Download RH: GRIFFIN ET AL. – EYEFLUKE INFECTION IN MOSQUITOFISH

RH: GRIFFIN ET AL. – EYEFLUKE INFECTION IN MOSQUITOFISH
MICROHABITAT SELECTION AND EYEFLUKE INFECTION LEVELS IN THE
WESTERN MOSQUITOFISH (GAMBUSIA AFFINIS)
Shane L. Griffin, Nichole Carpenter, Autumn Smith-Herron*, and Kristin K. Herrmann
Department of Biological Sciences, Tarleton State University, Box T-0100, Stephenville, Texas
76402. Correspondence should be sent to Shane L. Griffin at: [email protected]
ABSTRACT: A variety of trematode species infect the eyes of fish as second intermediate hosts.
In most cases the definitive host is a piscivorous bird. Studies of a few species have shown an
increase in transmission due to decreased visual acuity of the fish host. However, this may vary
depending on trematode microhabitat choice within the eye. Some trematode species are found in
the lens, some in the vitreous humor and others have been reported from the retina. Here we
report 3 genera of eyeflukes in 3 locations of the eye in the intermediate fish host, Gambusia
affinis. Clinostomum metacercariae were found attached to the outer sclera within the eye orbit,
and Diplostomum metacercariae were found in the lens. Posthodiplostomum metacercariae were
confirmed by histology to reside between the choroid and pigmented retina. Posthodiplostomum
metacercariae were found in both eyes of all 20 fish examined and in high intensities (up to 27
metacercariae per eye). High trematode intensities between the choroid and pigmented retina
found in this study may disrupt vision in this fish host. Our study is the first to document the
microhabitat of all 3 trematode metacercariae within the eye of G. affinis.
Eyeflukes are parasitic trematodes that commonly parasitize freshwater fish species
(Chappell, 1995). Eyefluke infections can result in deleterious effects on the second intermediate
fish host’s fitness and overall survivability. Infections can result in lens opacity or dislocation,
cataracts, detached retina or choroid, capsular rupture, and exophthalmia, which can lead to
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reduced vision or blindness in some cases (Rushton, 1937; Shariff et al., 1980; Grobbelaar et al.,
2015). Impaired vision may result in emaciation (Shariff et al., 1980) from a decreased ability of
a host to forage, as well as hinder reproduction and predator avoidance (Seppälä et al., 2004,
2005; Barber, 2007).
The complex life cycles of eyeflukes involve multiple stages, which incorporate both
sexual and asexual reproduction. Multiple transmission events take place throughout the life
cycle typically involving 2 intermediate hosts and a definitive host. Transmission to the first
intermediate snail host occurs by ingestion of eggs or penetration by the free-swimming
miracidia. In the snail host, miracidia undergo asexual reproduction and produce cercariae,
which eventually penetrate the epithelium of the second intermediate host, commonly a fish. In
some cases, as here, cercariae migrate to the eye and develop into metacercariae. Transmission to
the definitive host occurs via ingestion by a bird or predatory fish, depending on the trematode
species (Shoop, 1988).
Preliminary parasite data collected from local Texas river systems in 2013 indicated high
intensity eyefluke infections in several local genera of fishes including the Western
mosquitofish, Gambusia affinis (Poeciliidae) (K. K. Herrmann, pers. obs.). This common fish
species is favorable as the second intermediate host of various trematode species (Davis and
Huffman, 1977), which are found as metacercariae throughout the organs, tissues, and eyes.
Some eyefluke studies document microhabitat locations within the eyes of many species of fish
(Ferguson, 1943). In his synopsis of strigeid metacercariae of fishes, Hoffman (1960) recounts
studies documenting multiple species of eyefluke infecting various locations within the eyes. He
notes specifically, Diplostomum flexicaudum, Diplostomum spathaceum, and Diplostomum
lenticola infections of the lens, with D. spathaceum, Diplostomum scheuringi, and Tylodelphis
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clavata infecting the vitreous humor of various species of fish. Hoffman (1999) notes studies
reporting Diplostomum huronense infections of the lens and vitreous humor, as well as
Diplostomum pungiti infections of the retina and retinal area. Posthodiplostomum brevicaudatum
infection of the retina and choroid coat has also been reported (Wisniewski, 1958). However,
numerous studies on eyeflukes do not report a specific microhabitat.
The objective of this study was to investigate eyefluke infections within a single
population of G. affinis. In doing so, we aim to document infection intensities and
morphologically identify the genera of the metacercariae constituting these infections.
Information regarding eyefluke microhabitat commonly lacks specificity in many studies of
various host species including G. affinis. Therefore, we further examined the location of each
taxa to increase specificity in assessing potential microhabitat selection.
MATERIALS AND METHODS
Twenty G. affinis individuals were collected via seine net from the Paluxy River
(32.230460°N, -97.776282°W) in Glen Rose, Texas in June 2014. Individuals were identified to
species level (Thomas et al., 2007). Live fish were transported to the lab in an aerated container.
Individuals were kept in an aerated aquarium and fed ad lib. Upon processing, individuals were
euthanized in a Tricaine-Mesylate (MS-222) solution. Length, weight, and sex were recorded for
each individual prior to eye dissection. Eyes were carefully removed by severing all tissue and
the optic nerve to prevent damage. Left and right eyes were differentiated to determine potential
eye preference. Eyes were dissected in 0.075% saline solution. The eye layers visible using a
dissection microscope were completely separated, and the number of metacercariae and their
relative locations were recorded (Fig. 1). Eyeflukes were fixed and stained for identification.
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Specimens were deposited in the Sam Houston State University Parasitology Collection
(SHSUP001580- SHSUP001586). Statistical analyses were conducted using SPSS (v21).
An additional 7 G. affinis eyes were fixed in HistoTainer 10% neutral buffered formalin
(VWR) for histological analysis to confirm metacercariae location (Fig. 2). Of these, 4 eyes were
from individuals collected from the original study site on the Paluxy River that harbored high
infection intensities. The other 3 eyes were from individuals collected from the Bosque River
(31.976781°N, -98.031078°W) in Hico, Texas to serve as a control. According to preliminary
data from 2013, this site harbored 0% eyefluke prevalence in 48 fishes including G. affinis,
Cyprinella venusta, and Cyprinella lutrensis. Histology was conducted according to standard
histology procedures (Humason, 1962). Eyes were embedded in paraffin wax and sliced into 7
µm sections using a microtome. Tissue sections were floated in water and placed on microscope
slides coated in a protein adhesive. Sections were then stained in Hematoxylin & Eosin and
examined using an Olympus BX53 microscope paired with an Olympus DP72 camera (Olympus
Corporation, Tokyo, Japan).
RESULTS
All 20 fish were mature adult females ranging from 35.75 to 52.56 mm in total length
with a mean of 41.77 mm. Mean weight was 0.91 g and ranged from 0.60 to 1.84 g. From the 40
G. affinis eyes dissected, a total of 466 metacercariae were recovered. Of these, 10 encysted
metacercariae were found attached to the outer surface of the sclera within the eye orbit, 446
encysted metacercariae were found between the choroid and pigmented retina (Figs. 1, 2), and 10
unencysted metacercariae were found in the lens. Sclera metacercariae were identified as
Clinostomum sp. (Caffara et al., 2011). Choroid metacercariae were identified as
Posthodiplostomum sp. (Hughes, 1928; Hunter and Hunter, 1940; Hoffman, 1999). Unencysted
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lens metacercariae were identified as Diplostomum sp. (Palmieri et al., 1976; McKeown and
Irwin, 1995).
Infection intensities of Clinostomum and Diplostomum metacercariae each ranged from 0
to 2 individuals per eye, whereas Posthodiplostomum metacercariae ranged from 3 to 27
individuals per eye (Table I). There was no significant difference between the left and right eyes
in abundance of total eye parasites (t = -1.89, p = 0.075), Clinostomum, Posthodiplostomum or
Diplostomum metacercariae (t = 0.00, p = 1.0; t = -1.90, p = 0.072; t = 0.62, p = 0.54;
respectively). Data from both eyes were pooled to test for relationships between total length of
fish and intensity. Infection intensity was not correlated with fish length for total eye parasites
combined (r = 0.366, p = 0.056), or for Clinostomum or Diplostomum metacercariae (r = 0.301,
p = 0.098; r = -0.069, p = 0.386; respectively). However, total fish length is correlated with
combined intensity of Posthodiplostomum metacercariae in both eyes (r = 0.408, p = 0.037).
DISCUSSION
We examined a single population of G. affinis individuals, and documented 3 genera of
metacercariae inhabiting 3 locations within the eye. Posthodiplostomum metacercariae were
reported between the choroid and pigmented retina of both eyes in all 20 fish. Wisniewski (1958)
also documented Posthodiplostomum metacercariae embedded in the choroid coat of the eye, as
well as cercarial migration to that location, however this was in unspecified fish host species.
Studies by Janovy and Hardin (1987, 1988), and Janovy et al. (1997) report Posthodiplostomum
metacercariae in the eye and body cavity, but do not go on to specify which microhabitat within
the eye. A study on fathead minnows, Pimephales promelas, also documents Posthodiplostomum
infections in the eyes, as well as in the head, liver, and body cavity, but again does not specify
the location within the eye (Mitchell et al., 1982). Hunter and Hunter (1940) reported
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Posthodiplostomum minimum infections occurring in all visceral organs of various fish species,
and Rakauskas and Blaževičius (2009) documented Posthodiplostomum infections occurring on
the skin, fins, and gills of roach. Brock and Font (2009) reported Posthodiplostomum
metacercariae in G. affinis, however the metacercariae were found in the body cavity only. Our
study is the first to specifically document high intensities of Posthodiplostomum metacercariae in
the choroid of G. affinis. Such high intensity, up to 27 metacercariae per eye, most likely disrupts
the vision of the fish and could lead to lowered fitness as shown in experiments with other fish
host species (Rushton, 1937; Shariff et al., 1980; Grobbelaar et al., 2015).
We found Clinostomum metacercariae attached to the outer sclera within the eye orbit of
8 individuals. In his classification of the genus, Hoffman (1999) documents the occurrence of
Clinostomum metacercariae in various fishes, with a range broad enough that he hypothesized
members of this genus could likely infect most species of fish. Other studies on Clinostomum
reported metacercariae in multiple fishes, being found encysted in the musculature (Cort, 1913;
Meade and Bedinger, 1972; Dias et al., 2003), mesentery and viscera (Dias et al., 2003), and
subcutaneously (Hopkins, 1933; Al-Awadi et al., 2010). Mitchell et al. (1982) also documented
ocular infections of Clinostomum metacercariae in P. promelas. Unfortunately, the study only
generalized metacercariae location as being in the eye and did not report a specific microhabitat.
Overall, we found a lack of information in the literature regarding studies of Clinostomum
metacercariae specifically being attached to the sclera or located in the eye orbit in G. affinis.
In our study, Diplostomum metacercariae were documented in a single lens of 7
individuals and in both lenses of 1 individual. Infections of Diplostomum metacercariae occur in
the aqueous and vitreous humor (Hoffman, 1960; Shariff et al., 1980; Chappell, 1995;
Grobbelaar et al., 2015), retina (Shariff et al., 1980; Bortz et al., 1988; Chappell, 1995), brain
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(Hoffman, 1960; Grobbelaar et al., 2015), and mesentery and coelom (Hoffman, 1960).
However, Davis and Huffman (1977) and Aho et al. (1982) both document Diplostomum
infections occurring in the coelom of G. affinis, but neither found ocular infections. Numerous
studies have documented the lens microhabitat of Diplostomum metacercariae in various fishes
(see Hoffman, 1960; Shariff et al., 1980; Bortz et al., 1988; Dwyer and Smith, 1989; Chappell,
1995), however we again encountered a lack of studies reporting Diplostomum lens infections in
G. affinis.
Certain trematodes such as Diplostomum spp. are unencysted and free-moving while
inhabiting the eye of a teleost host (Grobbelaar et al., 2015). Diplostomum metacercariae
documented in the lens during this study were all found to exhibit this. Several hypotheses exist
as to why this phenomenon occurs, and generally involve the lens microhabitat serving as a
protective barrier against environmental factors. One hypothesis suggests the lens may provide
protection while passing through the definitive host’s stomach (Szidat, 1969). Further,
trematodes that inhabit the lens tend to exhibit lower host specificity from the reduced immune
response that occurs within the lens (Locke et al., 2010) and may therefore be found in a variety
of fish host species.
We found a significant correlation between metacercarial infection intensities and fish
host size only for Posthodiplostomum metacercariae. This is likely because Posthodiplostomum
metacercariae were found in high intensities in all fish sampled, whereas the sample size for
Clinostomum and Diplostomum was only 10 individuals each. Correlations of parasite intensity
in fishes generally constitute positive relationships with size and therefore age. For example,
Janovy et al. (1997) reported significant positive correlations in terms of fish host size when
analyzing ectoparasitic monogenean and larval trematode abundances. Similarly, Poulin (2000)
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showed positive correlations in all of the parasite-host relationships tested, although these
relationships were presumed to vary in their significance based on a multitude of biological
factors and statistical groupings. Another potential explanation for the lack of significance with
Clinostomum and Diplostomum metacercariae in terms of fish size is that the G. affinis used in
the study only represent a portion of the size range of the population, which does not allow for
assessing correlations between host size and infection intensity in an entire population. All
individuals in this study were more than 35 mm in length. This size limit was chosen to increase
the probability of obtaining infections that would allow for description of eyefluke microhabitat.
Furthermore, this size limitation may have contributed to the sex bias of all females in
our study. This situation poses questions regarding the sex ratio of the population sampled and
the effects of infection on G. affinis males. The body size of male individuals varies depending
on maturation relative to the breeding season with males maturing and achieving larger size later
in the season (Hughes, 1985). Regardless, males generally tend to be smaller (18 to 30 mm) than
females (30 to 65 mm; Kuntz, 1914). Thus, the size limit utilized in this study could explain the
lack of male G. affinis individuals. Alternatively, the population sampled could be heavily
female-biased, which can be common in poeciliid populations (Magurran, 2011). Further
research would be necessary to determine the sex ratio in the sampled population, the effects of
eyefluke infection on male individuals, as well as any potential differences between the sexes.
Future studies may also expand on our results to reveal any spatial and temporal patterns of all
helminth species parasitizing G. affinis.
ACKNOWLEDGMENTS
We thank C. Pyle and T.E. Barnes for their assistance in collection of G. affinis. We also
thank the Office of Student Research and Creative Activities for funding this project and
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providing an assistantship for SLG. Fish collection was conducted under Scientific Research
Permit (SPR-0403-284) issued by Texas Parks & Wildlife. This project was conducted in
accordance with the protocol and procedures established by the Animal Care and Use Committee
at Tarleton State University (Approval #: 05-005-2014).
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Figure 1. Dissection of a Gambusia affinis eye infected with Posthodiplostomum metacercariae
showing (A) posterior and (B) lateral views.
Figure 2. Histological sections of Gambusia affinis: (A) an uninfected eye and (B) an infected
eye showing an encysted Posthodiplostomum metacercaria in the choroid (S = sclera, C =
choroid, PR = pigmented retina, M = metacercaria, CW = cyst wall).
*Texas Invasive Species Institute, Sam Houston State University, Box 2506, Huntsville, Texas
77340.
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