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Life history of the fluted kidneyshell ptychobranchus subtentum.

INTRODUCTION

The United States has the greatest freshwater mussel diversity in the world; however, approximately 70% of this fauna are considered imperiled (Williams et al., 1993; Strayer et al., 2004). Devastation of the unionid fauna began in the 19th century as a result of mass deforestation (Hughes and Parmalee, 1999), and has continued to the present time. Impoundments, sedimentation, channelization, dredging, point and non-point pollution, and competition with exotic species like the zebra mussel (Driessena polymorpha) are commonly cited reasons for the decline (Watters, 2000; Strayer et al., 2004). The decline of unionids is a serious concern because mussels comprise a large magnitude of biomass (Negus, 1966) and play an important role in freshwater ecosystems (Strayer et al., 2004). Mussels are suspension feeders and their activity improves water quality by filtering contaminants, sediments, and nutrients (Anthony and Downing, 2001; Vaughn and Hakenkamp, 2001; Strayer el al., 2004). Furthermore, mussels serve as indicators of good water quality and healthy aquatic ecosystems (Anthony et al., 2001; Christian and Harris, 2005).

[FIGURE 1 OMITTED]

The mussel fauna of Tennessee includes 45 Cumberlandian species that are endemic to the upper Cumberland and Tennessee Rivet drainages (Ortmann, 1925). The fluted kidneyshell Ptychobranchus subtentum (Say, 1825), a Cumberlandian species, occurs primarily in small rivers and streams (Parmalee and Bogan, 1998; Cicerello and Schuster, 2003). However, Ortmann (1925) and Morrison (1942) noted that the fluted kidneyshell was able to survive in larger rivers in shoal habitats similar to the conditions of a smaller stream. Ptychobranchus subtentum usually occurs in a sand and gravel substrate in areas with swift current and depth of 0.6 m or less during normal flow (Parmalee and Bogan, 1998; Cicerello and Schuster, 2003).

Historically, the fluted kidneyshell occurred in portions of the mainstem of the Cumberland River, Tennessee River, and at least 16 Cumberland River tributaries and 21 Tennessee River tributaries (Fig. 1). Populations may also have occurred in other streams within the Cumberlandian region that were never sampled historically (USFWS, 2009). The tinted kidneyshell has been extirpated from the mainstem of the Cumberland and Tennessee rivers and about 68% of known historical streams (USFWS, 2009). Current distribution is limited to six streams in the Cumberland River system and six in the Tennessee River system. The fluted kidneyshell was listed as a candidate species by the US Fish and Wildlife Service in 1999 (USFWS, 1999).

Most North American freshwater mussel species are considered to be either tachytictic (short-term brooders) or bradytictic (long-term brooders). Typically, tachytictic species spawn in the spring or summer and release glochidia (larvae) in the same summer. Bradytictic species spawn in late summer or fall and brood their glochidia until the following spring or summer (Ortmann, 1909). Gordon and Layzer (1989) summarized published data on gravidity of fluted kidneyshells. Based on that summary supplemented by personal observations (J. Layzer), fluted kidneyshells are gravid from Sept. through May. This species is unique because its glochidia are contained in packets (conglutinates) that resemble blackfly pupae (Simulidae)(Barnhart et al., 2008). When a fish inhales a conglutinate, it ruptures and releases glochidia that attach to the fish (Barnhart and Roberts, 1997; Haag and Warren, 1997). Known host fishes for Ptychobranchus subtentum include the rainbow darter Etheostoma caeruleum, barcheek darter E. obeyense, fantail darter E. flabellare, redline darter E. rufilineatum, and banded sculpin Cottus carolinae (Luo, 1993).

Knowledge of most other life history aspects and population demographics of Ptychobranchus subtentum is lacking. This information is necessary for successful conservation and management of this species. Reproductive biology including fecundity, size at sexual maturity, suitable fish hosts, and sex ratio are important characteristics of the life history of a species (Yeager and Saylor, 1995; The National Native Mussel Conservation Committee, 1998). Our study focused on describing various life history traits of P. subtentum. Specific objectives were to: (1) determine whether the time to metamorphosis was related to the length of time glochidia were brooded, (2) determine additional possible host fishes, (3) determine fecundity, (4) determine sex ratio, and (5) determine age and growth characteristics in a fluted kidneyshell population in the upper Clinch River.

METHODS

STUDY AREA

The largest population of the fluted kidneyshell occurs in the upper Clinch River (Fig. 1), Hancock County, Tennessee (Starnes and Bogan, 1988; Parmalee and Bogan, 1998; USFWS, 2009). Our study site on the Clinch River was located at Kyles Ford, Hancock County, Tennessee (Fig. 1). While most fluted kidneyshell populations have been extirpated or are declining, the population in the Clinch River at Kyles Ford has increased from a density of 5.56/[m.sup.2] in 1994 (Ahlstedt and Tuberville, 1997), to 16.20/[m.sup.2] in 2004 (Ahlstedt et al., 2005). Since 2004, >4000 adult fluted kidneyshells have been collected from the Clinch River and translocated to three other rivers to reestablish populations (D. Hubbs, pers. comm.).

The Wolf River, Pickett County, Tennessee contains one of the few remaining populations of the fluted kidneyshell within the Cumberland River drainage. Although the population is small, recruitment is occurring (Moles et al., 2007).

SPAWNING AND GLOCHIDIAL DEVELOPMENT

In Aug. and Sept. 2005, and in May through Sept. 2006, we monitored mussel gravidity in the Clinch River at Kyles Ford. We snorkeled or used view buckets to collect epi- and endobenthic mussels. We measured total length (greatest distance between the anterior and posterior margins parallel to the hinge) of each Ptychobranchus subtentum encountered. We then pried the mussel open and examined the outer gills to determine gravidity (marsupial gills are inflated when gravid). We took samples from the water tubes of gravid individuals by carefully inserting the needle of a hypodermic syringe and injecting dechlorinated water (Waller and Holland-Barrels, 1988). The water ruptured the tubes and flushed out conglutinates, which we stored in 70% ethanol. We monitored embryo development by examining the samples with a dissecting microscope at 40x and 100x. White egg masses were considered unfertilized eggs, while fertilized eggs were enclosed in a membrane. All gravid and nongravid individuals were returned to the substrate unless otherwise stated.

FECUNDITY

On 23-24 Sept. 2005, we collected 28 gravid individuals from Kyles Ford, immediately euthanized them, and stored them in individual sealed plastic bags containing 70% ethanol. In the laboratory, we excised the marsupial gills and selected one gill per individual for analysis. For handling ease, we divided each gill into three equal sections based on the number of folds in the gill and then enumerated conglutinates for each section. We removed 10 conglutinates from each gill section and measured the length and width of each conglutinate with a dissecting microscope fitted with an ocular micrometer at 16x and 25x, dependent upon the size of the conglutinate. The length was measured from the tip of the "head" to the base of the conglutinate where the "tails" began and the width was measured across the "eye spots." We calculated an approximate volume for each conglutinate from its length and width by assuming a cylindrical shape. One measured conglutinate from each mussel was teased apart and all glochidia counted on a zooplankton counting wheel. We measured total length (parallel to the hinge), height (perpendicular to the hinge), and hinge length of three glochidia from each conglutinate with an ocular micrometer at 40x. The examined gills and conglutinates were chosen for each individual by using a random number generator.

We used least squares regression analysis to determine the relationship between number of glochidia and conglutinate volume. The number of glochidia per gill section was estimated for each individual by taking the mean conglutinate volume for each gill section and multiplying it by the number of glochidia per conglutinate (calculated from the regression of glochidia and conglutinate volume) and multiplying that product by the number of conglutinates in that gill section. The sum of glochidia in each section was doubled to estimate total fecundity. Empty conglutinates were not included in the fecundity estimate. We used least squares regression analysis to determine the relationships between fecundity and mussel total length and age. In addition, the number of conglutinates and conglutinate size were regressed against length.

HOST TRIALS

For our host trials, we used the basic methods reported by Howard (1914) as modified by Matteson (1948), Yokley (1972), Zale and Neves (1982), and Khym and Layzer (2000). In Sept. 2005, we collected gravid mussels from the Clinch River and transported them in wet mesh bags inside a cooler to Tennessee Wildlife Resources Agency's Normandy Hatchery, Normandy, Tennessee, where they were maintained in a flow-through raceway until needed. We collected fish by electrofishing with direct current and by seining. Fish were transported to the laboratory in a cooler with river water and an aerator. Prior to placing fish in holding tanks, we acclimated them at a rate of [less than or equal to] 2 C per h and treated them with a 167-mg/L solution of 37% formalin for 1 h to prevent the spread of parasites (Lasee, 1995). Each holding tank consisted of a re-circulating system that included a 340-L tank, a sand biofilter, a UV light sterilizer, a pump, and a chiller. Insulation covered all pipes and styrofoam covered the tanks to minimize water temperature fluctuations. Fish were fed bloodworms every other day and were supplied with PVC structures for cover.

We performed three transformation trials with Ptychobranchus subtentum. For each infestation, we opened gravid females, inserted a hypodermic needle into the water tubes of the marsupial gills and injected dechlorinated water to flush-out conglutinates. We teased conglutinates apart with dissecting probes to free glochidia. All glochidia were pooled together for infestation. Before infesting fish, a small subsample of glochidia was tested for viability by adding a small amount of sodium chloride; viable glochidia snap shut in the presence of salt (Lefevre and Curtis, 1912). We put fish and viable glochidia into a 19-L bucket containing approximately 9 L of water and an air stone to keep glochidia suspended for 35 to 45 min. We transferred infested fish to another bucket of water to allow unattached glochidia to fall off. After 5 min, fish were separated by species and transferred to 170-L tanks. These tanks were set up similar to the 340-L tanks, but screens were placed on the bottom of the tanks to decrease the chance of host fish preying on juvenile mussels. To eliminate water temperature as a variable, water temperature was maintained at 21 C (SE [+ or -] 0.04) throughout all trials.

Tanks were siphoned daily for 4 d post infestation then weekly until a juvenile was found and tanks were siphoned daily thereafter. Siphonate went through a 180-[micro]m mesh sieve. The contents of each sieve were examined with a polarized microscope and all glochidia and juveniles were counted. When no glochidia or juveniles were found for 5 consecutive days, the gills of all fish were examined for glochidia before being discarded. We considered the sum of glochidia and juveniles recovered from the siphonate of each tank as the initial infestation intensity. The total number of juveniles recovered was divided by the infestation intensity to determine the percent transformation.

In Trial 1, we wanted to determine if time to transformation was related to the length of time glochidia were brooded. We used 24 Sept. as day 1 (the first day we observed glochidia in the marsupium) and calculated the number of d to each infestation: 14 Nov. 2005 (52 d), 18 Jan. 2006 (117 d), and 6 Apr. 2006 (195 d). We used 25 rainbow darters and 25 fantail darters from the Cumberland River drainage, and 10 conglutinates from each of nine mussels for each infestation. We used the median test (Conover, 1971) to compare time to metamorphosis among infestation dates (months).

In Trial 2, we infested rainbow darters and fantail darters collected from a Cumberland River tributary with glochidia from mussels collected from the Clinch and Wolf rivers in Jan. 2006 to determine whether the time to metamorphosis varied between rivers. We infested 10 rainbow darters and 10 fantail darters with glochidia from two Ptychobranchus subtentum collected from the Wolf River (Cumberland River drainage), Pickett County, Tennessee. Forty conglutinates were removed from the Wolf River mussels (20 per mussel) and used to infest fish on the same day we infested fish with glochidia from the Clinch River for Trial 1 (18 Jan.). We used the median test (Conover, 1971) to compare time to metamorphosis between rivers.

In Apr. 2006, we collected and infested logperch Percina caprodes, gilt darter P. evides, banded darter Etheostoma zonale, blueside darter E. jessaei, bluebreast darter E. camurum, dusky darter P. sciera, wounded darter E. vulneratum, and stripetail darter E. kennicotti to determine their suitability as glochidial hosts (Trial 3). We used 10 conglutinates from each of nine mussels from the Clinch River to infest these species. We considered fish producing active juveniles as possible hosts.

SEX RATIO

In Jul. 2006, we collected mussels by wading throughout the mussel bed at Kyles Ford and haphazardly choosing a place to lay quadrats (0.25-[m.sup.2]) on the substrate. Individuals found at the surface of the substrate were considered epibenthic, while those found by excavating about 15 cm of substrate were considered endobenthic. We kept epibenthic and endobenthic mussels separate and extracted gonadal fluid samples to determine the sex of individuals. We opened the mussels and inserted a small wedge between the valves to hold them open. We inserted a needle (18-g) into the gonad and injected ~0.3 mL of dechlorinated water, and then withdrew ~0.1 mL of fluid (Bauer, 1987; Moles and Layzer, 2008).

We labeled each syringe, capped them and immediately put them on ice, and transported them to the laboratory where they were refrigerated at 9 C. Within 48 h of collection, we stained the samples with 10% aqueous methylene blue solution on a standard microscope slide (76 x 25 x 1 mm) (Saha and Layzer, 2008). We mixed gonadal fluid samples with the stain at a 1:1 ratio and allowed them to air-dry for 24 h before we examined them with a compound light microscope at 40x, 400x, and 1000x to determine sex. Sex was assigned based upon the presence of ova or sperm. Chi-square goodness-of-fit tests ([alpha] = 0.05) were used to analyze the sex ratio at Kyles Ford and to determine if either sex was more likely to be found epibenthically or endobenthically.

AGE AND GROWTH

For ageing, we collected fresh-dead shells from muskrat middens and supplemented these with shells of live individuals that were larger than those in middens at Kyles Ford. We also used five small fresh-dead shells collected in Oct. 2001. Additionally we collected live individuals, measured them with dial calipers to the nearest 0.01 mm and notched the ventral margin of their shells with a triangular file in Jul. 2005. We applied uniquely numbered tags to the posterior margin with cyanoacrylate glue. We then inserted the mussels into the substrate at the collection site. During the following Jul., we searched the site to collect a subsample of the tagged mussels, but we did not attempt to find all of them. We measured all recaptured individuals to determine growth for 1 y. We euthanized a subsample and used them for validating annulus formation.

We measured the length of each shell parallel to the hinge and cut thin-sections for ageing as described by Neves and Moyer (1988). We cut two consecutive sections, 175- and 200-[micro]m thick from the umbo to the ventral margin, perpendicular to the hinge, with a low-speed saw and a diamond wafering blade. Growth arrests extending from the umbo to the shell margin were counted with a dissecting microscope at 50x and 100x. We marked the distal edge of the thin section for each annulus encountered and transferred the marks onto the uncut valve. To determine length-at-annulus, we measured the distance between the anterior and posterior ends of each marked external growth line. Erosion of some shells resulted in a loss of distinguishable annuli. To correct for this, the number of annuli lost was estimated from shells that were not eroded. Lengths at these lost annuli were not estimated or used in analyses, but reported ages included an adjustment for annuli lost.

Length-at-annulus data were fitted to the von Bertalanffy growth equation:

[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]

where L[infinity] is the theoretical maximum length, k is a growth constant, t is a given age in y, and [t.sub.0] is the theoretical time when length is zero (von Bertalanffy, 1938). We used least squares non-linear regression to derive estimates of L[infinity], k, and [t.sub.0]. Based on these values, the estimates of [L.sub.t] were computed. We used the sign test (Conover, 1971) to compare growth increments of shells notched for validating annulus formation with growth increments derived from the von Bertalanffy growth equation.

For all statistical tests, significance was determined at [alpha] = 0.05 level. All parametric statistical analyses were performed using Statistical Analysis Systems software (SAS statistical software, version 8.2, SAS Institute, Inc., Cary, North Carolina).

RESULTS

SPAWNING AND GLOCHIDIAL DEVELOPMENT

On 18 Aug. 2005, we randomly collected 110 Ptychobranchus subtentum from Kyles Ford and examined their gills; none had inflated gills (Table 1). We first collected individuals with white inflated gills on 26 Aug.; examination of the contents of the marsupial gills indicated they contained masses of unfertilized eggs. Fertilization occurred between 26 and 31 Aug. Developing embryos and outer conglutinates began to form by 7 Sept., and the inflated gills had some brown color. On 24 Sept., the marsupial gills of all gravid individuals were dark brown and contained developed glochidia (Table 1). Individuals examined for gravidity varied in length from 37 mm to 103 mm, while gravid individuals varied in length from 46 mm to 103 mm. In 2006, the timing of ova deposition in the gills and embryo development was nearly identical (Table 1). Glochidia retention was monitored in May and Jun. 2006. In the Clinch River, we found that only the medial to posterior end of the marsupium of most individuals still contained glochidia on 20 May 2006 (Table 1). Gravid individuals placed in the Normandy raceway 25 Sept. 2005 were still gravid 16 May 2006. Although conglutinates were developed in Sept., they had more detail in May. By 20 Jun. the water temperature had reached 25 C and we found no gravid individuals in the Clinch River or at the Normandy Hatchery.

FECUNDITY

We determined fecundity for 28 individuals collected from Kyles Ford ranging in length from 50 to 101 mm. Developed conglutinates containing glochidia had eyespots anteriorly and two adhesive tails posteriorly, and resembled Simuliidae pupae (see photographs in Unio Gallery: http://unionid.missouristate.edu/gallery/Psubtentum/fluted.htm). The marsupium contained one conglutinate per water tube, except at the posterior and anterior ends of the gills after the last fold where water tubes were empty, regardless of mussel size.

The number of conglutinates varied among individuals. The mean number of conglutinates per female ([+ or -] 1 SE) was 208 [+ or -] 13.67 and ranged from 85 to 329. There was a positive linear relationship between number of conglutinates and mussel length ([r.sup.2] = 0.94, df = 26, P < 0.0001; Table 2). All conglutinates contained glochidia except for one that contained developing ova.

The mean number of glochidia per conglutinate ([+ or -] 1 SE) was 519 [+ or -] 41.2 and ranged from 166 to 915. There was a positive linear relationship between number of glochidia per conglutinate and volume of the conglutinate ([r.sup.2] = 0.83, df = 26, P < 0.0001; Table 2). Mean fecundity ([+ or -1] SE) was 2.47 x [10.sup.5] [+ or -] 2.64 x [10.sup.4] and ranged from 4.33 x [10.sup.4] for a 49 mm mussel to 5.02 x [10.sup.5] for a 91 mm mussel. There was a positive linear relationship between natural log transtormed fecundity and total length ([r.sup.2] = 0.81, df = 26, P <0.0001; Table 2), and natural log transformed fecundity and age ([r.sup.2] = 0.60, df = 26, P < 0.0001; Table 2). Mean glochidia length ([+ or -] 1 SE) was 228.6 [+ or -] 0.75 [micro]m, mean height ([+ or -] 1 SE) was 175.5 [+ or -] 3.5 [micro]m, and mean hinge length ([+ or -] 1 SE) was 86.0 [+ or -] 0.25 [micro]m (n = 85).

HOST TRIALS

The median day of metamorphosis of Clinch River glochidia on fantail darters varied among infestation dates from Nov. 2005 to Apr. 2006 ([chi square] = 13.72, df = 2, P = 0.001; Fig. 2). Time to first juvenile excystment was greater for fantail darters infested in Nov. (45 d post-infestation) than for those infested in Jan. (32 d) and Apr. (37 d). The temporal pattern in glochidial metamorphosis on rainbow darters was similar to that of the fantail darters, but it was not significant ([chi square] = 4.62, df = 2, P = 0.10; Fig. 2). The mean number of juveniles per fish ranged from 1.2 to 8.4 for fantail darters and 0.33 to 2.7 for rainbow darters among months (Table 3). The highest percent juvenile transformation occurred on fantail darters infested with glochidia from the Clinch River in Jan. (81%), and the lowest occurred on rainbow darters infested in Nov. (3.4%).

[FIGURE 2 OMITTED]

Glochidia of Ptychobranchus subtentum from both the Wolf River and Clinch River transformed on fantail darters and rainbow darters. The median day of glochidial metamorphosis was not significantly different between infestations with glochidia from the two rivers for fantail darters ([chi square] = 1.67, df = 1, P = 0.20) and rainbow darters ([chi square] = 1.28, df = 1, P = 0.26).

Metamorphosis of Clinch River glochidia occurred 24 to 48 d post-infestation on bluebreast darters, dusky darters, banded darters, stripetail darters, and logperch. Only those juveniles that transformed on bluebreast and dusky darters were active (Table 4), while those that seemingly transformed on banded darters, stripetail darters, and logperch were inactive and therefore we considered them to be non-viable. Juvenile transformation success was 63% for bluebreast darters and 19% for dusky darters (Table 4). Mean number of juveniles per fish varied from 0.5 to 12 (Table 3). Other species tested sloughed all glochidia within 6 d of infestation.

SEX RATIO

Our examination of gonadal fluid samples collected from 100 Clinch River individuals in Jul. 2006 indicated that 65 were female, 34 were male, and one individual (26-mm long) was immature. We did not observe any evidence of hermaphroditism in any gonadal fluid sample. The ratio of females to males (1.9:1) differed significantly from 1:1 ([chi square] = 9.71, df = 1, P = 0.0018). Thirty-four individuals were epibenthic, of which 26 were female and eight were male. Sixty-six individuals were endobenthic, of which 39 were female, 26 were male, and one was immature. Females and males were equally likely to be found either epibenthically or endobenthically ([chi square] = 2.69, df = 1, P = 0.10). The smallest individual with ova was 52 mm and the smallest individual with sperm was 50 mm, while a 26 mm individual had undeveloped gonads.

AGE AND GROWTH

One hundred thirty-one mussels were aged from thin-sections. Ages of individuals estimated from thin-sectioning ranged from 0 to 26 y, and total lengths ranged from 13.9 to 101.3 mm. Of the shells aged, 29 were eroded so that the location of at least the first annulus had to be estimated. We judged the number of missing annuli based on the extent of shell erosion and the number of annuli in this region for un-eroded shells. A correction factor of 1 to 4 y was added based on the extent of shell erosion and presumed missing annuli.

Mean annual growth in total length averaged 18.7 mm for the first year, 5.5 mm for year 2 to 6, 2.3 mm for year 7 to 19, and 0.88 mm for year 20 to 26 (Fig. 3). Mean lengths ([L.sub.t]) at annulus formation were fitted to the von Bertalanffy growth equation, [L.sub.t] (mm) = 121.5 mm x (1 - [e.sup.-0.058(t - 2.07)]) and used to predict values for length at age.

Twenty-six individuals that were measured and marked at the Clinch River in Jul. 2005 were recaptured in Jul. 2006. Growth varied among recaptured individuals and ranged from 0.0 to 6.2 mm. Mean growth ([+ or -] 1 SE) was 1.5 mm [+ or -] 0.33. Ten mussels recaptured ranging in length from 51.2 to 103.5 mm were sacrificed. Examination of thin-sections prepared from these individuals indicated that one new growth arrest occurred beyond the notch. However, growth increments of these 10 individuals were significantly less than predicted by the von Bertalanffy growth equation (Sign test, T = 10, P = 0.002).

[FIGURE 3 OMITTED]

DISCUSSION

SPAWNING AND GLOCHIDIAL DEVELOPMENT

We observed nearly identical temporal patterns in egg and embryo development between years in the Clinch River (Table 1). While temperature and photoperiod potentially are regulators of annual cycles, many of the mussels we found were completely buried and not exposed to changes in photoperiod. Therefore, we think the gametogenic cycle is more likely regulated by the annual temperature regime. Abnormal temperature regimes are known to inhibit gametogenesis (Heinricher and Layzer, 1999), and interstream differences in temperature regimes result in corresponding shifts in reproductive timing (Hastie and Young, 2003). However, actual sperm release may be triggered by a more tractable cue because fertilization of all eggs in females we examined occurred [less than or equal to] 5 d (Table 1). This suggests that sperm was not limiting even though females were nearly twice as abundant as males at our study site at Kyles Ford. Likely, there was an excess of sperm. Berg et al., (2008) suggested that sperm competition might occur within the reproductive tract of females because of the documented occurrence of multiple paternity within a single brood of glochidia (Christian et al., 2007). While sperm competition could have occurred within female fluted kidneyshells, we think there also would have been intense selective pressure among males to release sperm during the short time period that unfertilized eggs were available. We would expect a reproductive advantage to accrue to male genotypes that released sperm as soon as female marsupia were fully charged. Rain et al. (1996) hypothesized that pheromones are involved in the synchronous spawning of zebra mussels (Dreissena polymorpha). We suggest that pheromones associated with fully charged female Ptychobranchus subtentum (and possibly with broadcast male gametes) triggered synchronous sperm release among male fluted kidneyshells in the Clinch River.

Alter fertilization, it took about 4 wk for embryos to develop into viable Ptychobranchus subtentum glochidia (Table 1). Although the conglutinate portions that mimicked "appendages" were visible in Sept., they seemed to be more developed in May; it is unknown whether glochidia became more developed as well. The gradual color change of the marsupium from white to dark brown paralleled the development of conglutinates and the change in their contents from masses of opaque eggs to glochidia. Females held at the Normandy raceway, where no males were present, were still gravid in May, as were fluted kidneyshells in the Clinch River.

Developed conglutinates were comprised only of glochidia, which is characteristic of species with membrane bound conglutinates (Haag and Staton, 2003). All conglutinates we observed in the marsupial gills of Ptychobranchus subtentum were similar and resembled Simuliidae pupae as reported by Barnhardt et al. (2008). In contrast, Watters (1999) reported that two distinctly different types of conglutinates occurred in P. fasciolaris. Similarly, Hartfield and Hartfield (1996) indicated that conglutinates of P. greeni mimicked chironomid larvae while Haag and Warren (1997) observed conglutinates of P. greeni that resembled fertilized fish eggs. Ptychobranchus subtentum conglutinates included "eyespots" anteriorly and two adhesive "tails" posteriorly. The "eyespots" on the conglutinate are a primary site of glochidial release when a fish inhales a conglutinate (Barnhart and Roberts, 1997). The adhesive "tails" anchor conglutinates to the substrate as they tumble along the stream bottom, which may make them more appealing for fish to inhale (Barnhart and Roberts, 1997).

FECUNDITY

Mean fecundity of Ptychobranchus subtentum was about 250,000 glochidia. We would expect that this number of glochidia packaged in conglutinates resembling prey of benthic host fishes should lead to high natural infestation rates. Fecundity was related to length and age, but fecundity was best predicted by length (Table 2). Large individuals had the highest fecundity, supporting the conclusion made by Haag and Staton (2003) that large individuals within a mussel population are important to maintaining population viability.

HOST TRIALS

Water temperature during the encystment period influences the length of time to excystment (Howard and Anson, 1922; Zale and Neves, 1982; Watters and O'Dee, 1999). Although Ptychobranchus subtentum glochidia were viable in Nov., post infestation time to median day of excystment on fantail darters was significantly shorter in Jan. even though water temperatures did not differ (Fig. 2). While time to excystment of juveniles from rainbow darters infested in Jan. was shorter than from fish infested in Nov., the difference was not significant. Khym and Layzer (2000) found that the length of time from infestation to excystment was negatively related to the length of time Ligumia recta brooded glochidia. Clearly, water temperature is just one factor influencing the length of the metamorphosis period in the laboratory. While glochidia were viable in Nov., a longer brooding time (until Jan.) resulted in a shorter time to metamorphosis suggesting brooded glochidia undergo further development.

Host suitability did not seem to vary between drainages. Glochidia from both the Clinch River (Tennessee River drainage) and Wolf River (Cumberland River drainage) metamorphosed on fantail darters and rainbow darters collected from the Cumberland River drainage in an area devoid of Ptychobranchus subtentum (Table 3). Further, there was no significant difference between the temporal patterns in glochidial metamorphosis of these mussels.

The fluted kidneyshell exhibited considerable host specificity, primarily to benthic insectivorous percid fishes. Luo (1993) tested fish from eight families as possible hosts for Ptychobranchus subtentum, and only four darter species and the banded sculpin were potentially suitable hosts. Only two of the eight Percina and Etheostoma species tested in this study were potential hosts, the bluebreast darter and dusky darter (Table 3). Collections of these species from the wild with encysted glochidia are needed to confirm that they are natural hosts. Inactive juveniles excysted from banded darter, stripetail darter, and logperch. We are uncertain as to why these juveniles were nonviable and; therefore, these fish should be retested as possible hosts. These species were tested in Apr. at the same time percent transformation was lowest for rainbow darters and fantails darters (Table 3). Further, mortality of most fish species infested in Apr. was high (Tables 3 and 4). Surviving fish may have been in poor condition and less suitable as hosts.

SEX RATIO

The sample taken at Kyles Ford exhibited an unbalanced sex ratio where females were nearly twice as abundant as males. Berg et al. (2008) indicated that most freshwater mussel populations have sex ratios close to 1:1 and deviations from 1:1 have a greater ratio of males to females; however, female Ptychobranchus subtentum predominated at Kyles Ford. The cause of this unbalanced sex ratio is unknown. Haag and Staton (2003) found that populations in stressful environments were often characterized by highly skewed sex ratios. Judging from the increase in density during recent years (Ahlstedt et al., 2005) as well as obvious recruitment, the population of tinted kidneyshells at Kyles Ford does not seem to be under stress. While factors responsible for unbalanced sex ratios are unknown, determining the sex ratio of a population is critical tot modeling population dynamics and conservation of the species.

Although the sex ratio was 1.9:1 the percentage of gravid individuals ranged from 37--56%. If all females reproduced each year, the percentage of gravid individuals would have been higher based on the sex ratio. Bauer (1987) found that only 64% of female Margaritifera margaritifera take part in reproduction each year and suggested they go through a reproductive pausing period. Similarly, Moles and Layzer (2008) found that <37% of female Actinonaias ligamentina reproduced each year and proposed that they also go through a resting stage to acquire energy reserves to produce gametes in subsequent years. Based on the 1.9:1 sex ratio and our collection (N = 234) on 14 Sept. 2005, the day of the highest observed gravidity (56%), at most only 84% of female fluted kidneyshells reproduced in 2005 at Kyles Ford. This suggests that some female fluted kidneyshells also go through reproductive pausing.

AGE AND GROWTH

The fluted kidneyshell is a moderately thick-shelled riverine species and lives to at least 26 y in the upper Clinch River. Shells were frequently eroded to some degree so examination of thin-sections under magnification was essential for accurate aging and was a more reliable method than counting external rings. The presence of rings in freshwater mussel shells is well known, but there is much debate over whether the rings are deposited annually. Annual ring formation has long been assumed but rarely demonstrated (Haag and Commens-Carson, 2008) making annuli validation important. Annuli formation was validated for the Kyles Ford population of fluted kidneyshell. One growth arrest was formed beyond the notch at the shell margin phi there the previous year; however, growth increments of these individuals was less than those predicted by the von Bertalanffy growth equation. Haag and Commens-Carson (2008) found that mussels are sensitive to handling and it may cause a decrease in shell growth. Presumably, notching shells resulted in a higher degree of handling stress and negatively affected growth of the fluted kidneyshell. The von Bertalanffy growth equation, calculated from mean length-at-age data, was a good fit for the aged shells of Ptychobranchus subtentum (Fig. 3). The theoretical maximum length, 121.5 mm, obtained from the growth equation is close to the maximum length (120 mm) suggested by Parmalee and Bogan (1998). The growth rate K is variable among populations and may be indicative of environmental factors (Haag and Rypel, 2011). The growth rate of the robust population of fluted kidneyshells at Kyles Ford could serve as a benchmark for comparisons with other populations.

CONCLUSIONS

The robust population of fluted kidneyshells at Kyles Ford on the Clinch River provided a unique opportunity to study the life history of a healthy population. Whereas other known populations of the fluted kidneyshell have been either extirpated or are very small, the population at Kyles Ford has increased 4-fold over a 25 y long period (Alhstedt et al., 2005). The tinted kidneyshell's high fecundity, nearly complete fertilization of eggs and conglutinates mimicking simulid larvae to lure its hosts (sight-feeding, benthic insectivorous fishes) have been successful adaptations in the Clinch River. Despite these characteristics, most populations of the fluted kidneyshell have been extirpated or have declined. Many factors have been cited as contributing to the decline of mussel populations in the southeastern United States (Neves el al., 1997). However, Downing et al. (2010) found that only 48% of the studies they reviewed provided reasonable links between causes and observed mussel declines. Recovery of the fluted kidneyshell will be difficult. Nonetheless, the recent increase of fluted kidneyshells in the Clinch River at Kyles Ford and its life history attributes (e.g., high fecundity and use of some common, widely distributed fishes as hosts) suggests that if the specific causes of individual population declines are identified and corrected, the fluted kidneyshell can be recovered.

Acknowledgments.--We thank Erica Dyer, Kendall Moles, Samrat Saha, Matt Ashton, Andy Weber, and Ben Davis for their field and laboratory assistance. The U.S. Fish and Wildlife Service, and the Center for the Management, Utilization, and Protection of Water Resources, Tennessee Tech University provided funds for this project. We thank David Berg, Nathan Johnson, Jason Wisniewski and two anonymous referees for providing helpful comments on an earlier draft.

SUBMITTED 9 FEBRUARY 2010

ACCEPTED 5 AUGUST 2011

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V. MALISSA DAVIS (1)

Tennessee Cooperative Fishery Research Unit, Tennessee Technological University, Cookeville 38505

AND

JAMES B. LAYZER (2)

U.S. Geological Survey, Tennessee Cooperative Fishery Research Unit, Tennessee Technological University, Cookeville 38505

(1) Present address: Biology Department, Tennessee Technological University, Cookeville 38505

(2) Corresponding author: e-mail: jim_layzer@tntech.edu
TABLE 1.--Glochidial and conglutinate development of
Ptychobranchus subtentum collected from the upper Clinch River,
Tennessee from Aug. 2005 to Sept. 2006. N = total sample size,
number in parentheses represents mussels [less than or equal to]
46 mm long (minimum size found gravid), and percent gravid is
based on individuals [greater than or equal to] 46 mm long

                         Marsupial gills

Date             Appearance           Contents                  N

18 Aug 2005      Compressed           Empty                  110 (0)

26 Aug 2005      Inflated and white   Unfertilized eggs      103 (0)

31 Aug 2005      Inflated and white   Fertilized eggs        100 (2)

7 Sept. 2005     Inflated and some    Embryos and            101 (4)
                  brown               conglutinates
                                      forming

14 Sept. 2005    Inflated and some    Glochidia and          109 (2)
                 brown                conglutinates
                                      forming

24 Sept. 2005    Inflated and dark    Conglutinates formed   238 (4)
                 brown                around developed
                                      glochidia

20 May 2006      Inflated and dark    Conglutinates highly   13 (0)
                 brown                detailed with
                                      glochidia
                 With anterior end
                 spent

20 Jun. 2006     Compressed           Empty                  12 (0)

11 Jul. 2006     Compressed           Empty                  100 (1)

29 Aug 2006      Inflated and white   Fertilized eggs        92 (0)

12 Sept. 2006    Inflated and some    Embryos and            84 (0)
                 brown                conglutinates
                                      forming

19 Sept. 2006    Inflated and dark    Glochidia and          100 (2)
                 brown                conglutinates
                                      developing

                               Water
                            temperature
                 Percent   (C) at time of
Date             gravid      collection

18 Aug 2005        0%            26

26 Aug 2005        48%           25

31 Aug 2005        42%           25

7 Sept. 2005       52%           23

14 Sept. 2005      54%           24

24 Sept. 2005      56%           23

20 May 2006        46%           14

20 Jun. 2006       ()%           25

11 Jul. 2006       0%            22

29 Aug 2006        37%           24

12 Sept. 2006      50%           21

19 Sept. 2006      51%           21

TABLE 2.--Results of least squares regression analysis of
variables related to fecundity of Ptychobranchus  subtentum in
the Clinch River, Tennessee

Dependant variable         [b.sub.0]     [b.sub.1]

Number of conglutinates     -132.6            4.7
Number of glochidia            0.11          90.6
In Fecundity                  -1.39           3.18
In Fecundity               2,705              5.21

Dependant variable         Independent variable   [r.sup.2]      p

Number of conglutinates    Mussel length            0.94      <0.0001
Number of glochidia        Conglutinate volume      0.83      <0.0001
In Fecundity               In Mussel length         0.81      <0.0001
In Fecundity               In Mussel age            0.60      <0.0001

TABLE 3.--Results of laboratory infestations with Ptychobranchus
subtentum glochidia on fantail darters and rainbow darters.
Trial lcompared excystment period to length of brooding period.
For Trial 2, results offish infested with glochidia from the Wolf
River were compared to fish infested with glochidia from the
Clinch River on the same day in Jan. in Trial 1. For both trials
water temperature = 21C, SE [+ or -] 0.04

                                             Number of fish
                   Source of       Month
Fish species       mussels        infested   Initial    Final

Trial 1
 Fantail darter    Clinch River   Nov.          25       24
 Fantail darter    Clinch River   Jan.          25       25
 Fantail darter    Clinch River   Apr.          25       10
 Rainbow darter    Clinch River   Nov.          25       24
 Rainbow darter    Clinch River   Jan.          25       25
 Rainbow darter    Clinch River   Apr.          25       23
Trial 2
 Fantail darter    Wolf River     Jan.          10       10
 Rainbow darter    Wolf River     Jan.          10       10

                      Juvenile          Mean
                     excystment        number        Percent
                   period (d post-   juveniles/      juvenile
Fish species        infestation)        fish      transformation

Trial 1
 Fantail darter         44-62        6.1          65
 Fantail darter         32-55        8.4          81
 Fantail darter         37-50        1.2          16
 Rainbow darter         49-55        0.33          3.4
 Rainbow darter         32-54        2.7          55
 Rainbow darter         37-52        0.55          6
Trial 2
 Fantail darter         31-47        3.9          68
 Rainbow darter         30-49        0.8          89

TABLE 4.--Results of laboratory infestations of potential fish
hosts with Ptychobranchus subtentum glochidia (Trial 3).
Water temperature = 21 C, SE [+ or -] 0.04

                                                  Number    of fish
                         Source of      Month
Fish species              mussels      infested   Initial   Final

Banded darter (1)       Clinch River     Apr.       13        2
Bluebreast darter       Clinch River     Apr.        2        1
Blueside darter         Clinch River     Apr.       11        9
Dusky darter            Clinch River     Apr.        3        2
Gilt darter             Clinch River     Apr.        4        0
Logperch (1)            Clinch River     Apr.       10        2
Stripetail darter (1)   Clinch River     Apr.       13        3
Wounded darter          Clinch River     Apr.        3        2

                            Juvenile
                           excystment
                            period (d        Mean number
Fish species            post-infestation)   juveniles/fish

Banded darter (1)             24-50               2.5
Bluebreast darter             37-48              12
Blueside darter                --                 0
Dusky darter                  38-18               6.5
Gilt darter                    --                 0
Logperch (1)                   27                 0.5
Stripetail darter (1)         37-43               1
Wounded darter                 --                 0

                           Percent
                           juvenile
Fish species            transformation

Banded darter (1)             17
Bluebreast darter             63
Blueside darter               --
Dusky darter                  19
Gilt darter                   --
Logperch (1)                   2
Stripetail darter (1)          7
Wounded darter                --

(1) Juveniles were inactive
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