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The chestnut gall wasp (CGW), Dryocosmus kuriphilusYasumatsu, 1951 (Cynipini, Cynipoidea, Hymenoptera), is a globally invasive pest on chestnuts (Castanea spp., Fagaceae). Since its first reported occurrence in the state of Georgia in 1974, CGW has been reported to have spread to 16 additional states in the eastern United States. Despite its wide occurrence, investigations of the invasion and colonization history of CGW have not been conducted. To address this shortcoming, we screened one mitochondrial (cytochrome c oxidase subunit 1; COI) and one nuclear gene (internal transcribed spacer 2; ITS2) to examine the genetic variation across four discrete CGW populations from four states (New Jersey, Pennsylvania, Maryland, Tennessee). We tested whether CGW in the eastern U.S. originated from one or multiple introductions followed by subsequent dispersal. Additional insights came from comparing the sequences with two CGW populations sampled from its native range in mainland China, and one population each from Taiwan and Japan. Sequences revealed that both genes exhibit the same haplotype across all sampled specimens of all locations. The COI haplotype in this study is the dominant haplotype recorded in China, while the haplotype of ITS2 has been reported in both Japan and Europe. The uniformity of genetic structure suggests that CGW invasion in the U.S. could have either single or multiple origins, or both, accompanied with the common haplotype on each occasion. Further insights into the invasion history and expansion of CGW in the U.S. will require a combination of sampling more populations and utilizing a next generation sequencing strategy. [ABSTRACT FROM AUTHOR]

The animal gut microbiome can have a strong influence on the health, fitness, and behavior of its hosts. The composition of the gut microbial community can be influenced by factors such as diet, environment, and evolutionary history (phylosymbiosis). However, the relative influence of these factors is unknown in most bird species. Furthermore, phylosymbiosis studies have largely focused on clades that diverged tens of millions of years ago, and little is known about the degree of gut microbiome divergence in more recent species radiations. This study explores the drivers of microbiome variation across the unique and recent Hawaiian honeycreeper radiation (Fringillidae: Drepanidinae). Fecal samples were collected from 14 extant species spanning the main islands of the Hawaiian archipelago and were sequenced using three metabarcoding markers to characterize the gut microbiome, invertebrate diet, and plant diet of Hawaiian honeycreepers. We then used these metabarcoding data and the honeycreeper host phylogeny to evaluate their relative roles in shaping the gut microbiome. Microbiome variation across birds was highly individualized; however, source island had a small but significant effect on microbiome structure. The microbiomes did not recapitulate the host phylogenetic tree, indicating that evolutionary history does not strongly influence microbiome structure in the honeycreeper clade. These results expand our understanding of the roles of diet, geography, and phylogeny on avian microbiome structure, while also providing important ecological information about the diet and gut microbiota of wild Hawaiian honeycreepers. [ABSTRACT FROM AUTHOR]

Abstract Biomphalaria straminea is one of the three snails that act as the intermediate hosts of Schistosoma mansoni and is responsible for maintaining high rates of schistosomiasis in some areas of northeastern Brazil. The principal morphological characteristic of B. straminea is the marked vaginal wrinkling, although it is also present in congeneric species, a group known as the B. straminea complex. Based on the morphological and molecular analyses, we investigated the intraspecific variation of B. straminea. The specimens were obtained from 10 sites in Brazil, and the shells were analyzed, as well as 16 morphological structures of the reproductive system. The COI and ITS2 sequences were used for phylogenetic analysis, genetic divergence, and haplotype network (COI). We observed a large intraspecific variation in the morphological structures examined. The genetic divergence also demonstrated significant intraspecific variability in B. straminea: 0–3% in ITS2 sequences, and 0–6% in COI sequences. Analysis of the distribution of COI haplotypes recovered 16 haplotypes and haplotype diversity of 0.9088. These results indicate phenotypic variability that is not constrained to a locality or strictly controlled genetically by B. straminea, which may have driven a misidentified of these species.

Abstract Background The Anopheles hyrcanus group is distributed throughout the Oriental and Palaearctic regions and can transmit diseases such as malaria, Japanese encephalitis virus, and filariasis. This investigation marks the inaugural comprehensive study to undertake a phylogenetic analysis of the constituents of this malaria vector group in the northeastern region of Iran, juxtaposed with documented occurrences from different areas within Iran and worldwide. Methods Mosquitoes were collected using various methods from nine different locations in Golestan province from April to December 2023. The collected mosquitoes were identified morphologically using valid taxonomic keys. DNA was isolated using the Sambio™ Kit. COI and ITS2 primers were designed using Oligo7 and GeneRunner. PCR and purification were performed with the Qiagen kit. Subsequently, sequencing was carried out at the Mehr Mam GENE Center using an Applied Biosystems 3730XL sequencer. The nucleotide sequences were then analyzed and aligned with GenBank data using BioEdit. Kimura 2-parameter was Utilized for base substitutions. DNA models were selected based on AIC and BIC criteria. Bayesian and Maximum Likelihood trees were constructed, along with a haplotype network. Molecular diversity statistics computed using DnaSP software. Results In this study, a total of 819 adult mosquitoes were collected. An. hyrcanus was the second most abundant species, predominantly found in Kalaleh and Turkman counties. The sequenced and edited COI and ITS2 sequences were deposited in GenBank under specific accession numbers. Phylogenetic analyses using ML, BI, and NJ methods confirmed a monophyletic lineage for An. hyrcanus with strong support. Molecular analysis of Iranian An. hyrcanus found 11 diverse haplotypes, with the COI gene displaying low diversity. The ITS2 gene revealed two clades - one associating with Iran, Europe, and Asia; the other originating from southwestern Iran. The haplotype network showed two main groups - one from southwest Iran and the other from north Iran. Iran exhibited six distinct haplotypes, while Turkey showcased the highest diversity. Conclusions An. hyrcanus in southwestern Iran exhibits a distinct haplogroup, suggesting possible subspecies differentiation. Additional studies are required to validate this phenomenon.

Background: The Anopheles hyrcanus group is distributed throughout the Oriental and Palaearctic regions and can transmit diseases such as malaria, Japanese encephalitis virus, and filariasis. This investigation marks the inaugural comprehensive study to undertake a phylogenetic analysis of the constituents of this malaria vector group in the northeastern region of Iran, juxtaposed with documented occurrences from different areas within Iran and worldwide. Methods: Mosquitoes were collected using various methods from nine different locations in Golestan province from April to December 2023. The collected mosquitoes were identified morphologically using valid taxonomic keys. DNA was isolated using the Sambio™ Kit. COI and ITS2 primers were designed using Oligo7 and GeneRunner. PCR and purification were performed with the Qiagen kit. Subsequently, sequencing was carried out at the Mehr Mam GENE Center using an Applied Biosystems 3730XL sequencer. The nucleotide sequences were then analyzed and aligned with GenBank data using BioEdit. Kimura 2-parameter was Utilized for base substitutions. DNA models were selected based on AIC and BIC criteria. Bayesian and Maximum Likelihood trees were constructed, along with a haplotype network. Molecular diversity statistics computed using DnaSP software. Results: In this study, a total of 819 adult mosquitoes were collected. An. hyrcanus was the second most abundant species, predominantly found in Kalaleh and Turkman counties. The sequenced and edited COI and ITS2 sequences were deposited in GenBank under specific accession numbers. Phylogenetic analyses using ML, BI, and NJ methods confirmed a monophyletic lineage for An. hyrcanus with strong support. Molecular analysis of Iranian An. hyrcanus found 11 diverse haplotypes, with the COI gene displaying low diversity. The ITS2 gene revealed two clades - one associating with Iran, Europe, and Asia; the other originating from southwestern Iran. The haplotype network showed two main groups - one from southwest Iran and the other from north Iran. Iran exhibited six distinct haplotypes, while Turkey showcased the highest diversity. Conclusions: An. hyrcanus in southwestern Iran exhibits a distinct haplogroup, suggesting possible subspecies differentiation. Additional studies are required to validate this phenomenon. [ABSTRACT FROM AUTHOR]

Anopheles claviger (Meigen, 1804) (Diptera, Culicidae) is widespread in the western Palaearctic Region, but it was recorded in Karelia (Russia) for the first time. This record is one of the northernmost ones in the Palaearctic Region and Russia, updates the northern border of the An. claviger range. Mosquitoes were collected from July to September 2023 in the southern Karelia (the village of Gomselga, Kondopoga District, and Petrozavodsk) using Krishtal trap (from human) and Mosquito Magnet® trap (Pioneer design, Octenol as attractant). Seven females of An. claviger were collected in Gomselga; one specimen was sampled from Petrozavodsk City parks. Morphological identification of eight females was verified by COI and ITS2 sequences. Phylogenetic analysis of ITS2 and COI sequences confirmed the collected specimens to An. claviger s. s., clustering in both cases in a strongly supported clade clearly differentiated from the closely related species An. petragnani. The high diversity of An. claviger haplotypes from Karelia is in agreement with data from other geographical regions and shows that the records of this species in Gomselga and Petrozavodsk are not accidental. [ABSTRACT FROM AUTHOR]

Objective: To address the phylogenetic and phylogeographic relationship between different lineages of Anopheles (An.) subpictus species complex in most parts of the Asian continent by maximum utilization of Internal Transcriber Spacer 2 (ITS2) and cytochrome C oxidase I (COI) sequences deposited at the GenBank. Methods: Seventy-five ITS2, 210 COI and 26 concatenated sequences available in the NCBI database were used. Phylogenetic analysis was performed using Bayesian likelihood trees, whereas median-joining haplotype networks and time-scale divergence trees were generated for phylogeographic analysis. Genetic diversity indices and genetic differentiation were also calculated. Results: Two genetically divergent molecular forms of An. subpictus species complex corresponding to sibling species A and B are established. Species A evolved around 37-82 million years ago in Sri Lanka, India, and the Netherlands, and species B evolved around 22-79 million years ago in Sri Lanka, India, and Myanmar. Vietnam, Thailand, and Cambodia have two molecular forms: one is phylogenetically similar to species B. Other forms differ from species A and B and evolved recently in the above mentioned countries, Indonesia and the Philippines. Genetic subdivision among Sri Lanka, India, and the Netherlands is almost absent. A substantial genetic differentiation was obtained for some populations due to isolation by large geographical distances. Genetic diversity indices reveal the presence of a long-established stable mosquito population, at mutation-drift equilibrium, regardless of population fluctuations. Conclusions: An. subpictus species complex consists of more than two genetically divergent molecular forms. Species A is highly divergent from the rest. Sri Lanka and India contain only species A and B. [ABSTRACT FROM AUTHOR]

To address the phylogenetic and phylogeographic relationship between different lineages of Anopheles (An.) subpictus species complex in most parts of the Asian continent by maximum utilization of Internal Transcriber Spacer 2 (ITS2) and cytochrome C oxidase I (COI) sequences deposited at the GenBank. Seventy-five ITS2, 210 COI and 26 concatenated sequences available in the NCBI database were used. Phylogenetic analysis was performed using Bayesian likelihood trees, whereas median-joining haplotype networks and time-scale divergence trees were generated for phylogeographic analysis. Genetic diversity indices and genetic differentiation were also calculated. Two genetically divergent molecular forms of An. subpictus species complex corresponding to sibling species A and B are established. Species A evolved around 37-82 million years ago in Sri Lanka, India, and the Netherlands, and species B evolved around 22-79 million years ago in Sri Lanka, India, and Myanmar. Vietnam, Thailand, and Cambodia have two molecular forms: one is phylogenetically similar to species B. Other forms differ from species A and B and evolved recently in the above mentioned countries, Indonesia and the Philippines. Genetic subdivision among Sri Lanka, India, and the Netherlands is almost absent. A substantial genetic differentiation was obtained for some populations due to isolation by large geographical distances. Genetic diversity indices reveal the presence of a long-established stable mosquito population, at mutation-drift equilibrium, regardless of population fluctuations. An. subpictus species complex consists of more than two genetically divergent molecular forms. Species A is highly divergent from the rest. Sri Lanka and India contain only species A and B.

The present study fills a knowledge gap in the distribution and genetic variation of Morimus populations in the Balkans, by studiyng the representatives of the genus in Bulgaria – M. asper funereus Mulsant, 1862, M. verecundus bulgaricus Danilevsky, 2016 and M. orientalis Reitter, 1894. Additional information is provided for Albania and northern Greece. The mitochondrial cytochrome C oxidase subunit I (COI) marker and the nuclear internal transcribed spacer 2 (ITS2) were used for the genetic analyses. Three of the previously-defined mitochondrial lineages (Lb/HgA, L2 and L3) were detected in Bulgaria, as well as a new lineage (Str) from the Strandzha Mountains (south-eastern Bulgaria). A total of 24 distinct haplotypes, 20 of them in Bulgaria, were found. Bulgarian populations of Morimus demonstrated relatively high nucleotide diversity. The L3 COI lineage was confirmed as the most diverse and frequent in the Balkans. The L3 lineage is dominant in most of Bulgaria, but was not identified in the easternmost parts near the Black Sea coast, where the L2 and Str lineages were found. New data highlighted two dispersal routes of the L2 mitochondrial lineage on the Balkan Peninsula: 1) northwards along the Black Sea coast and 2) westwards, across the Balkans where only disjunct populations remain. North-western Bulgaria seems to be the eastern limit of the basal lineage Lb/HgA distribution. Our results show high levels of genetic exchange between most of the mitochondrially defined lineages, yet some of the easternmost populations probably remained isolated for comparatively longer periods.

Features of shell and genitalia as well as nucleotide sequences of selected mitochondrial and nuclear genes of specimens of Monacha cantiana from ten northern French and two Dutch populations were compared with the same features of British and Italian populations. They were found to be very similar to populations previously identified as belonging to the CAN-1 lineage of M. cantiana. This confirms previous suggestions that M. cantiana was introduced to western Europe (England, France and the Netherlands) in historical times. [ABSTRACT FROM AUTHOR]

The present study fills a knowledge gap in the distribution and genetic variation of Morimus populations in the Balkans, by studiyng the representatives of the genus in Bulgaria - M. asper funereus Mulsant, 1862, M. verecundus bulgaricus Danilevsky, 2016 and M. orientalis Reitter, 1894. Additional information is provided for Albania and northern Greece. The mitochondrial cytochrome C oxidase subunit I (COI) marker and the nuclear internal transcribed spacer 2 (ITS2) were used for the genetic analyses. Three of the previouslydefined mitochondrial lineages (Lb/HgA, L2 and L3) were detected in Bulgaria, as well as a new lineage (Str) from the Strandzha Mountains (south-eastern Bulgaria). A total of 24 distinct haplotypes, 20 of them in Bulgaria, were found. Bulgarian populations of Morimus demonstrated relatively high nucleotide diversity. The L3 COI lineage was confirmed as the most diverse and frequent in the Balkans. The L3 lineage is dominant in most of Bulgaria, but was not identified in the easternmost parts near the Black Sea coast, where the L2 and Str lineages were found. New data highlighted two dispersal routes of the L2 mitochondrial lineage on the Balkan Peninsula: 1) northwards along the Black Sea coast and 2) westwards, across the Balkans where only disjunct populations remain. North-western Bulgaria seems to be the eastern limit of the basal lineage Lb/HgA distribution. Our results show high levels of genetic exchange between most of the mitochondrially defined lineages, yet some of the easternmost populations probably remained isolated for comparatively longer periods. [ABSTRACT FROM AUTHOR]

Abstract Sharks of the genus Sphyrna are under intense exploitation globally. In Brazil’s northern coast, this genus represents a high proportion of fisheries landings and comprises four species. However, due to difficulty of specific identification when specimens are landed, most of the records are limited to the genus level. Here we analyzed the effectiveness of ITS2 (Internal Transcribed Spacer 2 of rDNA) fragment length protocol (Abercrombie et al., 2005) for identifying hammerhead shark species, comparing with the analysis of COI (Cytochrome oxidase subunit I) and ITS2 sequences. We evaluated samples of muscle tissue acquired in the main fishing ports of Maranhão: Carutapera, Raposa e Tutóia. Sampling was conducted between March 2017 to March 2018 and complemented with material deposited in collection (2015). COI results indicated the occurrence of endangered species which are prohibited to be landed. These include Sphyrna mokarran (67%), S. lewini (15%), S. tudes (3%), and S. tiburo (15%). For the ITS2 marker, we investigated the optimization of the protocol developed by Abercrombie (2005) for to improve the use in this geographical area througout design of a new primers.

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Niphargus carolinensis sp. nov. was sampled from the Carolina Mine located in North Rhine–Westphalia, Germany. The new species is described and compared to phylogenetically related species and species identified in nearby locations. The three phylogenetic markers (i.e., COI, 28S rRNA and ITS2) studied in the examined specimens had different sequences compared to those belonging to species present in locations neighboring the Carolina Mine, i.e., in a radius of 40 km. N. carolinensis sp. nov. is a small-to-medium-sized species that is poorly setose; has a relatively short antenna I, trapezoidal gnathopod propodites, long pereopod VI, and short uropod III; and is not differentiated sexually. The new species described herein is a case of narrow endemism and adds to the diversity of the genus Niphargus in Germany. This work is a contribution to knowledge on groundwater amphipod diversity and the systematics of the genus Niphargus close to the northern border of the distribution of this genus. [ABSTRACT FROM AUTHOR]

Trichophoromyia auraensis (Mangabeira, 1942) (Diptera, Psychodidae, Phlebotominae) has a wide geographic distribution in the western region of the Amazon biome, where it is a putative Leishmania vector. Here, we reported for the first time a population of this species in the Brazilian state of Maranhão, in the eastern Amazon, from which we DNA-barcoded and compared with previously processed specimens from Acre State, in the western Amazon. For this, we analyzed the DNA barcoding fragment (658 bp) of the mitochondrial cytochrome c oxidase subunit I (COI) gene and the nuclear internal transcribed spacer 2 (ITS2) of Trichophoromyia species using phylogenetic gene trees, and species delimitation algorithms. The analyses of COI barcodes showed high values of genetic distance (mean K2P = 5.17) and well-supported clades/MOTUs for the eastern and western populations of T. auraensis, which may indicate a possible complex of cryptic species. The western population of this taxon merged with the close-related sand fly Trichophoromyia velezbernali Posada-López, Galvis and Galati, 2018 from Colombia, which may be associated with the recent speciation history and introgression between these populations. These evidences should be evaluated with a more comprehensive sampling in terms of analyzed populations and molecular markers. [ABSTRACT FROM AUTHOR]

Background: Ixodes ricinus is an important vector of several pathogens, primarily in Europe. Recently, Ixodes inopinatus was described from Spain, Portugal, and North Africa and then reported from several European countries. In this study, a multiplex polymerase chain reaction (PCR) was developed to distinguish I. ricinus from I. inopinatus and used in the surveillance of I. inopinatus in Algeria (ALG) and three regions in the Czech Republic (CZ). Methods: A multiplex PCR on TROSPA and sequencing of several mitochondrial (16S rDNA, COI) and nuclear markers (TROSPA, ITS2, calreticulin) were used to differentiate these two species and for a subsequent phylogenetic analysis. Results: Sequencing of TROSPA, COI, and ITS2 separated these two species into two subclades, while 16S rDNA and calreticulin could not distinguish I. ricinus from I. inopinatus. Interestingly, 23 nucleotide positions in the TROSPA gene had consistently double peaks in a subset of ticks from CZ. Cloning of these PCR products led to a clear separation of I. ricinus and I. inopinatus indicating hybridization and introgression between these two tick taxa. Based on a multiplex PCR of TROSPA and analysis of sequences of TROSPA, COI, and ITS2, the majority of ticks in CZ were I. ricinus, no I. inopinatus ticks were found, and 10 specimens showed signs of hybridization. In contrast, most ticks in ALG were I. inopinatus, four ticks were I. ricinus, and no signs of hybridization and introgression were detected. Conclusions: We developed a multiplex PCR method based on the TROSPA gene to differentiate I. ricinus and I. inopinatus. We demonstrate the lack of evidence for the presence of I. inopinatus in Central Europe and propose that previous studies be re-examined. Mitochondrial markers are not suitable for distinguishing I. inopinatus from I. ricinus. Furthermore, our data indicate that I. inopinatus and I. ricinus can hybridize, and the hybrids can survive in Europe. [ABSTRACT FROM AUTHOR]

The accurate identification of pests and their natural enemies is critically important in pest management. Without requiring specialized taxonomic expertise, molecular identification has been widely used in the recognition of pests and their natural enemies, as well as in the assessment of their trophic interactions. However, systematic comparative studies on the selection of molecular markers are often lacking. Trichogramma are the most widely used parasitoid wasps for controlling various lepidopteran pests in both agriculture and forests. The most commonly used molecular markers for Trichogramma are cytochrome oxidase subunit 1 (COI) and internal transcribed spacer 2 (ITS2). It is still controversial which marker is superior in Trichogramma molecular identification. We surveyed Trichogramma COI and ITS2 sequences available on NCBI and BOLD systems, performed PCR and Sanger sequencing for seven laboratory-reared Trichogramma species, and compared these two markers in several aspects. We found that ITS2 covers more species than COI in the public dataset, but its sequence misidentification rate is also higher than that of COI; ITS2 generally shows a faster substitution rate than COI, but COI sequences can be more divergent than ITS2 in some Trichogramma species; ITS2 has a higher copy number and PCR success rate than COI, but the proportion of double peaks in chromatograms of Sanger sequencing is also higher than that of COI. In summary, the molecular marks ITS2 and COI have different characteristics and can complement each other in the molecular identification of Trichogramma. [ABSTRACT FROM AUTHOR]

Diglyphus Walker, 1844 (Hymenoptera: Eulophidae) is an economically important genus including species acting as biocontrol agents against agromyzid leafminer pests. A new species of Diglyphus, Diglyphus difasciatus Liu, Hansson & Wan, sp. nov., was discovered during the identification of agromyzid leafminers and their associated parasitoid wasps collected from 2016 to 2022 in China, based on morphological characteristics and molecular analyses of COI, ITS2 and 28S genes. Diglyphus difasciatus is similar to D. bimaculatus Zhu, LaSalle & Huang, distinguished by two interconnected infuscate vertical bands on the fore wing and the color of the scape. Molecular data support D. difasciatus and D. bimaculatus as two different species. The mean genetic distances between D. difasciatus and D. bimaculatus were 11.33%, 8.62%, and 0.18%, based on the COI, ITS2, and 28S genes, respectively. [ABSTRACT FROM AUTHOR]

• First report of Mediterranean recluse spider in Bangkok, Thailand, indicating potential invasive behavior in urban areas. • Phylogenetic analysis reveals a North African origin for L. rufescens , with human-mediated dispersal contributing to its spread. • Genetic approaches clarify Loxosceles evolution and species boundaries, emphasizing need for monitoring this medically important spider. The Mediterranean recluse spider, Loxosceles rufescens , has been discovered for the first time inhabiting human dwellings in Bangkok, Thailand. Expeditions across 39 localities revealed five establishments with L. rufescens populations. The highest density was recorded in a storage house on Yaowarat Road, located in the heart of Bangkok's Chinatown, where 315 individuals were found, including adults, juveniles, and spiderlings. This medically significant spider's presence in such a densely populated urban area raises concerns about potential envenomation risks. Thirteen specimens of L. rufescens were extracted for DNA and sequenced for molecular phylogenetic analyses. COI and ITS2 markers were used to investigate relationships within L. rufescens and across available Loxosceles species sequences. Results indicate COI is superior for resolving species-level genetic clusters compared to ITS2. Surprisingly, L. rufescens individuals from the same house were found in significantly distant COI lineages, suggesting mtDNA may not be suitable for studying intra-specific phylogeography in this case. Species delimitation methods ABGD and ASAP demonstrated promising results for both COI and ITS2, while bPTP and GMYC tended to overestimate species numbers. ITS2 exhibited high sequence similarity in L. rufescens , suggesting potential utility as a barcoding marker for identification of this globally distributed species. Genetic distance analyses revealed a potential barcoding gap (K2P) of 8–9 % for COI and <2 % for ITS2 in Loxosceles. This study contributes valuable sequence data for the medically important genus Loxosceles and highlights the need for integrative approaches in understanding its evolution and spread. The findings have important implications for pest management strategies and public health in urban environments. [Display omitted] [ABSTRACT FROM AUTHOR]

'Candidatus Phytoplasma prunorum' is causing ever increasing economic losses through the decline of apricot trees in European countries, e.g., Hungary. In this study, the pathogen was identified from plant tissues and insects by nested-PCR. The insect species were identified via morphology and molecular methods. The incidence of the pathogen was 29.6% in randomly selected apricot trees. Most of the infected trees with symptoms died within a year. These results show that phytoplasma is significantly present and causes damage in the investigated plantations. The only known insect vector of this phytoplasma is the plum psyllid, Cacopsylla pruni, which was regularly encountered in the sampled apricot orchards and in their surroundings. In a two-year study, several adults among the sampled specimens were observed to be infected by the pathogen. This observation further confirms the role of the plum psyllid in vectoring the phytoplasma. All the sampled plum psyllid adults belonged to the 'B' biotype. Besides C. pruni, Cacopsylla crataegi was abundant in the samples. Several adults of the latter species were also infected by the pathogen 'Ca. Phytoplasma prunorum'. The rates of occurrence of this phytoplasma in male and female adults of the two psyllid species appeared to be similar. The examined C. crataegi individuals showed genetic differences from each other and from specimens included in a previous investigation. [ABSTRACT FROM AUTHOR]

The chigger mite is a known vector for the zoonotic disease, Tsutsugamushi, which affects more than 4,000 patients each year in the Republic of Korea (ROK) since 2004. However, molecular data is only available for a few species of chigger mite, compared to the numbers of known species. This study focused on securing molecular data for six species (Leptotrombidium pallidum, Leptotrombidium scutellare, Leptotrombidium palpale, Leptotrombidium orientale, Neotrombicula gardellai, and Euschoengastia koreaensis) of chigger mites in the ROK from cytochrome c oxidase subunit I (COI) and internal transcribed spacer 2 (ITS2). In the results, there were each two genotypes in L. pallidum and L. palpale, and three genotypes in L. scutellae in the COI, whereas the ITS2 sequence was identical within the species. The other three species were identical from COI and ITS2. This study first revealed the genetic variation in the COI sequences of chigger mites in the ROK. Further studies could determine how this variation is related to ecological behavior and disease transmission. [ABSTRACT FROM AUTHOR]

Liriomyza huidobrensis, L. sativae and L. trifolii are polyphagous agromyzid leafminers that have recently arrived in Australia, posing a threat to Australian vegetable and ornamental crops. Adventive and endemic hymenopteran parasitoids of agromyzid leafminers already present in Australia should assist in the management of these invasive agromyzid species. Neochrysocharis formosa (Hymenoptera: Eulophidae: Entedoninae) is an idiobiont endoparasitoid commonly attacking Liriomyza spp. in many countries, but it has not been formally identified in Australia. This study provides the first confirmed Australian record of N. formosa as well as an unidentified Proacrias species, another entedonine species. Females of both species were reared from several adventive and endemic agromyzid leafminers in southern Australia. Laboratory cultures of both species established the presence of thelytokous reproduction. DNA barcodes (5′ COI, 3′ COI, ITS1, ITS2, 28S) were used to delineate species boundaries, with the 5′ end of the mitochondrial COI sequences pointing to multiple cryptic lineages among N. formosa. Additionally, 16S rRNA sequencing indicated that both species were infected with a Rickettsia bacterium, which is related to the Rickettsia present in thelytokous populations of N. formosa in Japan and China. These findings expand records for parasitoids attacking leafminers in Australia and highlight the potential for an endosymbiont to produce thelytokous strains that could provide for more efficient biocontrol agents for augmentative release. [ABSTRACT FROM AUTHOR]

Abstract Background Plasmodium knowlesi, a simian malaria parasite infection, increases as Plasmodium falciparum and Plasmodium vivax infections decrease in Johor, Malaysia. Therefore, this study aimed to identify the distribution of vectors involved in knowlesi malaria transmission in Johor. This finding is vital in estimating hotspot areas for targeted control strategies. Methods Anopheles mosquitoes were collected from the location where P. knowlesi cases were reported. Cases of knowlesi malaria from 2011 to 2019 in Johor were analyzed. Internal transcribed spacers 2 (ITS2) and cytochrome c oxidase subunit I (COI) genes were used to identify the Leucosphyrus Group of Anopheles mosquitoes. In addition, spatial analysis was carried out on the knowlesi cases and vectors in Johor. Results One hundred and eighty-nine cases of P. knowlesi were reported in Johor over 10 years. Young adults between the ages of 20–39 years comprised 65% of the cases. Most infected individuals were involved in agriculture and army-related occupations (22% and 32%, respectively). Four hundred and eighteen Leucosphyrus Group Anopheles mosquitoes were captured during the study. Anopheles introlatus was the predominant species, followed by Anopheles latens. Spatial analysis by Kriging interpolation found that hotspot regions of P. knowlesi overlapped or were close to the areas where An. introlatus and An. latens were found. A significantly high number of vectors and P. knowlesi cases were found near the road within 0–5 km. Conclusions This study describes the distribution of P. knowlesi cases and Anopheles species in malaria-endemic transmission areas in Johor. Geospatial analysis is a valuable tool for studying the relationship between vectors and P. knowlesi cases. This study further supports that the Leucosphyrus Group of mosquitoes might be involved in transmitting knowlesi malaria cases in Johor. These findings may provide initial evidence to prioritize diseases and vector surveillance.

Abstract Background Plasmodium vivax is transmitted by members of the Anopheles Hyrcanus Group that includes six species in the Republic of Korea: Anopheles sinensis sensu stricto (s.s.), Anopheles pullus, Anopheles kleini, Anopheles belenrae, Anopheles lesteri, and Anopheles sineroides. Individual Anopheles species within the Hyrcanus Group demonstrate differences in their geographical distributions, vector competence and insecticide resistance, making it crucial for accurate species identification. Conventional species identification conducted using individual genotyping (or barcoding) based on species-specific molecular markers requires extensive time commitment and financial resources. Results A population-based quantitative sequencing (QS) protocol developed in this study provided a rapid estimate of species composition ratios among pooled mosquitoes as a cost-effective alternative to individual genotyping. This can be accomplished by using species- or group-specific nucleotide sequences of the mitochondrial cytochrome C oxidase subunit I (COI) and the ribosomal RNA in ternal transcribed spacer 2 (ITS2) region as species identification alleles in a two-step prediction protocol. Standard genomic DNA fragments of COI and ITS2 genes were amplified from each Anopheles species using group-specific universal primer sets. Following sequencing of the COI or ITS2 amplicons generated from sets of standard DNA mixtures, equations were generated via linear regression to predict species-specific nucleotide sequence frequencies at different positions. Species composition ratios between An. sineroides, An. pullus and An. lesteri were estimated from QS of the COI amplicons based on the mC.260A, mC.122C and mC.525C alleles at the first step, followed by the prediction of species composition ratios between An. sinensis, An. kleini and An. belenrae based on QS of the ITS2 amplicons using the rI.370G and rI.389T alleles. The COI copy number was not significantly different between species, suggesting the reliability of COI-based prediction. In contrast, ITS2 showed a slightly but significantly higher copy number in An. belenrae, requiring an adjustment of its predicted composition ratio. A blind test proved that predicted species composition ratios either from pooled DNA specimens or pooled mosquito specimens were not statistically different from the actual values, demonstrating that the QS-based prediction is accurate and reliable. Conclusions This two-step prediction protocol will facilitate rapid estimation of the species composition ratios in field-collected Anopheles Hyrcanus Group populations and is particularly useful for studying the vector ecology of Anopheles population and epidemiology of malaria.

Abstract Objectives Hard ticks (Acari: Ixodidae) are ectoparasites of medical and veterinary importance. They are obligate blood-feeding vectors with the ability to transmit a wide variety of pathogens. Standard morphological keys are normally used for the identification of tick species. However, considering the importance of accurate species identification and the determination of bio-ecological characteristics of species, relying on morphological keys alone can be questionable. In this study, two DNA fragments (ITS2 and COI) were selected for phylogenetic evaluation of Iranian hard tick species belonging to the genera Dermacentor, Hyalomma, and Rhipicephalus. Results 1229 specimens of Dermacentor marginatus, D. niveus, Hyalomma anatolicum, Rhipicephalus bursa, and R. sanguineus s.l constituting 11 populations were collected from three different climatic and zoogeographical zones in Iran. Morphological studies revealed notable differences in important morphological characteristics between different populations of D. marginatus. The results of ITS2 sequence analysis provided additional evidence which supports the conspecificity of D. niveus and D. marginatus. Contrary to this finding, the sequence analysis of COI and phylogeny favored the separation of the two species. Given the greater importance of COI in identifying and discriminating species, a possibility heterospecificity between the two species should be considered.

Exploring the effectiveness of DNA barcoding in species identification is a prerequisite for biodiversity conservation and environmental monitoring. Aquatic oligochaetes could serve as excellent indicators in aquatic monitoring programmes. However, few studies have examined the effectiveness of DNA barcoding in these specific organisms. The mitochondrial cytochrome C oxidase (COI) gene of 83 specimens belonging to 40 species of 18 genera were sequenced in this study. The results showed that there was a barcode gap between species of Naididae and the intraspecific genetic distances of each species were smaller than interspecific genetic distances. The classification results of ABGD (Automatic Barcode Gap Discovery) were consistent with those of morphological identification, except for Tubifex tubifex and Lumbriculus variegatus. All species were successfully distinguished in the phylogenetic tree, based on the ITS2 region, which was coincident with the morphological result. Our results provided evidence that DNA barcoding can be used as an effective and convenient tool for species identification of the family Naididae and even for other aquatic oligochaetes. [ABSTRACT FROM AUTHOR]

Background: Plasmodium knowlesi, a simian malaria parasite infection, increases as Plasmodium falciparum and Plasmodium vivax infections decrease in Johor, Malaysia. Therefore, this study aimed to identify the distribution of vectors involved in knowlesi malaria transmission in Johor. This finding is vital in estimating hotspot areas for targeted control strategies. Methods: Anopheles mosquitoes were collected from the location where P. knowlesi cases were reported. Cases of knowlesi malaria from 2011 to 2019 in Johor were analyzed. Internal transcribed spacers 2 (ITS2) and cytochrome c oxidase subunit I (COI) genes were used to identify the Leucosphyrus Group of Anopheles mosquitoes. In addition, spatial analysis was carried out on the knowlesi cases and vectors in Johor. Results: One hundred and eighty-nine cases of P. knowlesi were reported in Johor over 10 years. Young adults between the ages of 20–39 years comprised 65% of the cases. Most infected individuals were involved in agriculture and army-related occupations (22% and 32%, respectively). Four hundred and eighteen Leucosphyrus Group Anopheles mosquitoes were captured during the study. Anopheles introlatus was the predominant species, followed by Anopheles latens. Spatial analysis by Kriging interpolation found that hotspot regions of P. knowlesi overlapped or were close to the areas where An. introlatus and An. latens were found. A significantly high number of vectors and P. knowlesi cases were found near the road within 0–5 km. Conclusions: This study describes the distribution of P. knowlesi cases and Anopheles species in malaria-endemic transmission areas in Johor. Geospatial analysis is a valuable tool for studying the relationship between vectors and P. knowlesi cases. This study further supports that the Leucosphyrus Group of mosquitoes might be involved in transmitting knowlesi malaria cases in Johor. These findings may provide initial evidence to prioritize diseases and vector surveillance. [ABSTRACT FROM AUTHOR]

Culex torrentium and C. vagans are morphologically similar mosquito species for which the name C. exilis has been used in Russia for a long time. We have compared the male genitalia in these species and analyzed the genetic differences between them, using specimens from different parts of their ranges. The results show that C. vagans can be readily distinguished from C. torrentium by the shape of the lateral arms of the phallosome. The genetic differences between the species are 3.5–3.6% for the COI gene and 14% for the ITS2 nuclear marker and reliably differentiated the two species. The ITS2 sequence of C. vagans is determined for the first time. The data obtained can be used to identify C. vagans and C. torrentium and to specify their ranges. [ABSTRACT FROM AUTHOR]

Background: Plasmodium vivax is transmitted by members of the Anopheles Hyrcanus Group that includes six species in the Republic of Korea: Anopheles sinensis sensu stricto (s.s.), Anopheles pullus, Anopheles kleini, Anopheles belenrae, Anopheles lesteri, and Anopheles sineroides. Individual Anopheles species within the Hyrcanus Group demonstrate differences in their geographical distributions, vector competence and insecticide resistance, making it crucial for accurate species identification. Conventional species identification conducted using individual genotyping (or barcoding) based on species-specific molecular markers requires extensive time commitment and financial resources. Results: A population-based quantitative sequencing (QS) protocol developed in this study provided a rapid estimate of species composition ratios among pooled mosquitoes as a cost-effective alternative to individual genotyping. This can be accomplished by using species- or group-specific nucleotide sequences of the mitochondrial cytochrome C oxidase subunit I (COI) and the ribosomal RNA internal transcribed spacer 2 (ITS2) region as species identification alleles in a two-step prediction protocol. Standard genomic DNA fragments of COI and ITS2 genes were amplified from each Anopheles species using group-specific universal primer sets. Following sequencing of the COI or ITS2 amplicons generated from sets of standard DNA mixtures, equations were generated via linear regression to predict species-specific nucleotide sequence frequencies at different positions. Species composition ratios between An. sineroides, An. pullus and An. lesteri were estimated from QS of the COI amplicons based on the mC.260A, mC.122C and mC.525C alleles at the first step, followed by the prediction of species composition ratios between An. sinensis, An. kleini and An. belenrae based on QS of the ITS2 amplicons using the rI.370G and rI.389T alleles. The COI copy number was not significantly different between species, suggesting the reliability of COI-based prediction. In contrast, ITS2 showed a slightly but significantly higher copy number in An. belenrae, requiring an adjustment of its predicted composition ratio. A blind test proved that predicted species composition ratios either from pooled DNA specimens or pooled mosquito specimens were not statistically different from the actual values, demonstrating that the QS-based prediction is accurate and reliable. Conclusions: This two-step prediction protocol will facilitate rapid estimation of the species composition ratios in field-collected Anopheles Hyrcanus Group populations and is particularly useful for studying the vector ecology of Anopheles population and epidemiology of malaria. [ABSTRACT FROM AUTHOR]

• Cryptic genetic lineages of Simulium chumpornense were evaluated. • Morphology and molecular data indicated the lineages are different species. • ITS2 sequences accurately identified cryptic species of S. chumpornense. • Two Leucocytozoon and one Trypanosoma species were found in S. khelangense. Species of the Simulium varicorne group in Thailand have veterinary significance as vectors of haemosporidian parasites. Accurate identification is, therefore, critical to the study of vectors and parasites. We used morphology and molecular markers to investigate cryptic genetic lineages in samples identified as Simulium chumpornense Takaoka & Kuvangkadilok, 2000. We also tested the efficiency of the nuclear internal transcribed spacer 2 (ITS2) marker for the identification of species in this group. Morphological examinations revealed that S. chumpornense lineage A is most similar to S. khelangense Takaoka, Srisuka & Saeung, 2022, with minor morphological differences. They are also genetically similar based on mitochondrial cytochrome c oxidase I (COI) sequences. Geographically, the sampling site where paratypes of S. khelangense were originally collected is <50 km from where S. chumpornense lineage A was collected. We concluded that cryptic lineage A of S. chumpornense is actually S. khelangense. COI sequences could not differentiate S. kuvangkadilokae Pramual and Tangkawanit, 2008 from S. chumpornense and S. khelangense. In contrast, ITS2 sequences provided perfect accuracy in the identification of these species. Molecular analyses of the blood protozoa Leucocytozoon and Trypanosoma demonstrated that S. khelangense carries L. shoutedeni, Leucocytozoon sp., and Trypanosoma avium. The Leucocytozoon sp. in S. khelangense differs genetically from that in S. asakoae Takaoka & Davies, 1995, signaling the possibility of vector-parasite specificity. [ABSTRACT FROM AUTHOR]

Global climate change and the accompanying rise in temperature could affect the biology and ecology of a number of vectors, including mosquitoes. High altitude areas that were previously unsuitable for the spread of mosquito vector populations could become suitable. The aim of this research was to study the distribution of mosquito species in higher altitude regions of Croatia. Samples were collected in three areas: Slavonian Mountains, Gorski Kotar, and Middle Velebit. Specimens were morphologically determined and confirmed by DNA barcoding and other genetic markers and showed the presence of 16 species belonging to six genera. The most abundant species were the Culex pipiens complex with 50% of the collected specimens. Both pipiens (Linnaeus, 1758) and molestus (Forskal, 1775) biotypes and their hybrids were identified within the complex, followed by Culex torrentium (Martini, 1925) (20.2%), Culiseta longiareolata (Macquart, 1838) (8.5%), and the invasive species Aedes japonicus (Theobald, 1901) (7.8% of the total number of collected specimens). The remaining 12 species made up 14.7% of the collected specimens. Intraspecific COI p-distances were within the standard barcoding threshold for OTUs, while interspecific genetic distances were much higher, confirming the existence of barcoding gaps. Mosquito fauna of Croatian mountains showed a moderate variety and made 30.8% of the total number of recorded mosquito species in Croatia thus far. [ABSTRACT FROM AUTHOR]

The mulberry silkworm, Bombyx mori, is known to produce silk and was introduced to the Philippines from China, Japan, Korea, and India. The silkworm germplasm in the Philippines is sourced from three centers located in regions that vary in climatic conditions: Philippine Textile Research Institute-Technology Center Misamis Oriental (PTRI-TCMO); Department of Science and Technology-Cordillera Administrative Region (CAR) in La Trinidad, Benguet; and the Don Mariano Marcos Memorial State University-Sericulture Research and Development Institute (DMMMSU-SRDI), La Union. To date, associated information with the centers' germplasm constitutes differences in biological and productive traits such as yield and quality of raw silk. It lacks genetic information on strain diversity, which provide basis for the efficient selection of parental strains with traits of economic interest. This study aimed to characterize, for the first time, the genetic diversity of 17 pure lines as parental strains. Genetic variation was evaluated using the cytochrome c oxidase 1 (COI) gene and internal transcribed spacer 2 (ITS2). BLAST analysis of the COI and ITS2 sequences confirmed the identity of B. mori strains. Phylogenetic trees constructed from COI sequences formed a monophyletic clade, while the trees based on ITS2 sequences formed two distinct clusters. The pairwise distances based on the Kimura 2-parameter (K2P) model between the B. mori strains ranged from 0-0.003 for COI and from 0-0.002 for ITS2, indicating low genetic variation for the corresponding molecular markers. Variation in the COI gene determined four nucleotide changes while variations in the ITS2 sequences determined four to six nucleotide changes, except for one strain with higher nucleotide change. This study reports that the use of COI and ITS2 as markers was not able to genotypically differentiate the 17 strains, which appear different in terms of phenotypic characters, suggesting the use of other molecular markers to characterize not only the 17 strains but all other strains in the germplasm. [ABSTRACT FROM AUTHOR]

Background: The Barotse floodplains of the upper Zambezi River and its tributaries are a highly dynamic environment, with seasonal flooding and transhumance presenting a shifting mosaic of potential larval habitat and human and livestock blood meals for malaria vector mosquitoes. However, limited entomological surveillance has been undertaken to characterize the vector community in these floodplains and their environs. Such information is necessary as, despite substantial deployment of insecticide-treated nets (ITNs) and indoor residual spraying (IRS) against Anopheles vectors, malaria transmission persists across Barotseland in Zambia's Western Province. Methods: Geographically extensive larval surveys were undertaken in two health districts along 102 km of transects, at fine spatial resolution, during a dry season and following the peak of the successive wet season. Larvae were sampled within typical Anopheles flight range of human settlements and identified through genetic sequencing of cytochrome c oxidase I and internal transcribed spacer two regions of mitochondrial and nuclear DNA. This facilitated detailed comparison of taxon-specific abundance patterns between ecological zones differentiated by hydrological controls. Results: An unexpected paucity of primary vectors was revealed, with An. gambiae s.l. and An. funestus representing < 2% of 995 sequenced anophelines. Potential secondary vectors predominated in the vector community, primarily An. coustani group species and An. squamosus. While the distribution of An. gambiae s.l. in the study area was highly clustered, secondary vector species were ubiquitous across the landscape in both dry and wet seasons, with some taxon-specific relationships between abundance and ecological zones by season. Conclusions: The diversity of candidate vector species and their high relative abundance observed across diverse hydro-ecosystems indicate a highly adaptable transmission system, resilient to environmental variation and, potentially, interventions that target only part of the vector community. Larval survey results imply that residual transmission of malaria in Barotseland is being mediated predominantly by secondary vector species, whose known tendencies for crepuscular and outdoor biting renders them largely insensitive to prevalent vector control methods. [ABSTRACT FROM AUTHOR]

The nominal genus Tasactes Faust, 1894, consisting of two originally included nominal species from Myanmar, is rediscovered for the first time since being erected. Adult weevils herein assigned to the taxonomically re-defined Tasactes were abundant in forest floor litter at five localities in China (Yunnan and Sichuan), plus one specimen is available from Shaanxi and three from Nepal. Phylogenetic analysis of a 2,275 bp matrix concatenated from one mitochondrial (COI) and two nuclear markers (ITS2 and 28S) revealed that the monophyletic Tasactes consists of eight evolutionary significant terminal clades, either allopatric (three) or sympatric (two on Cang Shan in Yunnan and three on Mount Emei in Sichuan). The genus Tasactes is nested within the monophyletic Stromboscerini, while the tribe is sister to monophyletic Dryophthorus. The two morphological diagnostic characters of Tasactes, which are unique within the tribe, are the transversely truncated antennal club and conically projecting velvety apex of the club. So defined, Tasactes renders the genus Orthosinus paraphyletic. Considering the taxonomic neglect and uncertainties surrounding nominal Stromboscerini, all herein reported members of this tribe, including the Tasactes, are not assigned to Linnaean species. This paper illustrates the "clogging taxonomy" phenomenon, in which obscure historical names render taxonomic assignment of newly sampled specimens precarious. All the data used herein (localities, sequences, specimen images) are available online in public datasets dx.doi.org/10.5883/DS-TASACT1 and dx.doi.org/10.5883/DS-TASACT2.

The area of invasive species Aedes albopictus and Aedes aegypti is expanding. Precise identification and understanding of the genetic diversity of invasive mosquito populations allows us to develop appropriate control methods. Endosymbiotic bacterium Wolbachia pipientis has different effects on their arthropod hosts and can influence the transmission and spread of the pathogens. The objective of the presented study was molecular-genetic identification of the Aedes mosquitoes collected in sampling sites on the Black Sea coast from 2007 to 2017; determination of genetic variability of Ae. aegypti, Ae. albopictus and their symbiotic bacteria Wolbachia; assessment of mosquitoes ability to be infected and to spread parasitic Dirofilaria. Another objective was obtaining the genetic characteristic of laboratory strain Ae. aegypti IMPITM. We investigated two markers of nuclear and mitochondrial DNA from Ae. albopictus and Ae. aegypti and compared them to DNA from Ae. cretinus and Ae. koreiсus sympatrically inhabiting the territory, as well as to one of Ae. aegypti from a laboratory line. The study of nuclear and mitochondrial DNA revealed a low level of variability in the invasive mosquitoes Ae. albopictus and Ae. aegypti collected at different collection sites and in different years. More than a half of Ae. albopictus were infected with Wolbachia, two strains of bacteria, wAlbA and wAlbB, occur in the Ae. albopictus population on the Black Sea coast. Total infection of Ae. aegypti and Ae. albopictus with dirofilariae was 1.8 %. Dirofilaria immitis was found only in mosquito abdomen, larvae of infective stage L3 were not found. D. repens larvae developed to the infective stage in the mosquitoes of both species.

The taxonomy of the syrphid genus Chrysotoxum Meigen, 1803 (Diptera: Syrphidae), is complex and currently under scrutiny. Two new species allied with Chrysotoxum vernale, one from the Western Mediterranean, Chrysotoxum hispanicum sp. n. and the other from the Eastern Mediterranean, Chysotoxum anatolicum sp. n., are described and illustrated. Chrysotoxum hispanicum sp. n. is distinguished from the similar C. vernale Loew by the size of the yellow abdominal fasciae and shape of surstyli. Chrysotoxum anatolicum sp. n., known only from females, possesses an almost entirely yellow-pigmented wing, unusual amongst the other studied species of the C. vernale group. Additionally, C. hispanicum sp. n. and C. anatolicum sp. n. are separated from each other, as well as from other species of the C. vernale group by COI and ITS2 gene markers. An identification key to the West Palaearctic species of the C. vernale group is provided. [ABSTRACT FROM AUTHOR]

Specimens obtained from ten populations of a Monacha species from the central Apennines were compared with six molecular lineages of Monacha cantiana s. l. (CAN-1, CAN-2, CAN-3, CAN-4, CAN-5, CAN-6) and two other Monacha species (M. cartusiana and M. parumcincta), treated as outgroup, by molecular (nucleotide sequences of two mitochondrial COI and 16S rDNA as well as two nuclear ITS2 and H3 gene fragments) and morphological (shell and genital anatomy) analysis. The results strongly suggest that these populations represent a separate species for which two names are available: the older Helix pantanellii De Stefani, 1879 and the junior M. ruffoi Giusti, 1973. The nucleotide sequences created well separated clades on each phylogenetic tree. Genital anatomy included several distinctive features concerning vaginal appendix, penis, penial papilla and flagellum; instead, shell characters only enabled them to be distinguished from M. cartusiana and M. parumcincta. Remarkably, populations of M. pantanellii show high morphological variability. Shell variability mainly concerns size, some populations having very small dimensions. Genital variability shows a more intricate pattern of all anatomical parts, being higher as regards the vagina and vaginal appendix. Despite this morphological variability, the K2P distance range of COI sequences between populations is narrow (0.2-4.5%), if we consider all but three of the 53 sequences obtained. This research confirmed that the species of Monacha and their molecularly distinguished lineages can only occasionally be recognised morphologically and that they have significant inter- and intrapopulation variability. The possibility of using an overall approach, including shell, genital and molecular evidence, was taken in order to establish a reliable taxonomic setting. [ABSTRACT FROM AUTHOR]

Satyrinae butterflies occurring in the Mediterranean apparently have reduced gene flow over sea straits, and for several species, recent wide‐scale biodiversity surveys indicate the existence of divergent mitochondrial lineages. Here, we apply an integrative approach and examine the phylogeography of the genus Lasiommata in the Western Palearctic. Our research comprised molecular analyses (mitochondrial and nuclear DNA) and geometric morphometrics (wings and genitalia) for two main species groups, and a comparative GMYC analysis, based on COI, of all the tribes within Satyrinae from this region. The GMYC approach revealed a particularly fast coalescence rate in the Parargina subtribe. The Lasiommata group was divided into 12 evolutionary significant units: six clades for the L. maera species group, five for the L. megera species group, and one for L. petropolitana, with divergences of about 1%. The patterns of COI were mirrored by ITS2 in L. maera, but the two markers were generally inconsistent in L. megera. On the contrary, morphological differences were coherent with the results of COI for L. megera, but less clearly so for L. maera.L. paramegaera and L. meadewaldoi were considerably differentiated for all the analyzed markers and likely proceeded faster in the process of speciation because of geographic isolation and reduced effective population size, rendering the rest paraphyletic. Our study illustrates the continuous nature of speciation and the difficulties of delimiting species. In Lasiommata, the recognition of taxa as diverging lineages or distinct, possibly paraphyletic species, mostly depends on the criteria adopted by different species concepts. [ABSTRACT FROM AUTHOR]

The pandemic of Zika virus in 2016 and other arboviruses prompted La Rioja Government in Spain to implement an entomological surveillance program of mosquitoes (Diptera; Culicidae) in the region of La Rioja. The morphological identification was supported by genetic analysis using the COI (cytochrome c oxidase subunit I) and the ITS2 (internal transcribed spacer 2) genes. In total, we identified 24 species arranged in 6 genera: Aedes (7 species), Anopheles (4 species), Coquillettidia (1 species), Culex (7 species), Culiseta (4 species), and Uranotaenia (1 species). Aedes sticticus and Aedes geniculatus are newly reported for La Rioja region. In total, 465 COI sequences were analyzed for Culicinae and Anophelinae and 54 ITS2 sequences for Anophelinae; all individuals identified as the same species clustered together in the Neighbor Joining trees. The levels of sequence divergence based on COI ranged between 0% and 2.62%, while the interspecific genetic divergence ranged from 3.05% to 20.07%. Within the genus Culiseta, certain specimens of Culiseta annulata, Culiseta litorea, and Culiseta subochrea were morphologically misidentified due to variation in the main diagnostic characters. The interspecific genetic divergence based on the ITS2 ranged from 0% to 2.98%. An accurate identification of mosquito vectors is the first step to establish a vector surveillance program for preventing pathogen transmission. [ABSTRACT FROM AUTHOR]

The Carabus subgenus Hygrocarabus contains two taxa: C. variolosus and C. nodulosus, the species or subspecies status of which is handled far from uniform in the literature. Both taxa show a similar morphology, the shape of the tip of the aedeagus provides a reliable morphological marker for identification. We analysed two mitochondrial gene parts (COI-5' and COI-3') and a nuclear one (ITS2). High diversity was found showing specific geographical patterns. Introgressive hybridisation was detected but interpreted not as an argument for subspecies status because high genetic distances indicated that it must have taken place in former times. In a laboratory hybridisation experiment, the male did not accept the female of the other taxon, supporting the conclusion that these are separate species. A series of refuges was expected for the period of ice ages. Although only the taxon C. variolosus is listed in Annex II and IV of the EU Habitats Directive, C. nodulosus also falls under this listing, as at the time of including the species into the Annexes in 2004, the two taxa were considered subspecies and hence the listing would include both, independent of later taxonomic revisions. [ABSTRACT FROM AUTHOR]

Melanopsidae were collected from 23 localities in central and south-eastern Europe, the Aegean Islands and Israel. This study considered Melanopsis from the Aegean Islands, the Peloponnese and Israel, and Esperiana esperi , Holandriana holandrii and Microcolpia from continental Europe. Shells and radulae were described and illustrated, showing continuous variation of shell characters and differences in radulae among genera. Phylogenetic analyses were conducted on single-locus datasets (COI , 18S, 28S, ITS2 and H3) and on a concatenated dataset. Molecular data showed a differentiation not paralleled in morphology, reflecting morphostatic evolution. In all species with ribbed shell-forms, the ribbed and unribbed forms did not differ genetically. Within Melanopsis , four clades representing five species were distinguished. Melanopsis buccinoidea is found in Khios and Lesbos Islands, M. astropaliae in Naxos, Samos and Tinos Islands, M. wagneri in Rhodes Island and the Peloponnese, and M. costata and M. saulcyi in Israel. Molecular results and the ICZN rule of priority restrict the range of M. buccinoidea to the North Aegean Islands. ' Melanopsis ' parreyssi belongs to the genus Microcolpia. Esperiana esperi belongs to a clade separate from Microcolpia. [ABSTRACT FROM AUTHOR]

Many continental species distributed in the Eurasian steppe occur as relict populations in the mountains of Western Europe. Their biogeographical responses to Quaternary climate changes have been poorly studied; however, they could have responded as cold-adapted species. We investigated the biogeographic history of a steppe beetle, Hycleus polymorphus , using mitochondrial and nuclear DNA sequences (COI , CAD , ITS2), and species distribution modelling (SDM) under present and past bioclimatic envelopes. We first performed a phylogenetic assessment to define species boundaries within the H. polymorphus species group. Specimens previously treated as Hycleus humerosus on morphological grounds are assigned to H. polymorphus , and those identified as Hycleus zebraeus assigned to Hycleus atratus. ITS2 data analyses revealed a strong phylogeographical structure of H. polymorphus populations, with four haplogroups corresponding to the (i) Italian Alps, (ii) French Alps and Pyrenees, (iii) South Balkan and Pontic mountains, and (iv) North Dinaric Alps. Based on these analyses and the SDM, we propose that during a glacial period, following the spread of steppic habitat, H. polymorphus underwent a range expansion from Asia to South-West Europe. Within the Mediterranean area, during the last interglacial the climatic suitability for the species was limited to mountains that acted as refugia and prompted allopatric divergence into four main lineages. [ABSTRACT FROM AUTHOR]

Alphaproteobacteria Wolbachia have been described as endosymbionts of approximately half of all aquatic insect species. These bacteria might affect not only reproduction but also the genetic diversity of its hosts. In the present study we identified Wolbachia endosymbiosis in freshwater true bug Aphelocheirus aestivalis F., 1794 (Heteroptera: Aphelocheiridae). Despite the fact that A. aestivalis is widely distributed in Europe, it occurs rather locally, often in isolated populations. Taking into account that Wolbachia, close relationships and past demographic phenomena could affect the genetic diversity of its host, we analyzed mitochondrial (COI and 16S) and nuclear (internal transcribed spacer 2) markers determined for A. aestivalis individuals collected from five populations. Moreover, we compared obtained COI sequences with those deposited in GenBank. Analyses revealed low genetic differentiation among samples tested, as well as low variation among determined COI sequences and those downloaded from the database. Although Wolbachia infection could correlate with decreasing mitochondrial diversity of its host, we suggest that low genetic variation observed in tested A. aestivalis samples (at both mitochondrial and nuclear levels) is a result of populations' close relationships, past demographic phenomena or is characteristic for this species. Detailed analysis of the wsp gene fragment revealed two distinct strains of Wolbachia infecting A. aestivalis. Both of them belong to supergroup A, also found in other arthropods. [ABSTRACT FROM AUTHOR]

Pseudechiniscus is a morphologically homogeneous genus of tardigrades. The morphological features commonly used for species discrimination in this genus are the dorsal sculpture, the shape and number of dorsal plates and trunk appendages. Species of the Pseudechiniscus suillus – facettalis complex are one of the most challenging tardigrades to identify. All species are similar in their general appearance and all lack trunk appendages. Moreover, not only the nominal Pseudechiniscus suillus , but also other members of the suillus – facettalis complex have been insufficiently described. In our study, we examined several populations from the Northern and the Southern Hemispheres that could be traditionally attributed to Pse. suillus. These populations were analysed using integrative taxonomy – a combination of classical morphology and morphometry with molecular data. Besides the differences in the dorsal sculpture and morphometry, we also found species-specific differences in ventral sculpture, which were originally used for discrimination of Pseudechiniscus species. Moreover, we provide an extensive discussion on all morphological and morphometric differences used in Pseudechiniscus taxonomy and indicate main taxonomic problems with this genus. Finally, we redescribe the nominal Pse. suillus from Italy. [ABSTRACT FROM AUTHOR]

Equine ocular setariasis arising mainly from ectopic infestation of Setaria digitata is a common vision impairing ophthalmic disease in India, and the identification of this filarial nematode is based solely on morphology. However, morphological characters alone are inadequate to detect and differentiate S. digitata from its congeners. The present communication reports the first phylogenetic characterization of equine S. digitata from India based on sequences derived from the mitochondrial cytochrome c oxidase subunit 1 (COI), the mitochondrial small subunit ribosomal DNA (12S rDNA), and the nuclear internal transcribed spacer 2 (ITS2). Three isolates were characterized for each gene, and respective sequences were submitted to NCBI database (MN078131, MN078132, and MN095798). The sequences were also compared with the other related sequences available from PubMed around the globe, and phylogenetic analysis was carried out in conjunction with nucleotide homologies. There was no intraspecific variation among the Indian isolates. The phylogenetic analysis of S. digitata, inferred from these genes, showed that the isolate sequences obtained from different host species created a separate monophyletic clade within the genus Setaria with minor sequence variations revealing similar molecular characteristics of S. digitata isolates throughout the globe. In addition, the studied Indian isolates were found closer to Sri Lankan isolates. The S. digitata and S. labiatopapillosa appeared as sister species. [ABSTRACT FROM AUTHOR]

This study investigated the relationship between two subspecies of the Citellophilus tesquorum flea, C. t. altaicus and C. t. sungaris, which are vectors of the bacterium Yersinia pestis that causes human plague across the vast territories of the Palearctic. Adult fleas were collected from 16 localities and 11 populations in 2019 and 2020. Specimens were morphologically verified for subspecies status and analysed for mitochondrial cytochrome c oxidase subunit I (COI) DNA, nuclear ribosomal cluster internal transcribed spacer 1 (ITS1) and ITS2, and Wolbachia-infection status. Our results demonstrated a genetic difference between C. t. altaicus and C. t. sungaris. According to mitochondrial data, the genetic distance between clades of C. t. altaicus and C. t. sungaris was comparable with the species divergence of the genus Callopsylla, which is closely related to Citellophilus. All studied populations of C. t. altaicus were Wolbachia-infected, whereas all studied populations of C. t. sungaris were symbiont-free. Data for ITS1 and ITS2 had much lower phylogenetic signals than mitochondrial data; however, diagnostic substitutions for C. t. altaicus and C. t. sungaris delimitation were also revealed. Analysis of a hardly accessible report on cross experiments allowed us to conclude the partial postzygotic isolation between these subspecies. Taken together, the molecular-genetic, geographical, morphological, and reproductive isolation findings verified that C. t. altaicus and C. t. sungaris subspecies could be considered as different species.

Billbugs (Sphenophorus spp.) are a complex of grass-feeding weevil species that reduce the aesthetic and functional qualities of turfgrass. Effective billbug monitoring and management programs rely on a clear understanding of their seasonal biology. However, our limited understanding of regional variation in the species compositions and seasonal biology of billbugs, stemming primarily from our inability to identify the damaging larval stage to species level, has hindered efforts to articulate efficient IPM strategies to growers. We used a combination of DNA barcoding methods and morphometric measures to begin filling critical gaps in our understanding of the seasonal biology of the billbug species complex across a broad geographic range. First, we developed a DNA barcoding reference library using cytochrome oxidase subunit 1 (COI) sequences from morphologically identified adult billbugs collected across Indiana, Missouri, Utah and Arizona. Next, we used our reference library for comparison and identification of unknown larval specimens collected across the growing season in Utah and Indiana. Finally, we combined our DNA barcoding approach with larval head capsule diameter, a proxy for developmental instar, to develop larval phenology charts. Adult COI sequences varied among billbug species, but variation was not influenced by geography, indicating that this locus alone was useful for resolving larval species identity. Overlaid with head capsule diameter data from specimens collected across the growing season, a better visualization of billbug species composition and seasonal biology emerged. This approach will provide researchers with the tools necessary to fill critical gaps in our understanding of billbug biology and facilitate the development of turfgrass pest management programs.