4 Propagation and Culture of Freshwater Mussels Cristi D. Bishop, Robert Hudson, and Jerry L. Farris INTRODUCTION The propagation and recovery of federally listed species has been supportedbywrittenpolicy from the U.S. Fish and Wildlife Service (USFWS) and National Marine Fisheries Service (NMFS). This policy outlines specific guidelines for the use of propagation as an essential tool for recovery and conservation of adeclining population through supportfrom the Endangered Species Act of 1973. Historical propagation efforts of other invertebrates, vertebrates, and plantspecies have indicated that this approach has circumvented the decline of certain species.Furthermore, one of the goals of species restoration is to develop sound policies based on best available technology, subsequently, the use of propagation in freshwater mussel recovery plansmay allow for alternative techniques beyond conventionalfish hostencystments.Implementingspecies recoveryasoutlined in the Endangered Species Act, requiresscientists to not only provide the technology for viable popu- lations (i.e., surviving and reproducing individuals), but also to account for their habitat and life historyrequirementssothatpopulationscan maintain enoughgeneticvariability to adaptto changesinthe environment. Unionidpropagation in the United States began in the early 1900s employing techniques very similar to thoseused today, which provided scientists abasis for understanding at least the limited requirements of this complexgroup of invertebrates. Understanding the life history of this unique group of invertebratesalsoforcesustoexaminethe life history, physiology,biochemistry, immunochemistry, and bioaccumulationofspecifichostfish.The specificmorphology of the fish gill as well as the blood supplyand gas exchange that it provides has been well publishedin the literature. This understandingofhost-fish physiology was aprerequisite for the development of artificial culturemedia (in vitro technique) in the early 1980s, which provided information about specific requirements for nutrients that supported development and growth of exposedglochidia. Propagation techniques that support juvenile transformation continue to be used with declining populations, including federally threatened and endangered (T&E) species identified in federal recovery plans. Techniques that include the use of host fish (in vivo) and in vitro methods have generated viable juveniles for various objectives including toxicity testing, in situ monitoring, and reintroduction efforts into recovering streams(Jacobson et al. 1993; Yeager, Cherry, and Neves 1994; Morgan, Welker, and Layzer 1997). Monitoring juvenile responses and survival has furn- ished evidence of differential sensitivity to various contaminants, holding conditions, and feeding regimes. Effective propagation has supported determination of possible impacts on early life stages (glochidia and juvenile), where LC50scan be calculated and compared to existing water quality criteriaand habitat assessments. Such information helps clarify reasons for declining populations and allowsmore accurateand effective evaluation of mitigation efforts of sensitive invertebrates. Juvenilepropagation techniques have been employed forvarioususes and aregenerally used 4284X—CHAPTER 4—19/10/2006—15:45—KARTHIA—XML MODEL C–pp. 65–94 65 © 2007 by the Society of Environmental Toxicology and Chemistry (SETAC) to reintroduce individuals into recovery streams, provide insight into the necessary requirements of arelatively unknowngroup of species, and assess impact by measuring endpointsthat demonstrate the relative sensitivities of this early life stage to various contaminants. The followingsections will address theseareasofresearch that have provided some indication for the reasons of continued decline for this group of uniqueaquatic invertebrates. B IVALVE L IFE H ISTORY:UNDERSTANDING E ARLY L IFE S TAGE L IMITATIONS The criticalevaluation of early life stage organisms is imperative to understand requirements for survival,growth,and reproduction of aspecies. It provides insight into the requirements for growth, effects of environmental perturbations, and sensitivities to contaminants. Scientistsfrom the U.S. Environmental Protection Agency (USEPA) that were developing guidance documents in the mid 1970s for toxicity test methods realized that early life stage testing was perhaps more critical to the protection of apopulation than older-age individuals; therefore, comparing sensitivities among different age groups is important for the determination of protection with water quality criteria. Toxicity test methods for adults, juveniles, and glochidiaare outlined in Chapter5. Measuring lethal and sublethal effects in early life stage individuals (e.g., glochidia or juveniles) provides an understanding of the range of sensitivities to various pollutantsthat are knowntoimpact thesurvival andreproduction of aspecies.The need forpropagation in testingarisesfromthe difficulty in finding naturally propagated juveniles in aquatic systems. The use of artificial techniques in culturing juveniles can offer additional opportunities for determining cause-effect relationships through laboratory or in situ toxicity tests. This will provide supportfor the much-needed protective measures (e.g., implementation of best management practices to reducesuspended solids, consider- ation of sensitive species in effluent discharge permits, and changesinthe pesticide registration process to include more sensitive, bivalve species) for this declining group of invertebrates.This decline prompted the U.S. Congress to amend the Endangered Species Act in 1988 to require federal agencies (e.g., USFWS) to provide implementation plans for the recovery of federally listed species. Recoveryplans generally include protectivemeasures to preventfurther population declines and supportbivalve conservationmeasures, which are achieved by improving the habitat, translocating the species,increasing the quality of captive propagation programs, and acquiring land adjacent to recovering streams. Recovery plans for freshwater mussels generally include habitat restoration, fish host suitability, and propagation of juveniles for reintroduction.Propagation efforts are currently limited to fish host techniques as aviable alternative to natural recruitment within an aquatic system. However, the utilization of artificial culture media may be the only way that somemusselswill survive extinction (personalcommunication, SAhlstedt 2003). Very often, artificial culturetech- niquescan produce significantly more juveniles than host-fish techniques. For example, in studies that used propagation techniques for fish hostsand artificial culture, therewere nearly ten and twenty timesmore juveniles cultured from various culturemedia. The uncertainties inherent in artificial cultureshouldnot restrict federal policy by limitingthe approach of propagationtofish host techniques only. THE NEED FOR ARTIFICIAL PROPAGATION The development of fish host techniques in the early 1900s, as well as the improvement of culture media for propagating mussels using artificial media (in vitro),provides auniqueopportunity for researcherstoidentify life historyrequirementsand survival mechanisms forthisgroup of declining bivalves.Artificialpropagation can be employedinresponse to environmentalspills and invasive exotic species (e.g., zebra mussels and black carp), as well as help stabilizedeclining populations through species recovery plans. Juveniletransformation has been reported for many species usingboth fish host (in vivo) and artificial media (in vitro).However, some species pose a significant challenge whenusingeither technique, including Elliptodeussloatianus,which produce Freshwater BivalveEcotoxicology66 4284X—CHAPTER 4—19/10/2006—15:45—KARTHIA—XML MODEL C–pp. 65–94 © 2007 by the Society of Environmental Toxicology and Chemistry (SETAC) lanceolate conglutinates, Lampsilis fragilis,and Pleurobema spp. (Milam et al. 2000;personal communication, PJohnson2003). To date,there are only two propagation techniques reported in the literature, one that includes host fish(Lefevre and Curtis 1912)and another using artificial culturemedia (Isom and Hudson 1982). This section will provide some insight into the current development of these techniques as well as include arguments for and against each one whendetermining which propagation method is most appropriate (Table 4.1). F ISH H OST T ECHNIQUES ( IN V IVO ) Techniques for the determination of fish hostshave been reportedand utilized by many researchers for decades (Howard1916; Coker et al. 1921; Penn 1939; Cope 1959; Giustietal. 1975; Jenkinson 1982; Hove and Neves 1991), while someunconventional hosts (e.g., amphibians) have alsobeen identified as supporting juvenile metamorphosis (Seshaiya 1969). Some species have shown rela- tively good successusing fishhosttechniques,whileothershaveproventobemoredifficult. Commonspecies as well as state and federally listed species are often difficult to transform due to the lackofknowledge of life history complexities and requirements (personalcommunication, JJones 2004). Two federally endangered species that are, at this point, particularly difficult are Dromus dromas and Cyprogenia stegaria. Techniques for determining fish host suitability include the use of aeration tanks, direct gill placement, and the use of anesthetics (i.e., MS222, which is tricaine methanesulfonate and trade name Finquel) to reduce handling stress on the fish (Zale and Neves 1982). While modifications of these have been reported from various researchers, the fundamental approach is the same. Aeration tanks are often used when there are viable glochidiawithseveral fish species and cohorts. However, if glochidiaare limited and/or the fish are smaller in sizeorhave small gill rakers, direct gill placement using pipettes is aviable alternative to aeration techniques for attachment onto the gill. Anesthetizing fish prior to encystment is undecided,inthat the possible effects of the anesthetic may inadvertently impair glochidia attachment and subsequent metamorphosis. The use of MS222 is not necessary if the pipetting onto the gill is done quickly, with little stress to the fish. Glochidial attachment can range from several days to several months depending on the mussel species,fish health (i.e., whether the individual is stressed or diseased duetootherenvironmental factors), water temperature, and perhaps othervariables presently unknown. Alternatively, fish survival can be jeopardized by excessive glochidial infestation as aresult of limiting gasexchange across the gill lamellae. While 50–100 glochidia/gill for fish that are 15–25 cm in length have been reportedasadequate (Hove et al. 2000), others have directly infested host fish with several thousand and achieved successful transformation and maintained fish viability once removed from the tanks (Milam et al. 2000; Winterringer 2004). Hove et al. (2000) described considerations for conducting fish suitability tests and included several issues that are important for the successful transformation of juveniles. Fish maintenance and holding prior to and during the encystment is fundamental to the fish host technique. The use of glass or high-gradepolycarbonate,flow-through tanks with adequate aerationand temperature controldevices aresuggestedtoreducefluctuations in waterquality andquantity.Providing some nutritiontohost fish during the encystment period has been noted several ways: feeding fish throughoutthe entire period and feeding fish only through the first half of the encystment period and eliminating food thereafter. Thelatter method reduces the amount of fish feces in the bottom of the tank,increasing the ability to selectively isolate juveniles more efficiently. Juveniles should be siphoned from the tank bottom and collected usingasieve series for isolation. Apolarized lens, which is attached to the objective lens of adissectingmicroscope, can be used to reflect, through understage lighting, only prismatic objects and block out sediment and feces that often eclipse the juvenile identification and counting process (Watters 1996). High quality foods (e.g., brine shrimp or live minnows) shouldbepromoted in the maintenance of fish hosts; however, acritical step in Propagation and Culture of Freshwater Mussels 67 4284X—CHAPTER 4—19/10/2006—15:45—KARTHIA—XML MODEL C–pp. 65–94 © 2007 by the Society of Environmental Toxicology and Chemistry (SETAC) TABLE 4.1 SummaryofSuccessful Juvenile Transformation Efforts Using Various Unionid Species: 1982–2003 Species Technique Purpose Reference A. plicata Fish host Reintroduction Hubbs (2000) Media Culture development Personal communication, BHudson and M Barfield (1993) Anodonta suborbiculata Fish host Host suitability Barnhart and Roberts (1997) (blue) Anodontoides ferussacianus Fish host Host suitability Hove et al. (1997) (blue) Toxilasma cylindrellus Fish host Unknown Hudson and Isom 1984 Cyclonaias tuberculata Fish host Host suitability Hove et al. (1997) (blue) E. angustata Media Toxicity testing Reintroduction Hudson, Barfield, and McKinney (1996) E. complanata Media Culture development Hudson, Barfield, and McKinney (1996) E. crassidens Media Unknown Personal communication, DSimbeck (2003) E. icenterina Fish host Toxicity testing Keller and Ruessler (1997) Fusconaia ebena Media Culture development Isom and Hudson (1982) Fusconaia flava Media Reintroduction Milam et al. (2000) Lampsilis cardium Fish host Toxicity testing Keller and Ruessler (1997) Media Milam et al. (2000) Myers-Kinzie2000 L. fasciola Fish host Reintroduction Morgan, Welker, and Layzer (1997) (blue) Media L. ovata Fish host Culture development Isom and Hudson (1982) L. rafinesqueana Fish host Host suitability Barnhart and Roberts (1997) and (blue) Shiver (2002) L. reeveiana Fish host Host suitability Barnhart and Roberts (1997) (blue) L. siliquoidea Media Reintroduction Milam et al. (2000) Media Survival and growth Myers-Kinzie(2000) L. streckeri Fish host Host suitability and reintroduction Winterringer(2003) L. subangulata Fish host Host suitability Personal Communication, CEchevarria(2004) L. teres Media Unknown Keller and Zam (1990) L. ventricosa Media Unknown Milam et al. (2000) Ligumia recta Media Culture development Isom and Hudson (1982),Milam et al. (2000) M. conradicus Fish host Reintroduction Morgan, Welker, and Layzer (1997) (blue) Megalonaias gigantia Media Unknown Personal Communication, BIsom and DSimbeck (2003) M. nervosa Fish host Reintroduction Hubbs (2000) Pleurobema coccineum Fish host Host suitability Hove et al. (1997) (blue) P. cordatum Media Culture development Hudson and Isom (1984) Ptychobranchus occidentalis Fish host Host suitability Barnhart and Roberts (1997) (blue) P. cataracta Media Unknown Dimmock and Wright (1993) P. grandis Fish host Toxicity testing Keller and Ruessler (1997) Fish host Reintroduction Milam et al. (2000) Media Personal Communication, BIsom (2003) S. undulatus Fish host Host suitability Hove et al. (1997) (blue) U. imbecillis Fish host Toxicity testing Keller and Zam (1991) (continued) Freshwater BivalveEcotoxicology68 4284X—CHAPTER 4—19/10/2006—15:45—KARTHIA—XML MODEL C–pp. 65–94 © 2007 by the Society of Environmental Toxicology and Chemistry (SETAC) reducing the possibility of fungaland bacterial growths includesthe prompt removal of uneaten food. Hove et al. (2000) stated that for bottom-feeding fish (e.g., minnowsand othercatostomids) that will feed on newly metamorphosed juveniles and sloughed glochidia, it is important to separate the fish from the bottom of the tank using aplastic net with asmall mesh size (1.6 mm) secured to the tank, which allowsthe juveniles to fall through the mesh but keeps fish from bottom feeding. Collection of wild host fish or even commercially spawned fish species requiressomeattention to detail,including the acclimation of fish once they have been broughtback to the propagation facility. We suggestthat fish be allowed to acclimate for several days prior to isolatingfor glochidia infestation. Conducting fish suitability trials should include multipleattempts using several indi- viduals of the same fish species with glochidia from different females to assure that metamorphosis occurs in at least two different test trials(Haag 2002). The range of fish species that co-occur with mussel populations is important to understand from amanagement perspective. Long-termrestoration goalsshouldinclude the successful recruitment of mussel fauna as well as viable host-fish populations in ariver reach. Subsequently, instream habitats shouldaccommodate both mussel and fish life historyrequirements to ensurethat sustain- able populations are beingsupported. Aviable mechanism for supporting juvenile reintroductions is to releaseinfested fish into the waterbody with known glochidia species.Several reports have indicated that the releaseofinfected fish can supportthe efforts of arecovery plan for both common and listed species (Milam et al. 2000;Genoa NFH, Chapter 5, Methods for Conducting ToxicityTests Using Corbicula Fluminea as Surrogate Species). Dependence on Fish Hosts—An Obligate Trait? Identification of fishhosts for unionid species has been reportedinthe literature sincethe turn of the twentieth century whenConnor (1905) identified Lepomis gibbosus (pumpkinseed) as asuccessful TABLE 4.1 (Continued) Species Technique Purpose Reference Warren (1996) Clem (1998) U. imbecillis Media Culture development Isom and Hudson (1982) Barfield et al. (1997) (blue) Toxicity testing Hudson and Shelbourne (1990) Wade et al. (1989) Fish host Physiological effects Dimmock and Wright (1993) Fish host Viability Fisher and Dimmock (2000) Media Unknown Keller and Zam (1990) Venustaconcha ellipsiformis Fish host Host suitability Riusech and Barnhart (2000) V. pleasii Fish host Host suitability Riusech and Barnhart (2000) V. iris Fish host Toxicity testing Jacobson et al. (1993) Fish host Behavior Yeager et al. (1994) Media Unknown Pers. comm. DSimbeck V. liensosa Fish host Toxicity testing Keller and Ruessler (1997) Fish host Host suitability Pers. comm. CEchevarria Media Unknown Keller and Zam (1990) V. taeniata Fish host Reintroduction Morgan, Welker, and Layzer (1997) (blue) V. vibex Fish host Host suitability Pers. comm. CEchevarria Propagation and Culture of Freshwater Mussels 69 4284X—CHAPTER 4—19/10/2006—15:45—KARTHIA—XML MODEL C–pp. 65–94 © 2007 by the Society of Environmental Toxicology and Chemistry (SETAC) fish host for Anodontacataracta (now Pyganodoncataracta ,Hoeh 1990). Watters (1994) reviewed publishedfish host and unionid relationships in North America with approximately95unionid species and over 150 fish species. Since then,there have been numerous publications that provide updates to reportedhost-fish requirements and include new fish species that are successful candi- dates to support juvenile transformation (Barnhart and Roberts 1997; Dee and Watters 1998;Hove et al. 2000;Winterringer 2004). Asummary of propagation techniques (e.g., fish host or artificial media), sinceWatters’ review was reportedin1994, has been provided as an update (Table 4.2). While the research suggests that mostspecies require ahost fish as an obligate trait of the bivalve life history, afew do not. Unionidsare alsotypically identified as either ageneralist, where its glochidia can transform on avarietyoffish species,oraspecialist, where only one or two host fish have been identified that aid in the successful metamorphosisofglochidia to the juvenile stage. Additionally, with unionid populations that are deemed specialists, someoftheseare also species listed as threatened or endangered by federal and state governments.Current debate, however, may suggest that declines in unionid populations are not necessarily due to aspecific host-fish requirement but rather to some other factor that inhibits survival, growth, and reproduction post- transformation. Many recovery projects for T&E species have identified various host fish, which TABLE 4.2 Advantages and Disadvantages of Host Fish (in Vivo)and Culture Media(in Vitro) Propagation Techniques Use FishHost (in Vivo)CultureMedia (in Vitro) Advantage Disadvantage Advantage Disadvantage Ability to use infested fish to place directly into arecovering system is an easy method for novelists With species whose host fish are unknown, a considerableeffort may be needed to determine this prior to any juvenile reintroduction Ability to obtain considerably more juveniles per unit effort Costs can be significantly higher Reintroduction/ monitoring Efforts for recovering streams can secure future recruitment by knowing and assuring that fish hosts are also residing in the stream segment Use of basin-specific host fish may be limited to the propagation effort Method can provide viable juveniles in lieu of unreported fish hosts Fungal and microbial infestationscan eliminate transforming glochidia if not closely monitored Determination of required host fish for each species Unhealthy fish may limit the production of transformed juveniles Not applicable Not applicable Host suitability Previously infested fish are reported to be immune to aglochidia infestation (Arey 1932) Toxicity exposures With some contaminants, juveniles transformed in vivo can be more sensitive With some contaminants, juveniles transformed in vitro can be more sensitive Freshwater BivalveEcotoxicology70 4284X—CHAPTER 4—19/10/2006—15:45—KARTHIA—XML MODEL C–pp. 65–94 © 2007 by the Society of Environmental Toxicology and Chemistry (SETAC) supportsuccessful and viable juveniles. Listed species such as Lampsilis streckeri (Winterringer 2004)and Lampsilis powelli have been identified as generalists. Independence from Fish Hosts Not all unionid species require fish hostsfor the transformation of juveniles. Strophitus undulatus and Utterbackia imbecillis are the two most reportedspecies that were found to transform from glochidiatojuvenile inside the marsupial pouch (Lefevre and Curtis 1912; Howard 1916; Allen 1924;personal communication, MBarfield2003), while Obliquaria species may also be (Lefevre andCurtis 1912)anonparasitic unionid. Adultfemalesofthese species apparentlyprovide essential nutrients for metamorphosis to take place; however, quantity and quality of thesecritical nutrients are unknown. A RTIFICIAL M EDIA C ULTURE ( IN V ITRO) History Freshwater mussel glochidia (i.e., larvae) are naturally transformed into juveniles, which include development of the internal organsnecessaryfor self-sustained existence as abenthic organism. Thisisaccomplished viaencystment in fishtissue(on thegillorfin).Interest in enhancing production of musselsthrough theuse of artificialculture wasseenearly in thetwentieth century whenEllis and Ellis (1926) reported the first successful culture of glochidia to transfor- mation followingthe excisionofthese from thegilltissueoftheir fishhost. Unfortunately, the details of their solutions were never published, and the host-fish attachmentprior to culture initiationmay have provided the stimulating factor(s), which allowed this developmental process prior to cultureinitiation. Much later, in the early 1980s, interest in the artificial cultureofglochidia was revived by Isom.Isom,who was familiar with the transformation workofEllis and Ellis (1926),contracted Bob Hudson to help attempt this artificial culture, based on his work with the cellculture of catfish(Hudson, Pardue, andRoberts1980). Isom and Hudson (1982) reported success in thetransformationofseveral species without theuse of afish hostatany time in theprocess, adistinct improvement over the Ellisand Ellisreportof1926.Thistechnique, which beganasamodificationofmodern cell-culturetechniques, made useofamixture of aminoacids, vitamins, andglucose in aUnionid Ringers solution (Ellis,Merrick,and Ellis 1930), alongwith the addition of fish plasma as asourceofprotein, growth stimulants, hormones, etc. Although this work began by mixingthese componentsfrom scratch(using the concentrations of each found in fish plasma as guidelines), Isom and Hudson (1982) alsoreported success using pre-mixed,commercially available cell culturemedia (Eagles essential and non-essential amino acids and Medium 199), which containsnearly all of these amino acids in concentrations as high or higher than those found in fish plasma. Even though this mixture has been used to produce thousands of juvenile musselsfor toxi- cology research(Wade,Hudson, and McKinney 1989; Johnson, Keller, and Zam 1993; Hudson et al. 1994;Hudson, Barfield, and McKinney 1996; Barfield, Clem, and Farris 1997; Clem 1998), it has been less than convenient for otherlabs to use because of the requirement that there be aready supply of fish from which blood can be drained and separated into plasma and non-plasma com- ponents. Because of this inconvenience andbecause the useoffish plasmaintroduces more variation in the results, research was initiated in 1990 to try to develop an alternative medium that was either aserum/plasma-freemedium or amedium using commercially available serum in minimal concentrations (Hudson and Shelbourne 1990). Keller and Zam (1990) first addressed the modification of the glochidial culture, demonstrating that other sera couldbesubstituted for the fish plasma, with horse serumproducing their best results. Hudson and Shelborne (1990) began amassive study for Don Wade of the Tennessee Valley Authority, at Presbyterian College, testing atotal of 64 different medium combinations. Later, Johnson, Keller, and Zam (1993) describe Propagation and Culture of Freshwater Mussels 71 4284X—CHAPTER 4—19/10/2006—15:45—KARTHIA—XML MODEL C–pp. 65–94 © 2007 by the Society of Environmental Toxicology and Chemistry (SETAC) culturemethods in their acutetoxicitytesting procedure; however, theseare nearly identical to those described by Isom and Hudson (1982),using fish plasma as the protein source, and the same cultureconstituents including identical antibiotics without any real improvements in technique. Other advances come as aresult of modification of the Hudson and Shelbourne (1990) work by Barfield, Clem,and Farris (1997),Milam et al. (2000). Culture MediaTechniques Glochidia are removed as described by Isom and Hudson (1982) or preferably by usingasyringe full of controlwater to flush them from thefemale marsupia. However, differing fromprior publications, glochidia are rinsed three to four times in autoclaved river water or reconstituted waterratherthandeionized water, andafinal rinsewithUnionid Ringers solution or Hank’s Balanced Salt solution.Approximately 300–900 glochidiaare seeded in a3-mLtotal medium in each 60-mm diameter, cell-culture dish. These dishes are incubated at 21–248 Cinanincubator having 4.6–5% CO 2 to maintainapH of about 7.3byuse of abicarbonate buffer (Isom and Hudson 1982; Milam et al. 2000). Modificationofthe Media The originalmedium was comprised of Eagles essential and non-essential amino acids in Unionid Ringers containing NaHCO 3 for pH control, vitamins, antibiotics,and glucose as theartificial portion,and fishplasmaasthe naturalprotein source,inafinalratio of two-thirdsartificial medium to one-third plasma (Isomand Hudson1982).Aspreviouslymentioned, othermedia were tested in 1990(Hudson and Shelbourne 1990)inamajor effort to improveresults in more species,resulting in comparisons of 64 different media combinations. Forthe last decade, David McKinney, Chief of Environmental Services at Tennessee Wildlife Resources Agency (TWRA), has maintained interest in the cultureand use of juveniles for toxicity testing, funding research that hasbeen presented in severalreports that have addedtothe originalmodification by Hudson and Shelbourne in 1990. Although the primary work has involved only one species, U. imbecillis, otherspecies have also been transformed usingartificial media (Table 4.3). The following sections discuss specific artificial media componentsand their variations. IonicBalance .Initially, Isom and Hudson (1982) used amodified UnionidRingersdescribed by Ellis, Merrick,and Ellis (1930);however,further testsshow that prepared,balanced salt solutions such as Earle’s or Hank’s balanced salt solution (Sigma) are usefulinrinsingglochidia as well as in their artificial transformation, even thoughthe yield may be slightly lower. Sera.Fish plasma was reportedbyIsom and Hudson (1982) as the choice proteinadditive; however, rabbit serumperformsaswell or nearly as well as the fish plasma whentransforming U. imbecillis,and rabbit performanceisbetter than porcine, horse, sheep,chicken, and fetal bovine sera (Hudson and Shelbourne 1990). These results differ from Keller and Zam (1990) who reported that horse serum wastheir most productive proteinadditive.Combinationsofthe above sera andplasma resulted in thehighest production beingfound in amedium containingafish plasma/rabbitserum combination (usually equaltoorbetter than fishplasmaalone), butfish/ porcine and rabbit/porcine combinations were not significantly different from fish plasma.Fish/ fetal bovine and fish/horse were significantly lower (Hudson and Shelbourne 1990). Since some laboratories do not have access to fish plasma, and sinceinfection rates are lower whenusing sterile sera obtained from biochemical supply companies,rabbitserum is considered to be thebest alternative. In onecase, Elliptio angustata cultured in rabbitserumsignificantly outperformed fish plasma in observed transformation rates (Hudson and Shelbourne 1990). Serum Replacements.Rabbit serum performs better than othercommercially available sera (Hudson and Shelbourne 1990); however,the goal to eliminate sera or plasma altogether resulted in the testing of several serum replacements. Hudson and Shelbourne (1990) evaluated cultures Freshwater BivalveEcotoxicology72 4284X—CHAPTER 4—19/10/2006—15:45—KARTHIA—XML MODEL C–pp. 65–94 © 2007 by the Society of Environmental Toxicology and Chemistry (SETAC) initiated using six serumreplacements and found that none produced even 20% of the yield of fish plasma cultures. These replacements were then tested in combination with fish plasma, and the results showed that the CPSR (Sigma), TCM, and TCH (Protide) in combination with fish plasma produced ahigheryield than the medium containingonlyfish plasmawithoutany additives. These and otherserumreplacementswere then tested in combination with othersera,and the resulting data indicatedthatrabbitserumincombination with TCM andTCH outperformed TABLE 4.3 MusselSpeciesTransformed Using Artificial MediaCulture Species (Subfamily) Media Ty pe Transformation Time Required Reference (Anodontinae) U. imbecillis Rabbit/TCH/TCM; Horse serum; Fish plasma (pl.) and all combinations 7days Isom and Hudson (1982), Wade, Hudson, and McKinney (1989), Hudson and Shelbourne(1990), Keller and Zam (1990), Dimock and Wright (1993), Barfield, Clem, and Farris (1997) and many others. P. cataracta Rabbit/TCH/TCM 7days Dimmock and Wright (1993) P. grandis Fish pl. 7days Personal communication, BIsom (2003) (Lampsilinae) L. ovata Fish pl. 22 days Isom and Hudson (1982) L. fasciola Fish pl. Unknown Personal communication, DSimbeck (2003) Ligumia recta Fish pl. 15 days Isom and Hudson (1982) L. siliquoidea Rabbit; Fish pl. and all combinations; Rabbit/TCH/TCM 11-20 days Milam and Farris (1998), Milam et al. (2000), Myers-Kinzie(2000) L. teres Horse serum Unknown Keller and Zam (1990) L. ventricosa Rabbit; Fish pl. and all combinations 12 days Milam et al. (2000) V. iris Fish pl. Unknown Personal communication, DSimbeck (2003) V. lienosa Horse serum Unknown Keller and Zam (1990) Toxolasma cylindrellus Fish pl. 20 days Hudson and Isom (1984) (Ambleminae) A. plicata Fish pl. 12–13 days Hudson, Barfield, and McKinney (1996) Fusconaia ebena Fish pl. 18 days Isom and Hudson (1982) Fusconaia flava Rabbit; Fish pl. and all combinations 9–11 days Milam et al. (2000) Megalonias gigantia Fish pl. 16 days Personal communication, BIsom, and DSimbeck (2003) Pleurobema cordatum Fish pl. 15 days Hudson and Isom (1984) (Unioninae) E. angustata Rabbit/TCH/TCM and Fish pl. 7days Hudson and Shelbourne (1990) Hudson, Barfield, and McKinney (1996) Elliptio crassidens Fish pl. Unknown Personal communication, DSimbeck (2003) E. complanata Rabbit/TCH/TCM and Fish pl. 15 days Hudson, Barfield, and McKinney (1996) Propagation and Culture of Freshwater Mussels 73 4284X—CHAPTER 4—19/10/2006—15:45—KARTHIA—XML MODEL C–pp. 65–94 © 2007 by the Society of Environmental Toxicology and Chemistry (SETAC) othersera/serum replacement combinations. The rabbit/TCH/TCM portion was one-third of the total medium at aratio of 1:1:1 each usinga12% stock solution of TCH and TCM. Antibiotics/Antimycotics.Multiple rinses of glochidiawashed from the marsupia, as described earlier, are essential to lower the rate of infectionsfrom bacteria and fungi.Toalso help insure alow rate of infection, antibiotics (e.g., penicillin and streptomycin) and an antimycotic (e.g., amphoter- icin B) that are usually found in mostcell cultures were the first to be used in developing the glochidial medium (Isom and Hudson 1982). Improvements of these techniques weredone by isolatingand culturing bacteria from glochidial cultures and measuring zones of inhibition from approximatelyone dozen commercially available antibiotics (Isom and Hudson 1982). The three antibiotics (i.e., carbenicillin, gentamicin sulfate, and rifampin) with the best inhibitory effects were reportedbyIsom and Hudson (1982) and are currently those used in all laboratories that have reportedglochidial cultures. Much later,bacteria that wereisolated from swabs of the gills of Amblema plicata , P. cataracta,and U. imbecillis ,wereidentified andmeasured forinhibiting effects(e.g.,growth)byfifteenantibiotics (Lovelessetal. 1999). Theassumption behindthis work is that contamination of artificial glochidial cultures mostlikelycomes from the parent’s gill tissue, which houses theseglochidia. The most effective antibiotics against the dozen bacteria isolated were neomycin, ciprofloxacin, and polymyxinB.Since these have never been evaluated for effect on actual transformation success, amixture of antibiotics including two of thesenew anti- bioticswas tested along with acontrol of standard antibiotics (Isom and Hudson 1982)for their effect on musseltransformation.Thismixture contained apenicillin-streptomycin-neomycin solution (5000 IU, 10 mg, and 5mgrespectively) and was added to cultures at arate of 30 m L (low),60 m L(medium), and 120 m L(high) per 3-mL culture dish alongwithpolymyxinBat arate of 4mg/mL, 10 mg/mL,and 20 mg/mL concentrations.The penicillin and streptomycin are not in the new antibiotic best performer list (Loveless et al. 1999); however, this mixture is commercially available (Sigma Aldrich) for laboratory use. Between 600 and 800juveniles wereevaluated on day six of the glochidial cultureand again on the second day after each dish had been placed in water. Each of thesewas compared with the control antibiotics (Isom and Hudson 1982); however,none outperformed the original control set in transformation success (Figure4.1). These same concen- trations of new antibiotics were used in combination with the control antibiotics and the results were similar (Figure 4.1). All treatments in Figure4.1 are significantly different from the control with the exception of the treatment having medium concentrations of the new solutions mixed in combination with the control antibiotics (contingencychi-square Z 0.498, 1df, p Z 0.48). One advantage of using this increased number of antibiotics may be that awider spectrum of bacteria couldbecontrolled better than by use of just the original antibiotics described by Isom and Hudson (1982). Often,fungalinfection will appearinone or more dishes during the cultureprocess. When this happens, fungalmycelia are removed using sterile forceps, and 75 m Lofnystatin (Sigma) is added to each culturedish.Ifafungal or bacterial infection is massive, the developing glochidia are pouredinto acylinderwith a112-m mmesh Nytex w screen bottom,resuspended in asterile 200-mL beaker, and rinsed several times in Earle’s or Hank’s balanced saltsolution. Following the rinsing, the glochidiaare resuspended in adish of balanced salt solution, aspirated out, and placed into aculture dish with fresh, complete media. Other Medium Components.Most othermedium components remain the same as first described by Isom and Hudson (1982).Eagles essential and non-essential amino acids are mixed with the UnionidRingers with the addition of taurineand ornithine. Cultures seem to transform well without the addition of these last two amino acids, but since these are found in fish plasma and sincesome species of mussels mayrequire thesetwo amino acids, they arestill used in most cultures. L-glutamine must be added weekly due to its inability to remain stable in solution. Other media (e.g., Medium 199) have also provedsuccessful in transformation, even though their composition varies slightly from the above-modified EaglesMedium (Isom and Hudson 1982; Keller and Zam 1990). Glochidiadeveloping artificiallyseem to be lower in lipid content than those developing Freshwater BivalveEcotoxicology74 4284X—CHAPTER 4—19/10/2006—15:45—KARTHIA—XML MODEL C–pp. 65–94 © 2007 by the Society of Environmental Toxicology and Chemistry (SETAC) [...]... Ahlstedt and M Fagg 2003) Individuals were monitored for survival © 2007 by the Society of Environmental Toxicology and Chemistry (SETAC) 42 84X CHAPTER 4 19/10/2006—15 :46 —KARTHIA—XML MODEL C – pp 65– 94 84 4284X CHAPTER 4 19/10/2006—15 :46 —KARTHIA—XML MODEL C – pp 65– 94 TABLE 4. 6 Number of Individuals Reintroduced into North American Watersheds to Meet Federal Restoration Goals Species Milam et al (2000) Jones,... parastitic freshwater mussel glochidia, Report to the Tennessee Valley Authority, p 25, 1990 Hudson, R G., Pardue, G B., and Roberts, J F., Ictalurid chromosome preparation by cell culture and squash methods, Prog Fish-Cult., 42 , 43 45 , 1980 © 2007 by the Society of Environmental Toxicology and Chemistry (SETAC) 42 84X CHAPTER 4 19/10/2006—15 :46 —KARTHIA—XML MODEL C – pp 65– 94 92 Freshwater Bivalve Ecotoxicology... beginning to understand © 2007 by the Society of Environmental Toxicology and Chemistry (SETAC) 42 84X CHAPTER 4 19/10/2006—15 :46 —KARTHIA—XML MODEL C – pp 65– 94 90 Freshwater Bivalve Ecotoxicology REFERENCES Allen, E., The existence of a short reproductive cycle in Anodonta imbecillis, Biol Bull., 46 , 88– 94, 19 24 Baird, M S., Life history of the spectaclecase, Cumberlandia monodonta Say, 1892 (Bivalvia,... rates by Hudson and Isom (19 84) and Gatenby et al (1997) Bivalves can utilize the organic carbon in the © 2007 by the Society of Environmental Toxicology and Chemistry (SETAC) 42 84X CHAPTER 4 19/10/2006—15 :46 —KARTHIA—XML MODEL C – pp 65– 94 82 Freshwater Bivalve Ecotoxicology organic fines that coat sediment particles, particularly the smaller ones with a higher surfaceto-volume ratio Sediment is usually... streckeri) Recovery Plan, U.S Fish and Wildlife Service, Jackson, Mississippi, p 14, 1991 © 2007 by the Society of Environmental Toxicology and Chemistry (SETAC) 42 84X CHAPTER 4 19/10/2006—15 :46 —KARTHIA—XML MODEL C – pp 65– 94 94 Freshwater Bivalve Ecotoxicology Wade, D C., Hudson, R G., and McKinney, A D., The use of juvenile freshwater mussels as a test species for evaluating environmental toxicity, Abstracts,... suitability studies, In Freshwater Mollusk Symposia Proceedings, pp 27– 34, Tankersley, R A., Warmolts, D I., Watters, G T., Armitage, B J., Johnson, P D., Butler, R S., Eds., Ohio Biological Survey Columbus, OH p xxiC2 74, 2000 Howard, A D., A new record in rearing fresh-water pearl mussels, Trans Am Fish Soc., 44 , 45 47 , 19 14 Howard, A D., A second generation of artificially reared fresh-water mussels, Trans... Chemistry (SETAC) 42 84X CHAPTER 4 19/10/2006—15 :46 —KARTHIA—XML MODEL C – pp 65– 94 88 Freshwater Bivalve Ecotoxicology (personal communication, C Echevarria 20 04) Two species were federally listed as endangered: Lampsilis subangulata and Pleurobema pyriforme Most of the specimens recovered from the dry tributary were maintained at the hatchery in recirculating tanks for approximately one year Bi-annual glycogen... discharges and their effect upon freshwater bivalves, Environ Toxicol Chem., 17, 1611–1619, 1998 © 2007 by the Society of Environmental Toxicology and Chemistry (SETAC) 42 84X CHAPTER 4 19/10/2006—15 :46 —KARTHIA—XML MODEL C – pp 65– 94 Propagation and Culture of Freshwater Mussels 93 Milam, C D., Farris, J L., Van Hassel, J., and Barfield, M L., Reintroduction of native freshwater mussels using in vivo... Toxicology and Chemistry (SETAC) 42 84X CHAPTER 4 19/10/2006—15 :46 —KARTHIA—XML MODEL C – pp 65– 94 80 Freshwater Bivalve Ecotoxicology relatively less sensitive to thermal and hypoxic stresses following metamorphosis than those juveniles transformed in vitro Acute toxicity exposures of juvenile U imbecillis to potassium chloride (reference solution) indicated that one-day-old individuals transformed via... Hydrobiologia, 287, 161–178, 19 94 Farris, J L., Milam, C D., and Harris, J L., Zebra mussel impacts on freshwater mussels in Arkansas, Final report to the Arkansas Game and Fish Commission, Little Rock, AR, p 68, 1998 © 2007 by the Society of Environmental Toxicology and Chemistry (SETAC) 42 84X CHAPTER 4 19/10/2006—15 :46 —KARTHIA—XML MODEL C – pp 65– 94 Propagation and Culture of Freshwater Mussels 91 Fisher, . refrigerator. Freshwater BivalveEcotoxicology76 42 84X CHAPTER 4 19/10/2006—15 :46 —KARTHIA—XML MODEL C–pp. 65– 94 © 2007 by the Society of Environmental Toxicology and Chemistry (SETAC) TABLE 4. 5 Variations. (LC50 Z 1. 34 mg Cu/L at apHof5.5) than at higherpHvalues (LC50Z 2.75 mg Cu/L at apHof7.2). Freshwater BivalveEcotoxicology80 42 84X CHAPTER 4 19/10/2006—15 :46 —KARTHIA—XML MODEL C–pp. 65– 94 © 2007. (19 84) and Gatenbyetal. (1997). Bivalves can utilize the organic carbon in the Propagation and Culture of Freshwater Mussels 81 42 84X CHAPTER 4 19/10/2006—15 :46 —KARTHIA—XML MODEL C–pp. 65– 94 ©