A molecular and morphological study of Ascaris suum in a human-pig contact scenario in northeastern Brazil / Estudo molecular e morfológico de Ascaris suum em um cenário de contato entre suínos e humanos no nordeste brasileiro

The aim of the present study was to assess morphologic and genetic data on ascariasis in swine (Sus scrofa domesticus) and humans in low-resource rural and periurban communities in the state of Piauí, Brazil. Our cross-sectional survey included 100 fecal samples obtained from swine and 682 samples from humans. Fifteen pigs were necropsied. Human and porcine fecal samples were examined to identify Ascaris eggs. Parasites obtained in the swine necropsies were studied using scanning electron microscopy (SEM), and the mitochondrial gene encoding the cytochrome oxidase 1 (cox1) enzyme was partially amplified and sequenced for molecular taxonomy and phylogenetic analyses. The overall prevalence of Ascaris eggs in the swine fecal samples was 16/100 (16%). No Ascaris eggs were identified in the human fecal samples. SEM of six worms recovered from pigs demonstrated morphological characteristics of A. suum. Cox1 sequences were compatible with A. suum reference sequences. Original and reference (GenBank) nucleotide sequences were organized into clusters that did not segregate the parasites by host species or and region. The largest haplogroups were dominated by haplotypes H01, H02 and H31. In the communities studied, there was no epidemiological evidence of the zoonotic transmission of ascariasis at the human-swine interface.(AU)

O presente estudo teve como objetivo acessar dados morfológicos e genéticos sobre a ascaridíase em suínos (Sus scrofa domesticus) e humanos, em comunidades rurais e periurbanas no estado do Piauí. O estudo transversal incluiu 100 amostras fecais de suínos e 682 amostras obtidas de humanos. Quinze suínos foram necropsiados. Amostras fecais suínas e humanas foram examinadas para detecção de ovos de Ascaris. Os parasitas adultos, obtidos nas necropsias, foram estudados através de microscopia eletrônica de varredura (MEV), e o gene mitocondrial codificante da enzima citocromo oxidase 1 (cox1) foi parcialmente amplificado e sequenciado para análises filogenéticas e de taxonomia molecular. A prevalência de Ascaris em amostras fecais de suínos foi 16/100 (16%), não sendo identificado nenhum caso de infecção por este parasita em humanos. A análise por MEV de parasitas recuperados de suínos demonstrou características morfológicas de Ascaris suum. As sequências nucleotídicas de cox1 foram compatíveis com A. suum. As sequências originais e de referência (obtidas no GeneBank) foram organizadas em clusters que não segregaram os parasitas por hospedeiro ou região geográfica. Os maiores haplogrupos foram dominados pelos haplótipos H01, H02 e H31. Nas comunidades estudadas, não foi evidenciada transmissão zoonótica de A. suum na interface suíno-humana.(AU)

Respiratory chain dysfunction in perifascicular muscle fibres in patients with dermatomyositis is associated with mitochondrial DNA depletion.

AIMS: Patients with dermatomyositis (DM) suffer from reduced aerobic metabolism contributing to impaired muscle function, which has been linked to cytochrome c oxidase (COX) deficiency in muscle tissue. This mitochondrial respiratory chain dysfunction is typically seen in perifascicular regions, which also show the most intense inflammatory reaction along with capillary loss and muscle fibre atrophy. The objective of this study was to investigate the pathobiology of the oxidative phosphorylation deficiency in DM. METHODS: Muscle biopsy specimens with perifascicular COX deficiency from five juveniles and seven adults with DM were investigated. We combined immunohistochemical analyses of subunits in the respiratory chain including complex I (subunit NDUFB8), complex II (succinate dehydrogenase, subunit SDHB) and complex IV (COX, subunit MTCO1) with in situ hybridisation, next generation deep sequencing and quantitative polymerase chain reaction (PCR). RESULTS: There was a profound deficiency of complexes I and IV in the perifascicular regions with enzyme histochemical COX deficiency, whereas succinate dehydrogenase activity and complex II were preserved. In situ hybridisation of mitochondrial RNA showed depletion of mitochondrial DNA (mtDNA) transcripts in the perifascicular regions. Analysis of mtDNA by next generation deep sequencing and quantitative PCR in affected muscle regions showed an overall reduction of mtDNA copy number particularly in the perifascicular regions. CONCLUSION: The respiratory chain dysfunction in DM muscle is associated with mtDNA depletion causing deficiency of complexes I and IV, which are partially encoded by mtDNA, whereas complex II, which is entirely encoded by nuclear DNA, is preserved. The depletion of mtDNA indicates a perturbed replication of mtDNA explaining the muscle pathology and the disturbed aerobic metabolism.

Morphological and molecular characterization of the marine-teleost parasitizing acanthocephalan Echinorhynchus hexagrammi (Syndermata: Palaeacanthocephala) from a new host, Liparis sp. (Actinopterygii: Scorpaeniformes).

Originally described from the masked greenling Hexagrammos octogrammus (Pallas, 1814), the palaeacanthocephalan Echinorhynchus hexagrammi Baeva, 1965 has so far been known from seven species in six families of marine teleosts distributed in the Sea of Okhotsk off Sakhalin and in the Northwestern Pacific off Hokkaido, Japan. In this study, we examined the phylogenetic position of E. hexagrammi based on material obtained from the intestine of an unidentified snailfish, Liparis sp., dredged in Akkeshi Bay, Hokkaido, Japan. We performed an analysis using two gene markers, the mitochondrial cytochrome c oxidase subunit I and the nuclear 28S rRNA, along with other sequences available in public databases. In the resulting tree, E. hexagrammi was more closely related to two species complexes, the E. bothniensis Zdzitowiecki and Valtonen, 1987 complex and the E. gadi Zoega in Müller, 1776 complex, rather than to E. brayi Wayland, Sommerville, and Gibson, 1999, E. cinctulus (Porta, 1905), E. salmonis Müller, 1784, and E. truttae Schrank, 1788. The morphology of the examined material herein identified as E. hexagrammi is briefly described. This study represents the first host record of E. hexagrammi from the snailfish family Liparidae.

Molecular identification of seven Sarcocystis species in red deer (Cervus elaphus) from Lithuania.

The diaphragm muscles of 77 free-ranging red deer (Cervus elaphus) were examined for Sarcocystis species in Lithuania. Sarcocysts were detected in 61 out of 77 (79.2%) animals investigated. A total of 60 isolated sarcocysts were identified to species using subunit I of cytochrome c oxidase (cox1) sequence analysis. Overall, seven species, S. entzerothi, S. hjorti, S. iberica, S. linearis, S. pilosa, S. truncata and S. venatoria, were confirmed in Lithuanian red deer. Sarcocystis entzerothi was reported in red deer for the first time. Previously this species was shown to use sika deer as well as roe deer and fallow deer as an intermediate host. Based on cox1, with the addition of the current data, altogether 13 Sarcocystis species have so far been shown to use red deer as an intermediate host. Species detected in red deer demonstrated considerable differences in intraspecific genetic variation at cox1. Genetic distances between different samples of S. hjorti and S. linearis were calculated using principal coordinates analysis (PCoA), implying molecular divergence of same Sarcocystis species using different hosts in the same geographical area and divergence of those employing same intermediate host species from different areas.

Systematics of Crepidostomum species from the Russian Far East and northern Japan, with description of a new species and validation of the genus Stephanophiala.

Current article touched upon the issue of the complicated taxonomic status of some species from the genus Crepidostomum collected from the freshwater fish in the rivers of Primorsky region, Sakhalin, and Hokkaido Islands. Primary morphological analyses showed affiliation of the worms to the species C. farionis (Müller, 1784) Lühe, 1909; C. metoecus Braun, 1900b; C. chaenogobii Yamaguti and Matsumura, 1942; C. nemachilus Krotov, 1959. We described the new species Crepidostomum achmerovi sp. nov. that is a sibling species of C. nemachilus. Molecular-genetic investigation have shown that C. nemachilus and C. achmerovi sp. nov. are closely related to C. metoecus in both 28S rDNA and cox1 mtDNA markers. Crepidostomum nemachilus forms a separate branch within the C. metoecus clade on the 28S BI tree with strong statistical support and separate clade in relation to C. metoecus clade on the cox1 BI tree. Values of p-distances between Crepidostomum species were at intergeneric level. Crepidostomum metoecus species complex including five species (C. metoecus, C. nemachilus, C. oschmarini, C. brinkmanni, and C. achmerovi sp. nov.) was reconsidered as independent genus Crepidostomum sensu stricto. Minimum Spanning Network showed that C. nemachilus, C. metoecus and C. achmerovi sp. nov. were separated by large number of mutational events and represent independent phyletic lines. An amended diagnosis is provided for the subfamily Crepidostomatinae, the genera Crepidostomum s. str. and Stephanophiala Nicoll, 1909, along with keys to species of both genera.

Species Delimitation of Scavenger Flies in the Valley of Mexico.

Identification of species involved in cadaveric decomposition, such as scavenger Diptera, is a fundamental step for the use of entomological evidence in court. Identification based on morphology is widely used in forensic cases; however, taxonomic knowledge of scavenger fauna is poor for many groups and for many countries, particularly Neotropical ones. A number of studies have documented the utility of a DNA barcoding strategy to assist in the identification of poorly known and diverse groups, particularly in cases involving immature states or fragmented organisms. To provide baseline knowledge of the diversity of scavenger Diptera in the Valley of Mexico, we generated a DNA barcode collection comprised of sequences of the cytochrome c oxidase subunit 1 (COI) gene for all families sampled at a nature reserve located in this region. We collected and identified specimens on the basis of morphology and a species delimitation analysis. Our analyses of 339 individuals delineated 42 species distributed across nine families of Diptera. The richest families were Calliphoridae (9 species), Sarcophagidae (7 species), and Phoridae (6 species). We found many of the species previously recorded for the Valley of Mexico, plus 18 new records for the region. Our study highlights the utility of DNA barcoding as a first-step strategy to assess species richness of poorly studied scavenger fly taxa.

Morphological and molecular data of new species of Characithecium and Diaphorocleidus (Monogenea: Dactylogyridae) from Neotropical characid fishes.

The present study describes three new species of monogenean parasites of characid fishes from the Upper Paraná River basin, Brazil: Characithecium paranapanemense n. sp. on Psalidodon paranae and Psalidodon bockmanni, Diaphorocleidus magnus n. sp. on Astyanax lacustris and Psalidodon fasciatus, and Diaphorocleidus neotropicalis n. sp. on Astyanax lacustris and P. bockmanni. An amendment for Diaphorocleidus is proposed, since additional characters observed in the new species required to extend the generic diagnostic features mainly to include: articulation process connecting the base of the MCO with accessory piece present or absent, and accessory piece with variable shapes (plate-like, pincer-shaped, wrench-shaped, sheath-shaped), divided or not into subunits. Characithecium paranapanemense n. sp. can be distinguished from other congeners by the morphology of its MCO and accessory piece. Diaphorocleidus magnus n. sp. differs from most of its congeners by the morphology of its accessory piece, the presence of articulation process connecting the base of the MCO with accessory piece, and the morphology of the sclerotized structures of the haptor. Diaphorocleidus neotropicalis n. sp. can be easily distinguished from its congeners by the morphology of the accessory piece, the sclerotized structures of the haptor and the morphology of the vagina. Molecular data of the new species (partial 28S rDNA and mitochondrial cytochrome oxidase I) were obtained and the first phylogenetic analysis based on 28S rDNA gene sequences for species of Characithecium and Diaphorocleidus are provided. Although Diaphorocleidus and Characithecium share some morphological similarities, phylogenetic analysis indicates that species of these two genera are not closely related.

Ancylostoma ceylanicum infections in humans in Vietnam.

Ancylostoma ceylanicum is recognized as the only zoonotic hookworm species that is able to mature into adult stage in the human intestine. While human infections caused by this hookworm species have been reported from neighboring countries and this hookworm is prevalent in dogs in Vietnam, human infection has never been reported in Vietnam. The present study, therefore, aimed to identify human infections with A. ceylanicum in Vietnam. A total of 526 fecal samples from the residents in Long An Province were collected and the presence of hookworm eggs was detected by the Kato-Katz method. The results indicated that the overall prevalence of human hookworm infection was 85/526 (16.2%). After filter paper culture, 3rd stage larvae were successfully obtained from 48 egg-positive samples. The larvae were identified for their species using semi-nested PCR-RLFP on the cox1 gene. As a result, two hookworm species were confirmed; single species infections with Necator americanus or A. ceylanicum, and mixed infections with both species were found in 47.9%, 31.3%, and 20.8% of the samples, respectively.

Microscopic and molecular detection of Deraiophoronema evansi (Lewis, 1882) in domestic Bactrian camels (Camelus bactrianus) of Mongolia.

Cameline filarosis is an important parasitic disease having an economic impact on the camel industry around the world. However, there has been no study on filarosis in Bactrian camels of Mongolia. Therefore, the aim of the present study was to detect and identify microfilariae of Deraiophoronema evansi (D. evansi) in Bactrian camels from three provinces, located in southern and southwestern Mongolia. Blood samples were obtained from 400 healthy two-humped camels of different ages and both sexes. All blood samples were analysed using a variety of diagnostic techniques. Microfilariae were detected in 30 Bactrian camels (7.5%) by the Knott technique, while 13 Bactrian camels (3.3%) tested positive in a direct smear test. D. evansi was detected in 18 Bactrian camels (4.5%) by PCR assay. Prevalence was shown to be high among Bactrian camels in the age group up to 5 years, while the lowest positive results were obtained for Bactrian camels in the 5-10-year age group and the over 10-year age group. To confirm the morphological identification, D. evansi-COI gene sequences were subjected to phylogenetic analyses. The D. evansi-COI gene sequences from Mongolian two-humped camels were identical to sequences from Iranian one-humped camels and were clustered together with these sequences in the phylogeny. This is the first report of molecular detection and identification of microfilariae of D. evansi in Bactrian camels of Mongolia.

Morphological and molecular characterization of Quinqueserialis (Digenea: Notocotylidae) species diversity in North America.

Estimates of trematode diversity are inaccurate due to unrecognized cryptic species and phenotypic plasticity within species. Integrative taxonomy (genetics, morphology and host use) increases the clarity of species delineation and improves knowledge of parasite biology. In this study, we used this approach to resolve taxonomic issues and test hypotheses of cryptic species in a genus of trematode, Quinqueserialis. Specimens from throughout North America were field collected from hosts and obtained from museums. We found three morphologically distinct groups and successfully sequenced specimens from two of these groups. DNA sequencing at the 28S and CO1 gene regions revealed that two of the three groups were genetically distinct. One genetic group included two morphological clusters demonstrating host-induced phenotypic plasticity within Quinqueserialis quinqueserialis. The other unique genetic group is a novel species, Quinqueserialis kinsellai n. sp., which is described herein. Our study illustrates the importance of integrating multiple sources of evidence when investigating trematode diversity to account for the influence of cryptic species or phenotypic plasticity. However, further sampling is needed to understand Quinqueserialis spp. diversity as some species have no genetic information associated with them.

Morphological and molecular characterization of Spirocerca lupi (Nematoda: Spirocercidae) from domestic dogs in Vietnam with reference to Spirocerca vulpis.

Spirocerca lupi is a common parasitic nematode associated with esophageal cancer of canids. Recent surveys have revealed an increasing number of canids infected with Spirocerca spp. in Africa, the Americas, Europe and Western Asia, and described a new species, Spirocerca vulpis, from red foxes (Vulpes vulpes). However, in Southeast Asia, research on Spirocerca spp. is scarce. Therefore, the aim of this study is to explore Spirocerca infection in domestic dogs in Vietnam and to identify the Spirocerca species by analyzing their morphometric and molecular data. We found Spirocerca spp. specimens in 51 (17.7%) out of 287 dogs examined with the intensity of infection ranging from one to 29 nematodes per dog. These nematodes were morphologically and molecularly identified as S. lupi. For morphology, the presence/absence of teeth, the ratio of glandular to muscular esophagus lengths, and the position of the vulva opening of S. lupi and S. vulpis varied between reports, suggesting caution in identification of Spirocerca species based exclusively on morphological characteristics. The molecular analysis based on a partial cox1 sequence revealed that S. lupi from Vietnam is genetically close to those from India and China, but far different from those of Israel, South Africa, Peru and Hungary. Given high genetic and morphological variations, more extensive surveys on Spirocerca spp. from various mammalian hosts at a greater scale are necessary to elucidate the divergence of this nematode.

Morphological and molecular characterization of Isospora amphiboluri (Apicomplexa: Eimeriidae), a coccidian parasite, in a central netted dragon (Ctenophorus nuchalis) (De Vis, 1884) in Australia.

An Isospora species, Isospora amphiboluri, originally described by Canon in 1967 and later by McAllister et al. (1995), was isolated from a central netted dragon (Ctenophorus nuchalis) housed at a wildlife rehabilitation centre in Perth, Western Australia. Sporulated oocysts of Isospora amphiboluri (n = 30) are spherical, 24.2 (26.5-23.0) µm in length and 23.9 (22.4-25.9) µm in width, with a shape index of 1.01. The bilayered oocyst wall is smooth and light-yellow in color. Polar granule, oocyst residuum and micropyle are absent. The sporocysts are lemon-shaped, 15.7 (15.2-18.0) × 10.2 (8.9-11.2) µm, with a shape index (length/width) of 1.53. Stieda and substieda bodies are present, the Stieda body being small and hemidome-shaped and the substieda half-moon-shaped. Each sporocyst contains four vermiform sporozoites arranged head to tail. The sporozoites are 11.7 (9.9-16.2) × 3.0 (2.4-3.5) µm, with a shape index (length/width) of 3.87. A sporocyst residuum is present. Sporozoites contain a central nucleus with a finely distributed granular residuum. Comparison of oocyst measurements and their features with other valid Isospora species from hosts in the Agamid family confirmed that this Isospora species is Isospora amphiboluri. Molecular characterization of I. amphiboluri at the 18S rRNA and MTCOI loci showed the highest similarity with I. amphiboluri from the central bearded dragon, 99.8% and 99.7% respectively. This is the first report of I. amphiboluri from a central netted dragon in Australia.

Multicopy targets for Plasmodium vivax and Plasmodium falciparum detection by colorimetric LAMP.

BACKGROUND: Loop-mediated isothermal amplification (LAMP) for malaria diagnosis at the point of care (POC) depends on the detection capacity of synthesized nucleic acids and the specificity of the amplification target. To improve malaria diagnosis, new colorimetric LAMP tests were developed using multicopy targets for Plasmodium vivax and Plasmodium falciparum detection. METHODS: The cytochrome oxidase I (COX1) mitochondrial gene and the non-coding sequence Pvr47 for P. vivax, and the sub-telomeric sequence of erythrocyte membrane protein 1 (EMP1) and the non-coding sequence Pfr364 for P. falciparum were targeted to design new LAMP primers. The limit of detection (LOD) of each colorimetric LAMP was established and assessed with DNA extracted by mini spin column kit and the Boil & Spin method from 28 microscopy infections, 101 malaria submicroscopic infections detected by real-time PCR only, and 183 negatives infections by both microscopy and PCR. RESULTS: The LODs for the colorimetric LAMPs were estimated between 2.4 to 3.7 parasites/µL of whole blood. For P. vivax detection, the colorimetric LAMP using the COX1 target showed a better performance than the Pvr47 target, whereas the Pfr364 target was the most specific for P. falciparum detection. All microscopic infections of P. vivax were detected by PvCOX1-LAMP using the mini spin column kit DNA extraction method and 81% (17/21) were detected using Boil & Spin sample preparation. Moreover, all microscopic infections of P. falciparum were detected by Pfr364-LAMP using both sample preparation methods. In total, PvCOX1-LAMP and Pfr364-LAMP detected 80.2% (81 samples) of the submicroscopic infections using the DNA extraction method by mini spin column kit, while 36.6% (37 samples) were detected using the Boil & Spin sample preparation method. CONCLUSION: The colorimetric LAMPs with multicopy targets using the COX1 target for P. vivax and the Pfr364 for P. falciparum have a high potential to improve POC malaria diagnosis detecting a greater number of submicroscopic Plasmodium infections.

Morphological and genetic characterization of Porrocaecum angusticolle (Molin, 1860) (Nematoda: Ascaridomorpha) from the common buzzard Buteo buteo (Linnaeus) (Accipitriformes: Accipitridae) in Czech Republic.

Porrocaecum angusticolle is a nematode species mainly parasitic in the birds of Accipitriformes and Strigiformes. However, some aspects of the morphology of P. angusticolle remain insufficiently known. In the present study, the detailed morphology of P. angusticolle was studied using light and, for the first time, scanning electron microscopy, based on newly collected specimens from the common buzzard Buteo buteo (Linnaeus) (Accipitriformes: Accipitridae) in Czech Republic. Some previously unreported morphological features of taxonomic significance were observed. The nuclear and mitochondrial DNA markers, including partial large ribosomal DNA (28S), complete internal transcribed spacer (ITS-1 + 5.8S + ITS-2), cytochrome c oxidase subunit 1 (cox1) and subunit 2 (cox2) of P. angusticolle were sequenced for molecular identification of this species. There was no intraspecific genetic variation detected in the 28S and ITS regions among different individuals of P. angusticolle, but low level of intraspecific nucleotide divergence was found in the cox1 (0.26-0.78%) and cox2 regions (1.0%). The 28S and cox2 of P. angusticolle were sequenced for the first time. Our molecular evidence supported the validity of both P. angusticolle and P. depressum. The newly obtained genetic data are helpful for further studies of DNA-based taxonomy, population genetics and phylogeny of the genus of Porrocaecum.

Cytochrome c oxydase I phylogenetic analysis of Haemogregarina parasites (Apicomplexa, Coccidia, Eucoccidiorida, Haemogregarinidae) confirms the presence of three distinct species within the freshwater turtles of Tunisia.

Species of Haemogregarina are apicomplexan blood parasites that use vertebrates as intermediate hosts. Due to limited interspecific morphological characters within the genus during the last decade, 18S rRNA gene sequences were widely used for species identification. As coinfection patterns were recently reported from nuclear molecular data for two sympatric freshwater turtles Mauremys leprosa and Emys orbicularis from Tunisia, our objectives were to design COI specific primers to confirm the presence of three distinct species in both host species. Blood samples were collected from 22 turtles, from which DNAs were extracted and used as templates for amplification. Following different rounds of PCR and nested PCR, we designed specific Haemogregarina COI primers that allowed the sequencing of nine distinct haplotypes. Phylogenetic Bayesian analysis revealed the occurrence of three well-differentiated sublineages that clustered together into a single clade. Based on pairwise genetic distances (p-distance), we confirmed the occurrence of three distinct but phylogenetically closely related species coinfecting M. leprosa and E. orbicularis in the same aquatic environments. Our results demonstrate that the use of fast evolving genes within Haemogregarina will help to investigate the parasite diversity within both intermediate vertebrate and definitive invertebrate hosts, and to assess the evolution, historical biogeography and specificity of haemogregarines.

Comparative Phylogeography and Integrative Taxonomy of Ochlerotatus caspius (Dipera: Culicidae) and Ochlerotatus dorsalis.

Given that accurately identifying pathogen vectors is vital for designing efficient mosquito control programs based on the proper surveillance of the epidemiologically important species, it has been suggested the complementary use of independently evolving genes and morphometric traits as a reliable approach for the characterization and delimitation of related species. Hence, we examined the spatial distribution of COI mtDNA and ITS2 rDNA variation from the historical perspective of Ochlerotatus caspius (Pallas, 1771) and O. dorsalis (Meigen, 1830), while simultaneously testing the utility of the two markers in integrative species delimitation when combined with phenotypic character analyses of larvae and adults. Despite the striking difference in haplotype diversity (high in COI mtDNA, low in ITS2 rDNA), no evident phylogeographic structure was apparent in the Palearctic O. caspius. The Holarctic O. dorsalis species was subdivided into two highly distinctive COI mtDNA phylogroups which corresponded to the Nearctic and Palearctic regions. Strong support for the independence of the two allopatric evolutionary lineages suggested that geographical barrier and climatic changes during Pleistocene caused vicariance of the ancestral range. COI mtDNA reliably distinguished O. caspius and O. dorsalis, while ITS2 rDNA yet again lacked the proper resolution for solving this problem. An integrative approach based on the larval and adult morphological traits have varying taxonomic applications due to their differential diagnostic values. Thus, by the implementation of an integrative taxonomic approach, we successfully detected species borders between the two epidemiologically relevant species and uncovered the presence of cryptic diversity within O. dorsalis.

Molecular characterization of Oxyspirura mansoni and Philophthalmus gralli collected from the eyes of domestic chickens in Bangladesh.

A variety of helminths have been found in domestic chickens in Bangladesh, but little is known about their gene sequences. Here, parasitic nematodes and trematodes were collected from the eyes of domestic chickens and analyzed for their morphological and morphometric characteristics, and characterized molecularly. The helminths were identified as Oxyspirura mansoni and Philophthalmus gralli. The ITS1 and ITS2 sequences of O. mansoni were 532 bp and 306 bp in length, respectively, and showed low identity (50.7-62.7%) with those of O. petrowi and O. conjunctivalis. Furthermore, the O. mansoni CO1 sequences (393 bp) showed five haplotypes (97.5-99.5% similarity) that formed a monophyletic clade. With respect to P. gralli, the ITS1 (452 bp) and ITS2 (736 bp) sequences showed 100% similarity with the reference sequences in GenBank. Both the ND1 and CO1 phylograms showed that P. gralli from Bangladesh, Costa Rica and Peru form a monophyletic clade, distinct from the clades of P. lucipetus and P. lacrymosus. Our data show that, Philophthalmus gralli isolates from Bangladesh, Costa Rica and Peru are genetically close to each other.

On the complementarity of DNA barcoding and morphology to distinguish benign endemic insects from possible pests: the case of Dirioxa pornia and the tribe Acanthonevrini (Diptera: Tephritidae: Phytalmiinae) in Australia.

Fruit flies are considered economically important insects due to some species being agricultural pests. However, morphological identification of fruit fly adults and larvae can be difficult requiring a high level of taxonomic expertise, with misidentifications causing problematic false-positive/negative results. While destructive molecular techniques can assist with the identification process, these often cannot be applied where it is mandatory to retain a voucher reference specimen. In this work, we non-destructively (and partial-destructively) processed larvae and adults mostly belonging to the species Dirioxa pornia (Walker, 1849), of the poorly studied nonpest fruit fly tribe Acanthonevrini (Tephritidae) from Australia, to enable molecular identifications whilst retaining morphological vouchers. By retaining the morphological features of specimens, we confirmed useful characters for genus/species-level identification, contributing to improved accuracy for future diagnostics using both molecular and morphological approaches. We provide DNA barcode information for three species of Acanthonevrini known from Australia, which prior to our study was only available for a single species, D. pornia. Our specimen examinations provide new distribution records for three nonpest species: Acanthonevroides variegatus Permkam and Hancock, 1995 in South Australia, Acanthonevroides basalis (Walker, 1853) and D. pornia in Victoria, Australia; as well as new host plant records for D. pornia, from kangaroo apple, apricot and loquat.

Emergence of the zoonotic tick Dermacentor (Indocentor) auratus Supino, 1897 (Acari: Ixodidae) in Singapore.

Though ticks pose a significant public health risk, until recently, little research had focused on the diversity of ticks and tick-borne diseases in Singapore. To date, only fourteen tick species in five genera have been recorded there. For the first time, Dermacentor auratus is recorded from Singapore from a range of hosts, including humans. DNA sequences are provided at 2 loci, for D. auratus, the cytochrome c oxidase I (COI) for DNA barcoding and the 16S large subunit ribosomal RNA (16S lsu rRNA). The health risk posed by D. auratus in Singapore is discussed.

Ixodes silvanus n. sp. (Acari: Ixodidae), a new member of the subgenus Trichotoixodes Reznik, 1961 from northwestern Argentina.

Females, nymphs, and larvae of Ixodes silvanus n. sp. collected from birds and from the vegetation in northwestern Argentina (Yungas Phytogeographic Province) are described herein. The new species belongs to the subgenus Trichotoixodes (Acari: Ixodidae). The female is diagnosed by a combination of the following characters: scutum with setae moderately long and more numerous in central field, fewer and moderately long setae on lateral fields, and inconspicuous setae in anterior field; basis capituli subtriangular dorsally; porose areas large and irregular in shape, lacking distinct margins; auriculae with straight edges diverging posterolaterally and ending with small blunt processes; hypostome narrow and pointed with dental formula 4/4 in the anterior third, then 3/3 and 2/2 near the base; coxae I with two spurs, sub-equal in size, internal slightly slimmer than external. The nymph is diagnosed by notum with numerous and long setae, ventral surface covered by numerous whitish setae, scutum with short scapulae and few and shallow punctations, setae on scutum few, short and irregularly distributed, basis capituli sub-triangular dorsally with posterior margin straight, cornua large and directed postero-laterally, auriculae large and projected laterally, lateral margin of basis capituli above auriculae with a lateral and triangular projection, hypostome pointed with dental formula 3/3 in the anterior third and then 2/2, and coxa I with two short, sub-equal, triangular spurs. The diagnostic characters of the larva are: basis capituli dorsally sub-triangular with lateral angles acute and posterior margin straight, auriculae as large triangular lateral projections, hypostome with apex bluntly pointed and dental formula 3/3 in the anterior third and then 2/2, coxa I with two short, sub-equal, triangular spurs, and pattern of dorsal and ventral body setae. This new species is phylogenetically related to Ixodes brunneus, Ixodes turdus and Ixodes frontalis, and the principal hosts for all its parasitic stages are birds.