Population Genetic Structure of Aedes (Stegomyia) aegypti (L.) at a Micro-Spatial Scale in Thailand: Implications for a Dengue Suppression Strategy

Background

The genetic population structure of Aedes (Stegomyia) aegypti (L.), the main vector of dengue virus, is being investigated in areas where a novel dengue suppression program is to be implemented. The aim of the program is to release and establish mosquito populations with impaired virus transmission capabilities. To model effects of the release and devise protocols for its implementation, information about the genetic structure of populations at a range of spatial scales is required.

Methodology/Principal Findings

This study investigates a potential release site in the Hua Sam Rong Subdistrict of Plaeng Yao District, Chachoengsao Province, in eastern Thailand which comprises a complex of five villages within a 10 km radius. Aedes aegypti resting indoors was sampled at four different times of year from houses within the five villages. Genetic markers were used to screen the mosquitoes: two Exon Primed Intron Crossing (EPIC) markers and five microsatellite markers. The raw allele size was determined using several statistical software packages to analyze the population structure of the mosquito. Estimates of effective population size for each village were low, but there was no evidence of genetic isolation by geographic distance.

Conclusions

The presence of temporary genetic structure is possibly caused by genetic drift due to large contributions of adults from a few breeding containers. This suggests that the introduction of mosquitoes into an area needs to proceed through multiple releases and targeting of sites where mosquitoes are emerging in large numbers.     

Molecular evidence of Wolbachia infection in natural populations of tropical odonates

Wolbachia are endosymbiotic bacteria that cause reproductive alterations in numerous arthropod species. Using a PCR-based method, we found that, out of 33 odonate species, four species were infected with Wolbachia. This finding represents the first record of Wolbachia infection in tropical odonates. Identical wsp gene sequences were found in the Wolbachia-infected common odonate species, Agriocnemis f. femina, collected from different locations in Thailand. The infection frequencies in several natural populations suggest that replacement of uninfected populations by Wolbachia-infected ones has recently occurred in this damselfly species.     

Simple,sensitive, and cost-effective detection of wAlbB Wolbachia in Aedes mosquitoes,using loop mediated isothermal amplification combined with the electrochemical biosensing method

BackgroundWolbachia is an endosymbiont bacterium generally found in about 40% of insects, including mosquitoes, but it is absent in Aedes aegypti which is an important vector of several arboviral diseases. The evidence that Wolbachia trans-infected Ae. aegypti mosquitoes lost their vectorial competence and became less capable of transmitting arboviruses to human hosts highlights the potential of using Wolbachia-based approaches for prevention and control of arboviral diseases. Recently, release of Wolbachia trans-infected Ae. aegypti has been deployed widely in many countries for the control of mosquito-borne viral diseases. Field surveillance and monitoring of Wolbachia presence in released mosquitoes is important for the success of these control programs. So far, a number of studies have reported the development of loop mediated isothermal amplification (LAMP) assays to detect Wolbachia in mosquitoes, but the methods still have some specificity and cost issues.Methodology/Principal findingsWe describe here the development of a LAMP assay combined with the DNA strand displacement-based electrochemical sensor (BIOSENSOR) method to detect wAlbB Wolbachia in trans-infected Ae. aegypti. Our developed LAMP primers used a low-cost dye detecting system and 4 oligo nucleotide primers which can reduce the cost of analysis while the specificity is comparable to the previous methods. The detection capacity of our LAMP technique was 1.4 nM and the detection limit reduced to 2.2 fM when combined with the BIOSENSOR. Our study demonstrates that a BIOSENSOR can also be applied as a stand-alone method for detecting Wolbachia; and it showed high sensitivity when used with the crude DNA extracts of macerated mosquito samples without DNA purification.Conclusions/SignificanceOur results suggest that both LAMP and BIOSENSOR, either used in combination or stand-alone, are robust and sensitive. The methods have good potential for routine detection of Wolbachia in mosquitoes during field surveillance and monitoring of Wolbachia-based release programs, especially in countries with limited resources.     

Mosquito vector‐associated microbiota: Metabarcoding bacteria and eukaryotic symbionts across habitat types in Thailand endemic for dengue and other arthropod‐borne diseases

Vector‐borne diseases are a major health burden, yet factors affecting their spread are only partially understood. For example, microbial symbionts can impact mosquito reproduction, survival, and vectorial capacity, and hence affect disease transmission. Nonetheless, current knowledge of mosquito‐associated microbial communities is limited. To characterize the bacterial and eukaryotic microbial communities of multiple vector species collected from different habitat types in disease endemic areas, we employed next‐generation 454 pyrosequencing of 16S and 18S rRNA amplicon libraries, also known as metabarcoding. We investigated pooled whole adult mosquitoes of three medically important vectors, Aedes aegypti, Ae. albopictus, and Culex quinquefasciatus, collected from different habitats across central Thailand where we previously characterized mosquito diversity. Our results indicate that diversity within the mosquito microbiota is low, with the majority of microbes assigned to one or a few taxa. Two of the most common eukaryotic and bacterial genera recovered (Ascogregarina and Wolbachia, respectively) are known mosquito endosymbionts with potentially parasitic and long evolutionary relationships with their hosts. Patterns of microbial composition and diversity appeared to differ by both vector species and habitat for a given species, although high variability between samples suggests a strong stochastic element to microbiota assembly. In general, our findings suggest that multiple factors, such as habitat condition and mosquito species identity, may influence overall microbial community composition, and thus provide a basis for further investigations into the interactions between vectors, their microbial communities, and human‐impacted landscapes that may ultimately affect vector‐borne disease risk.     

Bacteriophage WO-B and Wolbachia in natural mosquito hosts: infection incidence, transmission mode and relative density

Bacteriophages of Wolbachia bacteria have been proposed as a potential transformation tool for genetically modifying mosquito vectors. In this study, we report the presence of the WO-B class of Wolbachia-associated phages among natural populations of several mosquito hosts. Eighty-eight percent (22/25) of Wolbachia-infected mosquito species surveyed were found to contain WO-B phages. WO-B phage orf7 sequence analysis suggested that a single strain of WO-B phage was found in most singly (23/24) or doubly (1/1) Wolbachia-infected mosquitoes. However, the single Wolbachia strain infecting Aedes perplexus was found to harbour at least two different WO-B phages. Phylogenetic analysis suggested that horizontal transmission of WO-B phages has occurred on an evolutionary scale between the Wolbachia residing in mosquitoes. On an ecological scale, a low trend of co-transmission occurred among specific WO-B phages within Wolbachia of each mosquito species. Assessment of the density of WO-B phage by real-time quantitative polymerase chain reaction (RTQ-PCR) revealed an average relative density of 7.76 x 10(5)+/- 1.61 x 10(5) orf7 copies per individual mosquito for a single Wolbachia strain infecting mosquitoes, but a threefold higher density in the doubly Wolbachia-infected Aedes albopictus. However, the average combined density of WO-B phage(s) did not correlate with that of their Wolbachia hosts, which varied in different mosquito species. We also confirmed the presence of WO-B-like virus particles in the laboratory colony of Ae. albopictus (KLPP) morphologically, by transmission electron microscopy (TEM). The viral-like particles were detected after purification and filtration of Ae. albopictus ovary extract, suggesting that at least one WO-B-like phage is active (temperate) within the Wolbachia of this mosquito vector. Nevertheless, the idea of utilizing these bacteriophages as transformation vectors still needs more investigation and is likely to be unfeasible.     

Wolbachia infections of tephritid fruit flies: molecular evidence for five distinct strains in a single host species

Endosymbiotic bacteria of the genus Wolbachia are widespread among arthropods and can induce cytoplasmic incompatibility, thelytokous parthenogenesis, male-killing or feminization in their hosts. Here, we report phylogenetic relationships of Wolbachia in tephritid fruit flies based on wsp gene sequences. We also report, for the first time, five distinct strains of Wolbachia in Bactrocera ascita sp. B. Four of the five Wolbachia strains found in this species were in the same groups as those found in other tephritid fruit flies, suggesting possible horizontal transmission of Wolbachia from other fruit flies into B. ascita sp. B. The unreliability of wsp-specific group primers demonstrated in this study suggests that these primers might be useful only for preliminary identification of Wolbachia. Final determination of group affiliation needs to be verified with wsp sequence data. Received: 12 December 2001 / Accepted: 11 January 2002     

<Emphasis Type="Italic">Wolbachia</Emphasis> and bacteriophage WO-B density of <Emphasis Type="Italic">Wolbachia</Emphasis> a-infected <Emphasis Type="Italic">Aedes albopictus</Emphasis> mosquito

Wolbachia are maternally inherited symbiotic bacteria capable of inducing an extensive range of reproductive abnormalities in their hosts, including cytoplasmic incompatibility (CI). Its density (concentration) is likely to influence the penetrance of CI in incompatible crosses. The variations of Wolbachia density could also be linked with phage WO density. We determined the relative density (relative concentration) of prophage WO orf7 and Wolbachia (phage-to-bacteria ratio) during early developmental and adult stages of singly infected Aedes albopictus mosquito (Wolbachia A-infected) by using real-time quantitative PCR. Phage WO and Wolbachia did not develop at the same rate. Relative Wolbachia density (bacteria-to-host ratio) was high later in development (adult stages) whilst relative prophage WO density (phage-to-bacteria ratio) was low in the adult stages. Furthermore, 12-d-old adults of singly infected female mosquito had the highest Wolbachia density. In contrast, the larval stage 4 (L4) contained the highest prophage WO-B orf7 density. The association of hosts-Wolbachia-phage among diverse species is different. Thus, if phage and Wolbachia are involved in CI mechanism, the information of this association should be acquired for each specific type of organism for future use of population replacement or gene drive system.     

Closely Related Wolbachia Strains within the Pumpkin Arthropod Community and the Potential for Horizontal Transmission via the Plant

Phylogenetic studies have implicated frequent horizontal transmission of Wolbachia among arthropod host lineages. However, the ecological routes for such lateral transfer are poorly known. We surveyed the species of two arthropod communities, one on pumpkin and the other on loofah plants, for Wolbachia, constructed wsp gene phylogenies of those Wolbachia strains found to infect community members, and established ecological links among infected members. Four taxonomically diverse insects in the pumpkin arthropod community contained very closely related Wolbachia wsp sequences (<1.5% divergence by Kimura-2-parameter distances). These insects, namely, the whitefly Bemisia tabaci, the planthopper Nisia nervosa, the flea beetle Phyllotreta sp., and the fleahopper Halticus minutus, were all collected from pumpkin leaves. They were ecologically linked through feeding on the same leaf substrate. Unlike other infected leaf insects, the whitefly population appeared to have a permanent breeding relationship with pumpkin plants, and high and stable, but not fixed, monthly Wolbachia infection rates. Our findings suggest potential roles for the plant in Wolbachia transmission and for whiteflies in being an infection source for other pumpkin leaf-feeding insects.     

Combination of Mesocyclops thermocyclopoides and Bacillus thuringiensis var. israelensis: a better approach for the control of Aedes aegypti larvae in water containers.

The efficacy of a local Thai-strain of the copepod, Mesocyclops thermocyclopoides and the larvicide, Bacillus thuringiensis var. israelensis (Bti), used jointly and singly, was studied against Aedes aegypti in water containers. In a laboratory test, copepods alone produced mortality of 98-100% in 1st instar larvae of Ae. aegypti at copepod:larvae ratios ranging from 1:1 to 1:4. In an outdoor field simulated experiment that ran for 16 wk, after a single inoculation, the treatment of copepods and Bti combined yielded the better, more sustainable results than the agents used individually. Numbers of mosquito larvae per sample in the combined treatment were zero during the first 8 wk; larval numbers then increased but were maintained at a very low level for the next 4 wk after which the larval numbers increased moderately but still remained below numbers in the control. Bti alone kept the larvae at the zero level for the first 4 wk after which their numbers increased slightly and were at low levels up to 12 wk. Copepods alone maintained larval numbers at a low level as compared with those of the control. During the course of the experiment the larval numbers in the control were greater than 20 per sample. Statistically significant differences were noted among treatment means (F = 23.083, df = 3/60, P<0.01) over the total period of the study. The number of copepods in the joint treatment was significantly higher than in the copepod alone treatment for the first 8 wk (t = -4.97, df = 14, P<0.01). The density of copepods, however, for the whole 16-wk period was not significantly different in these two treatments (t = -1.51, df = 30, P>0.1).     

Endosymbiotic Wolbachia bacteria as biological control tools of disease vectors and pests

Wolbachia bacteria are common cytoplasmic symbionts of insects, mites and filarial nematodes. They can alter the reproduction of their hosts. The symbiont could be eliminated, transferred or used through genetic alteration to take advantage or remove their possible influences on pests and/or natural enemies. Their extensive effects on reproduction and host fitness have made Wolbachia the subject of growing attention as a potential biocontrol agent. Here, we summarize the relations of Wolbachia in the control of disease vectors and pests. Furthermore, the drawbacks of these bacteria are also discussed.