Biochemical and immunological characterization of collagen molecules from echinothurioid sea urchin Asthenosoma ijimai

Collagens collected from the test (the external hard covering of invertebrates) of the sea urchin, Asthenosoma ijimai, were characterized biochemically and immunologically. The amino-acid composition was typical of that of mammalian collagens. Crystals of segment-long-spacing showed that the molecules of sea urchin collagen were 300 nm long. Selective salt precipitation revealed that the collagen has the same solubility characteristics as type I collagen. The collagen was denatured at 23.1 degrees C. Anti-sea urchin collagen antisera were immunologically cross-reacted with collagens of the same species and the starfish Asterina pectinifera. However, the antisera showed no or slight responses to collagens of bovine type I, II, III, IV and V. The collagen molecules contained four alpha-chains, named alpha 1(SU), alpha 2(SU), alpha 3(SU) and alpha 4(SU), respectively. All of the four alpha-chains were eluted in the same fraction on gel filtration chromatography. Chains of alpha 1(SU) and alpha 2(SU) were extracted earlier than alpha 3(SU) and alpha 4(SU) during pepsin digestion. Other biochemical and immunological analyses clearly demonstrated that test of sea urchins contains two genetically different, but biochemically similar, species of collagens, one of which is composed of alpha 1(SU) and alpha 2(SU) chains, and the other of alpha 3(SU) and alpha 4(SU).     

Molecular phylogeny reveals the existence of two sibling species in the aphid pest Brachycaudus helichrysi (Hemiptera: Aphididae)

Piffaretti, J., Vanlerberghe‐Masutti, F., Tayeh, A., Clamens, A.‐L., C?ur d’Acier, A. & Jousselin E. (2012). Molecular phylogeny reveals the existence of two sibling species in the aphid pest Brachycaudus helichrysi (Hemiptera: Aphididae). —Zoologica Scripta, 41, 266–280. Brachycaudus helichrysi is a worldwide polyphagous aphid pest that seriously damages its primary hosts (Prunus spp.) and the various cultivated plants among its secondary hosts (e.g. sunflower). A recent study of the Brachycaudus genus suggested that this species might encompass two differentiated lineages. We tested this hypothesis, by carrying out a phylogenetic study of this aphid pest based on worldwide sampling and the evaluation of mitochondrial, nuclear and Buchnera aphidicola DNA markers. We show that this species is actually an amalgamation of two sibling taxa, B. helichrysi H1 and B. helichrysi H2, that seem to have overlapping geographic ranges and herbaceous host plant preferences. These two taxa displayed levels of genetic divergence as great as those generally found between sister species in the Brachycaudus genus, suggesting that they actually correspond to two distinct species. Our phylogenetic reconstructions revealed a degree of incongruence between the topologies obtained with the aphid gene data set and with data for a DNA marker from its primary endosymbiont. We identified possible reasons for this observation and discuss the ecological and genotypic data suggesting that B. helichrysi H1 and B. helichrysi H2 have different life cycles.     

A novel pigment protein in the ovary of echinothurid sea urchins,Asthenosoma ijimai and Araeosoma owstoni

• 1.1. A protein conjugated with a pigment, which showed a peak of absorbance at 385 nm, was identified and partially purified from the ovary of Asthenosoma ijimai and Araeosoma owstoni by butanol extraction, gel filtration, ion-exchange chromatography and adsorption chromatography. This protein was observed only in ovaries, but not in testes.
• 2.2. This protein of A. ijimai showed a molecular weight of 600 kDa on gel filtration. The isoelectric point of the protein was 4.7.
• 3.3. The possible presence of this protein was examined by gel filtration chromatography in the ovaries of 11 other species of sea urchins, Glyptocidaris crenularis, Diadema setosum, Temnopleurus hardwicki, Toxopneustes pileolus, Pseudocentrotus depressus, Hemicentrotus pulcherrimus, Strongylocentrotus intermedius, S. nudus, Echinostrephus aciculatus, Anthocidaris crassispina, Echinometra mathaei and Echinocardium cordatum. However, it was not detected.

     

Molecular evidence for the coexistence of two sibling species in Pylaiella littoralis (Ectocarpales,Phaeophyceae) along the Brittany coast

The great phenotypic variability and the lack of diagnostic characters in the genus Pylaiella render the systematic study of this genus problematic. In this study, we investigated the diversity of Pylaiella littoralis along the Brittany (France) coast using a DNA barcoding multilocus approach with mitochondrial (cox1, nad1, and atp9) and chloroplastic (rbcL and atpB) markers associated with a population genetics approach using 10 microsatellite markers. In addition, spatio‐temporal sampling was conducted along the Brittany coast. We sampled 140 individuals from four sites located between Saint‐Malo and Concarneau (380 km) from April to October. Mitochondrial sequence data revealed the occurrence of two sibling species, with a minimum of 2.4% divergence between them. Microsatellite genotypic data congruently revealed two well‐supported clusters matching the two mitochondrial clades of Pylaiella. Although gene flow is limited between species, occurrence of genetic admixtures in some populations suggested that reproductive isolation is not complete. Our study highlighted the complementarity of barcoding and population genetics approaches to shed light on the evolutionary processes that lead to speciation.     

Molecular analysis of larvae suggests the existence of a second species of Sulcascaris (Nematoda: Anisakidae: Anisakinae) in the Japanese moon scallop (Ylistrum japonicum) from Japanese waters

Herein, the morphological and genetic features of the larval specimens of anisakine nematode, isolated from Ylistrum japonicum (Pectinidae) collected from Japanese waters, were examined. Although the specimens were identified as members of the genus Sulcascaris, they were genetically divergent from Sulcascaris sulcata, which is currently the only member of the genus. The present study highlights the possibility that the Sulcascaris population inhabiting Japanese waters represents a unique taxonomic position within the genus Sulcascaris. Additionally, given that many pectinid scallops have been reported as intermediate host of S. sulcata, the present study implies that pectinid scallops may also represent the primary intermediate hosts for the present Japanese Sulcascaris species. Because S. sulcata infections can cause discoloration in the meat of scallops, regular monitoring of the prevalence and intensity of Sulcascaris infections is required to predict the impact of the infections on the market.     

Molecular evidence supporting the existence of two major groups in uropathogenic Escherichia coli

Abstract Molecular methods allow an extremely fine strain typing that can be used to establish the population structure of bacterial species. This methodology has been used to characterize a collection of 74 uropathogenic Escherichia coli obtained from three hospitals located in geographically distant towns in Spain, some representatives of the ECOR collection and other reference strains. Genomic DNA was analyzed by RAPD (Random Amplified Polymorphic DNA) that can characterize a bacterial strain to the level of defining individual clones. The 16S rDNA-23S rDNA spacers were amplified by PCR and submitted to restriction analysis. Finally, the presence or absence of G adhesins in Escherichia coli as well as the type of adhesin (three types are known) have been shown by PCR amplification followed by digestion with restriction enzymes. As expected a wide diversity was shown by RAPD and identical patterns were only found in the case of strains isolated from the same individual, an obvious case of relapse. Analysis of the spacers' restriction patterns showed the presence of two markedly differentiated clusters that we have named α and ß. Both RAPD and spacer restriction patterns originated similar clusters of strains showing a consistency in the evolution of the global genome with the sequence variation of the ribosomal spacers. Furthermore, most of the strains having G-adhesin, with only a few exceptions, corresponded to the α rRNA spacer group. The two spacer types detected were also consistent with some phenotypic markers such as sucrose and raffinose utilization. The α and β clusters could be intraspecific groups produced by partial sexual isolation or other barriers that are originating a divergent evolution.     

Hidden diversity in Antarctica: Molecular and morphological evidence of two different species within one of the most conspicuous ascidian species

The Southern Ocean is one of the most isolated marine ecosystems, characterized by high levels of endemism, diversity, and biomass. Ascidians are among the dominant groups in Antarctic benthic assemblages; thus, recording the evolutionary patterns of this group is crucial to improve our current understanding of the assembly of this polar ocean. We studied the genetic variation within Cnemidocarpa verrucosa sensu lato, one of the most widely distributed abundant and studied ascidian species in Antarctica. Using a mitochondrial and a nuclear gene (COI and 18S), the phylogeography of fifteen populations distributed along the West Antarctic Peninsula and Burdwood Bank/MPA Namuncurá (South American shelf) was characterized, where the distribution of the genetic distance suggested the existence of, at least, two species within nominal C. verrucosa. When reevaluating morphological traits to distinguish between genetically defined species, the presence of a basal disk in one of the genotypes could be a diagnostic morphological trait to differentiate the species. These results are surprising due to the large research that has been carried out with the conspicuous C. verrucosa with no differentiation between species. Furthermore, it provides important tools to distinguish species in the field and laboratory. But also, these results give new insights into patterns of differentiation between closely related species that are distributed in sympatry, where the permeability of species boundaries still needs to be well understood.     

Distribution of Anisakis species larvae from fishes of the Japanese waters

Human anisakiasis is caused by the consumption of raw, marinated or undercooked fish and squid infected with nematodes of the genus Anisakis Dujardin, 1845. In view of food safety, this study was carried out to examine the distribution of Anisakis species in marine fishes within Japanese waters. Seven fish species from six localities were collected and examined for Anisakis infection. Morphological and molecular (ITS region and mtDNA cox2 gene) characterization revealed the presence of two, among the three sibling species of Anisakis simplex, viz. A. simplex sensu stricto (s.s.) and A. pegreffii. Distribution data were collated with the results from the previous researches to better understand Anisakis distribution in Japanese waters. Distributions of Anisakis species were found to be locality-specific rather than host-specific, particularly between the two major species, A. simplex s.s. and A. pegreffii. Anisakis simplex s.s. is mainly found in fishes from northern Japan to Pacific sides, whereas A. pegreffii is in fishes from the Sea of Japan to East China Sea sides.     

Genetic evidence supporting the existence of two distinct species in the genusGasterosteus around Japan

Synopsis The genetic and morphological features ofGasterosteus aculeatus were investigated for 29 populations around Japan. Allozyme analyses recognized two groups (Pacific Ocean group and Japan Sea group) that had distinct characteristic features, and showed high genetic differentiation between them (D = 0.482). The Pacific Ocean group had a wide range, from North America to Japan, along the Pacific coast. The distribution of the Japan Sea group was limited around the Sea of Japan and the Sea of Okhotsk. The distribution of these groups were found to be sympatric on the Pacific coast of Hokkaido Island, Japan. From this area, genetic analyses demonstrated that the sympatric populations of the two groups formed independent breeding stocks, and it is considered that the two groups were reproductively isolated from each other. Additionally, each group had distinctive morphological features of lateral plates and caudal keels in the sympatric area. These results suggested that these two groups of the threespine stickleback comprise different species and that the Japan Sea group is taxonomically distinguishable fromG. aculeatus.     

Molecular, biogeographical and phenological evidence for the existence of three western European sibling species in the Colletes succinctus group (Hymenoptera: Apidae)

This paper examines a cryptic species complex comprising three nominal bee species: Colletes halophilus, Colletes hederae, and Colletes succinctus. Multiple individuals of each were sequenced for four gene fragments: mitochondrial cytochrome oxidase 1 (CO-1), internal transcribed spacer 2 (ITS-2) and flanking regions, elongation factor 1 alpha (EF-1α), and 28S. In addition, the distribution patterns and phenology of all species were examined. Fixed substitutions distinguishing the three species are present in EF-1α and ITS-2. Their distribution patterns differ clearly: Colletes halophilus is endemic to coastal habitats of the southern North Sea and the English Channel; Colletes hederae occurs from Slovenia in the southeast to southern England in the northwest; the range of Colletes succinctus covers most of Europe and reaches western Kazakhstan. Where the three species occur jointly in western Europe, their flight activities differ significantly. The genetic distinctions and the differences in distribution, phenology and flower specialisation clearly support their status as distinct species.     

Isozyme evidence for three sibling species in the Anopheles sundaicus complex from Indonesia

Electrophoretic studies of isoenzymes in three chromosomally distinct forms (A, B and C) of the mosquito Anopheles sundaicus Theobald (Diptera: Culiciae) were undertaken on wild samples collected from six geographically isolated populations in Indonesia. Analyses of 12 enzyme systems comprising 15 loci revealed significant allelic variations, genetic polymorphism, within and among the populations of the An. sundaicus complex. Phylogenetic dendrograms produced by analysis using the Biosys-1 program based on UPGMA methods show that all the populations of form A fall into one cluster, which is closely related to the form C cluster, whereas the populations of form B belong to a more distinct cluster. Allelic frequency and Wright's F statistics of Mpi (mannose phosphate isomerase) are sufficient to identify individuals of each cytological form. This isozyme data correlates with our previous cytological evidence for the existence of three isomorphic species within the taxon An. sundaicus in Indonesia. These three species of the An. sundaicus complex were found together sympatrically at one locality, Asahan in North Sumatra.     

Molecular detection of sibling species in anisakid nematodes

The number of sibling species of anisakid nematodes detected over the last two decades has been increased, fuelled by the use of genetic/molecular methodologies. In the present review, we summarize the biological species discovered within most of the nominal species belonging to the genera Anisakis, Contracaecum and Pseudoterranova by the use of allozyme (20-24 loci studied) and recently confirmed by us using mitochondrial cox-2 gene sequence analysis (mtDNA cox-2). Ecological evidence relating to the distributional range of the genetically detected sibling species and their host preferences, which represent data sets that can be utilized for species delimitation and definition, are summarized.     

Molecular differentiation of sibling species in the Galactomyces geotrichum complex

PCR-analysis, multilocus enzyme electrophoresis and molecular karyotyping were used to characterize 52 strains belonging to the genus Galactomyces. The resultant data revealed that a PCR method employing the universal primer N21 and microsatellite primer (CAC)₅ is appropriate for the distinction of four Ga. geotrichum sibling species, Ga. citri-aurantii and Ga. reessii. Better separation was achieved with the UP primer N21; each species displayed a specific pattern with very low intraspecific variation. We propose to use the primer N21 for the differentiation of the six taxa composing the genus Galactomyces. Multilocus enzyme electrophoresis revealed genetic homogeneity of each sibling species within the Ga. geotrichum complex. On the other hand, the four sibling species, having from 41 to 59% of nDNA homology and similar phenotypic characteristics, are clearly distinguished based on their electrophoretic profiles using two enzymes: mannose-6-phosphate isomerase (MPI) and phosphoglucomutase (PGM). Despite the same number of chromosomal bands, different karyotype patterns were found in Ga. geotrichum sensu stricto and its two sibling species A and B. Within each sibling species, chromosome length polymorphism was observed, in particular for small bands, allowing discrimination to the strain level.     

Molecular evidence of cryptic species within the Liriomyza huidobrensis (Diptera: Agromyzidae)

Phylogenetic relationships among populations of the polyphagous pea leafminer, Liriomyza huidobrensis (Blanchard), were investigated using DNA sequence data. Maximum parsimony analysis of 941 bp of mitochondrial cytochrome oxidase I and II genes showed that L. huidobrensis contains two well-defined monophyletic groups, one composed of specimens from California and Hawaii and one composed of specimens from South and Central America together with populations that have been recently introduced into other parts of the world. The differentiation between the two clades within L. huidobrensis is equivalent to that seen between other agromyzid species, suggesting that L. huidobrensis as currently defined contains two cryptic species. This finding is consistent with field observations of differences in pest status and insecticide resistance between L. huidobrensis populations. Until additional studies are complete, no changes in L. huidobrensis taxonomy are proposed. However, researchers and quarantine officials may wish to consider the findings of the current study in designing research, pest management, and quarantine programs for L. huidobrensis.     

A demonstration of allopatric sibling species within the Gymnophallidae (Digenea)

Meiogymnophallus minutus and Meiogymnophallus fossarum n. comb. both use the oystercatcher Haematopus ostralegus as their final host and Scrobicularia plana as the primary host. M. minutus uses only the estuarine cockle Cerastoderma edule as its second host, where it occurs enclosed in mantle epithelium in a wedge-shaped cavity beneath the umbo and causes no apparent damage to the host. In contrast, the metacercariae of M. fossarum occur in a variety of microhabitats in a wide variety of lagoon lamellibranchs, including Cerastoderma glaucum. In this host it occurs free in the extrapallial fluid usually beneath the periostracum along the edges of the two valves, where it severely inhibits shell growth, and also beneath the umbo. Comparative morphology, experimental cross infections, second host and microhabitat selection, together with ecological and geographical distribution, suggest that Meiogymnophallus minutus and M. fossarum n. comb. are allopatric sibling species. The former occurs in estuaries in northern and western Europe and the latter from lagoons along the French Mediterranean coast.

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Resumé Meiogymnophallus minutus et Meiogymnophallus fossarum n. comb. admettent comme hôte définitif l'Hûitriez-pie, Haematopus ostralegus, et comme premier hôte intermédiarie Scrobicularia plana. Les métacercaries de M. minutus parasitent C. edule; elles sont entourées par des proliférations palléales à l'intérieur d'une cavité en forme de coin fichée sous la charnière du Mollusqui; elles n'ingendrent aucun dommage chez leur hôte. Par contre, les métacercaires de M. fossarum si répartisseut dans plusiers types de microhabitats, chez différents espèces de Lamellibranches euryhalins, en particulier chez C. glaucum. Chez ce dernier, les métacercaires vivent librement dans le liquide extrapalléal, souvent logées à l'intérieur du sillon périostracol bordant les lobes palléaux ce qui entraine des perturbations dans la croissance de la cofuille. On trouve aussi des metacercaries sous la charnière mais elles ni y sont jamais enveloppées. La comparaison des caractères morpho-anatomiques de ces deux espécis, les infestations éxperimentales croisées, la sélection des deuxièmes hôtes ainsi que des microhabitats, la répartition géographique et la distribution écologique suggèrent qu'il n'agit de deux espèces jumelles allopatriques. La première se répartit duos les estuaries du nord et de l'ouest de l'Europe, la seconde le long des côtes méditerraneenes francaises.

     

Chromosomal evidence for sibling species of the malaria vector Anopheles cruzii.

An analysis of the ovarian polytene chromosomes of Anopheles cruzii from three localities in Southeast Brazil revealed the existence of two genetic entities within this morphologically uniform taxon. These cryptic species differed in the banding patterns of the X chromosome and 3L arm. A pattern of bands that cannot be explained by the fixation of any of the known inversions in chromosome X was revealed and named chromosomal form B to distinguish it from the standard pattern of this X chromosome, form A. Each chromosomal form is characterized by a different set of inversions. The lack of heterozygotes (A/B) for these X chromosome forms in populations where both forms coexist is evidence of absence or limited gene flow between the two groups.     

Extensive gene flow within sibling species in the deep-sea fish Beryx splendens

Molecular markers allow insights into the population biology and ecology of deep-sea organisms, which are usually hardly accessible to direct observation and poorly known. Such a study was undertaken here for the deep-sea fish Beryx splendens, a species of growing interest to fisheries. B. splendens populations were sampled on seamounts and continental margins in the southwestern Pacific (New Caledonia, New Zealand, southeastern Australia) and in the northeastern Atlantic. Two hundred and fifty individuals were characterised by their single-strand DNA conformation (SSCP) of a approximately 360-base-pair (bp) fragment of the mitochondrial cytochrome b gene, amplified by the polymerase chain reaction (PCR). Two major SSCP haplotypes were observed in New Caledonia, a and w, whose frequencies were negatively correlated along a north-to-south cline. All SSCP haplotypes in the total sample were sequenced on 273 bp. The phylogenetic tree of B. splendens haplotype sequences, rooted by two B. decadactylus sequences, showed that a and w belong to distinct mitochondrial clades, A and W, which are separated by approximately 4-6% nucleotide divergence. Thirty individuals from New Caledonia were characterised by their DNA fingerprint from arbitrary-primed PCR. The distribution of individual-pairwise similarity indices was strongly bimodal. The larger similarity values all corresponded to comparisons within a clade (A or W) while the lower values were all between clades. Therefore, there was a strict association between the mitochondrial type and the DNA (presumably, nuclear DNA) fingerprint of an individual. Altogether, these results point to the existence of two biological species (sp. A and sp. W) within the current taxon B. splendens. No within-species differentiation was detected at the regional scale (New Caledonia). A remarkable result is that the three cytochrome b haplotypes of northeastern Atlantic B. cf. splendens sp. A were also the three commonest in the southwestern Pacific populations of this species. Such a level of homogeneity in the distribution of haplotypes suggests there is, or recently has been, gene flow at the inter-oceanic scale.     

Molecular evidence for cryptic species within Cylicostephanus minutus (Nematoda: Strongylidae)

The nucleotide sequences of the first and second internal transcribed spacers of nuclear ribosomal DNA were determined for adults of Cylicostephanus minutus from different geographical origins. The lengths of first and second internal transcribed spacer sequences ranged from 370 to 372bp and 215 to 216bp, respectively. Pairwise sequence comparisons revealed that some individuals of C. minutus had identical first and second internal transcribed spacer sequences, whereas others differed by 3.0% and 7.4% in their first and second transcribed spacers, respectively. Some individuals with sequence differences originated from the same host. The levels of difference within C. minutus were higher than that between the morphologically distinct species, Cylicostephanus goldi and Cylicostephanus longibursatus (0.8% for the first internal transcribed spacer and 3.8% for the second internal transcribed spacer). The data provide support for the proposal that C. minutus represents a complex of at least two species. In order to study the population genetic structure of C. minutus, a PCR-linked single-strand conformation polymorphism technique was also established.