DNA barcoding of tropical black flies (Diptera: Simuliidae) in Thailand: One decade of progress

Abstract

Black flies (Diptera: Simuliidae) are important blood sucking insects because they are the vectors of disease agents transmitted to human and other animals. Rapid and correct species identifications are necessary for all aspects of the study of black flies. DNA barcodes based on mitochondrial cytochrome c oxidase I (COI) have been effectively used for the determination of black fly species. However, the success of this method requires a large and reliable COI sequence library. In this study, 171 DNA barcoding sequences from 17 black fly species were added to NCBI GenBank database, six of these species were reported for the first time. Efficacy of DNA barcodes for species identification was examined using 1,286 sequences representing 89 nominal species of black flies in Thailand. A considerable level of success (90%) was achieved but efficiency of COI sequences for species identification was very low in the following species-groups; Simulium asakoae, S. feuerborni, S. multistriatum and S. striatum. Incomplete lineage sorting or inadequate variation of this genetic marker for differentiation of recently diverged species are the more likely explanations, and thus, more variable genetic markers are needed. Other reasons for unsuccessful DNA barcoding are imperfect taxonomy and the misidentification of sources of reference sequences. Because many new black fly species in Thailand were described recently, a reassessment of the COI sequences reported previously is necessary.

Introduction

Black flies (Diptera: Simuliidae) are medically and veterinary important blood sucking insects. Globally, there are 2,401 species comprising 2,384 living and 17 fossil taxa (Adler, 2021). Some species of black flies are known as vectors of Onchocerca spp., which causes diseases in humans and other animals (Adler and McCreadie, 2019). Human onchocerciasis is the most well recognized disease in which black fly species play a role as vector of the disease agent. This disease is caused by Onchocerca volvulus which is transmitted by at least 27 black fly species/species complexes (Adler and McCreadie, 2019). There are at least 11 other Onchocerca species that can be transmitted to animals by 20 black fly species (Adler and McCreadie 2019) and some of these filarial parasites can occasionally be transmitted to humans (Takaoka et al., 2012).

In Thailand, 138 black fly species have been recorded (Srisuka et al., 2019, Srisuka et al., 2021; Thaijarern et al., 2019a; Takaoka et al.,2019, 2020, 2021; Aupalee et al., 2020). Seven taxa have been reported as human biters, namely Simulium asakoae Takaoka and Davies, S. chamlongi Takaoka and Suzuki, S. doipuiense Takaoka and Choochote complex, S. nigrogilvum Summers, S. nodosum Puri, S. tenebrosum Takaoka, Srisuka and Saeung complex and S. umphangense Takaoka, Srisuka and Saeung (Choochote et al., 2005; Pramual et al., 2016; Takaoka et al., 2017). Three of these species; S. asakoae, S. nigrogilvum and S. nodosum are potentially vectors of Onchocerca spp. transmitted among domestic and wild animals (Fukuda et al., 2003; Takaoka et al., 2003; Ishii et al., 2008; Saeung et al. 2020). The latter two species are also considered as pests of humans in some areas in northern Thailand (Adler and McCreadie, 2019; Pramual, 2021). Two species, S. asakoae and S. chumpornense of the subgenus Gomphostilbia Enderlein, are potentially vectors of blood protozoa of the genera Leucocytozoon and Trypanosoma (Jumpato et al., 2019; Thaijarern et al., 2019b; Pramual et al., 2020). Given the potential significance of black flies on human and animal welfare in Thailand, it is very important that taxa present in the country can be rapidly and correctly identified.

Morphological taxonomy has made good progress for black flies in Thailand (Takaoka et al., 2019), yet, species new to science are still being uncovered (e.g. Srisuka et al., 2021; Takaoka et al., 2021). Cytotaxonomy has also contributed significantly to the advancement of taxonomic knowledge of black flies in Thailand. There are 29 species that have been cytologically examined (Pramual, unpublished data) and 10 morphospecies are recognized as a species complex (Takaoka et al., 2019). Although morphology and cytology are highly valuable to black fly taxonomy, their efficacy is limited by morphological homogeneity of the family Simuliidae and because cytogenetic studies are only feasible for the larval stage. Therefore, molecular approaches are necessary for solving taxonomic problems of black flies in Thailand (Pramual and Wongpakam, 2014; Thaijarern et al., 2019a).

DNA barcode studies based on mitochondrial cytochrome c oxidase I (COI) sequences of black flies in Thailand were first reported for 13 species of the subgenus Gomphoistilbia by Pramual et al. (2011). A more comprehensive study was reported by Pramual and Adler (2014) with 41 species of all subgenera recorded in Thailand included. Since then, several new black fly species have been described. These comprise 53 novel species (3 in subgenus Asiosimulium Takaoka and Choochote, 35 in subgenus Gomphostilbia, 2 in subgenus Nevermannia Enderlein, 13 in subgenus Simulium Latreille s. str.) and four new country records (S. myanmarense Takaoka, Srisuka and Saeung, S. thuathienense Takaoka and Sofian-Azirun, S. daoense Takaoka and Adler and S. vanluni Ya'cob, Takaoka and Sofian-Azirun) that have been reported in Thailand during the last decade. Populations of following species previously recognized in Thailand but later described as new species (previous recorded); S. huaikaeoense Takaoka and Srisuka, Low (S. decuplum Takaoka and Davies), S. ngaoense Takaoka, Srisuka and Saeung (S. parahiyangum Takaoka and Sigit), S. chomthongense Takaoka, Srisuka and Choochote (S. caudisclerum Takaoka and Davies), S. wangkwaiense Takaoka, Srisuka and Saeung (S. quinquestriatum (Shiraki)) and S. tenebrosum Takaoka, Srisuka and Saeung complex (S. rufibasis Brunetti). Many of these newly described or newly recorded species also have associated DNA barcode sequences and a reassessment of the accuracy of DNA barcodes for species identification of black fly species in Thailand is needed. In addition, it will be also useful to examine the species status of specimens previously reported in public databases (e.g. NCBI GenBank) by comparing their sequence data with data that was reported in strict association with new species descriptions. This will reduce the confusion and erroneous records in the public databases, which are important reasons for unsuccessful DNA barcoding (Taylor and Harris, 2012). Finally, the present study also reported DNA barcode sequences of six black fly species for the first time and also added COI sequences for nine species previously reported in databases but with limited number of specimens.