Community composition of black flies during and after the 2020 vesicular stomatitis virus outbreak in Southern New Mexico, USA.

BACKGROUND: Vesicular stomatitis virus (VSV), a vector-borne pathogen of livestock, emerges periodically in the western US. In New Mexico (NM), US, most cases occur close to the Rio Grande River, implicating black flies (Simulium spp.) as a possible vector. In 2020, VS cases were reported in NM from April to May, although total black fly abundance remained high until September. We investigated the hypothesis that transience of local VSV transmission results from transient abundance of key, competent black fly species. Additionally, we investigated whether irrigation canals in southern NM support a different community of black flies than the main river. Lastly, to gain insight into the source of local black flies, in 2023 we collected black fly larvae prior to the release of water into the Rio Grande River channel. METHODS: We randomly sub-sampled adult black flies collected along the Rio Grande during and after the 2020 VSV outbreak. We also collected black fly adults along the river in 2021 and 2022 and at southern NM farms and irrigation canals in 2022. Black fly larvae were collected from dams in the area in 2023. All collections were counted, and individual specimens were subjected to molecular barcoding for species identification. RESULTS: DNA barcoding of adult black flies detected four species in 2020: Simulium meridionale (N = 158), S. mediovittatum (N = 83), S. robynae (N = 26) and S. griseum/notatum (N = 1). Simulium robynae was only detected during the VSV outbreak period, S. meridionale showed higher relative abundance, but lower absolute abundance, during the outbreak than post-outbreak period, and S. mediovittatum was rare during the outbreak period but predominated later in the summer. In 2022, relative abundance of black fly species did not differ significantly between the Rio Grande sites and farm and irrigation canals. Intriguingly, 63 larval black flies comprised 56% Simulium vittatum, 43% S. argus and 1% S. encisoi species that were either extremely rare or not detected in previous adult collections. CONCLUSIONS: Our results suggest that S. robynae and S. meridionale could be shaping patterns of VSV transmission in southern NM. Thus, field studies of the source of these species as well as vector competence studies are warranted.

Cytotaxonomic characterization and estimation of migration patterns of onchocerciasis vectors (Simulium damnosum sensu lato) in northwestern Ethiopia based on RADSeq data.

BACKGROUND: While much progress has been made in the control and elimination of onchocerciasis across Africa, the extent to which vector migration might confound progress towards elimination or result in re-establishment of endemism in areas where transmission has been eliminated remains unclear. In Northern Ethiopia, Metema and Metekel-two foci located near the Sudan border-exhibit continuing transmission. While progress towards elimination has been faster in Metema, there remains a problematic hotspot of transmission. Whether migration from Metekel contributes to this is currently unknown. METHODOLOGY/PRINCIPLE FINDINGS: To assess the role of vector migration from Metekel into Metema, we present a population genomics study of 151 adult female vectors using 47,638 RADseq markers and mtDNA CoI sequencing. From additional cytotaxonomy data we identified a new cytoform in Metema, closely related to S. damnosum s.str, here called the Gondar form. RADseq data strongly indicate the existence of two distinctly differentiated clusters within S. damnosum s.l.: one genotypic cluster found only in Metema, and the second found predominantly in Metekel. Because blackflies from both clusters were found in sympatry (in all four collection sites in Metema), but hybrid genotypes were not detected, there may be reproductive barriers preventing interbreeding. The dominant genotype in Metema was not found in Metekel while the dominant genotype in Metekel was found in Metema, indicating that (at the time of sampling) migration is primarily unidirectional, with flies moving from Metekel to Metema. There was strong differentiation between clusters but little genetic differentiation within clusters, suggesting migration and gene flow of flies within the same genetic cluster are sufficient to prevent genetic divergence between sites. CONCLUSIONS/SIGNIFICANCE: Our results confirm that Metekel and Metema represent different transmission foci, but also indicate a northward movement of vectors between foci that may have epidemiological importance, although its significance requires further study.

Significant reduction of blackfly densities in persistent onchocerciasis area following pilot implementation of an environment friendly approach (Slash and Clear).

The effectiveness of the "Slash and Clear" method in reducing blackfly populations in low transmission areas is established, but its impact in high transmission settings with large rivers and dense vectors is yet to be proven. A community-based intervention study was conducted in the Mbam Valley, Centre Cameroon, involving two sites: Bayomen (control) and Biatsota (intervention). In each arm, baseline blackfly densities were collected over one year using the human landing method. The intervention consisted of destroying the trailing vegetation where blackflies breed. Blackfly densities were collected post-intervention to assess the impact of the intervention. Post-intervention data showed a 50.8% reduction in blackfly density in Biatsota (mean number of collected blackflies from 1936 to 953), while a reduction of 26.7% was observed in Bayomen (mean number of collected blackflies from 2418 to 1774). The reduction rate attributable to the intervention was 32.9%. Statistical analysis confirmed that the reduction in blackfly density was significantly greater in the intervention site. This study demonstrates the feasibility and significant impact of the "Slash and Clear" method in high transmission areas. However, further research is required to assess its long-term effects and determine how this strategy can be scaled up and sustained until onchocerciasis elimination is achieved.

Ecologically and medically important black flies of the genus Simulium: Identification of biogeographical groups according to similar larval niches.

The black fly genus Simulium includes medically and ecologically important species, characterized by a wide variation of ecological niches largely determining their distributional patterns. In a rapidly changing environment, species-specific niche characteristics determine whether a species benefits or not. With aquatic egg, larval and pupal stages followed by a terrestrial adult phase, their spatial arrangements depend upon the interplay of aquatic conditions and climatic-landscape parameters in the terrestrial realm. The aim of this study was to enhance the understanding of the distributional patterns among Simulium species and their ecological drivers. In an ecological niche modelling approach, we focused on 12 common black fly species with different ecological requirements. Our modelling was based on available distribution data along with five stream variables describing the climatic, land-cover, and topographic conditions of river catchments. The modelled freshwater habitat suitability was spatially interpolated to derive an estimate of the adult black flies' probability of occurrence. Based on similarities in the spatial patterns of modelled habitat suitability we were able to identify three biogeographical groups, which allows us to confirm old assessments with current occurrence data: (A) montane species, (B) broad range species and (C) lowland species. The five veterinary and human medical relevant species Simulium equinum, S. erythrocephalum, S. lineatum, S. ornatum and S. reptans are mainly classified in the lowland species group. In the course of climatic changes, it is expected that biocoenosis will slightly shift towards upstream regions, so that the lowland group will presumably emerge as the winner. This is mainly explained by wider ecological niches, including a higher temperature tolerance and tolerance to various pollutants. In conclusion, these findings have significant implications for human and animal health. As exposure to relevant Simulium species increases, it becomes imperative to remain vigilant, particularly in investigating the potential transmission of pathogens.

Successful completion of onchocerciasis elimination mapping (OEM) in Niger, West Africa.

BACKGROUND: By 1987, onchocerciasis in Niger had been successfully controlled in the six endemic river basins. In 2017, onchocerciasis elimination mapping (OEM) was carried out to determine if there was any ongoing transmission in the country as a whole. METHODS: The recommended OEM procedures were implemented. RESULTS: Ten districts, that included 35 villages, required field investigation as sites of possible transmission. None of these were found capable of supporting black fly breeding, nor was there any evidence of the presence of Simulium sp. flies. CONCLUSIONS: The implementation of OEM indicates that there is no transmission of onchocerciasis currently taking place in these newly assessed sites in Niger.

Fusarium species associated with citrus blackfly (Aleurocanthus woglumi) from an agroecological polyculture in Brazil, including an augmented description of F. volatile.

The objectives of this study were to report Fusarium species associated with Aleurocanthus woglumi (Hemiptera: Aleyrodidae) collected from citrus leaves from an agroecological polyculture in Brazil, assess sexual reproductive mode of the species with unknown sexual stages, and provide an augmented description of F. volatile, for which we discovered a sexual stage. Nineteen Fusarium isolates were recovered from A. woglumi. These fungi belong to three species complexes, i.e., the F. chlamydosporum species complex (FCSC), the F. fujikuroi species complex (FFSC), and the F. incarnatum-equiseti species complex (FIESC). Based on multilocus phylogenetic analyses, the species were identified as F. annulatum, F. chlamydosporum, F. pernambucanum, F. sulawesiense, F. verticillioides, and F. volatile. Our results suggest that three species whose sexual stages are unknown (F. chlamydosporum, F. sulawesiense, and F. volatile) are also heterothallic. Intraspecific crosses of F. sulawesiense and F. volatile produced protoperithecia, whereas 66.7% of F. volatile crosses produced fertile perithecia. We provide an augmented description of the latter species to include characteristics of its sexual morph and those observed in the asexual morph that had not yet been described for the species. This study highlights the potential of researching insect-associated fungi to increase knowledge about the diversity, taxonomy, and versatility of Fusarium in ecosystems.

Co-occurrence of dual lineages within Simulium (Gomphostilbia) atratum De Meijere in the Indonesian Archipelago along Wallace's Line.

Mitochondrial cytochrome c oxidase subunit I (COI) sequences were utilized to infer the population genetic structure of Simulium (Gomphostilbia) atratum De Meijere, an endemic simulid species to Indonesia. Both median-joining haplotype network and maximum-likelihood tree revealed two genetic lineages (A and B) within the species, with an overlap distribution in Lombok, which is situated along Wallace's line. Genetic differentiation and gene flow with varying frequencies (FST = 0.02-0.967; Nm = 0.01-10.58) were observed between populations of S. (G.) atratum, of which population pairs of different lineages showed high genetic differentiation. Notably, the high genetic distance of up to 5.92 % observed within S. (G.) atratum in Lombok was attributed to the existence of two genetically distinct lineages. The co-occurrence of distinct lineages in Lombok indicated that Wallace's line did not act as faunistic border for S. (G.) atratum in the present study. Moreover, both lineages also exhibited unimodal distributions and negative values of neutrality tests, suggesting a pattern of population expansion. The expansion and divergence time estimation suggested that the two lineages of S. (G.) atratum diverged and expanded during the Pleistocene era in Indonesia.

Optimized strategy for real-time qPCR detection of Onchocerca volvulus DNA in pooled Simulium sp. blackfly vectors.

BACKGROUND: Onchocerca volvulus is a filarial parasite that is a major cause of dermatitis and blindness in endemic regions primarily in sub-Saharan Africa. Widespread efforts to control the disease caused by O. volvulus infection (onchocerciasis) began in 1974 and in recent years, following successful elimination of transmission in much of the Americas, the focus of efforts in Africa has moved from control to the more challenging goal of elimination of transmission in all endemic countries. Mass drug administration (MDA) with ivermectin has reached more than 150 million people and elimination of transmission has been confirmed in four South American countries, with at least two African countries having now stopped MDA as they approach verification of elimination. It is essential that accurate data for active transmission are used to assist in making the critical decision to stop MDA, since missing low levels of transmission and infection can lead to continued spread or recrudescence of the disease. METHODOLOGY/PRINCIPAL FINDINGS: Current World Health Organization guidelines for MDA stopping decisions and post-treatment surveillance include screening pools of the Simulium blackfly vector for the presence of O. volvulus larvae using a PCR-ELISA-based molecular technique. In this study, we address the potential of an updated, practical, standardized molecular diagnostic tool with increased sensitivity and species-specificity by comparing several candidate qPCR assays. When paired with heat-stable reagents, a qPCR assay with a mitochondrial DNA target (OvND5) was found to be more sensitive and species-specific than an O150 qPCR, which targets a non-protein coding repetitive DNA sequence. The OvND5 assay detected 19/20 pools of 100 blackfly heads spiked with a single L3, compared to 16/20 for the O150 qPCR assay. CONCLUSIONS/SIGNIFICANCE: Given the improved sensitivity, species-specificity and resistance to PCR inhibitors, we identified OvND5 as the optimal target for field sample detection. All reagents for this assay can be shipped at room temperature with no loss of activity. The qPCR protocol we propose is also simpler, faster, and more cost-effective than the current end-point molecular assays.

Real-Time Polymerase Chain Reaction Method for the Detection of Onchocerca volvulus in Post-Elimination Surveillance of Onchocerciasis in Ecuador.

Onchocerciasis has been declared eliminated in Ecuador and surveillance measures are of great interest. In this study, we examined the infectivity rates of Simulium exiguum by Onchocerca volvulus in previously hyperendemic areas in Esmeraldas province of Ecuador. These areas had previously undergone mass administration of ivermectin, which led to the interruption of transmission in 2009 and the certification of elimination in 2014. The study included three communities in Río Cayapas and one in Río Canandé, and a total of 2,950 adult S. exiguum were collected in 2018. We used quantitative polymerase chain reaction with O. volvulus O-150 plasmid control DNA to analyze 59 pools. Our findings revealed that the infectivity rates were zero, indicating that the transmission of O. volvulus remained suspended in the area.

Progress towards elimination of onchocerciasis transmission in Mali: A "pre-stop MDA" survey in 18 transmission zones.

BACKGROUND: Onchocerciasis control activities in Mali began in 1975 with vector larviciding carried out by the Onchocerciasis Control Programme (OCP), followed by the distribution of ivermectin from 1998 until the closure of the OCP in 2002. At that time, epidemiological evaluations, using skin snip microscopy and O-150 pool screening PCR in black flies, indicated that the disease had been largely controlled as a public health problem. Ivermectin distribution was nevertheless continued after 2002 in 34 of the 75 health districts in Mali as these were known to still be meso- or hyper-endemic for onchocerciasis. In addition, the onchocerciasis sites known to be hypo-endemic for onchocerciasis benefited from the distribution of ivermectin treatment as part of the mass drug administration (MDA) program for lymphatic filariasis. Various entomological and epidemiological evaluations have now indicated that Mali may have achieved successful interruption of onchocerciasis transmission. METHODS: A series of cross-sectional surveys to update vector breeding sites throughout the endemic areas, followed by a pre-stop ivermectin mass drug administration (Pre-stop MDA) survey, were undertaken in 2019-2020. Based on breeding site findings, historical epidemiological assessments, and vector collection site maps, 18 operational transmission zones (OTZ) were delineated within which a total of 104 first line villages were selected for evaluation. Dried blood spots (DBS) samples were collected from 10,400 children (5-9 years old) from these 104 first line villages and processed for the presence of OV16 antibody using a lab-based rapid diagnostic test. RESULTS: Within the 544 Simulium damnosum s.l. breeding sites visited in all five endemic onchocerciasis endemic regions of Mali 18.01% (98/544) were seen to be active with the presence of at least one stage of S. damnosum. The overall prevalence of OV16 positive children was 0.45% (47/10,400). However, two hotspots were identified: 2.60% (13/500) seroprevalence in the OTZ number 5 in Kayes Region and 1.40% (7/500) in the OTZ number 1 of Sikasso Region. CONCLUSION: These data show that onchocerciasis prevalence in the five endemic regions has declined to levels that indicate that Stop-MDA surveys should be now carried out in most of the OTZ except for one in the Kayes Region. This latter site will need additional ivermectin treatment before reevaluation, and an OTZ in the Sikasso Region requires revaluation before possibly reinitiating MDA.

Integrated taxonomy of black flies (Diptera: Simuliidae) reveals unexpected diversity in the most arid ecosystem of Europe.

The family Simuliidae includes more than 2000 species of black flies worldwide. Their morphological uniformity creates difficulty for species identification, which limits our knowledge of their ecology and vectorial role. We investigated the systematics of black flies in a semi-arid area of the Iberian Peninsula, an ecologically harsh environment for these organisms. Sampling adult black flies in three different habitats (by means of CDC traps) and in avian nest boxes and collecting immature stages in high-salinity rills provided a representative sample of the component species. A combination of approaches, including morphological, chromosomal, and molecular (based on the mitochondrial cytochrome C oxidase subunit I (COI) and internal transcribed spacer 2 (ITS2) genes) revealed five species: four common species (Simulium intermedium, S. petricolum, S. pseudequinum, and S. rubzovianum) and the first European record for S. mellah. Barcoding gap and phylogenetic analyses revealed that ITS2 is a key marker to identify the species, whereas the COI marker does not provide enough resolution to identify some species or infer their phylogenetic relationships. Morphological and chromosomal features are also provided to identify S. mellah unequivocally. Our study highlights the need for integrated studies of black flies in ecologically extreme habitats to increase our knowledge of their distribution, ecology, and potential risks for public health.

Efficacy of topical administration of prallethrin-permethrin-piperonyl butoxide (Bronco® Equine Fly Spray) for the treatment and control of flies and other nuisance insects of horses.

Numerous biting and nuisance insects are a noted cause of discomfort and stress to horses. Pyrethrins and pyrethroids have been used for many years in numerous formulations for the control of insect pests in animals, humans and environment. There are, however, few studies reporting their field efficacy in horses. The aim of the present study was to evaluate the repellent activity of a spray formulation based on prallethrin and permethrin synergized with piperonyl butoxide (BRONCO® Equine Fly Spray, Farnam Companies, Inc., USA) against annoying and harmful insects for horses in field conditions. Nine horses of mixed breed were divided into 2 groups (treatment and control). Pre-treatment insect counts were compared to daily counts for 4 days post-treatment (pt). One minute after the administration of the product (day 0), all the horses were negative for the presence of insects. All counts up to the 6-h pt check remained negative for Hippobosca equina, tabanid flies and Simulium spp., showing 100% efficacy. This remained above 90% throughout the study. For the H. equina, the repellent efficacy remained > 99.7% for all 4 days pt, for tabanid flies > 93.3% and for Simulium spp. > 97.4%. The efficacy against Musca spp. decreased from 82.2% at day 0 to 62.2% at day 3. Treatment was well-tolerated. In conclusion, despite the low number of tested horses, Bronco® has demonstrated high insecticide and repellent efficacy and a good persistence, maintained for up to 4 days post-treatment, against the most common species of insects harmful for horses.

A new species of Simulium (Asiosimulium) (Diptera: Simuliidae) from northeastern Thailand, with its phylogenetic relationships with related species in the subgenus Asiosimulium.

Simulium (Asiosimulium) khongchiamense sp. nov. is described based on females, males, pupae, and mature larvae collected from Khong Chiam District, Ubon Ratchathani Province, northeastern Thailand. It is characterized in the female by the medium-long sensory vesicle, scutum with 3 dark longitudinal vittae and elongate cercus; in the male by the number of upper-eye (large) facets in 17 or 18 vertical rows and 18 or 19 horizontal rows, hind basitarsus moderately enlarged and ventral plate with the posterior margin moderately concave medially; in the pupa by the head and thoracic integument sparsely covered with tubercles and gill of arborescent type with 32 or 33 filaments; and in the larva by the postgenal cleft deep, reaching the posterior margin of the hypostoma and sheath of the subesophageal ganglion dark pigmented. DNA analysis based on COI gene of all known species of the subgenus Asiosimulium, except for S. shanense and S. suchitrae, indicated that this new species can be clearly differentiated from all other related species (S. phurueaense, S. oblongum, S. saeungae, S. furvum, and S. wanchaii) with interspecific genetic distances ranging between 4.79% and 19.18%. This is the eighth species of the subgenus Asiosimulium. Taxonomic notes are given to distinguish this new species from the 7 known species members in its same subgenus. Additionally, keys to species of all members in the subgenus Asiosimulium are provided.

First report of filarial nematodes in the genus Onchocerca infecting black flies (Diptera: Simuliidae) in Iran.

Black flies are blood-sucking insects of public health importance, and they are effective vectors of pathogens and parasites, such as filarial nematodes of the genus Onchocerca. Our previous surveys have shown that individuals of Simulium turgaicum are annoying pests of humans and livestock in the Aras River Basin of Iran. In the present study, adult black flies of S. turgaicum were trapped from different ecotopes of five villages in Khoda-Afarin County, Iran. By using a sensitive nested PCR assay and targeting the nuclear 18S rDNA-ITS1 marker, filarial infections were found in 38 (1.89%) of 2005 black flies. Homology exploration of 360 bp of the sequences indicated that the filarial worms are members of the family Onchocercidae, with maximum alignment scores of 93-95%. Phylogenetic analysis showed that two Iranian Onchocerca isolates were clustered in the O. fasciata-O. volvulus lineage and were well separated from other filarial nematodes. Both the entomological evidence (empty abdomen of the specimens) and climatologic data (adequate accumulated degree days for development) suggest that the filarial DNA was probably that of infective larvae of vertebrates. This is the first report of an infection by Onchocerca species in S. turgaicum and the first record of onchocercids in black flies in Iran; however, more research is required to demonstrate transmission of these filarial worms by black flies in nature.

Trends in black fly density, parity and infection rates from riverside to villages of the Bafia Health District in Cameroon: implication for onchocerciasis vector control.

BACKGROUND: Reducing contact between humans and black flies can lead to interruption of onchocerciasis transmission. The Esperanza Window Trap (EWT) has been shown to be an effective tool for reducing black fly densities. Several shape-based improvements to this trapping system have been made to optimise its effectiveness, but optimisation of this trapping system has been based most often on the shape of the trap, collection in areas of high black fly density and the addition of attractants, without considering transmission potentials and parity rates. This study aims to investigate the differences in biting rates and transmission potential between three catch points along a transect to guide the choice of EWT placement. METHODS: Monthly black fly collection was carried out over a 1-year study period using the human landing method at three catch points along a transect from the riverside toward the centre of two first-line villages (Biatsota and Bayomen), in the Mbam valley in Cameroon. All female black flies caught were counted and dissected, and entomological indicators were computed and compared between the catch points and villages. RESULTS: A total of 80,732 black flies were caught, of which 57,517 were dissected; of the latter, 2743 (4.8%) were parous and 44 (1.6%) were infective. Regarding the distance to the river, a vector density gradient was observed, with the highest annual biting rates being recorded at the riverside. The highest annual transmission potentials were also recorded at the riverside (165 vs 255 infective larvae/man/year in Bayomen and Biatsota, respectively). Overall, the highest parity rates were recorded at the riverside in Biatsota (5.1%) where various human activities are frequent and at the centre of Bayomen village (6.3%). CONCLUSION: The results of this study reveal that entomological parameters were the highest at the riverside catch sites and indicate that riverside locations should be prioritised for EWTs or other trapping systems to achieve optimal performance in onchocerciasis control.

Antibiosis interaction of black soldier flies (Hermetia illucens) (Diptera: Stratiomyidae) with house fly (Musca domestica) (Diptera: Muscidae).

Black soldier fly (Hermetia illucens (Linnaeus, 1758)) larvae inhibit oviposition of house fly (Musca domestica Linnaeus, 1758) by releasing a semiochemical, although in some situations, inhibition is only partial. We hypothesized that there is a certain period in the life cycle of black soldier fly when it can cause antibiosis of the house fly. Choice and non-choice tests were used to separately examine the effectiveness of each developmental stage of black soldier fly (egg, larval (phase I, II, and III), prepupal and pupal stages) and crude extract of larvae on house fly oviposition. Tests using each developmental stage were compared with controls lacking black soldier fly. The effects of black soldier fly on the number of newly hatched house fly larvae were evaluated and there was a significant difference between the test and control in the number of eggs laid by house fly for all phases of the black soldier fly larval stage. Strong inhibition was found in some black soldier fly larval phases. Significant differences in the numbers of house fly eggs oviposited in food containers treated with crude extract were found when compared with a control, confirming that chemicals from black soldier fly larvae resulted in inhibition of oviposition of house fly. The results from experiment also indicated that chemicals from black soldier larvae influenced the number of house fly larvae newly hatched from eggs.

Morphological redescription of Simulium takahasii (Rubtsov), the first species of the subgenus Wilhelmia Enderlein (Diptera: Simuliidae) recognized in East Asia.

Simulium takahasii (Rubtsov), which was originally described from Japan, and recorded from Korea and China, is the first among the 19 species of the subgenus Wilhelmia Enderlein recorded from East Asia. It is striking in mating, blood-feeding and ovipositing in captivity and in experimentally transmitting Dirofilaria immitis (Leidy) and Brugia pahangi (Buckley & Edeson), and it is a severe biter of cattle and horses, rarely of humans. Nevertheless, updated information about its morphological characteristics was lacking, making comparisons with related species described from China difficult, since species of the subgenus Wilhelmia are almost indistinguishable from one another, in particular, in their female terminalia, male genitalia and most of larval features. In this study, as many morphological characteristics as possible of S. takahasii based on specimens from Japan are redescribed. New information about many features of this species including the length of the female sensory vesicle against the third palpal segment, number of male upper-eye (large) facets, arrangement of the eight pupal gill filaments, presence or absence of tiny dark setae on the dorsum of the larval abdomen and the number of rows and hooklets of the larval posterior circlet will be useful in evaluating the species status of several Wilhelmia species in China including the species regarded as S. takahasii.

From bites to barcodes: uncovering the hidden diversity of black flies (Diptera: Simuliidae) in Vietnam.

BACKGROUND: Prompt and precise identification of black flies (Simuliidae) is crucial, given their biting behaviour and significant impact on human and animal health. To address the challenges presented by morphology and chromosomes in black fly taxonomy, along with the limited availability of molecular data pertaining to the black fly fauna in Vietnam, this study employed DNA-based approaches. Specifically, we used mitochondrial and nuclear-encoded genes to distinguish nominal species of black flies in Vietnam. METHODS: In this study, 135 mitochondrial cytochrome c oxidase subunit I (COI) sequences were established for 45 species in the genus Simulium in Vietnam, encompassing three subgenera (Gomphostilbia, Nevermannia, and Simulium), with 64 paratypes of 27 species and 16 topotypes of six species. Of these COI sequences, 71, representing 27 species, are reported for the first time. RESULTS: Combined with GenBank sequences of specimens from Malaysia, Myanmar, Thailand, and Vietnam, a total of 234 DNA barcodes of 53 nominal species resulted in a 71% success rate for species identification. Species from the non-monophyletic Simulium asakoae, S. feuerborni, S. multistriatum, S. striatum, S. tuberosum, and S. variegatum species groups were associated with ambiguous or incorrect identifications. Pairwise distances, phylogenetics, and species delimitation analyses revealed a high level of cryptic diversity, with discovery of 15 cryptic taxa. The current study also revealed the limited utility of a fast-evolving nuclear gene, big zinc finger (BZF), in discriminating closely related, morphologically similar nominal species of the S. asakoae species group. CONCLUSION: This study represents the first comprehensive molecular genetic analysis of the black fly fauna in Vietnam to our knowledge, providing a foundation for future research. DNA barcoding exhibits varying levels of differentiating efficiency across species groups but is valuable in the discovery of cryptic diversity.

DNA barcoding of black flies (Diptera: Simuliidae) in Indonesia.

BACKGROUND: DNA barcoding is a valuable taxonomic tool for rapid and accurate species identification and cryptic species discovery in black flies. Indonesia has 143 nominal species of black flies, but information on their biological aspects, including vectorial capacity and biting habits, remains underreported, in part because of identification problems. The current study represents the first comprehensive DNA barcoding of Indonesian black flies using mitochondrial cytochrome c oxidase subunit I (COI) gene sequences. METHODS: Genomic DNA of Indonesian black fly samples were extracted and sequenced, producing 86 COI sequences in total. Two hundred four COI sequences, including 118 GenBank sequences, were analysed. Maximum likelihood (ML) and Bayesian inference (BI) trees were constructed and species delimitation analyses, including ASAP, GMYC and single PTP, were performed to determine whether the species of Indonesian black flies could be delineated. Intra- and interspecific genetic distances were also calculated and the efficacy of COI sequences for species identification was tested. RESULTS: The DNA barcodes successfully distinguished most morphologically distinct species (> 80% of sampled taxa). Nonetheless, high maximum intraspecific distances (3.32-13.94%) in 11 species suggested cryptic diversity. Notably, populations of the common taxa Simulium (Gomphostilbia) cheongi, S. (Gomphostilbia) sheilae, S. (Nevermannia) feuerborni and S. (Simulium) tani in the islands of Indonesia were genetically distinct from those on the Southeast Asian mainland (Malaysia and Thailand). Integrated morphological, cytogenetic and nuclear DNA studies are warranted to clarify the taxonomic status of these more complex taxa. CONCLUSIONS: The findings showed that COI barcoding is a promising taxonomic tool for Indonesian black flies. The DNA barcodes will aid in correct identification and genetic study of Indonesian black flies, which will be helpful in the control and management of potential vector species.

Fast-evolving nuclear genes as barcoding markers for black flies (Diptera: Simuliidae) in Thailand.

Rapid and accurate identification is a prerequisite for the study of all aspects of species, particularly for pests and vectors. Black flies are economically significant blood-sucking insects, as many species are pests and vectors that transmit parasites to humans and other animals. We examined the efficiency of two fast-evolving nuclear genes, elongator complex protein 1 (ECP1) and big zinc finger (BZF), for identifying 13 nominal species in three species-groups of black flies, the Simulium multistriatum, S. striatum, and S. tuberosum groups, in Thailand where the mitochondrial cytochrome c oxidase I (COI) gene has not been successful for differentiating many nominal species. ECP1 gene sequences were highly effective for identification, with >96% (181 of 188) of the specimens correctly identified. Unsuccessful identifications based on ECP1 were between S. nakhonense and S. chiangmaiense, which are members of the S. striatum species-group, whereas all identifications of nominal species of the S. multistriatum and S. tuberosum species-groups were successful. In contrast, BZF had successful rates for the S. striatum species-group, with >93% (71 of 76) of the specimens correctly identified. This gene also successfully assigned unknown larvae of the S. striatum group to species. Phylogenetic analyses and molecular species delimitations based on the BZF gene uncovered cryptic diversity in two nominal species, S. nakhonense and S. wangkwaiense, which will require resolution through further study.