Chigger mites are vectors of the bacterial disease scrub typhus, caused by Orientia spp. The bacterium is vertically transmitted in the vector and horizontally transmitted to terrestrial vertebrates (primarily wild small mammals), with humans as incidental hosts. Previous studies have shown that the size of the chigger populations is correlated with the density of small mammals in scrub typhus-endemic regions. Here, we explore interactions between the small mammals and chiggers in two oil palm plantations located in the Perak and Johor states of Peninsular Malaysia. The location in Perak also contained an aboriginal (Orang Asli) settlement. A ~5% sub-sample from 40,736 chigger specimens was identified from five species of small mammals (n = 217), revealing 14 chigger species, including two new records for Malaysia. The abundance and species richness of chiggers were significantly affected by habitat type (highest in forest border), state (highest in Perak), and season (highest in dry). The overall prevalence of Orientia tsutsugamushi DNA in small-mammal tissues was 11.7% and was not significantly affected by host or habitat characteristics, but in Johor, was positively associated with infestation by Leptotrombidium arenicola. These findings highlight the risk of contracting scrub typhus in oil palm plantations and associated human settlements.

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. Graphical

Understanding the factors associated with the species diversity and distribution of insect vectors is critically important for disease epidemiology. Black flies (Diptera: Simuliidae) are significant hematophagous insects, as many species are pests and vectors that transmit pathogens to humans and other animals. Ecological factors associated with black fly species distribution have been extensively examined for the immature stages but are far less well explored for the adult stage. In this study, we collected a total of 7706 adult black fly specimens from various locations in forests, villages and animal shelters in Thailand. The integration of morphology and DNA barcoding revealed 16 black fly taxa, including Simulium yvonneae, a species first found in Vietnam, which is a new record for Thailand. The most abundant species was the Simulium asakoae complex (n = 5739, 74%), followed by S. chumpornense Takaoka and Kuvangkadilok (n = 1232, 16%). The Simulium asakoae complex was dominant in forest (3786 of 4456; 85%) and village (1774 of 2077; 85%) habitats, while S. chumpornense predominated (857 of 1175; 73%) in animal shelter areas. The Simulium asakoae complex and S. nigrogilvum Summers, which are significant pests and vectors in Thailand, occurred at a wide range of elevations, although the latter species was found mainly in high (>1000 m) mountain areas. Simulium chumpornense, S. nodosum Puri and the S. siamense Takaoka and Suzuki complex occurred predominately in low (1000 m) mountain areas. A host blood meal analysis revealed that the S. asakoae; S. chamlongi Takaoka and Suzuki; S. nigrogilvum; S. chumpornense; and the S. striatum species group were biting humans. This is the first report of the latter two species biting humans. We also found that S. chumpornense was biting turkeys, and S. chamlongi was biting chickens, which are new host blood sources recorded for these species. In addition, we found that the S. feuerborni Edwards complex was biting water buffalo, which is the first report on the biting habits of this species.

Trombiculid mites (Acariformes) are unique among arthropods of medical importance in that only the larval instar (chigger) is parasitic, which can result in the transmission of zoonotic scrub typhus. The use of molecular approaches for chigger species discrimination has been very limited until recently, especially for those parasitizing bird hosts, where data remain scarce. Here, we aimed to generate DNA barcodes of chiggers parasitizing birds in Malaysia based on the mitochondrial cytochrome c oxidase subunit I (COI) gene following DNA extraction, PCR and sequencing. Fifty-four COI sequences from 8 bird-associated chigger species in Malaysia were combined with 50 GenBank sequences comprising 7 genera from various countries for DNA barcode and phylogenetic analysis. The correct identification rates for the 95 COI barcodes were 96.84% (Best Match) and 86.31% (Best-Close Match). DNA barcode analyses effectively clustered the 8 nominal species from this study into their respective genera. Genetic divergence of less than 3% was observed within Ascoschoengastia lorius, Neoschoengastia gallinarum, Parascoschoengastia heynemani, Leptotrombidium imphalum, and Blankaartia acuscutellaris, all of which formed a monophyletic clade, confirming their conspecific nature. Conversely, intraspecific divergences of 17.64%, 15.49%, and 11.63% were obtained for Toritrombicula densipiliata, Odontacarus audyi, and Leptotrombidium deliense. These divergences, supported by evidence of distinct entities through delimitation analyses, indicate potential cryptic diversity within these populations. In conclusion, this study represents the first molecular genetic analysis of bird chiggers in Malaysia, revealing varying levels of genetic divergence. Our findings highlight the utility of DNA barcoding for understanding chigger diversity and aiding in accurate identification.

Chigger mites were collected from 65 bird species in different regions of Thailand and Malaysia. In total, 21 species were recorded. The previously unknown fauna of chiggers parasitizing shorebirds of Thailand included one new species, Neacariscus (Whartonacarus) andamanensis n. sp. as well as Neacariscus (Neacariscus) pluvius (Wharton, 1945) previously recorded only in Oceania; Neacariscus (Whartonacarus) shiraii (Sasa, Kano and Obata, 1952) (known from Oceania and Japan); and Neacariscus (Whartonacarus) sulae (Oudemans, 1910) and Schoengastia archaea (Taufflieb, 1960), both described from West Africa. Toritrombicula kirhocephales Goff, 1982 described from Papua New Guinea was for the first time recorded in Asia (Thailand). Neoschoengastia gallinarum (Hatori, 1920) and Helenicula comata (Womersley, 1952) were for the first time recorded in Thailand. Ascoschoengastia lorius (Gunther, 1939) was for the first time recorded in Malaysia. Finally, Ericotrombidium cosmetopode (Vercammen-Grandjean and Langston, 1971) described from free larvae, was for the first time recorded on a host (bird Pellorneum ruficeps Swainson); and Leptotrombidium miculum (Traub and Audy, 1954) was for the first time recorded on a bird host. New host species were recorded for a large part of the collected chigger species.