INTERIM PERFORMANCE REPORT ENDANGERED SPECIES PROGRAM GRANT NUMBER F17AP01052 WILDLIFE PROJECTS – ALABAMA PROJECT Reproductive Characteristics and Host Fish Determination of Canoe Creek Clubshell, Pleurobema athearni (Gangloff et al. 2006) in Big Canoe Creek drainage (Etowah and St. Clair Counties), Alabama October 1, 2018 - September 30, 2020 ALABAMA DEPARTMENT OF CONSERVATION AND NATURAL RESOURCES WILDLIFE AND FRESHWATER FISHERIES DIVISION Prepared by: Todd B. Fobian Alabama Division of Wildlife and Freshwater Fisheries PROJECT Reproductive Characteristics and Host Fish Determination of Canoe Creek Clubshell, Pleurobema athearni (Gangloff et al. 2006) in the Big Canoe Creek drainage (Etowah and St. Clair Counties), Alabama Year 1 Interim Report State: Alabama Introduction Pleurobema athearni (Gangloff et al, 2006), Canoe Creek Clubshell is currently a candidate for federally threatened/endangered status by U.S. Fish and Wildlife Service (FWS). It is Coosa Basin endemic, with historical records only known from the Big Canoe Creek (BCC) system in Alabama (Gangloff et al. 2006, Williams et al. 2008). Recent surveys completed by ADCNR and USFWS established the species is extant at six localities in the basin, with two in Upper Little Canoe Creek (ULCC), one in Lower Little Canoe Creek (LLCC), and three in BCC proper. (Fobian et al. 2017). As culture methods improve, propagated P. athearni juveniles could soon be available to support reintroduction/augmentation efforts within historical range. Little is known about Pleurobema athearni reproduction, female brooding period, or glochidial hosts. Female P. athearni are presumed short term-brooders and likely gravid from late spring to early summer (Gangloff et al. 2006, Williams et al. 2008). Glochidial hosts are currently unknown although other Mobile River Basin Pleurobema species often utilize Cyprinidae (shiners) to complete metamorphosis (Haag and Warren 1997, 2003, Weaver et al. 1991, Williams et al. 2008). The focus of this study was to determine reproductive periodicity and fish host relationships. Methods Brood stock collection was completed in the LLCC, St. Clair/Etowah Counties Alabama by visual search with view buckets by Alabama Department of Conservation and Natural Resources (ADCNR) biologists (Table 1). Mussel brooders were acquired during multiple collection attempts in May and June 2019 (Table 2). All mussels encountered during searches were identified, photographed, and measured. A total of six gravid females were transported in a chilled insulated cooler with towels soaked in river water to the AABC (Alabama Aquatic Biodiversity Center, Marion, AL). Gravid females were placed in aerated 2 L plastic holding trays inside a 14° C incubator. Trays were filled with water collected from the brood stock site and 50% of the volume was replaced during the brooding period. The host trials were initiated on June 11, 2019. Pleurobema athearni glochidia were collected after self-release by the female for each trial. Post release brood stock were tagged with small numbered plastic bee tags adhered with super glue gel on the left valve and returned to site of capture. We assessed host suitability of 22 fish species across 5 families in laboratory trials. These species were selected based on previous published Pleurobema host studies (Haag and Warren 1997, 2003, Weaver et al. 1991, Williams et al. 2008) and likelihood of co-occurrence in typical P. athearni habitats. Fish were collected from three sites (Table 3) by backpack shocking and seining then transported to the AABC. Fishes were held in flow-through systems supplied with well water and were treated daily with kanamycin (75 µg/L) for three days prior to infection. Host trials used 1– 39 individuals of each potential host species depending on availability. All fishes used in host trials were euthanized (MS-222), preserved in ethanol, and identified following taxonomy in Boschung and Mayden (2004). We conducted separate host trials for each fish collection site (n = 3). The glochidia of two to three mussels were used for each trial depending on the amount required for the number and size of host fish tested (Table 3). We took care to avoid using non-viable glochidia and did not utilize glochidia liberated during transport or the long holding period. Pleurobema spp. are known to abort developing conglutinates or small numbers of glochidia prematurely (Williams et al. 2008), but even fully developed larvae are viable for less than 48 hours after release. Fish from Terrapin Creek (Cleburne County, Alabama), Carrol Creek (Tuscaloosa County, Alabama) and Marion Fish Hatchery (Perry County, Alabama) were utilized in host trials initiated between May 30, 2019-June 1, 2019 (Table 3). Viable glochidia were placed into a water bath at a density of ~4,000 glochidia/L. Fish were placed into the glochidia water bath for 15 min. with heavy aeration. Following inoculation, unattached glochidia were rinsed away by placing fish into clean water. Fish were then transferred to size appropriate containers and held individually. Host suitability was monitored in modified recirculating aquarium systems (Pentair Aquatic Eco-Systems AHAB®) for smaller fishes and flow through 50 gal conical tanks for larger individuals. The recirculating systems consisted of an array of 1, 3 and 9 L tanks. Water temperature was maintained at 18 ± 2ºC with Aqualogic Inc. Cyclone chillers. Recirculated water was filtered, and UV sterilized before returning to the tanks. AHAB tanks received approximately 0.5 L water/min and the outflow of each tank entered a filter cup with a 153 µm nylon screen to collect sloughed glochidia and juveniles. Large conical tanks were equipped with a double stand pipe to draw water from the bottom of the tanks and their effluent was filtered through a 153 µm nylon sock. Filters were examined daily after inoculation and every 2- 6 days thereafter until no glochidia or juveniles were recovered for at least 4 days. Before examining the filters, flow volume was increased to approximately 2 L/min in the AHAB aquaria and 15 L/min in the conical tanks for 15 min to ensure that all particles were collected in a filter cup or sock. Each filter was rinsed into a glass dish and the number of glochidia and juveniles from each fish were counted under a Nikon SMZ745T stereomicroscope. Juveniles mussels were distinguished from untransformed larvae by the presence of an active foot and fully formed adductor muscles. After no juveniles or glochidia had been recovered for 7 days, fishes were euthanized and examined for remaining encapsulated glochidia. Standard length was recorded, and the fish was preserved in ethanol or frozen. The number of glochidia that initially attached to each fish was calculated as the sum of the number of glochidia and juveniles recovered from the fish throughout the trial. Successful metamorphosis was calculated as the percentage live juveniles from the total attached glochidia. Hosts defined as “primary” if ≥ 50% of attached glochidia transformed to the juvenile stage, “marginal” if 0.1–50% transformed, and “unsuitable” if no transformation occurred (Haag and Warren 1997, 2003). Timing of juvenile metamorphosis was determined by minimum and maximum range and modal time to peak metamorphosis (DPI, days post infection) for juveniles recovered from individual fish. Results Brood Stock Collection Brood stock was collected on 15 May 2019 and 29 May 2019 at one site in LLCC St. Clair/Etowah Counties. Water temperature was 16.5º C and 21.0º C respectively. A total of 20 P. athearni ranging in size from 61–76 mm shell length were examined including six that were brooding developing embryos or glochidia (Table 2) Reproductive Life History Demibranchs of non-gravid animals were thin and slightly transparent, in contrast to gravid demibranchs which appeared thicker and either cream white, orange, or pink in color. Outer demibranchs of all gravid females were slightly inflated. Mussels were held at 16°C in aerated plastic trays with Little Canoe Creek water from 5/15/19 and slowly warmed to 22.5 °C by 5/30/19. Females released white or orange lanceolate- shaped conglutinates with developed glochidia scattered throughout unfertilized structural eggs (Figure 1 and 2). Glochidia color varied from white to orange, following the color of the structural eggs. Prior to volumetrically counting glochidia, released conglutinate material was mechanically disrupted by sending conglutinates through a 250µm nylon screen filter cup and subsampled to estimate viability. Viable glochidia used for infections was obtained from three females (Table 4). Viable glochidia (infection 1: n = 11,430 from three females; infection 2, n = 11,750 from two females; infection 3: n = 5,250 from two females) were placed into a water baths of 2.8, 2.9 and 1.32 L respectively (4,000–4,082 glochidia/L). Fish (infection 1: n = 140; infection 2: n = 75; infection 3: n = 26) were placed into the glochidia water bath for 15 min. with heavy aeration. Glochidia were unhooked and had a mean (± SD) length 135.2 ± 8.29 µm and height 134.7 ± 8.67 µm (Table 5, Figure 2). Tagged brood stock were returned to site of capture on 5 June 2019 by ADCNR biologists. Juveniles were recovered from host fishes 10–25 days after inoculation. The mean time (± SD) to peak juvenile recovery was 19 ± 3 days post-inoculation at 18º C ± 2º C (Figure 3). Successful metamorphosis of glochidia was observed on 10 of 22 species tested and 3 of 5 families (Table 6). Two of cyprinid species were primary hosts (Cyprinella trichroistia, Tricolor Shiner and Cyprinella callistia, Alabama Shiner). Eight species from three families were marginal hosts (Luxilus chrysocephalus, Striped Shiner; Campostoma oligolepis, Largescale Stoneroller; Notropis xaenocephalus, Coosa Shiner; Notropis stilbius, Silverstripe Shiner; Pimephales promelas, Fathead Minnow; Notemigonus crysoleucas, Golden Shiner; Lepomis megalotis, Longear Sunfish; and Percina palmaris, Bronze Darter) (Table 6). One marginal host (L. chrysocephalus, n = 1) had 34.6% metamorphosis while other marginal hosts had less than 7% metamorphosis (Table 6).