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This study assessed the differences in nutrition delivered to juveniles of two European freshwater mussel (Unionidae, Bivalvia) species with different levels of host specificity ( Unio crassus and Anodonta anatina ) developing on several potential host fish species. Juveniles were obtained, and the transformation success rate (the proportion of successfully transformed glochidia) was determined after experimental infestations under laboratory conditions. Juvenile lipid reserves were assessed via an epifluorescence method (Nile Red staining) and compared among different host fish species and individuals. The juveniles of both species developing on different hosts significantly differed in their lipid reserves as indicated by fluorescence activity. Moreover, juvenile growth during the early post-parasitic stage was different among host fish species, congruently with the results of lipid quantification. No clear general linkage was recorded between the quantitative (transformation success rate) and qualitative (lipid reserves) indicators of juvenile success. In the case of U. crassus , the host fish species that had the highest glochidium transformation success rate ( Phoxinus phoxinus ) also produced juveniles with significantly higher levels of lipid reserves compared with the other hosts. In contrast, a positive association between quantitative and qualitative indicators of juvenile success was not observed in the experiment with A. anatina suggesting that the conditions influencing the mortality of glochidium larvae during the parasitic phase and their energy uptake may have a different basis. The observed persisting effects of the fish-associated larval conditions on early juvenile stages might have critical consequences for the vitality and survival of juvenile freshwater mussels during the early post-parasitic stage, with implications for the effectiveness of breeding programs and strategies for host selection. A more thorough incorporation of the qualitative measures of the host fish value can lead to the further optimization of culturing protocols (improved nutrition reserves and growth of juveniles) and to setting more accurate management goals for host resources under field conditions.

Bilaterally asymmetrical glochidia (i.e. bivalved parasitic larvae bearing a large marginal appendage on a single valve) have been reported from five Asian freshwater mussel genera belonging to two separate subfamilies, the Gonideinae (i.e. Pseudodon, Solenaia, and Physunio) and Rectidentinae (i.e. Contradens and Trapezoideus). This classification requires that the bilaterally asymmetrical glochidium-bearing mussels are not monophyletic, and suggests that this atypical larval morphology evolved twice in the same geographic region. Although homoplastic glochidium characters are known (e.g. marginal appendages and size), we hypothesized that bilaterally asymmetrical glochidia represent a novel morphological synapomorphy. We tested the monophyly of the mussels bearing bilaterally asymmetrical glochidia using a molecular matrix consisting of representatives from all six freshwater mussel families and three molecular markers (28S, 16S, and COI). Bayesian inference, maximum likelihood, and ancestral state reconstruction were employed to estimate the phylogeny and larval trait transformations. The reconstructed phylogeny rejects the monophyly of the asymmetrical glochidium-bearing mussels and resolves two putative origins of asymmetrical glochidia; however, ancestral state reconstruction supports asymmetrical glochidia as a synapomorphy of only one supraspecific taxon of the Rectidentinae. In the Gonideinae, asymmetrical glochidia were autapomorphic of Pseudodon cambodjensis (Petit, 1865). That is, no other taxa resolved among the Gonideinae had bilaterally asymmetrical glochidia, including other Pseudodon species. We describe how the alleged intrageneric glochidial variation in Pseudodon, and in the other genera of the Gonideinae reported to have asymmetrical glochidia (i.e. Solenaia and Physunio), challenge the resolved convergence of asymmetrical glochidia. Our results are discussed in the context of freshwater mussel larval evolution, patterns in life-history traits, and the classification of freshwater mussels generally. © 2015 The Linnean Society of London

Comunicación dada en: euromal - 8th European Congress of Malacological Studies (10-14 September 2017, Kraków, Poland)., The global decline of freshwater mussels can be partially attributed to their complex life cycle. Their survival from glochidium to adulthood is like a long obstacle race, with juvenile mortality as a key critical point. Mass mortality shortly after entering into a juvenile state has been reported in both wild and captive populations, thus weakening the effec - tive bivalve population. A similar phenomenon oc - curs during metamorphosis in natural and hatchery populations of juvenile marine bivalves. Based on a morphological analysis of newly formed juveniles of the freshwater species Margaritifera margaritifera (Margaritiferidae) and Unio mancus (Unionidae), we show that a second metamorphosis, consisting of drastic morphological changes, occurs that leads to suspension feeding in place of deposit feeding by the ciliated foot. We hypothesise that suspension feed - ing in these two species develops and improves grad - ually due to several morphological features including the contact between cilia of the inner gill posterior filaments,¬ the¬ inner¬ gill¬ reflection,¬ the¬ appearance¬ of¬ the ctenidial ventral groove and the formation of the pedal palps. Regardless of the presence of available food, a suspension feeding mode replaces deposit feeding, and juveniles unable to successfully transi - tion morphologically or adapt to the feeding changes perish.

Glochidial morphology of two unionid species of the genus Schistodesmus Simpson, 1900 from Lake Poyang (China), Schistodesmus lampreyanus (Baird and Adams, 1867) and S. spinosus Simpson, 1900 were studied for the first time under light and scanning electron microscopy. Glochidial characters, including shape, size, hook, sensory hair tufts and exterior minute sculpture, are presented and compared with some allied species for the identification of glochidia. Schistodesmus has small and ovate subtriangular glochidia with styliform hook. Glochidia of the two species of Schistodesmus Simpson, 1900 differed from each other in relative abundance of microstylets and micropoints, and valve sculpture.

The possible occurrence of Unio crassus Philipsson, 1788 on the Iberian Peninsula has been a controversial issue since the nineteenth century. Several characters, including molecular, morphological and life cycle, show that the Iberian taxon is related to, but well differentiated from, the central European U. crassus. The valid name for this Iberian taxon is Unio tumidiformis Castro, 188512. Castro , JS . 1885 . Unionidae nouveaux du Portugal . Bull Soc Malacol France. , 2 : 277 – 294 . View all references. Live populations are distributed throughout the Guadiana, Mira and Sado river basins. Unio tumidiformis can be distinguished from other Unio species by its regular oval shape, high relative height and width, strong wavy umbonal rugae, supra-cardinal tooth on the right valve, and lower glochidium height. From the potential fish hosts tested, only Squalius alburnoides (Steindachner, 1866) resulted in juvenile drop-off, suggesting some degree of specialization in the parasite–host relationship. The endemic nature, population status and habitat of U. tumidiformis make this species important from a conservation point of view., The author Joaquim Reis received a grant from the Fundação para a Ciência e Tecnologia (SFRH/BD/12687/2003) and two Synthesis grants (ES-TAF-1005 and FR-TAF-985) for studying the MNCN and MNHN collections

The freshwater mussel (Bivalvia: Unionidae) fauna of North America is the richest of any continent, yet over 70% of these molluscs are listed as ‘endangered’, ‘threatened’ or of ‘special concern’ (Williams et al., 1993). Declines in richness and abundance at local and regional scales have continued largely due to anthropogenic activities (Nedeau, McCollough & Swartz, 2000). With few exceptions, the larvae of freshwater mussels, called glochidia, are obligate parasites of specific fish species, and mussel dispersal occurs primarily while glochidia are attached to the gills, fins or scales of a host fish. Their dependence on suitable host fish to metamorphose into juveniles and complete their life cycle (Watters, 1992) increases their susceptibility to environmental perturbation, as declines in host fish abundance, introduction of exotic fishes and alterations of fish communities can have drastic consequences on the ability of mussels to reproduce successfully. Host identification has been the focus of considerable research but, for many mussel species, the list of suitable host fish is incomplete or restricted to species in a limited portion of the mussel’s range. Identification of host fish is generally done under laboratory conditions; however, comparatively little is known about host use in the wild, and the relative importance of some host species to recruitment and dispersal of freshwater mussels (Martel & Lauzon-Guay, 2005) cannot be determined in the laboratory. Laboratory designation of host fish may not take into consideration the behavioural characteristics of the host fish, such as habitat use (Martel & Lauzon-Guay, 2005) and attraction to mantle lures or conglutinates, and host fish deemed suitable in the laboratory may serve a limited role in natural populations. On a larger scale, relationships between mussels and their host fish are further complicated as fish communities and host abundance differ among localities and watersheds (Vaughn & Taylor, 2000; Martel & Lauzon-Guay, 2005), and the ability of mussels to adapt to local host species (Rogers, Watson & Neves, 2001) demonstrates the need for a more complete understanding of host use in the wild. This information is critical for conservation and effective planning for natural recovery of many declining mussel species. The study of natural parasitism is greatly inhibited by the difficulty in obtaining reliable identification of encysted glochidia (Hoggarth, 1992). Identification to species using morphological characters is not always possible, and in some mussel communities it is not possible to identify glochidia below the level of subfamily (Weiss & Layzer, 1995). In an effort to provide a more reliable method for identification of glochidia, White, McPheron & Stauffer (1996) developed a dichotomous molecular identification key for the unionids of French Creek, PA, USA, that utilized restriction-fragment length polymorphisms (RFLPs) visualized on agarose gels for species identification. Using a similar approach that added a nested polymerase chain reaction (PCR), a dichotomous RFLP-based molecular key was developed for the unionids of Europe (Gerke & Tiedemann, 2001). However, these keys have not been rigorously applied to the study of natural parasitism in unionids, and there has been difficulty in effectively extracting DNA from a single glochidium using the published methods. The purpose of creating a molecular identification key to the ten species of freshwater mussels that occur in Maine was to determine host fish use in the wild for two species that are state-listed as ‘threatened’, the yellow lampmussel [Lampsilis cariosa (Say, 1817)] and tidewater mucket [Leptodea ochracea (Say, 1817)]. The goals were to examine host use in multiple localities, to determine if host fish identified in the laboratory are used as hosts in the wild and to determine if additional species are potential hosts by capturing naturally parasitized fish and identifying glochidia with the key. In addition, three other mussel species are currently state-listed as ‘special concern’, so identification of fish hosts for wild populations of mussels in Maine is essential information for regional conservation planning. To develop the key, all ten species were sampled at or within close proximity to localities in the three river drainages where Lampsilis cariosa and Leptodea ochracea occur (Fig. 1). Approximately 15 mg of mantle tissue was sampled from morphologically identified adult mussels using methods that are minimally invasive (Berg et al., 1995), and DNA was extracted from tissue samples using a QIAamp DNA Mini Kit (Qiagen Inc., Valencia, CA, USA) following the manufacturer’s instructions for the Tissue Protocol. The NADH dehydrogenase subunit 1 (ND1) gene of the mitochondrial (mtDNA) genome was amplified from each species using the unionid-specific primers Leu-uurF and LoGlyR (Serb, Buhay & Lydeard, 2003). Two specimens of each species from the widest possible distribution within the study area were selected for sequencing to examine intraspecific variation within the ND1 gene. PCR reactions consisted of 200 ng genomic DNA, 1 PCR buffer (20 mM Tris-HCl, pH 8.4, 50 mM KCl), 2 mM MgCl2, 0.2 mM dNTPs, 0.5 mM forward and reverse primers, 1.25 U Taq Polymerase (Invitrogen, Carlsbad, CA, USA), and sterile water to a total reaction volume of 50 ml. Reactions were amplified with a PTC programmable thermocycler with a heated lid (MJC Research, Inc., Watertown, MA, USA). Reaction conditions for doublestranded amplification consisted of an initial denaturation at 948C for 5 min, followed by 30 cycles of 948C for 45 s, 548C for 60 s, and 728C for 60 s; and a final extension of 728C for 5 min. PCR products were purified with Nanosep 30 k Centrifugal Devices (Pall Corp., Ann Arbor, MI, USA) following the manufacturer’s instructions. Products were sequenced in both directions using Big Dye (Perkin Elmer) terminator cycle sequencing (v. 3.1) and read with an ABI 3730 Capillary Sequencer. Glochidia were obtained from gravid female mussels by flushing the marsupium with water using a syringe. On naturally parasitized fish, individual glochidia were removed from ethanolpreserved gills with dissecting probes under 10 magnification using a dissecting microscope, and effort was made to minimize the amount of gill tissue attached to each glochidium. DNA was extracted from a single glochidium using a QIAamp DNA Mini Kit with a slightly modified Tissue Protocol. Detailed instructions of the Tissue Protocol included in the QIAamp DNA Correspondence: S.C. Kneeland; e-mail: stephen.kneeland@umit. maine.edu

Freshwater mussels (Unionidae) are among North America's most imperilled organisms. Mussels produce small larvae (glochidia) that parasitize aquatic vertebrates. We modified the Epicentre QuickExtract protocol to extract DNA from a single glochidium, collected directly from the marsupium of a female mussel, to use as template in polymerase chain reactions (PCRs). Yield per glochidium in a 40 µL extraction volume provided enough DNA for ≥ 15 PCRs per individual. We were successful in using this DNA for microsatellite analysis of up to three loci per individual. Offspring from one female showed evidence for multiple paternity within her brood. Our results are the first documentation of this phenomenon in freshwater mussels.

We measured shell length, hinge length, and height of glochidia from 21 freshwater mussel species occurring in the Sipsey River, Alabama, to test our ability to identify species based on these glochidial morphometrics. Glochidial size and shape differed widely among species; for all 3 dimensions, mean values for the largest species were 5 to 73 greater than for the smallest species. Within-species variation in glochidia size was low for all species with the exception of Pleurobema decisum, which was represented by 2 glochidial morphotypes; variation within each morphotype was similar in magnitude to variation within other species. We were able to classify 72 to 79% of total glochidia (n 5 870 or 750, respectively) to the correct species with discriminant function analysis. Percentage of correct classification ranged from 40 to 100% for individual species. Misclassifications were caused by overlap in shell dimensions between some species, but even species with poor clas- sification success were confounded with an average of only 2.2 to 3.0 other species. Unlike previous studies, we found that glochidia of closely related species were not necessarily more similar to each other than to glochidia of more distantly related species. For example, species in the tribe Quadrulini were widely divergent in glochidium size and represented some of both the smallest and largest glochidia in our study. These 3 shell measurements and subsequent application of discriminant func- tion analysis can be useful for identification of unknown glochidia or for rapidly narrowing the range of potential species identifications to smaller groups of species with similar glochidia.

The juvenile of Utterbackia imbecillis has recently become an important toxicity test organism. However, in temperate climates, gravid U. imbecillis are not readily available during many months of the year, especially if shoreline collection methods are used. This shortage of gravid mussels presents a logistical problem for use of juvenile U. imbecillis as toxicity test organisms. Therefore, this study was designed to test the feasibility of delaying and manipulating glochidial maturation in the laboratory through temperature control, because temperature changes seem to cue the maturation of glochidia in gravid mussels in the natural environment. The objectives of this study were to determine whether glochidial maturation can be stalled by holding adult mussels at cold temperatures, to determine if glochidial maturation could be cued by increasing water temperature at the time juveniles are needed for toxicity testing, and to predict the time required for cold-stored mussels to become gravid (time-to-gravid). Results of this study suggest that glochidial maturation can be stalled by holding mussels at winter temperature. Furthermore, glochidial maturation can later be cued by increasing the water temperature. Additionally, a survival analysis method is suitable for estimating the time-to-gravid for cold-stored mussels. These results should be useful for researchers requiring year-round availability of U. imbecillis for toxicity testing.

The glochidium larvae of freshwater mussels of the family Unionidae need to find suitable hosts to attach themselves and metamorphose into free-living juveniles. The specificity of the host-parasite relationship was investigated for the Iberian Unio tumidiformis Castro, 1885 by means of experimental infections and also by analyzing naturally infected fish. The process of encapsulation of glochidia was studied using scanning electron microscopy. Unio tumidiformis has proven to be an unusual host-specific unionid mussel, apparently parasitizing only fish of the genus Squalius Bonaparte, 1837. Successful encapsulation or complete metamorphosis was observed in five fish taxa: S. aradensis (Coelho, Bogutskaya, Rodrigues et Collares-Pereira), S. caroliterti (Doadrio), S. pyrenaicus (Günther), S. torgalensis (Coelho, Bogutskaya, Rodrigues et Collares-Pereira) and S. alburnoides (Steindachner) complex (only for the nuclear hybrids with at least one copy of the S. pyrenaicus genome). Complete metamorphose was achieved in 6 to 14 days at mean temperatures ranging from 21.8 to 26.1 °C. The current study provides support for cell migration being the main force of cyst formation and shows the influence of potential host's genome in response to the infection process to determine the success of the metamorphosis ©Institute of Parasitology, Biology Centre ASCR.

To metamorphose into juveniles and subsequently mature into adults, the glochidia larvae of freshwater mussels in the order Unionoida must temporarily parasitize the gills, fins, or other external structures of fish. Once attached to the fish, the glochidium is encapsulated by host fish epithelial tissue. The migration of epithelial cells of the bluegill sunfish Lepomis macrochirus over glochidia of Utterbackia imbecillis was examined by time-lapse video microscopy, and the morphology was examined by scanning electron microscopy. Initially, the leading edge epithelial cells migrating over the larvae became rounded and the cells moved as a sheet until the attached glochidium was completely covered. Cyst formation on host fish that had been repeatedly exposed to mussel larvae was significantly delayed and morphologically irregular compared to that on naive fish. Cyst formation on other species of fish that are less successful as hosts was examined. In general, it took longer for glochidia to become encapsulated on these less suitable potential hosts. The delay and irregularities in cyst formation on resistant fish and nonhost fish species may result in increased mortality and reduced success of metamorphosis of glochidia.

7 páginas, 2 figuras, 2 tables.., Genetic variability, shell morphology, anatomy, reproductive season, glochidium morphology and host fish of several Iberian populations of Unio mancus Lamarck, 1819 ( = U. elongatulus C. Pfeiffer, 1825) were examined. The genetics of three allopatric populations were studied, including two ‘races’ previously described for this species on the Iberian Peninsula. All shared the same haplotype of the COI mitochondrial gene. It is proposed that all known Iberian Mediterranean populations of the genus Unio correspond to Unio mancus. All mature specimens studied were dioecious. Glochidial release occurred between March and August. The barbel Barbus graellsii was the natural host of the U. mancus glochidia in the studied area. In artificial infestations performed in aquaria, seven fish species, all native, carried the glochidia throughout their complete metamorphosis. This process occurred in 141 degree/days. Newly released juveniles were spherical and had a thin marginal rim of new shell., We wish to thank to the Departamento de Medio Ambiente de la Diputación General de Aragón (DGA) for their support with the project Conservación de especies amenazadas de moluscos acuáticos de Aragón

The conglutinate of the creeper, Strophitus undulaus (Say, 1817) (Unionoidea: Unionidae), was studied with light and scanning electron microscopy. This conglutinate has a very simple structure consisting of a homogeneous, spongy material containing the glochidia. Each glochidium is contained within its egg membrane and is tethered to this membrane by the glochidial thread. Within the female's marsupium, the glochidia are contained within the conglutinate. When they are expelled to the water the glochidia emerge through pores to the outside and become infective. The mechanism for pushing the glochidia out of the conglutinate is osmotic. The marsupium apparently bathes the conglutinates in a hypertonic solution. When conglutinates are expelled to the surrounding hypotonic water, the spongy material of the conglutinate swells, pushing the glochidia out through the pores. Unlike other conglutinates studied, that of S. undulatus displays a brief period of innate motion which may be adaptive in attracting a suitable host.

The glochidium ofMargaritifera auriculariais described for the first time by using light microscopy, scanning electron microscopy and histological techniques. The larval mantle is formed by only two layers of cells; the inner one being much thicker, with microvilli. All cell masses of the glochidium are temporary aggregations that are the rudiments of organs of the subsequent juveniles which will be released after metamorphosis in the host tissues. In the glochidium there are three main masses of cells: (i) the muscle, which is in an anterior position; (ii) the oral plate in the centre of the larva; and (iii) the more ventrally and posteriorly situated ventral plate, or foot rudiment, flanged with lateral pits all bearing dense cilia. No rudimentary organs such as the pericardium, the kidney, the heart or nerve ganglia have developed. There are no visible hooks in the valve margins, but by using light microscopy we observed minute teeth covered by a rim of the periostracum. Near the margin of the shell there are two pairs of sensory hair tufts only observable by scanning electron microscopy. The glochidium ofM. auriculariais the largest of the family Margaritiferidae and intermediate between the glochidium of the known species of this family and those of Unionidae.

A survey of the fishes of the Black Warrior River was undertaken to determine fish host(s) of the federally threatened inflated heelsplitter, Potamilus inflatus. Seven hundred-twenty individual fishes representing 30 species were examined; mussel glochidia were found on 10 individual fishes representing nine species. Potamilus inflatus glochidia were only found infesting one freshwater drum (Aplodinotus grunniens), which is concordant with previous findings for the genus Potamilus. The morphology of P inflatus glochidia is described and compared to P purpuratus.

Glochidia are third-class levers in which the valves form the lever arms and the single adductor muscle produces the force. In this study the lengths of the lever arms and the areas of glochidial valves and adductor muscles were determined for 57 species of unionid glochidia. The position of the adductor muscle relative to the dorsal margin of the larval valve was also determined for each species. From these data and an analysis of the possible configurations of adductor muscle and valve dimensions, we determined that most of the glochidia within the Unionidae emphasize area of sweep during valve adduction. These glochidia possess long resistance arms and short force arms and generally had small-diameter adductor muscles. Other glochidia, however, were found to possess one or all of the following: short resistance arms, long force arms, and large-diameter adductor muscles. It is suggested that these glochidia are adapted for strength of valve adduction and that for these larvae a trade-off exists between strength of valve adduction and acceptable valve gape. Furthermore, this study suggests that the mode of attachment employed by glochidia has played a major role in the development of these bivalve larvae and has produced convergence in valve shape and adductor muscle size.

Previous work has demonstrated that calcium concretions, located in the gills of freshwater mussels, disappear during the reproductive season. While calcium is being mobilized, there is no increase in calcium content in the blood. The current study documents the passage of maternal calcium to glochidia, for the production of calcium carbonate shells while the larvae are brooding in the water channels of the lateral gills; 45Ca was injected into Anodonta grandis 2 mo before the onset of reproduction and the label was distributed through the animal but was concentrated largely in the calcium concretions. Following glochidia development, 45Ca was found incorporated in the shell of the embryos. To determine the extent of the maternal passage of calcium to the embryos, Ligumia subrostrata containing embryos undergoing shell formation were exposed to 45Ca, either by injection into the blood or by inclusion of label in the bathing medium. The amount of label found in the developing glochidia is proportional to the 45Ca content in the blood of the maternal animal. Isolated glochidia directly exposed to label will adsorb 45Ca onto their shells, but there is little addition of label to glochidia in intact maternal gills during the bath exposure experiments. The results indicate that the maternal contribution of 45Ca is over 90%, with direct contribution from the bathing medium accounting for less than 8%. These results indicate that the branchial water channel in which the embryos are developing is isolated from bath 45Ca. This study presents morphological evidence that the physiological isolation of glochidial shells from bath 45Ca is due to a rearrangement of water channel morphology, and that the chamber, in which the embryos develop, has no contact with the bathing medium.

During the past four years the writer has given considerable time to the field study of the Unioniche. A number of puzzling questions have arisen as to the relative importance of the various ecological factors that c@ntribute to the environmental complex of these animals. Among the most important of these factors, we may mention bottom conditions, water content, stream vol@ ume, stream fluctuations, relation to fish and natural enemies. In connection with this work, much difficulty was experienced in finding young mussels for study and experimentation. I have collected many specimens from the size of a nickel to a quarter1 but mussels under the size of a dime have been rare. A number of experienced field workers have spoken to me of a similar difficulty in finding juvenile specimens. In order to avoid any misunderstanding, I may state that by “? early juvenile life― I mean the period following the time when the mussel completes the parasitic stage and leaves the fish to lead an independent life, until it is about the size of a dime or about fifteen millimeters in length. This would cover, in most species, approximately the first year of independent existence. Other periods may be designated as later juvenile and adult life. The glochidium stage has received considerable study. Our knowledge of the parasitic period has been recently cleared up and extended by the careful investigations of LeFevre and Curtis.2 The later juvenile and adult life has received the attention of hundreds of students. The scattering and meager references in the literature, to the early juvenile stages, give us little infor mation on this important period in the life history of the mussel. During the past summer, while working on the Red River Mussel