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Abstract

In vitro culture has great potential for the propagation of freshwater mussels in both commercial and conservation aquaculture. The use of in vitro techniques precludes the need for host fish, thus decreasing costs and increasing efficiency. However, protocols are still lacking for many species that grow substantially during the parasitic stage. In this study, we tested the effects of taurine addition, serum type and source of lipids on the survival rate and increase in length of Margaritifera margaritifera larvae during the initial stage of culture (first 11 days of exposure to media). Our results show that taurine has no significant effect on the early survival rate of glochidia; however, the possible importance of this amino acid in subsequent stages is discussed. The use of an emulsified lipid mixture instead of traditional fish oil showed significantly higher rates of survival. Finally, the addition of serum showed variable effects, with both horse serum and newborn calf serum having higher survival in trials using mussel populations from different sources. These findings can contribute to the invention or improvement of in vitro protocols for species growing during infection and at the same time show the potential that early survival assessments could have for the development of in vitro methods in species with long parasitic stages.

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Book
Freshwater Mussel Propagation for Restoration Freshwater mussels are declining rapidly worldwide. Propagation has the potential to restore numbers of these remarkable organisms, preventing extinction of rare species and maintaining the many benefits that they bring to aquatic ecosystems. Written by practitioners with firsthand experience of propagation programs, this practical book is a thorough guide to the subject, taking readers through the process from start to finish. The latest propagation and culture techniques are explored as readers follow freshwater mussels through their amazing and complex life cycle. Topics covered include the basics of building a culture facility, collecting and maintaining brood stock, collecting host species, infesting host species with larval mussels, collecting and culturing juvenile mussels, releasing juveniles to the wild, and post-release monitoring. This will be valuable reading for any biologist interested in the conservation of freshwater mussel populations.
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Chapter
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Availability and uptake of metals in the accumulated sediments in stormwater treatment facilities was assessed by the metal accumulation patterns observed in freshwater mussels, as the first step in an Environmental Risk Assessment (ERA). Freshwater mussels, Elliptio complanata, were caged in various locations in several stormwater treatment facilities and control sites in southeastern Ontario. Mussels were sampled at 2, 5.5, 8, 11 and 14 weeks, and Ni, Cr, Cu, Cd and Pb concentrations in soft tissues were determined by ICP-MS. Selection of these metals was based on previous studies which had identified them in substantial quantities in stormwater pond sediments. A significant decrease in Ni concentrations and an increase in Pb concentrations relative to background levels were observed. Concentrations of Cu, Cd and Cr were generally not significantly different from background. Total metal concentrations in sediments were also determined, and compared with the observed mussel metal concentrations. No correlations were observed between total metals in sediment and the accumulated burden in the mussels. The results suggest that Pb is a possible concern in these stormwater facilities due to its availability. Ni, Cr and Cd did not appear to be in bioavailable forms and Cu had limited availability. The study was complementary to other work examining trace metals in stormwater management facilities, and provides further useful information about the habitat quality of these facilities, and the ecotoxicological risks that might be posed to resident species.
Article
Larvae of Utterbackia imbecillis normally undergo metamorphosis to the juvenile while attached to the gills or fins of a host fish; however, metamorphosis can also be induced in the laboratory in a modified cell culture medium. This study examined juveniles resulting from each of these rearing techniques to determine their relative physiological conditions. Juveniles reared in vitro grew more slowly and had higher mortality rates than did their fish-reared counterparts. Animals reared on their host fish accumulated triglycerides, cholesterol, glycogen, and protein during the parasitic metamorphic period. In contrast, animals reared in vitro showed an increase in the levels of triglycerides, but did not accumulate cholesterol, glycogen, or protein. These results suggest that fish-reared juvenile individuals of U. imbecillis are in more robust physiological condition than their in vitro-reared counterparts.
Article
1. Free amino acids of 17 species of aquatic invertebrates were determined by chromatographic methods.2. Qualitative differences in some amino acids were detected in organisms of different species. The concentration of alanine, arginine, aspartic acid, glutamic acid, glycine and taurine was measured and significant differences recorded.3. Taurine was found in high concentration in all the marine organisms studied but was not found in several fresh-water and terrestrial organisms.4. The possible role of taurine and other free amino acids in aquatic organisms is discussed.
Article
Freshwater mussels of the order Unionoida have life cycles that include larval attachment to and later metamorphosis on suitable host fishes. Information on the trophic relationship between unionoid larvae and their host fishes is scarce. We investigated the trophic interaction between fish hosts and encysted larvae of two species of freshwater mussels, Margaritifera margaritifera and Unio crassus, using stable isotope analyses of larvae and juvenile mussels as well as of host fish gill and muscle tissues before and after infestation. Due to different life histories and durations of host-encystment, mass and size increase in M. margaritifera during the host-dependent phase were greater than those of U. crassus. δ13C and δ15N signatures of juvenile mussels approached isotopic signatures of fish tissues, indicating a parasitic relationship between mussels and their hosts. Shifts were more pronounced for M. margaritifera, which had a five-fold longer host-dependent phase than U. crassus. The results of this study suggest that stable isotope analyses are a valuable tool for characterizing trophic relationships and life history strategies in host–parasite systems. In the case of unionoid mussels, stable isotopic shifts of the larvae are indicative of the nutritional versus phoretic importance of the host.
Article
The development of host fish techniques (in vivo) at the beginning of the 20th century, and the improvement of artificial culture media for in vitro culture of bivalves, provided an opportunity to identify the nutritional requirements needed to complete the life cycle of threatened freshwater bivalves. A freeze-dried extract of lambari, a Brazilian Tetra (Astyanax altiparanae), was used as an additive to the artificial culture medium M199 (Sigma®) to try to improve the survival of glochidia and the metamorphosis of Diplodon rotundus gratus and D. greeffeanus. We adjusted the pH of the culture medium to 6.8, the same value recorded at the collecting site of the bivalves. Glochidia were kept in an incubator at 18°C. 40% of D. rotundus gratus glochidia survived, and 20% reached metamorphosis after 22 days of incubation. For D. greeffeanus 50% of the glochidia survived with 15% undergoing metamorphosis after 22 days of incubation. These results suggest an increase in the post-metamorphic survival with the use of the medium M199 supplemented with the fish extract, with a survival percentage of 75% after 30 days at the juvenile stage. Therefore, this constitutes an effective process in the preparation of in vitro cultures for freshwater mussels, particularly for D. rotundus gratus and D. greeffeanus.
Article
The culture of juvenile Margaritifera margaritifera in cages is shown to be a useful method of raising the early post-parasitic stages in suitable rivers for scientific or conservation purposes. Survival rates of caged specimens are equal to those of free-living juveniles, and growth is equal or slower than under natural conditions. Factors affecting the viability of caged juveniles are: length of the shell, colonisation of cages by aquatic insects and amount of fine sediments accumulating in the cages.The influence of 12 water chemistry variables on the juveniles is analysed: growth and mortality largely depend upon water temperature; there is a negative relationship between growth and eutrophication.
Article
Analyses of several organic and inorganic elements in the plasma of different fish species in control and infested conditions with the freshwater mussel Hyriopsis (Limnoscapha) myersiana (Lea, 1856) were undertaken. The most suitable composition of plasma corresponded to the fish species which produced the highest number of transformed glochidia. The infestation assay showed that glochidia of H. myersiana had a short transformation period of 6–12 days to reach the juvenile stage. The highest recovery peak could be seen in the plasma of the common carp, with lower peaks in the nile tilapia, hybrid catfish and striped catfish. The significant increase at days 3 (P
Article
Data on threats affecting the life cycle of the endangered freshwater pearl mussel Margaritifera margaritifera L. are presented. There was no evidence to suggest that a change in density or age structure of host fish (brown trout) populations has contributed to the decline of mussels. The factor of greatest impact was pollution. Mortality of the adult stage exhibited a positive correlation with the concentration of nitrate in the water and, in particular, phosphate, calcium and BOD5 were correlated with decreasing survival and establishment of juvenile mussels. As fertility (i.e. production of glochidia) is maintained even in sparse populations, and in polluted rivers, the populations should recover if the causes of decline are removed. Eutrophication should therefore be prevented. It might accelerate recovery of sparse populations if the numbers of young mussels released by the hosts were increased by introducing artificially infected fish.
Article
From a conservation point of view, captive breeding of endangered mussel species can be a last-minute rescue tool in order to retain the evolutionary potential of priority populations which would not persist long enough to benefit from habitat restoration practices. Copyright
Article
It is proposed that the incorporation of a unique parasitic stage in the life-cycle of unionaceans which involves an obligate relationship between a vertebrate host, usually a fish, and a highly modified larval stage, the glochidium, has had far-reaching consequences with respect to overall morphology, extent of species' geographic ranges, and rate of speciation in the group.Glochidia are separable into three main types with respect to overall shape and attachment features, and are retained in variously modified brood pouches. When mature, glochidia are released in several different ways which reflect various adaptations involved in either attracting the fish host and/or increasing the probability of attachment. Glochidia do not seem capable of host selection, and the reaction of the host to the parasite seems to be the main factor in determining specificity. Release of glochidia is synchronized to correspond to periods of predictable host availability, such as during spawing migrations and nesting behaviour. Other adaptations include modifications of glochidial conglutinates to mimic host food items, and modifications of the unionacean mantle edges to attract hosts. In all cases, a good correlation exists between the type of lure used and host food preferences, but, despite these adaptations, host specificity among unionaceans seems low.Parasitism among unionaceans is postulated to be mainly advantageous in terms of predictability of dispersal by habitat-specific hosts, but parasitism is hypothesized to entail constraints in terms of the degree to which shell shape and life-habit can be diversified among unionaceans. The type of host parasitized is considered to affect the rate of diversification among populations and speciation among unionaceans: those that parasitize strictly freshwater hosts are more likely to exhibit highly individualistic populations in different drainages with respect to molecular genetic and soft-part characters, while those that parasitize anadromous or saltwater-tolerant hosts show little differentiation among widely distributed populations.
Article
1. Habitat degradation is a major reason for species extinctions. For parasite–host interactions, the decline of a parasite may not only be related to the parasite’s tolerance to habitat degradation but also indirectly through the host’s tolerance to the same disturbance. 2. Our objective was to explore the cause of population declines of the freshwater pearl mussel Margaritifera margaritifera by relating the age distribution, density and growth of the mussels with turbidity, sedimentation rates and density of the mussel’s host, trout Salmo trutta, in 26 Swedish streams. 3. An analysis of the age structure of nine mussel populations showed that maximum age differed by 60 years, with five populations having low proportions of juvenile mussels. Adult mussel density was higher at sites where juvenile mussels occurred than at sites lacking juvenile mussels. 4. Growth of adult mussels during the past 10 years was lower in the five streams lacking recent recruitment than in the four streams with recent recruitment, indicating that some environmental factor may be negatively impacting these populations. 5. A comparison among 24 populations indicated that turbidity and sedimentation may be responsible for recruitment failure in 58% of the populations. The age of the youngest mussel was positively related to turbidity and sedimentation, and juvenile mussel density was negatively related to turbidity and sedimentation. In contrast, trout density was not related to recruitment of mussels or sedimentation, but was positively related to turbidity in all streams, both with and without recent mussel recruitment. 6. Synthesis and applications. Recruitment failure of M. margaritifera appears to be related to its own vulnerability to turbidity and sedimentation rather than to its host’s response to this type of habitat degradation. The results from our study suggest that managers might be able to evaluate the potential viability of mussel populations by measuring stream turbidity. Restoration activities to improve the mussels’ environment should focus on reducing fine material transport into streams.
Article
The ways in which the parasitic glochidium larva of Anodonta cygnea L. make initial contact with their host are investigated, and it is suggested that the thread can act as an attachment organ.Glochidia discriminate during the initial stages of attachment, and select a suitable host. Selection is achieved by the glochidia recognizing and responding to certain substances which are associated with the surface of the fish. Although these substances remain unidentified, they are almost certainly not constituents of the intact epidermal mucus. They may, however, be formed by bacterial degradation of the mucus.Prior to the formation of a cyst, a glochidium maintains its hold by grasping the host tissue between the two shell valves. The valves are held together primarily by the adductor muscle, and the hooks help to grip the host.
Article
Several organic and inorganic sources from the plasma of different fish species and horse serum were utilized as additives to the artificial culture M199 medium to improve glochidial survival and transformation of Hyriopsis myersiana. After 2–3 days of culturing in the medium containing plasma of Nile tilapia or hybrid catfish, striped catfish or horse serum, the glochidia presented significantly (P<0.05) lower percentage survival compared to medium containing common carp plasma. The highest (93.77±3.0) and lowest (32.42±5.85) percentage survival rates of glochidia were found with common carp and striped catfish plasma, respectively. After 10 days, relevant signs of glochidia transformation, such as the foot and mantle edge, were observed. In all assays, the glochidia transformation reached 100% most probably due to the exchange of the medium at the fifth day and the addition of 1 ml of distilled water at the ninth day of culturing. The intense mobility of juveniles in the medium containing the common carp plasma indicated excellent culture conditions. The ideal density for this plasma corresponded to 150–200 glochidia per culture dish.The present results suggest that M119 medium complemented with the common carp plasma and the medium exchange during culturing period may constitute a functional process to prepare an in vitro culture for freshwater mussels, particularly H. myersiana. The most relevant amino acids for a successful development are CIT, GLX, LEU, PRO, THR and ALA particularly with the contents in the common carp plasma.
Article
The capacity for intracellular osmoregulation mediated by free amino acids was examined using isolated foot preparations of the three bivalve molluscs: the oligohalineCorbiculajaponica Prime, freshwater euryhalineC. leana Prime, and freshwater stenohalineAnodonta woodiana Lea species. In response to a salinity increase in the incubation medium, ninhydrin-positive substances accumulated in the isolated foot of the oligohaline and freshwater euryhaline species, but not in the freshwater stenohaline species. Possible explanations for such a difference were considered along with its evolutionary significance regarding the entrance of bivalves into the freshwater environment.
Article
This paper presents a simple and widely ap- plicable multiple test procedure of the sequentially rejective type, i.e. hypotheses are rejected one at a tine until no further rejections can be done. It is shown that the test has a prescribed level of significance protection against error of the first kind for any combination of true hypotheses. The power properties of the test and a number of possible applications are also discussed.
Article
Striking convergent evolution is found in the properties of the organic osmotic solute (osmolyte) systems observed in bacteria, plants, and animals. Polyhydric alcohols, free amino acids and their derivatives, and combinations of urea and methylamines are the three types of osmolyte systems found in all water-stressed organisms except the halobacteria. The selective advantages of the organic osmolyte systems are, first, a compatibility with macromolecular structure and function at high or variable (or both) osmolyte concentrations, and, second, greatly reduced needs for modifying proteins to function in concentrated intracellular solutions. Osmolyte compatibility is proposed to result from the absence of osmolyte interactions with substrates and cofactors, and the nonperturbing or favorable effects of osmolytes on macromolecular-solvent interactions.
Article
Nonfeeding larvae of the gastropod Haliotis rufescens maintained a constant amount of taurine during embryonic and larval development and, since no de novo synthesis of taurine was observed in these larvae, the maternal endowment of taurine to the egg was sufficient for larval development to metamorphosis. In contrast, feeding larvae of the bivalve Crassostrea gigas increased their taurine content by a factor of 43 during growth to metamorphosis (from 86 to 311 µm, valve length). Taurine was not present in algae used to feed the larvae, suggesting that de novo synthesis of taurine by the larvae met their requirements. In unfed larvae, cysteic acid, cysteine sulfinic acid and hypotaurine were labeled from a [35S]cysteine precursor, but taurine was not. Hyperosmotic treatment (from 33 to 44 salinity for up to 3 h) did not induce taurine synthesis in unfed larvae. However, larvae fed the alga Isochrysis galbana up-regulated their taurine synthesis from [35S]cysteine by a factor of 11 (fed, 11.7±2.2 fmol taurine larva-1 h-1; unfed controls, 1.08±0.33 fmol taurine larva-1 h-1; means ± s.e.m.). Fed larvae also synthesized taurine from [35S]methionine (18.4 fmol larva-1 h-1). I. galbana contained 5 fmol cell-1 of cysteine and methionine (combined) and, based on known feeding rates, we calculated that there were sufficient taurine precursors in the algae to supply the taurine requirements of growing larvae. The lack of significant de novo taurine synthesis reported for adult bivalve molluscs has led to the conclusion that taurine is a dietary requirement. Our findings for larval forms differ in that there is significant de novo synthesis of taurine during development.
Article
The interface of bone and aragonite nacre (Margaritifera, fresh water pearl mussel) was studied by in situ hybridization and a tartrate-resistant acid phosphatase (TRAP) histochemical assay. Columnar implants were inserted into rat femora for 4, 7, 14, 28 and 56 days. In medullary region, a burst of transient bone formation was observed, which propagated from the periphery towards the nacre implant. A fused interface of bone and nacre was observed at 14 days. Later, the new medullary bone was resorbed and bone marrow was re-established while a thin layer of bone tissue remained covering the implant surface. Expressions of collagen alpha1(I), osteocalcin, osteopontin mRNAs and TRAP in the surrounding tissue were monitored. Correlated with the histology events, a strong transient induction of collagen alpha1(I) and osteocalcin mRNAs as well as TRAP expression, exhibiting a peak signal intensity on day 7 and subsequent down-regulation after day 14 was observed. Osteopontin mRNA, in contrast, was expressed continuously. The degrading nacre surface appeared in direct contact with macrophages and multinucleated giant cells at both days 14 and 28. These cells expressed osteopontin mRNA intensively and some TRAP enzyme activity occasionally.
Simplifying methods for in vitro metamorphosis of glochidia
  • M Kern
Kern, M. (2017). Simplifying methods for in vitro metamorphosis of glochidia. Retrieved from https://bearworks.missouristate.edu/cgi/ viewcontent.cgi?article=4143&context=theses.
Investigations for the conservation and propagation of freshwater mussels(Doctoral dissertation)
  • C T Owen
Owen, C. T. (2009). Investigations for the conservation and propagation of freshwater mussels (Doctoral dissertation). University of Louisville, Louisville, KY.
Fluorescence techniques for evaluating lipid content of larval and juvenile mussels. In Freshwater Mollusk
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In vitro culture of parasitic glochidia of four unionacean mussels
  • Taskinen J.
Phases in the parasitism of the unionidae
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  • J A Barry
Howard, A. D., & Barry, J. A. (1922). Phases in the parasitism of the unionidae. The Journal of Parasitology, 9(2), 68-82. https ://doi. org/10.2307/3271139