REPRODUCTION AND PROPAGATION OF THE NEOSHO MUCKET, LAMPSILIS RAFINESQUEANA

Transcription

1 REPRODUCTION AND PROPAGATION OF THE NEOSHO MUCKET, LAMPSILIS RAFINESQUEANA A Thesis Presented to The Graduate College of Southwest Missouri State University In Partial Fulfillment of the Requirements for the Degree Master of Science by Melissa Ann Shiver May 31, 2002

2 REPRODUCTION AND PROPAGATION OF THE NEOSHO MUCKET, LAMPSILIS RAFINESQUEANA Biology Department Southwest Missouri State University, May 31, 2002 Master of Science Melissa Ann Shiver A B S T R A C T The Neosho mucket is a freshwater bivalve that occurs in the upper Arkansas river system in SE Kansas, NE Oklahoma, NW Arkansas and SW Missouri. Timing of reproduction was compared at two sites in the Spring River system: Shoal Creek at Joplin, MO and Spring River at Carthage, MO. Monthly samples of 6-30 individuals were examined at each site in 2001 for the presence of brooded larvae. Females spawned in May and brooded eggs and larvae from May through July. This timing is atypical of Lampsilis species, which typically brood continuously from fall through summer. Gonad fluid was collected in the field and examined microscopically for occurrence of eggs or sperm. Observations of gametes in gonad fluid samples indicate that Neosho muckets undergo gametogenesis in the late summer and early fall. This pattern appears consistent with the primitive lampsiline pattern of fall spawning. At Shoal Creek, 90% of females reproduced in 2001, but only 40% of the females at Spring River did so. Sterilizing trematodes (Rhipidocotyle sp.) were observed in gonad samples from some individuals in both populations. These samples lacked gametes. Shell size differed significantly between the two populations. Mean lengths of Spring River and Shoal Creek shells were 96.4 and 71 mm, respectively. Growth curves derived from shell annuli were compared among ages, sexes, and sites. Size was consistently smaller at the same inferred age in Shoal Creek. Host acquisition of immunity to glochidia was tested by repeated infestations of largemouth bass. Transformation success decreased from 87% to 55% when bass were exposed twice in succession. Transformation success of a related species, L. cardium, on bass decreased from 68% to 23% after previous exposure Neosho mucket glochidia. These results demonstrate that immunity develops after infestation and extends to closely related species. This abstract is approved as to form and content Chairman, Advisory Committee Southwest Missouri State University ii

3 REPRODUCTION AND PROPAGATION OF THE NEOSHO MUCKET, LAMPSILIS RAFINESQUEANA by Melissa Ann Shiver A Thesis Submitted to the Graduate College of Southwest Missouri State University in Partial Fulfillment of the Requirements for the Degree of Master of Science May 2002 Approved: Chairperson Member Member Associate Vice President for Academic Affairs and Dean of the Graduate College iii

4 ACKNOWLEDGMENTS I would like to thank my major advisor, Dr. Chris Barnhart for leading me through these past two years. He not only introduced me to freshwater mussels, but also inspired in me a keen interest in all invertebrates. He taught me that anything can be done at the last minute, and everything interesting is worth doing. I want to thank him for putting in the long hours to help me make this work possible. I would also like to thank my committee members, Dr. John Havel and Dr. Dan Beckman. A special thanks goes to Janet Sternburg who first introduced me to mussels and gently pushed me to return for my Master s degree. She provided the justification I needed to go back to school. I would also like to thank Sue Bruenderman, Andrew Roberts, Brian Obermeyer and Susan Rogers. Their support in providing information and resources made this work possible. I would like to thank those who helped me in the field, including Fawn Kirkland, Matt Stone, Shannon Bigham, Angela Delp, and Christian Hutson. Without them I would not have been able to reach the river. Thank you to Chesapeake Fish Hatchery and Missouri Department of Conservation for providing equipment, space and fish at the hatchery. I would like to thank James Peterson and Chris Schmitt, USGS, for their help in finding water quality information regarding my field sites. My largest support came from my parents, Marylyn and Fred Shiver, Jane Rackers, and Donald Spradley. I would not be the person I am without their confidence in me and the tremendous support they lend me in every day life. Funding for this work was provided by U.S. Fish and Wildlife Service, the Missouri Department of Conservation, and the Graduate College and Biology Department of Southwest Missouri State University. iv

5 TABLE OF CONTENTS Abstract... ii Title page... iii Acknowledgments... iv List of Tables...vi List of Figures... vii Introduction...1 Methods...7 Results...12 Discussion...41 Literature Cited...51 Appendix A...65 v

6 LIST OF TABLES Page Table 1. Summary of collections at Shoal Creek site...58 Table 2. Summary of collections at Spring River site...58 Table 3. Table 4. Table 5. Table 6. Survival following gonad sampling...59 Results of Kolmogorov-Smirnov test for ovum diameter and ratio of membrane to ovum diameter...60 Number of females whose ova were measured...61 Dimensions of Neosho mucket shells...62 Table 7. Size at successive growth lines of Neosho muckets...62 Table 8. Table 9. Table 10. Transformation of Neosho mucket glochidia on largemouth bass...63 Transformation of Neosho mucket on largemouth bass previously exposed to Neosho mucket glochidia...63 Transformation of plain pocketbook on largemouth bass previously exposed to Neosho mucket glochidia...64 vi

7 LIST OF FIGURES Page Figure 1. Lateral views of female and male Neosho muckets...3 Figure 2. Distribution map and field sites...19 Figure 3. Water temperature at the study sites...20 Figure 4. Field observations of brooding in Neosho muckets at two sites in the Spring River System, Missouri...21 Figure 5. Mortality versus frequency of gonad sampling...22 Figure 6. Photographs of cells and trematodes in gonad fluid samples...23 Figure 7. Figure 8. Figure 9. Frequency distributions of vitelline membrane diameter in gonad fluid samples...24 Frequency distributions of ovum diameters in gonad fluid samples...25 Frequency distributions of the ratio of membrane to ovum diameter in gonad fluid samples...26 Figure 10. Ovum diameter versus membrane diameter...27 Figure 11. Figure 12. Figure 13. Figure 14. Figure 15. Figure 16. Proportion of males with gametes and sperm morulae...28 Shell distributions of 191 Neosho muckets from two sites in the Spring River system, Missouri...29 Shell length to height ratio of male Neosho muckets at two sites in the Spring River system, Missouri...30 Shell length to height ratios of female Neosho muckets at two sites in the Spring River system, Missouri...31 Shell length to height ratios of Neosho muckets at Shoal Creek, Missouri...32 Shell length to height ratios of Neosho muckets at Spring River, Missouri...33 vii

8 Figure 17. Figure 18. Figure 19. Figure 20. Figure 21. Figure 22. Figure 23. Shell size at each growth line of male Neosho muckets...34 Shell size at each growth line of female Neosho muckets...35 Shell size at each growth line at Shoal Creek...36 Shell size at each growth line at Spring River...37 Transformation success of Neosho mucket glochidia on largemouth bass...38 Transformation success of Neosho mucket glochidia on previously exposed largemouth bass...39 Transformation success of plain pocketbook on largemouth bass previously exposed to Neosho mucket glochidia...40 viii

9 INTRODUCTION The Neosho mucket, Lampsilis rafinesqueana (Frierson 1927), a bivalve in the family Unionidae, is one of approximately 300 freshwater mussels that inhabit North America. This species is endemic to the Neosho, Spring, Verdigris, Illinois and Elk River systems in southeast Kansas, northeast Oklahoma, northwest Arkansas and southwest Missouri (Buchanan 1980, Obermeyer 1996, Obermeyer et al. 1997, Barnhart 2000). The Neosho mucket and many other freshwater mussels are threatened with extinction. Unionid mussels continue to be the most highly threatened and rapidly declining group of freshwater organisms in North America (Vaughn and Taylor 1999). This rapid decline is the result of a variety of factors for which man is mostly responsible. Overharvest is one of these factors. As early as the 1800 s, unionids were harvested and used for the manufacture of pearl buttons (Coker 1919). Later, beginning in the 1950's, the shell material was used as nuclei in the cultured pearl industry. The river habitat of mussels has been altered and degraded by construction of reservoirs, poor agricultural practices, gravel mining, deforestation, and contaminants. The results include erosion, increased siltation, decline of host fish, and even the introduced populations of exotics, such as the zebra mussel (LeFevre and Curtis 1910, 1912, Keller and Zam 1990, Bogan 1993, Vaughn 1998). Mussels are sedentary filter feeders, and they may remain in the same area for their entire lifespan, which may be as great as 100 years. Therefore, they are susceptible to habitat alteration (Vaughn and Taylor 1999). The Neosho mucket is currently classified at the state level as endangered in Kansas (Ken Brunson, Kansas Department of Wildlife and Parks, personal 1

10 communication) and Oklahoma (Oklahoma Department of Wildlife Conservation 2001) and a watch species in Missouri (Missouri Natural Heritage Program 2001). The Neosho mucket is currently classified as a candidate species for federal endangered status by the U.S. Fish and Wildlife Service (USFWS 2001). The general morphology of the Neosho mucket enables it to be identified easily in its habitat. Size of the Neosho mucket varies among sites, but the Neosho mucket is generally larger than other species in its habitat. The outer surface of the shell contains diagnostic characteristics (Utterback 1915, Oesch 1984). The shell is oblong, with height about times the length of the shell. The umbones are low and project only slightly or not at all above the dorsal curvature of the shell. Viewed from the side, the dorsal margin is gently rounded and the ventral margin is straight to gently curved. The anterior end is rounded. At the posterior end, the female is relatively taller than the male and inflated in the marsupial area (Figure 1). The shell is relatively thin and compressed in Shoal Creek specimens, but may be heavy and thick in other rivers, particularly in older specimens. Growth-rest lines are fairly prominent. The epidermis is usually light brown. Young specimens are marked with green, discontinuous, rays (chevrons). These markings provide positive identification if present (Figure 1). On the inside of the shell, the left valve has two stout, divergent, striated, triangular pseudocardinal teeth. The two lateral teeth are short, stout, and slightly curved, and the beak cavities are relatively shallow. The nacre is usually bluish-white to white and slightly iridescent towards the posterior (Utterback 1915, Oesch 1984, Couch 1997). 2

11 Figure 1. Lateral views of female (left) and male (right) Neosho muckets, showing the sexual dimorphism of the shells. Anterior ends are at left. Chevrons are shown more clearly on the female shell (figures Karen Couch 1997, used by permission). Compared to other bivalves, freshwater mussels mature slowly, reaching sexual maturity at anywhere from 2-12 years in age (Day 1984, Riusech and Barnhart 2000, Payne and Miller 2000). Up to millions of eggs are produced annually by each female (Bauer 1994). Two patterns of timing of reproduction may be seen within Unionidae. Short-term breeders (tachytictic 1 ) fertilize their eggs during spring or summer, and release their larvae within a few weeks. Spawning is over by the end of August. Longterm breeders (bradytictic 2 ) fertilize their eggs during late summer or fall and carry the fully developed glochidia through the winter until the following spring or summer (Ortmann 1911). Apparently all members of the genus Lampsilis and nearly all members of related genera (subfamily Lampsilinae) that have been examined are bradytictic, producing their eggs in the fall and releasing larvae the following spring or summer (Ortmann 1911). However, there is little information regarding the timing of reproduction of the Neosho mucket. Gravid females have been collected in June, July, 1 from the Greek roots tachy (fast), tict (to give birth) and ic (relating to). 2 as above, except brady (slow). 3

12 and August, but several females examined in January were not brooding (Barnhart and Roberts 1997, Barnhart 1999, 2000, and personal communication). Age and Growth The shell of freshwater mussels consists of three parts, the periostracum, the nacre, and the prismatic layer. The periostracum and the prismatic layer are secreted from the mantle only at the growing edge of the shell. The deposition of the innermost layer, the nacre, beneath these layers continues throughout the life of the mussel (Purchon 1977, Thorp and Covich 2001). Lines form in the shell during pauses in growth. These lines are called growth lines, and they potentially provide a record of age, size, and growth over time. This record of growth can reveal the history of environmental or manmade disturbances such as changes in temperature, water levels, nutrient availability, chemical events, etc. (McCuiag and Green 1983,White and Green 1998, Riusech 1999). This record might provide information that could aid in the management and conservation of species and give insight into the decline of species. Growth rings are also called annuli. In Latin, annu refers to annual, or yearly, while annul means a ring. The word annulus is often assumed to imply annual, although this is not implicit in the term. Growth lines are often assumed to form annually during the winter, when growth is slowed by low temperature, but there is debate about this. Growth lines might form more or less frequently than annually (Downing et al. 1992, Kesler and Downing 1997). For this reason, we will not assume that the rings are yearly, but merely that they mark pauses in growth of the animal. Because, the two sites are very 4

13 close in proximity, we can assume that the mussels at both sites experience the same periods of growth in relation to climate (McCuaig and Green 1983). Propagation Glochidia are obligate parasites on the gills or fins of fish and must successfully parasitize a host in order to metamorphose into the juvenile form. The female mussel increases her chances of coming in contact with fish by a special adaptation, the mantle flap lure. In Lampsilis and related genera, the lure is a modification of the mantle edge. When a fish strikes at the mantle flap lure, the marsupial gills are ruptured, releasing the glochidia into the surrounding water. The fish is then in direct contact with the glochidia, and a few glochidia are able to attach to the gills of the fish. Closure may be initiated by fibrinogen in the blood of the host (Henley and Neves 2001). The glochidia then complete their development as a parasite on the fish. After completing transformation they drop from the host fish (Jansen 1990). Transformation requires days to weeks, depending on temperature (Kat 1984). After transformation, the juvenile must fall into a substrate suitable for adult life in order to survive. Because the survival rate of glochidia is poor, a very large number of glochidia must be produced for successful reproduction. On the average, over 99.99% of glochidia fail to reach a suitable fish host (Young and Williams 1984, Jansen and Hanson 1991). The gravid females of Lampsilis do not release the entire contents of the gills in any one encounter with a fish (Haag and Warren 1999). This habit allows multiple encounters with the potential fish host and increases the likelihood that the glochidia will reach a suitable fish host and attach. 5

14 The host specificity of the glochidia ranges from generalists, which use a wide variety of fish, to specialists, which use only one or a few host fish (Haag and Warren 1999). Known fish hosts for the Neosho mucket are the largemouth bass (Micropterus salmoides), smallmouth bass (M. dolomieu), and the spotted bass (M. punctulatus) (Barnhart and Roberts 1997). Fish may be immune to glochidia. Two types of immunity are known. Fish species that are not suitable hosts demonstrate an intrinsic immunity to the glochidia. This immunity is nonspecific and may involve an inflammatory response (Arey 1932). Even suitable host species exhibit a variable tolerance for glochidia. Some individual fish transform a higher proportion of glochidia than others (Roberts and Barnhart 1999). Low temperature may suppress intrinsic immunity and allow higher transformation success in some species (Roberts and Barnhart 1997). The second type of host immunity is acquired following exposure to the glochidia, and is believed to involve a humoral response (similar to antibody production in vertebrates). (Bauer and Vogel 1987, O'Connell 1992, O'Connell 1999). Acquired immunity may depend upon the degree and frequency of infestation with glochidia, but little is known about this process (Watters and O Dee 1996). Another question is whether immunity is species-specific, or, alternatively whether immunity to one species could carry over to others. Freshwater mussel decline in recent years has prompted investigations to determine not only the causes of this decline, but a suitable solution to reverse that decline. If the proper fish host is known, it is possible to propagate glochidia in the lab by placing them on suitable host fish. This process increases the percentage of glochidia 6

15 that reach the fish host, and allows us to place the newly transformed mussels into a suitable habitat, thereby increasing the chances of survival. Some species can transform in an artificial nutrient medium (Isom and Hudson 1982, Keller and Zam 1990). The Neosho mucket is a suitable subject for my research for several reasons. First, conservation biologists have considerable interest in this species because of its limited range and recent declines in range and abundance. More information is needed in order to formulate recovery plans. Second, efforts to propagate this species are ongoing at Southwest Missouri State University. Thirdly, although this species is threatened, it remains fairly abundant in a few locations that are close to Springfield, Missouri, so that the species is accessible for field as well as lab study. The distribution of Neosho mucket populations is well known from recent surveys (Obermeyer 1996, Vaughn 1998) (Figure 2). Investigation of the reproductive biology of the Neosho mucket will facilitate ongoing efforts to propagate this mussel. The present study examines aspects of the reproductive biology of the Neosho mucket, including 1) the timing of reproductive events in the field, 2) differences in body size and growth rate between populations at two sites, and 3) the development of immunity by host fish, and whether host immunity to Neosho muckets affects tolerance of a related species, Lampsilis cardium. METHODS Study sites Field investigation of reproductive timing was conducted at two locations in the Spring River system, Shoal Creek (Newton County, Missouri), and Spring River (Jasper 7

16 County, Missouri). The two sites are separated by 26.5 km (16.5 mi) (Figure 1). Both are contained in the Spring River basin, which has a watershed area of 5,881 km 2 (2,271 mi 2 ). The Shoal Creek site is located just east, or upstream, of the old Highway 86 bridge crossing (T27N R33W S35), south of Joplin, MO. Watershed area at this site is 1,105 km 2 (427 mi 2 ). The Spring River site is located on the north side, or downstream of the Highway 96 bridge crossing (T29N R31W S34) at the downstream end of a small island, just east of Carthage, MO. The watershed area is 1,100 km 2 (425 mi 2 ). Twentythree field trips were made, 11 to each site, between February 2001 and February 2002 (Table 1-2). Field sampling I placed a temperature recorder (Onset Computer Optic Stowaway) at each site to record hourly temperature (± 0.1 o C) over a 1-year period (February 2001 February 2002). I visited both sites once a month over the 1-year period. During each visit my field helpers and I used snorkeling or a surface air supply to dive and observe mussels in the habitat. I examined > 5 females and 5 males during each visit. I sampled fluid from the gonads to determine when eggs and sperm were produced. I observed the marsupial gills to determine when the females brood glochidia. I numbered each individual captured using a Dremel rotary tool to engrave the shell. Length, height, and width were measured using digital calipers (precision ± 1 mm). The females were considered to be brooding if the marsupial gills contained eggs or embryos. I used 1cc, 18-gauge syringes to withdraw approximately 0.1 ml of fluid from gonads and/or from the swollen marsupia. Mussels were then placed back into the 8

17 substrate approximately where they were found. Dead shells were also collected and removed at each site when encountered. Gonad fluid analysis Samples of gonad fluid or marsupial contents were kept on ice, returned to the lab, and observed under a compound microscope, normally within 12 h. Gametes were photographed under magnification in order to record the development of egg and sperm throughout the annual cycle. I used a digital camera (Nikon Coolpix 900) and compound microscope (Nikon Eclipse E400). Digital pictures of the eggs were measured using Sigma Scan software (SPSS, Inc.). The longest and shortest diameters of each vitelline membrane or ovum were measured and averaged. These averages were used in all subsequent calculations. The distributions of ovum diameter and the ratio of membrane to ovum diameter were tested for seasonal variation using the Kruskal-Wallis test. The significance of differences between specific periods was examined with the Kolmogorov- Smirnov test (Sokal and Rohlf 1981). Age and Growth Sex of individuals was determined by several criteria. Females were distinguished from males by the swollen marsupial region of the posterior shell, pigmentation of the marsupial gill margin, and by observation of ova in the gonad samples of females or sperm in the gonad samples of males. The shell length was compared among sexes and among sites. The shell height/length ratio was compared among males and females. 9

18 Dead shells collected at each site were examined to determine growth in relation to shell size. Only shells with both valves were used. The shells were bleached and cleaned to facilitate a count of the growth lines. I used transmitted light to examine the major growth lines and distinguish them from other marks on the shell. Lines identified as major growth lines were continuous around the perimeter of the shell, darker, and broader than other marks. I laid a strip of tape across each shell from the umbone to the posterior edge of the shell, and made marks at the outer edge of each major growth line using a pencil. I taped this transect to another sheet of paper and measured the distance from the umbone to each growth line using a digital calipers (precision of ± 1 mm). Propagation Three transformation experiments were performed on largemouth bass. First, transformation success of Neosho mucket glochidia was determined on largemouth bass. Second, these "experienced" bass were infested a second time and compared with a second group of "naïve" fish that had not been previously exposed. Third, experienced and naïve bass were infested with a related species of mussel, the plain pocketbook (Lampsilis cardium). The bass were obtained from Chesapeake Fish Hatchery, Chesapeake, Missouri, and were all members of the same cohort of fish produced at the hatchery. Glochidia were flushed from the marsupium of each female using a hypodermic needle and syringe (Lasee 1991). Viability of glochidia was determined using salt to induce closure of the valves (LeFevre and Curtis 1912). The number of glochidia flushed from the females was determined using volumetric counts. Glochidia were 10

19 suspended in a known volume of water by agitating with a turkey baster. A volumetric pipette was used to remove 200-µl samples, which were placed in plastic Petri dishes. Each sample was examined under magnification and the glochidia were counted. At least 5 samples were counted and averaged for each suspension, and the total number of glochidia was estimated volumetrically. After the fish were swum with the glochidia, the remaining glochidia were concentrated by filtration and the number remaining was estimated as above. In the first transformation experiment, fish were anesthetized using Finquel (MS 222) and the glochidia were pipetted over the gills. In the other two experiments, the fish were infested by swimming for minutes in an aerated bucket that contained glochidia. Groups of 6-11 fish were placed in 25-gallon tanks on a shared, recirculating filtration system. Each tank comprised a replicate, with 3-4 replicates per treatment group. A temperature recorder was placed in one of the tanks to record the temperature every hour during the experiment. Live juveniles and empty shells were recovered by siphoning and filtration (125- µm sieve) every other day, and were counted to determine the number that attached and transformed and the number sloughed (Roberts 1997). The fish were starved throughout each experiment. When each experiment was over, the fish were fed for one month before the next experiment began. 11

20 RESULTS Field observations Temperature was remarkably similar at both study sites. The average temperature from 3/27/01-1/17/02 was and o C at the Spring River and Shoal Creek sites, respectively, a difference of only 0.32 o C (Figure 3). A total of 173 Neosho muckets were marked and examined, and 75 of these were recaptured at least once during the study. At the Shoal Creek site, 115 individuals were marked and 31 of these were recaptured at least once. The average time between the first and last observation of recaptured individuals was 81.5 days ± Gonad samples were taken from 105 Shoal Creek individuals, some of which were sampled more than once (Tables 1, 3). At the Spring River site, 58 individuals were marked and 43 of these were recaptured at least once. The average time between the first and last observation of recaptured individuals was 132 (± 81.1) days. Gonad samples were taken from 50 Spring River individuals, some of which were sampled more than once (Tables 2, 3). Brooding Brooding females were first observed on May 15 in Shoal Creek and May 29 in Spring River. Six of 16 females observed on these dates were brooding. Samples were drawn from the marsupial gills of two of these females. The brooded embryos of both appeared to be at the gastrula stage of development. One month earlier (April 19 and May 3, respectively) none of 19 females examined were brooding (Tables 1, 2). The latest that brooding females were observed was July 10 at Shoal Creek and July 25 at the Spring River site. On those dates, 6 of 18 females were brooding. One month later 12

21 (August 7 and August 20, respectively) none of 17 females observed were brooding (Tables 1, 2). During the peak of brooding, 40% of females (4 of 10) were brooding at the Spring River site and 91% of females (11 of 12) found were brooding at the Shoal Creek site (Figure 4). Sterilizing trematodes Gonad fluid samples from 9 of 155 individuals sampled (5.8%) contained cercariae of bucephalid trematodes (4 in the Spring River and 5 in Shoal Creek). Only the cercaria stage was seen (Figure 6f). Gametes were not found in any of these 10 samples. Only one of these individuals was sampled twice. Those individuals with trematodes were disproportionately likely to die during the study (see below). Mortality Eight of the 75 individuals (10.6%) that were recaptured during this study were found dead (1 of 31 from Shoal Creek, 7 of 44 from Spring River). Four of the 8 that were found dead had trematode infections in the gonad (Table 3). These were 4 of only 5 individuals with trematodes that were recaptured (dead or alive) during the study. Individuals that had trematodes were significantly more likely to be found dead during the study than individuals that lacked trematodes (Fisher's exact probability test, p= ). The number of times that gonad fluid samples were taken from an individual did not correlate significantly with the probability that an individual would later be found dead (Table 3, Figure 5; Spearman Coefficient of Rank Correlation: r s = 0.351, p>0.05). 13

22 Gametes in gonad samples Gonad fluid samples were examined to confirm sex and to characterize seasonal changes in gametes. The cells observed in gonad fluid samples were identified based upon morphology (Figure 6). Eggs and sperm were very distinctive. Each egg consists of an ovum surrounded by a vitelline membrane (Figure 6a). Ova usually were µm in diameter (Figures 56, 7). The vitelline membrane was usually closely appressed to smaller ova but expanded around larger ova, forming a vitelline space. Sometimes empty membranes were seen in gonad fluid samples (Figure 6e). Spermatids were approximately 5 µm in head length with a long flagellum (Figure 6b, 6c). Cells or cell clusters identified as sperm morulae were round, approximately 30µm diameter. They were partitioned into a number of conical sections, presumably destined to become spermatids (Figure 6b). Cells classified as granulocytes were approximately 10 µm in diameter, amoeboid, with many short, pointed pseudopods. The granulocytes frequently formed larger clumps (Figure 6d). Hyalinocytes were small (~10 µm), smooth, and spherical (Figure 6e). Both granulocytes and hyalinocytes were identified by comparison with published figures and descriptions (Kennedy et al. 1996). In total, 155 individuals were sampled for gonad fluid at least once, and 260 gonad fluid samples were examined. Of all samples, 117/260 (45%) had no gametes and no trematodes present. These included 74/136 (54%) gonad samples from males, and 36/124 (29%) of gonad fluid samples from females. Seven samples had trematodes and no gametes (no sample with trematodes had gametes). Of the 136 samples that contained gametes, 74 had eggs and 62 had sperm. One sample had both egg membranes and sperm. The individual from which this sample was taken was captured and sampled 5 14

23 times on different dates with the following results; 3/27/01- no gametes, 5/3/01 and 5/29/01- sperm, 6/27/01- both sperm and empty egg membranes (but no intact eggs), and 7/25/01- no gametes. The proportion of gonad samples that lacked gametes varied among collections but was not clearly seasonal (Table 1, 2, Appendix A). A few samples (3 of 155) contained digestive fluid, identified by color and the presence of diatoms, suggesting that the gut or digestive gland may sometimes have been sampled, rather than the gonad. Measurements of ovum diameter (OD), vitelline membrane diameter (MD), and the ratio of these measures (MD/OD) were grouped into six bimonthly periods: December-January, February-March, April-May, etc. (Figures 7, 8, 9). The medians of OD, MD, and MD/OD all varied significantly among these 6 periods (Kruskal-Wallis, p< 0.001). The number of ova sampled in these periods varied, at least in part, because of the variable number of females collected (Table 4). Two-sample comparisons among periods revealed significant changes during the year (Kolmogorov-Smirnov two-sample tests, Table 5). Ovum size distribution was constant early in the year (December-May), shifted to larger diameters in June-July and to smaller diameters in August-September and October-November (Figure 7). Another process observed in the female gonad samples was the enlargement of the vitelline membrane around each ovum. The expansion of this membrane forms the vitelline space in which the embryo develops. Eggs whose membrane had not yet expanded were not distinguished from eggs that may have lost their vitelline membrane during the process of sampling (empty membranes were observed in some samples). The proportion of ova that lacked an expanded membrane did not vary greatly with season 15

24 (MD/OD = 1.0, Figure 9). In a plot of MD vs. OD, it can be seen that larger ova (greater than 70 microns diameter) tended to have a more expanded membrane (Figure 10) Sperm and sperm morulae were characteristic of male gonad fluid samples. Sperm were seen less often in late summer/early fall, and more often in spring and winter months (Figure 11). Morulae were sometimes present in the fall, winter, and spring, but were absent in summer samples (Figure 11). Size and shape comparisons The shell length, height, and width of 191 individuals were measured (Table 6). Females and males from the Spring River were all significantly longer than those from Shoal Creek (t-tests, p <0.001)(Table 6, Figure 12). The overall mean shell length (both sexes combined) was 71 mm ± SD for Shoal Creek, and 96.4 mm ± 7.49 SD for Spring River. Males and females from the Shoal Creek population were significantly different in length (t-test, p <0.001) but the sexes did not differ in length at the Spring River site (t-test, p = 0.996) (Figure 12). The ratio of shell height to length was used as a simple metric for comparison of shell shape versus size, among sexes, and among sites. Combining sites, there was no significant correlation of shell shape with length in either males (Figure 13, p = 0.423) or females (Figure 14, p = 0.751). The shape of males differed significantly from that of females at both sites (p<0.001). Male shells were less tall, relative to length, than females (Figures 15, 16). There was no significant difference in the shape of males between sites or of females between sites (t-tests, females: p = , males: p = 0.379). 16

25 Age and growth A total of 28 shells were used for age and growth analysis: 7 males and 5 females from the Shoal Creek site, and 7 males and 9 females from the Spring River site. Measurements of shell size at successive growth lines showed that females grow more slowly than males, and that animals grew more slowly at the Shoal Creek site than at the Spring River site (Table 7, Figures 17, 18, 19, 20). Propagation In the first experiment, 4 replicate groups of 8-9 fish were infested with glochidia pooled from three female Neosho muckets from the Shoal Creek site. The fish ranged in size from 6.2 cm to 8.9 cm in total length. Transformed juveniles fell off from days after infestation. Peak drop-off occurred at 20 days (Figure 21). In total, 3,674 individuals were recovered (2,843 juveniles and 831 dead glochidia). 71% transformed to the juvenile stage (Table 8, Figure 21). The temperature of the water in the tanks was approximately 22.6 C. In the second experiment, 6 groups of fish (32 naïve fish, 31 experienced fish) were infested with glochidia from one female Neosho mucket taken from the Shoal Creek site. Transformed juveniles fell off from days. Peak drop-off occurred at 25 days. Approximately 78% (3,184/3,642) of the individuals recovered from the naïve group of fish were transformed juveniles and the rest were dead glochidia (Table 9, Figure 22). Approximately 53% (2,263/4,171) of individuals recovered from the experienced group were transformed (Table 9, Figure 22). There was a significant difference between the transformation success of the two groups (t-test, p< 0.05). 17

26 Approximately 39% (23,440/60,250) of the glochidia that attached to the fish were recovered. In the third experiment, 6 groups of 6-11 fish (19 naïve fish and 31 experienced fish) were infested with glochidia from three female plain pocketbook mussels from the Sac River. Fewer naïve fish were used in the test groups because they were of larger body size. Transformed juveniles dropped off between days. Peak drop-off of juveniles occurred at 35 days. Approximately 63% (3733/6152) of the plain pocketbook glochidia transformed on the naïve fish and only 23% (838/5093) transformed on the experienced fish (Table 10, Figure 23). There was a significant difference in transformation success between the two groups (t-test, p < 0.01). There was also a significant difference between the sizes (standard length) of fish between the two groups (t-test, p < 0.01). Approximately 62% (33,733/54,300) of the glochidia that attached to the fish were recovered. Temperature of the water was approximately 21.1 o C. 18

27 Figure 2. The past and current distribution of the Neosho mucket (data from Obermeyer 1996, Vaughn 1998). Stars indicate field site locations. 19

28 Temperature (C) Spring River Shoal Creek Temperature (C) Temp difference (C) Mar Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb Time (hours) Avg Figure 3. Water temperatures at the study sites ( o C) recorded hourly from February 2001 to March The third graph shows the difference in temperature between the two sites (Shoal Creek temperature - Spring River temperature). Horizontal line indicates the mean temperature difference (-0.32 o C) 20

29 Shoal Creek Spring River Percent females brooding Feb Jun Oct Feb Collection Date Figure 4. Percent of females brooding each month from February 2001 to February 2002 at two sites in the Spring River System, MO. The numbers beside the symbols indicate the total females observed. 21

30 Percent dead (%) ' Number of times gonad sampled Figure 5. Mortality versus frequency of gonad sampling. Symbols indicate the percent of marked individuals that were found dead, in relation to the number of times that those individuals had been gonad-sampled. Numbers beside symbols indicate the sample size in each category. There was no significant correlation (See Table 4). 22

31 A B C D E F Figure 6. Examples of cells and other objects seen in gonad fluid samples. A. Eggs. B. Spermatids and sperm morulae. C. Spermatids forming chains. D. Individual and clumped granulocytes. E. Empty egg membranes and round cells. F. Trematode cercaria. Scale lines in each panel are 100 µm. 23

32 Dec-Jan (N=144) Feb-Mar (N=32) Frequency (percent) Apr-May (N=185) Jun-July (N=23) Aug-Sept (N=29) Ovum diameter (µm) Oct-Nov (N=218) Figure 7. Frequency distributions of ovum diameter in gonad fluid samples of Neosho muckets in (Both sites were pooled). 24

33 30 20 Dec-Jan (N=144) Feb-Mar (N=32) Frequency (percent) Membrane diameter (µm) Apr-May (N=185) Jun-July (N=29) Aug-Sept (N=23) Oct-Nov (N=218) Figure 8. Frequency distributions of egg vitelline membrane diameter in gonad fluid samples of Neosho muckets in (Both sites were pooled). 25

34 Frequency (percent) Dec-Jan (N=144) Feb-Mar (N=32) Apr-May (N=185) Jun-July (N=29) Aug-Sept (N=23) Oct-Nov (N=218) Ratio (µm) Figure 9. Frequency distributions of the ratio of membrane to ovum diameter in gonad fluid samples during (Both sites were pooled). 26

35 Membrane diameter (µm) Ovum diameter (µm) Figure 10. Ovum diameter versus membrane diameter. Each point represents a single egg in a gonad fluid sample. Points along the diagonal line (MD = OD) represent eggs with either unexpanded membranes or missing membranes. 27

36 with sperm with morulae Proportion of males Dec-Jan Feb-Mar April-May June-July Aug-Sept Oct-Nov Sample date (bimonthly) Figure 11. Proportion of males that have sperm and the proportion of males that have morulae in their gonad fluid samples. Numbers beside symbols indicate the total number of males sampled. 28

37 Shoal males Number of individuals Shoal females Spring males Spring females Shell length (mm) Figure 12. Shell length distributions of 191 Neosho muckets from two sites, Shoal Creek, Joplin, MO and the Spring River, Carthage, MO. 29

38 0.8 Spring River males Shoal Creek males Shell height/length (mm) Shell length (mm) Figure 13. Comparison of shell length to height ratio in Neosho mucket males at two sites, Shoal Creek and the Spring River. 30

39 0.8 Spring River females Shoal Creek females Shell height/length (mm) Shell length (mm) Figure 14. Comparison of the shell length to height ratio of Neosho mucket females at two sites, Shoal Creek, Joplin, MO and the Spring River, Carthage, MO. 31

40 0.8 Males Females Shell height/length (mm) Shell length (mm) Figure 15. Comparison of shell length to height ratios of Neosho mucket females and males at the Shoal Creek site, Joplin, MO. 32

41 0.8 Males Females Shell height/length (mm) Shell length (mm) Figure 16. Comparison of shell length to height ratios of Neosho mucket females and males at the Spring River site, Carthage, MO. 33

42 Shell height (mm) Spring River Shoal Creek Growth lines Figure 17. Shell height (mm) measurements at each growth line of male Lampsilis rafinesqueana at Shoal Creek and Spring River. Symbols indicate means ± 95% confidence intervals. The curves were fitted by logistic regression. 34

43 Shell height (mm) Spring River Shoal Creek Growth lines Figure 18. Shell height (mm) measured at each growth line in female Lampsilis rafinesqueana at Shoal Creek and Spring River. Symbols indicate means ± 95% confidence intervals. The curves were fitted by logistic regression. 35

44 Shell height (mm) Females Males Growth lines Figure 19. Shell height (mm) measured at each growth line in female and male Lampsilis rafinesqueana at Shoal Creek. Symbols indicate means ± 95% confidence intervals. The curves were fitted by logistic regression. 36

45 Shell height (mm) Growth lines Females Males Figure 20. Shell height (mm) measured at each growth line in female and male Lampsilis rafinesqueana at Spring River. Symbols indicate means ± 95% confidence intervals. The curves were fitted by logistic regression. 37

46 Numbers of individuals Glochidia Juveniles Time (days) Figure 21. Transformation success of Neosho mucket juveniles on largemouth bass, Micropterus salmoides at 22 o C. 38

47 Naive Glochidia Juveniles Percent falloff Experienced Glochidia Juveniles Time (days) Figure 22. Comparison of transformation success of Neosho mucket juveniles between naïve largemouth bass and bass that have been previously infested with Neosho mucket glochidia. Temperature = 20 o C. 39

48 Naive Glochidia Juveniles Percent falloff of plain pocketbook Experienced Glochidia Juveniles Time (days) Figure 23. Comparison of transformation success of plain pocketbook juveniles between naïve largemouth bass and bass that have previously been infested with Neosho mucket glochidia. Temperature = 21 o C. 40

49 DISCUSSION Reproductive timing Mussels in the genus Lampsilis and other members of the subfamily Lampsilinae (sensu Ortmann 1911) are generally long-term brooders (bradytictic) spawning in late summer or fall and brooding during the winter, releasing glochidia the following spring or summer (Conner 1909, Ortmann 1911, LeFevre & Curtis 1912, Utterback 1915, Watters and O Dee 2000). The present study indicates that the Neosho mucket is different from its congeners in that it is a short-term brooder (tachytictic). Eggs appeared in the marsupial gills in May and were brooded until about the end of July (Figure 3). Only a few other reports and hints of tachyticty in Lampsilines have been published. Obliquaria reflexa is gravid only during the spring and summer months (LeFevre and Curtis 1912, Utterback 1915V). Utterback (1915VI) also suggests (without detail) that Truncilla donaciformis may be tachytictic. Finally, there is an unpublished report that Lampsilis anodontoides (= Lampsilis teres) may be tachytictic (Ellis 1928). Very few other Lampsilines are known to be short-term brooders. Therefore, this character appears likely to be derived in Lampsilis rafinesqueana. Somewhere along the lineage of this species, the period of spawning was shifted from fall to spring. This change presumably occurred by delaying spawning rather than advancing it, because the period of glochidial release (summer) is unchanged from the presumed ancestral pattern, and spawning can only occur after the previous brood has been released. Why is spawning delayed in this species, and does the shortened brooding period have any adaptive significance? One possibility is that the eggs and larvae are at risk during brooding in the gills, perhaps from microbial predators or bacteria. If so, perhaps 41

50 reducing the duration of brooding reduced mortality of larvae before their release from the gills. A second possibility has to do with the timing of allocation of resources. Assuming mussels are in negative energy balance during the winter, there may have been an advantage in spring spawning, because the female could assess energy reserves and make a "decision" about whether reserves were adequate to support the production of a brood. Another interesting aspect of the odd timing of spawning in Neosho muckets is the possibility that such a change from the ancestral condition (bradyticty) may have been a crucial step in the origin of this species. Presumably, sperm release and fertilization take place at the same time that females release eggs from the gonads into the marsupial gills. The timing of fertilization therefore must have been shifted to spring. Presumably, the ancestor of Neosho muckets spawned in the fall. A population that shifted its spawning to the spring would be reproductively isolated from the rest. It is interesting to speculate about the synchronization of male and female spawning during such a shift in reproductive timing. Little is known about mechanisms of spawning synchronization in Unionoids. If the sexes communicate chemically, it seems that the communication must be one-way and that males must lead, because they must be upstream to fertilize the female. In order for the female to shift the timing of spawning, the males only ability to track the process would be evolutionarily, with only those males that spawn synchronously with females leaving their genes in the next generation. Are Neosho muckets capable of brooding over the winter? I tested this question by holding 4 brooding Neosho muckets for 6 months (August 2001-January 2002) at 10 42

51 o C. The glochidia stayed in the gills, but all died. This result suggests that the Neosho mucket may have lost the ability to brood over the winter. However, more experiments are needed to determine whether this result can be confirmed. Mussels of the genus Lampsilis display a mantle flap lure to attract the host fish. The hosts of Neosho mucket are probably primarily black basses (Barnhart and Roberts 1997). Neosho mucket females possess a mantle flap lure, although the pigment eyespots present on the lure of many other species are indistinct or absent. During lure display the female generally emerges partly from the substrate and adopts a headstand posture with the posterior and the lure elevated (Kat 1984). I noted that most individuals of both sexes were more deeply buried in the substrate from late fall into early spring. Recaptures, mortality, and trematodes About half of the individuals that were marked were recaptured at least once (Tables 1, 2) Recaptures were more frequent at the Spring River site, probably because many of the marked animals were concentrated in a small area of relatively uniform and fine-grained substrate. At the Shoal Creek site, the marked animals were more widely distributed and the substrate was rocky and heterogeneous. Recaptures permitted us to observe mortality of marked animals. About 10% (8/75) of the individuals that were marked and recaptured were found dead during the course of the study. Presumably, dead individuals were no more or less likely to be found than live ones. Did the study increase mortality? Unfortunately, there is no control group for this question, because all of the monitored individuals were marked and otherwise disturbed during the study. 43

52 However, it is possible to test for possible effects of two factors on mortality within the study group: gonad sampling and trematode infestation. Trematode infection was significantly associated with mortality. The overall incidence of trematode infestation in this study was 5.8%, and the incidence in the recaptured animals was similar, 5.3%. The incidence of mortality of recaptured individuals with and without trematodes was 80% and 5.3%, respectively. Sterilizing trematodes (Bucephalidae, Rhipidocotyle sp.) are common parasites of bivalves (the intermediate host) and fish (the definitive host). They cause sterilization of the bivalve host by penetrating the gonad, allocating the host s reproductive energy for the asexual production of larval trematodes called cercariae (Lauckner 1983, Jokela, Uotila, and Taskinen 1993). The presence of trematodes may also have an impact on host response to environmental factors (Taskinen 1992). According to Lackner, trematodes eventually cause death in the mussel (Lackner 1983). Gonad fluid sampling Apparently very few previous studies utilized gonad fluid sampling (Bauer 1987). The procedure seemed to work well to sex individuals and monitor reproductive status, rather than sacrificing individuals. It was even possible sometimes to identify female samples without microscopy, because the ova are large enough to see by eye. However, a large fraction of gonad fluid samples did not contain gametes (54% of male samples and 29% of female samples). A higher percentage of samples might contain gametes if a larger volume of fluid were taken or if multiple samples were taken. Efficiency might also be improved by careful study of the position of the gonad and determination of the 44