Age and growth in three populations of Dosinia exoleta (Bivalvia: Veneridae) from the Portuguese coast

The present study aimed at estimating the age and growth in three populations of Dosinia exoleta from the Portuguese coast (Aveiro in the north, Setúbal in the southwest and Faro in the south). Two techniques were compared to ascertain the most suitable method for ageing D. exoleta. Growth marks on the shell surface and acetate peel replicas of sectioned shells were the techniques applied. Two hypotheses were tested: growth parameters present latitudinal variation along the Portuguese coast; growth parameters are influenced by the fishing exploitation. Shell surface rings proved inappropriate for ageing this species, whereas acetate peels provided realistic estimates of the von Bertalanffy growth parameters (K, L _∞ and t ₀). A latitudinal gradient in growth rate was detected, with a clear southward increase in the growth coefficient (K) of D. exoleta (Faro > Setúbal and Aveiro) indicating that warmer waters in southern Portugal provide optimal conditions for the growth of this species. Fishing exploitation in northern Portugal targets larger individuals and leaves behind a younger population of smaller individuals, decreasing the asymptotic shell length (L _∞ ) of D. exoleta from Aveiro. The overall growth performance was compared among populations of D. exoleta and with other venerid species worldwide.     

Next-generation sequencing,phylogenetic signal and comparative mitogenomic analyses in Metacrangonyctidae (Amphipoda: Crustacea)

Background

Comparative mitochondrial genomic analyses are rare among crustaceans below the family or genus level. The obliged subterranean crustacean amphipods of the family Metacrangonyctidae, found from the Hispaniola (Antilles) to the Middle East, including the Canary Islands and the peri-Mediterranean region, have an evolutionary history and peculiar biogeography that can respond to Tethyan vicariance. Indeed, recent phylogenetic analysis using all protein-coding mitochondrial sequences and one nuclear ribosomal gene have lent support to this hypothesis (Bauzà-Ribot et al. 2012).

Results

We present the analyses of mitochondrial genome sequences of 21 metacrangonyctids in the genera Metacrangonyx and Longipodacrangonyx, covering the entire geographical range of the family. Most mitogenomes were attained by next-generation sequencing techniques using long-PCR fragments sequenced by Roche FLX/454 or GS Junior pyro-sequencing, obtaining a coverage depth per nucleotide of up to 281×. All mitogenomes were AT-rich and included the usual 37 genes of the metazoan mitochondrial genome, but showed a unique derived gene order not matched in any other amphipod mitogenome. We compare and discuss features such as strand bias, phylogenetic informativeness, non-synonymous/synonymous substitution rates and other mitogenomic characteristics, including ribosomal and transfer RNAs annotation and structure.

Conclusions

Next-generation sequencing of pooled long-PCR amplicons can help to rapidly generate mitogenomic information of a high number of related species to be used in phylogenetic and genomic evolutionary studies. The mitogenomes of the Metacrangonyctidae have the usual characteristics of the metazoan mitogenomes (circular molecules of 15,000-16,000 bp, coding for 13 protein genes, 22 tRNAs and two ribosomal genes) and show a conserved gene order with several rearrangements with respect to the presumed Pancrustacean ground pattern. Strand nucleotide bias appears to be reversed with respect to the condition displayed in the majority of crustacean mitogenomes since metacrangonyctids show a GC-skew at the (+) and (-) strands; this feature has been reported also in the few mitogenomes of Isopoda (Peracarida) known thus far. The features of the rRNAs, tRNAs and sequence motifs of the control region of the Metacrangonyctidae are similar to those of the few crustaceans studied at present.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-566) contains supplementary material, which is available to authorized users.     

Phylogeny of Veneridae (Bivalvia) based on mitochondrial genomes

Veneridae is one of the most diverse families of bivalve molluscs. However, their phylogenetic relationships among subfamilies have been debated for years. To explore phylogenetic relationships of Veneridae, we sequenced 13 complete mitochondrial genome sequences from eight subfamilies and compared with available complete mitochondrial genome of other Veneridae taxa (18 previously reported sequences). Phylogenetic analyses using probabilistic methods recovered two highly supported clades. In addition, the protein‐coding gene order revealed a highly conserved pattern among the same subclade lineages. According to our molecular analyses, Tapetinae should be recognized as a valid subfamily, but the genera formed para‐polyphyletic clades. Chioninae was recovered not monophyletic that differs from a previously molecular phylogeny. Furthermore, the reconstructed chronogram calibrated with fossils recovered the Veneridae have originated during the early Permian (about 290 million years ago). Noticeably, programmed frameshift was found in the nad4 gene of Leukoma jedoensis, Anomalodiscus squamosus and Antigona lamellaris and cob gene of L. jedoensis. This is the first time that the presence of the programmed frameshift has been found in the protein‐coding genes of Heterodonta species. Our results improved the phylogenetic resolution within Veneridae, and a more taxonomic sampling analysis of the subfamily Chioninae is supposed to construct.     

Population genetics and comparative mitogenomic analyses reveal cryptic diversity of Amphioctopus neglectus (Cephalopoda: Octopodidae)

This study presented 96 cox1 and 76 cox3 genes of Amphioctopus neglectus populations. Three distinct lineages were formed from phylogenetic trees and networks constructed using haplotypes. Mitogenomes of A. neglectus-a and A. neglectus-b as the representatives of two lineages separated from population genetics were sequenced to compare with A. neglectus at the genome-level. Amphioctopus neglectus-a showed significant differences with A. neglectus, mainly reflected in gene length, intergenic regions and the secondary structure of tandem repeat motifs. Notably, two sequence deletions in mitogenomes of the two representative species were detected in different positions of major non-coding regions, which were the most distinct differences with A. neglectus. Pairwise genetic distances and the phylogenetic analysis supported the relationship of (A. neglectus-a + (A. neglectus + A. neglectus-b)). This study suggested that A. neglectus-a should be considered as a potential cryptic species of this complex, while A. neglectus-b needed further verification to be defined.     

Reproductive cycle and parasitism in the clam Anomalocardia brasiliana (Bivalvia: Veneridae)

The reproductive cycle and parasitism in the clam Anomalocardia brasiliana were studied in two different areas, an intermediate beach (Cidade) and a tidal flat (Araçá), in Southeast Brazil. Four gametogenic stages were described for males and females in both areas. Mature and spawning individuals were present throughout the period of study at Cidade and Araçá; however, major temporal differences were recorded between sites. Whereas only a few individuals in the resting stage were recorded at Cidade throughout the study period, resting stage individuals were frequent at Araçá. Moreover, a shorter period of gametogenesis was observed at Araçá than at Cidade. Oocyte number was the most effective parameter to differentiate gametogenic stages. An unidentified digenetic trematode was the only parasite infecting A. brasiliana at the study sites, and caused castration of all hosts. Parasite prevalence (proportion of infected hosts) was similar and low (ca 7.5%) at Cidade and Araçá and therefore is not expected to compromise the reproductive output of these populations. Comparison with data from previous studies suggests a latitudinal pattern for the reproductive biology of A. brasiliana, with resting stages occurring only at two high-latitude sites; however, no evidence was found for a latitudinal pattern of trematode prevalence in this species.     

Phylogenetic analysis of Veneridae (Bivalvia): Comparison of molecular and palaeontological data

An approximately 400-by-long portion of the 16s rRNA gene sequence has been determined for the venerid clamsChamelea gallina (Chioninae),Dosinia lupinus (Dosiniinae),Pitar rudis,Callista chione (Pitarinae),Tapes decussatus,T. philippinarum,Venerupis (=Paphia)aurea (Tapetinae), andVenus verrucosa (Venerinae). Neighbor-joining and maximum parsimony trees support the results of traditional classification methods at the subfamily level but do not support the concept of a genusTapes. The transversion divergence rate estimated on the basis of the palaeontological record for theC. gallina/V. verrucosa separation and for the Pitarinae is very close (0.14–0.16% per Myr, respectively) to that of ungulates and cetaceans, while the Tapetinae exhibit a much higher (0.36% per Myr) rate. Correspondence to: E. Olmo     

Larval development of the cockle Chione stutchburyi (Bivalvia: Veneridae) reared in the laboratory

Abstract

Larvae of the New Zealand cockle Chione stutchburyi (Wood, 1828) reared to settlement in the laboratory are described. They are characterised by dissimilar shoulders with a large, broadly rounded anterior shoulder and end, and broadly rounded umbo, typical of the Veneridae. Setting occurred after 20 days at a minimum length of 180/µm. Shell dimensions increased linearly during larval development but hinge length did not. The L:H ratio decreased from 1.27 at L = 100 to 1.1 at L = 200 and the L:D ratio from 1.95 at L = 100 to 1.59 at L = 200. An SEM study revealed that the larval hinge is characterised by a toothed provinculum forming a broad medial projection on the right valve, opposing two projections on the left valve. Conspicuous flanges on the anterior and posterior shoulders of the left valve fit into corresponding grooves of the right valve.     

The clam Meretrix lamarckii (Bivalvia: Veneridae) is a rich repository of marine lactic acid bacterial strains

Lactic acid bacteria (LAB) were isolated from the intestinal tract of the wild clam Meretrix lamarckii caught from the coastal waters of Kashima, Ibaraki, Japan. As many as 415 isolates were obtained using the culture method, of which 70 were considered presumptive LAB strains based on phenotypic tests. Phylogenetic analysis of these presumptive isolates of LAB based on the sequence of the 16S rRNA gene demonstrated that the species belonged to several genera of Lactobacillus, Lactococcus and Pediococcus. Interestingly, however, the species composition was different between the samples in July and October 2010. Further analyses based on the fermentation profiles revealed that the LAB from the clam caught in July 2010 were identified to be Lactobacillus curvatus, Lactobacillus plantarum, Lactococcus lactis subsp. cremoris and Pediococcus pentosaceus, whereas those in October 2010 were identified to be Lactobacillus plantarum, Lactococcus lactis subsp. lactis and P. pentosaceus. The diversity of LAB in the intestinal tract of the clam suggests that the filter feeder bivalves such as M. lamarckii are a rich repository of marine isolates of LAB.     

Parasitic castration of Eurhomalea lenticularis (Bivalvia: Veneridae) by a digenetic trematode: quantitative histological analysis

The clam Eurhomalea lenticularis may be parasitized by digenean trematodes of the family Plagiorchidae, specifically in the gonads (parasitic castration). A quantitative histological analysis of the parasitized gonads demonstrated a significant decrease in gonadal area, in the size of individual acini, and in the numbers of differentiated germ cells compared to unparasitized clams. Castration may be caused by mechanical compression due to trematode sporocyst growth. However, the uniform loss of germ cells in areas without sporocysts suggests that a more generalized mechanism is responsible. We suggest that parasitic castration has a primary effect on the host's neuroendocrine and gametogenic systems that regulate gamete production.     

Environmental and physiological controls on shell microgrowth pattern of Ruditapes philippinarum (Bivalvia: Veneridae) from Japan

The reproductive cycle and shell microgrowth patterns of thevenerid bivalve Ruditapes philippinarum from Matsukawa-ura,a small inlet facing the Pacific Ocean, northeastern Japan wereexamined. Histological examination of the gonads revealed thatspawning in this species occurred twice in 2005, once betweenlate June and early August and the other between late Septemberand early October. Comparison of the shell microgrowth patternswith the developmental stages of the gonad in each individualrevealed that in spawning individuals (mature and spawning stages),the mean lunar-day growth rates were significantly smaller thanthose in individuals which were not in spawning condition (spent,recovery and growing stages). In non-spawning individuals, themean lunar-day growth rates were positively correlated withseawater temperature up to 20°C. However, in spawning individuals,no correlation was observed between shell growth and seawatertemperatures >16°C. These facts suggest that physiologicalstress during reproduction has a negative influence on shellgrowth and results in spawning breaks. The present study indicatethat spawning breaks can be used to identify the timing of sexualmaturity and to count the number of spawning events occurringper year in extant and fossil bivalves.     

Ecotoxicological evaluation of tributyltin toxicity to the equilateral venus clam, Gomphina veneriformis (Bivalvia: Veneridae)

Tributyltin (TBT) is the most common pesticide in marine and freshwater environments. To evaluate the potential ecological risk posed by TBT, we measured biological responses such as growth rate, gonad index, sex ratio, the percentage of intersex gonads, filtration rate, and gill abnormalities in the equilateral venus clam (Gomphina veneriformis). Additionally, the biochemical and molecular responses were evaluated in G. veneriformis exposed to various concentrations of TBT. The growth of G. veneriformis was significantly delayed in a dose-dependent manner after exposure to all tested TBT concentrations. After TBT was administered to G. veneriformis, the gonad index decreased and the sex balance was altered. The percentage of intersex gonads also increased significantly in treated females, whereas no intersex gonads were detected in the solvent control group. Additionally, intersex gonads were detected in male G. veneriformis specimens exposed to relatively high TBT concentrations (20 μg L⁻¹). The filtration rate was also reduced in a dose-dependent manner in TBT-exposed G. veneriformis. We also noted abnormal gill morphology in TBT-exposed G. veneriformis. Furthermore, increases in antioxidant enzyme activities were observed in TBT-exposed G. veneriformis clams, regardless of dosage. Vitellogenin gene expression also increased significantly in a dose-dependent manner in G. veneriformis exposed to TBT. These results provide valuable information regarding our understanding of the toxicology of TBT in G. veneriformis. Moreover, the responses of biological and molecular factors could be utilized as information for risk assessments and marine monitoring of TBT toxicity.     

Reproductive biology of the rock-borer Petricola lithophaga (Retzius, 1788) (Bivalvia: Veneridae) in the Black Sea

For the first time, aspects of the reproduction of the little-known rock-borer mollusc Petricola lithophaga (Retzius, 1788) are described. Specimens were studied during the period from June 2012 to June 2014 (Black Sea, Sevastopol Bay). Histological methods were used to describe gametogenesis stages and spawning of the molluscs. The size and age of onset of sexual maturity, individual fecundity and other factors are defined.     

Use of shape to distinguish Chamelea gallina and Chamelea striatula (Bivalvia: Veneridae): Linear and geometric morphometric methods

The commercially fished striped venus clams Chamelea gallina and C. striatula (Bivalvia: Veneridae) are difficult to distinguish by inexperienced observers and the taxonomy of these species is still an issue of discussion. The differences in shape between C. gallina and C. striatula from Portuguese coastal waters were studied through conventional linear and geometric morphometric analysis, using both contour (elliptic Fourier analysis) and landmark-based methods. The relationships shell length vs. height, width, and total weight were significantly different between species. However, because there was a considerable overlap in the data sets, the species could not be distinguished using any combination of those linear measurements. Geometric morphometric methods provided shape variables that led to 0-6% misclassification rates between species; linear morphometric measures led to 16.8% error. Contour analysis revealed differences primarily in the shell umbo and lunular area. The umbo was more "sharp" and the lunula less pronounced in C. striatula than in C. gallina. Generalized procrustes superimposition (landmark analysis) showed that the main differences between species reside in the length of the pallial sinus. Thus, an index was developed (PI: Pallial Index = pallial sinus length/shell length), which was successfully used to separate the species (with 100% correct classification), i.e., specimens with PI lower than 0.119 belonged to C. gallina, whereas greater PI values were attributed to C. striatula. The use of these geometric morphometric methods allowed the detection of differences in shape between these two species and to develop an easy-to-use identification index. We encourage the development of analogous indices that apply the methods of geometric morphometrics to distinguish between other species whose identification is complicated.     

Embrionary and larval development of the marine clam Tivela mactroides (Bivalvia: Veneridae) in Zulia State, Venezuela

The marine clam, Tivela mactroides, from Ca?o Sagua beach, Venezuela, was spawned and reared under laboratory conditions to monitor its early development. Spawning was spontaneous but in some cases it had to be induced by the additon of eggs and sperm. After fertilization, the embryonic development occurred at 5 hr approximately. Trochophore larvae were observed between eight and ten hours later. Straight-hinged veliger stage appeared 15 hr after fertilization. Transition from veliger stage to the umbo stage occurred about eight days after fertilization. Pediveliger stage was observed 22 days after fertilization. Metamorphosis of T. mactroides was not successful under our laboratory conditions; probably the bacterial contamination and subsequent mortalities were important factors constraining the final phase of the larval cycle. However, in a few cases young individuals were observed. We suspect that this was due to unfavorable conditions (e.g.: bacterial contamination, unsuitable food availability, etc.) and the broad variation in developmental times, suggesting that these stages might be particularly sensitive to environmental changes. These results may not necessarily reflect what happens under natural conditions.     

Strict sex-specific mtDNA segregation in the germ line of the DUI species Venerupis philippinarum (Bivalvia: Veneridae)

Doubly Uniparental Inheritance (DUI) is one of the most striking exceptions to the common rule of standard maternal inheritance of metazoan mitochondria. In DUI, two mitochondrial genomes are present, showing different transmission routes, one through eggs (F-type) and the other through sperm (M-type). In this paper, we report results from a multiplex real-time quantitative polymerase chain reaction analysis on the Manila clam Venerupis philippinarum (formerly Tapes philippinarum). We quantified M- and F-types in somatic tissues, gonads, and gametes. Nuclear and external reference sequences were used, and the whole experimental process was designed to avoid any possible cross-contamination. In most male somatic tissues, the M-type is largely predominant: This suggests that the processes separating sex-linked mitochondrial DNAs (mtDNAs) in somatic tissues are less precise than in other DUI species. In the germ line, we evidenced a strict sex-specific mtDNA segregation because both sperm and eggs do carry exclusively M- and F-types, respectively, an observation that is in contrast with a previous analysis on Mytilus galloprovincialis. More precisely, whereas two mtDNAs are present in the whole gonad, only the sex-specific one is detected in gametes. Because of this, we propose that the mtDNA transmission is achieved through a three-checkpoint process in V. philippinarum. The cytological mechanisms of male mitochondria segregation in males and degradation in females during the embryo development (here named Checkpoint #1 and Checkpoint #2) are already well known for DUI species; a Checkpoint #3 would act when primordial germ cells (PGCs) are first formed and would work in both males and females. We believe that Checkpoint #3 is a mere variation of the "mitochondrial bottleneck" in species with standard maternal inheritance, established when their PGCs separate during embryo cleavage.     

Population dynamics of Megapitaria squalida (Bivalvia: Veneridae) at Magdalena Bay, Baja California Sur, Mexico

The population dynamics of an intertidal population of Megapitaria squalida was studied from September 2002 to February 2003 in Bahia Magdalena, Mexico. To obtain information about the artisanal and recreational fishery of M. squalida, local fishermen at different parts of the bay were interviewed. Clam densities were determined at one unexploited and two exploited sites along perpendicular transects from the shore line. Average clam density was 2.01+/-1.41 ind/m2, densities did not differ significantly between the three sampling sites. The length-total mass relation was Mtot = 0.0001*SL3.1644 (r2 = 0.9804, n = 92) and the length-tissue mass relation was Mtis = 0.0002*SL2.7144 (r2 = 0.945, n = 92). Von Bertalanffy-growth parameters were determined using marked individuals. Strong interindividual and also seasonal differences in growth were observed. Average annual values were: K = 0.655, Linfinity = 83 mm. Since M. squalida can reach a shell length of more than 135 mm in deeper waters, it is necessary to consider intertidal and subtidal populations separately. Average total mortality (Z) was 1.61 y(-1). No fishing mortality was calculated because total mortality Z at the unexploited control site was higher than Z at the exploited sites. It appears that M. squalida migrates into deeper water with increasing shell length, as average shell length increased at lower tidal levels and marked specimens showed a clear migration towards deeper waters. As yet no overexploitation could be found in the study area.     

The complete mitochondrial genome sequence of Sogatella furcifera (Horváth) and a comparative mitogenomic analysis of three predominant rice planthoppers

The white-backed planthopper (WBPH), Sogatella furcifera (Horváth), is one of the most destructive pests of rice crops in many Asian countries. Using long-PCR and shotgun library methods, we sequenced the entire mitochondrial genomes (mt-genomes) of two WBPH individuals. Total lengths of the mt-genome of the two WBPH individuals were 16,612 bp and 16,654 bp with an identical AT content of 76.19%. Among the 13 protein coding genes (PCGs), only nad5 used an atypical initiation codon GTG. Most of the tRNA genes had the typical cloverleaf secondary structure except that the dihydrouridine (DHU) arms in two trnS genes and the TΨC arm of trnG gene did not form a stable stem-loop structure. Similar to the brown planthopper (BPH), Nilaparvata lugens (Stål), and the small brown planthopper (SBPH), Laodelphax striatellus (Fallén), some extraordinary features were observed in the WBPH mt-genome. These include similar gene rearrangement pattern, unusually short length of the atp8 gene and variable numbers of tandem repeat (VNTR) structure in control region. Interestingly, the same tandem repeat unit with stable secondary structure appeared in two different planthoppers, WBPH and SBPH, which belong to two different genera of the Delphacidae. This peculiar feature provides a direct evidence for the close relationship between the two planthoppers and updates our understanding of the evolutionary characteristics of mitochondrial control region. Comparison with two other predominant rice planthoppers (BPH and SBPH) revealed that different PCGs of mitochondria exhibit different evolutionary patterns.     

Reproduction of an alien Ruditapes clam (Bivalvia: Veneridae) on recreational clam-gathering grounds in Tokyo Bay,Japan

The Manila clam Ruditapes philippinarum is an important fisheries resource and popular recreational native species in Japan. To counter the domestic shortage of Manila clams caused by depleted catches, similar clams were introduced into Japan from China and the Korean peninsula. The introduced clam is an unnamed morph closely related to R. philippinarum that originates from southern China. Its shell is morphologically different from that of the typical Manila clam and there are small but distinct genetic differences. There is evidence to suggest that the Chinese morph may hybridize with the Manila clam in the Ariake Sea, but not in Tokyo Bay. We carried out studies to determine the degree of overlap of the breeding seasons of both taxa by collecting samples of the Manila clam and the alien clam released onto recreational clam-gathering grounds in Tokyo Bay and their gonadal maturity was examined histologically. Spawning-stage individuals of the Manila clam appeared from April to August and those of the southern Chinese morph from July to August, indicating overlapping spawning periods and thus the potential for hybridization. We discussed the forming condition of the hybridized clam population in terms of reproduction and recruitment process of the clams.