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Kyasanur Forest Disease (KFD), also known as Monkey Fever, is an important tick-borne viral infection in India. It is mainly transmitted by the hard tick vector, Haemaphysalis spinigera . This re-emerging viral disease in humans has a mortality rate of 2–10% and was first reported in the Shimoga district of Karnataka in 1957. In recent years, KFD has been reported from various parts of the country, indicating possible geographical expansion and wider distribution of the tick vector. Assessing the distinct populations of H. spinigera and their local adaptations is important. However, the genetic structure of this vector population remains undocumented due to the lack of well-characterized genetic markers. Although the distribution of H. spinigera has been described, information on its genetic diversity and population structure is still limited. In this study, we characterized and validated a mitochondrial marker, 12S rRNA for assessing the genetic diversity of H. spinigera in Eastern and Western Ghats of India. Analysis of ninety individual ticks revealed a genetically diverse population comprising three haplotypes, which formed three distinct clades. These clades included haplotypes from nine geographical populations. Understanding the genetic structure of tick vectors is crucial for studying the transmission dynamics of tick-borne diseases. Our findings suggest that 12S rRNA is a suitable genetic marker for studying intraspecific variation in H. spinigera . To the best of our knowledge, this is the first to report the population structure of H. spinigera , highlighting its genetic diversity in the Western and Eastern Ghats of India.

Background Rhipicephalus ( Boophilus ) microplus (Canestrini, 1888), the Asian blue tick, is a highly invasive and adaptable ectoparasite. This tick species has successfully established itself in most regions of the world, with movement of cattle being a major driver for its spread. In the recent past, R. microplus ticks have been reported in three districts of Uganda. Information on its spread and distribution are vital in deepening our understanding of the ecological scenarios that lead to tick persistence and in the formulation of control strategies. This is especially important in the cattle-dense districts. Methods We randomly collected tick specimens from 1,461cattle spread across seven cattle dense districts located in the Central, Karamoja and West Nile regions of Uganda from January to September 2020. The ticks were identified using standard morpho-taxonomic keys and the R. microplus tick species identities were confirmed by sequencing of the ITS2 region, 12S rRNA and 16S rRNA genes and phylogenetic analyses. Results Adult ticks ( n = 13,019) were collected from 1,461 cattle. Seventeen tick species were identified based on morpho-taxonomic keys and the majority (47.4%; n =6184) of these were R. appendiculatus . In total, 257 R. microplus ticks were found infesting cattle in 18 study sites in the districts of Amudat, Kaabong, Napak (Karamoja region) and Arua (West Nile region). The identity of R. microplus was confirmed using molecular technics. No R. microplus tick was recorded in the districts of Lyantonde and Nakaseke (Central region). Arua district accounted for 82.1% ( n =211) of the R. microplus ticks recorded followed by Napak district at 16.3% ( n =42), while Amudat and Kaabong districts accounted for 1.5% ( n =4). Rhipicephalus microplus and R. decoloratus co-existed in 6 of the 13 study sites in Arua district, while in another 6 study sites, no R. decoloratus was recorded. In the Karamoja region districts R. decoloratus co-existed with R.microplus . Of the total 618 ticks belonging to four species of the subgenus Boophilus recorded in this study, R. decoloratus accounted for 50.04% ( n =334), followed by R. microplus at 41.58% ( n =257), R. geigyi at 2.75% ( n =17) and R. annulatus at 1.61% ( n =10). In the districts of Amudat, Kaabong and Napak, R. decoloratus was more dominant (76.1%; n =179) of the three Rhipicephalus ( Boophilus ) tick species recorded, followed by R. microplus (19.5%; n =46) and R. geigyi (4.2%; n =10). Contrariwise, R. microplus was more dominant (84%; n =211) in Arua district followed by R. decoloratus (10.7%; n =27), R. annulatus (3.9%; n =10) and R. geigyi (1.1%; n =3). Phylogenetic analyses of the ITS2 region, 12S rRNA and 16S rRNA genes revealed subgrouping of the obtained sequences with the previously published R. microplus sequences from other parts of the world. Conclusion Rhipicephalus microplus ticks were found infesting cattle in four districts of Uganda. The inability to find R. decoloratus , an indigenous tick, from six sites in the district of Arua is suggestive of its replacement by R. microplus . Rhipicephalus microplus negatively affects livestock production, and therefore, there is a need to determine its distribution and to deepen the understanding of the ecological factors that lead to its spread and persistence in an area.

Argas persicus (the fowl tick) is a species of soft tick commonly associated with poultry farms. It has a wide geographic distribution and colonizes different climate regions. Morphological identification of A. persicus has been reported worldwide, but genetic data regarding its molecular characterization is limited. The present study provides data for morphological identification and genetic characterization of A. persicus collected from domestic birds in traditional farms from east Algeria (Setif region). Additionally, A . persicus  samples originating from Gansu province in China were included for comparative molecular study. In total, 1518 ticks collected from 30 infested farms were examined and morphologically identified as A. persicus . Furthermore, the 14 tick samples obtained from China were morphologically identified as A. persicus. Molecular analysis of 30 ticks from Algeria (one tick from each infested farm) and the 14 Chinese samples based on PCR, sequencing, and phylogenetic analysis of three mitochondrial genetic markers (16S rRNA, 12S rRNA, and cox1) confirmed morphological results where all samples belonged to the A. persicus group. However, phylogenetic analysis showed that all Algerian samples and two Chinese samples belong to A. persicus sensu stricto (s.s.), while the remaining Chinese samples represented A. persicus sensu lato (s.l.) (divergent lineage). The present study confirms the occurrence of A. persicus s.s. both in Algeria and China, as well as provides novel molecular data for a distinct Chinese lineage of A. persicus .

The predator–prey-transmitted cestode Taenia hydatigena infects a wide range of definitive and intermediate hosts all over the world. Domestic and sylvatic cycles of transmission are considered as well. The parasite has considerable economic importance, particularly in sheep. Here, the molecular characters of T. hydatigena cysticerci in sheep from the Nile Delta, Egypt were investigated for the first time. For this purpose, 200 sheep carcasses and their offal were inspected at the municipal abattoir, Dakahlia governorate, Egypt. Cysticerci of T. hydatigena were collected and molecularly characterized employing the mitochondrial 12S rRNA gene. Cysticerci were found in 42 (21%) sheep, mostly attached to the omenti, mesenteries and livers. After molecular confirmation, nine isolates were sequenced displaying six different haplotypes. Analysis of the T. hydatigena 12S rRNA nucleotide sequences deposited in GenBank revealed 55 haplotypes out of 69 isolates, displaying high haplotype (0.797) and low nucleotide (0.00739) diversities. For the Tajima D neutrality index, a negative value (−2.702) was determined, indicating the population expansion of the parasite. Additionally, global data summarized in this study should be useful to set up effective control strategies against this ubiquitous parasite.

DNA barcoding is a powerful tool that provides rapid, accurate, and automatable species identification using standardized genetic region(s), such as for revealing the existence of cryptic species and/or rare species in biodiversity monitoring. DNA barcoding techniques require the development of sets of universal PCR primers for DNA barcoding. We tried to develop universal primer sets, and succeeded in designing not only universal primer sets for DNA barcoding regions of almost all insects, which were designed to include a hypervariable site between highly conserved sites, but also primer sets for longer fragment sequences for registration in a database. We confirmed successful amplification for 14 orders, 43 families, and 68 species with DNA barcoding in the mtDNA 16S rRNA region, and for 13 orders, 42 families, and 66 species with DNA barcoding in the mtDNA 12S rRNA region, including Apterygota and Pterygota (Paleoptera, Polyneoptera, Paraneoptera, and Oligoneoptera). A key feature is that the DNA fragments of the DNA barcoding regions amplified by these primer sets are both short at about 200-bp, and longer fragment sequences will increase the level of data registration in the DNA database. In addition, we evaluated the sensitivity of these newly developed primers using Epeorus aesculus (Heptageniidae), which inhabits a relatively wide range of river systems. The results of this study revealed the existence of a cryptic species or an undescribed species. Such resulting database enhancements will provide opportunities for increasingly accurate assessment of biodiversity and genetic diversity.

To successfully implement environmental DNA‐based (eDNA) diversity monitoring, the completeness and accuracy of reference databases used for taxonomic assignment of eDNA sequences are among the challenges to be tackled. Here, we have developed a workflow that evaluates the current status of GenBank for marine fishes. For a given combination of species and barcodes, a gap analysis is performed and potentially erroneous sequences are identified. Our gap analysis based on the four most used genes (cytochrome c oxidase subunit 1, 12S rRNA, 16S rRNA, and cytochrome b) for fish eDNA metabarcoding found that COI, the universal choice for metazoans, is the gene covering the highest number of Northeast Atlantic marine fishes (70%), while 12S rRNA, the preferred region for fish‐targeting studies, only covers about 50% of the species. The presence of too close and too distant barcode sequences as expected by their taxonomic classification confirms the existence of erroneous sequences in GenBank that our workflow can detect and eliminate. Comparing taxonomic assignments of real marine eDNA samples with raw and clean reference databases for the most used 12S rRNA barcodes (teleo and MiFish), we confirmed that both barcodes perform differently and demonstrated that the application of the database cleaning workflow can result in drastic changes in community composition. Besides providing a tool for reference database curation, this study confirms the need to increase 12S rRNA reference sequences for European marine fishes and evidences the dangers of taxonomic assignments by directly querying GenBank. We have developed a workflow that evaluates the current status of GenBank for marine fishes. For a given combination of species and barcodes, a gap analysis is performed and potentially erroneous sequences are identified. We have developed a workflow that evaluates the current status of GenBank for marine fishes. For a given combination of species and barcodes, a gap analysis is performed and potentially erroneous sequences are identified. Besides providing a tool for reference database curation, this study confirms the 14 need to increase 12S rRNA reference sequences for European marine fishes and evidences the dangers of taxonomic 15 assignments by directly querying GenBank.

The study of ichthyoplankton is paramount to understanding fish assemblages' reproductive dynamics. DNA metabarcoding has been applied as a rapid, cost‐effective, and accurate taxonomy tool, allowing the identification of multiple individuals simultaneously. However, there remain significant challenges when using DNA metabarcoding, such as molecular marker choice according to the taxonomic resolution and length of the fragment to be sequenced, primer bias, incomplete reference databases, and qualitative inference incongruences. Here, 30 ichthyoplankton pools collected from a Neotropical river were identified at a molecular level using DNA metabarcoding to compare the resolution, sensibility, specificity, and relative read abundance (RRA) recovery of three molecular markers: the standard COI fragment (650 pb, with each end analyzed individually) and two short 12S rRNA genes markers (≅200 bp – NeoFish and MiFish markers). The combined use of the three markers increased the genera detection rates by 25%–87.5%, allowing an increased taxonomic coverage and robust taxonomic identification of complex Neotropical ichthyoplankton communities. RRA is marker‐dependent, indicating caution is still needed while inferring species abundance based on DNA metabarcoding data when using PCR‐dependent protocols.

This study aimed to report the presence of Mesocestoides litteratus in dogs adopted from shelters in Türkiye. Gravid segments were examined microscopically in the faeces of dogs from different shelters located in Ankara and Kirikkale provinces in the central region of Türkiye. Then, genomic DNA obtained from these segments, a 446-bp fragment of the mitochondrial cytochrome C oxidase subunit 1 gene, and a 350-bp fragment of mitochondrial 12S rRNA were amplified and sequenced. BLASTn search was performed. During light microscopic examination, an egg-filled paruterine organ was observed in the middle part of the segment. Thin-shelled, oval, 35-µm-diameter parasite eggs containing an oncosphere with three pairs of hooklets were observed. The gravid segments were determined as Mesocestoides spp. based on the appearance of the typical paruterine organ. PCR results supported our diagnosis; moreover, according to the BLAST results, it was detected that the species infecting two dogs was 98.01–100% similar to M. litteratus . Praziquantel-containing medication was administered to the infected dogs at a dosage of 5 mg/kg. Foxes act as the final host of M. litteratus and the parasite is prevalent in wildlife; however, these animals may disperse the parasite in urban life. Veterinarians need to be made more aware of this parasite, especially if the dogs are owned from shelters.

Codfish is an essential part of the marine fishery resources, and contains several commercially important species, including Gadus species (G. morhua, G. macrocephalus, and G. chalcogrammus), and other five Gadiformes species, including Pollachius virens, Melanogrammus aeglefinus, Merluccius merluccius, Merluccius australis and Albatrossia pectoralis. According to our previous studies, Gadus species represents the most frequently used Gadiformes species for codfish products, with other five Gadiformes species also occasionally found. However, due to the legislative shortcomings, there is still not a harmonization around the definition of codfish in China, leading to great chaos. To help prevent this episode, the present study aimed to develop a one-tube fast method for codfish species identification, with particular attention to differentiate Gadus species, and the other five Gadiformes species, using the high resolution melting (HRM) analysis of a 12S rRNA mini barcode. The specificity of the newly designed primers was successfully confirmed for the eight common Gadiformes species. The novel HRM assay allowed the full discrimination between Gadus species and the other five Gadiformes species. 19 commercial codfish products were included in the Gadus cluster, cross-confirmed by the real-time PCR and DNA barcoding. The identification of less valuable U. japonicas is essentially a consumer fraud, while great health concern could also be highlighted by the identified Lagocephalus species. The established HRM of a 12S rRNA mini barcode method in the present study allows a fast and accurate identification of codfish species in processed fish products.

To explore the current state of genetic diversity of in the Chinese soft-shelled turtle (Pelodiscus sinensis) in the Dongting Lake basin, the genetic diversity of five sub-populations consisting of 71 turtles were analyzed through mitochondrial Cytb and 12S rRNA. Our results revealed 13 haplotypes for Cytb and 6 for 12S rRNA. The overall haplotype diversity and nucleotide diversity indices were 0.750 and 0.014, and 0.712 and 0.009, respectively. The Shaoyang sub-population showed the lowest genetic diversity according to both markers. The genetic distances between sub-populations ranged from 0.010 to 0.018 (Cytb), and from 0.002 to 0.014 (12S rRNA), with the largest distance observed between the Shaoyang and Junshan sub-populations. The Junshan sub-population was significantly different from the other sub-populations (p < 0.05), and gene exchange was weak, despite belonging to the same population. Genetic variation within the P. sinensis sub-population was much higher than that between sub-populations. There was no recent expansion event in the history of P. sinensis. Overall, the genetic diversity of P. sinensis was high, whereas it appeared to be homogenous, suggesting a potential decline in genetic diversity. This study provides valuable insights for the conservation and sustainable use of P. sinensis.