Freshwater Bivalve Ecotoxoicology - Chapter 4 doc

THE NEED FOR ARTIFICIAL PROPAGATION The development of fish host techniques in the early 1900s, as well as the improvement of culturemedia for propagating mussels using artificial media in

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 supported by written policy fromthe U.S Fish and Wildlife Service (USFWS) and National Marine Fisheries Service (NMFS) Thispolicy outlines specific guidelines for the use of propagation as an essential tool for recovery andconservation of a declining population through support from the Endangered Species Act of 1973.Historical propagation efforts of other invertebrates, vertebrates, and plant species have indicatedthat this approach has circumvented the decline of certain species Furthermore, one of the goals ofspecies restoration is to develop sound policies based on best available technology, subsequently,the use of propagation in freshwater mussel recovery plans may allow for alternative techniquesbeyond conventional fish host encystments Implementing species recovery as outlined in theEndangered Species Act, requires scientists to not only provide the technology for viable popu-lations (i.e., surviving and reproducing individuals), but also to account for their habitat and lifehistory requirements so that populations can maintain enough genetic variability to adapt tochanges in the environment

Unionid propagation in the United States began in the early 1900s employing techniques verysimilar to those used today, which provided scientists a basis for understanding at least the limitedrequirements of this complex group of invertebrates Understanding the life history of this uniquegroup of invertebrates also forces us to examine the life history, physiology, biochemistry,immunochemistry, and bioaccumulation of specific host fish The specific morphology of thefish gill as well as the blood supply and gas exchange that it provides has been well published inthe literature This understanding of host-fish physiology was a prerequisite for the development ofartificial culture media (in vitro technique) in the early 1980s, which provided information aboutspecific requirements for nutrients that supported development and growth of exposed glochidia.Propagation techniques that support juvenile transformation continue to be used with decliningpopulations, including federally threatened and endangered (T&E) species identified in federalrecovery plans Techniques that include the use of host fish (in vivo) and in vitro methods havegenerated viable juveniles for various objectives including toxicity testing, in situ monitoring, andreintroduction efforts into recovering streams (Jacobson et al 1993; Yeager, Cherry, and Neves1994; Morgan, Welker, and Layzer 1997) Monitoring juvenile responses and survival has furn-ished evidence of differential sensitivity to various contaminants, holding conditions, and feedingregimes Effective propagation has supported determination of possible impacts on early life stages(glochidia and juvenile), where LC50s can be calculated and compared to existing water qualitycriteria and habitat assessments Such information helps clarify reasons for declining populationsand allows more accurate and effective evaluation of mitigation efforts of sensitive invertebrates.Juvenile propagation techniques have been employed for various uses and are generally used

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to reintroduce individuals into recovery streams, provide insight into the necessary requirements of

a relatively unknown group of species, and assess impact by measuring endpoints that demonstratethe relative sensitivities of this early life stage to various contaminants The following sections willaddress these areas of research that have provided some indication for the reasons of continueddecline for this group of unique aquatic invertebrates

BIVALVELIFEHISTORY: UNDERSTANDINGEARLYLIFESTAGELIMITATIONS

The critical evaluation of early life stage organisms is imperative to understand requirements forsurvival, growth, and reproduction of a species It provides insight into the requirements for growth,effects of environmental perturbations, and sensitivities to contaminants Scientists from the U.S.Environmental Protection Agency (USEPA) that were developing guidance documents in the mid1970s for toxicity test methods realized that early life stage testing was perhaps more critical to theprotection of a population than older-age individuals; therefore, comparing sensitivities amongdifferent age groups is important for the determination of protection with water quality criteria.Toxicity test methods for adults, juveniles, and glochidia are outlined inChapter 5

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 pollutants that are known to impactthe survival and reproduction of a species The need for propagation in testing arises from thedifficulty in finding naturally propagated juveniles in aquatic systems The use of artificial techniques

in culturing juveniles can offer additional opportunities for determining cause-effect relationshipsthrough laboratory or in situ toxicity tests This will provide support for the much-needed protectivemeasures (e.g., implementation of best management practices to reduce suspended solids, consider-ation of sensitive species in effluent discharge permits, and changes in the pesticide registrationprocess to include more sensitive, bivalve species) for this declining group of invertebrates Thisdecline prompted the U.S Congress to amend the Endangered Species Act in 1988 to require federalagencies (e.g., USFWS) to provide implementation plans for the recovery of federally listed species.Recovery plans generally include protective measures to prevent further population declines andsupport bivalve conservation measures, which are achieved by improving the habitat, translocatingthe species, increasing the quality of captive propagation programs, and acquiring land adjacent torecovering streams Recovery plans for freshwater mussels generally include habitat restoration, fishhost suitability, and propagation of juveniles for reintroduction Propagation efforts are currentlylimited to fish host techniques as a viable alternative to natural recruitment within an aquatic system.However, the utilization of artificial culture media may be the only way that some mussels willsurvive extinction (personal communication, S Ahlstedt 2003) Very often, artificial culture tech-niques can produce significantly more juveniles than host-fish techniques For example, in studiesthat used propagation techniques for fish hosts and artificial culture, there were nearly ten and twentytimes more juveniles cultured from various culture media The uncertainties inherent in artificialculture should not restrict federal policy by limiting the approach of propagation to fish hosttechniques only

THE NEED FOR ARTIFICIAL PROPAGATION

The development of fish host techniques in the early 1900s, as well as the improvement of culturemedia for propagating mussels using artificial media (in vitro), provides a unique opportunity forresearchers to identify life history requirements and survival mechanisms for this group ofdeclining bivalves Artificial propagation can be employed in response to environmental spillsand invasive exotic species (e.g., zebra mussels and black carp), as well as help stabilize decliningpopulations through species recovery plans Juvenile transformation has been reported for manyspecies using both fish host (in vivo) and artificial media (in vitro) However, some species pose asignificant challenge when using either technique, including Elliptodeus sloatianus, which produce

lanceolate conglutinates, Lampsilis fragilis, and Pleurobema spp (Milam et al 2000; personalcommunication, P Johnson 2003).

To date, there are only two propagation techniques reported in the literature, one that includeshost fish (Lefevre and Curtis 1912) and another using artificial culture media (Isom and Hudson1982) This section will provide some insight into the current development of these techniques

as well as include arguments for and against each one when determining which propagation method

is most appropriate(Table 4.1)

FISHHOSTTECHNIQUES(INVIVO)

Techniques for the determination of fish hosts have been reported and utilized by many researchersfor decades (Howard 1916; Coker et al 1921; Penn 1939; Cope 1959; Giusti et al 1975; Jenkinson1982; Hove and Neves 1991), while some unconventional hosts (e.g., amphibians) have also beenidentified as supporting juvenile metamorphosis (Seshaiya 1969) Some species have shown rela-tively good success using fish host techniques, while others have proven to be more difficult.Common species as well as state and federally listed species are often difficult to transform due

to the lack of knowledge of life history complexities and requirements (personal communication,

J Jones 2004) Two federally endangered species that are, at this point, particularly difficult areDromus dromas and Cyprogenia stegaria

Techniques for determining fish host suitability include the use of aeration tanks, direct gillplacement, and the use of anesthetics (i.e., MS222, which is tricaine methanesulfonate and tradename Finquel) to reduce handling stress on the fish (Zale and Neves 1982) While modifications ofthese have been reported from various researchers, the fundamental approach is the same Aerationtanks are often used when there are viable glochidia with several fish species and cohorts However,

if glochidia are limited and/or the fish are smaller in size or have small gill rakers, direct gillplacement using pipettes is a viable alternative to aeration techniques for attachment onto the gill.Anesthetizing fish prior to encystment is undecided, in that the possible effects of the anestheticmay 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 musselspecies, fish health (i.e., whether the individual is stressed or diseased due to other environmentalfactors), water temperature, and perhaps other variables presently unknown Alternatively, fishsurvival can be jeopardized by excessive glochidial infestation as a result of limiting gas exchangeacross the gill lamellae While 50–100 glochidia/gill for fish that are 15–25 cm in length have beenreported as adequate (Hove et al 2000), others have directly infested host fish with several thousandand 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 includedseveral issues that are important for the successful transformation of juveniles Fish maintenanceand holding prior to and during the encystment is fundamental to the fish host technique The use ofglass or high-grade polycarbonate, flow-through tanks with adequate aeration and temperaturecontrol devices are suggested to reduce fluctuations in water quality and quantity Providingsome nutrition to host fish during the encystment period has been noted several ways: feedingfish throughout the entire period and feeding fish only through the first half of the encystment periodand eliminating food thereafter The latter method reduces the amount of fish feces in the bottom ofthe tank, increasing the ability to selectively isolate juveniles more efficiently Juveniles should besiphoned from the tank bottom and collected using a sieve series for isolation A polarized lens,which is attached to the objective lens of a dissecting microscope, can be used to reflect, throughunderstage lighting, only prismatic objects and block out sediment and feces that often eclipse thejuvenile identification and counting process (Watters 1996) High quality foods (e.g., brine shrimp

or live minnows) should be promoted in the maintenance of fish hosts; however, a critical step in

TABLE 4.1

Summary of Successful Juvenile Transformation Efforts Using Various Unionid Species:1982–2003

A plicata Fish host Reintroduction Hubbs (2000)

Media Culture development Personal communication, B Hudson and M

Barfield (1993) Anodonta

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, D Simbeck (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)

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, C Echevarria (2004)

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, B Isom and

D Simbeck (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 grandis Fish host Toxicity testing Keller and Ruessler (1997)

Fish host Reintroduction Milam et al (2000)

S undulatus Fish host Host suitability Hove et al (1997) (blue)

U imbecillis Fish host Toxicity testing Keller and Zam (1991)

(continued)

reducing the possibility of fungal and bacterial growths includes the prompt removal of uneatenfood Hove et al (2000) stated that for bottom-feeding fish (e.g., minnows and other catostomids)that will feed on newly metamorphosed juveniles and sloughed glochidia, it is important to separatethe fish from the bottom of the tank using a plastic net with a small mesh size (1.6 mm) secured tothe tank, which allows the juveniles to fall through the mesh but keeps fish from bottom feeding.Collection of wild host fish or even commercially spawned fish species requires some attention

to detail, including the acclimation of fish once they have been brought back to the propagationfacility We suggest that fish be allowed to acclimate for several days prior to isolating for glochidiainfestation Conducting fish suitability trials should include multiple attempts using several indi-viduals of the same fish species with glochidia from different females to assure that metamorphosisoccurs 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

a management perspective Long-term restoration goals should include the successful recruitment

of mussel fauna as well as viable host-fish populations in a river reach Subsequently, instreamhabitats should accommodate both mussel and fish life history requirements to ensure that sustain-able populations are being supported

A viable mechanism for supporting juvenile reintroductions is to release infested fish into thewaterbody with known glochidia species Several reports have indicated that the release of infectedfish can support the efforts of a recovery plan for both common and listed species (Milam et al.2000; Genoa NFH, Chapter 5, Methods for Conducting Toxicity Tests Using Corbicula Fluminea

as Surrogate Species)

Dependence on Fish Hosts—An Obligate Trait?

Identification of fish hosts for unionid species has been reported in the literature since the turn of thetwentieth century when Connor (1905) identified Lepomis gibbosus (pumpkinseed) as a successful

TABLE 4.1 (Continued)

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)

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)

V liensosa Fish host Toxicity testing Keller and Ruessler (1997)

Fish host Host suitability Pers comm C Echevarria

V taeniata Fish host Reintroduction Morgan, Welker, and Layzer (1997) (blue)

V vibex Fish host Host suitability Pers comm C Echevarria

fish host for Anodonta cataracta (now Pyganodon cataracta, Hoeh 1990) Watters (1994) reviewedpublished fish host and unionid relationships in North America with approximately 95 unionidspecies and over 150 fish species Since then, there have been numerous publications that provideupdates to reported host-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) A summary of propagation techniques (e.g., fish host or artificialmedia), since Watters’ review was reported in 1994, has been provided as an update (Table 4.2).While the research suggests that most species require a host fish as an obligate trait of thebivalve life history, a few do not Unionids are also typically identified as either a generalist, whereits glochidia can transform on a variety of fish species, or a specialist, where only one or two hostfish have been identified that aid in the successful metamorphosis of glochidia to the juvenile stage.Additionally, with unionid populations that are deemed specialists, some of these are also specieslisted as threatened or endangered by federal and state governments Current debate, however,may suggest that declines in unionid populations are not necessarily due to a specific host-fishrequirement 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

Fish Host (in Vivo) Culture Media (in Vitro)

Ability to use infested fish to place directly into a recovering system is an easy method for novelists

With species whose host fish are unknown, a considerable effort 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 infestations can 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 a glochidia infestation (Arey 1932)

Toxicity

exposures With some contaminants, juveniles transformedin vivo can be more sensitive With some contaminants, juveniles transformedin vitro can be more sensitive

support successful and viable juveniles Listed species such as Lampsilis streckeri (Winterringer2004) and Lampsilis powelli have been identified as generalists.

Independence from Fish Hosts

Not all unionid species require fish hosts for the transformation of juveniles Strophitus undulatusand Utterbackia imbecillis are the two most reported species that were found to transform fromglochidia to juvenile inside the marsupial pouch (Lefevre and Curtis 1912; Howard 1916; Allen1924; personal communication, M Barfield 2003), while Obliquaria species may also be (Lefevreand Curtis 1912) a nonparasitic unionid Adult females of these species apparently provideessential nutrients for metamorphosis to take place; however, quantity and quality of these criticalnutrients are unknown

ARTIFICIALMEDIACULTURE(INVITRO)

History

Freshwater mussel glochidia (i.e., larvae) are naturally transformed into juveniles, which includedevelopment of the internal organs necessary for self-sustained existence as a benthic organism.This is accomplished via encystment in fish tissue (on the gill or fin) Interest in enhancingproduction of mussels through the use of artificial culture was seen early in the twentiethcentury when Ellis and Ellis (1926) reported the first successful culture of glochidia to transfor-mation following the excision of these from the gill tissue of their fish host Unfortunately,the details of their solutions were never published, and the host-fish attachment prior to cultureinitiation may have provided the stimulating factor(s), which allowed this developmental processprior to culture initiation Much later, in the early 1980s, interest in the artificial culture of glochidiawas revived by Isom Isom, who was familiar with the transformation work of Ellis and Ellis(1926), contracted Bob Hudson to help attempt this artificial culture, based on his work with thecell culture of catfish (Hudson, Pardue, and Roberts 1980) Isom and Hudson (1982) reportedsuccess in the transformation of several species without the use of a fish host at any time inthe process, a distinct improvement over the Ellis and Ellis report of 1926 This technique,which began as a modification of modern cell-culture techniques, made use of a mixture ofamino acids, vitamins, and glucose in a Unionid Ringers solution (Ellis, Merrick, and Ellis1930), along with the addition of fish plasma as a source of protein, growth stimulants, hormones,etc Although this work began by mixing these components from scratch (using the concentrations

of each found in fish plasma as guidelines), Isom and Hudson (1982) also reported success usingpre-mixed, commercially available cell culture media (Eagles essential and non-essential aminoacids and Medium 199), which contains nearly all of these amino acids in concentrations as high orhigher than those found in fish plasma

Even though this mixture has been used to produce thousands of juvenile mussels for cology research (Wade, Hudson, and McKinney 1989; Johnson, Keller, and Zam 1993; Hudson

toxi-et al 1994; Hudson, Barfield, and McKinney 1996; Barfield, Clem, and Farris 1997; Clem 1998),

it has been less than convenient for other labs to use because of the requirement that there be a readysupply of fish from which blood can be drained and separated into plasma and non-plasma com-ponents Because of this inconvenience and because the use of fish plasma introduces morevariation in the results, research was initiated in 1990 to try to develop an alternative mediumthat was either a serum/plasma-free medium or a medium using commercially available serum

in minimal concentrations (Hudson and Shelbourne 1990) Keller and Zam (1990) first addressedthe modification of the glochidial culture, demonstrating that other sera could be substituted for thefish plasma, with horse serum producing their best results Hudson and Shelborne (1990) began

a massive study for Don Wade of the Tennessee Valley Authority, at Presbyterian College, testing

a total of 64 different medium combinations Later, Johnson, Keller, and Zam (1993) describe

culture methods in their acute toxicity testing procedure; however, these are nearlyidentical to those described by Isom and Hudson (1982), using fish plasma as the protein source,and the same culture constituents including identical antibiotics without any real improvements intechnique Other advances come as a result of modification of the Hudson and Shelbourne (1990)work by Barfield, Clem, and Farris (1997), Milam et al (2000).

Culture Media Techniques

Glochidia are removed as described by Isom and Hudson (1982) or preferably by using a syringefull of control water to flush them from the female marsupia However, differing from priorpublications, glochidia are rinsed three to four times in autoclaved river water or reconstitutedwater rather than deionized water, and a final rinse with Unionid Ringers solution or Hank’sBalanced Salt solution Approximately 300–900 glochidia are seeded in a 3-mL total medium

in each 60-mm diameter, cell-culture dish These dishes are incubated at 21–248C in an incubatorhaving 4.6–5% CO2to maintain a pH of about 7.3 by use of a bicarbonate buffer (Isom and Hudson1982; Milam et al 2000)

Modification of the Media

The original medium was comprised of Eagles essential and non-essential amino acids in UnionidRingers containing NaHCO3for pH control, vitamins, antibiotics, and glucose as the artificialportion, and fish plasma as the natural protein source, in a final ratio of two-thirds artificialmedium to one-third plasma (Isom and Hudson 1982) As previously mentioned, other mediawere tested in 1990 (Hudson and Shelbourne 1990) in a major effort to improve results in morespecies, resulting in comparisons of 64 different media combinations For the last decade, DavidMcKinney, Chief of Environmental Services at Tennessee Wildlife Resources Agency (TWRA),has maintained interest in the culture and use of juveniles for toxicity testing, funding research thathas been presented in several reports that have added to the original modification by Hudsonand Shelbourne in 1990 Although the primary work has involved only one species, U imbecillis,other species have also been transformed using artificial media(Table 4.3).The following sectionsdiscuss specific artificial media components and their variations

Ionic Balance Initially, Isom and Hudson (1982) used a modified Unionid Ringers described

by Ellis, Merrick, and Ellis (1930); however, further tests show that prepared, balanced saltsolutions such as Earle’s or Hank’s balanced salt solution (Sigma) are useful in rinsing glochidia

as well as in their artificial transformation, even though the yield may be slightly lower

Sera Fish plasma was reported by Isom and Hudson (1982) as the choice protein additive;however, rabbit serum performs as well or nearly as well as the fish plasma when transforming

U imbecillis, and rabbit performance is better than porcine, horse, sheep, chicken, and fetal bovinesera (Hudson and Shelbourne 1990) These results differ from Keller and Zam (1990) who reportedthat horse serum was their most productive protein additive Combinations of the above seraand plasma resulted in the highest production being found in a medium containing a fishplasma/rabbit serum combination (usually equal to or better than fish plasma alone), but fish/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 somelaboratories do not have access to fish plasma, and since infection rates are lower when using sterilesera obtained from biochemical supply companies, rabbit serum is considered to be the bestalternative In one case, Elliptio angustata cultured in rabbit serum significantly outperformedfish plasma in observed transformation rates (Hudson and Shelbourne 1990)

Serum Replacements Rabbit serum performs better than other commercially 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

initiated using six serum replacements and found that none produced even 20% of the yield of fishplasma cultures These replacements were then tested in combination with fish plasma, and theresults showed that the CPSR (Sigma), TCM, and TCH (Protide) in combination with fish plasmaproduced a higher yield than the medium containing only fish plasma without any additives.These and other serum replacements were then tested in combination with other sera, and theresulting data indicated that rabbit serum in combination with TCM and TCH outperformed

TABLE 4.3

Mussel Species Transformed Using Artificial Media Culture

Species (Subfamily) Media Type TransformationTime Required Reference

(Anodontinae)

U imbecillis Rabbit/TCH/TCM; Horse

serum; Fish plasma (pl.) and all combinations

7 days 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 7 days Dimmock and Wright (1993)

P grandis Fish pl 7 days Personal communication, B Isom (2003) (Lampsilinae)

L fasciola Fish pl Unknown Personal communication, D Simbeck

(2003)

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 ventricosa Rabbit; Fish pl and all

combinations 12 days Milam et al (2000)

(Unioninae)

E angustata Rabbit/TCH/TCM and Fish pl 7 days Hudson and Shelbourne (1990)

Hudson, Barfield, and McKinney (1996) Elliptio crassidens Fish pl Unknown Personal communication, D Simbeck

(2003)

E complanata Rabbit/TCH/TCM and Fish pl 15 days Hudson, Barfield, and McKinney (1996)

other sera/serum replacement combinations The rabbit/TCH/TCM portion was one-third of thetotal medium at a ratio of 1:1:1 each using a 12% stock solution of TCH and TCM.

Antibiotics/Antimycotics Multiple rinses of glochidia washed from the marsupia, as describedearlier, are essential to lower the rate of infections from bacteria and fungi To also help insure a lowrate of infection, antibiotics (e.g., penicillin and streptomycin) and an antimycotic (e.g., amphoter-icin B) that are usually found in most cell cultures were the first to be used in developing theglochidial medium (Isom and Hudson 1982) Improvements of these techniques were done byisolating and culturing bacteria from glochidial cultures and measuring zones of inhibition fromapproximately one dozen commercially available antibiotics (Isom and Hudson 1982) The threeantibiotics (i.e., carbenicillin, gentamicin sulfate, and rifampin) with the best inhibitory effects werereported by Isom and Hudson (1982) and are currently those used in all laboratories that havereported glochidial cultures Much later, bacteria that were isolated from swabs of the gills ofAmblema plicata, P cataracta, and U imbecillis, were identified and measured for inhibitingeffects (e.g., growth) by fifteen antibiotics (Loveless et al 1999) The assumption behind thiswork is that contamination of artificial glochidial cultures most likely comes from the parent’sgill tissue, which houses these glochidia The most effective antibiotics against the dozen bacteriaisolated were neomycin, ciprofloxacin, and polymyxin B Since these have never been evaluated foreffect on actual transformation success, a mixture of antibiotics including two of these new anti-biotics was tested along with a control of standard antibiotics (Isom and Hudson 1982) for theireffect on mussel transformation This mixture contained a penicillin-streptomycin-neomycinsolution (5000 IU, 10 mg, and 5 mg respectively) and was added to cultures at a rate of 30 mL(low), 60 mL (medium), and 120 mL (high) per 3-mL culture dish along with polymyxin B at a rate

of 4 mg/mL, 10 mg/mL, and 20 mg/mL concentrations The penicillin and streptomycin are not inthe new antibiotic best performer list (Loveless et al 1999); however, this mixture is commerciallyavailable (Sigma Aldrich) for laboratory use Between 600 and 800 juveniles were evaluated on daysix of the glochidial culture and again on the second day after each dish had been placed in water.Each of these was compared with the control antibiotics (Isom and Hudson 1982); however, noneoutperformed the original control set in transformation success(Figure 4.1).These same concen-trations of new antibiotics were used in combination with the control antibiotics and the resultswere similar (Figure 4.1) All treatments in Figure 4.1 are significantly different from the controlwith the exception of the treatment having medium concentrations of the new solutions mixed incombination with the control antibiotics (contingency chi-square Z0.498, 1 df, pZ0.48) Oneadvantage of using this increased number of antibiotics may be that a wider spectrum of bacteriacould be controlled better than by use of just the original antibiotics described by Isom and Hudson(1982)

Often, fungal infection will appear in one or more dishes during the culture process When thishappens, fungal mycelia are removed using sterile forceps, and 75 mL of nystatin (Sigma) is added

to each culture dish If a fungal or bacterial infection is massive, the developing glochidia arepoured into a cylinder with a 112-mm mesh Nytexwscreen bottom, resuspended in a sterile 200-mLbeaker, and rinsed several times in Earle’s or Hank’s balanced salt solution Following the rinsing,the glochidia are resuspended in a dish of balanced salt solution, aspirated out, and placed into

a culture dish with fresh, complete media

Other Medium Components Most other medium components remain the same as first described

by Isom and Hudson (1982) Eagles essential and non-essential amino acids are mixed with theUnionid Ringers with the addition of taurine and ornithine Cultures seem to transform well withoutthe addition of these last two amino acids, but since these are found in fish plasma and since somespecies of mussels may require these two amino acids, they are still 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 proved successful in transformation, even though their compositionvaries slightly from the above-modified Eagles Medium (Isom and Hudson 1982; Keller and Zam1990) Glochidia developing artificially seem to be lower in lipid content than those developing

from fish hosts Tankersley (2000) indicated that lipid levels in glochidia and juveniles vary witheach brood stock, being influenced by the parent mussel Furthermore, he indicated that the type ofculture medium influenced the lipid content of the developing glochidia and subsequent juveniles.Fisher (2002) demonstrated that glochidia developing in an artificial medium (i.e., the rabbit serummix) were lower in lipid content than in those transformed using fish hosts Consequently, Hudsoncompared transformation rates with media containing squid oil (Artemate, Argent ChemicalLaboratories) and cod liver oil, each added at 50 mL per culture dish While the rates did vary,the cod liver oil seemed to be better than the squid oil and also resulted in lower fungal infectionrates It is thought that the addition of this oil enhances the lipid storage of the glochidia (Tankersley2000).

Success of the in Vitro Cultured Juveniles

Lipid, triglycerides, cholesterol, glycogen, and protein concentrations in artificially producedjuveniles are lower than those transformed on fish (Fisher 2002) However, juvenile testing withsediments from the Pigeon, Conasauga, and Telico Rivers, and Lake Monticello (Hudson andRoberts 1997; Hudson and McKissic 1999) show a 50–60% survival of juveniles held in

a simple static culture for less than 90 days These juveniles were held in cylinders with a screenedbottom (112-mm mesh) and placed in sediment in a culture bowl and fed concentrated plankton two-to-three times per week U imbecillis have been grown in this system for over a year with

a resulting mean size of over 9 mm Starkey et al (2000) used a Partitioned Aquaculture System(PAS) at Clemson University to test growth of about 100 juvenile U imbecillis This system usesplanktonic algae as a treatment for fish wastes, where the wastes serve as nutrients for the plankton

as they flow with the water leaving the fish holding area through a slow-moving pond, which ispartitioned into lanes to allow time for blooming In an effort to control the algae bloom, Starkey

et al (2000) used adult Elliptio complanata as one of the algal harvesters They also placed 100juvenile U imbecillis in this concentrated, continuous flow of algae to determine their response to

0 10 20 30 40 50 60 70 80 90

Control (C) New-low New-med New-high Combo-low Combo-med Combo-high

Glochidial Treatment (day 6 in glochidial culture)

this high plankton load The juveniles responded with very high growth, from an average of 400 mm

in initial length when placed in the partitioned pond to 18.9 mm (47!increase) in just 117 days.Their most rapid growth occurred between days 47 and 61 with an average of 0.26 mm per day.Unfortunately, their survival rate was lower than desired, with only 7% survival at the end of the

117 days of the test Many other factors may have contributed to this mortality not associated withthe algae, since Hudson and Roberts (1997); Hudson and McKissic (1999) had 50–60% in100-day-old juveniles with a lower feeding regime in a simple laboratory static culture One factor may havebeen that the juveniles tested by Starkey et al (2000) came from static cultures and already mayhave been malnourished Barfield, Clem, and Farris (1997) showed that once diet-deprivedjuveniles were returned to a regular diet, they still had greater sensitivity to copper than thosethat had never been deprived of nourishment

The media described in the section titled Artificial Mediaculture (in Vitro) vary little in aminoacids and other artificial components The mixture used by Hudson and Shelbourne (1990) andothers is described in Table 4.4 The basic non-protein portion of the medium consists of thefollowing stock with no antibiotics/mycotics and can be stored in a refrigerator (less than 48C)for up to four weeks if glutamine is added fresh every time you make the final medium with serumand/or serum replacement A bacterial streak on a nutrient agar plate should be made weekly toensure sterility of this medium One can avoid making the above mixture by using premixedMedium 199 (Sigma); however, the development rate may be slower (Isom and Hudson 1982)

A protein source (i.e., serum or plasma) must be added to the above basic medium mixture Thevariety of sera, plasma, and serum replacements is discussed in Modification of the Media

(Table 4.5), with amounts stated in units needed to prepare one 3-mL culture dish Every dishwill receive the same volumes of the basic medium components, with the variation of each mediumconsisting mostly of different protein additives and/or their replacements

Table 4.5 shows the basic non-protein, non-antibiotic/mycotic, non-lipid medium mixture,which can be combined with different plasma or serum to produce all of the variations used inmost laboratories to date Although Keller and Zam (1990) reported success using horse serum, thiswas not listed since others report rabbit serum or fish plasma as their protein of choice (Isom andHudson 1982; Dimmock and Wright 1993; Milam et al 2000) The basic medium shown in

Table 4.3may be mixed and stored for four or more weeks at 0–48C The serum, serum ments, or plasma, the antibiotic/antimycotic solutions, and L-glutamine must be added fresh as themedium is being prepared for use The above components may be mixed in batch quantities,

replace-TABLE 4.4

Preparation of Stock Medium (MEM) for Glochidial Cultures

Unionid ringers 960 Ringers solution has as 2.2 g NaHCO 3 or 25 mM

Hepes buffer Store in refrigerator

a Glutamine will remain stable for only three weeks—hence, the medium must be re-made or fresh glutamine must be added every three weeks, even if stored in the refrigerator.

TABLE 4.5

Variations in Culture Media Used for Unionid Transformations

Each 60 by 15 mm Tissue Culture Dish Should Receive the Following:

bit/TCH/TCM

Note: Stock Solution Concentrations per mL: carbenicillin (2 mg), gentimycin (3 mg), rifampicin (50 mg), and amphotericin (0.2 mg) TCM and TCH are used in a concentration between 12 and 24%.

e Add to each dish individually.

Source: (From Hudson, R G and Shelbourne, C W., Improved in vitro culture of parastitic freshwater mussel glochidia, Report to the Tennessee Valley Authority, p 25, 1990 Including later changes).

whereas the cod liver oil must be added to each dish separately since it will not mix with thesolution Nystatin is added as needed for fungal infections All of the above remain fairly constant

in all medium mixtures, with the exception of the plasma or serum components

As seen inTable 4.3, most of the early transformation research was conducted using just fishplasma as a protein additive Although Hudson and Shelbourne (1990) found a mix of rabbit serumand fish plasma to be most effective, transformation has been carried out without the addition offish blood in most laboratories in order to reduce variability and for convenience Some of theAnodontinae have been successfully transformed with the simplest of media using just rabbit serumand TCH and TCM additives, as have some Elliptio species (Table 4.3) Successful attempts to usethe combination media listed in Table 4.4 have been limited to U imbecillis due to shippingproblems with other species Results from Hudson and Shelbourne (1990) would indicate that

a medium of fish plasma and rabbit serum may be the best beginning medium when first attempting

to culture a new species More work is needed to understand the nutritional needs of all mussels,especially in the area of lipid and protein additives

SHIPPINGMUSSELS ANDGLOCHIDIA

Specific techniques of shipping mussels are often not reported in the literature, and details of bothsuccessful and unsuccessful shipping are often noted from personal communications withresearchers who are actively involved in mussel transport Shipping gravid mussels is often necess-ary because mussels are not in the area where the propagation laboratory is located Additionally,researchers have found that the short-term brooders (i.e., Ambleminae) are prone to aborting theirembryos or glochidia following shipping (personal communication, J Jones 2004), although othershave found this to be less than 5% (personal communication, P Johnson 2003), resulting in partialdemibranch release during transportation If Ambleminae are transported in wet towels in an icechest, they often abort when returned to water Quadrula species seem to be especially prone toaborting glochidia when disturbed (pers comm S Ahlstedt) This presents a problem in thatabortion of glochidia and their deterioration results in poor success when attempting artificialculture

Transporting adults and glochidia should include chilling individuals using ice packs (e.g., BlueIce or frozen distilled water) The use of loose ice has the potential to melt during shipping andincreases the chance of fouling by specimens if they have been allowed to come in direct contactwith the ice/water Specimens can also be placed in burlap sacks soaked with river water andshipped without a substantial amount of water (personal communication, S Ahlstedt 2003).Under these conditions, mussels will be less likely to abort during transport but can abort afterthey have been warmed and placed into holding systems Others have found that long-term broo-ders (i.e., Lampsilines and Anodontines) hold their glochidia during shipping and handling Hubbsand Johnson have found that mussels carried in wet towels or mesh bags in containers with icepacks, but not touching the packs, travel best Some researchers (D Simbeck of Tennessee ValleyAuthority and R Hudson of Presbyterian College) have noticed that initiating artificial culturesseems to be more difficult from shipped parents than those that have been captured and carried inwater directly to the laboratory, even considerable distances Cultures also seem better whenparents are held a day or two in the lab and then cultured, a problem in the Ambleminae sincethey tend to abort when held in water

Zimmerman and Neves (2002) compared glochidia from two species over time in differenttemperature regimes and found that glochidia in the cooler temperatures (0–108C) did remain viablelonger than those at 258C (75% survival at 7.5 days for Villosa iris and at 14.4 days for Actinonaiaspectorosa) and were able to be transformed on fish following this time period Comparisons ofshipping glochidia in culture medium have been conducted using various packing methods with ice,limited studies indicted that the highest survival of glochidia were those that were shipped on a tray

suspended on ice; however, these results are difficult to repeat, and more work needs to be done inthis area.

Glochidia have been shipped free from the marsupia in river water or synthetic water, andexcised, gravid marsupia have also been shipped for use in propagation efforts Both types ofshipping resulted in snap-closure response to sodium chloride upon arrival in the laboratory.However, the most appropriate way to ship glochidia is free from the marsupia because itassures that the female was not sacrificed Alternatively, cold storage (less than 48C) of inflatedmarsupia has been shown to be effective in maintaining the condition of encapsulated glochidia fortoxicity testing Acute bioassays can be successfully carried out using excised glochidia that havebeen stored for up to four days

ADULTHOLDING

Holding and maintaining adult mussels in laboratory conditions is necessary to allow for transportacclimation, glochidia development, and in some cases, for reproduction to occur Villosa sp andLampsilis sp are particularly easy to maintain in the laboratory when given adequate foodquantity and quality Maintenance of these species results in relatively low mortality andconsiderable measured growth, indicating that these individuals are in reasonably good condition.Very often, females of Villosa, Pyganodon, Utterbackia, Tritogonia, Elliptio, and Pleurobemaspecies have repeatedly become gravid in holding conditions (personal communication, PJohnson 2003) Additionally, these laboratory conditions have provided refugia for numerousspecies for more than four years (Farris, Milam, and Harris 1998) Although reproduction was notmeasured in these studies, it remains an outstanding issue for laboratory maintenance of adultmussels and requires additional research to determine the effects (i.e., survival and reproduction)

of long-term holding facilities

PHYSIOLOGICAL TRANSFORMATION—PHASES AND COMPARATIVE

SENSITIVITIES OF DEVELOPMENT

Transformation of glochidia to juveniles on the fish gill and in media may range from 7 days togreater than 110 days, depending on mussel species, water temperature, and hostfish condition.Phases of development include the egg, glochidia, and juvenile stage While it is thought thatglochidia and egg phases are relatively protected from waterquality changes, juveniles may bemore susceptible to exposures once excysted from the fish gill or fin Several reports suggest thatcomparisons of glochidia and juvenile survival may be drastically different given a wide range ofcontaminant exposures (outlined in Chapter 5).Exposures to developing glochidia (i.e., alreadyattached to live fish gills) include one study that measured impacts from aqueous-bound copper onhostfish Results of this study indicate that encysted glochidia are relatively protected from acutecopper exposures and will successfully metamorphose into juveniles (Jacobson 1990) Since only

a single study with hostfish exposures has been reported, more work needs to be conducted

to support existing literature and provide additional exposure results with a variety of prioritypollutants (e.g., metals, organics, TSS, bacteria, etc.)

Comparisons of physiological conditions of juveniles transformed in vitro and in vivo indicatethat, in general, those individuals that metamorphose on fish hosts tend to be healthier than thosetransformed in culture media (Fisher 2002) These comparisons were conducted using U imbecilliswhere fish-reared individuals were found to absorb lipids and glycogen deposits from the fish,which provided a better survival and growth rate through several weeks Additionally, juvenilesreared from fish hosts had measurably higher concentrations of triglycerides, cholesterol, glycogen,and protein than juveniles cultured in media Overall, juveniles that transformed on fish hosts were