Unique among high passes: Insights into the genetic uniqueness among butterflies of Ladakh Trans-Himalaya through DNA barcoding.

BACKGROUND: The butterfly assemblage of Ladakh Trans-Himalaya demands a thorough analysis of their population genetic structure owing to their typical biogeographic affinity and their adaptability to extreme cold-desert climates. No such effort has been taken till date, and in this backdrop, we created a COI barcode reference library of 60 specimens representing 23 species. METHODS AND RESULTS: Barcodes were generated from freshly collected leg samples using the Sanger sequencing method, followed by phylogenetic clade analyses and divergence calculation. Our data represents 22% of Ladakh's Rhopaloceran fauna with the novel barcode submission for six species, including one Schedule II species, Paralasa mani. Contrary to the 3% threshold rule, the interspecific divergence between two species pairs of typical mountain genus Hyponephele and Karanasa was found to be 2.3% and 2.2%, respectively. The addition of conspecific global barcodes revealed that most species showed little increase in divergence value, while a two-fold increase was noted in a few species. Bayesian clade clustering outcomes largely aligned with current morphological classifications, forming monophyletic clades of conspecific barcodes, with only minor exceptions observed for the taxonomically complicated genus Polyommatus and misidentified records of Aulocera in the database. We also observed variations within the same phylogenetic clades forming nested lineages, which may be attributed to the taxonomic intricacies present at the subspecies level globally, mostly among Eurasian species. CONCLUSIONS: Overall, our effort not only substantiated the effectiveness of DNA Barcoding for the identification and conservation of this climatically vulnerable assemblage but also highlighted the significance of deciphering the unique genetic composition among this geographically isolated population of Ladakh butterflies.

Shark shuffle: segregated co-occurrence of multiple dusky and sandbar lineages at a human-altered habitat in the eastern Mediterranean Sea.

Requiem sharks (genus Carcharhinus) have previously been reported to form large aggregations around marine infrastructures in the eastern Mediterranean Sea. While this behaviour may offer fitness advantages at the individual level, the implications of extended residency at human-altered habitats for population persistence have yet to be assessed. In this work, we investigated the phylogeographic and demographic composition of sharks near a coal-fired power and desalination station in Israel. Our aim was to infer habitat use and the mechanisms underlying the aggregation behaviour, and to highlight potential conservation impacts. We sampled, measured, and released 70 individuals between 2016 and 2022 to assess genetic variability within the cytochrome C oxidase I (COI) region and to analyse the aggregation's structure based on the sharks' size and sex distribution. In addition, we performed meristic counts on a reference specimen collected dead at another power station in Israel to supplement species identification using the abovementioned techniques. Our findings indicate size-based sex segregation of adult female dusky and male sandbar sharks (Carcharhinus obscurus and Carcharhinus plumbeus, respectively), with each species comprising two COI haplotypes. In the dusky shark, one haplotype corresponded to an Indo-Pacific lineage, and the other matched an Atlantic lineage. In the sandbar shark, we observed a haplotype previously sampled in the Mediterranean Sea, the Red Sea, the Northwest Indian Ocean, and South Africa, and another haplotype that was unique to our study site and genetically closer to the former than to sequences sampled in other ocean basins. This study provides the first indication of sympatric aggregation amongst phylogeographically distinct dusky and sandbar shark lineages, suggesting that human-altered habitats in the eastern Mediterranean Sea may influence the distribution of these species. Based on the observed segregation pattern, we conclude that the site does not function as a nursery, parturition, or mating area, and discuss other plausible explanations that warrant further research. Finally, we highlight important directions for future research and the implications of our findings for management and conservation.

[Molecular tracing of Biomphalaria straminea in China].

OBJECTIVE: To investigate the origin of Biomphalaria straminea in China, so as to provide insights into assessment of schistosomiasis mansoni transmission risk and B. straminea control. METHODS: Guanlan River, Dasha River, Shenzhen Reservoir, upper and lower reaches of Kuiyong River, and Xinzhen River in Shenzhen, China, were selected as sampling sites. Ten Biomphalaria samples were collected from each site, and genomic DNA was extracted from Biomphalaria samples. DNA samples were obtained from 15 B. straminea sampled from 5 sampling sites in Minas Gerais State, Pará State, Federal District, Pernambuco State, and Sao Paulo State in Brazil, South America. Cytochrome c oxidase I (COI) and mitochondrial 16S ribosomal RNA (16S rRNA) genes were sampled using the above DNA templates, and the amplified products were sequenced. The COI and 16S rRNA gene sequences were downloaded from GenBank, and the sampling sites were acquired. All COI and 16S rRNA gene sequences were aligned and evolutionary trees of B. straminea were created based on COI and 16S rRNA gene sequences to identify the genetic similarity and evolutionary relationship between B. straminea samples from China and South America. RESULTS: A total of 60 COI gene sequences with a length of 529 bp and 3 haplotypes were obtained from B. straminea sampled from China. There were 165 COI gene sequences of B. straminea retrieved from GenBank, and following alignment with the above 60 gene sequences, a total of 33 haplotypes were obtained. Phylogenetic analysis showed that the three haplotypes of B. straminea from China were clustered into one clade, among which the haplotype China11 and three B. straminea samples from Brazil retrieved from GenBank belonged to the same haplotype. Geographical evolution analysis showed that the B. straminea samples from three sampling sites along eastern coasts of Brazil had the same haplotype with China11, and B. straminea samples from other two sampling sites were closely, genetically related to China11. A total of 60 16S rDNA gene sequences with approximately 322 bp in length were amplified from B. straminea in China, with 2 haplotypes identified. A total of 70 16S rDNA gene sequences of B. straminea were captured from GenBank. Phylogenetic analysis showed that Biomphalaria snails collected from China were clustered into a clade, and the haplotype China64 and the haplotype 229BS from Brazil shared the same haplotype. The 49 16S rDNA gene sequences of B. straminea from 25 sampling sites in southern Brazil, which were captured from GenBank, were included in the present analysis, and the B. straminea from 3 sampling sites shared the same haplotype with China64 in China. Geographical evolution analysis based on COI and 16S rRNA gene sequences showed that B. straminea sampled from eastern coastal areas of Brazil shared the same haplotypes in two gene fragment sequences with Biomphalaria snails collected from China. CONCLUSIONS: The Biomphalaria snails in China are characterized as B. straminea, which have a low genetic diversity. The Biomphalaria snails in China have a high genetic similarity with B. straminea sampled from eastern coastal areas of Brazil, which may have originated from the eastern coastal areas of Brazil.

Genetic Diversity of Rhipicephalus (Boophilus) microplus for a Global Scenario: A Comprehensive Review.

Rhipicephalus microplus poses a substantial threat to livestock health and agricultural economies worldwide. Its remarkable adaptability to diverse environments and hosts is a testament to its extensive genetic diversity. This review delves into the genetic diversity of R. microplus, employing three pivotal genetic markers: the cytochrome c oxidase I (COX1) gene, ribosomal genes, and microsatellites. The COX1 gene, a crucial tool for genetic characterization and phylogenetic clustering, provides insights into the adaptability of ticks. Ribosomal genes, such as internal transcribed spacer regions (ITS-1 and2) as well as 18S and 28S, are routinely utilized for species differentiation. However, their use is limited due to indels (insertions and deletions). Microsatellites and minisatellites, known for their high polymorphism, have been successfully employed to study populations and genetic diversity across various tick species. Despite their effectiveness, challenges such as null alleles and marker variations warrant careful consideration. Bm86, a well-studied vaccine candidate, exhibits substantial genetic diversity. This diversity directly influences vaccine efficacy, posing challenges for developing a universally effective Bm86-based vaccine. Moreover, the review emphasizes the prevalence of genes associated with synthetic pyrethroid resistance. Identifying single nucleotide polymorphisms in the acaricide-resistant genes of R. microplus has facilitated the development of molecular markers for detecting and monitoring resistance against synthetic pyrethroids. However, mutations in sodium channels, the target site for synthetic pyrethroid, correlate well with the resistance status of R. microplus, which is not the case with other acaricide target genes. This study underscores the importance of understanding genetic diversity in developing effective tick management strategies. The choice of genetic marker should be tailored based on the level of taxonomic resolution and the group of ticks under investigation. A holistic approach combining multiple markers and integrating additional molecular and morphological data may offer a more comprehensive understanding of tick diversity and relationships. This research has far-reaching implications in formulating breeding programs and the development of vaccine against ticks and tick-borne diseases (TTBDs) as well as strategies for the management of resistant ticks.

Preliminary study on buffy coat smear and molecular detection of microfilaria in domestic chickens (Gallus gallus domesticus) raised in Southern Thailand.

Background and Aim: Filarial nematode typically produces a larval stage (microfilariae) in the bloodstream of vertebrate hosts, where microfilariae reside in the blood or subcutaneous tissues. Filarial nematodes cause human diseases, such as river blindness and elephantiasis, which are widely studied. However, in avian species, they are overlooked because they are nonpathogenic. In Thailand, microfilaria can be found in wild birds and domestic chickens. Recently, an increase in the number of blood samples submitted to veterinary diagnostic laboratories may have increased the number of microfilariae. Therefore, knowledge about filarial species and reliable detection methods are important. Therefore, this study aimed to investigate the efficacy of buffy coat smear and polymerase chain reaction (PCR)-based methods for the detection of microfilaria in domestic chickens. In addition, parasites were identified using the sequence of the cytochrome c oxidase subunit 1 (COX1) gene. Materials and Methods: Giemsa-stained buffy coat smears from a previous study were reanalyzed. These available buffy coat smears were prepared from 55 domestic chickens raised as backyard free-ranging in Southern Thailand. Fifty-seven frozen genomic DNA extracted from chicken blood were used to detect the presence of the COX1 gene in Onchocercidae nematodes. The nested PCR protocol for amplification of the OnchoCOI_R2-OnchoCOI_R2 fragment of the COX1 gene was applied from a previous report. Sequences of COX1 were analyzed to identify Onchocercidae nematodes and if they were single or mixed infections. We constructed Bayesian phylogenetics to identify parasites and assessment of the relationship between filarial nematodes in avian species and other vertebrate hosts. Results: Buffy coat smears from 15 samples revealed microfilaria. Of these 15 samples, only eight were positive for COX1 nested-PCR amplification. The other two buffy coat-negative samples were also positive for nested-PCR. Sequencing of these 11 nested PCR-positive samples revealed that almost all of them were Onchocercidae nematodes. Bayesian phylogenetic analysis showed that chicken Onchocercidae spp. were grouped with other avian filarial nematodes. However, all chickens Onchocercidae spp. showed a double peak in the sequencing chromatogram, indicating mixed filarial infection (species or haplotypes). Therefore, no chicken Onchocercidae sequence was deposited on National Center for Biotechnology Information, GenBank. Conclusion: Giemsa-stained buffy coat smear was a reliable method for the detection of chicken microfilaria in routine veterinary diagnostic laboratories. Development of a new PCR-based method is necessary. This method may provide greater sensitivity and specificity of detection. In addition, the PCR method allowed us to access the genetic characteristics of nematodes, which helped us maximize our knowledge of nematodes. Further investigations, such as the pathogenicity of filarial nematodes in chickens and their potential vectors, are required.

Environmental DNA-based biodiversity profiling along the Houdong River in north-eastern Taiwan.

Background: This paper describes two datasets: species occurrences, which were determined by environmental DNA (eDNA) metabarcoding and their associated DNA sequences, originating from a research project which was carried out along the Houdong River (), Jiaoxi Township, Yilan, Taiwan. The Houdong River begins at an elevation of 860 m and flows for approximately 9 km before it empties into the Pacific Ocean. Meandering through mountains, hills, plains and alluvial valleys, this short river system is representative of the fluvial systems in Taiwan. The primary objective of this study was to determine eukaryotic species occurrences in the riverine ecosystem through the use of the eDNA analysis. The second goal was, based on the current dataset, to establish a metabarcoding eDNA data template that will be useful and replicable for all users, particularly the Taiwan community. The species occurrence data are accessible at the Global Biodiversity Information Facility (GBIF) portal and its associated DNA sequences have been deposited in the European Nucleotide Archive (ENA) at EMBL-EBI, respectively. A total of 12 water samples from the study yielded an average of 1.5 million reads. The subsequent species identification from the collected samples resulted in the classification of 432 Operational Taxonomic Units (OTUs) out of a total of 2,734. Furthermore, a total of 1,356 occurrences with taxon matches in GBIF were documented (excluding 4,941 incertae sedis, accessed 05-12-2023). These data will be of substantial importance for future species and habitat monitoring within the short river, such as assessment of biodiversity patterns across different elevations, zonations and time periods and its correlation to water quality, land uses and anthropogenic activities. Further, these datasets will be of importance for regional ecological studies, in particular the freshwater ecosystem and its status in the current global change scenarios. New information: The datasets are the first species diversity description of the Houdong River system using either eDNA or traditional monitoring processes.

A DNA barcode reference library of Croatian mosquitoes (Diptera: Culicidae): implications for identification and delimitation of species, with notes on the distribution of potential vector species.

BACKGROUND: Mosquitoes pose a risk to human health worldwide, and correct species identification and detection of cryptic species are the most important keys for surveillance and control of mosquito vectors. In addition to traditional identification based on morphology, DNA barcoding has recently been widely used as a complementary tool for reliable identification of mosquito species. The main objective of this study was to create a reference DNA barcode library for the Croatian mosquito fauna, which should contribute to more accurate and faster identification of species, including cryptic species, and recognition of relevant vector species. METHODS: Sampling was carried out in three biogeographical regions of Croatia over six years (2017-2022). The mosquitoes were morphologically identified; molecular identification was based on the standard barcoding region of the mitochondrial COI gene and the nuclear ITS2 region, the latter to identify species within the Anopheles maculipennis complex. The BIN-RESL algorithm assigned the COI sequences to the corresponding BINs (Barcode Index Number clusters) in BOLD, i.e. to putative MOTUs (Molecular Operational Taxonomic Units). The bPTP and ASAP species delimitation methods were applied to the genus datasets in order to verify/confirm the assignment of specimens to specific MOTUs. RESULTS: A total of 405 mosquito specimens belonging to six genera and 30 morphospecies were collected and processed. Species delimitation methods assigned the samples to 31 (BIN-RESL), 30 (bPTP) and 28 (ASAP) MOTUs, with most delimited MOTUs matching the morphological identification. Some species of the genera Culex, Aedes and Anopheles were assigned to the same MOTUs, especially species that are difficult to distinguish morphologically and/or represent species complexes. In total, COI barcode sequences for 34 mosquito species and ITS2 sequences for three species of the genus Anopheles were added to the mosquito sequence database for Croatia, including one individual from the Intrudens Group, which represents a new record for the Croatian mosquito fauna. CONCLUSION: We present the results of the first comprehensive study combining morphological and molecular identification of most mosquito species present in Croatia, including several invasive and vector species. With the exception of some closely related species, this study confirmed that DNA barcoding based on COI provides a reliable basis for the identification of mosquito species in Croatia.

Isospora and Lankesterella Parasites (Eimeriidae, Apicomplexa) of Passeriform Birds in Europe: Infection Rates, Phylogeny, and Pathogenicity.

Wild birds are common hosts to numerous intracellular parasites such as single-celled eukaryotes of the family Eimeriidae (order Eucoccidiorida, phylum Apicomplexa). We investigated the infection rates, phylogeny, and pathogenicity of Isospora and Lankesterella parasites in wild and captive passerine birds. Blood and tissue samples of 815 wild and 15 deceased captive birds from Europe were tested using polymerase chain reaction and partial sequencing of the mitochondrial cytochrome b and cytochrome c oxidase I and the nuclear 18S rRNA gene. The infection rate for Lankesterella in wild birds was 10.7% compared to 5.8% for Isospora. Chromogenic in situ hybridization with probes targeting the parasites' 18S rRNA was employed to identify the parasites' presence in multiple organs, and hematoxylin-eosin staining was performed to visualize the parasite stages and assess associated lesions. Isospora parasites were mainly identified in the intestine, spleen, and liver. Extraintestinal tissue stages of Isospora were accompanied by predominantly lymphohistiocytic inflammation of varying severity. Lankesterella was most frequently detected in the spleen, lung, and brain; however, infected birds presented only a low parasite burden without associated pathological changes. These findings contribute to our understanding of Isospora and Lankesterella parasites in wild birds.

Relationship between genetic diversity and morpho-functional characteristics of flight-related traits in Triatoma garciabesi (Hemiptera: Reduviidae).

BACKGROUND: Triatoma garciabesi, a potential vector of the parasitic protozoan Trypanosoma cruzi, which is the causative agent of Chagas disease, is common in peridomestic and wild environments and found throughout northwestern and central Argentina, western Paraguay and the Bolivian Chaco. Genetic differentiation of a species across its range can help to understand dispersal patterns and connectivity between habitats. Dispersal by flight is considered to be the main active dispersal strategy used by triatomines. In particular, the morphological structure of the hemelytra is associated with their function. The aim of this study was to understand how genetic diversity is structured, how morphological variation of dispersal-related traits varies with genetic diversity and how the morphological characteristics of dispersal-related traits may explain the current distribution of genetic lineages in this species. METHODS: Males from 24 populations of T. garciabesi across its distribution range were examined. The cytochrome c oxidase I gene (coI) was used for genetic diversity analyses. A geometric morphometric method based on landmarks was used for morpho-functional analysis of the hemelytra. Centroid size (CS) and shape of the forewing, and contour of both parts of the forewing, the head and the pronotum were characterised. Length and area of the forewing were measured to estimate the aspect ratio. RESULTS: The morphometric and phylogenetic analysis identified two distinct lineages, namely the Eastern and Western lineages, which coincide with different ecological regions. The Eastern lineage is found exclusively in the eastern region of Argentina (Chaco and Formosa provinces), whereas the Western lineage is prevalent in the rest of the geographical range of the species. CS, shape and aspect ratio of the hemelytra differed between lineages. The stiff portion of the forewing was more developed in the Eastern lineage. The shape of both portions of the hemelytra were significantly different between lineages, and the shape of the head and pronotum differed between lineages. CONCLUSIONS: The results provide preliminary insights into the evolution and diversification of T. garciabesi. Variation in the forewing, pronotum and head is congruent with genetic divergence. Consistent with genetic divergence, morphometry variation was clustered according to lineages, with congruent variation in the size and shape of the forewing, pronotum and head.

Cryptic Diversity in Sympatric Migonemyia migonei (Diptera: Psychodidae), Eventual Meaning for Leishmaniasis Transmission.

Migonemyia migonei (FranÒ«a, 1920) (Diptera: Psychodidae) belongs to the subfamily Phlebotominae, of epidemiological importance due to its role as a vector in leishmaniasis transmission cycles and its broad geographic distribution in South America. Few morphometric and genetic studies have demonstrated the existence of variability among geographically distant populations in Brazil. The aim of the study was to estimate the genetic distance within the morphospecies Mg. migonei through the analysis of cytochrome C oxidase subunit I (COI) sequences of specimens captured in Argentina and those available in online databases. The COI sequences from specimens collected in different localities of Argentina and sequences available in online databases were utilized. Genetic distances were analyzed and a median-joining haplotype network was constructed. Finally, phylogenetic reconstruction was performed according to Bayesian inference. The analyses led to the identification of at least two haplogroups: haplogroup I with sequences of specimens from Colombia, Brazil and Argentina, and haplogroup II with sequences of specimens from Argentina. Interestingly, specimens from Argentina whose haplotypes corresponded to both haplogroups, were collected in sympatry. The results suggest that Mg. migonei could be a species complex with at least two distinct members. This hypothesis could explain the known characteristics of adaptability and vector permissiveness of the species, as the putative cryptic species of the complex could differ in traits of epidemiological importance.

Turcinoemacheilus ekmekciae, a new dwarf loach from upper Tigris and Euphrates (Teleostei: Nemacheilidae).

Turcinoemacheilus ekmekciae, new species, from upper Euphrates and Tigris drainages is distinguished from other species of Turcinoemacheilus in Western Asia by having a dark stripe broader than the eye diameter along the lateral line, rarely possessing roundish blotches, 5-6 mandibular pores in mandibular canal, a comperatvely smaller head, a deeper body, and a greater pre-pelvic distance. Our specimens collected from the upper Great Zab, near the type locality of Turcinoemacheilus kosswigi, showed notable genetic divergence (a minimum K2P of 3.3%) from sequences reported as T. kosswigi in previous studies. Despite morphological similarities, this molecular difference suggests that the populations analysed in previous studies may represent a potential new species of Turcinoemacheilus, which we tentatively named as Turcinoemacheius cf. kosswigi. Molecular data also suggest that T. ekmekciae is characterized by a minimum K2P distance of 3.5% from Turcinoemacheilus minimus and T. cf. kosswigi. The three methods for species delimitation (assemble species by automatic partitioning [ASAP], Poisson tree processes [PTP], and multi-rate PTP [mPTP]) that were utilized for testing species assignments consistently identified our test group as a distinct species.

Molecular tracing of Biomphalaria straminea in China / 中国血吸虫病防治杂志

Objective To investigate the origin of Biomphalaria straminea in China, so as to provide insights into assessment of schistosomiasis mansoni transmission risk and B. straminea control. Methods Guanlan River, Dasha River, Shenzhen Reservoir, upper and lower reaches of Kuiyong River, and Xinzhen River in Shenzhen, China, were selected as sampling sites. Ten Biomphalaria samples were collected from each site, and genomic DNA was extracted from Biomphalaria samples. DNA samples were obtained from 15 B. straminea sampled from 5 sampling sites in Minas Gerais State, Pará State, Federal District, Pernambuco State, and Sao Paulo State in Brazil, South America. Cytochrome c oxidase I (COI) and mitochondrial 16S ribosomal RNA (16S rRNA) genes were sampled using the above DNA templates, and the amplified products were sequenced. The COI and 16S rRNA gene sequences were downloaded from GenBank, and the sampling sites were acquired. All COI and 16S rRNA gene sequences were aligned and evolutionary trees of B. straminea were created based on COI and 16S rRNA gene sequences to identify the genetic similarity and evolutionary relationship between B. straminea samples from China and South America. Results A total of 60 COI gene sequences with a length of 529 bp and 3 haplotypes were obtained from B. straminea sampled from China. There were 165 COI gene sequences of B. straminea retrieved from GenBank, and following alignment with the above 60 gene sequences, a total of 33 haplotypes were obtained. Phylogenetic analysis showed that the three haplotypes of B. straminea from China were clustered into one clade, among which the haplotype China11 and three B. straminea samples from Brazil retrieved from GenBank belonged to the same haplotype. Geographical evolution analysis showed that the B. straminea samples from three sampling sites along eastern coasts of Brazil had the same haplotype with China11, and B. straminea samples from other two sampling sites were closely, genetically related to China11. A total of 60 16S rDNA gene sequences with approximately 322 bp in length were amplified from B. straminea in China, with 2 haplotypes identified. A total of 70 16S rDNA gene sequences of B. straminea were captured from GenBank. Phylogenetic analysis showed that Biomphalaria snails collected from China were clustered into a clade, and the haplotype China64 and the haplotype 229BS from Brazil shared the same haplotype. The 49 16S rDNA gene sequences of B. straminea from 25 sampling sites in southern Brazil, which were captured from GenBank, were included in the present analysis, and the B. straminea from 3 sampling sites shared the same haplotype with China64 in China. Geographical evolution analysis based on COI and 16S rRNA gene sequences showed that B. straminea sampled from eastern coastal areas of Brazil shared the same haplotypes in two gene fragment sequences with Biomphalaria snails collected from China. Conclusions The Biomphalaria snails in China are characterized as B. straminea, which have a low genetic diversity. The Biomphalaria snails in China have a high genetic similarity with B. straminea sampled from eastern coastal areas of Brazil, which may have originated from the eastern coastal areas of Brazil.

First Study of Ascaris lumbricoides from the Semiwild Population of the Sumatran Orangutan Pongo abelii in the Context of Morphological Description and Molecular Phylogeny.

There is little evidence that the already described and accepted taxa of ascarids (Ascaris lumbricoides, A. suum, and A. ovis) infecting individuals of taxonomically distant groups (hominids, pigs, sheep, goats, and dogs) can be genetically or morphologically distinguished. However, despite described morphological differences, e.g., due to intraspecific variation, these are insufficient for species determination and may indicate differences amongst ascarids because of cross infections, hybrid production, and specific adaptations to hosts. Herein, the results of a molecular and morphological analysis of ascarids parasitising Sumatran orangutans (Pongo abelii Lesson, 1827) in native populations are presented. The research took place in the Bukit Lawang area, Indonesia, in 2009. Throughout the year, fresh faecal samples were collected regularly from 24 orangutans, and all were examined for the presence of nematode adults. Only five adult worms from two orangutan females were found during regular collection. Using the integrative taxonomic approach, the nematodes found were identified as A. lumbricoides. The significance of the find and its rarity is documented by the fact that this is the first confirmed finding of adult ascarids from an original orangutan site (not from a zoo) in more than 130 years (including the long-term study spanning the last 20 years focusing on orangutan parasites and natural antiparasitic drugs). More accurate morphometric parameters and genetic differences for the identification of ascarids were established. These parameters will be helpful for other findings in great apes and will also be suitable for further and precise determination of this parasite. The details distinguishing between male and female specimens are also stated and well defined. A comprehensive evaluation of the situation of Ascaris species parasitising orangutans, including a comparison with previously described orangutan parasite (i.e., A. satyri-species inquirenda), is discussed.

A One-Step Multiplex PCR Method to Rapidly Distinguish Two Strains of Diglyphus wani (Hymenoptera: Eulophidae) Against Agromyzid Leafminers.

Hymenopteran parasitoids generally show a haplo-diploid sex determination system. Haploid males are produced from unfertilized eggs, whereas diploid females develop from fertilized eggs (arrhenotokous). In some cases, diploid females develop from unfertilized eggs (thelytokous). Diglyphus wani (Hymenoptera: Eulophidae) is a biological control agent for agromyzid leafminers and have arrhenotokous and thelytokous strains. However, the morphological characteristics of two strains of D. wani are so similar that it is difficult to accurately distinguish them based on morphology. Here, a rapid molecular identification method was developed based on the mitochondrial gene cytochrome c oxidase I (COI) and one-step multiplex PCR. Two primer combinations, PC1 (Ar-F1/Th-F1/WR2) and PC2 (Ar-F1/Th-F4/WR2), were designed and repeatedly screened to distinguish two strains simultaneously, of which two special forward primers Th-F1/Th-F4 were used for the thelytokous strain and one special forward primer Ar-F1 was used for the arrhenotokous strain. In addition, a common reverse primer, WR2, was used for both strains. The PC1 and PC2 PCR assays were effective in distinguishing the two strains at different developmental stages and field colonies. This method provides a reliable, highly sensitive, and cost-effective tool for the rapid identification of the two strains of D. wani.

Resolving the phylogenetic relationship of Himalayan snow trout Schizothorax plagiostomus with other species of Schizothoracine using mitochondrial CO-I and Cyt b genes.

BACKGROUND: The classification of the sub-family Schizothoracinae has been debatable due to the overlap in morphological characters. There are discrepancies between classical taxonomy and molecular taxonomy, as well. In the present study, mitochondrial genes CO-I and Cyt b were sequenced to elucidate the phylogenetic status of three species of the genus Schizothorax. METHODS AND RESULTS: In total, 29 samples of three species viz., S. plagiostomus, S. progastus, and S. richardsonii, were collected from rivers of Uttarakhand, India. For phylogenetic analyses, 40 sequences of CO-I and 41 sequences of Cyt b of Schizothoracinae species were downloaded from NCBI. The highest genetic divergence based on CO-I (16.08%) is between S. plagiostomus and Ptychobarbus dipogon, while the lowest divergence (0.00%) is between 10 pairs of species. The highest divergence based on Cyt b (19.43%), is between S. niger and Gymnocypris eckloni, while the lowest divergence (0.00%) is between four pairs of species. The divergence (0.00% for CO-I and 2.38% for Cyt b) between S. chongi and S. kozlovi, seems a case of convergent molecular evolution of the CO-I gene and in this case, CO-I alone cannot be used to differentiate these two species. CONCLUSION: The simultaneous use of two molecular markers along with morphomeristic data is a better strategy for the classification of the sub-family Schizothoracinae. These results will be a resource dataset for determining the taxonomical status of Schizothoracine species and will help in the conservation and commercial production of these commercially important fish species.

A method for determining the origin of crude drugs derived from animals using MinION, a compact next-generation sequencer.

We evaluated whether MinION, an inexpensive portable sequencer, can be used to identify the origin of crude drugs derived from animals. Standard and nonstandard crude drugs with different species of origin were examined. In addition, standards mixed with nonstandard samples were used. As a target gene, cytochrome c oxidase I was amplified and sequenced. The fast mode results had a slightly lower match ratio than high-accuracy mode, but the animals of origin were correctly determined by BLAST for all samples. For antler velvet derived from Rangifer tarandus, even when the sequences were aligned based on Cervus elaphus, the animal of origin was determined correctly. Minor contents could be detected from mixtures of two animals, if the mixtures contained at least 19:1 mtDNA when the coverage allele-fraction threshold was 0.05. By contrast, in fast mode, two sequences could not be separated due to the low accuracy of the base-calling for each read. For fieldwork, the species of origin of crude drugs could be identified with only simple DNA extraction and library preparation. Therefore, MinION appears to be a convenient tool for identifying the origins of crude drugs derived from animals.

Celastrol and Rhynchophylline in the mitigation of simulated muscle atrophy under in vitro.

Muscular atrophy (MA) is a disease of various origins, i.e., genetic or the most common, caused by mechanical injury. So far, there is no universal therapeutic model because this disease is often progressive with numerous manifested symptoms. Moreover, there is no safe and low-risk therapy dedicated to muscle atrophy. For this reason, our research focuses on finding an alternative method using natural compounds to treat MA. This study proposes implementing natural substances such as celastrol and Rhynchophylline on the cellular level, using a simulated and controlled atrophy process. Methods: Celastrol and Rhynchophylline were used as natural compounds against simulated atrophy in C2C12 cells. Skeletal muscle C2C12 cells were stimulated for the differentiation process. Atrophic conditions were obtained by the exposure to the low concertation of doxorubicin and validated by FoxO3 and MAFbx. The protective and regenerative effect of drugs on cell proliferation was determined by the MTT assay and MT-CO1, VDAC1, and prohibitin expression. Results: The obtained results revealed that both natural substances reduced atrophic symptoms. Rhynchophylline and celastrol attenuated atrophic cells in the viability studies, morphology analysis by diameter measurements, modulated prohibitin VDAC, and MT-CO1 expression. Conclusions: The obtained results revealed that celastrol and Rhynchophylline could be effectively used as a supportive treatment in atrophy-related disorders. Thus, natural drugs seem promising for muscle regeneration.

Molecular Identification of Commercial Fish Maws by DNA Sequencing of 16S rRNA and Cytochrome c Oxidase I Genes.

ABSTRACT: Fish maws (dried swim bladders) have long been used for medicinal tonics and as a valuable food resource in Southeast Asia. However, it is difficult to identify the original species of fish maws sold in markets due to a lack of taxonomic characteristics. In the present study, 37 kinds of commercial fish maws from various medicinal material markets were examined, and gene sequences were successfully obtained from ca. 95% of the samples. Partial sequences of the 16S rRNA gene and cytochrome c oxidase I (COI) gene were obtained and used to investigate the origin of these commercial fish maws. Thirty-five specimens belonged to nine species: five croakers and four noncroakers. All species identification was supported by both high homogeneity (98 to 100%) and clear clustering with low within-group Kimura two-parameter divergence scores (0 to 0.04 for 16S rRNA and 0 to 0.07 for COI) and high between-group divergence scores (0.07 to 0.15 for 16S rRNA and 0.11 to 0.24 for COI). Croakers were the predominant species, accounting for 74% of the total fish maw specimens. The large demand for croakers has put some species at the risk of extinction due to overfishing. As a valuable food, fish maw has progressively become more popular and has been used as a substitute for shark fin. The identification results allowed us to learn more about the fish species available on the fish maw market and provided an indicator for possible control of threatened or endangered fish species. A probable correlation between the molecular characteristics and morphological features of fish maws was also found and could provide both consumers and merchants with an important reference for identifying the origin of fish maws.

A DNA barcode survey of insect biodiversity in Pakistan.

Although Pakistan has rich biodiversity, many groups are poorly known, particularly insects. To address this gap, we employed DNA barcoding to survey its insect diversity. Specimens obtained through diverse collecting methods at 1,858 sites across Pakistan from 2010-2019 were examined for sequence variation in the 658 bp barcode region of the cytochrome c oxidase 1 (COI) gene. Sequences from nearly 49,000 specimens were assigned to 6,590 Barcode Index Numbers (BINs), a proxy for species, and most (88%) also possessed a representative image on the Barcode of Life Data System (BOLD). By coupling morphological inspections with barcode matches on BOLD, every BIN was assigned to an order (19) and most (99.8%) were placed to a family (362). However, just 40% of the BINs were assigned to a genus (1,375) and 21% to a species (1,364). Five orders (Coleoptera, Diptera, Hemiptera, Hymenoptera, Lepidoptera) accounted for 92% of the specimens and BINs. More than half of the BINs (59%) are so far only known from Pakistan, but others have also been reported from Bangladesh (13%), India (12%), and China (8%). Representing the first DNA barcode survey of the insect fauna in any South Asian country, this study provides the foundation for a complete inventory of the insect fauna in Pakistan while also contributing to the global DNA barcode reference library.

Molecular identification of the potentially forensically relevant cluster flies Pollenia rudis (Fabricius) and Pollenia vagabunda (Meigen) (Diptera: Polleniidae) - non-recorded species in Algeria.

Cluster flies are represented by the genus Pollenia Robineau-Desvoidy, 1830 of the family Polleniidae Brauer and Bergenstamm, 1889. Their larvae are known to be internal parasites or predators of earthworms. Herein, we report for the first time the occurrence of the cluster flies Pollenia rudis Fabricius, 1794 and Pollenia vagabunda (Meigen, 1826) (Diptera: Polleniidae) on carcasses in Algeria and identify them through DNA barcoding. A region of the mitochondrial cytochrome c oxidase I gene (COI) was amplified and sequenced. Genetic distances were determined. A phylogenetic tree was constructed with the maximum parsimony method using 10 000 bootstrap replicates. A total number of 157 adults of P. rudis were collected together with 325 adults of Pollenia vagabunda. The occurrence of Pollenia on animal carcasses does not seem to be correlated with a particular stage of decomposition. All the sequences were correctly identified using the BLASTn tool from the GenBank database and the BOLD identification engine. Intra- and interspecific sequence divergence values were less than 1% and greater than 3%, respectively. COI barcodes obtained from this study were robust enough to identify and distinguish unambiguously between P. rudis and P. vagabunda. In the tree-based analysis, the cluster flies were all assigned to their respective species separately from each other confirming the morphological identification. These results provide DNA barcodes that contribute to the growth of reference databases and allow fast and accurate identification.