Molecular detection of blood protozoa and identification of black flies of the Simulium varicorne species group (Diptera: Simuliidae) in Thailand.

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.

The Biodiversity of Blackflies (Diptera: Simuliidae) in Brazil: New Distribution Records and Updated Checklist.

Currently, according to the most recent Simuliidae World Inventory, there are 97 valid species of blackflies recorded in Brazil, some of which act as vectors for zoonoses such as human onchocerciasis and mansonellosis in the northern and central-western regions of the country. Meanwhile, other species can cause serious socioeconomic problems due to the nuisance of female bites. Therefore, accurate knowledge of their distribution is crucial for the development and implementation of successful preventive strategies. With this aim, this study reviewed and updated the geographical distribution of the blackfly fauna throughout the Brazilian states. The data were compiled from three main sources: geographic information of material deposited at the Simuliidae Collection of the Instituto Oswaldo Cruz (CSIOC-IOC), a comprehensive review of scientific literature, and online biodiversity databases. We present a total of 71 new distribution records of 38 different Simuliidae species for 24 Brazilian states. Neither of these sets of records has been included in the Simuliidae World Inventory. Consequently, an updated Brazilian Simuliidae checklist, comprising a total of 98 valid species, is presented, highlighting these new distribution records. We also discuss six dubious records for the country and the implications of this updated data for the Simuliidae species richness of Brazil, its states, and biomes. This information is essential for future studies in the taxonomy, systematics, and biogeography of this family in Brazil.

Description of the male of Simulium triglobus Takaoka & Kuvangkadilok (Diptera: Simuliidae) from Thailand.

Simulium triglobus Takaoka & Kuvangkadilok from Thailand, in the Simulium (Simulium) multistriatum species-group, is unique among species in the family Simuliidae in having the female terminalia with three spermathecae (rather than one spermatheca). This species was described from Nan province, northern Thailand based on larvae, pupae and females but its male has remained unknown. In this study, the male of S. triglobus is described for the first time based on adult males reared from pupae collected from the type locality. The most distinctive characteristic of the male of S. triglobus is the shape of the ventral plate, which is hexagonal when viewed ventrally. No other members of S. multistriatum species group known thus far have such a unique ventral plate. In addition, the number of upper-eye (large) facets and color patterns of the legs can be used to differentiate this species in the male from other members of the S. multistriatum species-group. Mitochondrial cytochrome c oxidase I sequences enabled association of adult male specimens of S. triglobus with previously known life stages. Phylogenetic analysis based on these sequences revealed that specimens of S. triglobus formed a strongly supported monophyletic clade, being genetically distinct from other members of S. multistriatum species-group in Thailand.

Two new black fly species of the Simulium ( Simulium) rufibasis subgroup (Diptera: Simuliidae) from Taiwan.

Simulium ( Simulium) rufibasis Brunetti originally described from India was once considered a geographic generalist widely distributed in the Oriental Region. In this study, the species previously regarded as S. (S.) rufibasis in Taiwan was morphologically re-evaluated and found to be distinguished in the male and pupa from true S. (S.) rufibasis from India. This new species is described as S. ( S.) hehuanense sp. nov. based on a female, a male and their pupal exuviae in Taiwan. This new species is placed in the S. rufibasis subgroup of the S. tuberosum species-group, and is similar to S. (S.) yamatoense Takaoka, Adler & Fukuda from Japan and Korea, but it is barely distinguished by the slenderer forebasitarsi of the female and male. Another new, related species, S. (S.) xiulinense sp. nov., is described based on a male and its pupal exuviae. This new species is distinguished from S. (S.) hehuanense sp. nov. by the number of male upper-eye (large) facets in 20 or 21 vertical columns and 22 horizontal rows (19 vertical columns and 19 horizontal rows in the latter species), and presence of a pair of dorsolateral shiny spots on male abdominal segment 5 (absence in the latter species). Both new species inhabit streams at high elevations (ca, 2,600 m). They differ from S. (S.) sp. (probably S. (S.) arisanum Shiraki) from Taiwan, of the same subgroup, which breeds in streams at low elevations (433-685 m), by the pupal head and thorax covered with tubercles (bare in the latter species).

Simulium maiaherzogae sp. nov., a new species of black fly (Diptera, Simuliidae) from rock fields of southeastern Brazil.

A new species of black fly from rock fields of southeaster Brazil, Simulium maiaherzogae sp. nov., is described based on morphological features of larval, pupal and adult specimens. Morphological features of this new species were compared with members of Simulium (Trichodagmia) Enderlein 1934, showing a high similarity with S. scutistriatum Lutz 1909 and S. itajara Nascimento, et al. 2020. However, features of the male and female scutum and genitalia, pupal tubercles disposition, pupal gills, larval cephalic appendages, larval gill histoblast, numbers of posterior proleg hooks and rectal papillae lobules, allow to differentiate S. maiaherzogae sp. nov. Moreover, a more detailed analysis using Scanning Electron Microscopy (SEM) showed significant differences and numerical variations in meristic data between the new species and S. scutistriatum. The known distribution of this new species is restricted to the Rio do Salto sub-basin, headland of the Middle Paraíba do Sul River, near the Ibitipoca State Park, a region recognised for its biodiversity richness, but with rare simuliids records.

Two new black fly species of the Simulium (Gomphostilbia) asakoae species-group (Diptera: Simuliidae) from Taiwan.

Two new black fly species, Simulium (Gomphostilbia) kaohsiungense and S. (G.) shoufengense, are described from females, males and pupae in Taiwan. These new species are placed in the S. asakoae species-group. Simulium (G.) kaohsiungense sp. nov. is characterized by a combination of the elongate female sensory vesicle, male upper-eye (large) facets in 15 vertical columns and 15 or 16 horizontal rows, male hind basitarsus 1.02-1.15 times the width of the hind femur, and widened pupal terminal hooks. These characteristics distinguish this new species from most species of the S. asakoae species-group. Simulium (G.) shoufengense sp. nov. is similar in the female and male to S. (G.) asakoae Takaoka & Davies originally described from Peninsular Malaysia but is distinguished from the latter species in the pupa by the combined length of the primary and secondary stalks of the dorsal triplet of gill filaments as long as or longer than the common basal stalk of the gill, and dorsum of abdominal segments 1 and 2 not darkened. The species previously regarded in Taiwan as S. (G.) metatarsale Brunetti, originally described from India, is morphologically and molecularly identified as S. (G.) asakoae.

A male black fly of Simulium (Simulium) iwatense (Shiraki) (Diptera: Simuliidae) with genitalia-like appendages on abdominal segment 8.

A rare non-sex mosaic abnormality represented by genitalia-like appendages on the ventral surface of abdominal segment 8 of a male black fly collected in Hokkaido, Japan, is reported. The appendages consist of a pair of style-like projections each arising from a coxite-like base, inverted-Y shaped ventral plate-like structure, and isolated round structure. This male was morphologically and molecularly identified as an abnormal form of S. (S.) iwatense (Shiraki), the only species in the Simulium (Simulium) ornatum species-group in Japan, although certain morphological characteristics of this male including the reduced number of uppereye (large) facets and elongate cerci are different from those of S. (S.) iwatense.

Culturable bacteria in adults of a Southeast Asian black fly, Simulium tani (Diptera:Simuliidae).

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 ß-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.

A new species and four newly recorded species of black flies (Diptera: Simuliidae) from Bhutan.

Simulium (Simulium) thimphuense sp. nov. is described from a pupa and a mature larva in Bhutan. This new species is placed in the S. multistriatum species-group, and is characterized by the pupal gill with eight thread-like filaments divergent basally at an acute angle when viewed laterally and the cocoon slipper-shaped with several small openings anterolaterally. Four species of Simulium (Simulium) are newly recorded from Bhutan: S. barraudi Puri in the S. multistriatum species-group, S. nodosum Puri in the S. nobile species-group, S. chiangmaiense Takaoka & Suzuki in the S. striatum species-group and S. himalayense Puri in the S. variegatum species-group. Our study increases the number of black fly species known from Bhutan from 18 to 23.

Simulium (Gomphostilbia) aziruni: First record of a black fly (Diptera: Simuliidae) attracted to a human in Malaysia.

Most female black flies in the genus Simulium are blood-sucking flies and they can cause various parasitic diseases in human and animal. A total of 94 species of black flies have been reported in Malaysia, however, their biting behavior and role as vector of infectious agents remain understudied. To fill in this knowledge gap, we attempted to survey adult black flies from field populations in Peninsular Malaysia. In a survey carried out in 2017 at Tasik Kenyir, Terengganu, three females were caught while attracted and landed on human skin. Further morphological and molecular analyses showed that the specimens were identical to Simulium (Gomphostilbia) aziruni Takaoka, Hashim & Chen of the Simulium gombakense species-group. This is the first report on a black fly species attracted to human in Malaysia which serves as a steppingstone towards in-depth studies for black flies in this region.

A new species of the Simulium (Gomphostilbia) ceylonicum species-group (Diptera: Simuliidae) from Thailand.

A new black fly species, Simulium (Gomphostilbia) pangsidaense, is described on the basis of adult female, male, pupal exuviae and mature larvae from Pang Sida National Park, Sa Kaew Province, Eastern Thailand. This new species is placed in the Simulium ceylonicum species-group. It is distinguished from three Thai members of the S. ceylonicum species-group by the following characteristics: from S. (G.) curtatum Jitklang et al. and S. (G.) sheilae Takaoka & Davies by the wide pupal terminal hooks (triangular terminal hooks in the latter two species), and from S. (G.) sheilae and S. (G.) trangense Jitklang et al. by the number of male upper-eye facets in 13 vertical columns and 14 or 15 horizontal rows (10 or 11 vertical columns and 12 or 13 horizontal rows in the latter two species). This is the fourth member of the S. ceylonicum species-group recorded from Thailand.

Back from the past: Molecular and morphological support for Simulium mutucuna Nunes de Mello & Vieira da Silva, 1974 (Diptera: Simuliidae) as a valid species.

Simulium mutucuna, a species described based on a single female from Roraima state, was previously synonymized with Simulium paynei and currently is considered a synonym of Simulium rubrithorax. In the present paper we present morphological and molecular evidence supporting the validity of S. mutucuna based on analysis of specimens from Brazil, Venezuela and Mexico. We redescribe the female and describe, for the first time, the male, pupa and larva of S. mutucuna and discuss the morphological differences between this species and the others that are already considered as its senior synonyms. Currently, the distribution of S. mutucuna is restricted to Roraima state. The distribution record for S. rubrithorax in Brazil's North region needs to be removed, since the previous records were based on occurrence of S. mutucuna. Finally, we present new evidence of cryptic diversity in the S. paynei complex based on molecular information.

Foretelling the Phenotype of a Genomic Sequence.

Estimating phenotypic features (physical and biochemical traits) in a biological organism from their genomic sequence alone and/or environmental conditions has major applications in anthropological paleontology and criminal forensics, for example. To what extent do genomic sequences generally and causally determine phenotypic features of organisms, environmental conditions aside? We present results of two studies, one in blackfly (Insecta:Diptera:Simuliidae) larvae in two species (Simulium ignescens and S. tunja) with four phenotypic features, including the area and spot pattern of the cephalic apotome (in the form of a latin cross on the dorsal side of the head), the postgenal cleft (area under the head on the ventral side) and general body color in larva specimens; the second in strains of Arabidopsis thaliana. They establish that a substantial component of these phenotypic features (over 75 percent) are at least logically inferable, if not causally determined, by genomic fragments alone, despite the fact that these phenotypic features are not 100 percent determined entirely by genetic traits. These results suggest that it is possible to infer the genetic contribution in the determination of specific phenotypic features of a biological organism, without recourse to the causal chain of metabolomics and proteomic events leading to them from genomic sequences.

Pedrowygomyia (Diptera: Simuliidae): Discovery of a New Species After 30 yr.

Pedrowygomyia is a Neotropical genus of Simuliidae composed of four species; all were described in 1989 from high-elevation (above 3,000 m) areas in the Andean region. In this article, a new species for this genus, Pedrowygomyia hanaq n. sp., is described based on all stages of development. The new species was collected in the south-central Andes of Peru at an altitude above 4,000 m, and its known distribution is currently restricted to the type-locality. Based on the pupal stage, the new species appears to be more closely related to Pedrowygomyia punapi (Wygodzinsky & Coscarón) (Diptera: Simuliidae), a species known from Argentina, Bolivia, and Chile.

Three new species of the Simulium (Simulium) striatum species-group (Diptera: Simuliidae) from Thailand, with their phylogenetic relationships.

Three new species of black flies from Thailand, Simulium wangkwaiense, S. tadtonense and S. maeklongkeense, are described based on their adults, pupal exuviae and cocoons. All three new species are assigned to the Simulium (Simulium) striatum species-group, bringing its total number in Thailand to seven. Simulium wangkwaiense sp. nov. is the species formerly called S. quinquestriatum in Thailand. Certain male and pupal morphological characteristics are shown to separate all seven Thai species of this species-group. All of the three new species have been analyzed genetically for their phylogenetic relationships, with three known related species (except for S. thailandicum), by using the fast-evolving nuclear big zinc finger (BZF) gene.

Black flies (Diptera: Simuliidae) of the Aras River Basin: Species composition and floral visitation.

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.

A new species in Simulium (Trichodagmia) (Diptera: Simuliidae) from Chapada Diamantina region, Brazil: cryptic diversity revealed by morphological and molecular evidence.

We describe new species of black fly that had previously been identified as S. scutistriatum Lutz due to morphological similarities at the pupal stage. The description of the new species, Simulium (Trichogamia) itajara n. sp., is based on molecular and morphological evidences. The known distribution of the new species is currently restricted to the Paraguaçu River hydrographic basin in Chapada Diamantina National Park and the surrounded area in Bahia state, Brazil. The distribution record for S. scutistriatum in the northeast region of Brazil needs to be removed, since the previous records were based on occurrence of S. itajara n. sp.

Cryptic species of black flies (Diptera: Simuliidae) at high elevations in the Oriental Region: The Simulium vernum species group in Thailand.

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.

Ecology of black flies (Diptera: Simuliidae) in streams of northern and southern Thailand: Factors associated with larval and pupal distributions.

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.

Hidden biodiversity revealed by DNA barcoding in black fly genus Simulium.

BACKGROUND & OBJECTIVES: The black fly genus Simulium Latreille is one of the most important medical insect group of the family Simuliidae (Diptera) and many species of this genus are important pests of human and animals, while some of them also represent vectors of pathogens. Correct species identification is essential to the implementation of control measures for species of medical or agricultural importance. METHODS: In this study, the usefulness of DNA barcoding was discussed in distinguishing species of Simulium. RESULTS: Analysis showed hidden biodiversity, usually referred to in Simuliidae as cryptic species, which was detected in 15 species. Firstly, intraspecific divergences of eleven species was unexpectedly high and the maximum distances of them ranged from 5.1-16.8%. Based on the differential of K2P (Kimura 2-Parameter) distances, sequences were subdivided into two or three groups, respectively. Secondly, extremely low interspecific divergences were detected in eight groups of species, and shared haplotypes were also found among them. Furthermore, the subdivision within species and shared haplotypes among some species were all supported by the NJ (Neighbour-Joining) analysis. INTERPRETATION & CONCLUSION: Our results confirmed that DNA barcoding was a powerful tool for revealing hidden species diversity of black flies. Further work is needed to reveal ambiguous species delimitation in some problematic species groups.