The persistence of acquired immunity of largemouth bass Micropterus salmoides to

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1 Submitted to Fish and Shellfish Immunology 5/6/05 The persistence of acquired immunity of largemouth bass Micropterus salmoides to glochidia larvae of Lampsilis reeveiana (Bivalvia, Unionidae). Benjamin J. Dodd a,, M. Christopher Barnhart a, *, Constance L. Rogers-Lowery b, Todd B. Fobian a, Ronald V. Dimock Jr. b a Department of Biology, Missouri State University, Springfield, Missouri b Department of Biology, Wake Forest University, Winston-Salem, North Carolina Present address: Iowa Department of Natural Resources, Fisheries Research Station, U.S. Highway 34, Chariton, Iowa, U.S.A. * Corresponding author. Tel Fax address: chrisbarnhart@smsu.edu.

2 2 Abstract Host fish acquire immunity to the parasitic larvae (glochidia) of freshwater mussels (Unionidae). We investigated the persistence of acquired immunity of largemouth bass, Micropterus salmoides (Lacepède, 1802) to glochidia of the broken rays mussel, Lampsilis reeveiana (Call, 1887). Fish received 3 successive priming infections with glochidia to induce an immune response. Primed fish were held at O C and were challenged (re-infected) at intervals after priming. Metamorphosis success was quantified as the percent of attached glochidia that metamorphosed to the juvenile stage and were recovered alive. Metamorphosis success at 3, 7, and 12 months after priming was significantly lower on primed fish (26%, 40%, and 68% respectively) than on control fish (85%, 93%, and 92% respectively). A second group of largemouth bass was similarly primed and blood was extracted 0, 3, 7 and 12 months after priming. Immunoblotting was used to detect host serum antibodies to L. reeveiana glochidia proteins. Serum antibodies were evident in primed fish, but diminished over time and were nearly undetectable 7 and 12 months after priming. Primed fish had antibodies present in their serum within 4 days when re-infected 7 months after priming, but fish that received their first infection did not. The results are consistent with the hypothesis that antibodies are at least part of the mechanism of acquired immunity to glochidia. Acquired immunity of host fish potentially affects natural reproduction and artificial propagation of unionids, many of which are of conservation concern. Keywords: Glochidia; Antibodies; Acquired immunity; Unionidae; Largemouth bass

3 3 1. Introduction Freshwater mussels of the family Unionidae have an obligate, parasitic larval stage, the glochidium (pl. glochidia) that attaches to the gills or fins of host fish. Migration of host epithelial cells forms a cyst, in which the glochidium remains for days to months. During this time the glochidium gains nutrition from the host and metamorphoses to the juvenile stage [1, 2]. Juveniles leave the host and become free-living suspension-feeders. Unionid mussels are host specific and can utilize only a limited number of compatible host species. The number of compatible host species varies among mussel species from 1 to 37 [3]. Glochidia that attach to non-hosts (incompatible fish species) are typically sloughed, either live or dead, from one to several days after attachment. This incompatibility is apparently due to innate immune factors [4-10]. Compatible host species acquire immunity to mussel glochidia after one or more artificial infections [4, 11, 12]. Limited evidence suggests that host fish may also acquire significant immunity to mussel glochidia in nature [8, 13, 14, 15]. Although glochidia generally do not cause significant disease in host fish, the ability of hosts to acquire immunity could play important roles in mussel ecology and evolution. The underlying mechanisms of acquired immunity of host fish to glochidia are not fully understood. The timing and extent of cyst formation differ in primed and naïve fish and these differences may contribute to acquired immunity [4, 5, 12]. Several studies have reported antiglochidia factors (presumably antibodies) in the serum of infected host fish [7, 10, 16]. Antibodies from primed fish bind glochidia proteins and may be involved in acquired immunity [17]. However, specific cell-mediated and non-specific immune factors have not been ruled out as potential mechanisms. The persistence of the acquired immunity of host fish to glochidia is also not well understood [22]. Fish develop immunity to protist parasites in response to

4 4 experimental infections and antigen vaccinations, and immunity can persist for months to years [18-21]. North American mussel populations have declined dramatically over the last century. Many species are extinct and many others are thought to be in danger of extinction [23]. A better understanding of the persistence of acquired immunity of host fish to glochidia could aid conservation efforts. If immunity persists, mussels might rely primarily on immunologically naïve fish for recruitment [11]. Acquired immunity is also significant in the context of artificial propagation of mussels. Propagation of rare species of mussels is an objective in many federal recovery plans [24]. If acquired immunity is only temporary, the same host fish could be used repeatedly, which could reduce the labor and cost involved in collecting or propagating host fish. The primary objectives of this study were to determine the time course of acquired immunity and of serum antibody levels of largemouth bass (M. salmoides) after priming with glochidia of the broken rays mussel (Lampsilis reeveiana brevicula). During the course of the year-long study, we also investigated whether glochidia age affects metamorphosis success. 2. Materials and methods 2.1. Fish and mussels Six-month-old largemouth bass, 13.4 g ± 4.0 (SD) were provided by the Chesapeake State Fish Hatchery, Missouri. After transport to SMSU from the hatchery, the fish were treated prophylactically with Kanacyn antibiotic at 4 mg L -1 for 3 hours, 3 times at 48-hour intervals. The fish were thereafter kept in recirculating aquarium systems (21-23 C) and fed 1 to 2% of their body weight daily (AquaMax pellet feed, Purina Mills), except when monitoring glochidia infections, when they were fed every other day to reduce feces production.

5 5 Gravid broken ray mussels (Lampsilis reeveiana brevicula, hereafter referred to as L. reeveiana) were collected from Beaver Creek, Taney County, Missouri, USA during 2003 and Mussels were maintained unfed in aquaria (19-21 C) for up to 4 months. Both mussels and fish were kept on a 12:12 hour light dark photoperiod while in the laboratory Infection procedure We used glochidia from one female mussel per infection, and a different mussel for each infection. Glochidia harvest and host infection methods were similar to those used previously [17]. Fish were infected with glochidia by placing them as a group in a suspension containing 2,000 infective glochidia L -1, for 15 minutes. The volume of the suspension was 0.5 L fish -1. Aeration and stirring were used to keep the glochidia in suspension Parasitism We infected 80 largemouth bass with L. reeveiana glochidia 3 times in succession, at approximately 1-month intervals, to induce immunity ( priming ). Another group of 80 naïve fish was kept for use as controls. Subgroups of the primed and control fish were later challenged (re-infected) at intervals to evaluate the persistence of acquired immunity. The challenge infections were administered 101 d, 215 d, and 366 d after the last priming infection was administered (Figure 1a). The remaining primed and control fish were used to analyze serum antibodies (described below). After infection we immediately transferred the fish by dip net into individual 2.75 L tanks. These tanks were part of a recirculating system (AHAB Aquatic Habitats, Inc. Apopka, FLA). The system was modified for monitoring metamorphosis success of mussel glochidia on fish, as described previously [17]. We counted the glochidia and juveniles shed from each fish at 1 day post infection (dpi), and every 2 days thereafter until no more glochidia or juveniles were

6 6 recovered from any fish. An individual was classified as a live juvenile if foot activity was observed within 3-5 minutes of observation. For each fish, we determined intensity of infection (the number of glochidia initially attached), metamorphosis success (the percent of attached glochidia that metamorphosed to the juvenile stage and were recovered alive), number of live juveniles recovered, and the mean duration of successful parasitism (days from attachment to excystment of live juveniles). Although we held fish individually after each infection, we pooled the members of each group during the infection procedure. Two-tailed t-tests were used to compare the mean intensity of infection between primed and control fish. One-tailed t-tests were used to compare the mean metamorphosis success, mean number of juveniles, and mean duration of successful parasitism between primed and control fish. One-way ANOVA was used to compare the mean metamorphosis success and mean duration of successful parasitism of control fish among infections. Significant differences were found in metamorphosis success of different batches of glochidia on control fish. Therefore, we expressed the metamorphosis success on primed fish as a percent of their respective control means. Metamorphosis success was then compared among groups with Tukey s multiple comparison Effect of glochidia age Variation in control metamorphosis success was investigated in relation to glochidia age. Gravid females were collected during 2003 and 2004 and kept in the laboratory for up to 4 months in similar conditions as those described previously. Glochidia were extracted from 1 or 2 (pooled equal quantities of glochidia) females per infection, and different individuals were used for each infection. We infected naïve largemouth bass and monitored metamorphosis success for

7 7 each fish. Glochidia age was estimated as the number of days between the collection date of the first gravid female in September of a given year and the date the glochidia were used for infection. That is, we assumed that reproduction was synchronous and that the glochidia at the collection site were all of similar age. This assumption was based on previous field observations (see discussion). Glochidia were classified by age (1-120 days, days, or days). The mean metamorphosis success was compared among the three age classes using Tukey s multiple comparison. The results are expressed as mean ± 1 SD unless otherwise noted, and differences were judged significant if p was Serum antibodies Methods used for analyzing serum antibodies were similar to Dodd et al. [17]. Subgroups of the primed and control fish were sacrificed at intervals to evaluate serum antibody levels. Blood was extracted from primed and control fish at 42 d, 101 d, 215 d and 366 d after the last priming infection was administered (Figure 1b). Blood from all individuals in a group was pooled for each analysis. Serum was separated from blood cells by centrifugation and stored at minus 80 C until used for analysis. Immunoblotting techniques were used to determine whether serum from primed largemouth bass recognized glochidia proteins. Approximately 500 µl packed volume of glochidia was homogenized in 1500 µl total volume and solids were removed by centrifugation. Supernatant protein concentration was measured with Bradford s assay (Bio-Rad). Glochidia proteins and prestained molecular weight standards (Bio-Rad) were separated by SDS-PAGE and electrotransferred to 0.45 µm nitrocellulose membranes. The membranes were initially probed with pooled sera collected from primed or control largemouth bass. After rinsing in PBS-Tween, the membranes were incubated in mouse anti-bass antibodies and subsequently incubated in goat anti-mouse antibodies conjugated to horseradish peroxidase.

8 8 Antibody recognition was visualized using 4-chloro-1-napthol and hydrogen peroxide as the peroxidase substrate Immune memory We investigated whether antibody production in naïve fish differed from primed fish upon infection and re-infection, respectively. Two naive fish and two primed fish (3 infections) that were kept free of glochidia for 7 months after priming were infected with glochidia from the same mussel [17]. In this particular infection water temperature was C. We extracted blood from the primed fish at 4 dpi and the serum from the fish was pooled. Two naïve fish were also sacrificed and their serum was pooled and used for a control (serum was stored at - 80 C). Immunoblotting was later used to detect antibodies in serum [17]. 3. Results The intensity of infection (the number of glochidia initially attached) on experimental fish for the priming and challenge infections was 480 ± 139 and 607 ± 174, respectively (mean ± SD). Intensity differed significantly between primed and control fish only in the first challenge infection (2-tailed t-test, Table 1). Mean metamorphosis success (the percent of attached glochidia that metamorphosed to the juvenile stage and were recovered alive) on control fish during this study was approximately 82%. Metamorphosis success was significantly reduced on primed fish in all comparisons (Table 2) and averaged 48% of the controls. Metamorphosis success during the second priming infection ranged from 0 to 72% on primed fish, indicating substantial variation in the development of immunity among individual fish (Figure 3). The reduced metamorphosis success on primed fish was associated with elevated numbers of prematurely sloughed glochidia prior to appearance of metamorphosed juveniles (Figure 2).

9 9 The mean duration of successful parasitism (days from attachment to excystment of live juveniles) on control fish was approximately 20 dpi (Table 2, Figure 2). The duration of successful parasitism was significantly shorter on primed fish in all infections and averaged 80% of the control values. The difference lessened with time after priming (Table 2). Antibodies isolated from primed largemouth bass recognized glochidia proteins (Figure 4), while antibodies from naïve fish did not (data not shown). Control blots probed with largemouth bass serum and goat anti-mouse antibodies (no mouse anti-bluegill antibodies), mouse anti-bluegill and goat anti-mouse antibodies (no largemouth bass serum), goat anti-mouse antibodies only, and substrate only all produced negative results (data not shown). Largemouth bass produced serum antibodies to L. reeveiana proteins after one priming infection (Figure 4-2) and antibody levels were higher after 3 priming infections (Figure 4-3), which was illustrated by an increase in the number of detectable bands and darker staining. An intensely stained band was observed at kda and several less intense bands appeared at 120.1, 85.0, and 78.5 kda. In addition, 3 bands were observed at 44.5, 41.2 and 38.1 kda (Figure 4-3). However, the level of circulating serum antibodies decreased 3 months after priming (Figure 4-4) and bands were difficult to detect 7 months (Figure 4-5) and 12 months (Figure 4-6) after priming. Immune fish that were re-infected 7 months after priming produced a significant level of antibodies within 4 days post infection (Figure 4-7). This staining was similar to that observed in fish after completion of three priming infections (Figure 4-3). However, naïve fish that were infected for the first time showed no detectable antibodies in their serum at 4 days post infection (data not shown).

10 10 Variation in metamorphosis success among infections on naïve bass was related to the age of the glochidia (Table 3, Figure 5). Glochidia that were 0-120, , and days old had metamorphosis success of 87.5%, 88.7%, and 67.5%, respectively. The metamorphosis success of the oldest age class was significantly lower than the others (Tukey s multiple comparisons). We saw no change in the duration of successful parasitism with glochidia age (Table 3). 4. Discussion The adaptive immune system of fish has been studied primarily in the context of fish husbandry and in efforts to understand the evolution of the immune system. Our interest stems from the relationship between fish and mussels of the family Unionidae. The unionid lifecycle requires the attachment of a parasitic larva (glochidium) to a compatible host fish. Unionids are host-specific; glochidia that attach to a non-host fish do not tmetamorphose into juveniles and are usually sloughed within days after attachment. However, attachment of glochidia to a host species does not necessarily ensure metamorphosis either, because they may be subject to the adaptive immune defenses depending upon previous experience of the host [4, 11, 12, 17]. Acquired immunity of host fish to parasitic organisms can persist for months or years, and may be permanent in some cases. For example, mirror carp (Cyprinus carpio L.) that acquired immunity to the ciliate parasite Ichthyopthirius multifiliis (Ich) during artificial infection were still immune when challenged 8 months later [18]. Channel catfish (Ictalurus punctatus) that survived an intraperitoneal (IP) vaccination with the infective stage of Ich (theronts) were immune for at least 9 months [20] and 13 months [21] after injection. In addition, live IP vaccination with Cryptobia salmositica provided rainbow trout (Oncorhynchus mykiss) protection for at least 24 months [19].

11 11 Few studies have addressed the persistence of acquired immunity of host fish to glochidia. O Dee [22] indicated that one-year-old largemouth bass that acquired immunity to fatmucket (Lampsilis radiata luteola) glochidia retained their immunity over a 4-month period of simulated non-wintering (19 C) conditions after priming. The immunity partially degraded 4 months after priming. The present study showed that the metamorphosis success of L. reeveiana glochidia was significantly lower on primed fish (22 C) than on control fish for at least 12 months after priming. However, partial loss of immunity was evident at that time (Table 2). In the present study, the duration of successful parasitism was shorter on primed fish, a result also noted in other studies [12, 17]. This result suggests that the processes causing excystment are accelerated by priming. Shortened period of parasitism might affect juvenile condition because it could limit the nutrition that glochidia obtain from the host. Interestingly, we observed that juveniles from primed fish were more transparent than those from naïve fish. We speculate that this difference might relate to differences in protein or glycogen content. Further study and comparison of the condition of juveniles from primed and naïve fish is warranted. The persistence of host acquired immunity may be an important factor in the ecology, evolution, and management of unionid mussels. Limited evidence suggests that host fish also acquire immunity to glochidia in nature [8, 14, 15, 26]. If immunity persists, intraspecific and interspecific competition for naïve host fish may occur [17]. Such competition may have caused evolutionary diversification in host fish utilization [27], lure morphology and color, and luring behavior [28]. If older fish are more likely to be immune, age distribution of host fish populations may affect mussel recruitment. Also, mussel recruitment might be inhibited in dense mussel populations if host fish in the vicinity of such populations are more likely to be immune.

12 12 Artificial propagation of endangered mussels is becoming a common conservation practice. This process involves harvesting glochidia from gravid mussels, placing glochidia on host fish, and recovering and stocking juveniles back into the wild [29]. Ability to reuse host fish might reduce costs associated with host species that are difficult to collect. The fact that immunity waned over time in this study suggests that reuse of hosts the following year might be practical. However, the persistence of immunity to glochidia in other species of host fish has not been examined. There is also a need to investigate the effect of partial host immunity on the condition of juveniles that do successfully metamorphose. The adaptive immune system of teleosts includes lymphocytes similar to the B and T cells of mammals [30], which are capable of mounting specific humoral (antibody) and cellmediated immune responses. Fish B cells produce antibodies that are IgM-like tetramers [25]. Teleost antibodies may have multiple effects on parasites. For example, antibodies activate the complement cascade, which can lyse parasites [31, 32]. Antibodies can create conditions that can block invasion of the host or cause mobile parasites to leave the host [33]. In addition, antibodies can also immobilize parasites [34, 35]. However, the presence of antibodies does not necessarily mean they are involved in the protection of a fish [36]. In other cases, cell-mediated and non-specific responses appear to be the underlying mechanisms of protection [37, 38]. The mechanisms of acquired immunity of host fish to mussel glochidia are not well understood. Shedding of live and dead glochidia from immune and control fish was observed in this study and is common [17]. Cysts are formed around glochidia within hours after attachment [1, 39]. The shedding of live glochidia may be due to delayed or incomplete cyst formation [12], or by necrosis or sloughing of the cyst [4, 5]. We observed accelerated excystment of live juveniles as well as increased sloughing of glochidia. However, we also recovered dead

13 13 glochidia and observed dead glochidia still encysted on the hosts. These glochidia (recognized by open, empty valves) were likely killed by elements of the host immune system. In a cross immunity study, serum antibodies from primed largemouth bass recognized glochidia proteins of the priming species and closely related species, but not distantly related species. Nonetheless, cross-immunity to distantly related species was also observed, suggesting that factors other than antibodies were involved [17]. Li and Woo [19] examined the duration and mechanism of protection of a live Cryptobia salmositica vaccine administered IP to rainbow trout. Vaccinated fish were immune when challenged 1, 6, 12, and 24 months post vaccination, but the authors found no complement fixing antibodies circulating in the serum of vaccinated fish until after the challenge infections. However, vaccinated fish produced antibodies earlier than control fish when challenged. In the present study, circulating serum antibodies were not persistent, but reappeared faster in primed fish than in controls after challenge. This result suggests that memory B-cells persisted in the primed fish. Glochidia develop in the brooding chambers (marsupia) of parental mussels where they receive nutrition [40, 41] and calcium [42]. Lampsilis reeveiana is a long-term brooder, and glochidia may be held in female s marsupia for up to 10 months in the field or longer if kept in captivity. It is remarkable that glochidia within the marsupia remain viable for months, but degrade within days after release from the female. In the present study, glochidia that were held in parent mussels for 8 months had lower metamorphosis success than younger glochidia (Table 3, Figure 5). It is not known whether the decline of older glochidia is due to a failure of nutritive exchange late in the brooding season or to aging of the glochidium per se. Adult mussels

14 14 typically have reduced glycogen and lipid concentrations in their gills during the brooding season [43, 44]. Glochidia offer a very convenient system for investigation of the fish immune system. Although L. reeveiana and most other North American species are single-brooded, glochidia are available most of the year, and the present results show that viability can remain high for up to 7 months (Figure 5). Infections are benign, easily controlled and quantified, and metamorphosis success provides an ecologically relevant end-point. Future research should include a passive immunization study where antibodies to glochidia are isolated from the serum of immune fish and are injected intraperitoneally into naïve fish to show that antibodies may be a potential mechanism. Analysis of mucus antibodies is advisable. Investigation of the involvement of nonspecific or specific cell-mediated responses to glochidia is also needed. Acknowledgements Funding was provided by the United States Fish & Wildlife Service, Missouri Department of Conservation, Southwest Missouri State University, and Wake Forest University. We thank Dan Beckman, Elizabeth Bowen, Raymond Kuhn, John Heywood, Janice Horton, Janice Moll, Richard Myers, Joe Newton, and Clifford Starliper for their advice and assistance throughout this study. We greatly appreciate the assistance of Robert Brown, Christian Hutson, and Bri Kaiser in the lab and field. We would also like to thank Andy Cornforth and Dennis Whelan at Chesapeake State Fish Hatchery for providing fish for this study.

15 15 References [1] Arey LB. The nutrition of glochidia during metamorphosis: A microscopical study of the sources and manner of utilization of nutritive substances. Journal of Morphology 1932; 53: [2] Fisher GR, and Dimock RV Jr. Ultrastructure of the mushroom body: digestion during metamorphosis of Utterbackia imbecillis (Bivalvia: Unionidae). Invertebrate Biology 2002; 121(2): [3] Watters GT. An annotated bibliography of the reproduction and propagation of the Unionoidea (primarily of North America). Ohio Biological Survey Miscellaneous Contributions No.1. pp [4] Reuling, FH. Acquired immunity to an animal parasite. Journal of Infectious Diseases 1919; 24: [5] Arey LB. The formation and structure of the glochidial cyst. Biological Bulletin 1932; 62: [6] Meyers TR, Millemann RE. Glochidiosis of salmonid fishes. I. Comparative susceptibility to experimental infection with Margaritifera margaritifera (L.) (Pelecypoda: Margaritanidae). Journal of Parasitology 1977; 63(4): [7] Meyers TR, Millemann RE, Fustish CA. Glochidiosis of salmonid fishes. IV. Humoral and tissue responses of Coho and Chinook salmon to experimental infection with Margaritifera margaritifera (L.) (Pelecypoda: Margaritanidae). Journal of Parasitology 1980; 66(2):

16 16 [8] Bauer G. The parasitic stage of the freshwater pearl mussel (Margaritifera margaritifera L.) II. Susceptibility of brown trout. Archiv fur Hydrobiologie Supplement 1987; 76: [9] Waller DL, Mitchell LG. Gill tissue reactions in walleye Stizostedion vitreum vitreum and common carp Cyprinus carpio to glochidia of the freshwater mussel Lampsilis radiata siliquoidea. Diseases of Aquatic Organisms 1989; 6: [10] O Connell MT, Neves RJ. Evidence of immunological responses by a host fish (Ambioplites rupestris) and two non-host fishes (Cyprinus carpio and (Carassius auratus) to glochidia of a freshwater mussel (Villosa iris). Journal of Freshwater Ecology 1999;14(1): [11] Arey LB. Observations on an acquired immunity to a metazoan parasite. Journal of Experimental Zoology 1924; 38: [12] Rogers, CL, Dimock RV Jr. Acquired resistance of bluegill sunfish Lepomis macrochirus to glochidia larvae of the freshwater mussel Utterbackia imbecillis (Bivalvia: Unionidae) following multiple infections. Journal of Parasitology 2003; 89(1): [13] Young M, Williams J. The reproductive biology of the freshwater pearl mussel Margaritifera margaritifera (L.) in Scotland II. Laboratory Studies. Archiv Fuer Hydrobiologie 1984; 100(1): [14] Watters GT, O'Dee SH. Shedding of unmetamorphosed glochidia by fishes parasitized by Lampsilis fasciola Rafinesque, 1820 (Mollusca: Bivalvia: Unionidae): evidence of acquired immunity in the field? Journal of Freshwater Ecology 1996; 11:

17 17 [15] Hastie LC, Young MR. Freshwater pearl mussel (Margaritifera margaritifera) glochidiosis in wild and farmed salmonid stocks in Scotland. Hydrobiologia 2001; 445: [16] Bauer G, Vogel C. The parasitic stage of the freshwater pearl mussel (Margaritifera margaritifera L.) I. Host response to glochidiosis. Archiv fur Hydrobiologie Supplement 1987; 76: [17] Dodd BJ, Barnhart MC, Rogers-Lowery CL, Fobian TB, Dimock RV Jr. In press. Cross resistance of largemouth bass to glochidia of unionid mussels. Journal of Parasitology. [18] Hines RS, Spira DT. Ichthyophthiriasis in the mirror carp Cyprinu carpio (L.) V. Acquired Immunity. Journal of Fish Biology 1974; 6: [19] Li. S, Woo PTK. Efficacy of a live Crytobia salmositica vaccine, and the mechanism of protection in vaccinated rainbow trout Oncorhynchus mykiss, against cryptobiosis. Veterinary Immunology and Immunopathology 1995; 48: [20] Wang X, Dickerson HW. Surface immobilization antigen of the parasitic ciliate Ichthyophthirius multifiliis elicits protective immunity in channel catfish (Ictalurus punctatus). Clinical and Diagnostic Laboratory Immunology 2002; 9(1): [21] Burkart MA, Clark TG, Dickerson HW. Immunization of channel catfish, Ictalurus punctatus Rafinesque, against Ichthyophthirius multifiliis (Fouquet): killed versus live vaccines. Journal of Fish Diseases 1990; 13: [22] O Dee SH. Immune response of largemouth bass, Micropterus salmoides, to the fatmucket, Lampsilis radiata luteola (Unionidae), over repetitive infestations and overwintering. Ohio State University, M.S. thesis

18 18 [23] Williams JD, Warren ML, Cummings KS, Harris JL, Neves RJ. Conservation status of freshwater mussels of the United States and Canada. Fisheries 1993; 18(9):6-22. [24] NNMCC (National Native Mussel Conservation Committee). National strategy for the conservation of native freshwater mussels. Journal of Shellfish Research 1998; 17(5): [25] Iwama G, Nakanishi T. The fish immune system: organism, pathogen, and environment. Academic Press. San Diego, CA. pp [26] Young M, Williams J. The reproductive biology of the freshwater pearl mussel Margaritifera margaritifera (L.) in Scotland I. Field Studies. Archiv Fuer Hydrobiologie 1984; 99(4): [27] Graf DL Sympatric speciation of freshwater mussels (Bivalvia: Unionoidea): a model. American Malacological Bulletin 1997; 14(1): [28] Haag WR, Warren ML Jr. Effects of light and presence of fish on lure display and larval release behaviors in two species of freshwater mussels. Animal Behaviour 2000; 60: [29] Barnhart MC. Making mussels. Missouri Conservationist 2003; 64(8):4-9. [30] Clem LW, Miller NW, Bly JE. In: Warr G, Cohen N (Eds.), Phylogenesis of Immune Functions. CRC Press, Boca Raton, FL pp [31] Thomas PT, Woo PTK. An in vitro study on the haemolytic components from Cryptobia salmositica (Sarcomastigophora: Kinetoplastida). Journal of Fish Diseases 1989; 12: [32] Thomas PT, Woo PTK. Complement activity in Salmo gairdneri Richardson infected with Crytobia salmositica (Sarcomastigophora:

19 19 Kinetoplastida) and its relationship to the anaemia in cryptobiosis. Journal of Fish Diseases 1989; 12: [33] Clark TG, Dickerson HW. Antibody-mediated effects on parasite behavior: Evidence of a novel mechanism of immunity against a parasitic protist. Parasitology Today 1997; 13(12): [34] Clark TG, Dickerson HW, Gratzek JG, Findly RC. In vitro response of Ichthyophthirius multifiliis to sera from immune channel catfish. Journal of Fish Biology 1987; 31: [35] Clark TG, Dickerson HW, Findly RC. Immune response of channel catfish to ciliary antigens of Ichthyophthirius multifiliis. Developmental and Comparative Immunology 1988; 12: [36] Salati F. Vaccination against Edwardsiella tarda. In AE Ellis (Editor), Fish Vaccination. Academic Press, London; 1988, pp [37] Mehta M, Woo PTK. Acquired cell-mediated protection in rainbow trout, Oncorhynchus mykiss, against the haemoflagellate, Cryptobia salmositica. Parasitology Research 2002; 88: [38] Buchmann K. Immune mechanisms in fish skin against monogeneans a model. Folia Parasitologica 1999; 46:1-9. [39] Arey LB. A microscopical study of glochidial immunity. Journal of Morphology 1932; 53: [40] Wood EM. Development and morphology of the glochidium larva of Anodonta cygnea (Mollusca: Bivalvia). Journal of Zoology, London 1974; 173:1-13.

20 20 [41] Schwartz ML, Dimock RV Jr. Ultrastructural evidence for nutritional exchange between brooding unionid mussels and their glochidia larvae. Invertebrate Biology 2001; 120: [42] Silverman H, Kays WT, Dietz TH. Maternal calcium contribution to glochidial shells in freshwater mussels (Eulamellibranchia: Unionidae). Journal of Experimental Zoology 1987; 242: [43] Jadhav ML, Lomte VS. Seasonal variation in biochemical composition of the freshwater bivalve, Lamellidens corrianus. Rivista Idrobiologia 1982; 21:1-17. [44] Pekkarinen M. Seasonal changes in calcium and glucose concentrations in different body fluids of Anodonta anatina (L.) (Bivalva: Unionidae). Netherlands Journal of Zoology 1997; 47:31-45.

21 21 Table legends Table 1. Description of host fish, priming, and challenge infections. A group of 40 fish was exposed to 3 successive priming infections, along with controls. Subsets of these primed fish and naïve controls were later used for three challenge infections. DPII = days post initial infection. N=number of host fish. Fish body mass and the intensity of infection (number of initially attached glochidia per fish) are indicated as means ± SD. Asterisks indicate significant differences between primed and control fish (two-tailed t-test, p 0.05) Table 2. Priming and challenge infection results. Metamorphosis success indicates percent of attached glochidia that were recovered as live juveniles. The duration of successful parasitism indicates days from attachment to excystment of live juveniles. Numbers are means ± standard deviation (see Table 1 for sample sizes and DPII). Asterisks indicate significant differences between control and primed groups. Different letter superscripts indicate significant differences among priming infections. Table 3. The effect of glochidia age on the metamorphosis success and duration of successful parasitism of L. reeveiana glochidia on naïve largemouth bass. Values are mean ± standard deviation.

22 22 Figure legends Figure 1. Experiments used to test the duration of acquired resistance of largemouth bass to L. reeveiana glochidia (a) and the presence of serum antibodies (b). Priming and challenge refer to the timing of infections with glochidia. Extraction refers to the timing of sacrifice and blood extraction. Numbers in parentheses are host sample sizes (experimental, control). Figure 2. The development and duration of acquired resistance of largemouth bass to Lampsilis reeveiana glochidia. Graphs show the time course of recovery of glochidia and juveniles from bass during the 3 priming infections, and the challenge infections which took place 3, 7, and 12 months post priming. Recovery graphs for corresponding control (naïve) fish are adjacent to the graphs for experimental fish. Bars indicate the mean number of glochidia or juveniles recovered per host fish, and error bars represent standard error. Figure 3. Frequency distribution of L. reeveiana metamorphosis success on individual largemouth bass. Fish (n=40) received three successive priming infections, then a portion of the primed fish was challenged (re-infected) 3, 7, and 12 months post priming to test the duration of acquired resistance to glochidia. Figure 4. Antigens in L. reeveiana glochidia extract that bound with whole serum of previously infected largemouth bass. SDS-Page and Western Blot were used to separate the proteins of L. reeveiana glochidia and to identify the antigens recognized by bass serum, respectively. Molecular weight (MW) standards (kda) were run simultaneously to identify the size of the glochidia proteins (1). The following lanes represent antigens that were bound by bass serum

23 23 after: one infection (2), three infections (3), three infections with three months rest (4), three infections with seven months rest (5), three infections with twelve months rest (6), and four days post re-infection following three infections with seven months rest (7). Figure 5. The effect of glochidia age on the metamorphosis success of Lampsilis reeveiana glochidia on naïve largemouth bass. Values are means ± SE. Data are shown in Table 3.

24 24 Table 1. Infection DPII Hosts n Mass (g) Intensity Priming 1 -- Naïve ± ± 134 Control -- Naïve ± ± 101 Priming 2 25 Primed ± 3.7* 487 ± 132 Control -- Naïve ± ± 123 Priming 3 60 Primed ± 3.7* 428 ± 136 Control -- Naïve ± ± 60 Challenge Primed ± 4.1* 474 ± 119* Control -- Naïve ± ± 105 Challenge Primed ± ± 96 Control -- Naïve ± ± 108 Challenge Primed ± ± 94

25 25 Table 2. Infection Duration of successful parasitism (days) Metamorphosis success (%) Priming ± ± 6.6 Control 19.7 ± ± 9.8 Priming ± 1.2* 38.1 ± 17.4* % of control 90.3 ± 6.0 a 62.6 ± 28.6 a Control 26.0 ± ± 4.2 Priming ± 2.5* 28.0 ± 21.4* % of control 67.1 ± 9.6 b 34.0 ± 26.0 b Control 18.6 ± ± 5.5 Challenge ± 1.0* 26.5 ± 21.5* % of control 80.2 ± 5.1 c 31.3 ± 25.4 Control 18.2 ± ± 2.3 Challenge ± 2.2* 40.0 ± 31.7* % of control 83.0 ± 12.3 a, c 43.3 ± 34.3 a, b Control 19.5 ± ± 2.7 Challenge ± 2.0* 68.1 ± 15.7* % of control 84.3 ± 10.1 a, c 74.1 ± 17.1 a

26 26 Table 3. Infection Glochidia age (days) Number of mussels Number of host fish Metamorphosis success (%) Duration of successful parasitism (days) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.9

27 27 Figure 1. Days post initial infection a b 0 Priming (40) Priming (40) 25 Priming (40, 10) Priming (40) 60 Priming (40, 9) Priming (40) 102 Extraction (14, 9) 161 Challenge Extraction (12, 10) (10, 8) 275 Challenge Extraction (7, 7) (4, 4) 426 Challenge Extraction (6, 7) (1, 2)

28 28 Figure st Priming Glochidia Juveniles nd Priming 200 Control Number per fish rd Priming Challenge (3 month) Control Control Challenge (7 month) Control Challenge (12 month) Control Days post infection

29 29 Figure Priming Infections 1 (n=40) 4 3 Challenge Infections 3 month (n=12) Number of fish (n=40) month (n=7) (n=40) month (n=6) Metamorphosis success (%)

30 Figure 4. 30

31 31 Figure Transformation success (%) Age of glochidia (days)