Pattamaporn Kittayapong

Background: Aedes aegypti is a primary vector of several arboviral diseases, including dengue, chikungunya, Zika, and yellow fever. Eliminating Ae. aegypti can disrupt virus transmission cycles, thereby reducing the incidence of these diseases. Releasing sterile Ae. aegypti males has been implemented to suppress their natural populations, using three approaches, i.e., ionizing radiation-induced sterilization (Sterile Insect Technique, SIT), Wolbachia -induced cytoplasmic incompatibility (Incompatible Insect Technique, IIT), and a combination of both approaches (SIT/IIT). Methodology/principal findings: This study investigated effects of different X-ray radiation doses on the ecological fitness of the Thai wild-type and Wolbachia trans-infected Ae. aegypti . Both wild-type and w AlbB trans-infected Ae. aegypti were irradiated with different X-ray radiation dosages, i.e., 30, 50, and 70 Gy, at the pupal stage. Our results demonstrated reduced fitness parameters in both Ae. aegypti strains when irradiation dosages increased, with greater adverse effects observed in the Wolbachia- infected group. Interestingly, 30 Gy irradiation appeared to prolong the lifespan of w AlbB trans-infected Ae. aegypti . Male fertility in both wild-type and w AlbB trans-infected mosquitoes irradiated at 50 Gy and 70 Gy was nearly zero. Wolbachia density generally declined with increasing radiation, and higher doses were associated with reduced mating competitiveness. At 30 Gy, wild-type males outperformed w AlbB trans-infected males in mating competitiveness; but it was reversed at 50 Gy and 70 Gy. Conclusions/significance: Based on the evaluation of ecological fitness parameters and mating competitiveness across various X-ray dosages under laboratory conditions, 30 Gy is recommended for sterilizing both Thai wild-type and w AlbB trans-infected Ae. aegypti . Our findings aim to improve the efficiency of sterile mosquito release programs, thereby suppressing Ae. aegypti vector populations and mitigating the risk of arboviral disease transmission .

Sterile Insect Technique (SIT), Incompatible Insect Technique (IIT) or a combination of the two has become alternative promising vector control approaches. In order to apply these approaches, the targeted mosquitoes need to be sterilized and released. So far, the irradiation of mosquitoes has been conducted at the pupae or adult stages. In this study, we investigated the possibility of applying X-ray irradiation at the egg stage and also assessed the effect on the development and sterility of both wild-type and Wolbachia trans-infected Aedes aegypti mosquito vectors. The eggs of both wild-type and Wolbachia trans-infected lines were irradiated using X-ray at the doses of 1, 3, 5 and 7 Gy. Development of immature stages was observed. For wild-type Ae. aegypti, X-ray irradiation at the doses from 3 Gy decreased the development of the first-instar larvae and increased the development of the third-instar larvae but there was no effect on pupae. For Wolbachia trans-infected ones, a irradiation dose as low as 1 Gy could increase the development of the forth-instar larvae while an irradiation dose of 7 Gy induced significantly high mortality to the pupae (p < 0.05). To assess sterility, males and females that emerged from irradiated eggs were mated with the non-irradiated ones. Our results showed that an irradiation dose of 7 Gy significantly caused more than 90% sterility in both wild-type males and females (p < 0.05). However, this irradiation dose could be reduced to 5 Gy to sterilize both males and females infected with Wolbachia. Our findings revealed, for the first time, that applying a low-dose X-ray irradiation at the egg stage could sterilize both wild-type and Wolbachia trans-infected Ae. aegypti when they become adults. Egg irradiation could make the implementation of SIT, IIT or combined SIT/IIT for vector control much more feasible as the sterile eggs are easier to distribute and operate when compared to other developmental stages of mosquitoes.

Background: Arboviral diseases such as dengue, chikungunya and Zika are public health concerns worldwide. Prevention and control of these diseases still depend on controlling Aedes aegypti mosquito vectors. Sterile insect technique (SIT) and incompatible insect technique (IIT) are environmental friendly approaches that show promising impacts. In order to plan an implementation of SIT/IIT technology, background knowledge, attitudes and practices (KAP) related to mosquito-borne diseases, mosquito vectors and their prevention and control, as well as acceptance and willingness to pay (WTP) for the technology, in the targeted communities are needed. Methodology/Principal findings: In this paper, we conducted questionnaire surveys on KAP and WTP in 400 sampled households in seven communities located in two districts in Bangkok, Thailand. Multivariate logistic regressions analysis was used to determine the association among knowledge, attitudes and practices regarding dengue, chikungunya, and Zika. Our findings indicated that participants had high knowledge on dengue (85.25%), and they were more concerned with the severity of dengue than chikungunya and Zika. Participants with ages lower than 35 years old (p = 0.047) and incomes higher than 5,000 THB (p = 0.016) had more knowledge of mosquito vectors. Moreover, 47% of respondents had positive attitude toward sterile mosquitoes and their application in vector control even though 45.5% of them had never heard about the technology. However, the majority of them were not willing to pay (52%); and if they had to pay, the maximum would be 1–2 THB (US$ 1 = ~34 THB) per sterile mosquito, since most of them expected to receive the service as public support from the government. Conclusions/significance: Our study was the first to study KAP and WTP related to SIT/IIT technology. It provided unique insights into how communities view the technology. It also suggested potential for successful implementation with proper education as well as highlighted the need for cost-sharing strategies with government subsidization for SIT/IIT deployment. Municipal officials and community health volunteers were key communication channels and targeted public education was needed, especially on under-recognized diseases like chikungunya and Zika. These findings should be useful for health authorities in planning to integrate SIT/IIT technology with traditional approaches for disease vector control and prevention.

Novel and alternative vector control approaches using a sterile male-based release to suppress Aedes aegypti mosquito vectors have recently been tested in the field in many countries. These approaches included the sterile insect technique (SIT), incompatible insect technique (IIT), and a combination of both techniques. In this study, we conducted a series of experiments to compare the quality between radiation-based and Wolbachia-induced sterile males in terms of flight ability, sterility, mating competitiveness, survival rate, and longevity. Aedes aegypti mosquitoes irradiated at 50 Gy (SIT) and those trans-infected with wAlbB Wolbachia (IIT) were used for quality comparison. Our results showed that irradiated and Wolbachia trans-infected males were not significantly different in flight ability (p > 0.05) and both could induce sterility in wild-type females. In addition, although irradiation at 50 Gy or Wolbachia trans-infection reduced male mating competitiveness, combined irradiation and Wolbachia wAlbB trans-infection increased male competitiveness at the one-to-one ratio. Increasing the number of sterile males released could compensate for reduced competitiveness but it does not make them more competitive. Irradiation did not affect the survival and longevity of irradiated males, but it showed significant negative impacts on females (p < 0.05); while the opposite was observed in the case of Wolbachia infection, i.e., with significant increase in the survival rate of Wolbachia trans-infected males (p < 0.05), but both survival and longevity were reduced in Wolbachia trans-infected females with no significant impacts (p > 0.05). In conclusion, neither irradiation nor Wolbachia trans-infection significantly affected the quality of sterile males except their mating competitiveness; but this could compensate by increasing the number of sterile males released. Sterility could be induced by either 50 Gy irradiation or wAlbB trans-infection. Mating competitiveness results showed that a higher number of sterile males produced by irradiation need to be released in comparison to those produced by Wolbachia trans-infection. Our results should be useful for planning SIT, IIT, or a combination for Ae. aegypti vector control.

Background: Sterile Insect Technique (SIT), Incompatible Insect Technique (IIT) and a combination become the alternative promising vector control approaches. In these approaches, the targeted mosquitoes need to be sterilized and released. So far, the irradiation of mosquitoes has been conducted at the pupae or adult stages. In this study, we investigated the possibility of the X-ray irradiation at the egg stage and also assessed the effect on the development and sterility of both wild-type and Wolbachia trans-infected Aedes aegypti mosquito vectors. Methodology/Principal Findings: The eggs of Aedes aegypti , both wild-type and Wolbachia trans-infected lines, were irradiated using X-ray at the doses of 1, 3, 5 and 7 Gy. Development of immature stages was observed. For wild-type Aedes aegypti , X-ray irradiation at the doses from 3 Gy decreased the development of the first instar larvae and increased the development of the third instar larvae but there was no effect on pupae when compared to those of the controls ( p < 0.05). For Wolbachia trans-infected ones, the irradiation dose as low as 1 Gy could increase the development of the forth-instar larvae while the irradiation dose of 7 Gy induced significantly high mortality in pupae. To assess sterility, males and females emerged from irradiated eggs were mated with the normal ones. Results showed that the irradiation doses for sterilization of Wolbachia trans-infected Ae. aegypti was lower than those of the wild-type ones. The egg hatch rate of both males and females could significantly be reduced from the irradiation dose of 5 Gy in Wolbachia trans-infected Ae. aegypti and 7 Gy in the wild-type ones. Conclusions/Significance: Our findings revealed the possibility to sterilize both wild-type and Wolbachia trans-infected Ae. aegypti by applying a low-dose X-ray irradiation at the egg stage. Egg irradiation could make an implementation of SIT, IIT or combined IIT/SIT for vector control much more feasible as the sterile dry eggs are easier to distribute and operate when compared to other developmental stages of mosquitoes. Author summary: Releasing sterile males to control Aedes aegypti mosquitoes and the diseases they transmit is one of the alternative environmental friendly approaches. So far, sterilization of mosquitoes has been targeted on the pupae or adult stages. In this study, we investigated the possibility to apply X-ray irradiation at the egg stage which is more feasible and easier to operate. Aedes aegypti eggs, both infected and uninfected with the Wolbachia bacteria, were irradiated by X-ray at the doses of 1, 3, 5 and 7 Gy. Results showed that the irradiation dose of 7 Gy significantly caused more than 92% sterility in both wild-type males and females. However, this irradiation dose could be reduced to 5 Gy to sterilize both males and females infected with Wolbachia . In conclusion, the X-ray irradiation dose of 5 Gy could be an optimum dose for irradiation of Wolbachia -infected Ae. aegypti eggs as it could induce high level of sterility in males and nearly complete sterility in females while the irradiation dose of 7 Gy could induce sterility in both males and females uninfected with Wolbachia . This approach should be useful for the sterile male release programs to control Aedes -transmitted diseases.

Background: Arboviral diseases such as dengue, chikungunya and Zika are public health concerns worldwide. Prevention and control of these diseases still depend on controlling Aedes aegypti mosquito vectors. Sterile insect technique (SIT) and incompatible insect technique (IIT) are environmental friendly approaches that show promising impacts. In order to plan an implementation of SIT/IIT technology, the background knowledge, attitude and practice related to these diseases and their mosquito vectors in the targeted communities are needed. Methodology/Principal findings: In this paper, we conducted the questionnaire surveys on general knowledge, attitude and practice (KAP) related to mosquito-borne diseases, mosquito vectors, as well as prevention and control in 400 sampling households in seven communities located in two districts in Bangkok, Thailand. The acceptance and willingness to pay (WTP) for sterile mosquitoes to be used as an alternative vector control approach was also investigated. Our findings indicated that the surveyed participants had high knowledge on dengue (85.25%) and they were more concerned with the severity of dengue (81%) than chikungunya (42.5%) and Zika (37%). Participants with the ages lower than 35 years old ( p = 0.047) and the incomes higher than 5,000 THB ( p = 0.016) had more knowledge on mosquito vectors. Moreover, 47% of respondents had positive attitude toward sterile mosquitoes and their application in vector control even though 45.5% of them had never heard about the technology. However, the majority of them were not willing to pay (52%); and if they had to pay, the maximum would be 1-2 THB per sterile mosquito, as most of them expected to receive free service from the government. Conclusions/significance: The baseline information obtained from this questionnaire survey could be used for planning the sterile mosquito release by public health authorities in Bangkok, Thailand where dengue, chikungunya and Zika were still prevalent. Author summary: A questionnaire survey was conducted in seven communities in Bangkok, Thailand to obtain the baseline information on knowledge, attitude and practice (KAP) related to mosquito-borne diseases, i.e., dengue, chikungunya and Zika, including mosquito vectors and how to control them. The questionnaire also asked about the acceptance and willingness to pay (WTP) for sterile mosquitoes used in controlling mosquito populations. Our results showed that, from the total of 400 sampling households, about 85% of participants were familiar with dengue, the mosquito vectors as well as their prevention and control. Furthermore, participants with lower ages and higher incomes had more knowledge on mosquito vectors ( p < 0.05). Even though the majority of participants showed positive perception about sterile mosquitoes release in terms of the environment, economic, social and quality of life, but more than half of them were not willing to pay for sterile mosquitoes as they would like to receive them free of charge from the government. In the case that they have to pay, the cost that they could afford was 1-2 THB per sterile mosquito. These findings should be useful for public health authorities in planning to apply the sterile mosquito release as an alternative mosquito control approach in Bangkok, Thailand.

Arbovirus diseases, such as dengue, chikungunya, and Zika, are important public health problems. Controlling the major vector, Aedes aegypti, is the only approach to suppressing these diseases. The surveillance of this mosquito species needs effective collecting methods. In this study, a simple MosHouse sticky trap was evaluated in a semi-field condition. Our results demonstrated the efficiency of this trap in collecting Ae. aegypti males, and no significant difference (p > 0.05) in the numbers of males was detected when compared with the widely used BG- Sentinel trap. However, there were significantly lower numbers of females (p < 0.05) collected using the MosHouse trap when compared to the BG-Sentinel trap. We also found a significant difference (p < 0.05) in the collected numbers between irradiated and non-irradiated males. More irradiated males were collected in the MosHouse traps. The improvement of male collection was achieved with the addition of a sugar stick and sticky flags. Significantly higher numbers of males were collected in the MosHouse trap with sticky flags compared to the original one when they were released independently of females, but both were collected in higher numbers when they were released together (p < 0.05). In conclusion, our experiments demonstrated that the MosHouse trap could sample Ae. aegypti, especially males, as efficiently as the established BG-Sentinel trap, while the cost was more than 50 times lower, showing the potential of the MosHouse trap for improved Ae. aegypti male and female surveillance with very large numbers of traps at affordable costs. In addition, significantly (p < 0.001) increased male sampling was achieved by adding an external sticky flag on the MosHouse trap, providing an avenue for further development of the novel male-trapping strategy.

Background: Wolbachia 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 findings: We 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/Significance: Our 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.

Background: Wolbachia 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 issues. Methodology/Principal Findings: We describe here the development of a LAMP combined with the DNA strand displacement-based electrochemical sensor (BIOSENSOR) method to detect wAlbB Wolbachia in trans-infected Ae. aegypti . Our developed LAMP primers were more specific to wAlbB detection than those of the previous published ones if the assays were conducted with low-cost and non-specific detecting dyes. The detection capacity of our LAMP technique was 3.8 nM and the detection limit reduced to 2.16 fM when combined with the BIOSENSOR. Our study demonstrates that the 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/Significance: Our 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. Author Summary: Mosquito-borne diseases such as dengue, chikungunya, Zika, and yellow fever are transmitted to humans mainly by the bites of Aedes aegypti mosquitoes. Controlling these diseases relies mostly on the use of insecticides, in which the efficiency has been reduced through development of insecticide resistance in mosquitoes. Wolbachia is the endosymbiotic bacteria that are naturally found in 40% of insects, including mosquitoes. The bacteria could protect their hosts from viral infections and could also cause sterility in host populations, therefore, providing an opportunity to use them for disease control. Application of a Wolbachia -based strategy needs simple, rapid and sensitive methods for detecting the bacteria in released mosquitoes. In this paper, we develop the combined methods of LAMP and BIOSENSORS for detecting wAlbB Wolbachia in mosquitoes. Our positive LAMP reaction can be visualized by color change from violet to blue at a sensitivity of ≥ 60 pg of genomic DNA. When used in combination with the BIOSENSOR method, the sensitivity increased a million fold without losing specificity. Our study indicates that both developed methods, either used in combination or stand-alone, are efficient and cost-effective, hence, it could be applied for routine surveys of Wolbachia in mosquito control programs that use Wolbachia -based approaches.