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We explored how the ratio of blood-feeding behaviour-nonfeeding, bird feeding (ornithophily) and mammal feeding (mammalophily)-and voltinism (univoltine and multivoltine) vary over a latitudinal gradient from Alaska to Florida. These two fixed species traits were divided into five mutually exclusive combinations of trait states-nonfeeding/univoltine, ornithophilic/univoltine, mammalophilic/univoltine, ornithophilic/multivoltine and mammalophilic/multivoltine-within each of three datasets (north, east and west). We found a significant association between location (north, east and west) and trait state, which was driven by the large percentage of nonfeeding females in the north. When this trait state was removed, no significant differences were found for the remaining trait states and locations. Although the distribution of trait states did not differ between east and west datasets, the distribution with relation to 1° changes in latitude within each of these datasets showed distinct differences. In the east, both ornithophilic/univoltine and mammalophilic/univoltine species significantly increased with latitude, in proportion to the total species present. In contrast, the proportion of mammalophilic/multivoltine species decreased as latitude increased. Ornithophilic/multivoltine species in the east and the trait states in the west did not show any significant relationship to latitude., (© 2024 The Author(s). Medical and Veterinary Entomology published by John Wiley & Sons Ltd on behalf of Royal Entomological Society.)

Background: Leucocytozoonosis, a parasitic disease of birds, is caused by haemosporidian protozoan parasites of the genus Leucocytozoon, which infect diverse avian species, including poultry. These parasites are transmitted by several black fly species, but knowledge of the factors determining the diversity and prevalence in these vectors, which is crucial for fully understanding disease epidemiology, is largely unexplored. In this study, we investigated factors associated with the prevalence and diversity of Leucocytozoon species in black flies from Thailand., Methods: Adults of two black fly taxa (Simulium asakoae Takaoka and Davies complex and S. khelangense Takaoka, Srisuka and Saeung) were collected using sweep nets at nine locations in northern and northeastern regions of Thailand. Specimens were identified morphologically and the results corroborated by DNA barcoding. Molecular methods using specific primers for amplification of the mitochondrial cytochrome b (cyt b) gene of Leucocytozoon were used to detect the parasite in black flies. Species and lineages of Leucocytozoon were determined using the MalAvi database of malaria parasites and related haemosporidians in avian hosts. Regression analysis was used to examine relationships between Leucocytozoon diversity and prevalence, black fly abundance and habitat characteristics., Results: A total of 11,718 adult black flies were collected, of which 4367 were members of the S. asakoae complex and 7351 were S. khelangense. For molecular detection of Leucocytozoon, we randomly selected 300 individual female black flies of the S. asakoae complex and 850 females of S. khelangense pooled into groups of five individuals (= 170 pools). A total of 34 of the 300 specimens of the S. asakoae complex and 118 of the 170 pools of S. khelangense were positive for Leucocytozoon. Fifty-four lineages (haplotypes) were identified, all of which belonged to those reported in domestic chickens, Gallus gallus, with one exception that was identified in S. khelangense and found to be closely related to the Leucocytozoon lineages reported in owls; this is the first record of the latter lineage in Asian black flies. Among these haplotypes, nine and 45 were exclusively found in the S. asakoae complex and S. khelangense, respectively. No lineage was shared between these black fly taxa. Analysis of similarity (ANOSIM) revealed significant Leucocytozoon lineage composition between the two black flies. Phylogenetic analysis found that Leucocytozoon lineages in the S. asakoae complex and S. khelangense are largely isolated, agreeing with the ANOSIM result. The overall prevalence of Leucocytozoon in the S. asakoae complex was 11.3% and ranged from 9% to 13% in each collection. Leucocytozoon prevalence in S. khelangense was 21%, varying from 13% to 37% in each collection. The Shannon H' index indicated greater Leucocytozoon diversity in S. khelangense (H' = 3.044) than in the S. asakoae complex (H' = 1.920). Regression analysis revealed that Leucocytozoon diversity was positively related to black fly abundance and negatively related to maximum air temperature., Conclusions: The results of this study show that the prevalence and diversity of Leucocytozoon lineages in the S. asakoae complex and S. khelangense from Thailand were associated with the abundance of these black flies and with air temperature. The Leucocytozoon lineages identified also showed some degree of black fly taxon specificity, possibly related to different abundance peaks of these vectors. The environmental conditions that favor the development of black flies are possibly a driver of Leucocytozoon prevalence, diversity and vector-parasite co-evolution., Competing Interests: Declarations Ethics approval and consent to participate Not applicable. Consent for publication All authors read and approved the final manuscript. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Current biomechanical models suggest that butterflies and moths use their proboscis as a drinking straw pulling nectar as a continuous liquid column. Our analyses revealed an alternative mode for fluid uptake: drinking bubble trains that help defeat drag. We combined X-ray phase-contrast imaging, optical video microscopy, micro-computed tomography, phylogenetic models of evolution and fluid mechanics models of bubble-train formation to understand the biomechanics of butterfly and moth feeding. Our models suggest that the bubble-train mechanism appeared in the early evolution of butterflies and moths with a proboscis long enough to coil. We propose that, in addition to the ability to drink a continuous column of fluid from pools, the ability to exploit fluid films by capitalizing on bubble trains would have expanded the range of available food sources, facilitating diversification of Lepidoptera.

As an island about 150 km from the mainland, Taiwan would be expected to have endemic species. About 64 % of its 36 species of black flies are considered endemic, more than twice the level of endemicity that has been recorded for all insects on the island. To begin assessing the validity of the high level of endemism for the Simuliidae, we used giant chromosome banding patterns and cytochrome c oxidase I (COI) sequences, against a well-defined morphological backdrop, to evaluate three of Taiwan's black flies, Simulium chungi Takaoka & Huang, S. pingtungense Huang & Takaoka, and S. sakishimaense Takaoka. Molecular data revealed high similarity of populations of S. sakishimaense in Taiwan and at the type locality on Ishigaki Island, Japan, about 180 km to the east. Thus, populations referred to as S. sakishimaense in Taiwan are conspecific with typical S. sakishimaense in Japan, confirming their non-endemicity in Taiwan. Simulium sakishimaense might have reached Ishigaki by island hopping via Taiwan from the Chinese mainland. Chromosomes and the COI gene agree with morphology that S. sakishimaense is a member of the S. multistriatum species group although the chromosomal banding patterns do not indicate that it is distinct from S. fenestratum Edwards on the mainland. Although molecular sequences indicate S. sakishimaense is monophyletic, this taxon falls within the same Operational Taxonomic Unit as nine other members of the S. multistriatum group, including S. fenestratum. Simulium pingtungense, in agreement with morphology, is molecularly distinct from the 10 other analyzed members of the S. striatum species group, tentatively suggesting that it is endemic to Taiwan, pending analysis of samples from mainland China. Simulium chungi in Taiwan is chromosomally and molecularly unique, with larvae resembling those of S. saskishimaense. It is not, however, a member of either the S. multistriatum or S. striatum species groups. For now, the S. chungi species group remains a legitimate taxon consisting of two species. Strengthening the case for endemic taxa in Taiwan awaits analysis of key samples from the Chinese mainland., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Most studies on black flies focus on the taxonomy and ecology of their aquatic stages. Despite posing a public health threat, the adults remain poorly studied in many countries, including Malaysia. The present study represents the first investigation of the distribution of wild-caught black flies from various ecological landscapes and climatic conditions in Malaysia. CO 2 -baited Malaise traps were set randomly at 41 sampling sites across Peninsular Malaysia from 2020 to 2023. In total, 532 black flies belonging to 14 species of four subgenera were captured. To ensure taxonomic rigor, specimens were identified to species morphologically and molecularly. The subgenus Gomphostilbia was the most abundant (71.43 %), followed by Simulium (14.28 %) and Davieselleum and Nevermannia each representing 7.14 % of the total captures. These species represented 14.74 % of the total species recorded from Malaysia. The most frequently collected species were Simulium roslihashimi (24.39 %), followed by S. aureohirtum, S. vanluni, and S. (Gomphostilbia) sp. 1 with 7.32 % each. The highest relative abundance was found for S. vanluni (86.09 %) and S. roslihashimi (7.14 %). Most species were found at elevation below 300 m (78.57 %); fewer were at elevation higher than 1,000 m (21.43 %). Two principal components accounted for 85.3 % of the total intersite variance. Simulium roslihashimi was found at almost every site, with a maximum relative humidity of 90 %. Simulium aureohirtum and S. vanluni were found at sites with relative humidity up to 73 %, but S. aureohirtum was found at higher temperatures (31 °C) compared with S. roslihashimi (28 °C) and S. vanluni (29 °C). The present study establishes the groundwork for further studies of wild adults in Malaysia and identifies the need to use more traps over the range of seasons and environmental conditions, particularly near breeding sites., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Invasive species are a threat to ecosystems worldwide, but determining if a species is adventive or native is not always straightforward. The black flies that inhabit the Galapagos Islands, long known as Simulium ochraceum, are blood-feeding pests of humans and livestock. They first came to the attention of residents in 1989, suggesting a recent arrival. Earlier colonization, however, has been suggested, based largely on polymorphic genetic loci. To address questions of origin, provenance, and length of residency, we conducted a macrogenomic analysis of the polytene chromosomes of the S. ochraceum complex from seven sites in the Galapagos Islands and 30 sites in mainland Ecuador, Central America, and the Caribbean. Among 500 analyzed larvae, we discovered 88 chromosomal rearrangements representing 13 cytoforms, at least seven of which are probably full species. All evidence points to a single, cohesive cytoform with full species status in the Galapagos, conspecific with mainland populations, and widely distributed in the Neotropical Region. It has an identical, nearly monomorphic banding sequence with 10 novel fixed inversions and a subtle but unique Y-linked chromosomal rearrangement across all populations sampled in the Galapagos, the mainland, and the Caribbean. We recalled the name Simulium antillarum from synonymy with S. ochraceum and applied it to the Galapagos black flies, and we established that wolcotti is a junior synonym of antillarum. The time(s) and mode(s) of arrival of S. antillarum in the Galapagos remain uncertain, although the wide geographic distribution, including islands in the Caribbean, suggests that the species is an adept colonizer. Regardless of how long it has been in the archipelago, S. antillarum might have assumed a functional role in the streams of San Cristobal, but otherwise has had a detrimental effect on humans and livestock and potentially on the unique birds and mammals of the Galapagos Islands., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Simulium asakoae Takaoka and Davies has been confirmed to bite humans and has been incriminated as a vector of blood protozoan parasites of the genera Leucocytozoon and Trypanosoma, as well as an unknown filarial parasite in Thailand. However, its attraction to humans has remained uninvestigated in Malaysia. Recently, 27 black flies were collected in Pahang, Malaysia, of which 25 were captured in CO 2 -baited Malaise traps and two were collected from humans during trapping activity. All specimens were morphologically identified as S. asakoae. Cytochrome c oxidase I sequences of the two specimens caught on humans showed 100% similarity with those of S. asakoae in the NCBI GenBank, confirming their morphological identity. Blood-meal analysis using a HAL·HAR TM kit did not show the presence of domestic or wild animal DNA. However, human DNA was amplified from one engorged fly in the cytochrome b gene amplification assay, providing the first evidence of blood-feeding by S. asakoae in Malaysia.

To probe its environment, the flying insect controllably flexes, twists, and maneuvers its antennae by coupling mechanical deformations with the sensory output. We question how the materials properties of insect antennae could influence their performance. A comparative study was conducted on four hawkmoth species: Manduca sexta, Ceratomia catalpae, Manduca quinquemaculata, and Xylophanes tersa. The morphology of the antennae of three hawkmoths that hover while feeding and one putatively non-nectar-feeding hawkmoth (Ceratomia catalpa) do not fundamentally differ, and all the antennae are comb-like (i.e., pectinate), markedly in males but weakly in females. Applying different weights to the free end of extracted cantilevered antennae, we discovered anisotropy in flexural rigidity when the antenna is forced to bend dorsally versus ventrally. The flexural rigidity of male antennae was less than that of females. Compared with the hawkmoths that hover while feeding, Ceratomia catalpae has almost two orders of magnitude lower flexural rigidity. Tensile tests showed that the stiffness of male and female antennae is almost the same. Therefore, the differences in flexural rigidity are explained by the distinct shapes of the antennal pectination. Like bristles in a comb, the pectinations provide extra rigidity to the antenna. We discuss the biological implications of these discoveries in relation to the flight habits of hawkmoths. Flexural anisotropy of antennae is expected in other groups of insects, but the targeted outcome may differ. Our work offers promising new applications of shaped fibers as mechanical sensors. STATEMENT OF SIGNIFICANCE: Insect antennae are blood-filled, segmented fibers with muscles in the two basal segments. The long terminal segment is muscle-free but can be flexed. Our comparative analysis of mechanical properties of hawkmoth antennae revealed a new feature: antenna resistance to bending depends on the bending direction. Our discovery replaces the conventional textbook scenario considering hawkmoth antennae as rigid rods. We showed that the pectinate antennae of hawkmoths behave as a comb in which the bristles resist bending when they come together. This anisotropy of flexural resistance offers a new mode of environmental sensing that has never been explored. The principles we found apply to other insects with non-axisymmetric antennae. Our work offers new applications for shaped fibers that could be designed to sense the flows., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Two species of black flies (Simuliidae) in Thailand, Simulium chumpornense Takaoka and Kuvangkadilok, 2000, and S. khelangense Takaoka, Srisuka & Saeung, 2022, are potent vectors of avian blood protozoa of the genera Leucocytozoon and Trypanosoma and are pests of domestic avian species. Although the adults are abundant throughout Thailand, information on their breeding habitats is limited, and the immature stages of S. khelangense are unknown. We collected the larvae and pupae of S. khelangense from the Mekong River, the first-ever record of Simuliidae from this large continental river. Mitochondrial cytochrome c oxidase I and internal transcribed spacer 2 were used to associate the larvae and pupae with known adults. Both genetic markers strongly supported their identity as S. khelangense . The larvae and pupa of S. khelangense are described. The pupal gill filaments, larval abdominal protuberances, and setae distinguish this species from other members of the S. varicorne species group. The immature stages of S. chumpornense inhabit a wide variety of flowing waters, from small streams (3 m wide) to enormous continental rivers (400 m wide); thus, S. chumpornense is a habitat generalist. In contrast, S. khelangense was found only in the large Mekong River and is, therefore, a habitat specialist. Both species can exploit their principal habitats and produce abundant adult populations.

The first record of a European black fly, Simulium ichnusae Rivosecchi & Contini, is documented for Africa, where it was found at elevations above 1000 m in the Djurdjura Mountains of Algeria. Considered an endemic species of Sardinia for 60 years, S. ichnusae must now be regarded as pseudoendemic. Larvae, pupae, females, and males from the Djurdjura Mountains compare well with those in the original description of the species. Chromosomally, S. ichnusae differs from the standard banding sequence of the S. vernum group by a transposed nucleolar organizer-the only known member of the group with this condition-and three fixed inversions, all of which are shared by other members of this species group. The addition of S. ichnusae to the Algerian fauna increases the number of nominal species of black flies known from the country to 34. The discovery of S. ichnusae suggests that additional trans-Mediterranean species await discovery in the coastal Mediterranean mountains and emphasizes that claims of endemism depend on thorough surveys and taxonomic accuracy.

The polytene chromosomes, larva, pupa, and male are described for the black fly Simulium chaouikaidi n. sp., a member of the Simulium venustum group from Morocco. Chromosomally, this new species is one fixed inversion removed from the standard banding sequence for the subgenus Simulium and has microscopically undifferentiated sex chromosomes and minimal autosomal polymorphism. It is homosequential with two other species in the S. venustum species group: S. bergi Rubtsov and S. paramorsitans Rubtsov. Structurally, each known life stage of the new species is distinct from that of all other simuliids in North Africa. This new species is the southernmost member of the S. venustum group in the Old World and is apparently endemic to the Rif Mountains. Its restricted distribution includes Bou Hachem Natural Park where its habitat is afforded some protection.

Discoveries of endemic species highlight areas of biogeographic and conservation interest. Endemic species, however, are often morphologically disguised as more common and widespread species. The larval polytene chromosomes revealed a new species of black fly, Prosimulium fungiforme , from the Djurdjura Mountains of northern Algeria, and its female, male, pupa, and larva are described. The species is chromosomally unique; none of its 11 chromosomal rearrangements are shared with other species. Although the new species structurally resembles Prosimulium rufipes (Meigen) with which it previously has been confused, it can be distinguished from all other known species of Prosimulium in the Western Palearctic based on at least one character in each described life stage. Symbiotic organisms included two species of microsporidia, at least one of which is probably undescribed, one unknown protozoan pathogen novel in simuliids, and the trichomycete fungus Harpella melusinae Léger and Duboscq. Associated simuliid species included at least one new species of the genus Helodon . The new species of Prosimulium is tentatively considered endemic to the mountains of northern Algeria but might be expected in the mountains of eastern Morocco and northern Tunisia and perhaps in Sicily. If its endemic status holds, it would be the only nominal species of black fly unique to Algeria.

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Ruiz-Arrondo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Hovering hawkmoths expend significant energy while feeding, which should select for greater feeding efficiency. Although increased feeding efficiency has been implicitly assumed, it has never been assessed. We hypothesized that hawkmoths have proboscises specialized for gathering nectar passively. Using contact angle and capillary pressure to evaluate capillary action of the proboscis, we conducted a comparative analysis of wetting and absorption properties for 13 species of hawkmoths. We showed that all 13 species have a hydrophilic proboscis. In contradistinction, the proboscises of all other tested lepidopteran species have a wetting dichotomy with only the distal ∼10% hydrophilic. Longer proboscises are more wettable, suggesting that species of hawkmoths with long proboscises are more efficient at acquiring nectar by the proboscis surface than are species with shorter proboscises. All hawkmoth species also show strong capillary pressure, which, together with the feeding behaviors we observed, ensures that nectar will be delivered to the food canal efficiently. The patterns we found suggest that different subfamilies of hawkmoths use different feeding strategies. Our comparative approach reveals that hawkmoths are unique among Lepidoptera and highlights the importance of considering the physical characteristics of the proboscis to understand the evolution and diversification of hawkmoths., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

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., (© 2023. Springer Nature Limited.)

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., (© 2023. BioMed Central Ltd., part of Springer Nature.)

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., (© 2023. The Author(s).)

Viscosity determines the resistance of haemolymph flow through the insect body. For flying insects, viscosity is a major physiological parameter limiting flight performance by controlling the flow rate of fuel to the flight muscles, circulating nutrients and rapidly removing metabolic waste products. The more viscous the haemolymph, the greater the metabolic energy needed to pump it through confined spaces. By employing magnetic rotational spectroscopy with nickel nanorods, we showed that viscosity of haemolymph in resting hawkmoths (Sphingidae) depends on wing size non-monotonically. Viscosity increases for small hawkmoths with high wingbeat frequencies, reaches a maximum for middle-sized hawkmoths with moderate wingbeat frequencies, and decreases in large hawkmoths with slower wingbeat frequencies but greater lift. Accordingly, hawkmoths with small and large wings have viscosities approaching that of water, whereas hawkmoths with mid-sized wings have more than twofold greater viscosity. The metabolic demands of flight correlate with significant changes in circulatory strategies via modulation of haemolymph viscosity. Thus, the evolution of hovering flight would require fine-tuned viscosity adjustments to balance the need for the haemolymph to carry more fuel to the flight muscles while decreasing the viscous dissipation associated with its circulation.

One of the most popular tools for species discovery and resolution is the DNA barcode, typically based on the cytochrome c oxidase I (COI) gene. However, other non-genic barcodes are available for Diptera. The banding sequence of polytene chromosomes in some dipteran cells, particularly of the larval silk glands, can provide a unique species barcode. We used the sequence of bands to reveal a new species of black fly in the Simulium ( Boreosimulium ) annulus species group from California, USA. To further characterize the species and provide more integrated taxonomy, we morphologically described all life stages above the egg, formally named the species Simulium ustulatum n. sp., and provided a conventional COI barcode. The COI barcode confirmed the chromosomal and morphological evidence that the species is a new member of the S. annulus group, and enabled identification of the larva and female, which are structurally similar to those of other species. The chromosomal barcode shows that this species has the most rearranged complement, compared with the eight other North American members of its species group, with up to 12 times the number of fixed rearrangements. Up to six chromosomal rearrangements, including autosomal polymorphisms and sex-linked phenomena, are shared with other members of the group. The most unique and conspicuous chromosomal feature of this new species is a large, pale-staining chromocenter from which the six chromosomal arms radiate. The distribution of this univoltine species in lowland rivers of California's Central Valley could make it vulnerable, given climate change and increasing land development.

Insect antennae are hollow, blood-filled fibers with complex shape. Muscles in the two basal segments control antennal movement, but the rest (flagellum) is muscle-free. The insect can controllably flex, twist, and maneuver its antennae laterally. To explain this behavior, we performed a comparative study of structural and tensile properties of the antennae of Periplaneta americana (American cockroach), Manduca sexta (Carolina hawkmoth), and Vanessa cardui (painted lady butterfly). These antennae demonstrate a range of distinguishable tensile properties, responding either as brittle or strain-adaptive fibers that stiffen when stretched. Scanning electron microscopy and high-speed imaging of antennal breakup during stretching revealed complex coupling of blood pressure and cuticle deformation in antennae. A generalized Lamé theory of solid mechanics was developed to include the force-driven deformation of blood-filled antennal tubes. We validated the theory against experiments with artificial antennae with no adjustable parameters. Blood pressure increased when the insect inflated its antennae or decreased below ambient pressure when an external tensile load was applied to the antenna. The pressure-cuticle coupling can be controlled through changes of the blood volume in the antennal lumen. In insects that do not fill the antennal lumen with blood, this blood pressure control is lacking, and the antennae react only by muscular activation. We suggest that the principles we have discovered for insect antennae apply to other appendages that share a leg-derived ancestry. Our work offers promising new applications for multifunctional fiber-based microfluidics that could transport fluids and be manipulated by the same fluid on demand. STATEMENT OF SIGNIFICANCE: Insect antennae are blood-filled, segmented fibers with muscles in the two basal segments. The long terminal segment is muscle-free but can be flexed. To explain this behavior, we examined structure-function relationships of antennae of cockroaches, hawkmoths, and butterflies. Hawkmoth antennae behaved as brittle fibers, but butterfly and cockroach antennae showed strain-adaptive behavior like fibers that stiffen when stretched. Videomicroscopy of antennal breakup during stretching revealed complex coupling of blood pressure and cuticle deformation. Our solid mechanics model explains this behavior. Because antennae are leg-derived appendages, we suggest that the principles we found apply to other appendages of leg-derived ancestry. Our work offers new applications for multifunctional fiber-based microfluidics that could transport fluids and be manipulated by the fluid on demand., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Mermithids are parasites of black flies that cause host mortality along with physical and behavioural changes in infected hosts. However, there is a lack of knowledge on the distribution of mermithids infecting black fly larvae and the factors that influence these distributions in Asia, including Malaysia. A total of 13,116 mid- to late-instar black fly larvae belonging to 42 species were collected from 138 streams across East and West Malaysia and screened for the presence of mermithid parasites. Overall, 121 mermithids were obtained from 107 (0.82%) larvae of nine (21.4%) black fly species. The average number of mermithids per black fly host was 1.10 ± 0.04 (SE), ranging from one to three mermithids per host. Mermithid infection was highest in Simulium trangense, with a frequency of occurrence of 6.5%, followed by S. cheongi (5.8%) and S. angulistylum complex (2.9%). Infection was lowest in S. brevipar and S. tahanense, with a frequency of occurrence of 0.7% each. Regression analysis indicated that mermithid infections in larval black flies were significantly associated with cooler and shallower streams with more canopy cover, dense riparian vegetation, high dissolved oxygen, and lower conductivity and complete pH. Forward logistic regression further indicated that infections in S. cheongi were associated with shaded, cooler, slightly acidic streams with higher conductivity and dissolved oxygen. These findings suggest that mermithid infections in larval black flies in Malaysia are not randomly distributed and are influenced by the breeding habitat of their hosts., (Copyright © 2022. Published by Elsevier B.V.)

The species status of two cytoforms of two species complexes in the Simulium (Simulium) tuberosum species-group, which are almost indistinguishable in the pupal stage, was morphologically and genetically evaluated. Cytoform 'L' of the S. (S.) tani Takaoka & Davies complex, previously recognized as S. (S.) suzukii Rubtsov in Taiwan, is described as a new species, S. (S.) jianshiense. It is morphologically distinguishable as adults from two members of the S. (S.) suzukii complex: cytoform 'C' from Hokkaido, selected to represent the type of S. (S.) suzukii sensu stricto, and cytoform 'D' from Okinawa Island and, based on our chromosomal analysis, also from Amami Island. This new species is genetically separated from both cytoforms of the S. (S.) suzukii complex with a genetic distance of 5.31-6.67%. Cytoform 'D' is distinguished from cytoform 'C' by the color of the male forecoxa and relative length of the female sensory vesicle to the third maxillary palpomere. Accordingly, the name S. (S.) ryukyuense Ogata, once regarded as a synonym of S. (S.) suzukii, is revalidated and applied to cytoform 'D'. The genetic distance between S. (S.) ryukyuense and S. (S.) suzukii sensu stricto is 1.24-1.60%., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Hypothesis: Self-repair in living organisms, without tissue regeneration or regrowth, is rare. Recent discovery that butterflies can self-repair the proboscis after the two halves (galeae) have been separated raised a question about the physical mechanism allowing them to reunite the parts. We discovered that butterflies pump saliva during repair of their proboscises. We then hypothesized that saliva spreading along the food canal of the proboscis would create capillary forces capable of bringing the galeae together., Experiment: To test the hypothesis, we distinguished capillary forces from muscular action of the galeae by sedating butterflies and video tracking retraction of the saliva menisci during galeal separation. To theoretically show capillary adhesion, the elastic moduli of the galeae were measured, and the galeal profiles were extracted from videos as a function of time. The values were then fitted with a mathematical model based on an augmented Euler-Bernoulli beam theory whereby each galea was treated as a beam bent by capillary forces due to saliva. We also evaluated friction forces that prevented disjoining of the galea at the tip of their separation., Findings: The results showed that butterflies use saliva to repair their proboscises via capillary adhesion, and theoretically supported the role of saliva in providing the necessary capillary forces to bring the galeae together. Tangential shear forces acting parallel to the galea at the tip of their separation are caused primarily by friction between the cuticular linking structures., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Simulium hasekei new species is described from the Alps of Austria. It is characterized by a broad trapezoidal ventral plate in the male, long straight arms of the genital fork in the female, a well-developed anterodorsal projection on the weakly woven cocoon, and a deep rounded postgenal cleft and strong paralateral hypostomal teeth in the larva. The species is chromosomally most similar to the Holarctic S. bicorne Dorogostaisky, Rubtsov Vlasenko and S. fontinale Radzivilovskaya and the Nearctic S. craigi Adler Currie. It is known from one small ponor brook, in association with S. aureum (Fries) s. s., S. beltukovae (Rubtsov), and Simulium vernum Macquart s. s.

By any measure, such as abundance, species diversity or geographic range, the Simulium ornatum species group is one of the most successful Palearctic taxa of black flies. To explore potential diversity in this group in the Tian Shan range of Central Asia, we focused on Kyrgyzstan, in which three nominal morphospecies have been recorded. Among our samples, we morphologically identified S. mesasiaticum Rubtsov and a second possible species tentatively identified as S. ferganicum Rubtsov. By analyzing banding patterns of the larval polytene chromosomes, we discovered two fixed inversions, two sex-linked rearrangements, and 19 autosomal rearrangements, including supernumerary B chromosomes. The chromosomal data indicate minimal diversity of only one or two species across the surveyed area of nearly 50,000 km 2 . Mitochondrial DNA (CO1) sequences fell into three distinct clusters, possibly representing separate species. The chromosomal, molecular, and morphological data indicate that Kyrgyz populations are unique within the S. ornatum group, but the data sets are not entirely congruent. Thus, reconciling data sets and assigning existing names is tentative. Simulium mesasiaticum is linked with undifferentiated sex chromosomes, one of the three CO1 clades, and higher elevations, whereas S. ferganicum is tenuously associated with differentiated sex chromosomes, a separate CO1 clade, and lower elevations. These associations leave one Kyrgyz larva, which is in a third CO1 clade, unlinked to a formal species name. Our analyses also indicate that S. ornatum Meigen sensu stricto , contrary to previous reports, does not occur in Kyrgyzstan and should be deleted from the country's faunal list.

Although the microbiome of blood-feeding insects serves an integral role in host physiology, both beneficial and pathogenic, little is known of the microbial community of black flies. An investigation, therefore, was undertaken to identify culturable bacteria from one of Malaysia's most common black flies, Simulium tani Takaoka and Davies, using 16S rDNA sequencing, and then evaluate the isolates for antibiotic resistance and virulence genes. A total of 20 isolates representing 11 bacterial species in four genera were found. Five isolates showed β-hemolysis on Columbia agar, and virulence genes were found in three of these isolates. Some degree of resistance to six of the 12 tested antibiotics was found among the isolates. The baseline data from this study suggest rich opportunities for comparative studies exploring the diversity and roles of the microbiome of S. tani and other Southeast Asian black flies., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Sexually anomalous individuals, typically intersexes or gynandromorphs, bear a mixture of male and female traits. Twelve sexually anomalous individuals of the black fly Simulium (Gomphostilbia) trangense Jitklang, Kuvangkadilok, Baimai, Takaoka & Adler were discovered among 49 adults reared from pupae. All 12 sexually anomalous adults were parasitized by mermithid nematodes, although five additional parasitized adults had no overt external anomalies. Sequence analysis of the 18S rRNA gene revealed that the mermithids, possibly representing a new species, are related to Mesomermis spp., with genetic distances of 5.09-6.87%. All 12 anomalous individuals had female phenotypical traits on the head, thorax, forelegs, midlegs, and claws, but male features on the left and right hind basitarsi. One individual had mixed male and female genitalia. The findings are in accord with the trend that mermithid infections are associated with sexually anomalous adult black flies.

Hypodermins A (HA), B (HB), and C (HC) of warble flies are modulatory antigens involved in host inflammation and immune responses during migration of the warble fly larvae through host connective tissues. In the current study, molecular characteristics of the genes encoding HA, HB, and HC were revealed from cDNA constructs of third-instar larvae of Hypoderma bovis. The open reading frame (ORF) of each hypodermin gene was amplified with modified gene-specific primers, and the resulting PCR products were cloned into pGEM-T Easy Vector to produce recombinant plasmids (rHA, rHB, and rHC). The ORF sequences of rHA, rHB, and rHC genes are 705 bp, 771 bp, and 783 bp long and encode proteins of 234, 256, and 263 amino acids with predicted sizes of 25.74 kDa, 27.79 kDa, and 28.51 kDa, respectively. The rHC gene was subcloned into the pET 100/D-TOPO Expression Vector, and the recombinant HC was purified using affinity chromatography. Western blotting indicated that rHC was recognized by the sera of cattle naturally infested with H. bovis. The rHC and a synthetic peptide (sHC) containing its linear B cell-specific epitope were evaluated as serological markers in indirect ELISA (iELISA) for the diagnosis of bovine hypodermosis. Both sHC and rHC iELISAs had sensitivity values equal to or higher than 90 % and specificity values of 100 %. A total of 200 serum samples from cattle in the Central Anatolia Region of Turkey were also analyzed by rHC and sHC-iELISAs to reveal the seroprevalence of bovine hypodermosis. The results of both iELISAs were consistent with one another and revealed a hypodermosis prevalence of 62 %. Our study provides the first data on molecular characterization of hypodermin genes of H. bovis and indicates the efficacy of recombinant antigen and peptide-based iELISA for serodiagnosis of bovine hypodermosis., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Proboscises of many fluid-feeding insects share a common architecture: they have a partially open food canal along their length. This feature has never been discussed in relation to the feeding mechanism. We formulated and solved a fluid mechanics model of fluid uptake and estimated the time required to completely fill the food canal of the entire proboscis through the openings along its length. Butterflies and moths are taken as illustrative and representative of fluid-feeding insects. We demonstrated that the proposed mechanism of filling the proboscis with fluid through permeable lengthwise bands, in association with a thin film of saliva in the food canal, offers a competitive pathway for fluid uptake. Compared with the conventional mechanism of fluid uptake through apically restricted openings, the new mechanism provides a faster rate of fluid uptake, especially for long-tongued insects. Accordingly, long-tongued insects with permeable lengthwise bands would be able to more rapidly exploit a broader range of liquids in the form of films, pools, and discontinuous columns, thereby conserving energy and minimizing exposure to predators, particularly for hovering insects., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

We examined the general architecture of interactions between stream-dwelling larval black flies (Diptera: Simuliidae) and their common parasites in 1736 collections across North America. Mermithid nematodes (family Mermithidae), microsporidia (phylum Microsporidia), and the fungus Coelomycidium simulii Debaisieux (phylum Blastocladiomycota) infected larval black flies. We found similar continental distributions for these three parasite taxa across North America. At least one of these taxa was represented in 42.2% of all black fly collections. Species interactions in ecological networks typically imply that each link between species is equally important. By employing quantitative measures of host susceptibilities and parasite dependencies, we provide a more complete structure for host-parasite networks. The distribution of parasite dependencies and host susceptibilities were right-skewed, with low values indicating that most dependencies (parasites) and susceptibilities (hosts) were weak. Although regression analysis between host frequency and parasite incidence were highly significant, frequency analysis suggested that the distributions of parasites differ significantly among the four most common and closely related (same subgenus) species of hosts. A highly significant pattern of nestedness in our bipartite host-parasite network indicated that specialized parasites (i.e., those that interact with few host species) tend to occur as subsets of the most common hosts., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Black flies are insects of medical, veterinary, and environmental significance. Historically, they have attacked humans and caused simuliotoxicosis in livestock in the Aras River Basin in northwest Iran. However, information on the species and their bionomics is limited in the region. Adult flies were collected from diverse ecotopes of the Aras River Basin. After morphological identification, representative specimens of each morphological group were subjected to mtDNA COI gene sequence analysis for species diagnosis and to infer relationships. Flies also were examined for pollinia. A total of 1880 specimens representing 12 morphotaxa in two genera (Simulium and Metacnephia) were identified: Simulium turgaicum (n=1834), S. kiritshenkoi (n=12), S. bezzii (n=7), S. brevitarse (n=7), S. pseudequinum (n=5), S. aureum species group (n=4), S. vernum species group (n=3), S. transcaspicum (n=1), three unidentified species of the subgenus Simulium (n=5), and Metacnephia possibly persica (n=2). Fifty two haplotypes were detected for the 65 COI sequences analyzed. Intraspecific genetic divergence was 0.19-8.83%, whereas the mean interspecific genetic distances among the morphotaxa were 1.41-19.58%. Molecular analyses recovered three well-supported lineages within S. turgaicum. One lineage included black flies collected from agricultural fields, a second lineage involved black flies captured from animals, and a third lineage included specimens that had visited flowers, as evidenced by presence of pollinia. The relative abundance (97%) and observations of the S. turgaicum complex biting humans are important epidemiological factors. Future studies are needed to define the potential epidemiological risk of simulids in Khoda-Afarin County of Iran., Competing Interests: Declaration of Competing Interests All authors declare that they have no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

High-mountain summits in tropical regions are habitat islands that provide refugia for northern taxa and opportunities for speciation. These focal hotspots of biodiversity are also threatened by increasing anthropogenic pressures and changing climatic conditions. The largest species group of black flies, the Simulium vernum group, reaches its southern limit in the mountains of Southeast Asia, providing an opportunity to examine macrogenetic diversification at the southern edge of the group's range. We investigated the polytene chromosomes of two morphologically similar species in the S. vernum group, S. chomthongense Takaoka, Srisuka & Choochote, and S. khunklangense Takaoka & Srisuka, from their type localities near the summit of Doi Inthanon, Thailand's highest mountain. Chromosomal analyses of 112 larvae demonstrated reproductive isolation between S. chomthongense and S. khunklangense and revealed a cryptic species of S. chomthongense. The three species have the same fixed banding sequence in five of the six chromosome arms, but they are distinguished by the successive addition of overlapping inversions in the IIIL arm. The two cytospecies (A and B) of S. chomthongense s. l. are the third example of endemic or nearly endemic cryptic species pairs near the top of Doi Inthanon, suggesting the possibility of sympatric speciation and the importance of conserving high-elevation stream habitats., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The ecology of black flies in Thailand was investigated, based on 19,451 larvae and pupae collected from 65 stream sites in 10 northern provinces during the rainy, cool, and hot seasons, and 1,906 larvae and pupae collected from 18 sites in 9 southern provinces during the cool season. Twenty-seven black fly species were identified from northern Thailand, of which 26 were found in the cool season, when richness was greatest. Significant regressions between species richness and elevation fit a unimodal model in the rainy season but a linear model in the cool and hot seasons. Twenty-two species occurred in all seasons. Species in the subgenera Gomphostilbia and Nevermannia were most common in the hot season, whereas species in the subgenus Simulium were predominant in the cool season. Some species (e.g., S. nakhonense) were geographically widespread, whereas others (e.g., S. chaliowae and S. weji) were restricted to particular localities. Eighteen species and species complexes were found in southern Thailand. The S. tani complex was the most widely distributed taxon, occurring at 66.7% of the sites in the South. Ecological analyses revealed that water temperature, elevation, conductivity, dissolved oxygen, and stream size were among the significant factors associated with the distributions of black flies in both regions of Thailand-the same factors associated with simuliid distributions in other areas of the world., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing financial interests or personal relationships that influenced the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The widespread nominal black fly Simulium (Simulium) rufibasis Brunetti was reexamined morphologically, chromosomally, and molecularly to determine the status of populations in Japan and Korea with respect to S. rufibasis from the type locality in India and to all other known species in the S. (S.) tuberosum species-group. Morphological comparisons established that the species previously known as S. rufibasis in Japan and Korea is distinct from all other species. Consequently, it was described and illustrated as a new species, Simulium (S.) yamatoense. Simulium yokotense Shiraki, formerly a synonym of S. rufibasis, was morphologically reevaluated and considered a species unplaced to species-group in the subgenus Simulium. Chromosomal analyses of S. yamatoense sp. nov. demonstrated that it is unique among all cytologically known species of the S. tuberosum group and is the sister species of the Taiwanese species tentatively known as S. (S.) arisanum Shiraki. Populations of S. yamatoense sp. nov. included two cytoforms, based on the sex chromosomes. Cytoform A, including topotypical representatives, was found in Kyushu, Japan, whereas cytoform B was found in Korea and Honshu, Japan. Molecular analysis based on the COI mitochondrial gene generally corroborated morphological and chromosomal data that S. yamatoense sp. nov. is a distinct species and, like the chromosomal data, indicate that it is most closely related to S. arisanum, with interspecific genetic distance of 2.92-4.63%., (© The Author(s) 2019. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Accurate species identification provides the foundation for successful pest management and vector control of black flies. Accordingly, we examined the mitochondrial DNA cytochrome oxidase I (COI) gene sequences of four morphologically and chromosomally identified species of black flies (Simulium vernumgroup sp., S. bergi Rubtsov, S. bezzii (Corti), and S. kiritshenkoi Rubtsov) in Northeast Anatolia Region of Turkey where simuliid pest problems and simuliotoxicosis cases have been reported among cattle. COI gene sequences of these species and closely related species available in GenBank were used to provide species-level diagnoses and infer relationships. Both subgenera (Nevermannia and Simulium) were monophyletic, and subclades generally corresponded with species groups. Intraspecific genetic divergence was 0.2-1.6%, whereas the mean interspecific genetic divergence among the four species was 11.2-14.5%. The COI analysis produced results congruent with morphological concepts of the nominal species S. bergi and S. bezzii. Probable misidentifications in GenBank were revealed, especially for species in the S. ornatum and S. vernum groups, complicating identification capability. Sequence variation in the COI barcode region also might not be adequate for species delineation and identification among some species in these two species groups., (Copyright © 2019. Published by Elsevier B.V.)

Simuliid larvae are common inhabitants of mountain rivers throughout the world, where they can serve as ecological indicators. Black flies were sampled during three seasons in four rivers in the upper basin of the Bogotá River in the Colombian Andes, and physical, chemical, and hydrological data were recorded. Multivariate methods were used to determine the relationships between the presence and abundance of simuliid species and environmental characteristics. Fourteen species were found: eight in the genus Gigantodax (Enderlein, Diptera, Simuliidae) and six in the genus Simulium (Latreille, Diptera, Simuliidae). Dissolved oxygen, dissolved solids, redox potential, chemical oxygen demand, and nutrients contributed to an explanation of species distributions. Species in clean waters and in more polluted waters had narrow niches; those in low to moderately polluted waters had broader niches. Species in the lower reaches of the watercourses had greater turnover, perhaps because the most sensitive species had disappeared and been replaced by more tolerant species. Thus, simuliids can be used as predictors of environmental characteristics of Andean rivers and can be useful in the evaluation and management of these watersheds., (© The Author(s) 2019. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In the conservation of endangered species, suppression of a population of one native species to benefit another poses challenges. Examples include predator control and nest parasite reduction. Less obvious is the control of blood-feeding arthropods. We conducted a case study of the effect of native black flies (Simulium spp.) on reintroduced Whooping Cranes (Grus americana). Our intent was to provide a science-driven approach for determining the effects of blood-feeding arthropods on endangered vertebrates and identifying optimal management actions for managers faced with competing objectives. A multiyear experiment demonstrated that black flies reduce nest success in cranes by driving incubating birds off their nests. We used a decision-analytic approach to develop creative management alternatives and evaluate trade-offs among competing objectives. We identified 4 management objectives: establish a self-sustaining crane population, improve crane well-being, maintain native black flies as functional components of the ecosystem, and minimize costs. We next identified potential management alternatives: do nothing, suppress black flies, force crane renesting to occur after the activity period of black flies, relocate releases of cranes, suppress black flies and relocate releases, or force crane renesting and relocate releases. We then developed predictions on constructed scales of 0 (worst-performing alternative) to 1 (best-performing alternative) to indicate how alternative actions performed in terms of management objectives. The optimal action depended on the relative importance of each objective to a decision maker. Only relocating releases was a dominated alternative, indicating that it was not optimal regardless of the relative importance of objectives. A rational decision maker could choose any other management alternative we considered. Recognizing that decisions involve trade-offs that must be weighed by decision makers is crucial to identifying alternatives that best balance multiple management objectives. Given uncertainty about the population dynamics of blood-feeding arthropods, an adaptive management approach could offer substantial benefits., (Published 2018. This article is a U.S. Government work and is in the public domain in the USA.)

A recently described species of black fly, Simulium wayani Takaoka and Chen, from the island of Timor was chromosomally mapped to provide insights into its evolutionary and biogeographic history. The morphologically based species status of S. wayani is supported by a suite of fixed chromosomal rearrangements and unique sex chromosomes derived primarily from a large pool of polymorphisms in the S. ornatipes complex in Australia. The banding patterns of its polytene chromosomes indicate that S. wayani is closely related to a pair of homosequential cryptic species (S. norfolkense Dumbleton and S. ornatipes cytoform A2) in the S. ornatipes Skuse complex on mainland Australia; all three species uniquely share the same amplified band in their chromosomal complement. The low level of polymorphism and heterozygosity in S. wayani, relative to Australian populations of the S. ornatipes complex, suggests few colonization events from the larger land mass., (Copyright © 2019 Elsevier B.V. All rights reserved.)

More than any other group of macro-organisms, true flies (Diptera) dominate the freshwater environment. Nearly one-third of all flies-roughly 46,000 species-have some developmental connection with an aquatic environment. Their abundance, ubiquity, and diversity of adaptations to the aquatic environment position them as major drivers of ecosystem processes and as sources of products and bioinspiration for the benefit of human society. Larval flies are well represented as ecosystem engineers and keystone species that alter the abiotic and biotic environments through activities such as burrowing, grazing, suspension feeding, and predation. The enormous populations sometimes achieved by aquatic flies can provide the sole or major dietary component for other organisms. Harnessing the services of aquatic Diptera for human benefit depends on the ingenuity of the scientific community. Aquatic flies have played a role as indicators of water quality from the earliest years of bioassessment. They serve as indicators of historical and future ecological and climate change. As predators and herbivores, they can serve as biological control agents. The association of flies with animal carcasses in aquatic environments provides an additional set of tools for forensic science. The extremophilic attributes of numerous species of Diptera offer solutions for human adaptation to harsh terrestrial and extraterrestrial environments. The potential pharmaceutical and industrial applications of the symbiotic microbial community in extremophilic Diptera are better explored than are those of dipteran chemistry. Many flies provide valuable ecological and human services as aquatic immatures, but are also pests and vectors of disease agents as terrestrial adults. The scientific community, thus, is challenged with balancing the benefits and costs of aquatic Diptera, while maintaining sustainable populations as more species face extinction.

Blood clotting at wound sites is critical for preventing blood loss and invasion by microorganisms in multicellular animals, especially small insects vulnerable to dehydration. The mechanistic reaction of the clot is the first step in providing scaffolding for the formation of new epithelial and cuticular tissue. The clot, therefore, requires special materials properties. We have developed and used nano-rheological magnetic rotational spectroscopy with nanorods to quantitatively study nucleation of cell aggregates that occurs within fractions of a second. Using larvae of Manduca sexta, we discovered that clot nucleation is a two-step process whereby cell aggregation is the time-limiting step followed by rigidification of the aggregate. Clot nucleation and transformation of viscous blood into a visco-elastic aggregate happens in a few minutes, which is hundreds of times faster than wound plugging and scab formation. This discovery sets a time scale for insect clotting phenomena, establishing a materials metric for the kinetics of biochemical reaction cascades. Combined with biochemical and biomolecular studies, these discoveries can help design fast-working thickeners for vertebrate blood, including human blood, based on clotting principles of insect blood.

A new species of black fly, Prosimulium kazukii, is described based on female, male and pupal specimens collected from central Honshu, Japan. It is placed in the Prosimulium magnum species-group, and is characterized in the female and male by yellow thoraces, and in the pupa by the frons and dorsal surface of the thorax without distinct tubercles. By these characters, it is distinguished from all four related species (P. apoina Ono, P. kalibaense Ono, P. sarurense Ono, and P. yezoense Shiraki) of the same species-group in Japan. The female of this new species was previously known as an aberrant form of P. yezoense.

The ability of aquatic insects to colonize Earth's most remote freshwater habitats, such as those of islands and deserts, is limited to select taxa. Among black flies, the premiere colonization specialist is Simulium ruficorne Macquart, the only species known from both the Afrotropical and Palearctic regions. We investigated the cytogenetics of S. ruficorne to gain insight into its wide geographic distribution and ability to colonize oceanic islands and deserts. On the basis of larval polytene chromosomes from 14 locations, we documented 17 novel and previously known chromosomal rearrangements and established five cytoforms (A1, A2, B, C, and D), of which probably four (A1/A2, B, C, and D) are distinct species and two (A1 and A2) represent sex-chromosome polymorphism involving a heteroband in the long arm of chromosome III. The chromosome restructuring phenomena associated with the five cytoforms are consistent with the trend in the Simuliidae that one and the same rearrangement can assume different functions in the various descendants of a common ancestor in which the rearrangement was polymorphic. The most widely distributed cytoforms are A1 and A2, which are found in North Africa, the Canary Islands, and Majorca. Simulium ruficorne, the only known black fly in the Hoggar Mountains of the central Sahara Desert, represents a cohesive population of cytoform A1 little differentiated from other North African populations of A1 and A2. Cytoform B inhabits the West African mainland, cytoform C is on Tenerife, and cytoform D is on Cape Verde. We suggest that dispersal and colonization specialists, such as S. ruficorne, are multivoltine inhabitants of temporary streams, and must relocate as their habitats deteriorate. Simulium ruficorne, therefore, should have adaptations that contribute to successful dispersal and colonization, perhaps largely physiological in nature, such as tolerance of high temperatures and droughts., Competing Interests: The authors have declared that no competing interests exist.

The Simulium rufibasis subgroup is one of three subgroups of the Simulium (Simulium) tuberosum species-group; it is characterized by a pair of clustered stout hairs on the ventral surface of female abdominal segment 7. A member of the S. rufibasis subgroup in Taiwan was investigated morphologically and genetically using the universal cytochrome c oxidase subunit I (COI) barcoding gene and polytene chromosomal banding pattern. The Taiwanese material is morphologically similar to S. rosliramlii Takaoka & Chen from Vietnam and represents the second species of the S. rufibasis subgroup known from Taiwan. It also represents a novel molecular lineage that is distinct from three other primary lineages identified as S. doipuiense, S. doipuiense/S. rufibasis, and S. weji previously reported from Thailand. The mitochondrial evidence for a distinct lineage in Taiwan is supported by chromosomal analysis, which revealed unique sex chromosomes. For nomenclatural stability, we associate the name S. arisanum Shiraki with the Taiwanese entity. Originally described from females from Taiwan, S. arisanum until now has remained an enigmatic species.

The Simulium aureum group (Diptera: Simuliidae), also known as subgenus Eusimulium Roubaud, is a monophyletic, Holarctic taxon of bird-feeding black flies with a reduced chromosome number of two and remarkably similar external structure in all life stages. We analyzed the banding patterns of the polytene chromosomes to understand the composition of this species group along the northern coast of the Black Sea where two little-known nominal species have their type localities. Our analyses link the names Simulium krymense (Rubtsov) and Simulium maritimum (Rubtsov) with unique chromosomal characters, indicate that both are male chiasmate, and reveal the presence of Simulium angustipes Edwards along the northern coast of the Black Sea for the first time. We show that S. krymense has a banding sequence most similar to the hypothesized ancestral form of the S. aureum group, and that the entire group is derived from within the Simulium vernum group, rendering the latter group and its encompassing subgenus, Nevermannia Enderlein, paraphyletic.

The life of Roger Ward Crosskey stands as a paragon of scholarly dedication to the profession of entomology. His early interests in natural history prepared him for a diverse entomological career of about 65 years. He began as a medical entomologist working on the onchocerciasis problem with the Colonial Service in Northern Nigeria, and officially transitioned to dipterology, first studying the Tachinidae as a taxonomist with the Commonwealth Institute of Entomology, and later the Simuliidae as a Senior Principal Scientific Officer (Merit) with The Natural History Museum in London. His contributions to the fight against onchocerciasis (river blindness) and to the study of Diptera, particularly the Simuliidae, are an enduring legacy. An indexed chronological listing of his 247 publications is provided, in addition to lists of the taxa that he named and the taxa named in his honor.

Among the most prominent, large-scale patterns of species richness are the increases in richness with decreasing latitude and with increasing habitat heterogeneity. Using the stream-dwelling larval and pupal stages of North American black flies (Diptera: Simuliidae), we address 3 broad questions about species richness: (i) Does a significant latitude-richness relationship exist? (ii) How does habitat heterogeneity influence gamma diversity? (iii) What is the sign (positive or negative) of the latitude-richness and the heterogeneity-richness relationships? We found no evidence that habitat heterogeneity influences gamma diversity. The estimated peak species richness for black flies in North America was at 50-53°N, which also corresponds with peak generic richness. All plesiomorphic, extant lineages of the Simuliidae in the Western Hemisphere are found in cool mountainous environments of North America, suggesting that peak richness at 50-53°N might be a signature of this phylogenetic pattern and a reflection of underlying historical processes., (© 2017 Institute of Zoology, Chinese Academy of Sciences.)