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BackgroundConventional gold standard characterization of chigger mites involves chemical preparation procedures (i.e. specimen clearing) for visualization of morphological features, which however contributes to destruction of the arthropod host DNA and any endosymbiont or pathogen DNA harbored within the specimen.Methodology/Principal findingsIn this study, a novel work flow based on autofluorescence microscopy was developed to enable identification of trombiculid mites to the species level on the basis of morphological traits without any special preparation, while preserving the mite DNA for subsequent genotyping. A panel of 16 specifically selected fluorescence microscopy images of mite features from available identification keys served for complete chigger morphological identification to the species level, and was paired with corresponding genotype data. We evaluated and validated this method for paired chigger morphological and genotypic ID using the mitochondrial cytochrome c oxidase subunit I gene (coi) in 113 chigger specimens representing 12 species and 7 genera (Leptotrombidium, Ascoschoengastia, Gahrliepia, Walchia, Blankaartia, Schoengastia and Schoutedenichia) from the Lao People’s Democratic Republic (Lao PDR) to the species level (complete characterization), and 153 chiggers from 5 genera (Leptotrombidium, Ascoschoengastia, Helenicula, Schoengastiella and Walchia) from Thailand, Cambodia and Lao PDR to the genus level.A phylogenetic tree constructed from 77 coi gene sequences (approximately 640 bp length, n = 52 new coi sequences and n = 25 downloaded from GenBank), demonstrated clear grouping of assigned morphotypes at the genus levels, although evidence of both genetic polymorphism and morphological plasticity was found.Conclusions/SignificanceWith this new methodology, we provided the largest collection of characterized coi gene sequences for trombiculid mites to date, and almost doubled the number of available characterized coi gene sequences with a single study. The ability to provide paired phenotypic-genotypic data is of central importance for future characterization of mites and dissecting the molecular epidemiology of mites transmitting diseases like scrub typhus.

Adult mosquitoes in the Anopheles maculatus group were surveyed from different regions of Thailand and five different species were morphologically identified, including Anopheles maculatus, Anopheles sawadwongporni, Anopheles notanandai, Anopheles dravidicus, and Anopheles willmori. Blood-feeding activity and host preference of two species, Anopheles maculatus and Anopheles sawadwongporni, were observed during a one-year period at Pu Teuy Village, Sai Yok District, Kanchanaburi Province, west-central Thailand. Both species were more prevalent during the wetter period of the year and each had a greater predilection to feed on cattle than humans. Primary feeding activity occurred between 20:00-23:00 and a smaller peak at 01:00-03:00. Findings are discussed relative to the importance of these two vectors for malaria transmission in Pu Teuy. Journal of Vector Ecology 34 (1): 62-69. 2009.

The Anopheles minimus Complex Theobald (Diptera: Culicidae) is composed of the 3 sibling species A, C, and E. The malaria vectors An. minimus A and C are distributed over the Southeast Asian region, whereas species E is restricted to the Ryukyu Japanese Islands. Because species A and C can be sympatric and present specific behaviors and have a role in malaria transmission, it is important to differentiate them. The literature mentioned the presence of a presector pale spot on the wing costa of An. minimus A, whereas species C may exhibit both presector and humeral pale spots. However, the reliability of their diagnostic power has not been established over large temporal and geographic surveys. From the analyses of 9 populations throughout Southeast Asia, including published data and field populations from 2 sites in Thailand, we showed that the wing patterns present spatial and temporal variations that make these two morphological characters unreliable for the precise identification of An. minimus A and C. Therefore, molecular identification remains the most efficient method to obtain an unambiguous differentiation of these 2 species. Correct species identification is essential and mandatory for any relevant study on the Minimus Complex and for the application of successful control strategies.