Zoonotic onchocerciasis is a human infection caused by Onchocerca species of animal origins and transmitted by black fly vectors. The reported incidence of this disease has increased throughout the world. This study aims to clarify the vectorial roles of black fly species in zoonotic filarial transmission in Tak province, western Thailand. The integrated approach of morphological and DNA sequence-based analyses was used to identify species of both wild-caught female black flies and infective filarial larvae found in the infected black flies. All of 494 female black flies captured were identified as Simulium nigrogilvum, through scanning electron microscopy (SEM) and DNA sequence analyses based on the cytochrome c oxidase subunit I (COI) and subunit II (COII), and the fast-evolving nuclear elongation complex protein 1 (ECP1) genes. Four females of S. nigrogilvum harbored one to three third-stage larvae (infective larvae) in their thoraces, with an infection rate of 0.81% (4/494). All infective larvae were similar in morphology and size to one another, being identified as Onchocerca species type I (= O. sp. type A), a bovine filaria, originally reported from Japan, and also as O. sp. found in S. nodosum in Thailand, based on their body lengths and widths being 1,068-1,346 µm long by 25-28 µm wide, and morphological characters. Comparisons of cytochrome c oxidase subunit I (COI) and 12S rRNA sequences of mitochondrial DNA (mtDNA) and phylogenetic analyses with those of previous reports strongly supported that all larvae were O. sp. type I. This report is the first indicating the presence of O. sp. type I in Thailand and its vector being S. nigrogilvum.
Although several studies have reported pharmacological and immunological activity, as well as the role of black flies in transmitting pathogens to vertebrate hosts through salivary glands (SG) during blood feeding, SG proteomes of the anthropophilic black flies in Thailand have never been reported. Therefore, this study determined the SG proteomes of female S. nigrogilvum and S. nodosum. Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional (2-DE) gels containing separated SG proteins of individual species were subjected to liquid chromatography-tandem mass spectrometry (LCMS/MS) and an orthologous protein search from eukaryotic organism, nematocera and simuliidae databases for total protein identification. SDS-PAGE and protein staining revealed at least 13 and 9 major protein bands in the SGs of female S. nigrogilvum and S. nodosum, respectively, as well as several minor ones. The 2-DE demonstrated a total of 56 and 41 protein spots for S. nigrogilvum and S. nodosum, respectively. Most of the proteins obtained in both species were enzymes involved in blood feeding, including proteases, apyrases, hyaluronidases, aminopeptidase and elastase. The results obtained in this study provided a new body of knowledge for a better understanding on the role of salivary gland proteins in these black fly species in Thailand.
An 11-year-old boy living in Otsu City, Shiga Prefecture, Kansai Region, Western Honshu, Japan had zoonotic onchocercosis. The patient developed a painful swelling on the little finger of his left hand. The worm detected in the excised mass had external transverse ridges but did not have inner striae in the cuticle. On the basis of the parasite's histopathological characteristics, the causative agent was identified as a female Onchocerca dewittei japonica (Spirurida: Onchocercidae). The species of the filarial parasite was confirmed by sequencing the cox1 gene of the parasite. The Japanese wild boar Sus scrofa leucomystax is a definitive host for O. dewittei japonica, which is then transmitted by blackflies as the vector to humans. The current case described occurred in the Kansai Region, Western Honshu, where such infections were previously not reported.
Simulium (Nevermannia) maeaiensesp. n. is described on the basis of female, male, pupal and larval specimens collected from Chiang Mai Province, Thailand. This species is assigned to the feuerborni species-group of the subgenus Simulium (Nevermannia), and is distinctive among this species-group in having the female cibarium furnished with numerous dark minute conical processes on the lower part, the female genital fork with a strongly sclerotized horizontal bar on each arm, and six long pupal gill filaments arising nearly at the same level from the common basal stalk and lying in a horizontal plane. Identification keys to seven species of the feuerborni species-group reported from Thailand are provided for females, males, pupae and mature larvae.
To investigate the breeding habitat preference of black flies, a comprehensive black fly survey was conducted for the first time in Peninsular Malaysia. Preimaginal black flies (pupae and larvae) were collected manually from 180 stream points encompassing northern, southern, central and east coast of the Peninsular Malaysia. A total of 47 black fly species were recorded in this study. The predominant species were Simulium trangense (36.7%) and Simulium angulistylum (33.3%). Relatively common species were Simulium cheongi (29.4%), Simulium tani (25.6%), Simulium nobile (16.2%), Simulium sheilae (14.5%) and Simulium bishopi (10.6%). Principal Component Analysis (PCA) of all stream variables revealed four PCs that accounted for 69.3% of the total intersite variance. Regression analysis revealed that high species richness is associated with larger, deeper, faster and higher discharge streams with larger streambed particles, more riparian vegetation and low pH (F=22.7, d.f.=1, 173; P<0.001). Relationship between species occurrence of seven common species (present in >10% of the sampling sites) was assessed. Forward logistic regression analysis indicated that four species were significantly related to the stream variables. S. nobile and S. tani prefer large, fast flowing streams with higher pH, large streambed particles and riparian trees. S. bishopi was commonly found at high elevation with cooler stream, low conductivity, higher conductivity and more riparian trees. In contrast, S. sheilae was negatively correlated with PC-2, thus, this species commonly found at low elevation, warmer stream with low conductivity and less riparian trees. The results of this study are consistent with previous studies from other geographic regions, which indicated that both physical and chemical stream conditions are the key factors for black fly ecology.
Human zoonotic onchocercosis is caused by Onchocerca dewittei japonica, parasitic in wild boars (Sus scrofa leucomystax) in Japan. Previously, microfilariae longer than those of Onchocerca dewittei japonica were observed in skin snips from wild boars during the study of O. dewittei japonica. Moreover, the third-stage larvae (L3) of these longer microfilariae were obtained from the blackfly Simulium bidentatum after experimental injections. Based on morphometric and molecular studies, similar L3 were found in blackflies during fieldwork in Oita, Japan. However, except for O. dewittei japonica, adult worms of Onchocerca have not been found in wild boars. In this study, we discovered adult females of a novel Onchocerca species in the skin of a wild boar in Oita, and named it Onchocerca takaokai n. sp. Females of this new species had longer microfilariae and differed from O. dewittei japonica in terms of their morphological characteristics and parasitic location. The molecular characteristics of the cytochrome c oxidase subunit 1 and 12S rRNA genes of the new species were identical to those of the longer microfilariae and L3 previously detected, but they differed from those of O. dewittei japonica at the species level. However, both species indicated a close affinity among their congeners and Onchocerca ramachandrini, parasitic in the warthog in Africa, was basal in the Suidae cluster of the 12S rRNA tree.
Black soldier fly (BSF) larva is an attractive animal feed replacer due to its noticeable nutritional content. However, the conventional rearing method often resulted in BSF with undesirably high heavy metal residues that are harmful to animals. In this work, putrefied Sesbania grandiflora (S. Grandiflora) leaves were employed as feed to rear BSF larvae. The resultant BSF prepupae were found to contain 43.5% protein and 16.7% fat, reflecting a comparable protein content and a 2-fold reduction in crude fat than those reared using conventional kitchen waste. Moreover, high quantities of arginine (25.4 g/kg dry matter basis (DM)), carnitine (32.9 g/kg DM), and short-chain fatty acids, including lauric (40.00%), palmitic (19.20%), and oleic (12.10%) acids, have also been noticed in the BSF prepupae. Furthermore, the BSF larvae have been recorded with 0.185 mg/kg chromium, 0.380 mg/kg selenium, and mercury below the detection limit, which is far lower than those reared using conventional kitchen and agricultural wastes (≈1.7 mg/kg chromium, 1.2 mg/kg selenium, and 0.2 mg/kg mercury). Overall, the study shows that the nutritional quality of BSF prepupae is extensively improved when using S. Grandiflora as their feed. The resultant BSF prepupae may serve as an alternative feed for animal rearing.
The transmission of zoonotic filarial parasites by black flies has so far been reported in the Chiang Mai and Tak provinces, Thailand, and the bites of these infected black flies can cause a rare disease-human zoonotic onchocerciasis. However, species identification of the filarial parasites and their black fly vectors in the Chiang Mai province were previously only based on a morphotaxonomic analysis. In this study, a combined approach of morphotaxonomic and molecular analyses (mitochondrial cox1, 12S rRNA, and nuclear 18S rRNA (SSU HVR-I) genes) was used to clarify the natural filarial infections in female black flies collected by using human and swine baits from two study areas (Ban Lek and Ban Pang Dang) in the Chiang Mai province from March 2018 to January 2019. A total of 805 and 4597 adult females, belonging to seven and nine black fly taxa, were collected from Ban Lek and Ban Pang Dang, respectively. At Ban Lek, four of the 309 adult females of Simulium nigrogilvum were positive for Onchocerca species type I in the hot and rainy seasons. At Ban Pang Dang, five unknown filarial larvae (belonging to the same new species) were detected in Simulium sp. in the S. varicorne species-group and in three species in the S. asakoae species-group in all seasons, and three non-filarial larvae of three different taxa were also found in three females of the S. asakoae species-group. This study is the first to molecularly identify new filarial species and their vector black fly species in Thailand.
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.
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.
The nuisance bites of blackflies and transmission of Onchocerca volvulus, which causes onchocerciasis, constitutes a threat to public health and an impediment to food production in rural and riverine communities in Nigeria. The entomological profile of onchocerciasis at Adani, Nigeria, was investigated from August 2010 to January 2011 to determine the transmission of O. volvulus after 15 years of ivermectin distribution in the area. A total of 548 adult female blackflies of the Simulium damnosum complex were caught using human baits and dissected. Of this number, 248 flies were caught in the wet season (August to October), while 300 flies were caught in the dry season (November to January). The relative abundance of flies at Adani varied from 21 in December to 243 in January. The monthly catches between September and October and between December and January were significantly different. The monthly population density of the flies ranged from 0.5Flies/Man/Hour (FMH) in December to 5.5FMH in January. The diurnal biting pattern of the S. damnosum complex at the site showed a bimodal peak of activity with the evening peak being higher than the morning peak except in October when the morning peak was higher than the evening peak. The morning peaks were observed between 7.00 am and 10.00 am, whereas the evening peaks occurred between 4.00 pm and 6.00 pm. The morning and evening biting peaks in all the months were not significantly different. Nulliparous flies accounted for 75.7% of the total catch, whereas 24.3% of the flies caught were parous. The infection, infective bites and transmission of O. volvulus during the study period were zero. This study suggests that transmission of O. volvulus has been halted and the flies are presently more nuisance biters than disease vectors since no stage of O. volvulus was found in the flies dissected.
Simulium (Nevermannia) ledangense sp. nov. is described from females, males, pupae and mature larvae from Peninsular Malaysia. This new species is assigned to the Simulium feuerborni species-group of the subgenus Nevermannia, and is characterized by the pupa having a very long stalk of the ventral paired gill filaments, which is almost five times longer than the interspiracular trunk and female tergites of segments 2 and 5 to 7 shiny. Taxonomic notes are given to distinguish this new species from three known species of the S. feuerborni species-group from Malaysia.
Three new species of black flies, Simulium (Nevermannia) langbiangense, S. (N.) phami, and S. (N.) bachmaense, are described on the basis of females, males, pupae and larvae collected in Vietnam. All three species are assigned to the Simulium feuerborni species-group of Simulium (Nevermannia). Simulium (N.) langbiangense sp. nov. is characterized by the female sensory vesicle with a large opening, and a short common basal stalk of the six pupal gill filaments, S. (N.) phami sp. nov. is most striking in having the pupal gill with five filaments, a character not reported in species of the S. feuerborni species-group, and S. (N.) bachmaense sp. nov. is characterized by the female genital fork with a triangular lobe-like projection pointed posteromedially on each arm, and the small larval postgenal cleft. This represents the first record of the S. feuerborni species-group from Vietnam.
Two new species of black flies, Simulium (Gomphostilbia) roslihashimisp. n. and Simulium (Gomphostilbia) lurauensesp. n., are described on the basis of reared adult, pupal and larval specimens collected from Peninsular Malaysia. These two new species are placed in the ceylonicum species-group within the subgenus Gomphostilbia. Simulium (Gomphostilbia) roslihashimisp. n. is most distinctive with the male having almost entirely yellow antennae, and Simulium (Gomphostilbia) lurauensesp. n. is characterized in the female by having the elongate sensory vesicle and the yellowish-white hairs on the base of the costal vein and on the stem vein, in the male by the greater number of large upper-eye facets and the spindle-shaped hind basitarsi which are much narrower than the hind tibiae and femora and in the pupa by the small terminal hooks. Keys to species of the ceylonicum species-group reported from Peninsular Malaysia are provided for females, males, pupae and mature larvae.
Species of the Simulium (Simulium) melanopus species-group in Sabah are taxonomically revised by examining type specimens of S. (S.) crassimanum S. (S.) laterale, and S. (S.) nigripilosum, all described from females by Edwards in 1933, and newly collected samples from the vicinity of Mt. Kinabalu. The females of these three species are redescribed, and their males and pupae are described for the first time based on adults reared from pupae. Simulium (S.) liewi Takaoka, 2007 and S. (S.) kinabaluense Smart & Clifford, 1969 are synonymized with S. (S.) crassimanum and S. (S.) laterale, respectively. Simulium (S.) cheedhangi Takaoka, Sofian-Azirun & Ya'cob, 2015 is newly recorded from Sabah. Two new related species, S. (S.) lardizabalae and S. (S.) timpohonense, are described from males reared from pupae. Keys to identify eight species of the S. melanopus species-group in Sabah are provided for females, males, pupae and mature larvae.
Surveys of pupae and larvae of black flies (Diptera: Simuliidae) were carried out in Thua Thien Hue Province of central Vietnam, and Lam Dong Province of southern Vietnam in 2014. A total of 26 species belonging to the genus Simulium were collected, consisting of eight known species, one newly recorded species, and 17 new species (of which three species of the subgenus Nevermannia were described in 2014). The remaining 14 new species (nine of the subgenus Gomphostilbia and five of the subgenus Simulium) are described here based on females, males, pupae and mature larvae. The total number of species of black flies in Vietnam is now 46. Keys to identify all 26 species recorded from the two provinces of Vietnam are given for females, males, pupae and mature larvae.
A new species of black fly, Simulium (Simulium) luculentum, is described from specimens collected in Thailand, and is assigned to the Simulium variegatum species-group of the subgenus Simulium Latreille. This new species is distinguished from most members of this species group in the female by the mid femur and tibia entirely yellowish; and in the pupa by the gill with six filaments, which are somewhat inflated basally. This species represents the fifth member of the S. variegatum species-group recorded from Thailand.
The black-winged fly, Felderimyia fuscipennis (Diptera: Tephritidae), is an insect pest of bamboo shoot, mainly distributed in Thailand, Malaysia and Yunnan Province and Guangxi Autonomous Region, China. The complete sequence of the mitogenome of F. fuscipennis has been determined in this study. The whole mitogenome sequence is 16,536 bp in length, which totally contains 13 protein-coding genes (PCGs), 2 rRNA genes, 22 tRNA genes, and a non-coding region (putative control region, CR). The phylogeny indicates that F. fuscipennis of subfamily Trypetinae was monophyletic and clearly separated from both Dacinae and Tephritinae with high bootstrap value supported.
Black flies form a group of small blood-sucking insects of medical and veterinary importance. This study aimed to investigate the community structure, biodiversity and spatial and temporal distribution of adult black flies in tropical rain forests, by using malaise traps in Doi Inthanon National Park, northern Thailand. Malaise traps were placed along six elevational gradients (400 m to 2500 m, above sea level) at Doi Inthanon National Park, Chiang Mai province, from December 2013 to November 2014. A total of 9406 adult female black flies belonging to five subgenera-Daviesellum (2%), Gomphostilbia (23%), Montisimulium (11%), Nevermannia (16%) and Simulium (48%)-were collected. Among 44 taxa found, S. tenebrosum complex had the highest relative abundance (11.1%), followed by the S. asakoae species-group (9.6%), the S. striatum species-group (7.7%), S. inthanonense (6.6%), S. doipuiense complex (6.4%), S. chomthongense complex (5.3%), S. chumpornense (5.1%) and S. nigrogilvum (4.1%). Two human-biting species-S. nigrogilvum and species in the S. asakoae species-group-were found in all of the collection sites with 100% species occurrence. Species richness was highest at mid elevation (1400 m), which is represented by 19 black fly species. The peak and lowest seasonal abundance was observed in the rainy and hot season, respectively. Seasonal species richness was highest in the cold season, except for that from elevation sites at 700 m, 1700 m and 2500 m. This study revealed that the malaise trap is effective in providing important data for further monitoring of the effects of environmental changes and conservation planning on the biodiversity of black flies in Doi Inthanon National Park.
Simulium chayamaritae Takaoka and Srisuka from Thailand belongs to the Simulium darjeelingense species-group of Simulium (Simulium) (Diptera: Simuliidae). The female of this species is described for the first time based on a female reared from a pupa collected from Chiang Mai, Thailand. It is characterized by the sensory vesicle elongate and the inner margins of the arms of the genital fork divergent, then convergent apically. It is similar to the female of S. eshimai Takaoka and Adler of the same speciesgroup from Vietnam. Taxonomic notes are given to separate it from two other species of the S. darjeelingense species-group from India and Malaysia, and 28 of 31 other species of the subgenus Gomphostilbia recorded from Thailand.