Here, the data presented herein are preliminary evidence of native Wolbachia in An. minimus, a major malaria vector in an endemic area of mainland Southeast Asia. The identified Wolbachia in An. minimus, herein called wAnmi, was phylogenetically clustered in subgroup B, similar to wAlbB, which has been associated with the suppression of development of P. falciparum in An. stephensi [12]. Along the Thailand-Myanmar border, malaria transmission continues in many areas [25] [26], including the Umphang District of Tak Province in western Thailand. Most collection sites were in the Ban Nong Luang Village of Umphang District located in a valley primarily surrounded by forested mountains. Since some villagers engage in hunting-gathering and agriculture in areas near the forest (Fig. 1A), the risk of malaria infection in individuals is high. In the Umphang Valley, there are reportedly seven putative malaria vectors: An. minimus, An. dirus, An. baimaii, An. sawadwongporni, An. maculatus, An. pseudowillmori and An. aconitus [17]. In the Thasongyang District northern Umphang Valley, female An. minimus, An. maculatus, An. annularis, and An. barbirostris have been shown to carry P. vivax sporozoites, confirming their role in malaria transmission [27]. Regarding the number of Anopheles in the Umphang Valley, An. minimus was the most abundant (> 50%), followed by the potential malaria vectors An. peditaeniatus (~ 20%) and An. maculatus (~ 10%) [17]. Hence, this study included samples from An. minimus, An. peditaeniatus, An. maculatus and An. aconitus, the major malaria vectors, for the detection of Wolbachia.
In Thailand, only one survey of Wolbachia in mosquitoes was conducted to amplify the filamenting temperature-sensitive mutant Z (ftsz) and Wolbachia surface protein (wsp) genes. All 23 mosquito species in the genera Aedes, Culex, and Mansonia were positive for the ftsz and wsp genes, whereas none of the 19 Anopheles species were positive [8]. Failure to detect Wolbachia-specific genes in Anopheles spp. was consistent with the results of studies in European, African, and American specimens [10] [9]. Nevertheless, detection of the Wolbachia 16S rRNA region was accomplished. The W-Spec primers were designed to specifically amplify a 438-bp sequence at the 3’ region of the 16S rRNA gene in Wolbachia [21]. The W-Spec primers allowed the detection of Wolbachia in temperate North American arthropods, including the family Culicidae but excluding other mosquito families. Subsequently, Baldini et al reported the first evidence of Wolbachia in the reproductive organs of male and female An. gambiae, a major malaria vector in sub-Saharan Africa. In the same DNA samples, the W-Spec primer-based PCR was able to amplify the 16S rRNA fragment, whereas Wolbachia-specific surface protein and fructose-biphosphate aldolase-based PCR failed [13], implying good sensitivity of the W-Spec primers. Moreover, Shaw et al. further improved the sensitivity of W-Spec primer-based PCR by using nested primers (16SNF and 16SNR). The use of nested PCR allowed the detection of Wolbachia in An. coluzzii [15], An. gambiae in Mali [16] and An. arabiensis in Tanzania [28]. Additional studies were able to amplify the Wolbachia 16S rRNA fragment in DNA samples extracted from head-thorax or thorax-abdomen, implying the possibility of Wolbachia infection in nonreproductive organs [29] [12]. Collectively, Wolbachia infection in somatic and germ cells can be detected using nested PCR, which amplifies the conserved region of the Wolbachia 16S rRNA gene.
Nested PCR is regarded as a highly sensitive tool for detecting targets of interest that are present in very low amounts. We sometimes failed to amplify the 438-bp fragment using W-SpecF and W-SpecR in the initial PCR; however, there were 412-bp amplicons observed in the nested PCR [15], implying good sensitivity of the nested PCR. As such, false-negative results may occur in cases of low-intensity Wolbachia infection because the quantity of the target of interest is below the limit of nested PCR. Given that DNA samples were extracted from the head and thorax of female Anopheles, detection failure in nested PCR is possibly due to low-intensity infection or the reproductive organ specificity of Wolbachia. Therefore, assays with high sensitivity, such as quantitative PCR, may aid in the detection of low-intensity Wolbachia infection [30] [31, 32]. Moreover, DNA preparation from the whole body of mosquitoes ensures the inclusion of Wolbachia strains that specifically infect germ cells.
The high sensitivity of nested PCR may cause low specificity, especially when primers bind to the conserved region of a common gene. Since Wolbachia is capable of infecting the majority of insect species [33], and the W-Spec primers amplify the conserved region of the Wolbachia 16S rRNA-coding gene, false-positive results due to environmental contamination from other insects may occur. In our study, 438-bp DNA amplicons with low-fluorescence intensity were present in the initial PCR, but we failed to reamplify these amplicons in the subsequent nested PCR using the 16SNF and 16SNR primers, suggesting the possibility of nonspecific amplification in the initial run. To minimize environmental contamination, PCR preparations were performed in a clean hood. Despite great care in the pre-PCR steps, we sometimes observed DNA bands in the negative control lane. Thus, DNA sequencing of the PCR product was necessary to confirm Wolbachia-specific amplification.
To the best of our knowledge, the present data are the first preliminary evidence of native Wolbachia in An. minimus. However, this study has limitations. First, the presence of the Wolbachia 16S rRNA gene in DNA samples from Anopheles is not direct evidence of natural Wolbachia infection because environmental contamination during mosquito capture, DNA extraction and PCR preparation is possible. Intracellular localization of Wolbachia in Anopheles spp. is a more definitive indicator of Wolbachia infection than sequencing. This could be done by using in situ hybridization [15] [34]. Moreover, given the availability of remaining DNA samples obtained during a previous two-year survey [17], this study included the DNA samples regardless of sample size. Thus, the prevalence of Wolbachia in Anopheles spp. in Umphang Valley could not be statistically estimated. Identification of the Wolbachia strain in An. minimus requires further confirmation using high-sensitivity assays and whole mosquitoes for DNA extraction.
Wolbachia has been under investigation for its potential application in blocking malaria transmission. As proof-of-concept, field trials in Australia demonstrated that the release of laboratory-reared mosquitoes infected with Wolbachia resulted in the rapid spread of Wolbachia among wild uninfected mosquito populations [35]. Population invasion by a particular Wolbachia strain depends on the level of cytoplasmic incompatibility, host fitness (survival, fecundity and fertility), and vertical transmission. Therefore, the following issues need to be assessed: the potential of the native Wolbachia identified in An. minimus to render resistance to Plasmodium parasites and interfere with malaria transmission, its ability to cause cytoplasmic incompatibility, and its effects on host fitness.