A survey of gregarines of the odonates of Otsego County, New York Crystal Wiles1 and Florian Reyda2

INTRODUCTION Gregarines are one-celled protozoan parasites, and are members of the phylum Apicomplexa, in the order Eugregarinorida (Schmidt et al. 2009). These single-celled creatures parasitize a wide variety of invertebrate such as oysters, spiders, and . The gregarines of and (Insecta: ), all of which belong to the gregarine family Actinocephalidae (Percival et al. 1995), are the focus of this study. Odonates become infected with gregarines by consuming water that contains oocysts, or by eating prey that have oocysts attached to their bodies (Vivier & Deportes 1990). The diversity of gregarines is unimaginably vast, as is the diversity of their invertebrate hosts (Hays et al. 2007, Janovy et al. 2007a). There are ~1 million different described species, and every year more are discovered (Brusca & Brusca 2003). Gregarines are host-specific, meaning each gregarine species typically only infects one kind of insect to complete its life cycle (Wenrich 1935). In addition, each insect species could host one or more gregarine species. Based on the high host specificity of gregarines, once could predict that there may be as many or more species of gregarines than species of insects. This is especially true for the gregarines of the Actinocephalidae family that parasitize odonates. Original work to describe the gregarine parasites in the odonates of North America was done in the early 1900s by Ellis and Crawely (e.g., Ellis 1914). In recent years, studies in the Midwest have described several species from odonates (e.g., Percival et al. 1995). In these studies, previously described gregarine species from Europe were also found in North American odonates, however new species of gregarine parasites were discovered as well (Clopton et al. 1993; Hays et al. 2007; Percival et al. 1995). The studies did not include parasites of odonates in New York. This project was therefore undertaken to discover the diversity of gregarine parasites found in New York’s and populations to further our understanding of the vast diversity of gregarine parasites, and overall biodiversity, in New York.

METHODS Dragonflies and damselflies were collected from ponds throughout Otsego County, New York, including several at the Thayer Farm of State University of New York College (SUNY) at Oneonta Biological Field Station during the summer of 2009. There were seven sites in total used for the survey. Site 1 was a pond located at the SUNY Oneonta College Camp station,

1 BFS Summer Intern, summer 2010. Previous affiliation: SUNY Oneonta. Current affiliation: Oklahoma State University. Funding from The College at Oneonta Research Foundation.

2 Assistant Professor of Invertebrate Zoology and Researcher, Biology Department and Biological Field Station, SUNY Oneonta located at 42.50° N 75.06° W. The pond there is an enclosed man-made pond. Site 2 included the Chain Ponds of the Thayer Farm, located at 42.79° N 74.91° W. All the ponds at this site are located on farm fields that are still in use. The Chain Ponds are a group of four ponds that are located in a marsh like area connected together by a small stream. Site 3, Lower Pond, was a small pond located between sites 2 and 4, in between the farm fields at 42.79° N 74.90° W. Site 4 was Big Pond, the largest of the ponds at Thayer Farm located at 42.80° N 74.91° W. Site 5 was Beaver Pond on Rum Hill, located at 42.80° N 72.93° W. The pond is located off of an old road/hiking trail that starts on Vibbard Road, Springfield, New York. Site 6 was Moe Pond, a large pond located at the Upper Site of the SUNY Oneonta Biological Field Station just outside of Cooperstown, New York at 42.7° N 74.95° W. A dip net was used to collect aquatic larval (nymph) stages, and an aerial net was used to collect adult stages. Once collected, odonates were identified to genus or species using a taxonomic key (Merritt et al. 2008), and then dissected. Insects were dissected on glass slides, by pulling the intestine out of the body and dragging the intestine across the slide. Any host animals found infected with parasites was preserved in vials or jars to serve as vouchers for future reference. Aquatic larval hosts were preserved in 95% ethanol while adult hosts were frozen dry or stored in 95% ethanol in vials. Vouchers of the insect hosts and their parasites were deposited in the SUNY Oneonta Parasite Collection, administered by Florian Reyda. Gregarines found were stained on glass slides with Semichon’s Carmine, dehydrated in a graded ethanol series, made transparent with xylene, and mounted in Canada balsam under a coverslip. Once the balsam had dried, the parasites were examined with a compound microscope to identify the gregarine parasites with a taxonomic key (Clopton 2009). Identifications of gregarine parasites were done in the summer of 2010. Infection data were recorded in a field notebook and the prevalence (percentage of animals examined infected with each gregarine species) and mean intensity (average number of individuals of a gregarine species per infected host individual) were calculated.

RESULTS

The adult odonates collected were infected more frequently than the nymphs in June and July, but in August nymphs were more frequently infected than were adults.

The species of odonate collected at each site and the prevalence and mean intensity of gregarine infections are shown in Table 1. In total, twenty-three species of odonates were examined in this survey, including twelve species of dragonflies and eleven species of damselflies. Of these twenty-three odonate species, ten species were found to be infected with at least one species of gregarine. Of all the gregarine species encountered, only one, Nubenocephalus secundus was recognized as a previously described species (Table 1). Among the sites surveyed, the pond at the College Camp was found to harbor both the highest prevalence and mean intensities of gregarine infections.

Table 1. Odonate species examined, and gregarine species encountered, and their prevalence and mean intensity. *The host was found to be infected but the gregarine was not kept.

Site Odonate Species (#) Prevalence Mean Intensity Gregarine Species 1 - College Camp Epitheca sp. Charpentier, 1840 4 0% 0 Ischnura verticalis Say, 1839 8 63% 1.9 Nubenocephalus secundus Hays 2007 internum Montgomery, 1943 1 100% ≥ 100 Unidentified gregarine species Symptrum rubicundulum Say, 1839 2 50% ≥ 50 Unidentified gregarine species 2 - Chain Ponds Anax junius Drury, 1773 1 100% 3 Unidentified gregarine species Anax sp. Leach, 1815 1 0% 0 Dorocordulia s p. Needham, 1901 2 0% 0 Enallagma civile Hagen, 1861 1 0% 0 Enallagma sp. Charpentier, 1840 2 0% 0 Epitheca sp. Charpentier, 1840 1 0% 0 Gynacantha sp. Rambur, 1842 2 0% 0 Ishnura verticalis 9 22% 4 Nubenocephalus secundus Hays 2007 sp. Selys, 1850 2 0% 0 Nehalennia irene Hagen, 1861 2 0% 0 Sympetrum internum 1 100% 2 Unidentified gregarine species 10%0 3 - Lower Pond Enallagma sp. 3 0% 0 Ishnura verticalis 15 13% 10.5 Unidentified gregarine species Nehalennia sp. 2 0% 0 Sympetrum rubicundulum 20%0 4 - Big Pond Argia fumipennis Burmeister, 1839 1 100% 1 Unidentified gregarine species Argia sp. Rambur, 1842 1 0% 0 Enallagma sp. 4 0% 0 Epitheca sp. 4 0% 0 Gomphus sp. Leach, 1815 3 0% 0 Gynacantha sp. 1 0% 0 Ishnura verticalis 20 5% 13 Nubenocephalus secundus Hays 2007 Libellula sp. Linnaeus, 1758* 1 100% 1 Unidentified gregarine species Nehalennia sp. 8 0% 0 Nehalennia gracilis Morse, 1895 1 0% 0 Pantala sp. Hagen, 1861 1 0% 0 Somatochlora sp. Selys, 1871 2 0% 0 Sympetrum internum 1 100% ≥ 50 Unidentified gregarine species 5 - Beaver Pond Didymops transversa Say, 1839 1 100% 1 Unidentified gregarine species Enallagma civile 30%0 Enallagma cyathigerum Charpentier, 1840 1 100% 41 Unidentified gregarine species Ishnura verticalis 10%0 Nehalennia sp. 2 0% 0 Sympetrum internum 1 100% 9 Unidentified gregarine species Sympetrum rubicundulum 1 100% 22 Unidentified gregarine species 6 - Moe Pond Amphigrion sp. 5 0% 0 Argia sp. 1 0% 0 Chromagrion sp. 1 0% 0 Enallagma sp. 5 0% 0 Epitheca sp. 1 0% 0 Hesperagrion sp. 1 0% 0 Ishnura verticalis 20%0 Nehalennia sp. 1 0% 0

DISCUSSION

It is interesting that the odonates from the pond at the College Camp had higher infection rates, in terms of prevalence and intensity, than at other collection sites. (i.e., there was a higher proportion of individuals infected at this site and the hosts had a greater number of parasites in their intestine than what was seen at the other sites). One host collected at the College Camp pond, Sympetrum internum, the cherry-faced meadowhawk, had hundreds of gregarines in its intestine. In contrast, the infected hosts at the other sites harbored only a few gregarine individuals. This difference may be because the College Camp pond is an enclosed water body, whereas the other four collecting sites all have outflows, perhaps leading to a higher concentration of oocysts, the infective stage, in the water. Further research would need to be carried out to see if this was the case. The gregarine species collected in the different dragonfly and damselfly species included, to our knowledge, only a single previously known gregarine species, Nubenocephalus secundus from the eastern fork-tail damselfly Ischnura verticalis. This gregarine was previously reported from eastern fork-tails in Texas (Hays et al. 2007). All of the other gregarines listed in Table 1, however, are only identified to genus. Some of the gregarines listed in Table 1 as unidentified species may represent previously reported gregarines that we were unable to identify to species because typically, only the trophozoite stage of the gregarine life cycle was examined in this study. Species-level identification of gregarines is typically done based on the shape and morphology of several ontogenetic stages of the gregarine life cycle. While efforts were made to examine reproductive stages such as the sporont and gamont stages, these were not helpful because these stages typically lack distinguishing characteristics. A key stage in making species- level identifications of gregarines is the oocyst stage, which is generally invariable within the species, making it a good taxonomic character (Janovy et al. 2007b). Oocysts are the first stage in the gregarine life cycle that protect the developing parasite and infect the next host in the cycle. Observations of oocysts require laboratory-rearing of odonate hosts, and were therefore beyond the scope of this study. Another obstacle to making species-level identifications of gregarines was the problematic nature of some species descriptions in the literature. Unfortunately, many of the currently known gregarine species have either incomplete descriptions, describing only subsets of the gregarines various life cycle stages, or are not up to date with current description methods (see comments by Janovy et al. 2007a). Incomplete descriptions of some gregarine species therefore made the task of identifying the collected gregarines nearly impossible. Yet, some of the gregarine species found seem to be new species because they are completely unlike any gregarine that is in the current taxonomic key. Thus there is a need for continued work on this intriguing host-parasite system. This survey is the first step towards understanding the parasites in the Odonata of New York. Prior to the survey, diversity of these parasites in dragonflies and damselflies of New York odonates was completely unknown. Further study will focus on the parasites of the eastern fork- tail damselfly, Ischnura verticalis and characterize its gregarine species diversity.

CONCLUSIONS

Although New York has a long and rich tradition of natural history studies, there are still several, if not dozens, of species that are undescribed and new to science in the state. This list likely includes most of the gregarine species encountered in the current study.

ACKNOWLEDGEMENTS

Thanks to Dr. Jeffery Heilveil for help with insect identification. Many thanks are given to Cleo Szmygiel, Katie Eyring, Liza Hendricks, Robert Wiles, and Dr. Caira’s 2009 parasitology class (University of Connecticut) for help collecting insects. Thanks to Dr. Bill Harman, Matt Albright, and Holly Waterfield of the Biological Field Station for logistical help. Thanks to Dr. Tamara Cook’s lab (Sam Houston State University) for the use of their DIC microscope. This work was funded in part by a Student Research Grant awarded to C.W. by the SUNY Oneonta Research Foundation.

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