Sampling of Culicoides with nontraditional methods provides unusual species composition and new records for southern Spain.

BACKGROUND: Culicoides midges have been well-studied in Spain, particularly over the last 20 years, mainly because of their role as vectors of arboviral diseases that affect livestock. Most studies on Culicoides are conducted using suction light traps in farmed environments, but studies employing alternative trapping techniques or focusing on natural habitats are scarce. METHODS: In the present study, we analyze Culicoides captured in 2023 at 476 sites in western Andalusia (southern Spain) using carbon dioxide-baited Biogents (BG)-sentinel traps across different ecosystems. RESULTS: We collected 3,084 Culicoides midges (3060 females and 24 males) belonging to 23 species, including the new species Culicoides grandifovea sp. nov. and the first record of Culicoides pseudolangeroni for Europe. Both species were described with morphological and molecular methods and detailed data on spatial distribution was also recorded. The new species showed close phylogenetic relations with sequences from an unidentified Culicoides from Morocco (92.6% similarity) and with Culicoides kurensis. Culicoides imicola was the most abundant species (17.4%), followed by Culicoides grandifovea sp. nov. (14.6%) and Culicoides kurensis (11.9%). Interestingly, Culicoides montanus was the only species of the obsoletus and pulicaris species complexes captured, representing the first record of this species in southern Spain. A total of 53 valid Culicoides species have been reported in the area, with 48 already reported in literature records and 5 more added in the present study. Information on the flight period for the most common Culicoides species is also provided. CONCLUSIONS: To the best of our knowledge, our study represents the most comprehensive effort ever done on nonfarmland habitats using carbon-dioxide baited suction traps for collecting Culicoides. Our data suggests that using carbon dioxide traps offers a completely different perspective on Culicoides communities compared with routinely used light traps, including the discovery of previously unrecorded species.

Population genetics of the biting midge Culicoides oxystoma Kieffer (Diptera: Ceratopogonidae) from Thailand and its genetic relationships with global populations.

Culicoides oxystoma Kieffer is a vector of viruses, filarial nematodes and protozoa of the genus Leishmania transmitted to humans and other animals. Understanding genetic diversity, genetic structure and genetic relationships among geographically widespread populations will provide important information related to disease epidemiology. In this study, genetic diversity, genetic structure and genetic relationships between Thai C. oxystoma and those reported from other countries were inferred based on mitochondrial cytochrome oxidase I (COI) and nuclear internal transcribed spacer 1 (ITS-1) sequences. A high level of genetic diversity was found in C. oxystoma from Thailand. The maximum K2P intraspecific genetic divergence for COI gene and ITS-1 sequences were 4.29% and 6.55%, respectively. Despite high genetic diversity, no significant genetic differentiation was found within the 13 Thai populations. This could be a result of unspecialized habitat requirement of the larval habitat, abundance and continuous distribution of host blood sources, potential for long distance movement with host via trading. Mitochondrial genealogy analysis of the global population of C. oxystoma revealed three (A, B and C) genetically divergent lineages. Specimens from Thailand were included in the main lineage (A) with those from all other countries except those from Senegal that formed lineage B and those of Lineage C that was exclusively found in Bangladesh. The nuclear (ITS-1) genetic markers genealogy indicated that Thai C. oxystoma belong to the same genet.

Transcriptomic Investigation of the Virus Spectrum Carried by Midges in Border Areas of Yunnan Province.

Yunnan province in China shares its borders with three neighboring countries: Myanmar, Vietnam, and Laos. The region is characterized by a diverse climate and is known to be a suitable habitat for various arthropods, including midges which are notorious for transmitting diseases which pose significant health burdens affecting both human and animal health. A total of 431,100 midges were collected from 15 different locations in the border region of Yunnan province from 2015 to 2020. These midges were divided into 37 groups according to the collection year and sampling site. These 37 groups of midges were then homogenized to extract nucleic acid. Metatranscriptomics were used to analyze their viromes. Based on the obtained cytochrome C oxidase I gene (COI) sequences, three genera were identified, including one species of Forcipomyia, one species of Dasyhelea, and twenty-five species of Culicoides. We identified a total of 3199 viruses in five orders and 12 families, including 1305 single-stranded positive-stranded RNA viruses (+ssRNA) in two orders and seven families, 175 single-stranded negative-stranded RNA viruses (-ssRNA) in two orders and one family, and 1719 double-stranded RNA viruses in five families. Six arboviruses of economic importance were identified, namely Banna virus (BAV), Japanese encephalitis virus (JEV), Akabane virus (AKV), Bluetongue virus (BTV), Tibetan circovirus (TIBOV), and Epizootic hemorrhagic disease virus (EHDV), all of which are capable, to varying extents, of causing disease in humans and/or animals. The survey sites in this study basically covered the current distribution area of midges in Yunnan province, which helps to predict the geographic expansion of midge species. The complexity and diversity of the viral spectrum carried by midges identified in the study calls for more in-depth research, which can be utilized to monitor arthropod vectors and to predict the emergence and spread of zoonoses and animal epidemics, which is of great significance for the control of vector-borne diseases.

Species-level Microbiota of Biting Midges and Ticks from Poyang Lake.

Objective: The purpose of this study was to investigate the bacterial communities of biting midges and ticks collected from three sites in the Poyang Lake area, namely, Qunlu Practice Base, Peach Blossom Garden, and Huangtong Animal Husbandry, and whether vectors carry any bacterial pathogens that may cause diseases to humans, to provide scientific basis for prospective pathogen discovery and disease prevention and control. Methods: Using a metataxonomics approach in concert with full-length 16S rRNA gene sequencing and operational phylogenetic unit (OPU) analysis, we characterized the species-level microbial community structure of two important vector species, biting midges and ticks, including 33 arthropod samples comprising 3,885 individuals, collected around Poyang Lake. Results: A total of 662 OPUs were classified in biting midges, including 195 known species and 373 potentially new species, and 618 OPUs were classified in ticks, including 217 known species and 326 potentially new species. Surprisingly, OPUs with potentially pathogenicity were detected in both arthropod vectors, with 66 known species of biting midges reported to carry potential pathogens, including Asaia lannensis and Rickettsia bellii, compared to 50 in ticks, such as Acinetobacter lwoffii and Staphylococcus sciuri. We found that Proteobacteria was the most dominant group in both midges and ticks. Furthermore, the outcomes demonstrated that the microbiota of midges and ticks tend to be governed by a few highly abundant bacteria. Pantoea sp7 was predominant in biting midges, while Coxiella sp1 was enriched in ticks. Meanwhile, Coxiella spp., which may be essential for the survival of Haemaphysalis longicornis Neumann, were detected in all tick samples. The identification of dominant species and pathogens of biting midges and ticks in this study serves to broaden our knowledge associated to microbes of arthropod vectors. Conclusion: Biting midges and ticks carry large numbers of known and potentially novel bacteria, and carry a wide range of potentially pathogenic bacteria, which may pose a risk of infection to humans and animals. The microbial communities of midges and ticks tend to be dominated by a few highly abundant bacteria.

Decoding the genome of bloodsucking midge Forcipomyia taiwana (Diptera: Ceratopogonidae): Insights into odorant receptor expansion.

Biting midges, notably those within the Ceratopogonidae family, have long been recognized for their epidemiological significance, both as nuisances and vectors for disease transmission in vertebrates. Despite their impact, genomic insights into these insects, particularly beyond the Culicoides genus, remain limited. In this study, we assembled the Forcipomyia taiwana (Shiraki) genome, comprising 113 scaffolds covering 130.4 Mbps-with the longest scaffold reaching 7.6 Mbps and an N50 value of 2.6 Mbps-marking a pivotal advancement in understanding the genetic architecture of ceratopogonid biting midges. Phylogenomic analyses reveal a shared ancestry between F. taiwana and Culicoides sonorensis Wirth & Jones, dating back approximately 124 million years, and highlight a dynamic history of gene family expansions and contractions within the Ceratopogonidae family. Notably, a substantial expansion of the odorant receptor (OR) gene family was observed, which is crucial for the chemosensory capabilities that govern biting midges' interactions with their environment, including host seeking and oviposition behaviors. The distribution of OR genes across the F. taiwana genome displays notable clusters on scaffolds, indicating localized tandem gene duplication events. Additionally, several collinear regions were identified, hinting at segmental duplications, inversions, and translocations, contributing to the olfactory system's evolutionary complexity. Among the 156 ORs identified in F. taiwana, 134 are biting midge-specific ORs, distributed across three distinct clades, each exhibiting unique motif features that distinguish them from the others. Through weighted gene co-expression network analysis, we correlated distinct gene modules with sex and reproductive status, laying the groundwork for future investigations into the interplay between gene expression and adaptive behaviors in F. taiwana. In conclusion, our study not only highlights the unique olfactory repertoire of ceratopogonid biting midges but also sets the stage for future studies into the genetic underpinnings of their unique biological traits and ecological strategies.

Large-Scale Cytochrome C Oxidase Subunit I Gene Data Analysis for the Development of a Multiplex Polymerase Chain Reaction Test Capable of Identifying Biting Midge Vector Species and Haplotypes (Diptera: Ceratopogonidae) of the Culicoides Subgenus Avaritia Fox, 1955.

The emergence of culicoid-transmitted bluetongue and Schmallenberg viruses in several European countries demonstrated the ability of indigenous biting midge species to transmit pathogens. Entomologic research programs identified members of the Obsoletus Group (Culicoides subgenus Avaritia) as keyplayers in disease epidemiology in Europe. However, morphological identification of potential vectors is challenging due to the recent discovery of new genetic variants (haplotypes) of C. obsoletus sensu stricto (s.s.), forming distinct clades. In this study, 4422 GenBank entries of the mitochondrial cytochrome c oxidase subunit I (COI) gene of subgenus Avaritia members of the genus Culicoides were analyzed to develop a conventional multiplex PCR, capable of detecting all vector species and clades of the Western Palearctic in this subgenus. Numerous GenBank entries incorrectly assigned to a species were identified, analyzed and reassigned. The results suggest that the three C. obsoletus clades represent independent species, whereas C. montanus should rather be regarded as a genetic variant of C. obsoletus s.s. Based on these findings, specific primers were designed and validated with DNA material from field-caught biting midges which achieved very high diagnostic sensitivity (100%) when compared to an established reference PCR (82.6%).

Full genome characterization and evolutionary analysis of Banna virus isolated from Culicoides, mosquitoes and ticks in Yunnan, China.

Introduction: Banna virus (BAV), a potential pathogen that may cause human encephalitis, is the prototype species of genus Seadornaviru within the family Reoviridae, and has been isolated from a variety of blood-sucking insects and mammals in Asia. Methods: Culicoides, Mosquitoes, and Ticks were collected overnight in Yunnan, China, during 2016-2023 using light traps. Virus was isolated from these collected blood-sucking insects and grown using Aedes albopictus (C6/36) cells. Preliminary identification of the virus was performed by agarose gel electrophoresis (AGE). The full genome sequences of the BAVs were determined by full-length amplification of cDNAs (FLAC) and sequenced using next-generation sequencing. Results: In this study, 13 strains BAV were isolated from Culicoides, Mosquitoes and Ticks. Their viral genome consisted of 12 segments of double-stranded RNA (dsRNA), and with three distinct distribution patterns. Sequence analysis showed that Seg-5 of four strains (SJ_M46, SJ_M49, JC_M19-13 and JC_C24-13) has 435 bases nucleotide sequence insertions in their ORF compared to other BAVs, resulting in the length of Seg-5 up to 2128 nt. There are 34 bases sequence deletion in Seg-9 of 3 strains (WS_T06, MS_M166 and MS_M140). Comparison of the coding sequences of VP1, VP2, VP5, VP9 and VP12 of the 13 BAV strains, the results show that VP1, VP2 and VP12 are characterised by high levels of sequence conservation, while VP9 is highly variable, under great pressure to adapt and may be correlated with serotype. While also variable, VP5 appears to be under less adaptive pressure than VP9. Additionally, phylogenetic analysis indicates that the 13 BAV strains locate in the same evolutionary cluster as BAVs isolated from various blood-sucking insects, and are clustered according to geographical distribution. Conclusion: The data obtained herein would be beneficial for the surveillance of evolutionary characteristics of BAV in China and neighboring countries as well as extend the knowledge about its genomic diversity and geographic distribution.

Isolation and identification of Tete virus group (Peribunyaviridae: Orthobunyavirus) from Culicoides biting midges collected in Lichuan County, China.

In July 2018, a virus (JXLC1806-2) was isolated from Culicoides biting midges collected in Lichuan County, Jiangxi Province, China. The virus isolate showed significant cytopathic effects within 48 hours after inoculation with mammalian cells (BHK-21). JXLC1806-2 virus could form plaques in BHK-21 cells, and the virus titer was 1×105.6 pfu/mL. After inoculation with the virus, suckling mice developed disease and died. The nucleotide and amino sequence analysis showed that the JXLC1806-2 virus genome was composed of S, M and L segments. Phylogenetic analysis showed that the S, M and L genes of JXLC1806-2 virus belonged to the Tete serogroup, Orthobunyavirus, but formed an independent evolutionary branch from the other members of the Tete serogroup. The results showed that the JXLC1806-2 virus, which was named as Lichuan virus, is a new member of Tete serogroup, and this is the first time that a Tete serogroup virus has been isolated in China.

Vesicular Stomatitis Virus Elicits Early Transcriptome Response in Culicoides sonorensis Cells.

Viruses that are transmitted by arthropods, or arboviruses, have evolved to successfully navigate both the invertebrate and vertebrate hosts, including their immune systems. Biting midges transmit several arboviruses including vesicular stomatitis virus (VSV). To study the interaction between VSV and midges, we characterized the transcriptomic responses of VSV-infected and mock-infected Culicoides sonorensis cells at 1, 8, 24, and 96 h post inoculation (HPI). The transcriptomic response of VSV-infected cells at 1 HPI was significant, but by 8 HPI there were no detectable differences between the transcriptome profiles of VSV-infected and mock-infected cells. Several genes involved in immunity were upregulated (ATG2B and TRAF4) or downregulated (SMAD6 and TOLL7) in VSV-treated cells at 1 HPI. These results indicate that VSV infection in midge cells produces an early immune response that quickly wanes, giving insight into in vivo C. sonorensis VSV tolerance that may underlie their permissiveness as vectors for this virus.

Common but not connected: high genetic structure and cryptic genetic diversity in the ubiquitous biting midge Culicoides peregrinus Kieffer.

The biting midge Culicoides peregrinus Kieffer is a significant pest and vector species, and knowledge of its genetic diversity and genetic structure is critically important for designing an effective control program. However, such information is limited to only small sample-size DNA barcoding studies. Therefore, in this study, we used mitochondrial cytochrome c oxidase I (COI) to examine genetic structure and diversity of C. peregrinus from northeastern Thailand. In addition, we also inferred genetic relationships between C. peregrinus from Thailand and those reported from other countries across the geographic range of the species. Maximum intraspecific genetic divergence (3.83%) within Thai specimens was relatively high compared to other Culicoides species. Genetic structure analysis revealed that 71% (32 from 45) of population comparisons were highly significantly different. A high level of genetic structure among populations, even between those in close geographic proximity (22 km geographic distance) suggested that there has been little or no movement between local populations. This is possibly due to the ability to exploit diverse types of breeding site and a generalist feeding habit which enables C. peregrinus to complete its life cycle within cattle pens. Genetic relationships between Thai C. peregrinus and those reported from other countries revealed three genetically divergent lineages (A, B and C) associated with geographic origins. Specimens from Thailand + China formed lineage A, those from Australia formed lineage B and India + Bangladesh belonged to lineage C. These genetically divergent lineages also agree with morphological variation of the wing pale marking spots. Further investigation using independent genetic loci from nuclear genes will be very useful to resolve taxonomic status of these divergent lineages.

Evaluation of potential reference genes in the biting midge Culicoides sonorensis for real-time quantitative PCR analyses.

Studies examining differentially expressed genes and gene silencing by RNA interference (RNAi) require a set of stably expressed reference genes for accurate normalization. The biting midge Culicoides sonorensis is an important vector of livestock pathogens and is often used as a model species for biting midge research. Here, we examine the stable expression of six candidate reference genes in C. sonorensis: actin, ß-tubulin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ribosomal protein subunit (RPS) 18, vacuolar ATPase subunit A (VhaA), and elongation factor 1-beta (EF1b). Gene expression was assessed under seven conditions, including cells treated with double-stranded RNA (dsRNA), 3rd and 4th instar larvae treated with dsRNA, six developmental stages, four adult female body parts or tissue groups, and females injected with bluetongue virus or vesicular stomatitis virus. Stable gene expression was assessed using RefFinder, NormFinder, geNorm, and BestKeeper. The ranked results for each analysis tool under each condition and a comprehensive ranking for each condition are presented. The data show that optimal reference genes vary between conditions and that just two reference genes were necessary for each condition. These findings provide reference genes for use under these conditions in future studies using real-time quantitative PCR to evaluate gene expression in C. sonorensis.

Wing morphometrics of biting midges (Diptera: Culicoides) of veterinary importance in Madagascar.

Biting midges are vectors of arboviruses such as bluetongue virus, bovine ephemeral fever virus, Akabane virus, African horse sickness virus, epizootic haemorrhagic disease virus and Schmallenberg virus. Fast and accurate identification of biting midges is crucial in the study of Culicoides-borne diseases. Morphological identification of biting midges has revealed the presence of cryptic species. A total of 20 species are reported in Madagascar. In this study, we assessed wing morphometric analysis for identification of seven species namely C. dubitatus Kremer, Rebholtz-Hirtzel and Delécolle, C. enderleini Cornet and Brunhes, C. kibatiensis Goetghebuer, C. miombo Meiswinkel, C. moreli Clastrier, C. nevilli Cornet and Brunhes, and C. zuluensis de Meillon. Culicoides enderleini, C. miombo, C. moreli, C. nevilli and C. zuluensis are vectors diseases. A molecular approach, based on the cytochrome oxidase I gene (Cox1), was used for species delimitation. The molecular analysis presented seven different clades grouped two-by-two according to morphological characters. A total of 179 wing images were digitised. We found morphometric variation among seven species based on 11 landmarks and two outlines. Wing shape variation plots showed that species overlapped with species belonging to the same group. The cross-validation revealed a relatively high percentage of correct classification in most species, ranging from 91.3% to 100% for landmarks; 60% to 82.6% for outlines-1 and 77.1% to 91.3% for outlines-2. Our study suggests that wing geometric morphometric analysis is a robust tool for reliable "Moka Fohy" identification in Madagascar. This inexpensive and simple method is a precise supplement to morphological identification, with reaches the accuracy of Cox1 barcoding.

Immigration and seasonal bottlenecks: high inbreeding despite high genetic diversity in an oscillating population of Culicoides sonorensis (Diptera: Ceratopogonidae).

Most population genetic studies concern spatial genetic differentiation, but far fewer aim at analyzing the temporal genetic changes that occur within populations. Vector species, including mosquitoes and biting midges, are often characterized by oscillating adult population densities, which may affect their dispersal, selection, and genetic diversity over time. Here, we used a population of Culicoides sonorensis from a single site in California to investigate short-term (intra-annual) and long-term (inter-annual) temporal variation in genetic diversity over a 3 yr period. This biting midge species is the primary vector of several viruses affecting both wildlife and livestock, thus a better understanding of the population dynamics of this species can help inform epidemiological studies. We found no significant genetic differentiation between months or years, and no correlation between adult populations and the inbreeding coefficient (FIS). However, we show that repeated periods of low adult abundance during cooler winter months resulted in recurring bottleneck events. Interestingly, we also found a high number of private and rare alleles, which suggests both a large, stable population, as well as a constant influx of migrants from nearby populations. Overall, we showed that the high number of migrants maintains a high level of genetic diversity by introducing new alleles, while this increased diversity is counterbalanced by recurrent bottleneck events potentially purging unfit alleles each year. These results highlight the temporal influences on population structure and genetic diversity in C. sonorensis and provide insight into factors effecting genetic variation that may occur in other vector species with fluctuating populations.

Uncovering the Underlying Mechanisms Blocking Replication of Bluetongue Virus Serotype 26 (BTV-26) in Culicoides Cells.

At least 12 serotypes of 'atypical' bluetongue virus (BTV-25 to BTV-36) have been identified to date. These atypical serotypes fail to infect/replicate in Culicoides-derived cell lines and/or adult Culicoides vectors and hence can no longer be transmitted by these vectors. They appear to be horizontally transmitted from infected to in-contact ruminants, although the route(s) of infection remain to be identified. Viral genome segments 1, 2 and 3 (Seg-1, Seg2 and Seg-3) of BTV-26 were identified as involved in blocking virus replication in KC cells. We have developed Culicoides-specific expression plasmids, which we used in transfected insect cells to assess the stability of viral mRNAs and protein expression from full-length open reading frames of Seg-1, -2 and -3 of BTV-1 (a Culicoides-vectored BTV) or BTV-26. Our results indicate that the blocked replication of BTV-26 in KC cells is not due to an RNAi response, which would lead to rapid degradation of viral mRNAs. A combination of degradation/poor expression and/or modification of the proteins encoded by these segments appears to drive the failure of BTV-26 core/whole virus-particles to assemble and replicate effectively in Culicoides cells.

Genome-wide analyses of Glutathione S-transferase gene family and expression profiling under deltamethrin exposure in non-biting midge Propsilocerus akamusi.

Glutathione S-transferases (GSTs) are major enzymes in detoxification phase II, and have been functioned in resistance to various insecticides or oxidative stress. Herein, we selected the non-biting midge, Propsilocerus akamusi, widespread in Asian aquatic ecosystems, to uncover the gene location, structure, and phylogenetics relationship of GSTs at genome scale first time. Thirty-three cytosolic and four microsomal GST genes were identified and located on the four chromosomes. The cytosolic GSTs involved in the eight subclasses and five GST genes were unclassified. The expansion of GST genes in P. akamusi experienced duplication events on the delta, theta, xi, iota, and unclassified subclasses. The RNA-Seq analyses and RT-qPCR validation showed that the expression of PaGSTt2 gene is significantly elevated, with deltamethrin concentration increasing. The tertiary structure of PaGSTt2 enzyme was reconstructed, which was different from the other theta gene in the active site. In addition, the GST genes of six chironomids were first described based on the assembled genomes to explore the difference of those in the adaptation to kinds of environments. The GST frame for P. akmusi and its expression profiles provide valuable resources to understand their role in insecticide resistance of this species, as well as those of other biting midges.

DNA Barcoding Culicoides Biting Midges (Diptera: Ceratopogonidae) in Northeast Brazil.

Biting midges of the genus Culicoides are small insects associated with the transmission of several pathogens, which requires the correct identification of the species, for implementation of effective strategies against these insects. However, many species are difficult to identify only by morphological characters. Therefore, the use of molecular methods can help in the taxonomy and systematics of this group. Here, the DNA barcode approach was evaluated for nine species of Culicoides from the State of Maranhão, Brazil. We generated 39 sequences from a 476 bp (base pairs) fragment of the cytochrome c oxidase subunit I (COI) mitochondrial gene. To assess the usefulness of COI barcodes for the identification of these species, paired genetic distances from intra and interspecific comparisons and phylogenetic trees were generated in MEGA and RAxML/BEAST softwares, respectively. In addition, species delimitation was performed using the PTP, GMYC, and ABGD algorithms. The intra and interspecific genetic distances showed a clear distinction between them, demonstrating that, for the taxa studied, there can hardly be ambiguous identifications with barcodes. In the same sense, the phylogenetic reconstruction resulted in well-supported clades for all morphospecies analyzed.

Effects of storage conditions and digestion time on DNA amplification of biting midge (Culicoides) blood meals.

BACKGROUND: Molecular analysis of blood meals is increasingly used to identify the hosts of biting insects such as midges and mosquitoes. Successful host identification depends on the availability of sufficient host DNA template for PCR amplification, making it important to understand how amplification success changes under different storage conditions and with different durations of blood meal digestion within the insect gut before being placed into the storage medium. METHOD: We characterised and compared the digestion profile of two species of Culicoides over a 96-h period using a novel set of general vertebrate primers targeting the 16S rRNA gene. A set number of individuals from each species were killed over 13 time points post-blood feeding and preserved in 95% ethanol. Samples were stored either at ambient room temperature or in a - 20 °C freezer to examine the effect of storage condition on the PCR amplification success of host DNA. RESULTS: We found that amplification success across the 96-h sampling period post-feeding was reduced from 96 to 6% and 96% to 14% for Culicoides nubeculosus and Culicoides sonorensis, respectively. We found no effect of storage condition on PCR amplification success, and storage in 95% ethanol was sufficient to maintain high rates of amplifiable host DNA for at least 9 months, even at room temperature. CONCLUSIONS: These findings highlight the limited time frame during which an individual may contain amplifiable host DNA and demonstrate the importance of timely sample capture and processing post-blood feeding. Moreover, storage in 95% ethanol alone is sufficient to limit host DNA degradation. These results are relevant to the design of studies investigating the biting behaviour and disease transmission potential of Culicoides and other biting Diptera.

DNA Barcoding of Culicoides Latreille (Diptera: Ceratopogonidae) From Thailand Reveals Taxonomic Inconsistencies and Novel Diversity Among Reported Sequences.

Recent focus on Culicoides species diversity in Thailand was prompted by a need to identify vectors responsible for the transmission of African Horse Sickness in that country. To assist rapid genetic identification of species, we sampled mitochondrial cytochrome c oxidase subunit I (COI) DNA barcodes (N = 78) from 40 species of Culicoides biting midge from Thailand, including 17 species for which DNA barcodes were previously unavailable. The DNA barcodes were assigned to 39 Barcode Identification Numbers (BINs) representing terminal genetic clusters at the Barcode of Life Data systems (BOLD). BINs assisted with comparisons to published conspecific DNA barcodes and allowed partial barcodes obtained from seven specimens to be associated with BINs by their similarity. Some taxonomic issues were revealed and attributed to the possible misidentification of earlier reported specimens as well as a potential synonymy of C. elbeli Wirth & Hubert and C. menglaensis Chu & Liu. Comparison with published BINs also revealed genetic evidence of divergent population processes and or potentially cryptic species in 16 described taxa, flagged by their high levels of COI sequence difference among conspecifics. We recommend the BOLD BIN system to researchers preparing DNA barcodes of vouchered species for public release. This will alert them to taxonomic incongruencies between their records and publicly released DNA barcodes, and also flag genetically deep and potentially novel diversity in described species.

Mitochondrial genome sequencing, mapping, and assembly benchmarking for Culicoides species (Diptera: Ceratopogonidae).

BACKGROUND: Mitochondrial genomes are the most sequenced genomes after bacterial and fungal genomic DNA. However, little information on mitogenomes is available for multiple metazoan taxa, such as Culicoides, a globally distributed, megadiverse genus containing 1,347 species. AIM:  Generating novel mitogenomic information from single Culicoides sonorensis and C. biguttatus specimens, comparing available mitogenome mapping and de novo assembly tools, and identifying the best performing strategy and tools for Culicoides species. RESULTS: We present two novel and fully annotated mitochondrial haplotypes for two Culicoides species, C. sonorensis and C. biguttatus. We also annotated or re-annotated the only available reference mitogenome for C. sonorensis and C. arakawae. All species present a high similarity in mitogenome organization. The general gene arrangement for all Culicoides species was identical to the ancestral insect mitochondrial genome. Only short spacers were found in C. sonorensis (up to 30 bp), contrary to C. biguttatus (up to 114 bp). The mitochondrial genes ATP8, NAD2, NAD6, and LSU rRNA exhibited the highest nucleotide diversity and pairwise interspecific p genetic distance, suggesting that these genes might be suitable and complementary molecular barcodes for Culicoides identification in addition to the commonly utilized COI gene. We observed performance differences between the compared mitogenome generation strategies. The mapping strategy outperformed the de novo assembly strategy, but mapping results were partially biased in the absence of species-specific reference mitogenome. Among the utilized tools, BWA performed best for C. sonorensis while SPAdes, MEGAHIT, and MitoZ were among the best for C. biguttatus. The best-performing mitogenome annotator was MITOS2. Additionally, we were able to recover exogenous mitochondrial DNA from Bos taurus (biting midges host) from a C. biguttatus blood meal sample. CONCLUSIONS: Two novel annotated mitogenome haplotypes for C. sonorensis and C. biguttatus using High-Throughput Sequencing are presented. Current results are useful as the baseline for mitogenome reconstruction of the remaining Culicoides species from single specimens to HTS and genome annotation. Mapping to a species-specific reference mitogenome generated better results for Culicoides mitochondrial genome reconstruction than de novo assembly, while de novo assembly resulted better in the absence of a closely related reference mitogenome. These results have direct implications for molecular-based identification of these vectors of human and zoonotic diseases, setting the basis for using the whole mitochondrial genome as a marker in Culicoides identification.

DNA Barcoding of genus Culicoides biting midges (Diptera: Ceratopogonidae) in the Brazilian Amazon.

Culicoides biting midges are capable to transmit Oropouche virus, Bluetongue virus and Mansonella spp. This study aimed to assess the utility of DNA barcode as an alternative method in the Culicoides species identification. The study was conducted in Jamari National Forest. Biting midges were collected using HP light traps during four months, February, April, August and October 2018. Insects were morphologically identified to the species level, and rest of the body were subjected to DNA extraction and PCR targeting a fragment of the cytochrome c oxidase subunit I (COI) gene, which were analyzed and deposited in GenBank. A phylogenetic gene tree was reconstructed using RAxML software, and the sequences were assigned at Molecular Operational Taxonomic Unit (MOTU) level by species delimitation algorithms. According to morphological approach, 18 species of 2 subgenera and 7 species groups were identified. A total of 191 new COI barcodes from 18 species were generated. Of these, fifteen species have been deposited for the first time in all datasets in the world. These sequences allowed the correct identification of 188 and 187 specimens according to the BM and BCM criteria, respectively. The intraspecific genetic distances ranged from 0 to 16.5%, while the interspecific ones ranged from 2.1 to 27.1%. The nominal species Culicoides glabellus and C. tetrathyris splitted into three and two MOTUs, respectively, except for mPTP, indicating a cryptic diversity in these species. Also, sequences of C. pseudodiabolicus formed two MOTUs using all algorithms, except for PTP and ABGD, suggesting the existence of two potential species. In contrast, some barcodes of C. quasiparaensis and C. paraensis merged into a single MOTU, which can be explained by the complex characteristics of the paraensis group, since these species have similar morphological characters. Here, we provided the first COI barcodes for biting midges in Rondônia and Brazil, and demonstrates that these are sufficient to discriminate between some species.