2009 SOUTHEASTERN NATURALIST 8(4):599–608 Pathology of the Trematode Alloglossidium renale in the Freshwater Grass Shrimp Palaemonetes kadiakensis Stephen C. Landers1,* and Rebecca D. Jones1 Abstract - The trematode Alloglossidium renale was commonly observed in the freshwater shrimp Palaemonetes kadiakensis in Pike County, AL, with a prevalence of 30.2% and 12.7% at two different collection sites. Sectioned antennal glands demonstrated tissue damage near the parasite, including antennal gland tubule cell compression and destruction of host cells. Normal tubules as well as those showing cellular changes were functional and contained parasite ova. Grass shrimp outlive this infection, as evidenced by masses of ova and debris within infected antennal glands, but no viable parasite. The non-lethal nature of the parasite helps to explain the high intensity of infection observed at one of the collection sites. This study furthers our understanding of the effect of this parasite on its host, and extends the reported range of Alloglossidium renale to Alabama. Introduction Alloglossidium renale Font and Corkum is a parasite (Trematoda: Macroderoididae) found within the antennal glands of Palaemonetes kadiakensis Rathbun (Mississippi Grass Shrimp). It was first described in Louisiana (Font and Corkum 1975), and has since been the subject of many articles (Brooks 2003; Carney and Brooks 1991; Font 1980; Font and Corkum 1976, 1977; Lefebvre and Poulin 2005; Smythe and Font 2001). The complete life cycle is not known (the molluscan intermediate host has not been identified), though it has been determined that the parasite becomes mature within the antennal glands of shrimp and that no vertebrate host is required. The shrimp stage is considered either an adult or a progenetic metacercaria by different authorities (Lefebvre and Poulin 2005). Within the Order Plagiorchiformes, the genus is considered to be derived due to its 2-host life cycle, having likely evolved from a 3-host life cycle (Carney and Brooks 1991, Smythe and Font 2001) During the summer of 2007, numerous freshwater shrimp from Pike County, AL, were observed with dark masses behind the antennal scales where the antennal glands are located. The masses were identified as A. renale, prompting a histological investigation into the effects of the worm on its host. The objectives of this study were to determine the pathological effects of A. renale on the host antennal gland and to determine if such infections impair kidney function and host size. 1Department of Biological and Environmental Sciences, Troy University, Troy, AL 36082. *Corresponding author - slanders@troy.edu. 600 Southeastern Naturalist Vol. 8, No. 4 Materials and Methods Palaemonetes kadiakensis were collected along the grassy edges of the Conecuh River (31º48'15"N, 86º2'49"W) and Olustee Creek (31º56'38"N, 86º7'6"W) by dip net in Pike County, AL, between June 2007 and August 2008. The animals were returned to Troy University and maintained in water from the collection site. Animals were measured (anterior end of rostrum– posterior end of uropods), and infection with A. renale was recorded. Worms for whole-mount preparation were fixed in 5–10% formalin and stained with Gill’s hematoxylin. Except for the initial collection of 3 worms, the fixative was used at room temperature and was not heated. Worms and antennal glands prepared for paraffin sections were fixed in 5–10% formalin before infiltration with Paraplast. Ten-μm sections were cut on a rotary microtome and stained with Gill’s hematoxylin and fast green. Worms and antennal glands prepared for plastic sections were fixed at room temperature in 2–3% glutaraldehyde buffered in 0.1M sodium cacodylate, pH 7.2, and post-fixed in 2% buffered osmium tetroxide before infiltration with Spurr’s resin. Sections (1–2 μm) were cut on an ultramicrotome with a diamond knife and stained with toluidine blue (1% toluidine blue, 1% sodium tetraborate). Some specimens for plastic sections were fixed in unbuffered 2% glutaraldehyde only. Specimens were photographed with a Nikon DXM 1200 digital camera mounted on a Nikon E600 light microscope. Images were adjusted for brightness, contrast, and gamma levels using Adobe Photoshop Elements 6.0. Results Live observations Alloglossidium renale infections were visible through the host exoskeleton at the base of the antennal scale. The worms occasionally moved within the antennal glands. When dissected from the host, the worms moved actively, contracting and expanding the oral sucker. Some worms regurgitated material from the digestive caeca when removed from the host, and others released a string of ova from the genital pore. The ova were sticky and remained in a string until disturbed. The prevalence data and host measurments for the 2 collection sites are reported in Table 1. Identification Alloglossidium renale was identified by 1) its specific habitat in the antennal gland of freshwater Palaemonetes kadiakensis in North America, 2) its progenetic development in an invertebrate definitive host, and 3) comparison Table 1. Occurrence of Alloglossidium renale in Palaemonetes kadiakensis from Pike County, AL. Average host length Location # infected Total % infected Infected (mm) Uninfected (mm) Conecuh River 23 76 30.2 23.4 19.7 Olustee Creek 31 243 12.7 31.0 29.4 All sites 54 319 16.9 27.8 27.5 2009 S.C. Landers and R.D. Jones 601 of whole stained specimens with the species description (Font and Corkum 1975). Specifically, the diameter of the testis relative to the body width, and the absence of a metacercarial wall were diagnostic characteristics used in the key provided in the previous reference. Further, the body length:width ratio (less than 3:1), testis:ovary ratio, ovary shape, and absence of a metacercarial wall were diagnostic characteristics for A. renale in a comparative study of numerous species within the Macroderoididae (Smythe and Font 2001). The average values of A. renale structures (Table 2) were smaller than those reported in the original species description, though the maximum sizes were within those ranges. The average length and width were within the ranges reported by Carney and Brooks (1991). The ova were smaller than the species description, though the maximum sizes fell within the published range. Voucher specimens of gravid A. renale have been deposited in the US National Parasite Collection in Beltsville, MD (USNPC 101574). Pathology of Alloglossidium renale infections (Figs. 1–12) Paraffin and plastic sections of Alloglossidium renale within the host or isolated antennal glands were analyzed. The best fixation and histology were observed with buffered glutaraldehyde and osmium, with plastic sections. Sections revealed either one or two parasites folded within an antennal gland (though we have observed 5 worms within one antennal gland). The worms were surrounded by antennal gland tubules with intact lumena (Fig. 1). In some cases, the trematode had grown to a large enough size to reduce the amount of remaining host renal tissue (Figs. 2–3, 5). The tissue loss was apparently due to parasite feeding and compression due to parasite growth. Compression of host cells was obvious when tubule nuclei were concentrated against the parasite (Fig. 1). There was no metacercarial cyst wall or extracellular secretion separating the parasite from the host. Evidence of parasite material traveling throughout the antennal gland was found, including ova, and dark spherical inclusions similar to material within the parasite uterus (Figs. 6–9). Cellular differences were evident in some but not all host cells near or touching the parasite (Figs. 7–9). Normal tubule cells were typically cuboidal, had a microvillar border, and in plastic sections revealed a large open nucleus with scattered chromatin granules (a homogeneously tinted nucleus was produced if osmium was not used as a post-fixative). Close to the parasite, many cells had a more squamous shape, denser cytoplasm, and a compact nucleus (Figs. 7–9). A gradient of tubule cell morphologies was observed in some specimens, with healthy cuboidal cells transitioning to compact squamous cells closer to the parasite. This transition affected the Table 2. Measurements from gravid (ova producing) Alloglossidium renale (in microns). Body Body Oral Ventral Anterior Posterior Ova Ova length width sucker sucker testis testis Ovary length width Average 992 423 108 92 84 100 214 22 12 n 14 14 12 8 4 3 4 10 10 Range 600–1560 225–545 90–135 47–125 50–120 75–125 195–230 19–25 9–14 602 Southeastern Naturalist Vol. 8, No. 4 Figures 1–5. Sections through shrimp antennal glands. CP: cirrus pouch, DC: digestive caeca, M: host muscle, PC: parenchymal cells, OS: oral sucker, U: uterus with ova, V: vitellarium, VS: ventral sucker. 1–3. Paraffin sections. 4–5. Plastic sections. Figure 1. Sagittal section through the worm revealing host antennal gland tubules being consumed (top of photo at oral sucker) and tubule compression and necrosis (compacted tubule cell nuclei at arrow). Bar = 220 μm. Figures 2–3. An infected antennal gland in which little of the organ remains. This antennal gland contained two worms. Only a thin layer of tissue exists between the worm and the host musculature (arrow). Arrowhead indicates ovum. Figure 2 Bar = 200 μm. Figure 3 Bar = 100 μm. Figure 4. Uninfected right antennal gland showing normal tubule cells. Host musculature is on the right. Bar = 100 μm. Figure 5. Left antennal gland (infected) from same host animal as in Figure 4. Parasite tegument with spines (small dots on surface) indicated by an arrow. Ova trapped between the host and parasite indicated by arrowhead. Bar = 100 μm. 2009 S.C. Landers and R.D. Jones 603 cell shape, nuclear shape, chromatin distribution, and cytoplasmic density. Very thin (<5.0 μm) squamous cells commonly abutted the parasite. Some cells in contact with the parasite had a microvillar border, and others had lost their brush border. Parasite ova were present in numerous areas of infected antennal glands, including tubules presumably leading downstream to the nephridiopore. Most significantly, they were found within the antennal gland tubules in areas showing pathological effects as well as normal tubules, indicating that infected antennal glands are able to filter and transport fl uid throughout the remaining tubule system (Fig. 7–9). Ova were also present between the parasite and host tissue (Figs. 3, 5), as well as within the digestive caeca of the trematode. The later location was evidently due to the parasite ingesting its released ova while feeding upon the antennal gland. Worm remnants or evidence of past infection were present in some antennal glands (Figs. 10–12). Sections of antennal glands containing parasite remnants revealed masses of ova, host cells, and debris within the host tubules. This condition was observed twice, in the same shrimp, with both antennal glands appearing functional and exhibiting little tubule damage. In one antennal gland with evidence of infection (Fig. 10), sections throughout Figure 6. Plastic section of A. renale within the host. Arrows indicate material within the antennal gland tubule (left arrow) that is similar to material within the parasite uterus (right arrow). This individual worm had many dark spherical inclusions, lipid droplets, and ova within the uterus. Arrowheads indicate parasite surface. Bar = 50 μm. 604 Southeastern Naturalist Vol. 8, No. 4 Figures 7–8. Plastic sections of A. renale within the host. OV: ova, U: uterus with ova, V: vitellarium. Figure 8 is an enlargement from Figure 7. Ova within antennal gland tubules are labeled. Near the parasite, the tubule cells are fl attened and have a densely-stained cytoplasm. Nuclei in the fl attened cells (large arrows) are compact and dense compared to healthy tubules farther away from the parasite. Healthy tubule cells are cuboidal, stain lightly with toluidine, and have open spherical nuclei with scattered chromatin. The brush border is indicated in both normal and compressed cells (small arrows). Bars = 100 μm. the organ revealed no indication of a parasite except for ova, which were found scattered in the organ and in a small spherical mass. A portion of the gland was not sectioned (removed during initial trimming), though a worm, if present, would not have been missed by our examination. The mass of ova was associated with host tubule cells, recognizable by their nuclei. Debris filled the gland tubules, and the lumen was distended, though no trace of the worm was observed. The other infected antennal gland revealed a large mass of parasite tissue and ova (Figs.11–12). This large mass was surrounded by condensed and uncondensed tubule cell nuclei. The mass contained accumulations of ova, lipid droplets, and apparently necrotic parasite tissue that was difficult to interpret. The antennal gland was distended to accommodate the infection, while clusters of ova were observed in tubules downstream from the worm. Discussion Alloglossidium renale is now recorded from southeastern Alabama in the freshwater shrimp Palaemonetes kadiakensis. This is a new distribution record, adding to the known distribution in Louisiana (Font and Corkum 2009 S.C. Landers and R.D. Jones 605 1975) and North Carolina (Carney and Brooks 1991). In Pike County, there was a disparate prevalence between the two collecting sites. This disparity was not surprising given what has previously been reported for locational and seasonal variations in the occurrence of A. renale (see Font and Corkum 1976). The disparity was probably related to the distribution of the presumed intermediate snail host in the life cycle. We have not collected or analyzed Palaemonetes paludosus, another reported host for this parasite (Carney and Brooks 1991). The destruction and trauma to antennal gland cells was attributed to 1) the scraping of the parasite’s spiny tegument against the host cells, 2) parasite growth and compression of the tubules, and 3) ingestion of host tissue. We did not critically differentiate between the regions of the antennal gland (antennal gland tubules [labyrinth], bladder, coelomosac) for all of the cells in our sections (Bell and Lightner 1988, Miller 1990, Parry 1955, Peterson and Loizzi 1973). Most of the cells surrounding the worm were consistent with tubule cells, but may represent other antennal gland cell types as well. It is likely that all cells involved in the antennal gland complex were Figures 9–10. Plastic sections of A. renale or ova within the host. M: host muscle, U: uterus with ova. Figure 9. Compressed antennal gland tubules (containing ova) are between the parasite and host muscle tissue. A non-compressed, lessdamaged tubule area (top right) also contains ova. Small arrow inidcates brush border of tubule cells; Large arrows indicate nuclei of damaged tubule cells; Arrowheads indicate parasite surface. Bar = 100 μm. Figure 10. Small mass of ova and debris within antennal gland tubules. This section revealed the largest mass observed in this antennal gland, which is evidence of an earlier viable parasite. Arrowhead indicates nucleus of a host tubule cell. Bar = 50 μm. 606 Southeastern Naturalist Vol. 8, No. 4 potentially affected. A similar loss of antennal gland tissue and tissue damage due to abrasion was reported for Alloglossoides caridicola in a crayfish host (Turner 1985). Font and Corkum (1976) reported that the host shrimp can outlive the parasite. We observed evidence of this as either a necrotic mass within Figures 11–12. Plastic sections of a large A. renale remnant within the host. Figure 11. Mass of ova and debris (right) from a necrotic A. renale within the host antennal gland. Ova (arrowhead) are found within the surrounding tubules. Bar = 100 μm. Figure 12. A higher magnification and different section of the remnant in Figure 11. The mass is surrounded by host tubule cells (nuclei at arrowheads). Individual cells are not recognizable in the mass. Bar = 50 μm. 2009 S.C. Landers and R.D. Jones 607 the host or as a mass of ova without any remnant of a worm. In these two specimens, damage to the host organ was minimal, with apparently healthy tubules surrounding a mass of debris with ova. These observations indicated that the worm does not always destroy the host antennal gland before its death. Ova present in multiple locations throughout the antennal gland tubules (labyrinth) indicated that infected glands were able to filter fl uid from the hemocoel and transport that fl uid downstream to the urinary bladder. Ova within the nephridial tubule system occur in other antennary gland parasites, such as Allocorrigia filiformis, where they were located in the interstitial space and resulted in nodule formation and a host melanin reaction in Procambarus clarkii (Turner 1984). We did not observe a visible melanin reaction when non-osmicated, non-stained plastic sections were examined. Alloglossidium infections were not only non-lethal, but in some cases had minimal affects on the host despite the tissue damage that occurs. The data indicating similar host lengths for infected and non-infected shrimp (Table 1) supported this conclusion. Many unsolved questions remain to be answered concerning A. renale and its brief life history. As reviewed by Font and Corkum (1976), the parasites can mature and produce ova within 6 weeks. The death of the worms occurred before the seasonal mortality of the host, leading the authors (Font and Corkum 1976) to propose a close seasonal adaptation between parasite and host. Our study supports this earlier work, and suggests little effect at all on the hosts when comparing body length of infected and non-infected shrimp. An interesting future avenue of research may be to investigate the effect of the host on A. renale, to uncover any host response that limits the parasite. Additionally, given the brief and predictable life cycle, and the accessibility of the parasite (visible externally through the shrimp), this trematode may be a good model organism for studying the biology of senescence. Acknowledgements The authors thank Dr. Alvin Diamond for help in collecting Palaemonetes kadiakensis and for reviewing this manuscript, and Sarah Braune for help in the laboratory. Portions of this research were presented at the annual meeting of the Association of Southeastern Biologists: Jones, R.D., and S.C. Landers 2008. Morphological analysis of the trematode parasite Alloglossidium. Southeastern Biology 55:230–231. Literature Cited Bell, T.A., and D.V. Lightner, 1988. A handbook of normal penaeid shrimp histology. Special Publication No. 1. World Aquaculture Society, Baton Rouge, LA. 114 pp. Brooks, D.R. 2003. Lessons from a quiet classic. Journal of Parasitology 89:878–885. Carney, J.P., and D.R. Brooks. 1991. Phylogenetic analysis of Alloglossidium Simer, 1929 (Digenea: Plagiorchiiformes: Macroderoididae) with discussion of the origin of truncated life cycle patterns in the genus. Journal of Parasitology 77:890–900. 608 Southeastern Naturalist Vol. 8, No. 4 Font, W.F. 1980. 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