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

Izwan-Anas N, Ya'cob Z, Low VL, Lourdes EY, Ramli R, Bolongan G, et al.

Acta Trop, 2021 Jun;218:105904.
PMID: 33775626 DOI: 10.1016/j.actatropica.2021.105904

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.
Occurrence of five distinct clades of mermithid nematodes (Nematoda: Mermithidae) infecting black fly larvae (Diptera: Simuliidae) in tropical streams in Malaysia

Lourdes EY, Low VL, Izwan-Anas N, Dawood MM, Sofian-Azirun M, Takaoka H, et al.

Parasitol Int, 2023 Jun;94:102733.
PMID: 36693472 DOI: 10.1016/j.parint.2023.102733

Mermithids are the most common parasites of black flies and are associated with host feminization and sterilization in infected hosts. However, information on the species / lineage of black fly mermithids in Southeast Asia, including Malaysia requires further elucidation. In this study, mermithids were obtained from black fly larvae collected from 138 freshwater stream sites across East and West Malaysia. A molecular approach based on nuclear-encoded 18S ribosomal RNA (18S rRNA) gene was used to identify the species identity / lineage of 77 nematodes successfully extracted and sequenced from the specimens collected. Maximum likelihood and neighbor-joining phylogenetic analyses demonstrated five distinct mermithid lineages. Four species delimitation analyses: automated simultaneous analysis phylogenetics (ASAP), maximum likelihood Poisson tree processes with Bayesian inferences (bPTP_ML), generalized mixed yule coalescent (GMYC) and single rate Poisson tree processes (PTP) were applied to delimit the species boundaries of mermithid lineages in this data set along with genetic distance analysis. Data analysis supports five distinct lineages or operational taxonomic units for mermithids in the present study, with two requiring further investigation as they may represent intraspecific variation or closely related taxa. One mermithid lineage was similar to that previously observed in Simulium nigrogilvum from Thailand. Co-infection with two mermithids of different lineages was observed in one larva of Simulium trangense. This study represents an important first step towards exploring other aspects of host - parasite interactions in black fly mermithids.
Wild-caught adult black flies (Diptera: Simuliidae) from various ecological landscapes in Malaysia

Izwan-Anas N, Halim MRA, Low VL, Adler PH, Ya'cob Z

Acta Trop, 2024 Aug 28;259:107374.
PMID: 39214235 DOI: 10.1016/j.actatropica.2024.107374

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. CO2-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.
First blood-meal record of Simulium asakoae (Diptera: Simuliidae) in Malaysia, with notes on its distribution in Asia and status as a potential vector

Izwan-Anas N, Ya'cob Z, Low VL, Lourdes EY, Teoh BT, Mansor MS, et al.

Trop Biomed, 2024 Sep 01;41(3):251-256.
PMID: 39548777 DOI: 10.47665/tb.41.3.003

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 CO2-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·HARTM 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.
From bites to barcodes: uncovering the hidden diversity of black flies (Diptera: Simuliidae) in Vietnam

Putt QY, Ya'cob Z, Adler PH, Chen CD, Hew YX, Izwan-Anas N, et al.

Parasit Vectors, 2023 Aug 07;16(1):266.
PMID: 37545007 DOI: 10.1186/s13071-023-05892-0

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

Hew YX, Ya'cob Z, Adler PH, Chen CD, Lau KW, Sofian-Azirun M, et al.

Parasit Vectors, 2023 Jul 22;16(1):248.
PMID: 37480109 DOI: 10.1186/s13071-023-05875-1

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.