Southeastern Naturalist 243 K.M. Harriger, J.R. Knight, and M.G. Wegener 22001155 SOUTHEASTERN NATURALIST 1V4o(2l.) :1244,3 N–2o5. 32 Host-Fish Identifications for Two Gulf Coast-Drainage Freshwater Mussels, Lampsilis straminea (Southern Fatmucket) and Quadrula succissa (Purple Pigtoe) Kathryn M. Harriger1,*, John R. Knight1, and Matthew G. Wegener1 Abstract - Host-fish requirements are unknown for many freshwater mussels, which impedes conservation efforts. We conducted host-fish trials for 2 mussels, Lampsilis straminea (Southern Fatmucket) and Quadrula succissa (Purple Pigtoe). Our objectives were to verify techniques by successfully transforming glochidia from a long-term–brooding mussel with known hosts (L. straminea) and to identify hosts for a short-term–brooding mussel with no confirmed hosts (Q. succissa). Results of the trials confirmed known hosts for L. straminea, identified 4 new hosts for L. straminea, and identified 3 ictalurid hosts for Q. succissa. Based on results of this research, additional host trials are needed for Q. succissa and should focus on ictalurids. This study provided information necessary for the management and conservation of mussels with previously unknown hosts in southeast Alabama and west Florida Gulf Coast drainages, while confirming available resources and techniques used by the Florida Fish and Wildlife Conservation Commission for future host-fish trials. Introduction Freshwater mussels (Unionidae) are highly imperiled in the United States. The Southeast has a high diversity of mussels, including 98% of all threatened and endangered mussels in the United States (Williams et al. 2014). Declines in mussel fauna have been attributed to several anthropogenic factors, particularly in the Southeast. Those factors include degradation of water quality, destabilization of stream habitats, population fragmentation, impoundment of lotic waters, and declines in native fish populations (Dudgeon et al. 2006, Neves et al. 1997). Unionid mussels are unique in that almost all species are obligate parasites on the gills and fins of native fishes during the larval (glochidial) stage of development. Glochidia transform into juvenile mussels while encysted on their host and fall off as juveniles when metamorphosis is complete. Each mussel species uses a specific suite of fish host(s) and can be classified as a host specialist or generalist (Haag 2012). Knowledge of host requirements is particularly important to mussel conservation since declining host-fish populations could result in decreased dispersal and reproductive success in mussels (McNichols et al. 2011, Neves et al. 1997). Few host-fish relationships have been identified for mussels in eastern Gulf Coast drainages, and this information is critical for successful conservation efforts, including mussel propagation, reintroduction, translocation, and habitat restoration. 1Florida Fish and Wildlife Conservation Commission, Blackwater Research and Development Center, Holt, FL 32564. *Corresponding author - kate.harriger@myfwc.com. Manuscript Editor: Paul M. Stewart Southeastern Naturalist K.M. Harriger, J.R. Knight, and M.G. Wegener 2015 Vol. 14, No. 2 244 Research is needed to determine the host requirements of different mussel species. Life-history strategies (e.g., long- or short-term brooding) vary by mussel species, which presents logistical hurdles in such research. Long-term brooders spawn in late summer, and their fertilized eggs develop into mature glochidia in the marsupial gills of female mussels (Lefevre and Curtis 1912, Ortmann 1911). Mature glochidia are released during winter or the following spring or summer (Williams et al. 2014). Short-term brooders spawn in spring to early summer and release mature glochidia within a few weeks of spawning (Garner et al. 1999, Lefevre and Curtis 1912, Ortmann 1911, Williams et al. 2014). Collecting mature glochidia from gravid short-term brooders can be more difficult than from long-term brooders because of the short period in which short-term brooders are gravid. Mature glochidia are easily harvested from long-term brooders by flushing the mussels’ gills with a stream of water from a syringe (Eads et al. 2006, Haag et al. 1999), but short-term brooders usually must be held in a laboratory setting for a period of time before they release mature glochidia (Haag and Warren 2003, White et al. 2008). Handling stress is often an additional issue during glochidial collection due to the release of conglutinates by mussels (particularly short-term brooders) before glochidia are mature (Lefevre and Curtis 1912). Many of northwest Florida’s mussels are imperiled and in need of conservation efforts. Fifteen mussel species are federally listed as threatened or endangered in the eastern Gulf Coast drainages from the Escambia River to the Suwannee River in northern Florida (USFWS 1993, 1998, 2012; Williams et al. 2014). Eight of these species are endemic to Gulf Coast drainages (Escambia, Yellow, and Choctawhatchee rivers) in southern Alabama and northwest Florida (USFWS 1998, 2012; Williams et al. 2014). Host-fish requirements are only known for 2 of these species: Fusconaia burkei (Walker in Ortmann and Walker) (Tapered Pigtoe) and Pleurobema strodeanum (Wright) (Fuzzy Pigtoe) (USFWS 2012, White et al. 2008, Williams et al. 2014). Until this information is known for all of the threatened species, effective conservation efforts in the area will be limited. Therefore, research on host-fish requirements for endangered and threatened mussels in northwest Florida is considered a priority for these mussels’ conservation. To address this need, a facility was designed at the Florida Fish and Wildlife Conservation Commission (FWC) Blackwater Research and Development Center (BRDC) in Holt, FL, for host-fish research and identification of host requirements for northwest Florida’s imperiled freshwater mussels. The first objective of this study was to conduct preliminary host-fish trials using a long-term–brooding mussel with known hosts to verify techniques and determine that the facility was suitable for conducting host-fish research. The common, longterm– brooding mussel Lampsilis straminea (Conrad) (Southern Fatmucket) was used in the preliminary trials. The second objective was to identify host fishes for a short-term–brooding mussel with unknown hosts: Quadrula succissa (Lea) (Purple Pigtoe). Methods were created and adapted for future use in research on federally threatened and endangered mussel species in Florida. Southeastern Naturalist 245 K.M. Harriger, J.R. Knight, and M.G. Wegener 2015 Vol. 14, No. 2 Methods Mussel collection We collected gravid mussels from the Escambia and Yellow rivers in spring 2012 and 2013 using visual and tactile snorkel surveys (Fig. 1). Gravid L. straminea were collected near the Molino, FL, boat ramp (30.723649°N, 87.305758°W) on the Escambia River in April 2012, and were identified in the field by the presence of Figure 1. Study area in northwest Florida (Escambia, Blackwater, and Yellow rivers). Host fishes were collected throughout the Blackwater River, and mussels were collected from Mystic Springs and Molino boat ramps (white circles) on the Escambia River in 2012 and 2013. Southeastern Naturalist K.M. Harriger, J.R. Knight, and M.G. Wegener 2015 Vol. 14, No. 2 246 inflated gills. We monitored Q. succissa for gravidity in the Yellow and Escambia rivers throughout spring 2013, but mussels used in the trials were from the Molino and Mystic Springs, FL, boat ramps (30.85427°N, 87.31126°W) on the Escambia River. We identified gravid Q. succissa by the presence of inflated gills and a change in gill color from grayish to cream-colored (Williams et al. 2008). Gravid mussels were kept cool during transport and were held at 10 °C in the laboratory to prevent premature release of glochidia (Zale and Neves 1982). We recorded water temperature during each Q. succissa survey to investigate the relationship between water temperature and glochidia maturation. This relationship was not determined for L. straminea. Host-fish collection We collected potential host fishes from the Blackwater River (Santa Rosa County, FL) throughout April in 2012 and 2013 via boat electrofishing (Fig. 1). The Blackwater River watershed is a system naturally lacking mussel populations (Williams et al. 2008), which ensured that potential hosts had not developed passive immunity to glochidia from a previous glochidial infection (Reuling 1919, Rogers and Dimock 2003, Zale and Neves 1982). Each fish collected throughout the study was less than 25.4 cm total length due to size limitation in the aquarium system setup. Fish sampling in 2012 targeted 2 known hosts, Micropterus salmoides (Lacepède) (Largemouth Bass) and Lepomis macrochirus (Rafinesque) (Bluegill), based on previous research for L. straminea (Keller and Ruessler 1997). We also collected and investigated the host potential for 10 other fish species that occupied similar habitats to L. straminea (Table 1). Fish sampling in 2013 targeted ictalurid and cyprinid fishes (Table 2) since they are common hosts for other species of Quadrula (Haag and Warren 2003; Hove et al. 2011, 2012; Howard 1913). Juvenile Ictalurus punctatus (Channel Catfish) were difficult to collect in early spring, so we obtained young-of-the-year Channel Catfish from the Richloam State Fish Hatchery in Webster, FL. Potential host fishes were held in a flow-through aquarium system (10-L tanks, Pentair Aquatic Habitats, Apopka, FL) and acclimated to the system for approximately one month prior to the study. This system was located in a climate-controlled building to maintain a constant water temperature (~22.5 °C) to assure survival of the potential hosts. All water was obtained from an onsite well and aerated before it entered the system. Host-fish trials Methods for the host-fish trials followed these general steps: 1) glochidial collection, 2) verification of glochidial viability, 3) glochidial infestation of host fishes, and 4) monitoring of juvenile mussel transformation. Methods varied for completing steps 1–3 for the L. straminea and Q. succissa trials, but were the same for step 4. We adapted the methods in the Q. succissa study from the L. straminea methods to streamline the infestation process. Additionally, the gravid gills of Q. succissa were smaller and less inflated than those of L. straminea, which precluded the gill-flushing methods used in the L. straminea trials. Southeastern Naturalist 247 K.M. Harriger, J.R. Knight, and M.G. Wegener 2015 Vol. 14, No. 2 Lampsilis straminea. We collected glochidia from 2 gravid L. straminea by piercing the gills with a 3-ml syringe of water and flushing glochidia into a beaker of water, similar to methods used by O’Dee and Watters (2000). We exposed a subsample of glochidia (n < 50) to salt crystals (NaCl) to determine viability, which was identified by rapid snapping in response to the salt (Lefevre and Curtis 1912, Zale and Neves 1982). Glochidia were used in host-fish trials only if greater than 80% of the glochidia in the subsample were viable. We allowed viable glochidia for the L. straminea trials to settle to the bottom of a 250-ml beaker of water and then collected the dense suspensions of glochidia from the bottom of the beaker using a 1-ml syringe. Methods for infecting potential host fishes with L. straminea glochidia were adapted from O’Brien and Brim Box (1999). We introduced up to 4 fish of various species to glochidia inside each container of water with aeration to ensure that glochidia remained suspended. Small fishes were exposed to 0.1 ml of a dense suspension of glochidia inside a 2.4-L container filled with 1 L of water; larger fishes were exposed to 0.2 ml of a dense suspension of glochidia inside a 4.7-L container filled with 2 L of water. We did not calculate the concentration of glochidia in the suspension for this preliminary host-fish trial. Fishes were exposed to glochidia for 30 minutes and returned to their respective aquaria. Quadrula succissa. We collected glochidia from Q. succissa by placing the mussels into containers of water at room temperature (~21 °C) and allowing them to release glochidia naturally (Haag and Warren 2003). We used 8 mussels for the first trial and 15 mussels for the second trial. However, the actual number of mussels that contributed to the glochidia used in each trial was unknown because mussels were not held individually. Host-fish infection methods were the same as those used for L. straminea, except for the use of fewer, larger buckets (to streamline the infection process). For each trial, we divided the fishes into 2 groups (small fishes and larger fishes), and each group was held in a 26.5-L bucket with 15.1 L of water. Approximately 1500 viable glochidia were used in each bucket during the first trial, and approximately 1900 viable glochidia were used in each bucket in the second trial. Once fishes had been infected by glochidia, the methods were the same in the 2012 and 2013 trials. Each aquarium had a filter cup (5.1-cm PVC pipe with 150- μm nylon screen covering one end) placed at the outflow to catch glochidia and transformed juvenile mussels. We attempted to conduct replicate trials for all fish species, although they were not possible for some stream fishes due to small sample sizes or mortality prior to a trial. We examined the contents of filter cups under a dissecting microscope approximately every 48 hours to count the number of viable juvenile mussels, nonviable juvenile mussels, and glochidia (only presence/absence of glochidia was noted for L. straminea) that were present. Flow rate in the aquarium system was increased for approximately 20 minutes prior to examining filter cups to ensure that the aquaria were adequately flushed. We identified viable juvenile mussels by foot movement (Haag and Warren 1997) and considered juvenile mussels that showed Southeastern Naturalist K.M. Harriger, J.R. Knight, and M.G. Wegener 2015 Vol. 14, No. 2 248 no movement to be nonviable. We considered fishes that produced viable juvenile mussels to be hosts. If no glochidia or juvenile mussels were found in a filter cup after 3 consecutive examinations, the associated potential host fish was removed from the trial (Fritts et al. 2012). Data analysis During the 2013 Q. succissa trial, the suitability of each fish species as a host was quantitatively evaluated. For individual fish, we first calculated percentage success of juvenile mussel metamorphosis (%M = [viable juveniles/(viable juveniles + nonviable juveniles + glochidia)] × 100) and then the mean %M across replicates for each fish species (modified from Johnson et al. 2012). Nonviable juvenile mussels were included in the calculation because viable and nonviable juveniles were differentiated in this study, and the term “viable juvenile” rather than “juvenile” was used to avoid confusion. Metamorphosis success may have been overestimated because fishes were not rinsed immediately after the glochidial infection. We were unable to calculate %M for the L. straminea trial because counts on glochidia were not recorded. Results Lampsilis straminea A total of 3608 viable juvenile L. straminea were produced during the trial. Six of the 12 fish species produced viable juvenile L. straminea (Table 1). Largemouth Bass and Bluegill, both known host fish for L. straminea, were confirmed as hosts in this study. Newly identified hosts included Micropterus. sp. cf. punctulatus (Provisional Bass species; Bagley et al. 2011), Lepomis punctatus (Spotted Sunfish), Ambloplites ariommus (Shadow Bass), and Fundulus olivaceus (Blackspotted Topminnow). Largemouth Bass (n = 4) produced 73% (2648 individuals) of the total viable juvenile mussels (hereafter referred to as juvenile mussels) produced during the trial (Table 1). Shadow Bass and the Provisional Table 1. Fish species, sample size (n), percent of fish that produced juvenile mussels (%), total number of juvenile mussels collected (Juv), and days to juvenile mussel transformation (Period) during the 2012 host-fish study for Lampsilis straminea (16 May–18 June 2012). Fish species n (%) Juv Period Cyprinella venusta Girard (Blacktail Shiner) 3 (0) 0 - Moxostoma poecilurum Jordan (Blacktail Redhorse) 2 (0) 0 - Fundulus olivaceus (Storer) (Blackspotted Topminnow) 1 (100) 4 19 Ambloplites ariommus Viosca (Shadow Bass) 1 (100) 534 14–26 Lepomis auritus (L.) (Redbreast Sunfish) 1 (0) 0 - Lepomis gulosus Cuvier (Warmouth) 2 (0) 0 - Lepomis macrochirus Rafinesque (Bluegill) 6 (83) 60 14–26 Lepomis megalotis (Rafinesque) (Longear Sunfish) 3 (0) 0 - Lepomis punctatus (Valenciennes) (Spotted Sunfish) 2 (50) 5 14–19 Micropterus sp. cf. punctulatus (Bagley et al.) (Provisional Bass species) 2 (100) 357 14–19 Micropterus salmoides (Lacepède) (Largemouth Bass) 4 (100) 2648 14–28 Percina nigrofasciata (Agassiz) (Blackbanded Darter) 2 (0) 0 - Southeastern Naturalist 249 K.M. Harriger, J.R. Knight, and M.G. Wegener 2015 Vol. 14, No. 2 Bass species also produced large numbers of juvenile mussels despite the small sample size of both species (n = 1 and n = 2, respectively). The Blackspotted Topminnow (n = 1) was the only non-centrarchid identified as a host, and it produced only 4 juveniles during the trial. Due to small sample size and limited number of juvenile mussels produced, continued research is needed to confirm the suitability of Blackspotted Topminnow as a host to L. straminea. Quadrula succissa A total of 314 juvenile Q. succissa were produced over the course of both trials. Juveniles were produced only from infections of ictalurids (Table 2). Channel Catfish was the only host identified in the first trial, and Channel Catfish, Ameiurus natalis (Yellow Bullhead), and Noturus funebris (Black Madtom) were identified as hosts in the second trial (Table 2). Channel Catfish (n = 2) had the greatest metamorphosis rate (mean %M = 42.1, range = 25.0–59.3%), followed by Yellow Bullhead (n = 2, %M = 13.0) and Black Madtom (n = 2, %M = 4.5%). Ten juvenile mussels were collected from one Black Madtom on day 12 during the trial (Table 2). Quadrula succissa with mature glochidia were collected between 7 June and 3 July 2013 in the Escambia and Yellow rivers; water temperatures during that time ranged from 24 to 29 °C. Quadrula succissa were not gravid during surveys in May and September 2013. Surveys were not conducted from early July through August due to high water levels. Discussion Host-fish trials were successful in producing juvenile mussels from host fishes for both short- and long-term–brooding mussels. Techniques used for conducting host-fish trials were validated by confirming known hosts for L. straminea. The techniques were further validated by identifying additional hosts for L. straminea Table 2. Results of the 2013 host-fish study for Quadrula succissa (trial A: 28 June–22 July 2013, and trial B: 3 July–2 August 2013) including fish species, total number of juvenile mussels collected (Juv), days to juvenile mussel transformation (Period), and mean metamorpho sis success (%M) with standard deviation (SD). One individual represented each fish sp ecies in each trial. Trial A Trial B Fish species Juv Period Juv Period %M (SD) Cyprinella venusta (Blacktail Shiner) 0 - 0 - - Notropis texanus Girard (Weed Shiner) 0 - 0 - - Pteronotropis hypselopterus (Günther) (Sailfin Shiner) 0 - 0 - - Ameiurus natalis (Lesueur) (Yellow Bullhead) 0 - 158 14–23 13.0 (6.4) Ictalurus punctatus (Rafinesque) (Channel Catfish) 12 14–17 134 12–21 42.1 (24.3) Noturus funebris Gilbert and Swain (Black Madtom) 0 - 10 12 4.5 (18.4) Noturus leptacanthus Jordan (Speckled Madtom) 0 - 0 - - Fundulus olivaceus (Blackspotted Topminnow) 0 - 0 - - Lepomis macrochirus (Bluegill) 0 - 0 - - Micropterus sp. cf. punctulatus Provisional Bass species 0 - 0 - - Southeastern Naturalist K.M. Harriger, J.R. Knight, and M.G. Wegener 2015 Vol. 14, No. 2 250 that are similar to known hosts for other species of Lampsilis. Three of the new hosts for L. straminea were predatory fish: Provisional Bass species, Largemouth Bass, and Shadow Bass. Lampsilis straminea produces a mantle lure that imitates a small prey fish (Williams et al. 2008), so it is logical that piscivorous fish, such as Largemouth Bass, would be attracted to the lure and serve as hosts for L. straminea. Other Lampsilis spp. host-fish trials show Micropterus spp. producing more juvenile mussels than other fishes (Haag et al. 1999; Haag and Warren 1997, 2003; Keller and Ruessler 1997; O’Brien and Brim Box 1999). Other newly documented hosts were Spotted Sunfish and Blackspotted Topminnow, though these were likely only marginal hosts due to the small number of juvenile mussels produced. Additionally, Blackspotted Topminnows are not expected to be an ecologically important host due to surface-feeding habits that would probably make natural glochidial infestations rare (Haag and Warren 1997). The current study also identified 3 ictalurid hosts for Q. succissa, a short-term brooder, for which no hosts had been previously identified, although ictalurids have been identified as hosts for several other species of Quadrula. Ictalurids, such as Channel Catfish and Pylodictis olivaris (Rafinesque) (Flathead Catfish), tended to produce more juvenile mussels than Noturus spp. and Ameiurus spp. (Haag and Warren 2003; Hove et al. 2011, 2012; Howard 1913), and results herein corroborate these studies. Results of the Q. succissa trials indicate that at least 3 ictalurids are hosts, but the suitability of each ictalurid as a host is not yet clear. At least 3 criteria should be examined when determining the suitability of a host fish: the number of juvenile mussels produced, metamorphosis success, and percent of a fish species that successfully produced juvenile mussels (Johnson et al. 2012, O’Brien and Brim Box 1999). Examining the suitability of a host fish under these criteria helps determine the importance of the host relative to other fish hosts (Eads et al. 2010, McNichols et al. 2011, O’Brien and Brim Box 1999). Based on the above criteria, Channel Catfish may be the most suitable host fish tested in our trials since it was the only fish species to consecutively produce juveniles in the trials, and also had the greatest mean %M (42.1%). The importance of this species is currently unclear since there was high variation in %M. It is difficult to understand the importance of the other ictalurids (Yellow Bullhead and Black Madtom) as hosts to Q. succissa at this time since these trials had limited success. Additional host trials are needed to better understand host-fish requirements for Q. succissa and should focus on ictalurids tested in this study along with Ameiurus nebulosus (Lesueur) (Brown Bullhead), Ameiurus catus (L.) (White Catfish), Ictalurus furcatus (Valenciennes) (Blue Catfish), and Flathead Catfish. Methods herein may have contributed to ambiguous results in Q. succissa trials. Glochidial concentrations in trials were lower than many other studies (i.e., 2000/L in Dodd et al. 2006, 4000/L in Johnson et al. 2012) and may have contributed to lower glochidial infestation rates. Furthermore, fish species were combined in containers during host infestation, whereas many studies keep fish species separated (Johnson et al. 2012, O’Brien and Brim Box 1999). Combining fish species Southeastern Naturalist 251 K.M. Harriger, J.R. Knight, and M.G. Wegener 2015 Vol. 14, No. 2 may have altered fish behavior and negatively affected glochidial infestation rates. Additionally, gills of the host fish in this study were not examined for encysted glochidia after host trials were completed. While methods in this study were somewhat altered from common standards, useful data were provided with apparent hosts and additional insight into other possible vectors of juvenile recr uitment. This study lays a foundation for future host-fish research at the BRDC and provides information that will be valuable to the conservation of mussels in Florida Gulf Coast drainages. We successfully conducted host-fish trials for both long- and short-term–brooding mussels, expanded host-fish knowledge for L. straminea, and identified new hosts for Q. succissa. This information will be useful in propagation programs for these species, if needed, and will help direct future host-fish research for these species. Acknowledgments We thank Paul Johnson, Jim Williams, and Gary Warren for their insights in setting up our aquarium system and working with brood mussels. 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