Download Epidermal club cells do not protect fathead minnows against

Biological Journal of the Linnean Society, 2009, 98, 884–890. With 2 figures
Epidermal club cells do not protect fathead minnows
against trematode cercariae: a test of the anti-parasite
hypothesis
CLAYTON T. JAMES1†, BRIAN D. WISENDEN2 and CAMERON P. GOATER1*
1
Department of Biological Sciences, University of Lethbridge, 4401 University Drive, Lethbridge,
Alberta, T1K 3M4, Canada
2
Biosciences Department, Minnesota State University Moorhead, 1104 7th Avenue South, Moorhead,
MN 56560, USA
Received 22 April 2009; accepted for publication 16 June 2009
bij_1327
884..890
Epidermal club cells of fishes in the superorder ostariophysi have puzzled evolutionary biologists because they were
historically linked to chemical alarm signalling and relied on group selectionist explanations. Alternative hypotheses to explain the existence of these cells include the possibility of an anti-pathogenic or anti-parasitic function.
If this is so, individual fish should invest in increased numbers of club cells after exposure to parasites, and club
cells should contain components that reduce the infectivity of skin-penetrating larvae. Infectivity of cercariae of the
trematode Ornithodiplostomum sp. was significantly reduced when exposed to the skin extract of fathead minnows
(an ostariophysan), but also to skin extract of mollies (a non-ostariophysan that lacks club cells), respectively,
compared to controls. Moreover, club cell density was not affected by exposure to cercariae. Taken together, these
results are inconsistent with an anti-parasite function for these cells and instead suggest a generic role in response
to injury. © 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 98, 884–890.
ADDITIONAL KEYWORDS: alarm substance – anti-predation – Ornithodiplostomum – Ostariophysi –
infectivity – Pimephales.
INTRODUCTION
Given the extent and variety of costs that parasites
impose on individual hosts, parasite-mediated selection should favour host defences that limit exposure
to infective stages, or limit their negative effects
(Poulin, 2007). The detection and/or avoidance of
parasites appears to be limited to conspicuous and
pathogenic infective stages, especially for aquatic
hosts (Wisenden, Goater & James, 2009). Furthermore, immune defences are expressed at the expense
of growth and reproduction, often with important
fitness consequences (Zuk & Stoehr, 2002). Thus,
natural selection may favour anti-parasite strategies
that defend against parasites at the point of host–
*Corresponding author. E-mail: [email protected]
†Present address: Alberta Conservation Association, Box
900-26, Peace River, Alberta, T8S 1T4, Canada
884
parasite contact or entry into the host, reducing the
associated costs of parasites at the site of infection.
In fish, the epidermis represents an initial site for
complex immune responses against water borne
parasites and pathogens (Whitear, 1986; Kearn,
1999). Immunological components, such as lymphocytes, macrophages, and several types of granulocytes
found in fish mucus (produced by goblet cells located
in the epidermis), have been shown to reduce the
infectivity, growth, and reproduction of various parasites at the site of infection (Shephard, 1994). Another
cellular component of fish epidermis that has been
linked to host immunity comprise the epidermal club
cells of fishes in the superorder ostariophysi (Chivers
et al., 2007; Halbgewachs et al., 2009).
Epidermal club cells that contain the chemical
alarm cue known as ‘Schrekstoff’ are a defining characteristic of the ostariophysi (Pfeiffer, 1977). It is now
well established that damaged skin serves as a
© 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 98, 884–890
ANTI-PARASITE FUNCTION OF CLUB CELLS
mechanism for risk assessment by fish (Chivers &
Smith, 1998). These cells pose a metabolic cost
(Wisenden & Smith, 1997), although the mechanism
by which individuals that invest in these cells recover
fitness benefits is not clear because the cells release
their contents only when ruptured by a predator. The
lack of obvious fitness benefits to justify investment
in epidermal club cells poses a challenge for evolutionary theory (Williams, 1992). One idea that neatly
resolves the evolutionary enigma is the possibility
that epidermal club cells contain anti-pathogenic or
anti-parasitic agents (Smith, 1992; Magurran, Irving
& Henderson, 1996). Recently, Chivers et al. (2007)
provided several lines of empirical evidence in
support of the anti-parasite hypothesis.
Ostariophysan fish are frequently infected with
the resting stage of aquatic trematodes, known as
metacercariae. These encyst on and in a range of
species-specific tissues, typically awaiting ingestion
by an appropriate final host. Two general arguments
lead to the prediction that club cells may deter
trematode infections of fish. First, most cercariae of
fishes actively penetrate the host’s epidermis.
Although cercariae vary widely in size across
species, most are large relative to club cells and, as
such, likely pass through or disrupt these cells
during the penetration process. Second, the putative
active chemical in ostariophysan club cells that
elicits alarm reactions is a nitrogen oxide (NO) side
group, such as the one contained in hypoxanthine3-N-oxide (Pfeiffer et al., 1985; Brown et al., 2000).
NO plays an important role in host resistance to the
free-living stages of some helminth parasites (James,
1991). For example, NO has been shown to inhibit
metabolic activity of the schistosomula of Schistosoma mansoni (James & Hibbs, 1990). It is conceivable that NO in club cells may play a similar
anti-cercarial role in the epidermis of fish. In
experimental studies, Chivers et al. (2007) showed
that minnows exposed to cercariae had a higher
density of epidermal club cells compared to unexposed controls. Furthermore, Poulin, Marcogliese
& McLaughlin (1999) showed that rainbow trout
exposed to trematode cercariae induced a fright reaction in naïve, unexposed conspecifics. This finding
implies that trematode cercariae rupture club cells
when penetrating the host epidermis and ultimately
come in contact with the contents of these cells.
However, whether these results are generalizable to
other host/parasite systems remains unknown.
In the present study, we tested two predictions of
the anti-parasite hypothesis. First, on the basis of the
study by Chivers et al. (2007), we predicted that exposure to cercariae should result in increased investment in epidermal club cells. Second, if club cells
contain anti-parasite properties, we predicted that
885
immersion of cercariae in skin extract should reduce
cercariae infectivity.
MATERIAL AND METHODS
HOST/PARASITE SYSTEM
Interactions between fathead minnows and metacercariae of trematodes in the genus Ornithodiplostomum have been studied extensively in one of our
laboratories (C.P.G.). One advantage of this minnow/
trematode interaction is that the life-cycles of
Ornithodiplostomum spp. can be maintained in the
laboratory using the F1 generation of field-collected
snails (Physa spp., first intermediate host), uninfected
fathead minnows (second intermediate host), and
domestic chickens (surrogate final host) as experimental hosts (Sandland & Goater, 2000; James et al.,
2008).
In these experiments, we exposed parasite-naïve
fathead minnows to cercariae of Ornithodiplostomum
sp. This unidentified species, and its congener
Ornithodiplostomum ptychocheilus, are the two most
common and abundant trematodes in populations of
fathead minnows in Alberta, typically infecting all
fish in a population with up to several hundred
encysted metacercariae (Sandland, Goater & Danlychuk, 2001). Subsequent to penetration of the
epidermis of minnows, metacercariae of Ornithodiplostomum sp. encyst in the liver and body cavity,
where they remain for the remainder of the life of the
host, or until they are ingested by appropriate fisheating birds. We used Ornithodiplostomum sp. in the
present study partly as a result of the availability of
experimentally infected Physa gyrina that consistently released large numbers of cercariae, and also
because our earlier work had indicated a strong
negative affect on growth of experimentally-infected
minnows (James et al., 2008).
EXPERIMENT 1: EFFECT
OF FISH SKIN EXTRACT
ON CERCARIAE INFECTIVITY
Fathead minnows (30 days old) used in the experiment were obtained from the US EPA National
Health and Environmental Effects Research Laboratory (Duluth, MN, USA) on 19 July 2007. The
fish were held in four separate 190-L tanks for 3
days before the experiment and fed ad libitum on
Tetramin® flake food.
The overall aim of the present study was to evaluate the infectivity of known-aged cercariae that had
been immersed in one of: (1) water containing blank
dechlorinated tap water (control); (2) water containing minnow skin extract (containing ostariophysan
club cells and other skin components); or (3) water
containing molly skin extract (generic fish skin
© 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 98, 884–890
886
C. T. JAMES ET AL.
lacking ostariophysan club cells). Although the epidermis of some non-ostariophysan fishes such as
Poecilids are known to contain alarm substances
(Smith, 1982), and some non-ostariophysans also
contain club cells (Chivers et al., 2007), the present
study was designed to control for the anti-parasite
properties of generic fish skin that are not attributable to ostariophysan club cells.
Ornithodiplostomum sp. cercariae were obtained in
accordance with the methods of Sandland & Goater
(2000) for O. ptychocheilus. Eight, 1-day-old chickens
were fed metacercariae from the viscera of wild
fathead minnows collected in June from Gold
Spring Lake, southern Alberta, Canada (49°05′50″N,
111°59′49″W). Trematode eggs were collected from
chick faeces 5 days post-infection. The resulting
miricidia were pipetted into small vials containing
laboratory-reared juvenile pond snails, Physa gyrina,
measuring 3–5 mm in maximum length. Snails began
to release cercariae at approximately 28 days postexposure. To obtain cercariae, 12 infected snails were
placed into a flask for 3 h. The numbers of cercariae
in three, 1-mL aliquots were counted and averaged to
estimate the total numbers of cercariae released over
3 h. We used this estimate to evaluate the volume of
water containing 100 cercariae.
Skin extract was prepared from wild adult female
fathead minnows (mean ± SE = 5.74 ± 0.08 cm,
N = 55) collected from Deming Lake (MN, USA)
(47°10′13.24″N, 95°10′06.67″W) on 11 July 2007.
Mollies were obtained from a commercial supplier
and, upon arrival in the laboratory, were immediately
euthanized to prepare skin extract. Each fish was
euthanized by cervical dislocation (MSUM IACUC
protocol 07-R-009-N-N-C/D). Skin fillets were removed from both sides of the fish, measured for
area and then immersed in 100 mL of chilled dechlorinated tap water. An area of of minnow skin
(72.15 cm2) was collected and homogenized using a
hand blender and filtered through polyester fibre.
This solution was diluted to 720 mL with dechlorinated water. Similarly, we collected 70.06 cm2 of molly
skin, blended it with a hand blender and diluted with
dechlorinated tap water to 700 mL. Thus, the concentration of both skin extract solutions comprised 2 cm2
of skin per 20-mL dose. Twenty-mililitre aliquots of
each type of skin extract and dechlorinated water
(control treatment) were pipetted separately into
118-mL plastic containers (N = 35 containers per
treatment) with sealable lids and frozen at -20 °C
until needed.
The experiment was set up as a single factor analysis of variance (ANOVA) with three levels of cue type:
water control, fathead minnow extract, and molly
skin extract. On the day of exposure, the containers
were removed from the freezer and thawed. One
hundred cercariae were pipetted directly into each
container and, 30 min later, minnows were added.
Containers were arranged so that all treatments
were evenly distributed spatially and minnows were
assigned at random to containers. After exposure, all
minnows were removed from the Petri plates, and
separated into 37-L tanks based on treatment.
Minnows were fed Tetramin® flake food daily for 5
weeks under a 12 : 12 dark/light cycle to allow metacercariae time to develop into the encysted stage
(Sandland & Goater, 2000). Minnows were euthanized
with an overdose of methane tricaine sulphonate
(MS222), dissected, and the numbers of metacercariae
in the body cavity counted using standard methods.
EXPERIMENT 2: EFFECT
OF CERCARIAE EXPOSURE ON
THE DENSITY OF EPIDERMAL CLUB CELLS AND
EPIDERMAL THICKNESS
This experiment tested the prediction that individual
fish should invest in more club cells following exposure to cercariae. The source of fish, snails, and
cercariae was the same as that described for Experiment 1. The exposure protocol was also the same. A
total of 90, 30-day-old minnows (average length ± SE,
29.72 ± 0.22 mm) were divided into three groups of
30. One group (high-dose) was exposed three times to
batches of 50, 2-h-old cercariae every 2 days commencing on 30 July 2007 and ending on 3 August
2007. A second group (low dose) was exposed on the
same days to ten cercariae; the third group (control)
received water only. Fish were assigned at random to
Petri dishes that contained 40 mL of water with zero,
ten or 50 2-h-old cercariae. The exposure period was
2 h. On the last day of the experiment (i.e. 3 days
after the third and final exposure), 20 fish from each
treatment were euthanized and a section of tissue
from the nape region was removed from each minnow,
preserved in 10% buffered formalin and prepared for
histological examination. The remaining ten fish in
each treatment were maintained on flake food for 4
weeks to allow assessment of the success of metacercariae encystment.
Tissue samples were sent to the North Dakota
State University Veterinary Diagnostic Laboratory
for histological preparation. For each epidermal
tissue sample, three paraffin-imbedded sections (7 mm
thick) were removed and stained with Periodic Acid
Schiff’s reagent and then counterstained with Lillie’s
haematoxylin. Three sections of each tissue sample
were evaluated for the number of club cells and
epidermal thickness. Epidermal thickness was determined as the distance between the basement membrane of the epidermis and the outer surface of the
epithelium (Wisenden & Smith, 1997). The three
measurements of club cell density and epidermal
© 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 98, 884–890
ANTI-PARASITE FUNCTION OF CLUB CELLS
90
14
80
12
887
70
Club cell density per mm
Metacercariae recovered
Con
60
50
40
30
20
Low
High
10
8
6
4
2
10
0
0
0
Water
Molly
Minnow
Figure 1. Mean ± SE number of encysted metacercariae
in minnows exposed to: (1) cercariae immersed in
dechlorinated water control; (2) cercariae immersed in
skin extract of non-ostariophysan mollies; and (3) cercariae immersed in skin extract of the ostariophysan
fathead minnow. Identical letters above bars indicate no
significant difference (Tukey test, P < 0.05).
thickness were averaged for each section to provide
an estimate of an individual’s investment in club
cells. The observer was blind to all treatments.
STATISTICAL
ANALYSIS
Data were tested for normality prior to analyses
using Kilmogorov–Smirnov tests. Differences among
host lengths for all experiments were compared using
one-way ANOVAs. The effect of cue type on metacercariae intensity in the first experiment was analysed
using an analysis of covariance (ANCOVA) with
minnow length as a covariate. For the second experiment, alarm cell density and epidermal thickness
were compared with one-way ANCOVAs with host
length as the covariate. Where appropriate, post-hoc
comparisons of means between two samples were
performed using Tukey tests.
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Epidermal thickness (mm)
Figure 2. Number of epidermal club cells per millimeter
in the nape region as a function of epidermal thickness for
fathead minnows exposed to: (1) 0 cercariae; (2) 30 cercariae; and (3) 150 cercariae. Con, control.
of the skin extract treatments, although there was no
difference between the two skin extract groups (Tukey
test, P < 0.05).
The average total length of minnows in the second
experiment did not differ among groups (one-way
ANOVA: F1,57 = 0.39, P = 0.68) and host length was
not significantly correlated with club cell density
(F1,56 = 2.35, P = 0.13) or epidermal thickness
(F1,56 = 3.39, P = 0.07) and was dropped from the
analyses. All fish exposed to cercariae were infected
with metacercariae after the 4-week development
period (Fig. 2). Cercarial dose did not significantly
affect mean club cell density (one-way ANCOVA:
F2,56 = 0.37, P = 0.691) or epidermal thickness
(F2,56 = 0.19, P = 0.828). There was no effect of cue
treatment on club cell density when epidermal
thickness was used as a covariate (ANCOVA cue
type: F2,54 = 1.36, P = 0.266; epidermal thickness:
F1,54 = 52.98, P < 0.001, cue type ¥ epidermal thickness: F2,54 = 1.18, P = 0.316; Fig. 2).
DISCUSSION
RESULTS
Eighty-nine percent of 105 minnows in the first
experiment survived to 4 weeks post-infection, with
29–32 minnows represented for each treatment. All
surviving minnows were infected with encysted metacercariae. Mean host length did not differ among the
three treatment groups (one-way ANOVA: F2, 88 = 0.61;
P = 0.55). The mean number of metacercariae
was significantly affected by cue type (ANOVA:
F2,78 = 147.67, P < 0.001, Fig. 1). Water controls had
significantly more encysted metacercariae than either
The results from both experiments were inconsistent
with the predictions of the anti-parasite hypothesis.
Although immersion of cercariae in water containing
skin components of fathead minnows led to markedly
reduced infectivity, a similar reduction occurred for
cercariae immersed in skin components of nonostariophysan mollies. Thus, a component of fish skin
extract reduces cercarial infectivity, although that
component is likely not associated with the contents
of club cells. Furthermore, fathead minnows did not
alter their investment in club cells after repeated
exposure to Ornithodiplostomum sp. cercariae over
© 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 98, 884–890
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C. T. JAMES ET AL.
6 days. These results indicate that club cells do
not provide specific protection against these skinpenetrating parasites.
Diplostomules of Ornithodiplostomum sp. traverse
the epidermis to reach their ultimate site of encystment. Thus, they must come into contact with various
components of the epidermis in addition to club cells.
Populations of lymphocytes, macrophages, and granulocytes have been identified in the fish epidermis
(Whitear, 1986). These secretions, alone and in combination, could play a direct role in reducing the
infectivity of Ornithodiplostomum sp. diplostomules.
Mucus or goblet cells are also known to secrete
a variety of anti-parasite compounds, such as
lysozymes, lectins, and proteolytic enzymes (Buchmann & Brescianni, 1998), which have been shown to
protect fish from parasitic fungi, bacteria, and monogenean trematodes. Fathead minnow skin contains
large numbers of mucus cells (Wisenden & Smith,
1997), although their functional role as a defense
against parasites has not been evaluated. Because all
fish possess goblet cells within their epidermis (Shephard, 1994), components in fish mucus most likely
serve nonspecific anti-parasitic functions that may
interfere with cercariae during attachment to the host
or penetration through the epidermis (Haas, 1994).
An alternative explanation for the observed reduction in cercarial infectivity is that a component of skin
extract interferes with the normal host-finding, hostcontacting, and/or migration behaviors of cercariae
and diplostomules. In vivo and in vitro experimental
studies involving a closely-related trematode, Diplostomum spathaceum, indicate that the process of cercariae penetration and diplostomule migration within
fish intermediate hosts is highly complex (Haas et al.,
2007). For example, migration of individual diplostomules from the point of penetration into subdermal tissues, and ultimately into peripheral veins,
requires navigation along concentration gradients of
chloride ions, glucose, and glucosamine. We expect
that similar complex migratory mechanisms occur
for Ornithodiplostomum sp. diplostomules as they
traverse the epidermis. Thus, the skin extracts used
in the present study may have disoriented cercariae
and/or diplostomules by interfering with the detection
of navigation cues that would be present in the intact
epidermis.
The results obtained in Experiment 2 contrast
those previously reported by Chivers et al. (2007),
involving cercariae of another species of trematode.
In their study, the density of epidermal club cells in
fathead minnows repeatedly exposed to cercariae of
the turtle trematode Teleorchis was significantly
higher than those exposed to water controls. Several
factors are known to affect the rate of production of
club cells, which, alone or in combination, could
explain these contrasting results. Minor differences
in the time course of the experiment, in host age
(Carreau-Green et al., 2008), and in host condition
(Wisenden & Smith, 1997) could have contributed to
the lack of a skin response in the present study.
James et al. (2008) showed that fathead minnows
infected with Ornithodiplostomum sp. lost weight
relative to controls, suggesting a metabolic cost as the
cercariae develop and metamorphose into fullyencysted metacercariae. Loss of body condition as
a result of developing metacercariae also occurred
in European minnows that were experimentally
exposed to cercariae of the brain-encysting trematode,
Diplostomum phoxini (Ballabeni & Ward, 1993). A
developmental cost of this type is unlikely for
Teleorchis-exposed minnows because the metacercariae of these reptile trematodes are unlikely to
develop beyond the penetration phase.
One intriguing possibility for the contrasting
results obtained in the two studies is that club cell
proliferation, and perhaps other forms of epidermal
immunity, may be most detectable in hosts exposed to
novel pathogens and parasites. Supportive evidence
for this hypothesis would include the identification
of mechanisms used by specialist parasites such as
Ornithodiplostomum sp. that migrate through the
epidermis without direct damage to club cells. Migrating schistostomules of Schistosoma spp. navigate
between cells in the epidermis of experimentally
exposed mice on their way to blood vessels located
within the dermis (McKerrow & Slater, 2002). By
contrast, the schistosomules of Trichobilharzia, a
common blood fluke of water birds, cause localized
cellular and tissue damage in the epidermis of
nonhost mammals that provokes a generalized host
immune response leading to cercarial dermatitis, or
‘swimmers itch’ (Bahgat & Ruppel, 2002). One extension of the novelty hypothesis is that strong and
consistent alarm cell responses may only exist for
those parasites or pathogens that remain for
extended periods in the epidermis of fish, such as
some pathogenic water molds (e.g. Saprolegnia) or
those metacercariae that cause ‘black spot’ in the
epidermis of their intermediate hosts (Chivers et al.,
2007). The data obtained in the present study add to
the hypothesis that epidermal club cells of the ostariophysi are part of a complex, generalized response
system that confronts pathogens and parasites such
as cercariae (especially dead ones in the case or
Teleorchis; Chivers et al., 2007), fungal hyphae
(Saprolegnia; Chivers et al., 2007), or bacteria in the
case of secondary bacterial infection after epidermal
damage from mechanical abrasion or exposure to
ultraviolet light. Thus, club cells in both ostariophysian and perciformes (perch and darters) fishes likely
serve this function (Chivers et al., 2007). The data
© 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 98, 884–890
ANTI-PARASITE FUNCTION OF CLUB CELLS
obtained in the present study signal the need for
additional studies on the suite of epidermal barriers
and defences against parasite attack.
ACKNOWLEDGEMENTS
Funding for this research was provided by an operating grant to C.P.G. from the Natural Sciences and
Engineering Research Council of Canada and faculty
research grants to B.D.W. from the Minnesota State
University Moorhead College of Social and Natural
Sciences.
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