritiherbivore e
13 3
PseudochironomusPseudochironomus spp.detritiherbivore e
11
detritiherbivore e
EndochironomusEndochironomus albipennis
22
StictochironomusStictochironomus spp.
TribelosTribelos intextus
11
detritivore e
DicrotendipesDicrotendipes gr. nervosus detritiherbivore e
33
PolypedilumPolypedilum gr. nubeculosum detritiherbivore e
15 5
detritiherbivore e
13 3
CryptochironomusCryptochironomus spec. detritiherbivore e
21 1
PolypedilumPolypedilum gr. bicrenatum
ChironomusChironomus spp.
detritivore e
MicrotendipesMicrotendipes chloris agg.
detritivore e
GlyptotendipesGlyptotendipes spp.
detritiherbivore e
16 6
77
12 2
DemicryptochironomusDemicryptochironomus
vulneratus
detritivore
e
11
HarnischiaHarnischia spec.
carnivore e
11
CryptotendipesCryptotendipes spec.
detritiherbivore e
11
ParachironomusParachironomus gr. arcuatus
detritiherbivore e
11
EinfeldialFleuria EinfeldialFleuria
55
detritiherbivore e
PsectrocladiusPsectrocladius gr. sordidellusl
limbatellus
herbivore
e
10 0
Orthocladiinae: :
CricotopusCricotopus sylvestris agg.
detritiherbivore e
22
CricotopusCricotopus intersectus agg.
detritiherbivore e
11
CricotopsCricotops bicinctus
detritiherbivore e
11
ChaetocladiusChaetocladius piger agg.
detritivore e
11
ProdiamesaProdiamesa olivacea
detritiherbivore e
11
105 5
ChapterChapter 5
OrthocladiusOrthocladius orthocladius
detritii herbivore
11
Tanypodinae: :
CorynoneuraCorynoneura scutellata agg. detritiherbivore e
22
ClinotanypusClinotanypus nervosus
carnivore e
22
Procladiuss.\. Procladiuss.\.
carnivore e
16 6
AblabesmyiaAblabesmyia spec.
carnivore e
33
ConchapelopiaConchapelopia spec.
carnivore e
11
MacropelopiaMacropelopia spec.
carnivore e
11
TanypusTanypus punctipennis cf.
carnivore e
44
CladotanytarsusCladotanytarsus spec.
detritiherbivore e
12 2
TanytarsusTanytarsus spec.
detritiherbivore e
55
RheotanytarsusRheotanytarsus spec.
herbivore e
11
Tanytarsini: :
106 6
StempellinellaStempellinella spec.
detritiherbivore e
11
ParatanytarsusParatanytarsus spec.
detritiherbivore e
66
MicropsectraMicropsectra spec.
detritiherbivore e
22
CHAPTERR 6
CONCLUDINGG REMARKS
107 7
ChapterChapter 6
Parameterss characterizing nutritional value of sediments
Thiss thesis showed that the nutritional value of sediments for detritivores
dependss on the presence of different fractions, e.g. algal material. In this paragraph it is
attemptedd to define the ideal parameters for measuring nutritional value of sediments,
allowingg regulation of communities of detritivores to be analyzed properly.
Thee effect of food quantity and quality was separated here by standardization of
foodd components on either organic matter content or total dry weight. Strict separation
off organic matter abundance and composition may seem artificial, because in studies on
benthic-pelagicc coupling food quantity and composition are mostly related. For detritivoress sedimenting algal blooms represent both an increase in food availability and food
quality.. However, after sedimentation of phytodetritus is mixed with inorganic or
refractivee organic material. This mixing poses several practical problems for measuring
nutritionall value, because the admixture of mineral particles and refractive organic
matterr in the ingestible size ranges is lowering the apparent quality of food, since these
fractionss load the guts of detritivores with indigestible material. Standardization of
biochemicall parameters on organic matter content of the sediment separates the effect of
foodd quality from the effect of food dilution by mineral particles and from the multiple
rolee of organic matter abundance (Chapters 3 and 5). So, a simplified conclusion is that
sedimentaryy organic matter needs to be chemically characterized to assess nutritional
valuee of sediments. However, mineral particle size distribution should also be measured
inn relation to the ingestion capacity of the detritivore concerned. Parameterization of
foodd quality is still bound to rely on such complexity.
Thee biochemical nature of food suitable for detritivores was proven to be
diverse.. PUFA-content (polyunsaturated fatty acids) was one of the factors strongly
associatedd with food quality in Chapters 3 and 5. Short-chained PUFA as well as
pigmentss are biomarkers for the presence of algae (Napolitano 1999). This labile
organicc matter of algal origin is likely to stimulate growth of chironomid larvae. Nutritionall value of sediments was also found to be related to bacterial biomass, indicating
thatt detritivores are versatile and opportunistic feeders. The finding that biomarkers of
bothh algal and bacterial material are related to nutritional value of sediments can be
explainedd by the sequence of degradation and microbial enrichment of organic matter.
Afterr cell-death algae will be broken down fast, through chemical and microbial
108 8
ConcludingConcluding Remarks
processes.. Degradation of algal material will proceed fastest when algal material has
justt sedimented and still contains the most labile components such as PUFA and
chlorophylll (Napolitano 1999), leading to the correlation between mineralization rate
andd nutritional value of sediments. Microbial degradation advances conversion of organicc matter into refractive material. Simultaneously, microbial biomass supplements the
foodd sources of detritivores (Martin et al. 1980, review Phillips 1984a, review Bowen
1987,, review Graf 1992, Wolf et al. 1997). Microorganisms do not contain PUFA such
ass algae and macrophytes do (Napolitano 1999), but a number of studies suggests that
microorganismss enrich organic matter with other essential compounds, such as vitamins
andd amino acids (Phillips 1984a, Wolf et al. 1997). Fungi are thought to supplement
invertebratess with enzymes capable of breaking down cellulose (Martin et al. 1980,
Sinsabaughh et al. 1985, Barlocher & Porter 1986, Chamier & Willoughby 1986,
Chamierr 1991, McGrath & Matthews 2000). This microbial activity was represented by
specificc fatty acids which are biomarkers for bacteria (Napolitano 1999).
Althoughh it is most likely that heterotrophic microorganisms constitute a part of
thee nutrition for detritivores as is supported by numerous studies (see references above),
thee correlations found between detritivores and bacterial abundance may also have
resultedd from the interaction between macrofauna abundance and microbial activity.
Bioturbationn by benthic fauna stimulates microbial activity through mechanical
disturbancee and subsequent physical and chemical alteration of organic matter in sedimentss (Johnson et al. 1989, Van de Bund et al. 1994). However, changes in numbers of
bacteriaa have been reported to be fauna! species specific. In laboratory studies with
sedimentss from eutrophic lakes Tubifex tubifex and Chironomus plumosus affected
bacteriall abundance negativily between densities of 0 and 20,000 ind m"2, but Monopodiadia affinis and C. riparius did not affect bacterial abundance (densities between 0 and
10,0000 ind m"2) (Johnson et al. 1989, Van de Bund et al. 1994). Mechanical stirring and
ingestionn of sediment bacteria were mentioned to reduce bacterial numbers. Evidently,
theree is no simple one-way relationship between bacteria and detritivores. Since many
studiess confirm the nutritional role of microorganisms in food for detritivores it is
assumedd that microorganisms enriched the food sources of detritivores also during the
presentt study similarly as in the microbial-enrichment studies (see references above).
109 9
ChapterChapter 6
Inn the present study the nutrition of invertebrates in field sediments was
approachedd statically, assuming no feed back between food and consumer. Naturally,
thee nutritional value in natural sediments is not static but rather is in dynamic equilibriumm with detritivores. Grazing of macroinvertebrates may decrease organic matter
contentss (Hillebrand et al. 2000) and bioturbation enhances bacterial productivity
stimulatingg degradation of organic matter (Johnson et al. 1989, Van de Bund et al.
1994).. Therefore, it is expected that the nutritional state of sediment at a certain point in
timee is strongly related to the period prior to sampling.
Thee present thesis shows that the nutritional value of sediments regulates
detritivoree communities and that in turn the nutritional value of sediments depends on
labilee biomass, e.g. algal material and bacteria. This implies that future studies focusing
onn food quality (effect of food composition) should include biomarkers for these
organisms,, such as PUFA for algae and branched fatty acids for bacteria. These
biomarkerss have to be properly normalized. Yet, it is not possible to ignore the role
playedd by co-ingestion of mineral particles with organic matter and the feedback of
detritivoress on microbial regrowth in sediments.
Feedingg strategies of Chironomus riparius and other detritivores
Thiss section reviews the response of Chironomus riparius and other detritivores
too organic matter as a food source in sediments. Furthermore, the use of C. riparius as
modell organism for detritivore assemblages in eutrophic watersystems is discussed.
Thee model organism in the present study, C. riparius, is a detritivore of the
collector-gathererr type. Collector-gatherers maximize ingestion rate in order to maximizee food uptake (Cummins & Klug 1979, review Lopez & Levinton 1987). A consequencee of the short passing time of sediment in the gut is that the organism is not able to
performm intensive digestion processes (Bjarnov 1972). Less than 10% of the organic
matterr ingested can be assimilated by detritivores (Cummins 1973). Therefore, it is
likelyy that detritivores only use the most easily absorbed components present in the
ingestedd sediment. Assimilation efficiencies of > 70% have been reported in invertebratess using algae as food (Johannsson & Beaver 1983, Fitzgerald & Garner 1993,
Cowiee & Hedges 1996). Also bacteria have shown to be efficiently digested by
detritivoress (Harper et al. 1981, review Phillips 1984a, Lopez & Levinton 1987).
110 0
ConcludingConcluding Remarks
Especiallyy extracellular polysaccharides of bacteria can be assimilated with high
efficiencyy (Couch et al. 1996). Absence of an intensive digestion system in most
detritivoress may have led to the correlations of labile organic matter with resident
speciess of detritivores in the field and larval growth of C. riparius (Chapter 5), because
off a relative uniform exploitation of only labile components. However, extrapolation of
nutritionall demands of C. riparius to other taxa found in the shallow and eutrophic
ecosystemss sampled in Chapter 5 should be performed with care. The macro fauna
communityy did not only consist of chironomid larvae, but also of taxa which may have
differentt nutritional demands. Differences in nutritional demands may be based on
differencess in body size, body composition, and physiological adaptations. Chironomini
aree indicators of eutrophic watersystems (Saether 1975, Resh & Rosenberg 1984,
Johnsonn 1995) and, therefore, could demand a high organic matter abundance compared
too some other oligotrophic detritivores. Since invertebrates mostly have similar
demandss for certain sets of food components in order to be able to produce new animal
tissuee (Downer 1981, Phillips 1984a, Blomquist et al. 1991), demands for food
componentss are expected to be similar. Even though C. riparius larvae may have
nutritionall demands higher than some other taxa, the use of C. riparius larvae as test
organismss in bioassays has been shown to discriminate nutritional values of a wide
rangee of sediments (Chapter 3).
Organismss such as collector-gatherers that live in an environment with organic
matterr of overall low nutritional value would greatly profit of an ability to detect and
selectt habitats based on nutritional value. Detection and selection of sites based on
nutritionall value would increase chances to develop and can increase population growth
(Hartt & Robinson 1990, Fonseca & Hart 1996). However, a high number of taxa is
limitedd in their ability to migrate and therefore, the distribution of sediment infauna is
assumedd to result mainly from larval settling and less so through migration (Butman
1987).. For tube-inhabiting chironomid larvae changing site is not attractive, because
buildingg a tube takes time and energy (Armitage et al. 1995). Moreover, spending time
onn the surface of the sediment instead of in the tube increases predation risk (Hershey
19855 & 1987, Ten Winkel 1987, Macchiusi & Baker 1991, Baker & Ball 1995). So,
manyy detritivores will limit active migration. Yet, under unfavourable conditions they
mayy move from their habitat to increase chances to survive. For instance, if individuals
111 1
ChapterChapter 6
stayy at an overcrowded site chances to mature are low due to competition for food
sourcess (Rasmussen 1985).
Migrationn is not restricted to Is' instar chironomid larvae, but also takes place
duringg later life stages of C. riparius (Groenendijk et al. 1998). In Chapter 4 3rd instar C.
ripariusriparius larvae were also found to be able to migrate from sites with unfavourable food
concentrations.. Similar results were found for 3rd instars of Chironomus tentans (Sibley
ett al. 1998). If food concentrations were lowered under a threshold concentration C.
ripariusriparius larvae did not settle (Chapter 4). At higher, but still limiting food conditions,
larvaee settled after physical contact with the substrate and build tubes, but emigration
occurredd after a number of hours. Observations of C. riparius larvae at food levels
beloww the threshold concentration suggests the presence of olfaction in C. riparius
similarr as was observed in C. tentans (Baker & Ball 1995). Delayed emigration after
settlementt in food levels above the threshold concentration may have been stimulated
byy depletion of food at the site of settlement.
Sincee nutritional value of sediments is affected by ingestion of mineral particle
sizess a negative preference for substrates with small mineral particles would be
expectedd (Chapter 4). For C. riparius preference for a certain particle size distribution
wass not observed, but particle size preference tests were performed at food levels in
whichh the chironomid larvae reached maximum growth rate. In contrast with expectations,, Sibley et al. (1998) found consistent selection preference of 1st instar of
ChironomusChironomus tentans for the smaller particle size range of two substrates at surplus foo
levels,, but food level preference was stronger than and independent of preference for
particlee size distribution. Preference of C. tentans found for both small mineral particle
sizee substrates and high organic matter abundance (Sibley et al. 1998) is in accordance
withh the situation in the field. In natural systems fine grained substrates are typically
associatedd with higher concentrations of organic matter and chironomids are often
stronglyy correlated with patches of accumulated organic matter (Armitage et al. 1995).
Thee study of Sibley et al. (1998) and Chapter 4 of this thesis suggest that chironomid
larvaee prefer a substrate primarily for its food content. Inhibition of food intake by
ingestionn of small mineral particles along with organic matter is not likely to be a main
factorr determining preference of detritivorous chironomid larvae.
112 2
ConcludingConcluding Remarks
Inn conclusion, the main characteristics of the model species C. riparius feeding
onn detritus have been captured in the present thesis. It has been proven that key
parameterss like half-saturation values for food-limited growth and threshold food
concentrationss for settlement or migration can be quantified. Expanding this knowledge
fromm the model species to co-existing detritivore species is likely to clarify observations
onn complex invertebrate assemblages exploiting sediment detritus.
Regulationn of invertebrate communities dominated by detritivores
Inn this thesis the nutritional value and physical characteristics of sediments have
beenn discussed and reviewed as factors regulating communities of detritivores.
However,, communities of detritivores are also regulated by biological factors such as
predationn (top-down control) and interspecific competition. This section will discuss
thesee ecological factors for detritivores in relation to the bottom-up control by limiting
nutritionall value of sediments.
Top-downTop-down control
Top-downn control of detritivores in sediments is to be expected, since predators
suchh as fish and water mites are known to have a strong impact e.g. on chironomid
communitiess (Ten Winkel 1987) and invertebrate predators have been noted in the
sedimentss sampled in the present study (Chapter 5). In spite of the potential control of
detritivoress by predators in the sampled sediments a number of correlations between
nutritionall value and densities of detritivores were observed. Positive correlations
betweenn carnivores and detritivores, and between detritivores and detritus are compatiblee when predation and detrital feeding are more or less in equilibrium. Equilibrium
predatory-preyy theories emphasize the role of predators in maintaining prey populations
att or near an equilibrium (Resh & Rosenberg 1984). These theories define predation as
positivelyy dependent on prey density. As the density of prey decreases, the predator
capturess and eats proportionally fewer individuals (Ten Winkel 1987). In some cases
thee predators feed opportunistically and captures prey species according to numerical
representation,, e.g. the predators switch the majority of the attacks to an abundant
species.. Although densities of prey may very well be suppressed by predation at peak
densitiess of prey (Gee 1989) an equilibrium between densities of predators and prey
explainss the correlations between prey and predator numbers noted in Chapter 5.
113 3
ChapterChapter 6
Anotherr explanation for the correlation between predators and herbivorous or
detritivorouss invertebrates in sediment is that predation may not be able to reduce
densitiess of detritivores to low densities. In a soil food-web bacterial- and fungalfeedingg nematodes have been shown not to be top-down controlled by predatory
nematodess and hence, the secondary production of the bacterial- and fungal-feeding
nematodess is high compared to the production rate of the predatory nematodes (Wardle
&& Yeates 1993). Similarly, predation was not able to restrain microbivore biomass
respondingg to enhanced resources (Mikola & Setala 1998). The benthic macrofauna
assemblagess in soft sediments (Chapter 5) consisted for the greatest portion of
chironomidss and oligochaetes which may indeed be taxa with high secondary
productionn rates (Bonacina et al. 1996, Benke 1998, Specziar & P. Biro 1998). Still
theirr biomass is dispersed in large volumes of sediments and therefore may be hard to
retrievee for carnivores compared to for instance pelagic fauna. Thus, the top-down
controll of detritivores in both soils and sediments is likely to be much less stringent than
inn free-living consumers.
Chironomidss and oligochaetes spend most time in the sediment while other taxa
aree mostly active at the surface of the sediment. Behavioural differences between taxa
leadd to preferential predation on specific species. For instance, specific mobility and
verticall distribution of meiofauna in the sediments lead to preferential predation on
benthicc copepods by several fish species, shrimps, and mysids (review Coull 1990). For
chironomidss it is known that inhabiting a tube decreases risk of predation by a number
off predators (Healey 1984, Hershey 1985a & b, Macchiusi & Baker 1991, Ten Winkel
1987).. The same may apply for oligochaetes, which react to the presence of predators
byy migrating deeper in the sediment. Harpacticoid copepods is the meiofaunal group
thatt is most preyed upon by epibenthic predators, even when they are greatly
outnumberedd by nematodes (review Gee 1989, review Coull 1990, Nilsson 1992).
Preferencee for harpacticoid copepods were mentioned to possibly be an active choice by
predators,, because harpacticoids are a food source of superior nutritional quality.
However,, more strongly it was suggested that high activity of the copepods on the
sedimentss surface versus the deep burrowing of nematodes during presence of predators
hass lead to preferential preying on copepods. Thus, chironomids and oligochaetes may
showw clear correlations with the nutritional value of the sediment because other taxa are
114 114
ConcludingConcluding Remarks
preferredd to prey upon. If specific taxa are strongly selected by the predator obviously
correlationss between predator and prey disappear. For example, extreme predation
pressuree of the snail Urosalpinx cinera was able to drive the hard surface inhabiting
barnaclee Balanus balanoides to extinction (Katz 1985).
Iff the prey is in equilibrium with its predator correlations between predator and
preyy densities are likely to be observed. Yet, this does not imply that top-down control
off detritivores by carnivores is very strong, because of the embedding of both predator
andd prey in an amorphic mass of sediment.
InterspecificInterspecific competition
Interspecificc competition may also hinder a clear response of species to resource
availability.. Competition for resources may result in exclusion of species, some species
takingg faster or more efficient advantage of food than others (such as discussed before).
Exampless of competitive exclusion is evident from studies on all kinds of ecosystems.
AA classical case is the mutual exclusion of two barnacle species in competing for space
inn the intertidal zone (Connell 1961). Also, competitive exclusion at microscale was
demonstratedd with microcosm experiments during which one species of protozoa in soil
wass shown to be able to use food more rapidly and efficiently than the other species, the
latterr eventually being excluded from the microcosm (Hanson 1964).
Coexistencee of species with similar food sources is made possible by resource
partitioningg and character displacement. A textbook example of resource partitioning is
foundd among the seed eating ant species living in the desert (Davidson 1977). When ant
speciess were separated from other species grain sizes that were used as food source
overlappedd among ant species. However, the different species of ants were able to coexistt because of their adapted selection for sizes of seeds when sharing the same
habitats.. Coexistence of two competitors can also be realized by partitioning of the food
sourcess through character displacement. Character displacement is found in deposit
feedingg mud snails coexisting in brackish waters (Fenchel 1975). Two species of snails
aree able to co-exist by differentiating food sources and adapting the ingestion apparatus
too these different particle sizes.
Inn the present study several detritivorous taxa co-exist and showed similar
correlationss to the nutritional value of sediments. There is no proof of the mechanisms
115 5
ChapterChapter 6
off competition here, but it is plausible that resource partitioning between detritivores
occurs.. Later instar chironomid larvae indeed are known to be able to ingest larger
particless than oligochaetes because of bigger menta of later instar chironomid larvae.
Usingg different particle sizes reduces competition for food sources thereby potentially
enablingg coexistence of chironomids and oligochaetes. An even more clear distinction
betweenn food sources of detritivores found in the sampled sediments is provided for
shredderss (e.g. Gammarus) and collector gatherers (Chironomus spp.). Both feeding
modess belong to detritivores but shredders consume Coarse Particulate Organic Matter
(CPOM)) and collector-gatherers Fine Particulate Organic Matter (FPOM) (Cummins &
Klugg 1979). In some cases the presence of one detritivorous taxa benefits another by
conditioningg of the environment. For instance, chydorids take advantage of the presence
off chironomid larvae by feeding on chironomid larvae faeces, although early instar
chironomidd growth was inhibited by presence of chydorids (Van de Bund 1994). Thus,
competitionn and commensalisms is likely to almost concur in detritivores. Another
examplee of commensalism are shredders which produce FPOM thereby stimulating
growthh rates of the FPOM-feeding collectors (Cummins et al. 1973).
Resourcee partitioning makes it probable that more than just one species of
detritivoress is able to take advantage of detritus. In the sediments used in this thesis
shredderss and collector-gatherers probably do not compete for food because the taxa
aimm at different forms of detritus. Also, other taxa of detritivores are expected to coexist
inn sediments without competing with other detritivorous taxa as was already postulated
forr oligochaetes and chironomid larvae based on differences in mentum widths. The
evidencee for resource partitioning is sufficient to explain correlations of densities of
severall coexisting detritivorous taxa with parameters of nutritional value of sediments.
Inn conclusion, positive correlations found in this study between detritivores and
predatorss on the one hand and correlations between densities of detritivores and
sedimentt food quality on the other hand can be explained by an equilibrium between
predators,, detritivores, and food quality. This equilibrium may be stimulated by the
physicall distribution of all components in large volumes of sediment. Resource
partitioningg or commensalism is indicated to modify competition for food among
detritivorouss animals in sediment. The diverse options to use detritus as food enhance
speciess diversity of the detritivorous community.
116 6
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129 9
130 0
Summary Summary
Particulatee organic matter in natural waters originates from algae and macrophytes,, but can also include fragments of dead animals and terrestrial run-off. This
materiall tends to accumulate at the bottom and there supports distinct assemblages of
detritivoress that use the organic matter as both habitat and food source. Detritivores
formm a major link in the food web, processing the nutritionally low quality dead organic
matter,, and constituting one of the main food sources for predators, like carnivorous
invertebrates,, fish, and birds.
Fieldd studies on the response of benthic macrofauna to sedimentation of algal
bloomss suggest that food limitation controls benthic macrofauna during most of the
year.. Although these field studies clearly recognize the role of food as factor regulating
populationn dynamics of sediment-feeding freshwater invertebrates, literature on the
influencee of food composition on detritivores is limited. Also the physical characteristicss of sediments are likely to affect the suitability of sediment for invertebrates. Detritivoress ingest mineral particles along with organic detritus and it was expected that inorganicc material obstructs food uptake and consequently hampers growth of detritivores.
Thee present study aimed to clarify the influence of the nutritional value of sedimentss on benthic detritivores. Focal points were the influence of sedimentary organic
matterr and its biochemical composition on the survival and growth of benthic detritivoress and the interaction between particle size distribution and nutritional value of sediments. .
Inn Chapter 2 the quantitative effects of organic matter abundance and compositionn on the detritivorous Chironomus riparius was assessed. For this purpose a set of
artificiall food items were biochemically analyzed for C, N, P, carbohydrates, proteins,
andd total fat content, and offered in concentration series to first instar larvae of the
modell species in standardized mineral substrate. Larval length and survival were recordedd after one week of growth. Saturation growth curves were fitted, and for each food
itemm the slope and the maximum length were estimated. In general, maximum length
attainedd by larvae reared on fish foods and on food items of animal origin was higher
thann the maximum length reached on food items of plant origin, whereas slopes at
limitingg food concentrations of the growth curves for larvae reared on foods of plant
originn were steeper than slopes of curves for larvae reared on fish food or on food of
animall origin. Results indicated that the optimal food composition depended on the
131 1
Summary Summary
amountt of food available. For instance, high N, P, and lipid contents stimulated growth
att high food levels, whereas the amount of carbohydrates appeared to be important in
definingg growth at low food levels. This interaction of food quantity and quality was
suggestedd to be the result of limiting energy availability at low food levels versus
limitingg food quality at high food levels.
Thee results of Chapter 2 were verified for the field by sampling sediment and
measuringg growth and survival of the midge C. riparius on these sediments in the
labora-toryy (Chapter 3). The sediments were also analyzed for a set of biochemical
varia-bles.. Correlations were sought between the biochemical variables and larval
growthh and survival. Positive correlations of larval growth and survival with
polyunsaturatedd fatty acids (PUFA), pigments, and microbial mineralization rate were
foundd when biochemical variables were standardized on dry weight. When the variables
weree standardized on organic matter weight additional significant positive correlations
withh P, carbohydrates, proteins, and fatty acids of bacterial origin appeared. Clearly,
organicc matter composition constituted an important factor influencing detritivore
growth,, with newly produced organic material supporting highest larval growth.
Thee generally sub-optimal growth of chironomid larvae measured in natural
sedimentss was not caused by the widely diverging physical characteristics of the
substratess tested, because amendment with food permitted undisturbed larval developmentt (Chapter 3). Biochemical variables needed to be standardized on organic matter
contentt in order to reveal a number of correlations between biochemical variables and
larvall growth. The close correlation of organic matter content and small particle sizes
securedd that standardization on organic matter basis focussed on the size fraction availablee as food source to the chironomid larvae. The necessity to standardize suggested
thatt mineral sediment particles, indiscriminately ingested with food, may have reduced
growthh potential of sediments. Therefore, Chapter 4 focused on the effect of particle
sizee distribution on chironomid larvae growth using artificial mineral substrates.
Minerall particle size had no effect on larval growth at saturating food supply. However,
att limiting food levels growth of third instar larvae was hampered by ingestion of small
minerall grains, thereby confirming the hypothesis posed in Chapter 3 that mineral
sedimentt particles ingested along with food can reduce growth potential of sediments.
Inn Chapter 4 preference of C. riparius larvae for particle size distributions of
132 2
Summary Summary
substratess and for food levels were also explored. When offered a choice between two
differentt particle size substrates larvae clearly preferred the substrate supplied with
food,, independent of the particle size ranges. During preference experiments in which
larvaee were offered two food levels, a threshold concentration was found below which
larvaee continued crawling and did not settle to construct tubes. When allowed to choose
betweenn two food levels both above the threshold concentration, the food concentrations
weree only distinguished if food concentration differences were large enough. In food
concentrationss higher than the threshold concentration larvae were observed to settle
withinn a few hours and to construct tubes. Depletion of food at the place of residence
andd the ability of larvae to find food through olfaction were mentioned as possible
triggerss for chironomid larvae to leave the substrate above the threshold concentration.
Growthh and preference experiments in Chapter 4 showed that organic matter content
andd particle size distribution are interacting factors determining larval growth of C.
ripariusriparius and abundance in situ.
Chapterr 5 examined if the nutritional value of sediments is a factor structuring
naturall macrofauna communities and which food components are responsible for the
regulationn of macrofauna communities. Macrofauna species in a range of soft-bottom
sedimentss sampled in The Netherlands were enumerated and their occurrence in the
sedimentt samples was correlated to the results of bioassays performed with C. riparius
andd to biochemical sediment composition. Growth of Chironomus riparius was used as
aa direct measure of nutritional value. Newly produced organic matter, represented by
variabless similar to the variables found to support growth of C. riparius in Chapter 3
(PUFA,, bacterial fatty acids, pigments, and labile organic matter fraction), was
associatedd with abundances of a number of detritivorous taxa and therefore is most
likelyy a key factor for the nutritional
valuee of sediments. Growth of C.
ripariusriparius larvae correlated well with
abundancess of detritivorous taxa but
nott with taxa that have other modes
off feeding. Therefore, growth of C.
ripariusriparius was found to effectively
indicatee the nutritional value of
133 3
Summary Summary
sedimentt for bulk feeders. The use of bioassays with midge larvae excluded indirect
effectss caused by covariation of organic matter content with other factors determining
thee habitat for macrofauna species, e.g. oxygen regime or stability of the substrate. It
wass postulated that non-food cq. habitat parameters selected the species inhabiting sites.
Yet,, nutritional value determined the overall density of detritivores. Therefore, it was
concludedd that sediment food quality in soft-bottom sediments is a major structuring
factorr for faunal composition.
Inn the concluding remarks (Chapter 6) it was attempted to define the ideal parameterss for measuring nutritional value, in order to analyze regulation of communities of
detritivoress properly. The present thesis showed that the nutritional value of sediments
dependss on labile biomass, e.g. algal material and bacteria. This implies that future
studiess focusing on food quality (effect of food composition) should include biomarkers
forr these organisms, such as PUFA as biomarker for algae and branched fatty acids for
bacteria.. Biomarkers have to be properly normalized, e.g. on organic matter content.
Standardizationn of biochemical variables on organic matter content is useful to
circumventt covariation of organic matter with factors other than food.
Inn the second part of Chapter 6 it was concluded that the main characteristics of
feedingg of the model species C. riparius on detritus have been captured in the present
thesis.. Key parameters determining nutritional value of organic matter in sediments,
half-saturationn values for food-limited growth of chironomid larvae, and threshold food
concentrationss for settlement or migration can be quantified. Expansion of knowledge
onn the model species to co-existing detritivore species was recommended in order to
clarifyy the observations on complex invertebrate assemblages exploiting sediment
detritus. .
Finally,, Chapter 6 discussed the biological factors predation and interspecific
competitionn in relation to bottom-up control of detritivore populations. Positive
correlationss that were found in this study between detritivores and predators on the one
handd and correlations between densities of detritivores and sediment food quality on the
otherr hand can be explained by an equilibrium between predators, detritivores, and food
quality.. It was suggested that this equilibrium is stimulated by the distribution of all
componentss in large volumes of sediment.
134 4
Samenvatting Samenvatting
Particulairr organisch materiaal in natuurlijke wateren is afkomstig van fytoplanktonn en hogere waterplanten, maar bestaat ook uit dood dierlijk en terrestrisch
materiaal.. Dit organische materiaal accumuleert op waterbodems en vormt daar het
habitatt en voedsel voor levensgemeenschappen van detritivoren. Detritivoren vormen
eenn belangrijke schakel in het voedselweb, omdat ze één van de omvangrijkste voedselbronnenn zijn voor predatoren als vissen en vogels. Bovendien verwerken detritivoren
hett weinig voedzame organische materiaal in sedimenten.
Veldstudiess suggereren dat benthische macrofauna gemeenschappen gedurende
hett grootste deel van het jaar gelimiteerd wordt door voedsel. Alhoewel de rol van
voedsell als regulerende factor van detritivoren alom erkend wordt, is weinig literatuur
overr de invloed van voedselsamenstelling op detritivoren beschikbaar. Detritivoren
wordenn naast voedsel ook door de fysische samenstelling van sedimenten beïnvloed.
Detritivorenn nemen minerale delen op samen met organisch materiaal. Tijdens de
huidigee studie werd verwacht dat het anorganische materiaal de voedselopname limiteertt en zo de groei van detritivoren belemmert. Deze studie concentreerde zich op de
invloedd van voedselwaarde van sedimenten. Punten van aandacht waren het effect van
voedselsamenstellingg op de groei en overleving van detritivoren en de interactie tussen
korrelgrootte-verdelingg en de voedselwaarde van sedimenten.
Inn hoofdstuk 2 werden de effecten van voedselhoeveelheid en -samenstelling op
dee detritivore larven van Chironomus riparius bekeken. Een set artificiële voedsels
werdd biochemisch geanalyseerd op C-, N-, P-, carbohydraten-, proteïne-, en vet-inhoud.
Hett voedsel werd in concentratie-series aangeboden aan eerste stadium larven op
gestandaardiseerdd substraat. Na 1 week werden lengte en overleving van de larven
geregistreerd.. Voor elk voedseltype werd een groei-verzadigingscurve gefit en werden
maximalee groei en helling van de curve geschat. Op visvoer en voedsel van dierlijke
oorsprongg was de maximale groei hoger dan op plantaardig voedsel. Groei-verzadigingscurvess van plantaardig voedsel hadden daarentegen stijlere hellingen vergeleken
mett curves van dierlijk voer. De resultaten indiceerden dat optimale voedselsamenstellingg afhankelijk is van de beschikbare voedselhoeveelheid. Hoge N-, P- en
vetgehaltess stimuleerden bijvoorbeeld hoge groei bij hoge voedselhoeveelheden, maar
carbohydratenn stimuleerden juist groei bij lage voedselhoeveelheden. De interactie
tussenn voedselkwaliteit en -kwantiteit zou het resultaat kunnen zijn van limiterende
135 5
Samenvatting Samenvatting
voedselkwaliteitt bij hoge voedselhoeveelheden versus limiterend energie-nivo bij lage
voedselhoeveelheden. .
Inn hoofdstuk 3 werden de resultaten van hoofdstuk 2 getoetst op de veldsituatie.
Groeii en overleving van C. ripahus larven op natuurlijke sedimenten werd gemeten en
gecorreleerdd aan de biochemische samenstelling. Groei en overleving correleerde
positieff met meervoudig onverzadigde vetzuren (PUFA), pigmenten, en microbiële
afbraak,, als deze variabelen werden uitgedrukt als een percentage van het sediment
drooggewichtt (DW). Als de biochemische variabelen werden gestandaardiseerd op
organischh materiaal gehalte werden daar significante correlaties met P, carbohydraten,
proteïness en bacteriële vetzuren aan toegevoegd. Samenstelling van organisch materiaal
bleekk van grote invloed op de groei van detritivoren, waarbij vers, labiel organisch
materiaall de hoogste larvale groei tot stand bracht.
Dee suboptimale groei van chironomide larven in natuurlijke sedimenten werd
niett veroorzaakt door de fysische samenstelling van de substraten, want verrijking van
dee sedimenten met artificieel voer leidde tot ongestoorde groei. Biochemische variabelenn moesten op organische stof hoeveelheid gestandaardiseerd worden om een aantal
correlatiess tussen chemische variabelen en larvale groei te onthullen. Dit suggereert dat
mineralee delen, die samen met voedsel worden opgenomen, het ontwikkelingspotentiëel
vann sedimenten voor muggenlarven verlagen. In hoofdstuk 4 werd daarom bekeken of
mineralee korrelgrootte-verdel ing van substraten de groei van muggenlarven beïnvloedt.
Bijj verzadigende voedselconcentraties had de korrelgrootte-verdeling geen effect op de
larvalee groei. Bij limiterende voedselhoeveelheden werd de groei van derde stadium
larvenn belemmerd door inname van kleine minerale delen. Dit bevestigde de hypothese
inn hoofdstuk 2, dat opname van minerale delen samen met voedsel de voedzaamheid
vann sedimenten verlaagt.
Inn hoofdstuk 4 werd ook de voorkeur van C. ripahus larven voor korrelgrootteverdelingg en voedselconcentraties onderzocht. Als de larven konden kiezen tussen 2
verschillendee korrelgrootte substraten dan lieten de larven duidelijk voorkeur zien voor
hett substraat waaraan voedsel was toegevoegd, onafhankelijk van de korrelgrootte van
dee 2 substraten. Er werd een grenswaarde voor de voedselconcentratie gevonden waaronderr de larven zich niet vestigden, maar op het oppervlakte van het substraat bleven
kruipen.. Als de larven de keuze kregen tussen 2 voedselconcentraties boven de grens-
136 6
Samenvatting Samenvatting
waarde,, dan werden de concentraties alleen onderscheiden als het verschil tussen de
voedselconcentratiess groot genoeg was. Mogelijke stimulansen om een substraat te
verlatenn zouden uitputting van het voedsel en aanwezigheid van reukzin bij de muggenlarvenn kunnen zijn. De groei- en preferentie-experimenten uit hoofdstuk 4 lieten zien
datt voedselhoeveelheid en korrelgrootte-verdeling interacterende factoren zijn die
larvalee groei en abundantie in situ van C. riparius beïnvloeden.
Inn hoofdstuk 5 werd onderzocht of de voedzaamheid van sedimenten macrofaunaa gemeenschappen beïnvloedt en welke voedselcomponten voor regulatie van de
gemeenschappenn verantwoordelijk zijn. Hiervoor werd macrofauna van een aantal
zachtee waterbodems bemonsterd en gedetermineerd. Macrofauna abundanties werden
gecorreleerdd aan de biochemische samenstelling van de sedimenten en aan resultaten
vann bioassays die waren uitgevoerd met C. riparius. Groei van muggenlarven werd
gebruiktt als maat voor voedingswaarde van sedimenten. Bioassays met muggenlarven
sluitenn indirecte effecten door covariatie van organisch materiaal met andere factoren,
gerelateerdd aan het habitat van macrofauna (bv. zuurstof), uit. Vers en labiel organisch
materiaal,, gerepresenteerd door dezelfde variabelen die de groei van muggenlarven
bevorderenn (hoofdstuk 3), was gerelateerd aan abundanties van een aantal soorten
detritivorenn en was daarom waarschijnlijk een sleutelfactor voor de voedingswaarde van
sedimenten.. Groei van C. riparius larven correleerde goed met dichtheden van een
aantall soorten detritivoren, maar niet met soorten met andere voedingswijzen. De resultatenn van de veldstudie suggereerden dat habitat-factoren de soorten selecteerden die in
staatt waren bepaalde sedimenten te bewonen en dat voedselkwaliteit de macrofauna
dichthedenn bepaalde.
Inn het afsluitende hoofstuk 6 werd gepoogd om de ideale parameters voor
voedsell waarde van sedimenten te definiëren, zodat regulatie van detritivoor gemeenschappenn accuraat kan worden geanalyseerd. Het huidige proefschrift toont aan dat de
voedingswaardee van sedimenten afhankelijk is van labiel biomassa, namelijk algenmateriaall en bacteriën. Dit impliceert dat in studies omtrent voedsel biomarkers van algen
(PUFA)) en microorganismen (bacteriële vetzuren) moeten worden gebruikt. Inname van
mineralee delen samen met voedsel door detritivoren maakt standaardisatie van de
biomarkerss op organisch materiaal noodzakelijk. Bovendien omzeilt standaardisatie op
organischh materiaal covariatie van organisch materiaal met habitat factoren.
137 7
Samenvatting Samenvatting
Inn het tweede deel van hoofdstuk 6 werd geconcludeerd dat parameters die
voedselwaardee beschrijven (biochemische componenten,
half-verzadigingsconcen-
tratiess en grenswaarden) kunnen worden gekwantificeerd. Kennis omtrent voedsel in
relatiee met het model soort zou moeten worden uitgebreid om verspreiding van
detritivorenn in het veld beter te kunnen verklaren.
Inn het derde deel van hoofdstuk 6 werden biologische factoren als predatie en
competitiee tussen soorten besproken, in relatie met bottom-up control van detritivoren
gemeenschappen.. Positieve correlaties tussen detritivoren en predatoren tegenover
correlatiess tussen detritivoren en voedselkwaliteit, zoals die werden gevonden in de
huidigee studie, kunnen verklaard worden door evenwicht tussen voedselkwaliteit en
dichthedenn van predatoren en detritivoren. Er werd gesuggereerd dat het evenwicht
tussenn voedselkwaliteit, detritivoren en predatoren verstevigd wordt door de
verspreidingg van deze componenten in grote hoeveelheden sediment.
138 8
Dankwoord Dankwoord
Mijnn promotie-tijd in Amsterdam was een leerzame periode, zowel op sociaal
alss op wetenschappelijk vlak. Zoals iedere promovendus had ik mijn pieken en dalen,
hebb ik kleine successen geboekt, maar ook fouten gemaakt.
Jaapp Postma en Marco hebben de eerste opvang in groot boos Amsterdam
verzorgd.. Hun energie heeft mij de eerste 2 jaar doorgeholpen. Jaap had de trein al
rijdendee toen ik aankwam op de vakgroep, 's Ochtends maakten Marco's trouwe kopjes
koffiee mij wakker, en de rest van de dag hield Marco's geluidswal van grappen en
grollenn mij verdoofd aan het werk. Na Marco kwam Sieppie me helpen als analiste, die
ookk van gezelligheid houdt, maar minder van soxhlet en modder. Mijn eerste studente
Maaikee heeft samen met Marco emmers modder en zand gezeefd. Ondanks de
toenemendee stank in het nat-lab hebben ze de enorme klus geklaard. Annelies was de
derdee analist die aan mij werd toegewezen. Zij is een toegewijde en secure werker.
Armeliess was altijd in voor een rondje wandelen, tijdens welke we vaak maatschappelijkee problemen, maar ook de natuur bespraken. Helen wees tijdens deze rondjes
trouww elk konijn aan dat op onze weg kwam. Helen heeft ook getracht me UvA-politiek
bijj te brengen en stond me altijd met
raadd en daad bij. Helen, je kan zeggen
watjee wilt, maar de vakgroep bouwt op
**
;•
je.. Wim heeft me vooral de laatste fase
vann de promotie doorgetrokken. Net als
elkee promovendus van onze vakgroep
werdd ik onder perenbomen gezet en
moestt ik aan manuscripten ruiken,
maarr gelukkig vond hij sommige
bevindingenn ook "lollig". Jaap Dorgelo wil ik bedanken voor zijn advies en luisterend
oorr als promovendus decaan. Michiel heeft zich in de laatste fase spontaan opgeworpen
alss begeleider en strategisch adviseur, wat ik erg gewaardeerd heb.
Hulpp kwam af en toe uit onverwachte hoek. Jos Brouwer (UU) en Eric Boschker
(NIOO,, Yerseke) hebben mij belangeloos geholpen bij het identificeren van vetzuurpieken.. Steven Arisz heeft mij geweldig geholpen door me wegwijs te maken op zijn
GCC en deze dagenlang, zo niet wekenlang, aan mij uit te lenen. Op het GC-lab is ook
eenn fijne vriendschap ontstaan. Frans Kerkum (RIZA, Lelystad) heeft mij geholpen met
139 9
Dankwoord Dankwoord
hett bemonsteren van sedimenten. Deze reisjes waren altijd gezellige uitjes.
Promovendii moeten regelmatig stoom afblazen, bij voorkeur bij andere
promovendi.. Gelukkig leende menigeen op de vakgroep zich daar dankbaar voor. Met
Saskiaa kon ik lekker raaskallen, want haar kan het niet gek of luidruchtig genoeg. Dat
doett me meteen herinneren dat ik me bij mijn (voormalige) kamergenotes moet verontschuldigenn voor elk overlast dat ik hun heb
bezorgd.. Vrouwenkamer 4.09 was altijd een gezelligg kippenhok. Vraagbaak Eric, het stukje is af.
Helpdeskk Harm, het was fijn de laatste periode van
dee promotie de smart te kunnen delen. De vakgroep
heeftt me enorm geholpen alert te blijven onder alle
omstandigheden,, aangezien het op AEE elke dag
kermiss is. Ik wil specifiek alle studenten bedanken
voorr alle gezelligheid die ze met zich mee brengen.
Hett thuisfront bestaat uit mijn familie en
vrienden.. Ik heb hele lieve vrienden, die geduldig
mijnn verhalen aanhoren. Twee van hen, Anna en
Hilde,, ronden hun promotie bijna gelijktijdig met
mijj af. Ik zie uit naar hun proefschriften, die
ongetwijfeldd onbegrijpelijke taal bevatten voor eenvoudige aquatisch ecologen. Ik wens
zee succes met de laatste loodjes en het verkrijgen van voldoende gram. Mijn zus Thera
heeftt mij het goede voorbeeld gegeven, alhoewel ik nu pas snap welke boontjes zij heeft
moetenn doppen tijdens haar promotie-tijd. Dankjewel voor de belangstelling die je altijd
hebtt getoond voor de voortgang van mijn promotie. Mijn ouders bieden mij een veilige
basis,, waar ik altijd terecht kan. Zij zijn mijn trouwste supporters, mijn hele leven al.
Overr mijn liefje Ronald zou ik wereldkundig willen maken dat hij mij thuis in
dee watten heeft gelegd, maar dat hij ook op werkvlak klaar stond om mij te helpen. Ik
houu van jou.
&£ &£
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