Designation of the neotype of Triatoma dimidiata (Latreille, 1811) (Hemiptera, Reduviidae, Triatominae), with full integrated redescription including mitogenome and nuclear ITS-2 sequences

The taxonomic status of Triatoma dimidiata (Latreille, 1811) is, by far, the most discussed within Triatominae. Molecular studies have recovered at least three independently evolving lineages in T. dimidiata across its range. The original description of T. dimidiata (as Reduvius dimidiatus) included few taxonomic characters, and no types were assigned. To define and describe the cryptic diversity within T. dimidiata sensu lato (s.l.), a neotype must be designated. For this purpose, all 199 specimens identified as T. dimidiata from the collections of the Smithsonian Institution – National Museum of Natural History and the American Museum of Natural History, ranging from Peru to Mexico, were studied. Only one specimen (from Tumbes, Peru) matched the combination of characters as listed in the original description, and it is herein formally designated as the neotype for T. dimidiata. The neotype is morphologically described and DNA sequences of its whole mitochondrial genome and the nuclear second internal transcribed spacer region (ITS2), commonly used in triatomine molecular systematics studies, are presented and compared to other publicly available sequences of T. dimidiata s.l. in GenBank. Our results suggest that T. dimidiata sensu stricto (s.s.) is somewhat rare and, therefore, unlikely to serve as a major vector of Chagas disease.

Molecular characterization of Anopheles fluviatilis species complex in the Islamic Republic of Iran

A species-specific polymerase chain reaction [PCR] assay was used to identify the species composition of the Anopheles fluviatilis complex in the Islamic Republic of Iran. All the amplified DNA samples from specimens collected from different areas yielded a fragment of 450 bp size, a PCR product corresponding to that of the species denoted as Y. The sequence data from 21 ITS2 [second internal transcribed spacer] regions were compared with those publicly available in the GenBank database and confirmed that the specimens were 100% identical to species Y of India. Species Y is presumably the same as species T that has no role in transmission of malaria in India, whereas An. fluviatilis is known as a secondary vector of malaria in the Islamic Republic of Iran

Second internal transcribed spacer (ITS-2) as genetic marker for molecular characterization of Sarcoptes scabiei in rabbits from several areas of East Java, Indonesia

Objectives The purpose of this study is to use the second internal transcribed spacer (ITS-2) to determine the molecular characteristics of Sarcoptes scabiei in rabbits from several areas of East Java. Methods Collecting S. scabiei mites from rabbits with clinical signs of scabies; DNA extraction with minikit QIAamp DNA; polymerase chain reaction amplification; nucleotide sequence analysis; homology and phylogenetic tree using the Neighbor-Joining method in the program molecular evolutionary genetics analysis-7 (MEGA-7). Results Sequence analysis of ITS-2 S. scabiei from five regions in East Java showed an identity >91.23% with isolates from China (KX695125.1). The phylogenetic analysis of ITS-2 S. scabiei from Mojokerto rabbits has a close relationship with AB82977.1; Surabaya and Nganjuk rabbits are closely related to KX695125.1; while Sidoarjo and Pasuruan rabbits are closely related to EF514469.2. and AB369384.1. Conclusions The homology analysis of all samples showed identity of more than 91.23% with isolate China (KX695125.1). The sequences of ITS-2 gen of S. scabiei from rabbits in several areas were relatively close to S. scabiei obtain various hosts from National Centre for Biotechnology Information (NCBI) data.

Intragenomic sequence variations in the second internal transcribed spacer (ITS2) ribosomal DNA of the malaria vector Anopheles stephensi

Second Internal Transcribed Spacer (ITS2) ribosomal DNA (rDNA) sequence is a widely used molecular marker for species-identification or -delimitation due to observed concerted evolution which is believed to homogenize rDNA copies in an interbreeding population. However, intra-specific differences in ITS2 of Anopheles stephensi have been reported. This study reports the presence of intragenomic sequence variation in the ITS2-rDNA of An. stephensi and hypothesizes that observed intra-specific differences in this species may have resulted due to ambiguous DNA sequence-chromatogram resulting from intragenomic heterogeneity. Anopheles stephensi collected from different parts of India were sequenced for complete ITS2 and the variable region of 28S-rDNA (d1-d3 domains). Intragenomic variations were found in ITS2 region of all An. stephensi sequenced, but no such variation was observed in d1 to d3 domains of 28S-rDNA. Cloning and sequencing of ITS2 through the d3 domain of the 28S region of rDNA from representative samples from northern, central, and southern India confirmed the presence of intragenomic variation in ITS2 due to transitions at three loci and two bp indel in a di-nucleotide microsatellite locus. Multiple haplotypes were observed in ITS2 raised from such variations. Due to the absence of detectable intragenomic sequence variation in the d1 to d3 domain of 28S rDNA of An. stephensi, this region can serve as an ideal reference sequence for taxonomic and phylogenetic studies. The presence of intragenomic variation in rDNA should be carefully examined before using this as a molecular marker for species delimitation or phylogenetic analyses.

Genetic variability, cryptic species and phylogenetic relationship of six cyathostomin species based on mitochondrial and nuclear sequences

Cyathostomins are important intestinal nematode parasites of equines and include 50 accepted species. Their taxonomy has been frequently revised and the presence of cryptic species suggested. Furthermore, usually molecular- and morphology-based phylogenetic analyses give divergent results. In this study, the nucleotide sequences of the nuclear second internal transcribed spacer (ITS-2) and the mitochondrial partial cytochrome c oxidase subunit I (COI) were determined for adults of six cyathostomin species (Coronocyclus coronatus, Coronocyclus labiatus, Cylicocyclus nassatus, Cylicostephanus calicatus, Cylicostephanus longibursatus, Cylicostephanus minutus) collected from different equine species within two geographic regions. Maximum likelihood trees were calculated for ITS-2, COI, and concatenated data. No obvious differentiation was observed between geographic regions or equine host species. As previously reported, Coronocyclus coronatus and Cylicostephanus calicatus revealed a close relationship. Cryptic species were detected in Cylicostephanus minutus and Cylicostephanus calicatus. Cylicocyclus nassatus and Coronocyclus labiatus showed diverse mitochondrial and nuclear haplotypes occurring in different combinations, while Cylicostephanus longibursatus was comparatively homogenous. In conclusion, a combined analysis of nuclear and mitochondrial haplotypes improved resolution of the phylogeny and should be applied to the remaining cyathostomin species and across additional equine host species and geographic regions.

Intragenomic sequence variations in the second internal transcribed spacer (ITS2) ribosomal DNA of the malaria vector Anopheles stephensi

Second Internal Transcribed Spacer (ITS2) ribosomal DNA (rDNA) sequence is a widely used molecular marker for the species-identification or -delimitation due to observed concerted evolution which is believed to homogenize rDNA copies in an interbreeding population. However, intra-specific differences in ITS2 of Anopheles stephensi have been reported. This study reports the presence of intragenomic variation in the ITS2 region of An. stephensi and hypothesized that observed intra-specific differences in this species may have been resulted due to ambiguous DNA-chromatogram resulting from intragenomic heterogeneity. Anopheles stephensi collected from different parts of India were sequenced for complete ITS2 and the variable region of 28S rDNA (d1-d3 domains). Intragenomic variations at fixed loci were found in ITS2 of all An. stephensi sequenced, but no such variation was observed in d1 to d3 domains of 28S rDNA. Cloning and sequencing of ITS2 through the d3 domain of the 28S region of rDNA from representative samples from northern, central and southern India confirmed the presence of intragenomic variation in ITS2. The variations are due to three transitions and two bp indel in a di-nucleotide microsatellite locus at fixed loci. Multiple haplotypes were observed in ITS2 raised from such variations. Due to the absence of detectable intragenomic sequence variation in the d1 to d3 domain of 28S rDNA of An. stephensi, this region can serve as an ideal reference sequence for taxonomic and phylogenetic studies. The presence of intragenomic variation in rDNA should be carefully examined before using this as a molecular marker for species delimitation or phylogenetic analyses.

Gastrointestinal Nematode Infections in Antelopes from Morocco: A Coprological Survey

This study examined the gastrointestinal parasitological status of three endangered Sub-Saharan antelope species (Addax nasomaculatus, Oryx dammah, Gazella dorcas) hosted at Souss-Massa National Park in Morocco. A total of 254 faecal samples (80 samples from the addax population, 81 from the oryx population and 93 from the dorcas population) were analysed to determine the prevalence and the intensity of the parasites in host faeces (expressed as the mean EPG: egg per gram), using microscopic methods (Flotation and McMaster) and the molecular identification of parasites using PCR and sequencing of the second internal transcribed spacer region of the rDNA (ITS-2). The prevalence results in the addax, oryx and dorcas gazelle were 43.7%, 2.4%, and 61.3%, respectively, for Nematodirus spp.; 21.2%, 12.3%, and 16.13%, respectively, for Trichuris spp.; and 36.2%, 39.5%, and 53.7%, respectively, for other, undistinguished strongylids. The means of EPG values for parasites in addax, oryx and dorcas gazelle were 8.9, 2.4, and 61.3, respectively, for Nematodirus spp.; 4.3, 2.4, and 4.8, respectively, for Trichuris spp.; and 18.1, 16.6, and 50.1, respectively, for other undistinguished strongylids. Sequencing of the ITS-2 rDNA region of the isolated parasites allowed the identification of Camelostrongylus mentulatus and Nematodirus spathiger in these three antelope species. We can conclude that the studied antelopes are infected at tolerating levels with the first record of Camelostrongylus mentulatus and Nematodirus spathiger in those antelopes in Morocco.

Coprological survey of protostrongylid infections in antelopes from Souss-Massa National Park (Morocco)

SummaryProtostrongylids, small nematode lungworms, are an integral part of the wild ruminant helminth community, which can damage animals’ health when they are held in captivity or semi-captive conditions. The Sahelo-Saharan antelope species dorcas gazelle (Gazella dorcas), the scimitar-horned oryx (Oryx dammah), and the addax (Addax nasomacculatus), reintroduced to Souss-Massa National Park in Morocco, could be host to many species of Protostrongylids. This study was conducted from January to July 2015 to identify infecting parasite species, and determine their prevalence and abundance in all three antelope species. A total of 180 individual fecal samples were collected, morphologically examined by the Baermann technique, and molecularly identified by PCR amplification and sequencing of the second internal transcribed spacer region of the rDNA (ITS-2).Two parasite species were found in the three antelope populations: Muellerius capillaris and Neostrongylus linearis. The prevalence scores recorded for M. capillaris were 98.40 % in the addax, 96.70 % in dorcas gazelle, and 28.40 % in the oryx. The prevalence rates of N. linearis were 60 % in the addax, 23.40 % in dorcas gazelle, and 90 % in the oryx. Excreted larvae were quantified by LPG (larvae per gram) counting: for M. capillaris, the LPG mean values were 92.94 in the addax, 133.09 in dorcas gazelle, and 1.48 in the oryx; and for N. linearis, the LPG mean values were 6.02 in the addax, 1.37 in dorcas gazelle, and 32.81 in the oryx. These findings indicate that the three species of antelopes are infected with Muellerius capillaris and Neostrongylus linearis to varying degrees in intensity and prevalence.

Phylogenetic Relationships within the Nematode Subfamily Phascolostrongylinae (Nematoda: Strongyloidea) from Australian Macropodid and Vombatid Marsupials

Background: The strongyloid nematode subfamily Phascolostrongylinae are parasites of the large intestine and stomach of Australian macropods (Macropodidae) and wombats (Vombatidae). Based on morphological classifications, the Phascolostrongylinae is comprised of seven genera belonging to three tribes (Phascolostrongylinea, Macropostrongyloidinea, and Hypodontinea). The phylogenetic relationships among the genera of the Phascolostrongylinae were tested using the first and second internal transcribed spacer (ITS+) sequences of the ribosomal DNA. Results: Monophyly was encountered in the tribe Phascolostrongylinea comprising two genera, Phascolostrongylus and Oesophagostomoides, found exclusively in the large intestine of wombats. The tribe Hypodontinea, represented by the genera Hypodontus and Macropicola from the ileum and large intestine of macropods was also found to be monophyletic, but with low support. The tribe Macropostrongyloidinea comprising the genera Macropostrongyloides and Paramacropostrongylus was paraphyletic with the species occurring in the stomach grouping separately to those found in the large intestines of their hosts. Finally, Macropostrongyloides dissimilis from the stomach of the swamp wallaby and Paramacropostrongylus toraliformis from the large intestine of the eastern grey kangaroo were distinct from their respective congeners. Conclusion: The current study provided strong support for the generic composition of the tribe Phascolostrongylinea and low support for the tribe Hypodontinea. However, the relationships within the tribe Macropostrongyloidinea are more complex and its monophyly was not supported by the current ITS+ dataset. The unexpected finding of M. dissimilis and P. toraliformis being distantly related to their respective congeners suggests a requirement for future taxonomic revision which may warrant separation of these species at the generic level.

An Assessment of the Molecular Diversity of Ticks and Tick-Borne Microorganisms of Small Ruminants in Pakistan

This study investigated ticks and tick-borne microorganisms of small ruminants from five districts of the Federally Administered Tribal Area (FATA) of Pakistan. Morphological (n = 104) and molecular (n = 54) characterization of the ticks revealed the presence of six ixodid ticks: Rhipicephalus (Rh.) haemaphysaloides, Rh. microplus, Rh. turanicus, Haemaphysalis (Hs.) punctata, Hs. sulcata and Hyalomma anatolicum. Phylogenetic analyses of nucleotide sequence data for two mitochondrial (16S and cytochrome c oxidase 1) and one nuclear (second internal transcribed spacer) DNA regions provided strong support for the grouping of the six tick species identified in this study. Microfluidic real-time PCR, employing multiple pre-validated nuclear and mitochondrial genetic markers, detected 11 potential pathogens and endosymbionts in 72.2% of the ticks (n = 54) tested. Rickettsia (R.) massiliae was the most common pathogen found (42.6% of ticks) followed by Theileria spp. (33.3%), Anaplasma (A.) ovis and R. slovaca (25.9% each). Anaplasma centrale, A. marginale, Ehrlichia spp., R. aeschlimannii, R. conorii and endosymbionts (Francisella- and Coxiella-like) were detected at much lower rates (1.9–22.2%) in ticks. Ticks from goats (83.9%) carried significantly higher microorganisms than those from sheep (56.5%). This study demonstrates that ticks of small ruminants from the FATA are carrying multiple microorganisms of veterinary and medical health significance and provides the basis for future investigations of ticks and tick-borne diseases of animals and humans in this and neighboring regions.