INTERIM PERFORMANCE REPORT

ENDANGERED SPECIES PROGRAM

GRANT NUMBER

F17AP01052

WILDLIFE PROJECTS –

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 (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 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 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 ( 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).

Tentative Schedule of Tasks during Project Extension Amendment period October 1, 2019 –

September 30, 2020:

Spring and Summer field data collection:

• Repeat 2019 host trial methods with additional families, species, and larger sample sizes.

Statistical analyses of data and write up: July 2020-December 2020

• Analyze host fish metamorphosis data and complete final report.

Literature Cited

Boschung, H. and R. Mayden. 2004. Fishes of Alabama. Smithsonian Books, Mobile, AL. 736 pp.

Fobian, T.B., M.L. Buntin, J.T. Holifield, J.T. Garner and P.D. Johnson. 2017. Freshwater Mollusks

(Mollusca: Unionidae and Gastropoda) in Big Canoe Creek (Etowah and St. Clair Counties),

Alabama. Final Project Summary to the U.S. Fish and Wildlife Service. 41pp.

Gangloff, M.M., Williams, J.D. and Feminella, J.W. 2006. A new species of freshwater mussel

(Bivalvia: Unionidae), Pleurobema athearni, from the Coosa River Drainage of Alabama,

USA. Zootaxa, 1118(1), pp.43-56.

Haag, W.R. and Warren Jr, M.L., 1997. Host fishes and reproductive biology of 6 freshwater mussel

species from the Mobile Basin, USA. Journal of the North American Benthological Society,

16(3), pp.576-585.

Haag, W.R. and Warren Jr, M.L., 2003. Host fishes and infection strategies of freshwater mussels in

large Mobile Basin streams, USA. Journal of the North American Benthological Society,

22(1), pp.78-91.

Mirarchi, R.E. ed. 2004. Alabama Wildlife: Volume 2: Imperiled Aquatic Wildlife. University of

Alabama Press, Tuscaloosa, AL. 256 pp.

Mobile Basin Mollusk Recovery Plan (MBMRP). 2010. Plan for the population restoration and

conservation of freshwater mollusks of the Mobile Basin. Vol +. 102 pp.

Weaver, L.R., Pardue, G.B. and Neves, R.J., 1991. Reproductive biology and fish hosts of the

Tennessee clubshell Pleurobema oviforme (Mollusca: Unionidae) in Virginia. American

Midland Naturalist, pp.82-89.

Williams, J.D., A.E. Bogan, J.T. Garner. 2008. Freshwater Mussels of Alabama and the Mobile

Basin in Georgia, , and Tennessee. The University of Alabama Press, Tuscaloosa,

AL. 908 pp. Wynn, E.A., O’Neil, P.E., McGregor, S.W., Powell, J.R. and Gangloff M. 2016. Watershed

Assessment of the Big Canoe Creek System for Recovery and Restoration of Imperiled

Aquatic Species. Geological Survey Alabama. Tuscaloosa, Alabama. Bulletin 185.

Table 1. Location of Pleurobema athearni brood stock collection site Site Stream Location County Coordinates Code LCC -48 Little Canoe Cr St. Clair / Etowah County 33.928989° Downstream of Steele -86.165388° Station Rd.

Table 2. Site, date and number of Pleurobema athearni examined for gravidity at brood stock

collection site in 2019.

Developing Stream Number Number Embryos or Temperature Site Date Examined Gravid Glochidia (C)

LCC -48 5/15/19 3 2 DE 16.5

LCC -48 5/22/19 4 0 -- 20.7

LCC -48 5/29/19 11 3 2G and 1DE 21.0

LCC -48 6/5/19 2 1 n/a 22.0

Table 3. Overview of Pleurobema athearni infection details in 2019. Infection # 1 2 3 Date 5/30/19 5/31/19 6/1/19 N Female Mussels 3 (#1, 2 and 4 Red) 2 (#2 and 4 Red) 2 (#2 and 4 Red) (ID #) Fish Mixed Sp./Terrapin Cr., Mixed Sp./Terrapin Golden Species/Location Mobile Cr. and Fathead Shiners/AABC Logperch/Carrol Cr. Minnows/AABC and Redeye Bass/AABC N Fish 140 75 26 N Infective 11,430 11,750 5,250 Glochidia Bath Volume (L) 2.8 2.9 1.32 Glochidia per L 4,082 4,000 4,000 Bath Volume per 0.02 0.04 0.05 fish (L) Glochidia per fish 140 157 202

Table 4. Overview of Pleurobema athearni female, identification number, glochidia release, size and conglutinate color, used for host fish infections in 2019 trials.

Mussel Date Total Infective Unfertilized Length Width Height Conglutinate ID # Glochidia Glochidia Eggs (mm) (mm) (mm) Color

#1 Red 5/30/19 5,500 5,400 21,900 75 29 54 Orange

#2 Red 5/30/19 2,800 1,080 1,680 61 20 42 Orange

#4 Red 5/30/19 5,200 4,950 3,750 76 22 49 White

#2 Red 5/31/19 7,750 1,750 5,500 61 20 42 Orange

#4 Red 5/31/19 24,300 10,200 12,000 76 22 49 White

#2 Red 6/1/19 10,650 3,000 5,850 61 20 42 Orange

#4 Red 6/1/19 16,500 2,250 7,750 76 22 49 White

#3 Red 5/31/19 Few early Few early Primarily 67 22 45 Orange ~10 embryos embryos Eggs conglutinates present

Total 2019 5,500 5,400 21,900 75 29 54 Orange #1 Red

Total 2019 21,200 5,830 13,030 61 20 42 Orange #2 Red

Total 2019 46,000 17,400 23,500 76 22 49 White #4 Red

Table 5. Mean glochidia dimensions of Pleurobema athearni collected from LLCC in 2019 (± S D) #Glochidia/#Females Length Height L/H ratio n = 36 / n = 3 135.2 ± 8.29 134.7 ± 8.67 1.01 ± 0.07

Table 6. Summary of Pleurobema athearni metamorphosis. *indicate low sample size Location/Species Mean Metamorphosis n Encysted Juveniles Juveniles/ [% ± Std Dev (range)] glochidia produced fish

Terrapin Creek, Cleburne County AL

Cyprinella trichroistia (Tricolor Shiner) 78.5 ± 15.9 (28.6–100) 39 1,594 1,316 33.7

Cyprinella callistia (Alabama Shiner) 73.6 ± 3.9 (70.8–76.3) 2* 86 63 31.5

Luxilus chrysocephalus (Striped Shiner) 34.6 1* 107 37 37

Campostoma oligolepis (Largescale Stoneroller) 7.4 ± 12.5 (0–31.3) 6 146 5 1.3

Notropis xaenocephalus (Coosa Shiner) 2.9 ± 7.6 (0–20) 7 52 1 0.1

Notropis stilbius (Silverstripe Shiner) 2.4 ± 6.3 (0–16.7) 7 36 1 0.1

Lepomis megalotis (Longear Sunfish) 0.9 1* 114 1 1

Percina palmaris (Bronze Darter) 0.8 ± 2.2 (0–6.3) 8 203 1 0.1

Hypentelium etowanum (Alabama Hogsucker) 0 1* 54 0 0

Lepomis cyanellus (Green Sunfish) 0 2* 66 0 0

Moxostoma erythrurum (Golden Redhorse) 0 2* 326 0 0

Lepomis macrochirus (Bluegill) 0 3* 89 0 0

Etheostoma coosae (Coosa Darter) 0 3* 15 0 0

Cottus carolinae (Banded Sculpin) 0 3* 4 0 0

Fundulus stellifer (Southern Studfish) 0 4* 26 0 0

Etheostoma stigmaeum (Speckled Darter) 0 2* 3 0 0

Etheostoma jordani (Greenbreast Darter) 0 15 62 0 0

Percina nigrofasciata (Blackbanded Darter) 0 2* 19 0 0 mixed Cyprinid species n/a 50 n/a 120 2.4

Carrol Creek, Tuscaloosa County, AL

Percina kathae (Mobile Logperch) 0 5 71 0 0

AABC, Perry County, AL

Micropterus coosae (Redeye Bass) 0 5 515 0 0

Pimephales promelas (Fathead Minnow) n/a 25 n/a 11 0.4

Notemigonus crysoleucas (Golden Shiner) n/a 26 n/a 2 0.1

Figure 1. Lanceolate Pleurobema athearni conglutinates, orange (above) or white (below).

A B

C D Figure 2. Conglutinate from Pleurobema athearni with developed glochidia scattered throughout unfertilized structural eggs (A and B above). Glochidia were unhooked and had a mean (± SD) length 135.2 ± 8.29 µm and height 134.7 ± 8.67 µm (C and D below).

20 18 16 14

12 Glochidia 10 Juveniles 8 6 4 2 Mean Number Glochidia/Juveniles Number Mean 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Day Post Infection

Figure 3. Pleurobema athearni glochidia and juvenile release from Cyprinella trichroistia (n = 5) collected from Terrapin Creek, Cleburne County, AL.