Morphological and molecular diagnosis of anisakid nematode larvae from cutlassfish (Trichiurus lepturus) off the coast of Rio de Janeiro, Brazil

Anisakid nematode larvae from Trichiurus lepturus off coast of Rio de Janeiro were studied using light, laser confocal and scanning electron microscopy, in addition to a molecular approach. Mitochondrial cytochrome c-oxidase subunit 2 (mtDNA cox-2), partial 28S (LSU) and internal transcribed spacers (ITS-1, 5.8S, ITS-2) of ribosomal DNA were amplified using the polymerase chain reaction and sequenced to evaluate the phylogenetic relationships between the nematode taxa. The morphological and genetic profiles confirmed that, of the 1,030 larvae collected from the 64 fish examined, 398 were analysed, of which 361 were Hysterothylacium sp. and 37 were Anisakis typica. Larvae of Hysterothylacium sp. were not identified to the species level due to the absence of similar sequences for adult parasites; however, the ITS sequence clustered in the phylogenetic tree with sequences of H. deardorffoverstreetorum, whereas an mtDNA cox-2 and LSU concatenated phylogenetic analysis demonstrated the presence of two clades, both of them under the same name as the larval H. deardorffoverstreetorum. Data on the occurrence of parasites during the winter and summer months were compared using the t-test. The greatest prevalence and intensity of infection were recorded for larval Hysterothylacium, with a prevalence of 51.56% and an intensity of up to 55 parasites per fish. The larval Anisakis exhibit a higher abundance and intensity of infection in the winter months, and those of Hysterothylacium during the summer. However, the t-test indicated no significant differences between the abundance and intensity of infection recorded during the months of collection for either of these larval nematodes. All sequences generated in this study were deposited in GenBank.     

Hysterothylacium patagonense n. sp. (Nematoda: Anisakidae) from freshwater fishes in Patagonia,Argentina, with a key to the species of Hysterothylacium in American freshwater fishes

A new anisakid nematode, Hysterothylacium patagonense n. sp., is described from the intestine of the temperate bass Percichthys trucha (Cuvier & Valenciennes), a freshwater fish from Lake Aluminé, Patagonia, Argentina. It is characterised mainly by the absence of lateral alae, the length ratio of the caecum and ventricular appendix (1:0.9–1.8), the length of the spicules (0.952–1.292 mm) and the presence of minute spines on the tip of the tail. In the same locality and in Lake Huechulafquen, advanced third-stage and fourth-stage larvae of this parasite were also recorded from the digestive tract of the introduced salmonids, Salvelinus fontinalis (Mitchell), Oncorhynchus mykiss (Walbaum) and Salmo trutta L. This is the second Hysterothylacium species described from freshwater fishes in South America. A key to Hysterothylacium species parasitising American freshwater fishes is provided.     

Infection status of the sea eel (Astroconger myriaster) purchased from the Noryangjin fish market with anisakid larvae]

Although the sea eel (Astroconger myriaster) is suspected as one of the most important fish host for human anisakiasis in Korea, no report has been made on the infection status of the sea eel with anisakid larvae. In the present study, 26 sea eels (Astroconger myriaster) were purchased from the Noryangjin fish market in Seoul, and anisakid larvae were collected from their viscera, muscle, head and skin. The collected larvae were classified by their morphological types. A total of 1,351 anisakid larvae were collected from 15 of 26 fish examined. Among them, 1,269 were recovered from the viscera, 66 from the muscle, and 16 from the head and skin. Morphologically, most of the anisakids were classified into 6 known larval types, Anisakis type I (564 larvae) of Berland (1961), Contracaecum type A (409) and type D (5) of Koyama et al. (1969), Contracaecum type C' (83) and type D' (117) of Chai et al. (1986), and Contracaecum type V (1) of Yamaguti (1935). Remaining 172 specimens were new in the available literature, hence, designated as Contracaecum type A' (new type). The present results revealed that the sea eels caught in the Korean waters are heavily infected with anisakid larvae, not only in their viscera but also in the muscle, and Anisakis type I was the most common among the 7 larval types.     

Description and genetic characterisation of Hysterothylacium (Nematoda: Raphidascarididae) larvae parasitic in Australian marine fishes

Nematodes belonging to the genus Hysterothylacium (family Raphidascarididae) infect various species of marine fish in both the larval and adult stages. Humans can be accidentally infected upon eating infected seafood. In spite of their importance, relatively little is known of their occurrence and systematics in Australia. An examination of various species of marine teleosts in Australian waters revealed a high prevalence of Hysterothylacium larval types. In the present study, seven previously undescribed Hysterothylacium larval morphotypes (V to VII and IX to XII) were discovered. In total we found 10 different morphotypes and we genetically characterised nine morphotypes identified. A morphological dichotomous identification key has been established to differentiate these morphotypes. Since some larvae of Hysterothylacium from marine fishes cannot be differentiated morphologically from other nematode larvae, such as Paraheterotyphlum, Heterotyphlum, Iheringascaris and Lapetascaris, the first and second internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA (rDNA) of these larvae were characterised to confirm their taxonomic status. This genetic characterisation implied that some distinct morphotypes belong to different developmental stages of the same species. In addition, it revealed that some morphotypes can comprise distinct genotypes. No match was found between ITS-1 and ITS-2 sequences obtained from larvae in the present study and those from adults available in the GenBank, highlighting the lack of knowledge on occurrence of adult nematodes infecting Australian fish.     

Description of adults and nymph, and redescription of the larva, of Ornithodoros marinkellei (Acari:Argasidae), with data on its phylogenetic position

The argasid tick Ornithodoros marinkellei Kohls, Clifford, and Jones, 1969 was described 4 decades ago based on larval specimens collected from bats (Pteronotus spp.) in Colombia and Panama. Thereafter, larval O. marinkellei parasitizing bats were reported from Venezuela, Guyana, and Brazil. Herein, we describe the adults and nymph, and redescribe the larva of O. marinkellei based on specimens recently collected in the western Brazilian Amazon region. In contrast to all other known adult argasids, the idiosoma of both males and females of O. marinkellei is covered with sclerotized plaques. The idiosoma of the nymph of O. marinkellei is entirely micromamillated, and differs from the adults by the absence of plaques. The larva of O. marinkellei is morphologically similar to the larvae of the 2 other species belonging to the subgenus Subparmatus , i.e., Ornithodoros viguerasi Cooley and Kohls, 1941 and Ornithodoros mormoops Kohls, Clifford, and Jones, 1969 . Because of the long and narrow dorsal plate, the larva of O. marinkellei is readily distinguished from O. viguerasi and O. mormoops. Comparison of our larvae from Brazil with O. marinkellei paratype specimens from Colombia confirmed their taxonomic identification. However, a few morphological differences, particularly in the size of the gnathosoma, were observed. Further studies are necessary to clarify whether O. marinkellei is a complex of different species, or a single species represented by morphologically polymorphic, and geographically distinct populations. Partial mitochondrial 16S rDNA gene sequences were generated for O. marinkellei specimens from Brazil, and compared with available homologous sequences in GenBank. Phylogenetic analyses revealed O. marinkellei to be distinct from the remaining argasid species available in GenBank, including other bat-associated tick species that are found in sympatry with O. marinkellei in the Neotropical region.     

Phylogeographic studies on natural populations of the South American fruit fly, <Emphasis Type="Italic">Anastrepha fraterculus</Emphasis> (Diptera: Tephritidae)

Anastrepha fraterculus is an important pest of commercial fruits in South America. The variability observed for morphological and behavioural traits as well as genetic markers suggests that A. fraterculus represents a complex of synmorphic species rather than a single biological species. We studied the correlation between geographical distribution and genetic variation in natural populations from Argentina and south Brazil. Fragments of the mitochondrial gene COII were sequenced in 28 individuals. The matrix of aligned sequences was phylogenetically analysed by parsimony, yielding a cladogram of haplotypes. Based on Templeton’s nested method, no clade showed any geographic pattern for the gene COII, indicating lack of significant association between haplotypic variability and geographic distribution. The analysis of nucleotide substitution distances by Neighbour-Joining algorithm showed that geographically distant populations exhibit low genetic distances. The corresponding trees clustered the populations without showing any geographic pattern. This result suggests that the populations studied are not reproductively isolated.     

Larval anisakids parasitizing the blue whiting, Micromesistius poutassou, from Motril Bay in the Mediterranean region of southern Spain

A total of 301 blue whiting, Micromesistius poutassou Risso, 1826, ranging in length from 17 to 28 cm, from Motril Bay (Mediterranean coast, south Spain) were examined for anisakid nematodes, as these fish are common items in the Spanish Mediterranean diet. Three anisakid species were morphologically identified with a total prevalence of 10.63%. Anisakis simplex s.l. Rudolphi, 1809 had a prevalence value of 6.65%, compared with 2.66% for A. physeteris Baylis, 1923 and 2.33% for Hysterothylacium aduncum Rudolphi, 1802. Variations in prevalence values with season and host size are discussed. Allozyme markers (leucine aminopeptidase-1) were used to identify anisakid nematodes assigned to the A. simplex complex and all examined larvae were found to correspond genetically to A. pegreffii Nascetti et al., 1986.     

A new morphological type of nematode larvae of the family Anisakidae (Ascaridida)

Larvae of anisakid nematodes of a new morphological type from the anadromous mikizha Parasalmo mykiss (Salmonidae) are described. The larvae have 6 sclerotized cogs on the cephalic end of the body. These larvae are referred to the tribe Lappetascarini Rasheed, 1965 as forms with uncertain generic membership.     

Insights into species diversity of the genus Hydropsyche Pictet, 1834 (Hydropsychidae,Trichoptera) from the Lake Kinneret catchment (Israel)

Species identification based on larval stages of aquatic insects in Israel is difficult, given the paucity of taxonomic studies. In order to gain deeper understanding of biological processes in Israel's freshwater ecosystems and to develop a bioindication system, species have to be known and characterised at all life stages. Here, we applied an integrative morphological and genetic approach to characterise caddisflies of the genus Hydropsyche Pictet, 1834 from the Lake Kinneret catchment. Seventeen sites in the Lake Kinneret catchment were sampled in spring/summer 2015 and 2016. For 77 hydropsychid larvae and two adult specimens of H. yahfufah Malicky, 2001, we analysed a 658 bp fragment of the mitochondrial COI gene and compared the results of different genetic species delimitation methods to observed morphological variation. Our results suggest the presence of at least five species. This study is an important first step towards assessing stream biodiversity of a yet largely understudied region and highlights advantages of integrative taxonomic approaches.     

Systematic studies on Anopheles galvaoi Causey,Deane & Deane from the subgenus Nysssorhynchus blanchard (Diptera: Culicidae)

Anopheles galvaoi, a member of the subgenus Nyssorhynchus, is redescribed based on morphological characters of the adults male and female, fourth-instar larva and pupa. Female, male genitalia, larval and pupal stages are illustrated. Data about medical importance, bionomics, and distribution are given based on literature records. Adult female of An. galvaoi can be easily misidentified as An. benarrochi Gabald n and An. aquasalis Curry. A few characters are indicated for identifying female and immatures of An. galvaoi. Phylogenetic relationships among An. galvaoi and six other species of the Oswaldoi Subgroup are estimated using COII mtDNA and ITS2 rDNA gene sequences. Lectotype of An. galvaoi, an adult female from Rio Branco, State of Acre, is invalidated.     

Multiple primer PCR for the identification of anisakid nematodes from Taiwan Strait

There were six major larval anisakid species found in commercial marine fishes caught in the Minnan fishing ground in the Taiwan Strait: Anisakis physeteris, Anisakis pegreffii, Raphidascaris trichiuri, Contracaecum aduncum, Contracaecum muraenesoxi, Contracaecum sp. For rapid identification of the parasite species above, a single and a multiple primer PCR (multiplex PCR) method, using specific primers based on aligned sequences of the internal transcribed spacer ITS-1, 5.8S, and ITS-2 of nuclear ribosomal DNA, were jointly used for the rapid identification of these anisakid larvae. The primers yielded distinct PCR products for each of the anisakid nematodes, providing rapid and accurate tools for identifying anisakid nematodes with distinct geographical distribution.     

Identification of adults and larvae in Spiochaetopterus (Polychaeta, Chaetopteridae): consequences for larval transport and recruitment

This work is based on recent studies describing new species in Spiochaetopterus (Annelida Polychaeta). New morphological features were brought to light in studies of adults from widely separate areas. A parallel study of planktonic larvae in three areas (northern Atlantic Ocean, Gulf of California, Mediterranean Sea) led to the isolation of several larval categories, each related to an already described species. The large number of species already described shows that the founder species has been subjected to an intense adaptive radiation and that in spite of the ability of larvae to disseminate, each species now inhabits only a limited geographical area. Teleplanic larvae are thus not as effective as is generally supposed in controlling the adult area. The new paradigm implies that individuals from widely separate areas belong to species other than Spiochaetopterus costarum. Although larvae of each species are capable of covering long distances, larval transport may be interrupted at various stages of the life cycle for a variety of reasons. The most powerful causes of failure are not linked to the lifespan of planktonic larvae, but rather to constraints on the ability to realize physiological functions, including fecundation. The putative cosmopolitanism of S. costarum, before new species were described, was the result of both inadequate morphological examination and the use of questionable ecological arguments linked to larval dispersal.     

Parasites as biological tags in population studies of demersal and pelagic fish species

Among the different techniques applied in a holistic approach for fish stock identification, the use of parasites as "biological tags" is becoming increasingly important. In this presentation, our recent studies on the use of some parasite species, identified by genetic markers, and the parasite/fauna composition, in stock identification of demersal (Merluccius merluccius), small pelagic (Trachurus trachurus), and large pelagic fish species (Xiphias gladius) are reviewed. Different species of Anisakis and Hysterothylacium were genetically identified by the application of genetic (allozyme) markers. Statistically significant differences in the spatial distribution of distinct species of Anisakis were found in the fish considered. As to the species of Hysterothylacium genetically detected, different relative proportions were detected in several Mediterranean and Atlantic samples of swordfish (X. gladius). This study demonstrates the potential value of these anisakid nematodes, at both larval and adult stages, as "biological tags" for these fish species in European waters.     

Anisakid nematodes from stranded pygmy sperm whales, Kogia breviceps (Kogiidae), in three localities of the Yucatan Peninsula, Mexico

The present paper reports the presence of 3 adult and juvenile anisakid nematode species: Anisakis simplex, A. brevispiculata, and Pseudoterranova ceticola, which were recovered from the digestive tract of stranded pygmy sperm whales (Kogia breviceps) from 3 localities along the coast of the Yucatan Peninsula. The presence of these anisakid adult nematodes suggests that larval stages may occur in cephalopods or fishes used for human consumption, which represents a potential danger to public health. The occurrence of the 3 anisakid species in coastal waters of the Yucatan Peninsula represents new geographical records for Mexico.     

云南边境地区果实蝇属种类DNA条形码鉴定

【目的】果实蝇属Bactrocera中有国际上重要的检疫性害虫, 基于形态的物种鉴定有一定的局限性。另一方面, 云南边境地区为东南亚地区实蝇入侵我国的重要通道。因此, 对该地区实蝇分子鉴定方法的研究对于该属物种的快速准确鉴定具有重要意义。本研究旨在探讨DNA条形码技术在果实蝇属物种鉴定中的有效性。【方法】使用线粒体基因COI和COII序列的通用引物对果实蝇属20个物种60份样品进行PCR扩增、测序和序列分析; 采取距离方法和建树方法评价2种序列的鉴别能力。【结果】COI和COII序列平均长度分别为682 bp和339 bp, 种内和种间遗传差异较大, 有较明显的遗传距离间隔（barcoding gap）, 鉴定成功率分别为91.2%和90.7%。另外, 分子系统树表明华实蝇亚属Sinodacus不是单系群。【结论】COI和COII序列均能够将绝大多数果实蝇属物种进行准确鉴别, 应用COI或COII序列进行果实蝇属物种鉴定具有一定的可行性。     

Multiplex PCR for the identification of Anisakis simplex sensu stricto, Anisakis pegreffii and the other anisakid nematodes

A multiplex PCR method was established for the rapid identification of Anisakis simplex sensu stricto, A. pegreffii, A. physeteris, Pseudoterranova decipiens, Contracaecum osculatum and Hysterothylacium aduncum. The sequence alignment of the internal transcribed spacer 1 region (ITS-1) between A. simplex s. str. and A. pegreffii showed a high degree of similarity, but only two C-T transitions were observed. To differentiate A. simplex s. str. from A. pegreffii, an intentional mismatch primer with an artificial mismatched base at the second base from the primer 3' end was constructed. This intentional mismatch primer, which produced a PCR band only from A. pegreffii DNA, was able to differentiate the two morphologically indistinguishable sibling species of A. simplex. Specific forward primers for other anisakid species were also designed based on the nucleotide sequences of the ITS region. The multiplex PCR using these primers yielded distinct PCR products for each of the anisakid nematodes. The multiplex PCR established in this study would be a useful tool for identifying anisakid nematodes rapidly and accurately.     

The Hoosier cavefish,a new and endangered species (Amblyopsidae,Amblyopsis) from the caves of southern Indiana

We describe a new species of amblyopsid cavefish (Percopsiformes: Amblyopsidae) in the genus Amblyopsis from subterranean habitats of southern Indiana, USA. The Hoosier Cavefish, Amblyopsis hoosieri sp. n., is distinguished from A. spelaea, its only congener, based on genetic, geographic, and morphological evidence. Several morphological features distinguish the new species, including a much plumper, Bibendum-like wrinkled body with rounded fins, and the absence of a premature stop codon in the gene rhodopsin. This is the first new cavefish species described from the United States in 40 years and exemplifies how molecular data can alert us to the presence of otherwise cryptic biodiversity.     

A comparison of honeybee (Apis mellifera) queen,worker and drone larvae by RNA‐Seq

Honeybees (Apis mellifera) have haplodiploid sex determination: males develop from unfertilized eggs and females develop from fertilized ones. The differences in larval food also determine the development of females. Here we compared the total somatic gene expression profiles of 2-day and 4-day-old drone, queen and worker larvae by RNASeq. The results from a co-expression network analysis on all expressed genes showed that 2-day-old drone and worker larvae were closer in gene expression profiles than 2-day-old queen larvae. This indicated that for young larvae (2-day-old) environmental factors such as larval diet have a greater effect on gene expression profiles than ploidy or sex determination. Drones had the most distinct gene expression profiles at the 4-day larval stage, suggesting that haploidy, or sex dramatically affects the gene expression of honeybee larvae. Drone larvae showed fewer differences in gene expression profiles at the 2-day and 4-day time points than the worker and queen larval comparisons (598 against 1190 and 1181), suggesting a different pattern of gene expression regulation during the larval development of haploid males compared to diploid females. This study indicates that early in development the queen caste has the most distinct gene expression profile, perhaps reflecting the very rapid growth and morphological specialization of this caste compared to workers and drones. Later in development the haploid male drones have the most distinct gene expression profile, perhaps reflecting the influence of ploidy or sex determination on gene expression.