Reproductive phenology of E. complanata in Otsego County, NY

Reproductive phenology of E. complanata in Otsego County, NY Thomas Franzem 1, Paul Lord 2, Andrew Gascho-Landis 3, and Robin LaRochelle 4 ABSTRACT There is uncertainty about the timing of Elliptio complanata glochidia release in central New York. We conducted surveys from late April to early July to determine sex ratios and timing of glochidia brooding and release in a population of Elliptio complanata on the fringes of the Susquehanna watershed. Through mark and recapture and visceral mass and gill samples we found that this population has a slightly higher percentage of males than females. The percentage of males and females was much higher than the percentage of apparent hermaphrodites. By monitoring glochidia presence in the gills of mussels, we were able to determine a time frame for peak glochidia production and release. We concluded that the peak time for production in this population begins in late May, reaches its peak in early June, and tapers off in late June early July. The findings of this study have answered some of our questions about E. complanata s reproductive phenology, however, it raised questions too. During future projects, we plan on incorporating detection of hermaphrodites and multiple broods into our study design to hopefully broaden our understanding of E. complanata s lifecycle. Key words: Elliptio complanata, glochidia, visceral mass. 1 Undergraduate Research Aide, SUNY Oneonta. 2 Instructor, SUNY Oneonta. 3 Assistant Professor, SUNY Cobleskill. 4 Undergraduate, SUNY Cobleskill. INTRODUCTION The Eastern elliptio (Elliptio complanata; hereinafter referred to as Elliptio) is the most abundant and widespread unionoid in New York. Elliptio plays a crucial role in the environment by filtering water and consuming algae and zooplankton, making nutrients available to other animals that would otherwise be unavailable (Nedeau 2008). Despite its wide distribution and importance to freshwater ecosystems, there are gaps in our understanding of the life cycle of Elliptio. For example, the timing of the release of glochidia (the larval stage of freshwater mussels) is poorly understood. Glochidia are obligate parasites on fish; this is responsible for the dispersal of Elliptio. Researchers have documented different times for unionid glochidia release. For example, Matteson, as cited in Downing (1989), determined glochidia release to be mid to late July while Nedeau (2008) gives the entire summer season as the time of glochidia release. Lellis et al (2013) concluded that American eels (Anguilla rostrata) are the most effective and only host species for Elliptio glochidia in the Susquehanna watershed. Recent research prior to 2015 found no evidence of reproduction of Elliptio anywhere in the Susquehanna River, the Chemung River, the Canisteo River, the Tiougnioga River, the Chenango River, the Unadilla River, Butternut Creek, Catatonk Creek, or Wharton Creek (Lord & Pokorny, 2012; Lord et al., 2010; Lord & Harman, 2010; Lord & Harman, 2009, Harman et al., 2008). It is likely that the lack of reproduction and recruitment of Elliptios throughout the Susquehanna watershed is due to dams and impoundments impeding the movements of the

catadromous A. rostrata. Prior to 2015, there were no recent records of recruitment of Elliptios in the NY Susquehanna drainage. Recently observed recruitment is on the fringes of the NY Susquehanna drainage where watershed waters comingle with other watershed waters. (Landry, pers comm.) This focused our attention on the reproductive phenology of Elliptio. Attempts in the lab at SUNY-Oneonta to identify a glochidia release date for Elliptio have failed (Lentz, pers comm). The purpose of this study is to (1) determine sex ratios for Elliptio, (2) ascertain when females are brooding glochidia, and (3) establish when females are releasing glochidia. A better understanding of their life cycle and biology will inform future conservation efforts and facilitate research on this species and other freshwater mussel species. This study s results supports efforts to reintroduce A. rostrata to the Susquehanna. The stream reach we studied began at a DEC access point (UTM 18T491010 4702099) off NYS Route 23 in West Oneonta, NY. Starting approximately 30 m north of the Route 23 Bridge, divers worked upstream for approximately 146 m. The river was, on average, 15.4 meters wide. Depth ranged from less than a half a meter at the start of the reach to 1.5 + meters in some parts; the depth increased and decreased no more than.5m in response to precipitation. Concentrated in the center, the substrate was a mix of cobble, gravel, and sand. Both banks were silt. The water flowed through most of the stream at low velocity, however the downstream end of the reach was the beginning of a riffle. Surveys METHODS Full surveys were conducted on 23 April 2016, 18 May 2016, 1 Jun 2016, and 8 July 2016. In addition, four smaller surveys were conducted on 9 June 2016, 16 June 2016, 20 June 2016 and 27 June 2016 to attain water readings, sample for glochidia, and collect and examine 8+ marked mussels. Starting at the DEC access point, divers searched for mussels as they moved upstream. When an Elliptio was found, the position was marked with a GPS by a dive tender and a rebar stake was hammered into the riverbed to relocate mussels ready for replanting. We collected 20+ mussels per full survey. The mussels were brought to shore, where each captured mussel was tagged with two unique plastic Hallprint tags (Type FPN) (one on each valve). Cyanoacrylate glue (Seachem Flourish TM Glue; Hartmann et al., 2016) was used to attach the tags. Elliptios were pried open 7mm with a clam knife (Dexter Russel 3 Clam Knife) and fingers. A 7mm diameter wooden dowel was inserted between the valves. To extract sperm and eggs, we used the sampling techniques outlined by Saha and Layzer (2008). A syringe (Exel Int. 3ml Disposable Syringe) with 0.5mL of sterile water was inserted into the visceral mass. The point of injection was below the foot towards the posterior of the mussel. We injected the water and removed 1ml of visceral fluid. The syringes were labeled with the mussels tag number and stored in a cooler. The mussels were inspected for swollen or enlarged gills. If they were even slightly swollen, gill fluid was extracted using the same technique to extract the visceral fluid. After sampling, the mussels were provided to a dive tender. Using a combination of GPS points and rebar stakes to determine the location from which each tagged mussel was

originally extracted from the sediments, the Elliptio was handed to a diver. The diver dug a small hole using a hand rake and inserted the mussel s anterior-end into the riverbed and filled the hole, careful to leave the posterior-end of the mussel uncovered. On subsequent visits to the mussel bed, the tag numbers of recaptured mussels were recorded. We examined the recaptured animals for swollen gills; a gill sample was taken if the gills appeared swollen. We did not take visceral mass samples on the recaptured mussels unless there was evidence of hermaphroditism in an Elliptio. Laboratory Work The visceral mass and gill samples were brought to the laboratory and examined using a microscope. We first examined the samples using 10x magnification and inspected for eggs and glochidia. If neither of these were found, a cover slip was placed on the slide, the magnification was switched to 100x, and we examined the sample for sperm. Glochidia Sampling After glochidia was detected in some samples, we deployed a Wildco stream drift net (100 µm mesh) immediately downstream of the mussel bed (Culp et al. 2008) every time we sampled. Nets were placed in the water for 30 minutes between 10 am and 3 pm. During each sampling occasion, stream flow was measured at the net and the water height on the net was recorded for calculating the volume of water passing through the net during each sampling time. Samples were stained with phloxine B (Culp et al. 2011), numbered, and then brought back to the laboratory where they were examined for glochidia. Water Quality Measurements Starting on 18 May 2016 we recorded water quality readings for temperature, dissolved oxygen (DO), ph, salinity, conductivity, and total dissolved solids (TDS) from a YSI multiprobe. Sex Ratios RESULTS We determined the sex of 56 captured mussels: 23 were females, 27 were male, and 6 were apparent hermaphrodites (Figure 1). Two of the apparent hermaphrodites showed evidence of protandry and four were apparently protogynous. Glochidia Brooding On 23 April, we captured, tagged, and replanted twenty-one mussels; none were gravid. The following survey we captured twenty mussels, four were recaptures and four were gravid females. On 1 June 2016, we found twelve gravid females out of twenty-one mussels sampled (11 were recaptures). The following survey on 9 June 2016 was not a full survey; only nine mussels (all recaptures) were sampled. Of these nine, four were female. Three of the females were gravid, with either mature glochidia or developing glochidia. On 20 June, thirteen mussels

were captured; six were recaptures. The five untagged animals were not tagged during this survey, but visceral mass and gill samples were taken nonetheless. Of the thirteen captured, five were gravid. On 8 July 2016 we conducted our last full survey. Twenty mussels were captured; fifteen were recaptures. Of these twenty mussels only one was gravid (Table 1). The water temperature during the peak time for glochidia release ranged from 18.64 C on 1 June and 20.66 C on 20 June, however, the temperature dropped to 14.77 C on 9 June. Figure 1. Number of Eastern Elliptio (Elliptio complanata) males, females, and apparent hermaphrodites in Otego Creek survey area in 2016. Table 1: Summary of surveys conducted of Otego Creek in West Oneonta (Otsego County) NY. Count of males and females only of newly captured Eastern elliptios (Elliptio complanata) and does not include recaptures; the number of gravid individuals includes both new and recaptured animals. Survey Dates Total Captured Recaptures Male (Not counting recaptures) Female (Not counting recaptures) Apparent Hermaphrodites (Not counting recaptures) Number of Gravid Individuals 23-Apr-16 21 0 7 10 4 0 18-May-16 20 4 9 5 2 4 1-Jun-16 21 11 4 6 0 12 9-Jun-16 9 9 - - 0 8 20-Jun-16 13 8 4 1 0 5 8-Jul-16 19 15 3 1 0 1

Water Quality Measurements Water temperature showed an increasing trend; it was coldest on 18 May with a temperature of 8.67 C and warmest on 8-Jul at 21.45 C. DO steadily decreased across the time frame. Except for a spike in late June, salinity remained relatively consistent over our surveys. Conductivity generally increased from May to July. ph stayed within a range of 6.75 to 7.81 throughout the survey. TDS increased sharply between 20 June and 27 June, but other than that it remained relatively consistent (Table 2). Table 2: Water quality data collected associated with Eastern Elliptio (Elliptio Complanata) collected at Otego Creek in West Oneonta (Otsego County), NY. Survey Dates Temperature Dissolved Oxygen Conductivity Salinity ph TDS 18-May-16 8.67 C 13.20mg/L 107µs/cm 0.07 7.74 NA 1-Jun 18.64 C 9.10mg/L 159µs/cm 0.08 7.65 0.117 9-Jun-16 14.77 C 8.99mg/L 131µs/cm 0.08 6.75 0.106 16-Jun-16 19.39 C 8.38mg/L 163µs/cm 0.09 7.81 0.118 20-Jun-16 20.66 C 8.24mg/L 181µs/cm 1.09 7.51 0.128 27-Jun-16 19.22 C 7.49mg/L 187µs/cm 0.1 7.4 1.37 8-Jul-16 21.45 C 9.01mg/L 192µs/cm 0.1 7.53 1.34 Sex Ratios DISCUSSION The sex ratio we found is consistent with what we expected (Figure 1); Downing et al (1989) found that across size classes the percentage of males was higher than females and hermaphrodites. Of the six apparent hermaphrodites we found, four of them appeared protogynous, however there is no evidence of protogyny in this species. Downing (1989) concludes that Elliptio is a protandrous sequential hermaphrodite. In his study he defined hermaphrodites in this species as animals that had between 10% and 90% female gonad tissue. It is likely that during visceral mass samples we extracted female gonad tissue and on a subsequent survey recaptured the animal and extracted male gonad tissue when we sampled the visceral mass. It is possible that other captured Elliptios were hermaphrodites that we didn t detect; nonetheless the percentage of apparent hermaphrodites we found is similar to what Downing found. Apparent hermaphrodites were all found to be brooding glochidia at some point. Based on this, it is likely that these apparent hermaphrodites were functionally female; most hermaphroditic individuals are functionally gonochoristic (Haag 2012). Hermaphroditism in this population would be an interesting topic for future research. Glochidia Brooding We conclude, based on our data, that the peak time for brooding and release of glochidia in this population of mussels begins in mid to late May and continues for several weeks before tapering off in late June early July (Figure 2). We assume that some Elliptios are brooding and

releasing glochidia before and after this time frame, but the bulk of glochidia production and release for this population is within this period. Figure 2. Percent of female Eastern elliptio (Elliptio Complanata) brooding glochidia in Otego Creek survey area in 2016. Water temperature can be a cue for spawning and can alter the length of the brooding period and time of glochidia release in many species of freshwater mussels (Watters and O Dee 2000). It appears that different populations of Elliptio may release glochidia at different times. Furthermore, timing of glochidia brooding and release can change year-to-year within a population. Bearing this in mind, the population we studied has an average temperature preference of 18.37 C for glochidia production and release. Elliptio is able to produce at least two broods across a season (Price and Eads 2011). Detecting multiple glochidia broods were beyond the goals of this study. It is possible that some individuals in this population of Elliptio can produce multiple broods, however we did not detect any; we did not collect a gravid individual, recapture it to find it s not gravid, then capture it a third time and find it s gravid again. It could be they produce secondary broods later in the season. Detection of multiple broods should be incorporated into future studies on this population.

LITERATURE CITED Culp, J. J., W. R. Haag, D. A. Arrington, & T. B. Kennedy. 2011. Seasonal and speciesspecific patterns in abundance of freshwater mussel glochidia in stream drift. J. North Am. Benthol. Soc. 30(2):436-445. Downing, J. A., J. P. Amyot, M. Pérusse, and Y. Rochon. 1989. Visceral sex, hermaphroditism, and protandry in a population of the freshwater bivalve Elliptio complanata. J. North American Benthological Society. 8(1):92-99. Haag, W. R. 2012. North American Freshwater Mussels: Natural History, Ecology, and Conservation. New York (NY): Cambridge University Press. Hartmann, J. T., S. Beggel, K. Auerswald & J. Geist. 2016. Determination of the most suitable adhesive for tagging freshwater mussels and its use in an experimental study of filtration behaviour and biological rhythm. J of Molluscan Studies. Landry, Jenny, A.Personal communication with P. Lord. 2015. Lellis, W. A., B. St. John White, J. C. Cole, C. S. Johnson, E. van Snik, and H. S. Galbraith. 2013. Newly documented host fishes for the Eastern Elliptio Mussel Elliptio complanata. J. of Fish and Wildlife Management. 4(1):75-85. Lentz, Vicky. Discussion with P. Lord. 2016. Nedeau, E. J. 2008. Freshwater Mussels and the Connecticut River Watershed. Connecticut River Watershed Council, Greenfield, Massachusetts. xviii+132 pp. Price, J. E., and C. B. Eads. 2011. Brooding patterns in three freshwater mussels of the genus Elliptio in the Broad River, South Carolina. American Malacological Bulletin 29:121 126. Saha, S. & J. B. Layzer. 2008. Evaluation of a nonlethal technique for determining sex of freshwater mussels. J. North Am. Benthol. Soc. 27:84 89. Watters, G. T. and S. H. O Dee. 2000. Glochidia release as a function of water temperature: beyond bradyticty and tachyticty. In: R. A. Tankersley, D. I. Warmolts, G. T. Watters, B. J. Armitage, P. D. Johnson, and R. S. Butler, eds., Proceedings of the Conservation, Captive Care, and Propagation of Freshwater Mussels Symposium, 1998. Ohio Biological Survey, Columbus Ohio. Pp. 135-140