Morphological and genetic analyses of Simulium (Gomphostilbia) okinawense Takaoka and S. (G.) tokarense Takaoka (Diptera: Simuliidae) from the Nansei Islands, Japan: redescription and transfer from the S. ceylonicum species-group to the S. asakoae species-group.

Simulium (Gomphostilbia) okinawense Takaoka and S. (G.) tokarense Takaoka, both from the Nansei Islands, Japan, were morphologically reexamined and genetically analysed by using the COI gene sequences. The female, male, pupa and mature larva of the two species are redescribed. Morphological reexamination shows that both species are more similar to species in the S. asakoae species-group than to those in the S. ceylonicum species-group, by having a medium-long female sensory vesicle, yellow tuft hairs (S. (G.) okinawense) or yellow tuft hairs mixed with a few to several dark hairs (S. (G.) tokarense) at the base of the radial vein in the female and male, and medium-long larval postgenal cleft. However, the body of the male ventral plate (viewed ventrally) is parallel-sided (S. (G.) okinawense) or parallelsided or slightly narrowed (S. (G.) tokarense) and not emarginated basally, differing from those of most species in the S. asakoae species-group. Our genetic analysis shows that S. (G.) tokarense is in the S. asakoae species-group, and S. (G.) okinawense formed a separate sister clade with other members of the S. asakoae species-group with high bootstrap support. From the results of morphological and genetic analysis combined, S. (G.) okinawense and S. (G.) tokarense are transferred from the S. ceylonicum species-group to the S. asakoae species-group.

Molecular circumscription of the blackfly Simulium oyapockense from South America.

Problems related to the identity of Simuliidae species are impediments to effective disease control in Amazonia. Some of these species, such as Simulium oyapockense Floch & Abonnenc, 1946 (Diptera: Simuliidae), are vectors of the organisms that cause onchocerciasis and mansonellosis diseases. This blackfly species has a wide distribution in South America, and it is suspected of being a complex of cryptic species. The aim of this study is to characterize the nominal species S. oyapockense using partial COI gene sequences. Seven populations of S. oyapockense (morphologically identified) were analysed, including one from its type-locality. The other six populations were collected in Brazil and in Argentina. A taxon collected in Amazonas state, Brazil, with adults similar to S. oyapockense but with distinct pupae, was also included in the analysis (Simulium 'S'). The nominal species S. oyapockense is circumscribed, and its geographical distribution is restricted to areas north of the Amazon River. Populations of S. oyapockense s.l. collected south of the Amazon River comprise a species complex that needs to be evaluated using integrative taxonomy. Simulium 'S' represents a species with unique morphological and molecular characteristics. Distinguishing cryptic species is a prerequisite for reducing the taxonomic impediment, especially in medically important taxa.

Description of the female of Simulium (Wallacellum) tuyense Takaoka (Diptera: Simuliidae) from the Philippines.

Simulium (Wallacellum) tuyense Takaoka, one of the 14 Philippine species in the subgenus Wallacellum Takaoka, is a rare species in having the pupal antennal sheath with a row of stout spines on the apex of each protuberance corresponding to flagellomeres 2-9. The female of this species is described for the first time based on specimens from Mindoro, the Philippines. It is characterized by an elongate sensory vesicle, triangular ovipositor valve, genital fork with two projections, and paraproct not produced anteriorly. This species is morphologically indistinguishable in the female, as in the male and pupa, from S. (W.) yonakuniense Takaoka from Yonakuni Island, Japan and Lanyu Island, Taiwan. Taxonomic notes on the female of this species relevant to those of related species are provided. Collection data to validate earlier distribution records of S. (W.) tuyense and S. (W.) suyoense Takaoka from Mindanao are presented.

Chironomids are commensals of the larvae and pupae of Blephariceridae and Simuliidae from the North Caucasus (Diptera: Chironomidae: Orthocladiinae).

Illustrated morphological descriptions of chironomid larvae from subfamily Orthocladiinae Cardiocladius sp. 1, which as commensals live between ventral suckers of Blephariceridae larvae, as well as larvae and pupae of Eukiefferiella claripennis group inhabited of Simuliidae pupal cocoons, are given. DNA barcodes of these chironomid species and sequences of their hosts, three species of Liponeura Loew (Blephariceridae) and one species of Simulium aff. variegatum (Simuliidae), are provided.

Morphological revision of Simulium (Gomphostilbia) ogatai (Rubtsov) in the S. ceylonicum species-group (Diptera: Simuliidae) from Japan.

Simulium (Gomphostilbia) ogatai (Rubtsov) from Japan, the only Palaearctic species in the S. ceylonicum species-group, is morphologically revised. Its female is described for the first time and its male and pupa are redescribed based on specimens from Gifu, Honshu. It is characterized in the female and male by the base of the radius with a tuft of yellow hairs, in the female by the head slightly wider than the thorax, 5.89 times as wide as the greatest width of the frons, and short sensory vesicle 0.21-0.24 times as long as the third palpal segment; in the male by the number of upper-eye (large) facets in 15 vertical columns and 16 horizontal rows, and hind basitarsus spindle-shaped, enlarged, 3.79 times as long as wide, and 0.83 and 1.00 times as wide as the greatest width of the hind tibia and femur, respectively; and in the pupa by the terminal hooks simple and cone-like, and the cocoon with an anterodorsal projection. Taxonomic notes for this species are given relevant to several related species in the Oriental Region, and its assignment to the S. ceylonicum species-group is confirmed.

A new species of black fly, formerly cytoform C of the Simulium angulistylum complex (Diptera: Simuliidae), from a high mountain in northeastern Thailand.

Cytoform C, one of three cytoforms in the Simulium angulistylum Takaoka & Davies complex from a high mountain in northeastern Thailand was morphologically and molecularly investigated. All known life stages (larva, pupa, adult male and female except egg) were morphologically similar to, but distinguishable from S. angulistylum s. str. and S. isanense Takaoka, Srisuka & Saeung in the adults by the relative length of the fore and hind basitarsi and relative length of the tooth to the claw. It is also morphologically distinct from other species of the S. epistum species-group. Here, it is formally described as a new species, S. prayooki. Molecular genetic data based on mitochondrial cytochrome c oxidase subunit I (COI) also supported the morphological similarity between the new species and the two related known species (S. angulistylum s. str. and S. isanense) as phylogenetic analysis retrieved them all from a single clade and with a low level of interspecific genetic divergence (1.74%). This might possibly have resulted from incomplete lineage sorting as they are likely to share a recent common ancestor. Despite limitation of molecular genetic differentiation, the new species was distinctly different from two other cytoforms of S. angulistylum complex based on polytene chromosome banding patterns and ecology of the immature stages. Thus, this study highlights the necessity of using an integrated approach for fully understanding black fly biodiversity.

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.

Descriptions of two new species of the Simulium (Simulium) striatum species-group (Diptera: Simuliidae) from Taiwan and Japan, and a revised description of S. (S.) quinquestriatum (Shiraki).

Species in the Simulium (Simulium) striatum species-group (Diptera: Simuliidae) in Taiwan and Japan are morphologically reevaluated. Simulium (S.) yanpingense sp. nov. is described as the third member in this species-group from Taiwan and a revised description of S. (S.) quinquestriatum (Shiraki) is provided. The species previously regarded as S. (S.) quinquestriatum in Japan is described as a new species, S. (S.) tanegashimaense. Simulium (S.) yanpingense sp. nov. is similar to S. (S.) pingtungense Huang & Takaoka from Taiwan, in many characteristics including the haired basal portion of the female radius, dark leg color, and male scutum with brassy hairs, but it is distinguished from the latter species by the number of male upper-eye (large) facets, which are in 17 (rarely 18) vertical columns and 17 or 18 horizontal rows in this new species but in 19 vertical columns and 20 horizontal rows in S. (S.) pingtungense. Simulium (S.) tanegashimaense sp. nov. is similar to S. (S.) quinquestriatum originally described from Taiwan by having the male scutum with yellow fine hairs, but is distinguished from the latter species by the number of male upper-eye (large) facets, which are in 17 (rarely 16) vertical columns and 17 horizontal rows in this new species but in 19 or 20 vertical columns and 19 or 20 horizontal rows in S. (S.) quinquestriatum. The morphological differences between these two new species and their related species from India, Malaysia, Thailand and Vietnam are noted.

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.

Atypical behavior of a black fly species connects cavity-nesting birds with generalist blood parasites in an arid area of Spain.

BACKGROUND: The feeding behavior of bloodsucking insects determines the transmission, distribution, host spectrum and evolution of blood parasites in the wild. Conventional wisdom suggests that some vector groups (e.g. black flies, family Simuliidae) are consistently exophagous daytime biters. We aimed to understand more about the exceptions to this pattern by combining targeted trapping and molecular identification of parasites in vectors. METHODS: In this study, we collected black flies in nest boxes used by European rollers Coracias garrulus in southeastern Spain. We molecularly analyzed 434 individual insects, identifying the black fly species caught in the nest boxes, their potential vertebrate blood meals, and the haemosporidian parasite lineages that they carried. RESULTS: Only one black fly species, Simulium rubzovianum, appeared to enter the nest boxes of rollers. Among the trapped specimens, 15% contained vertebrate DNA, which always belonged to rollers, even though only half of those specimens were visibly engorged. Furthermore, 15% of all black flies contained Leucocytozoon lineages, indicating previous feeding on avian hosts but probably not on infected adult rollers. The known vertebrate hosts of the recorded Leucocytozoon lineages suggested that large and/or abundant birds are their hosts. Particularly represented were cavity-nesting species breeding in the vicinity, such as pigeons, corvids and owls. Open-nesting species such as thrushes and birds of prey were also represented. CONCLUSIONS: Our data strongly suggest that S. rubzovianum bites uninfected roller nestlings and infected individuals of other species, potentially incubating adults, inside nest boxes and natural cavities. This simuliid does not appear to have a strong preference for specific host clades. Contrary to the general pattern for the group, and possibly enhanced by the harsh environmental conditions in the study area, this black fly appeared to intensively use and may even have a preference for confined spaces such as cavities for feeding and resting. Preferences of vectors for atypical microhabitat niches where hosts are less mobile may enable social and within-family transmission and parasite speciation in the long term. At the same time, a lack of host preference in concentrated multispecies communities can lead to host switches. Both processes may be underappreciated driving forces in the evolution of avian blood parasites.

Form and function of labral fan-ray microtrichia and posterior proleg barbs in Neotropical black flies larvae (Diptera: Simuliidae).

The microtrichia of the labral fan primary rays, considered as the finest structure of the larval filter mechanism in Simuliidae, and the posterior proleg hooks, a structure used for substrate attachment, were describe using Scanning Electron Microscopy for the first time in 14 Neotropical species of black flies. Four patterns of primary rays microtrichia and two types of hooks barbs disposition were found. The present study provides new morphological information for Neotropical black fly species, compares it with previous information for worldwide species. Also, shows that the presence of posterior proleg hooks barbs is common among black fly species, that these barbs are not related with the larval habitat or with the primary ray microtrichia pattern, and that flow conditions may influence the last-instar larvae microtrichial patterns, at least in most of the Neotropical species studied here.

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.

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.

A new species of Simulium (Gomphostilbia) (Diptera: Simuliidae) from Northern Thailand, with its genetic relationship in the S. asakoae species-group.

A new black fly species, Simulium (Gomphostilbia) kiewlomense, is described from females, males, pupae and mature larvae in Thailand. This new species is placed in the S. asakoae species-group and is characterized by having a combination of the elongate female sensory vesicle, widened male hind basitarsus, which is much wider than the hind femur, small pupal terminal hooks, and light greenish larval abdominal segments 1-3. Taxonomic notes are given to separate this new species from other related species. A DNA analysis using the COI gene shows that this new species has two genoforms with 1.21% difference. This is the 28th species of the S. asakoae species-group in Thailand, strengthening the evidence for high species diversity of this species-group.

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.

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.

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.

Comparisons of Respiratory Pupal Gill Development in Black Flies (Diptera: Simuliidae) Shed Light on the Origin of Dipteran Prothoracic Dorsal Appendages.

During the transformation of immature aquatic dipteran insects to terrestrial adults, the prothoracic pupal respiratory organ enables pupae to cope with flood-drought alternating environments. Despite its obvious importance, the biology of the organ, including its development, is poorly understood. In this study, the developing gills of several Simulium Latreille (Diptera: Simuliidae) spp. were observed using serial histological sections and compared with data on those of other dipteran families published previously. The formation of some enigmatic features that made the Simulium gill unique is detailed. Through comparisons between taxa, we describe a common developmental pattern in which the prothoracic dorsal disc cells not only morph into the protruding respiratory organ, which is partially or entirely covered with a cuticle layer of plastron, but also invaginate to form a multipart internal chamber that in part gives rise to the anterior spiracle of adult flies. The gill disc resembles wing and leg discs and undergoes cell proliferation, axial outgrowth, and cuticle sheath formation. The overall appendage-like characteristics of the dipteran pupal respiratory organ suggest an ancestral form that gave rise to its current forms, which added more dimensions to the ways that arthropods evolved through appendage adaptation. Our observations provide important background from which further studies into the evolution of the respiratory organ across Diptera can be carried out.