SWAP (Southwestern Assoc Parasitol) presentations from Janovy's lab in 2009
Following a great trip to the Oklahoma City Zoo and the Oklahoma City Memorial, seven students from our lab presented their undergraduate research at the Southwestern Association of Parasitologists meeting, April 16-18, 2009, at the University of Oklahoma Biological Station, Lake Texoma. Mohammed Vhora won the undergraduate student paper competition and will attend the American Society of Parasitologists meeting in August, at the University of Tennessee, where he will also present, before going on to graduate school at Oklahoma State University. Kristin Brotan’s paper was part of her Degree with Highest Distinction thesis. Mohammed’s and Zach’s projects were mentored by Gabe Langford, and Mark’s was inspired by questions arising from Alaine Knipes’ doctoral dissertation. Joey Smith got back in Lincoln on April 14, from three months in Argentina on a research adventure then left the next day for SWAP. Shay Hampton didn’t make the trip this year, but presented her poster at the UCARE undergraduate research conference on the UNL campus. Congrats to Mohammed and Kristin, and thanks to all the hard-working scholar undergrads from our lab!!
JJ
Student paper abstracts:
Molecular phylogeny of North American Rhabdias spp.:
ecological and physiological factors in lungworm host specificity.
G.J. Langford and J. Janovy, Jr., School of Biological Sciences, University of Nebraska-Lincoln.
Members of the nematode genus Rhabdias are among the most commonly encountered helminths of amphibians and reptiles around the world. In recent years lungworms have increased in popularity as a model system for the study of host-parasite interactions. Unfortunately, lungworms can be difficult to identify due to a lack of unique morphological characters in many species and the unknown potential for species complexes. Mis-identification can lead to problems when describing the life cycle and host specificity of morphologically similar species. The goal of this study was to elucidate the relationships of several species of lungworms using a molecular phylogeny and discuss implications for lungworm host specificity and lifecycles. PCR was used to amplify approximately2,100 base pairs of rDNA that included the 39 end of 18S gene, the ITS region (ITS1, 5.8S, ITS2), and the 59 end of the 28S gene. Maximum parsimony and maximum likelihood trees were constructed using PAUP, and a Bayesian inference tree was constructed with Mr. Bayes. Host specificity data was collected using both experimental infections and samples from anurans and snakes collected in nature. Our results suggest that host specificity varies widely within lungworms and is constrained more so by ecological than physiological factors. The phylogeny suggests anuran lungworms form a tight clade that is distantly related to snake lungworms. Sequences from related genera (e.g. Entomelas) are needed before the relationship between snake and anuran lungworms can be resolved.
Co-occurrence of Haematoloechus complexus (Trematoda: Haematoloechidae) and Rhabdias joaquinensis (Nematoda: Rhabdiasidae) in the plains leopard frog (Rana blairi). Mohammed S. Vhora, Gabriel Langford, and John Janovy, Jr. School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska.
Haematoloechus spp. and Rhabdias spp. are common parasites that infect the lungs of anurans. These parasites are often reported from the same host species, however no published studies have addressed the co-occurrence of these parasites in single hosts. In this study we attempted to determine if Haematoloechus compexlus and Rhabdias joaquinensis co-occurred in the plains leopard frog (Rana blairi) sampled from eastern Nebraska. In October 2008, we sampled 42 plains leopard frogs using minnow traps and hand capture (at night) from Elk Creek, Lancaster County, Nebraska. The trachea and lungs were removed and examined for Haematoloechus spp. and Rhabdias spp. using standard dissecting techniques, worms were designated whether they were found in the left or right lung. The parasites collected were preserved and identified as H. complexus and R. joaquinensis. We found a high prevalence and mean intensity of both H. complexus and R. joaquinensis, and our results suggest these lung parasites co-occur in this frog population. Given that worms co-occur it seems unlikely competitive exclusion is acting on this system, however more subtle types of competition cannot be ruled out.
Patterns of Intraspecific Morphological Variation Among
Localized Populations of Dactylogyrus
simplex and D. bychowski
(Platyhelminthes, Monogenoidea)
in
Mark Bi and John Janovy, Jr., School of Biological Sciences,
Dactylogyrus simplex and D. bychowski occur on the gills of fathead minnows (Pimephales promelas).
Fathead minnows in three converging Nebraska streams, Elk Creek, Oak Creek, and West Oak Creek, are believed to constitute distinct populations. In this study, we searched for patterns of intraspecific morphometric variation among D. simplex and D. bychowski from the three fathead populations. Fifteen distinct point-to-point measurements of sclerotized parts were recorded in a total of 85 D. simplex collected between the months of August and October and 102 D. simplex and 51 D. bychowski collected between April and May. Variation among sites was analyzed using one-way ANOVA. For D. simplex, analyses were stratified by season in order to control for effects of temperature. In the fall D. simplex sample, 9 measurements, 4 of the hamuli and 5 of the marginal hooks, differed significantly among populations. In the spring D. simplex sample, 8 measurements, 6 of the hamuli and 2 of the marginal hooks, differed significantly among populations. Four measurements showed significant variation across both seasons and, of those, 2 displayed a consistent pattern of variation among locations. Among the D. bychowski populations, 3 hamulus measurements and 4 hook measurements differed significantly. Patterns of morphological variation observed in this study suggest Dactylogyrus populations in this watershed are isolated, as are the host populations, and are structurally divergent.
Osmoregulation in gregarines: the effects of varying
incubation medium tonicity on cell size and shape in Gregarina niphandrodes (Apicomplexa)
from adult Tenebrio molitor (Tenebrionidae, Coleoptera). Jessica H. Ebers and J. Janovy, Jr., University of Nebraska-Lincoln,
Lincoln, Nebraska.
This study was designed to determine the extent to which a protistan parasite, Gregarina niphandrodes, from adult mealworms (Tenebrio molitor, Coleoptera, Tenebrionidae) is capable of osmoregulation. The effects of varying incubation medium tonicities on cell size and shape were measured during exposures of up to 4 hr. Parasite cell viability was assessed by use of trypan blue dye exclusion tests. Single gregarines were placed in depression slides, each containing an isotonic, hypertonic or hypotonic insect saline or sucrose solution, and widths and lengths of protomerites and deutomerites were taken at regular time intervals. Results showed that Gregarina niphandrodes are resistant to environmental changes in tonicity, when incubated in insect salines of varying concentrations, but were unable to osmoregulate in sucrose solutions. Dye exclusion tests revealed that gregarines were able to survive for a minimum of four hours in the given solutions. These findings suggest that, unlike other common parasites thought to be unable to osmoregulate, gregarines may in fact have the ability to do so. In addition, the studies demonstrate that gregarine membrane function can be studied using diverse molecule types to vary incubation medium tonicity.
Preliminary
attempts to maintain gregarines in-vitro.
J. Smith and J. Janovy, Jr., School of Biological Sciences, University of Nebraska-Lincoln
The present study describes attempts to maintain gregarines using various in vitro culture techniques. Culture media consisted of either Tenebrio muscle saline or TNM-FH insect medium (Sigma), with or without various additives such as glucose, amylopectin, Tribolium destructor larval gut tissue and streptomycin or methicillin. Condition of gregarines was assessed by measuring cytoplasmic density and homogeneity as well as length and width of protomerites and deutomerites after 24 or 48 hours. Results indicate that gregarines display significant variation of the measured characteristics when maintained in different media. Gregarines maintained in culture consisting of insect medium displayed greater cytoplasmic density and homogeneity than gregarines obtained directly from T. destructor gut as well as other cultures used. The results suggest that in vitro culture methods for gregarines are suitable avenues for exploring cell functions that may contribute to obligate parasitism.
Physiological response of Gregarina confusa
to a varied host diet.
Kristin K. Brotan, Chelsea P. Bloom, and John Janovy, Jr. School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, NE 68588-0118
Previous research in this lab has shown that the diet of larval Tribolium destructor has a major effect on cytoplasmic contents of its parasites, Gregarina confusa, particularly in terms of paraglycogen storage and cell volume. In this research, the effects of single dietary components, including amino acids, proteins, monosaccharides, disaccharides, and polysaccharides, as well as B vitamins, multivitamins, and combinations of amino acids and carbohydrates, were studied using starvation and re-feeding techniques described by Schreurs and Janovy (2008, J. Parasitol., 94:567-570). Beetle larvae were isolated from culture and starved for 5-7 days, then divided into experimental and control groups. Experimental groups received the test diet. Twenty-four hours after being re-fed, experimental and control groups were dissected, and data were collected on the length, width, and cytoplasmic density of each of the gregarines using digital photography followed by image analysis using ScionImageTM software. Results indicate that the carbohydrate polymer amylopectin, when included in a host’s diet, allows the parasite to quickly accumulate its stored cytoplasmic polysaccharide. None of the other dietary components allowed this polysaccharide accumulation when provided either singly or in combination. These studies now allow detailed analysis of the processes by which gregarines obtain the nutrients necessary to complete their life cycles.
Manipulation of host temperature and diet: experiments on three species of flour beetles and their gregarine parasites. Z. Baumfalk, G. J. Langford, and J. Janovy, Jr., School of Biological Sciences, University of Nebraska-Lincoln.
The purpose of this experiment was to clear flour beetles of their gregarine parasites through host starvation and temperature manipulation. Three flour beetle larvae were examined in this study: Tenebrio molitar, Tribolium destructor and Tribolium freemani. To clear gregarine infections beetles were placed in 37°C incubators with no food for 48 hours. Following exposure we found that Tenebrio molitar were virtually cleared of their gregarine parasites. Tribolium destructor and Tribolium freemani also displayed a similar (yet incomplete) reduction in prevalence and mean intensity, indicating varying levels of resilience to heat and starvation in gregarine parasites. A wide array of ecological questions arose during these experiments. Specifically, in Tenebrio molitar we wondered (1) if the host could be re-infected, (2) if temperature played a role in re-infection, and (3) if re-infection was not observed, then how was the host-parasite relationship altered to prevent reestablishment of parasites. To determine if hosts could be re-infected and if host temperature played a role in re-infection, T. molitar larvae were simultaneously re-fed sterilized food and placed into 4 treatments: 27° C + frass, 27° C + no frass, 33° C + frass, or 33° C + no frass. Only larvae exposed to the 27° C + frass treatment reestablished infections. We noted that re-infected larvae exhibited significantly greater mean intensities than time-0 and time-t controls. Further experiments are planned to explore the increase in mean intensity following re-exposure, and determine what stage of the gregarine lifecycle is arrested during host re-exposure to frass in a high heat environment.
SWAP (Southwestern Assoc Parasitol) presentations from Janovy's lab in 2004
The southwest regional parasitology meetings were held at the University of Oklahoma Biological Station (UOBS), Lake Texoma, OK, April 22-24, 2004. There were 17 papers and three posters presented, for an excellent meeting. Three regular papers were presented from this lab (abstracts below), Matt presented a poster, and Thursday evening Jaclyn Helt gave a special presentation based on a seminar she'd given earlier at UNL.
Samana Schwank
CYST REGULATION AND FORMATION IN GREGARINE PARASITES (APICOMPLEXA: EUGREGARINORIDA). Samana Schwank, School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska 68588.
The reproductive strategies of apicomplexans have never been fully explored and gregarines (Apicomplexa: Eugregarinorida) provide an excellent model to study the reproductive mechanisms in parasites. Gregarines are intestinal parasites of insects. In this study beetles (Tenebrio molitor) were infected with different numbers of parasites (Gregarina niphandrodes) and the gametocysts were collected daily and counted from the frass. Some beetles carried a "high" parasite intensity (oocysts from 10-20 gametocysts used to infect each beetle at the start of the experiment) and others carried a "low" intensity (oocysts from 2-5 gametocysts/beetle used in infections); other beetles remained as controls. The frass was inspected over a twenty-eight day period, or until such time as shedding ceased. Gametocysts were counted, measured, and oocysts produced by them at dehiscence were counted. In particular, I wished to see if there was any correlation between the numbers of parasites carried by the host and the number of gametocysts shed. I was also looking to see if parasite intensity affected the timing of gametocyst shedding. In heavily infected beetles, there was a two-phased shedding of gametocysts, indicating some inhibition of development in heavy infections.
Jill Detwiler
THE GREGARINE-TENEBRIONID MODEL SYSTEM: DO EXPERIMENTAL INFECTIONS ALTER OUR VIEW OF PARASITE EVOLUTION? J. DETWILER, School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, NE 68588. Host specificity is a fundamental life history trait of parasites that describes the number of host species a parasite can exploit. Although a focus of many parasitological investigations, the mechanisms responsible for host specificity remain unclear. Phylogenetic, ecological, and physiological avenues and constraints could be explanations for a parasite species' ability to colonize the range of hosts it occupies. In this study, we are attempting to determine whether phylogenetic constraints play a role in the observed host specificity in the gregarine/beetle model system. Historically, the gregarine/beetle model system is characterized by stenoxenous associations between host and parasite, but that assumption may not be valid across taxa, and has not been studied experimentally across a wide range of hosts. A series of experimental cross infections among six tenebrionid species and five species of gregarines were performed to establish the extent of host stadium specificity. The results demonstrate that gregarines from five Tribolium species and Palorus subdepressus can infect both the larva and adult stages of the host. This result is in contrast to previous studies with Tenebrio molitor where gregarine species were host stadium specific and could not successfully colonize into the other host stage. In our study, infection patterns of host specificity were mapped onto the host phylogeny resulting in no clear pattern, which indicates that host specificity is not driven by phylogenetic constraints in this host-parasite system. In future studies, alternative mechanisms for host specificity should be evaluated to determine the factors dictating which parasite species occupy which host species in this system. By experimentally determining host specificity, we can better understand the evolutionary avenues and constraints to evolutionary processes such as species diversity and ultimately, parasite evolution.
Matt Bolek
THE LITTLE FROG GETS ITS WORMS FIRST: LIFE CYCLE VARIATION AND COLONIZATION OF YOUNG OF THE YEAR LEOPARD FROGS BY TWO TREMATODES. Matthew G. Bolek, Scott D. Snyder, and John Janovy Jr. University of Nebraska-Lincoln, School of Biological Sciences, Lincoln, NE, 68588, and University of Nebraska at Omaha, Department of Biology, Omaha, NE 68182. Studies on lung flukes (Haematoloechidae) indicate that frogs become infected by ingesting dragonfly intermediate hosts, while studies on bladder flukes (Gorgoderidae) indicate that frogs become infected by ingesting tadpole or frog intermediate hosts. Young of the year (YOY) frogs are rarely infected with these parasites because they are gape limited predators that are unable to feed on large intermediate hosts such as dragonflies and other frogs. We examined the role of diet and intermediate hosts in the recruitment of lung flukes and bladder flukes to YOY frogs from Nebraska. Our results indicate that in Nebraska where there are few frog species that can support these parasites, parasites alter their life cycles by being generalists at the intermediate host level or skip the intermediate host by infecting tadpoles directly. More importantly, both parasites are accomplishing the same thing, infecting very young frogs. Our data on distantly related trematodes suggests that a parasite's life cycle may vary depending on regional environment and available definitive hosts. Our study suggests that a particular life cycle variant will be favored by regional environmental conditions and host availability. These local factors influence parasite transmission probabilities at one or more stages during their complex life cycle, allowing us to generate hypotheses about the mechanisms that drive evolution of parasite life cycles.
and
FROM A FROG'S PERSPECTIVE, IS A DRAGONFLY A DRAGONFLY? EVOLUTIONARY AVENUES FOR AN CONSTRAINTS ON THE TRANSMISSION OF FROG LUNG FLUKES (HAEMATOLOECHUS SPP.). Matthew G. Bolek, Scott D. Snyder, and John Janovy Jr. University of Nebraska-Lincoln, School of Biological Sciences, Lincoln, NE, 68588, and University of Nebraska at Omaha, Department of Biology, Omaha, NE 68182. Leopard frogs in Nebraska, Rana pipiens and R. blairi, are commonly infected with Haematoloechus complexus but exhibit ecological host specificity and are rarely infected with H. varioplexus and H. longiplexus, which primarily infect bullfrogs, R. catesbeiana. This ecological host specificity has been largely assumed to be due to differences in frog ecology. However, recent studies indicate that Haematoloechus species vary in their second intermediate host specificity. It was hypothesized that these differences in Haematoloehus intermediate host specificity may play a significant role in lung fluke transmission to frogs. We have completed the life cycles of H. complexus, H. varioplexus, and H. longiplexus in the laboratory by raising and infecting 1st intermediate snail hosts, 2nd intermediate arthropod hosts and frog definitive hosts. These data show that these congeners differ in their 1st and 2nd intermediate host specificity as well as their frog definitive host specificity. Our data indicates that 2nd intermediate host specificity and frog feeding behavior can affect host specificity in the frog definitive hosts. Additionally, we have constructed a molecular phylogeny by sequencing the internal transcribed spacer region of nuclear rDNA of 11 North American and European Haematoloechus species from 18 different populations. Life history data of the 11 North American and European Haematoloechus species mapped on to this phylogeny suggest evolutionary avenues for and constraints on transmission that differ at each stage in the life cycle. Our data indicates that life cycle evolution in trematodes is complex and differs in closely related species at different stages in their life cycle.
SWAP (Southwestern Assoc Parasitol) presentations from Janovy's lab in 2005
The southwest regional parasitology meetings were held at the University of Oklahoma Biological Station (UOBS), Lake Texoma, OK, April 21-23, 2005. There were 28 papers and posters presented, for an excellent meeting. Fourregular papers were presented from this lab (abstracts below).
Alaine Knipes
POPULATION STRUCTURE AND RECRUITMENT OF MONOGENEA AND TREMATODA IN THREE CYPRINID FISH OF THE SALT VALLEY WATERSHED, LANCASTER COUNTY, NEBRASKA. A. Knipes, School of Biological Sciences, University of Nebraska Lincoln
A study of the occurrence and distribution of parasites in hosts in a system allows inference of mechanisms that determine such distributions. A comparison of 11 parasite species on three host species in three distinct sites provided insight into the ways in which fish hosts recruit their parasistes in nature. Fathead minnows (Pimephales promelas), sand shiners (Notropis ludibundis), and creek chubs (Semotilus atromaculatus) were collected between July and November 2004, from three converging tributaries; Elk Creek, West Oak Creek and Oak Creek, Lancaster County, Nebraska. Fish were examined for Uvulifer ambloplitis and monogenean gill parasites (genus Dactylogyrus). Consistent with previous studies, mean abundance and prevalence of U. ambloplitis indicated that fish are not moving between the sites, and thus the sites contain distinct host populations. Mean abundances, prevalences, and parasite population distribution parameters for Dactylogyrus species also differed between sites. Thus both biotic and abiotic factors of the host and parasite environments are considered to be among the mechanisms controlling recruitment of parasites by their fish hosts. The results also suggest that stable differences in host-parasite encounter dynamics may occur at relatively small geographic scales.
Erica Peterson
CARBOHYDRATE ACCUMULATION DURING GROWTH AND PAIRING IN TWO GREGARINA MINUTA-LIKE PARASITES OF FLOUR BEETLES (TRIBOLIUM SPP.). E. Peterson, School of Biological Sciences, University of Nebraska Lincoln.
Some gregarine species (Apicomplexa: Conoidasida: Eugregarinorida) are good model systems for examining interactions between parasites and their environments due to ease of manipulation and access to diverse host-parasite combinations. The hypothesis to be tested in these studies was that adult and larval hosts provide distinct environments for parasites, and that the effects of these environments can be seen in parasite physiology. Parasite carbohydrate (CHO) accumulation and growth were measured in two different life cycle stages of two naturally-infected beetle species. There were differences between the two cell compartments in CHO accumulation and differences between gamonts in accumulation in the same (corresponding) compartment. The data highlight the complexity of single-celled parasitic organisms by demonstrating their differing physiological behavior as singlets and mated pairs. Variations within simple unicellular parasites due to differing life stages of the host indicate that environments consisting of various nutrients and hormones impact parasitic development, a concept which can be applied to more complex host-parasite systems.
Kati Brazeal and Mackenzie Waltke
THE RELATIONSHIP BETWEEN CLIFF SWALLOW (HIRUNDO PYRRHONOTA) DEMOGRAPHIC CLASSES AND FEATHER MITE (TROUESSARTIA SP.) ABUNDANCE. Kathleen R. Brazeal and Mackenzie Waltke, School of Biological Sciences, University of Nebraska Lincoln.
This study investigated the association between host factors and parasite abundance in feather mites (Trouessartia sp.) living on the barbs and shafts of primary wing feathers of cliff swallows (Hirundo pyrrhonota). These mites were used for this study because their relationship with their host has not clearly been defined although casual observations suggest a tightly regulated system in which parasites are strictly confined to a particular region of the primaries. Birds were mist netted at 2 sites near Keystone, NE. The relative mite abundances were estimated for each bird in the sample and analyses looked for correlations with host sex, age, and weight. Relationships were found between mite abundance and age and weight of the birds, but not with sex. Therefore mites do not infect one sex more frequently than the other, but older and heavier birds tend to be more heavily infected. This work has shown an ecological relationship between feather mites and cliff swallows that needs further examination.
Mattew Bolek
BABY FROGS AND HUNGRY MAGGOTS: WHY IS MYIASIS SO RARE IN NORTH AMERICAN AMPHIBIANS? Matthew G. Bolek and John Janovy Jr. School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska 68588.
Myiasis involves the invasion of living animals by larvae of dipterous flies, and primarily occurs in mammals. Larvae of certain species of blow flies (Calliphoridae) can cause myiasis in frogs and toads but there are few reports from North American amphibians. Of these, most are from toads (bufonids). In the present study, we observed primary myiasis in a population of juvenile wood frogs from southestern Wisconsin and compared our observations to previous studies on myiasis from toads. We found that: 1) flies lay eggs on healthy wood frogs; 2) eggs hatch with first instar maggots penetrating the skin; 3) maggots develop to mature third instars within 13-16 hr of egg hatching; 4) maggots kill the host within 7-47 hr of egg hatching; and 5) maggots consume the entire frog carcass reducing it to bones within 42-59 hr of egg hatching. Our observations on the time of death and how quickly carcasses of wood frogs become consumed by these maggots compared to previous studies on toads suggest that finding infected juvenile wood frogs may be uncommon. Therefore, myiasis may be much more common than is currently observed.
Mattew Bolek et al.
HOW LITTLE FROGS AND BIG FROGS GET THEIR WORMS: LIFE CYCLE PLASTICITY IN FROG BLADDER FLUKES. Matthew G. Bolek, Scott D. Snyder, and J. Janovy, Jr., School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska 68588 and Department of Biology, University of Nebraska Omaha, Omaha, Nebraska.
The bladder fluke Gorgoderina attenuata has large geographical variation and infects 13 different amphibian and reptile species. Life cycle studies on the East Coast indicate that frogs become infected with this bladder fluke when they ingest tadpole or frog second intermediate hosts containing metacercariae. Young of the year (YOY) frogs are rarely infected with these parasites because they are gape-limited predators that cannot feed on large hosts such as tadpoles or other frogs. We surveyed 3 species of frogs of different age groups for the presence of bladder flukes from Nebraska. YOY frogs of 2 species are commonly infected with these parasites, whereas only adults of a third species are commonly infected with this bladder fluke, suggesting different routes of infections in these frog species. Using molecular techniques and laboratory infections we show that this bladder fluke has 3 different strategies of infecting frogs in Nebraska. First, when tadpoles ingest cercariae, they skip the metacercaria stage and reach maturity in these hosts. Second when cercariae are ingested by aquatic dragonflies and damselflies they become metacercariae; when ingested by frogs the worms mature in the bladder. Finally, when adult frogs feed on tadpoles infected with larval worms they also become infected. Our study suggests that a particular life cycle variant will be favored by regional environmental conditions where factors influence probabilities of transmission at 1 or more stages during the complex life cycle, which allows us to generate hypotheses about the mechanisms that drive evolution of parasite life cycles.
