A new gymnophallid trematode from the intestine of mice infected with metacercariae from the razor clam Sinonovacula constricta

Meiogymnophallus sinonovaculae n. sp. (Digenea: Gymnophallidae) is described from metacercariae found in the razor clam, Sinonovacula constricta, in the Republic of Korea, and adults recovered from the small intestine of experimentally infected mice. The worms are characterized by paired clusters of vitelline follicles, a well-developed pars prostatica with abundant prostate cells, a voluminous undivided seminal vesicle (in adults and metacercariae), and large, grouped, domelike sensory papillae on the ventral surface anterior to the ventral sucker (in metacercariae). This new species resembles the type species Meiogymnophallus affinis, but differs in having compact, elliptical, and 4-5-lobed vitellaria, and an excretory vesicle with bicornuated anterior arms reaching to the oral sucker. This is the second documentation of the presence of a species of Meiogymnophallus in the Republic of Korea.     

Characterization of new microsatellite loci from the razor clam (Sinonovacula constricta) and transferability to related species

Simple sequence repeats (SSRs) have been widely used in marine animals, but were seldom used directly to test intra- and inter-specific transferability in the populations of related species. 19 new pairs of microsatellite primers were obtained from Sinonovacula constricta. The allele number of these markers ranged from 3 to 15. The observed and expected heterozygosities changed from 0.033 to 1.000 and from 0.033 to 0.912, respectively. Three loci (SC3-8, SC3-19, and SC4-17) significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction, and none of the loci showed linkage disequilibrium. In addition, the transferability of all polymorphic SSRs was assessed in three closely related species. The results showed that four loci were informative in Solen strictus (SC2-11, SC2-12, SC4-19 and SC2-2) and Cultellus attenuates (SC2-2, SC4-9, SC4-19 and SC3-11), respectively, and two loci (SC2-2 and SC2-11) were informative in Solen grandis. These loci will be useful for the evaluation of genetic diversity, connectivity among related species and stock management.     

Chromosome‐level genome assembly of the razor clam Sinonovacula constricta (Lamarck, 1818)

Bivalves, a highly diverse and the most evolutionarily successful class of invertebrates native to aquatic habitats, provide valuable molecular resources for understanding the evolutionary adaptation and aquatic ecology. Here, we reported a high‐quality chromosome‐level genome assembly of the razor clam Sinonovacula constricta using Pacific Bioscience single‐molecule real‐time sequencing, Illumina paired‐end sequencing, 10X Genomics linked‐reads and Hi‐C reads. The genome size was 1,220.85 Mb, containing scaffold N50 of 65.93 Mb and contig N50 of 976.94 Kb. A total of 899 complete (91.92%) and seven partial (0.72%) matches of the 978 metazoa Benchmarking Universal Single‐Copy Orthologs were determined in this genome assembly. And Hi‐C scaffolding of the genome resulted in 19 pseudochromosomes. A total of 28,594 protein‐coding genes were predicted in the S. constricta genome, of which 25,413 genes (88.88%) were functionally annotated. In addition, 39.79% of the assembled genome was composed of repetitive sequences, and 4,372 noncoding RNAs were identified. The enrichment analyses of the significantly expanded and contracted genes suggested an evolutionary adaptation of S. constricta to highly stressful living environments. In summary, the genomic resources generated in this work not only provide a valuable reference genome for investigating the molecular mechanisms of S. constricta biological functions and evolutionary adaptation, but also facilitate its genetic improvement and disease treatment. Meanwhile, the obtained genome greatly improves our understanding of the genetics of molluscs and their comparative evolution.     

Identification and characterization of a clam ferritin from Sinonovacula constricta

Ferritin, a major iron storage protein of most living organisms, plays a crucial role in iron metabolism. Here we reported the isolation and characterization of a cDNA of ferritin gene from Sinonovacula constricta (denoted as ScFER). The full-length cDNA of ScFER was of 996 bp, consisting of a 5'-UTR of 120 bp, a 3'-UTR of 360 bp, and a complete open reading frame of 516 bp encoding a polypeptide with 171 amino acid residues. The predicted molecular mass of deduced amino acid of ScFER was 19.76 kDa and the theoretical pI was 5.07. Quantitative real-time PCR was employed to analyze the expression profiles of ScFER mRNA in muscle, mantle and visceral mass after iron exposure. The peak expression level of ScFER in the three tissues was 1.79-fold, 1.31-fold and 3.51-fold increases in muscle, mantle and visceral mass, respectively. The polyclonal antibodies generated from the recombinant product of ScFER could be specifically identified not only the recombinant product, but also the native protein from muscle. All these results strongly suggested that ScFER was involved in the iron metabolism regulation in S. constricta.     

Polymorphisms of LAP3 gene and their association with the growth traits in the razor clam Sinonovacula constricta

Molecular Biology Reports - Leucine aminopeptidase 3 (LAP3) is an important proteolytic enzyme that catalyzes the hydrolysis of leucine residues from the amino termini of protein or peptide...     

A rickettsiales-like infection in the Pacific razor clam, Siliqua patula

An intracellular procaryotic rickettsiales-like organism was discovered in the digestive absorptive epithelium of the Pacific razor clam, Siliqua patula, from the Pacific Coast of Washington State. Of 80 animals examined, 30% contained the organisms, and all positive animals were between 1.5 mm and 3.0 cm shell length. The organisms appeared as basophilic inclusions averaging 5.0 μm in diameter. Ultrastructurally, the inclusions were membrane-bound vacuoles containing procaryotic forms which averaged 0.8 μm by up to 1.8 μm in length. These organisms had a double layer of trilaminar membrane which appeared to form blebs and outer membrane vesicles. The organisms were pleomorphic by both histological and electron microscopical examination. The host response to infection appeared to consist only of the rounding and detachment of the infected cells. Specific immunofluorescent tests for Rickettsia rickettsia, R. prowazekii, Coxiella burnetti, Chlamydia psittaci, and Legionella pneumophila were all negative when tested on impression smears of infected clam digestive gland.     

Isolation and characterization of polymorphic microsatellite loci in the razor clam Ensis siliqua

Five polymorphic microsatellite loci in the razor clam Ensis siliqua are described. A collection consisting of 34 individuals from Finisterre, Spain, was analysed. Loci were isolated from the sequences of intersimple sequence repeat (ISSR) markers. Detailed analysis of 42 ISSR markers led to the design of 16 primer pairs. Five of these yielded consistent and polymorphic products. The number of alleles ranged from five to 23 per locus with the observed heterozygosity ranging from 0.46 to 0.94. Linkage equilibrium was observed in all loci and three of them showed significant deviations from Hardy–Weinberg equilibrium.     

The razor clam Sinonovacula constricta uses the strategy of conversion of toxic ammonia to glutamine in response to high environmental ammonia exposure

Molecular Biology Reports - High ammonia can inhibit the survival and growth, and even cause mortality of razor clam (S. constricta). The accumulation of ammonia to lethal concentrations in some...     

Biosynthesis of long-chain polyunsaturated fatty acids in the razor clam Sinonovacula constricta: Characterization of four fatty acyl elongases and a novel desaturase capacity

As an unusual economically important aquaculture species, Sinonovacula constricta possesses high levels of long-chain polyunsaturated fatty acids (LC-PUFA). Previously, our group identified fatty acyl desaturases (Fad) with Δ5 and Δ6 activities in S. constricta, which was the first report of Δ6 Fad in a marine mollusc. Here, we further successfully characterize elongases of very long-chain fatty acids (Elovl) in this important bivalve species, including one Elovl2/5, two Elovl4 isoforms (a and b) and a novel Elovl (c) with Elovl4 activity. In addition, we also determined the desaturation activity of S. constricta Δ6 Fad toward 24:5n-3 to give 24:6n-3, a key intermediate in docosahexaenoic acid (DHA) biosynthesis. Therefore, S. constricta is the first marine mollusc reported to possess all Fad and Elovl activities required for LC-PUFA biosynthesis via the ‘Sprecher pathway’. This finding greatly increases our understanding of LC-PUFA biosynthesis in marine molluscs. Phylogenetic analysis by interrogating six marine molluscan genomes, and previously functionally characterized Elovl and Fad from marine molluscs, suggested that DHA biosynthetic ability was limited to a few species, due to the general lack of Δ4 or Δ6 Fad in most molluscs.     

Polymorphic microsatellite loci for population studies of the razor clam, <Emphasis Type="Italic">Sinonovacula constricta</Emphasis>

The razor clam (Sinonovacula constricta) is an important aquacultured bivalve in China. The natural populations of this species are decreasing quickly. To facilitate studies on genetic diversity and population structure of wild populations, microsatellites were isolated from a CA enriched genomic library. Eight microsatellite loci were polymorphic in 30 individuals from Chongming in Shanghai, China. The number of alleles per polymorphic locus varied from 6 to 13 and the values of observed heterozygosity and expected heterozygosity ranged from 0.350 to 1.000 and from 0.602 to 0.902, respectively. These microsatellites are being used in studying population differentiation and genetic diversity for effective conservation and management genetic resources of S. constricta.     

Population genetic structure of banana corm weevil Cosmopolites sordidus (Germar) in India

The population genetic structure of Cosmopolites sordidus (Germar), an economically important pest of bananas, was studied using the sequence data of the internal transcribed rDNA (ITS1+ITS2) and the mitochondrial ‘COI-tRNA^Leu-COII’ region from seventy nine individuals collected from six sampling locations in India. The ITS data revealed 70% within population variation and non-significant genetic differentiation estimates suggesting lack of phylogeographic sub-structuring. 49% within population variation and highly significant genetic differentiation values were obtained with the mitochondrial data. The Mantel test revealed lack of correlation between genetic and geographic distance with both the markers. Demographic expansion of the populations was confirmed by the star shaped haplotype networks, demographic tests and mismatch distribution curves using both the markers. Molecular diversity indices show a high haplotype diversity (Hd) but low nucleotide diversity (π) suggesting that the populations are closely related. Considering the low self-dispersal ability of the weevils, these results suggest that the range expansion of this banana pest in India has taken place mainly through transport of infested corms and plant material resulting in the weevils forming localised populations which are not genetically distinct from each other. The high gene diversity (Hd) has enabled the weevils to adapt to varying environmental conditions which could explain the range expansion of this pest in India. The observed discrepancy in the genetic differentiation estimates using these two markers can be attributed to the evolutionary dynamics of the nuclear and mitochondrial genomes.     

Evaluation of the utility of COI and ITS markers as tools for population genetic studies of temperate gorgonians

The aim of this study was to analyse the polymorphism of two molecular markers, mitochondrial COI and nuclear ITS 2, to determine the genetic structure of Mediterranean gorgonian populations. Four gorgonians, Eunicella cavolinii, Eunicella singularis, Paramuricea clavata and Corallium rubrum were analysed for COI, while only E. cavolinii was analysed for ITS. These species have been recently affected by mass mortality events probably linked to climate change. In the context of these strong disturbances, reliable data on the genetic structure of affected populations could be critical in order to propose management and conservation strategies for these species. A hierarchical sampling plan encompassing different spatial scales (from tenths to hundreds of kilometres) was designed to test these markers. Both markers failed to make evident any genetic structure of these populations. The evolutionary features and utility of these markers are critically discussed and future research directions are proposed.     

Molecular identification of four Panonychus species (Acari: Tetranychidae) in Korea,including new records of P. caglei and P. mori

Four species of the genus Panonychus are identified in Korea including two previously known species P. citri (McGregor) and P. ulmi (Koch) and two newly identified species P. mori Yokoyama and P. caglei Mellot. Morphological diagnostics were observed among the four species in the color of dorsal tubercles and in the shape of male aedeagus. The dorsal tubercles of P. citri, P. ulmi, P. mori and P. caglei are red, white, whitish red and light red, respectively. The aedeagi of male adults are diagnostic in sigmoid‐shaped at the distal end and its shape and length was different among four species. Molecular comparison of the internal transcribed spacer 2 (ITS2) sequence of nuclear DNA and cytochrome‐c oxidase subunit I (COI) nucleotide sequence of mitochondrial DNA among the four species showed divergences 8–12% and 9–12%, respectively. Molecular analysis of the ITS2 and COI sequences revealed their divergences were slightly different among four species. In addition, species‐specific primer sets were designed at the base on ITS2 sequences to precisely diagnose these four species at the molecular level.     

Chloroplast DNA variation and genetic structure of Miscanthus sinensis in southwest China

Miscanthus sinensis is a dominant perennial C4 grass with the potential to being a feedstock crop in North America, Europe, and China. Variation in chloroplast DNA sequence was used to obtain information regarding the genetic diversity and structure of populations of M. sinensis in southwest China. Chloroplast DNA, trnL-F and rpl20-rps12 sequences from seventy-five individuals representing 14 populations of M. sinensis were used to study the sequence variation. Seven haplotypes and 16 polymorphic sites (2.7%) were identified. Five substitutions, 6 indels, and 5 existing substitutions and indels sites, were detected through splicing these two gene segments. The genetic diversity within the studied populations (diversity of haploids, h = 0.561, nucleotide diversity, π = 0.00504) was low, this may be affected by the relatively larger effect of genetic drift on the chloroplast DNA, reflecting smaller effective populations than nuclear DNA. Genetic variance within the populations was higher than that between the populations, suggesting that higher gene flow may exist within these populations. The results of parsimony network in seven haplotypes indicated that H1 and H2 may be ancient haplotypes, which may help guide future research on the origin of M. sinensis. Our results provide information on the genetic diversity and structure of M. sinensis and may assist future studies on the phylogeography of M. sinensis.     

Comparative studies of the population genetic structure of the Brachionus calyciflorus species complex from four inland lakes in Wuhu,China

To study how the population genetic structure in zooplankton respond to environmental conditions, using comparative limnology, the genetic diversity and genetic differentiation of the B. calyciflorus complex collected from four inland lakes in Wuhu City, China were investigated based on the 16S rRNA gene and nuDNA ITS sequences. The results displayed a high genetic diversity, and the nucleotide diversity of the 16S rRNA gene was higher than that of the ITS sequence. The phylogenetic analyses grouped the four populations into two cryptic species (Bc-JT and Bc-FL) with strong support. The two cryptic species were found in lakes with different trophic levels, demonstrating significant ecological specialization. The origins of clone TW12 were not consistent in the phylogenetic trees between two genetic markers, which might be attributed to the effects of male-mediated gene flow on the phylogenetic relationships of rotifers. The nucleotide diversity of the cryptic species Bc-JT was higher than that of Bc-FL, indicating that eutrophication might decrease the genetic diversity of cryptic species. The total phosphorus concentration in water bodies might be the most important factor affecting the genetic diversity of species.     

Molecular identification and phylogenetic analysis of economically important acaroid mites (Acari: Astigmata: Acaroidea) in Korea

Molecular diagnosis is highly valuable for the species identification of microscopic mites. Here, we collected some economically important mites of the superfamily Acaroidea from various stored products in Korea. Those nucleotide sequences of ribosomal second internal transcribed spacer (ITS2) and the mitochondrial cytochrome oxidase subunit I (COI) regions were determined by PCR using universial primers. In nucleotide sequence comparison at GenBank database, seven species including Rhizoglyphus robini, R. echinopus, Sancassania sp. Acarus siro, Tyrophagus putrescentiae, T. similis in Acaridae and Suidasia medanensis in Suidasiidae were identified. Particularly, COI sequences of R. robini, R. echinopus, Sancassania sp. and T. similis were firstly determined. Our results suggest that the phylogenetic relationships inferred from the ITS2 region, rather than COI region, were similar to those derived based on their morphological classification. Our study provides molecular information for the identification and phylogenetic relationship of acaroid mites in Korea.     

Histological survey of symbionts and other conditions in razor clam Ensis arcuatus (Jeffreys, 1865) (Pharidae) of the coast of Galicia (NW Spain)

Symbionts and abnormal conditions of razor clam Ensis arcuatus were surveyed in three commercially important natural beds of Galician estuaries (NW Spain). Samples of 15-20 E. arcuatus were collected every 2 months from January 2003 until July 2004 and processed for histological examination. Prokaryote-like colonies, renal coccidians, gregarines, Trichodina sp. ciliates, haplosporidian-like plasmodia, turbelaria, trematode metacercariae, cestode-like larvae and basophilic inclusion bodies were observed in razor clam tissues without causing host damage. Bucephalid digenean sporocysts and germinoma were seen in some samples causing moderate or severe damage to the host depending on the intensity of infection and both could be a cause for concern if prevalence reached epizootic levels in Galician E. arcuatus populations. None of the parasites detected is OIE notifiable and, in general, the commercially exploited beds studied seem to be devoid of serious pathogens.     

厦门和汕头沿海短沟对虾群体遗传结构及分化

采用聚丙烯酰氨凝胶电泳对2002年10月采集的厦门（XM02）和汕头（ST）沿岸短沟对虾野生群体和2003年8月采集的厦门沿岸短沟对虾野生群体（XM03）进行群体遗传结构及其分化的研究。共检测了EST、ACP、ALP、MDH、ME、SOD、POD、ADH和LDH等9种酶共23个基因座位。结果表明：3个群体平均每位点等位基因有效数目（Ae）为1．207～1．244,多态位点百分数（P0．95）为39．13％-43．48％,群体平均观察杂合度（H0）为0．1231-0．1494,期望杂合度（He）为0．132-0．1443;XM02、XM03和ST的遗传偏离指数（d）分别为0．041、-0．045、0．181,表明群体内的近交衰退不明显,遗传变异水平较高,种质资源尚好。群体之间的遗传距离（D）为0．0010-0．0064,基因流（Nm）为18．98-26．57,表明厦门和汕头短沟对虾应属同一群体,推测台湾海峡中部较深水域或东侧的较高温度水域为其越冬和繁殖的场所。