Wolbachia infection-responsive immune genes suppress Plasmodium falciparum infection in Anopheles stephensi.

Wolbachia, a maternally transmitted symbiotic bacterium of insects, can suppress a variety of human pathogens in mosquitoes, including malaria-causing Plasmodium in the Anopheles vector. However, the mechanistic basis of Wolbachia-mediated Plasmodium suppression in mosquitoes is not well understood. In this study, we compared the midgut and carcass transcriptomes of stably infected Anopheles stephensi with Wolbachia wAlbB to uninfected mosquitoes in order to discover Wolbachia infection-responsive immune genes that may play a role in Wolbachia-mediated anti-Plasmodium activity. We show that wAlbB infection upregulates 10 putative immune genes and downregulates 14 in midguts, while it upregulates 31 putative immune genes and downregulates 15 in carcasses at 24 h after blood-fed feeding, the time at which the Plasmodium ookinetes are traversing the midgut tissue. Only a few of these regulated immune genes were also significantly differentially expressed between Wolbachia-infected and non-infected midguts and carcasses of sugar-fed mosquitoes. Silencing of the Wolbachia infection-responsive immune genes TEP 4, TEP 15, lysozyme C2, CLIPB2, CLIPB4, PGRP-LD and two novel genes (a peritrophin-44-like gene and a macro domain-encoding gene) resulted in a significantly greater permissiveness to P. falciparum infection. These results indicate that Wolbachia infection modulates mosquito immunity and other processes that are likely to decrease Anopheles permissiveness to Plasmodium infection.

Alpha-mannosidase-2 modulates arbovirus infection in a pathogen- and Wolbachia-specific manner in Aedes aegypti mosquitoes.

Multiple Wolbachia strains can block pathogen infection, replication and/or transmission in Aedes aegypti mosquitoes under both laboratory and field conditions. However, Wolbachia effects on pathogens can be highly variable across systems and the factors governing this variability are not well understood. It is increasingly clear that the mosquito host is not a passive player in which Wolbachia governs pathogen transmission phenotypes; rather, the genetics of the host can significantly modulate Wolbachia-mediated pathogen blocking. Specifically, previous work linked variation in Wolbachia pathogen blocking to polymorphisms in the mosquito alpha-mannosidase-2 (αMan2) gene. Here we use CRISPR-Cas9 mutagenesis to functionally test this association. We developed αMan2 knockouts and examined effects on both Wolbachia and virus levels, using dengue virus (DENV; Flaviviridae) and Mayaro virus (MAYV; Togaviridae). Wolbachia titres were significantly elevated in αMan2 knockout (KO) mosquitoes, but there were complex interactions with virus infection and replication. In Wolbachia-uninfected mosquitoes, the αMan2 KO mutation was associated with decreased DENV titres, but in a Wolbachia-infected background, the αMan2 KO mutation significantly increased virus titres. In contrast, the αMan2 KO mutation significantly increased MAYV replication in Wolbachia-uninfected mosquitoes and did not affect Wolbachia-mediated virus blocking. These results demonstrate that αMan2 modulates arbovirus infection in A. aegypti mosquitoes in a pathogen- and Wolbachia-specific manner, and that Wolbachia-mediated pathogen blocking is a complex phenotype dependent on the mosquito host genotype and the pathogen. These results have a significant impact for the design and use of Wolbachia-based strategies to control vector-borne pathogens.

Mating harassment may boost the effectiveness of the sterile insect technique for Aedes mosquitoes.

The sterile insect technique is based on the overflooding of a target population with released sterile males inducing sterility in the wild female population. It has proven to be effective against several insect pest species of agricultural and veterinary importance and is under development for Aedes mosquitoes. Here, we show that the release of sterile males at high sterile male to wild female ratios may also impact the target female population through mating harassment. Under laboratory conditions, male to female ratios above 50 to 1 reduce the longevity of female Aedes mosquitoes by reducing their feeding success. Under controlled conditions, blood uptake of females from an artificial host or from a mouse and biting rates on humans are also reduced. Finally, in a field trial conducted in a 1.17 ha area in China, the female biting rate is reduced by 80%, concurrent to a reduction of female mosquito density of 40% due to the swarming of males around humans attempting to mate with the female mosquitoes. This suggests that the sterile insect technique does not only suppress mosquito vector populations through the induction of sterility, but may also reduce disease transmission due to increased female mortality and lower host contact.

Different mechanisms of X-ray irradiation-induced male and female sterility in Aedes aegypti.

BACKGROUND: Aedes aegypti (Ae. aegypti) is the major vector that transmits many diseases including dengue, Zika, and filariasis in tropical and subtropical regions. Due to the growing resistance to chemical-based insecticides, biological control methods have become an emerging direction to control mosquito populations. The sterile insect technique (SIT) deploys high doses of ionizing radiation to sterilize male mosquitoes before the release. The Wolbachia-based population suppression method of the incompatible insect technique (IIT) involves the release of Wolbachia-infected males to sterilize uninfected field females. Due to the lack of perfect sex separation tools, a low percentage of female contamination is detected in the male population. To prevent the unintentional release of these Wolbachia-infected females which might result in population replacement, a low dose of X-ray irradiation is deployed to sterilize any female escapees. However, it remains unclear whether these irradiation-induced male and female sterilizations share common mechanisms. RESULTS: In this work, we set out to define the minimum dose of X-ray radiation required for complete female sterilization in Ae. aegypti (NEA-EHI strain). Further results showed that this minimum dose of X-ray irradiation for female sterilization significantly reduced male fertility. Similar results have been reported previously in several operational trials. By addressing the underlying causes of the sterility, our results showed that male sterility is likely due to chromosomal damage in the germ cells induced by irradiation. In contrast, female sterility appears to differ and is likely initiated by the elimination of the somatic supporting cells, which results in the blockage of the ovariole maturation. Building upon these findings, we identified the minimum dose of X-ray irradiation on the Wolbachia-infected NEA-EHI (wAlbB-SG) strain, which is currently being used in the IIT-SIT field trial. Compared to the uninfected parental strain, a lower irradiation dose could fully sterilize wAlbB-SG females. This suggests that Wolbachia-carrying mosquitoes are more sensitive to irradiation, consistent with a previous report showing that a lower irradiation dose fully sterilized Wolbachia-infected Ae. aegypti females (Brazil and Mexican strains) compared to those uninfected controls. CONCLUSIONS: Our findings thus reveal the distinct mechanisms of ionizing X-ray irradiation-induced male or female sterility in Ae. aegypti mosquitoes, which may help the design of X-ray irradiation-based vector control methods.

A mass rearing cost calculator for the control of Culex quinquefasciatus in Hawai'i using the incompatible insect technique.

BACKGROUND: Hawai'i's native forest avifauna is experiencing drastic declines due to climate change-induced increases in temperature encroaching on their upper-elevation montane rainforest refugia. Higher temperatures support greater avian malaria infection rates due to greater densities of its primary vector, the southern house mosquito Culex quinquefasciatus, and enhance development of the avian malaria parasite Plasmodium relictum. Here we propose the use of the incompatible insect technique (IIT) or the combined IIT/sterile insect technique (SIT) for the landscape-scale (i.e., area-wide) control of Cx. quinquefasciatus, and have developed a calculator to estimate the costs of IIT and IIT/SIT applications at various sites in Hawai'i. METHODS: The overall cost of the infrastructure, personnel, and space necessary to produce incompatible adult males for release is calculated in a unit of ~ 1 million culicid larvae/week. We assessed the rearing costs and need for effective control at various elevations in Hawai'i using a 10:1 overflooding ratio at each elevation. The calculator uses a rate describing the number of culicids needed to control wild-type mosquitoes at each site/elevation, in relation to the number of larval rearing units. This rate is a constant from which other costs are quantified. With minor modifications, the calculator described here can be applied to other areas, mosquito species, and similar techniques. To test the robustness of our calculator, the Kaua'i-specific culicid IIT/SIT infrastructure costs were also compared to costs from Singapore, Mexico, and China using the yearly cost of control per hectare, and purchasing power parity between sites for the cost of 1000 IIT/SIT males. RESULTS: As a proof of concept, we have used the calculator to estimate rearing infrastructure costs for an application of IIT in the Alaka'i Wilderness Reserve on the island of Kaua'i. Our analysis estimated an initial investment of at least ~ $1.16M with subsequent yearly costs of approximately $376K. Projections of rearing costs for control at lower elevations are ~ 100 times greater than in upper elevation forest bird refugia. These results are relatively comparable to those real-world cost estimates developed for IIT/SIT culicid male production in other countries when inflation and purchasing power parity are considered. We also present supplemental examples of infrastructure costs needed to control Cx. quinquefasciatus in the home range of 'i'iwi Drepanis coccinea, and the yellow fever vector Aedes aegypti. CONCLUSIONS: Our cost calculator can be used to effectively estimate the mass rearing cost of an IIT/SIT program. Therefore, the linear relationship of rearing infrastructure to costs used in this calculator is useful for developing a conservative cost estimate for IIT/SIT culicid mass rearing infrastructure. These mass rearing cost estimates vary based on the density of the targeted organism at the application site.

A standalone incompatible insect technique enables mosquito suppression in the urban subtropics.

The strong suppression of Aedes albopictus on two Guangzhou islands in China has been successfully achieved by releasing males with an artificial triple-Wolbachia infection. However, it requires the use of radiation to sterilize residual females to prevent population replacement. To develop a highly effective tool for dengue control, we tested a standalone incompatible insect technique (IIT) to control A. albopictus in the urban area of Changsha, an inland city where dengue recently emerged. Male mosquitoes were produced in a mass rearing facility in Guangzhou and transported over 670 km under low temperature to the release site. After a once-per-week release with high numbers of males (phase I) and a subsequent twice-per-week release with low numbers of males (phase II), the average numbers of hatched eggs and female adults collected weekly per trap were reduced by 97% and 85%, respectively. The population suppression caused a 94% decrease in mosquito biting at the release site compared to the control site. Remarkably, this strong suppression was achieved using only 28% of the number of males released in a previous trial. Despite the lack of irradiation to sterilize residual females, no triple-infected mosquitoes were detected in the field post release based on the monitoring of adult and larval A. albopictus populations for two years, indicating that population replacement was prevented. Our results support the feasibility of implementing a standalone IIT for dengue control in urban areas.

Wolbachia wAlbB inhibit dengue and Zika infection in the mosquito Aedes aegypti with an Australian background.

Biological control of mosquito vectors using the endosymbiotic bacteria Wolbachia is an emerging strategy for the management of human arboviral diseases. We recently described the development of a strain of Aedes aegypti infected with the Wolbachia strain wAlbB (referred to as the wAlbB2-F4 strain) through simple backcrossing of wild type Australian mosquitoes with a wAlbB infected Ae. aegypti strain from the USA. Field releases of male wAlbB2-F4 mosquitoes resulted in the successful suppression of wild populations of mosquitoes in the trial sites by exploiting the strain's Wolbachia-induced cytoplasmic incompatibility. We now demonstrate that the strain is resistant to infection by dengue and Zika viruses and is genetically similar to endemic Queensland populations. There was a fourfold reduction in the proportion of wAlbB2-F4 mosquitoes that became infected following a blood meal containing dengue 2 virus (16.7%) compared to wild type mosquitoes (69.2%) and a 6-7 fold reduction in the proportion of wAlbB2-F4 mosquitoes producing virus in saliva following a blood meal containing an epidemic strain of Zika virus (8.7% in comparison to 58.3% in wild type mosquitoes). Restriction-site Associated DNA (RAD) sequencing revealed that wAlbB2-F4 mosquitoes have > 98% Australian ancestry, confirming the successful introduction of the wAlbB2 infection into the Australian genomic background through backcrossing. Genotypic and phenotypic analyses showed the wAlbB2-F4 strain retains the insecticide susceptible phenotype and genotype of native Australian mosquitoes. We demonstrate that the Wolbachia wAlbB2-F4, in addition to being suitable for population suppression programs, can also be effective in population replacement programs given its inhibition of virus infection in mosquitoes. The ease at which a target mosquito population can be transfected with wAlbB2, while retaining the genotypes and phenotypes of the target population, shows the utility of this strain for controlling the Ae. aegypti mosquitoes and the pathogens they transmit.

Pilot trial using mass field-releases of sterile males produced with the incompatible and sterile insect techniques as part of integrated Aedes aegypti control in Mexico.

BACKGROUND: The combination of Wolbachia-based incompatible insect technique (IIT) and radiation-based sterile insect technique (SIT) can be used for population suppression of Aedes aegypti. Our main objective was to evaluate whether open-field mass-releases of wAlbB-infected Ae. aegypti males, as part of an Integrated Vector Management (IVM) plan led by the Mexican Ministry of Health, could suppress natural populations of Ae. aegypti in urbanized settings in south Mexico. METHODOLOGY/PRINCIPAL FINDINGS: We implemented a controlled before-and-after quasi-experimental study in two suburban localities of Yucatan (Mexico): San Pedro Chimay (SPC), which received IIT-SIT, and San Antonio Tahdzibichén used as control. Release of wAlbB Ae. aegypti males at SPC extended for 6 months (July-December 2019), covering the period of higher Ae. aegypti abundance. Entomological indicators included egg hatching rates and outdoor/indoor adult females collected at the release and control sites. Approximately 1,270,000 lab-produced wAlbB-infected Ae. aegypti males were released in the 50-ha treatment area (2,000 wAlbB Ae. aegypti males per hectare twice a week in two different release days, totaling 200,000 male mosquitoes per week). The efficacy of IIT-SIT in suppressing indoor female Ae. aegypti density (quantified from a generalized linear mixed model showing a statistically significant reduction in treatment versus control areas) was 90.9% a month after initiation of the suppression phase, 47.7% two months after (when number of released males was reduced in 50% to match local abundance), 61.4% four months after (when initial number of released males was re-established), 88.4% five months after and 89.4% at six months after the initiation of the suppression phase. A proportional, but lower, reduction in outdoor female Ae. aegypti was also quantified (range, 50.0-75.2% suppression). CONCLUSIONS/SIGNIFICANCE: Our study, the first open-field pilot implementation of Wolbachia IIT-SIT in Mexico and Latin-America, confirms that inundative male releases can significantly reduce natural populations of Ae. aegypti. More importantly, we present successful pilot results of the integration of Wolbachia IIT-SIT within a IVM plan implemented by Ministry of Health personnel.

Assessing Aedes aegypti candidate genes during viral infection and Wolbachia-mediated pathogen blocking.

One approach to control dengue virus transmission is the symbiont Wolbachia, which limits viral infection in mosquitoes. Despite plans for its widespread use in Aedes aegypti, Wolbachia's mode of action remains poorly understood. Many studies suggest that the mechanism is likely multifaceted, involving aspects of immunity, cellular stress and nutritional competition. A previous study from our group used artificial selection to identify a new mosquito candidate gene related to viral blocking; alpha-mannosidase-2a (alpha-Mann-2a) with a predicted role in protein glycosylation. Protein glycosylation pathways tend to be involved in complex host-viral interactions; however, the function of alpha-mannosidases has not been described in mosquito-virus interactions. We examined alpha-Mann-2a expression in response to virus and Wolbachia infections and whether reduced gene expression, caused by RNA interference, affected viral loads. We show that dengue virus (DENV) infection affects the expression of alpha-Mann-2a in a tissue- and time-dependent manner, whereas Wolbachia infection had no effect. In the midgut, DENV prevalence increased following knockdown of alpha-Mann-2a expression in Wolbachia-free mosquitoes, suggesting that alpha-Mann-2a interferes with infection. Expression knockdown had the same effect on the togavirus chikungunya virus, indicating that alpha-Mann-2a may have broad antivirus effects in the midgut. Interestingly, we were unable to knockdown the expression in Wolbachia-infected mosquitoes. We also provide evidence that alpha-Mann-2a may affect the transcriptional level of another gene predicted to be involved in viral blocking and cell adhesion; cadherin87a. These data support the hypothesis that glycosylation and adhesion pathways may broadly be involved in viral infection in Ae. aegypti.

Lab-scale characterization and semi-field trials of Wolbachia Strain wAlbB in a Taiwan Wolbachia introgressed Ae. aegypti strain.

Dengue fever is one of the most severe viral diseases transmitted by Aedes mosquitoes, with traditional approaches of disease control proving insufficient to prevent significant disease burden. Release of Wolbachia-transinfected mosquitoes offers a promising alternative control methodologies; Wolbachia-transinfected female Aedes aegypti demonstrate reduced dengue virus transmission, whilst Wolbachia-transinfected males cause zygotic lethality when crossed with uninfected females, providing a method for suppressing mosquito populations. Although highly promising, the delicate nature of population control strategies and differences between local species populations means that controlled releases of Wolbachia-transinfected mosquitoes cannot be performed without extensive testing on specific local Ae. aegypti populations. In order to investigate the potential for using Wolbachia to suppress local Ae. aegypti populations in Taiwan, we performed lab-based and semi-field fitness trials. We first transinfected the Wolbachia strain wAlbB into a local Ae. aegypti population (wAlbB-Tw) and found no significant changes in lifespan, fecundity and fertility when compared to controls. In the laboratory, we found that as the proportion of released male mosquitoes carrying Wolbachia was increased, population suppression could reach up to 100%. Equivalent experiments in semi-field experiments found suppression rates of up to 70%. The release of different ratios of wAlbB-Tw males in the semi-field system provided an estimate of the optimal size of male releases. Our results indicate that wAlbB-Tw has significant potential for use in vector control strategies aimed at Ae. aegypti population suppression in Taiwan. Open field release trials are now necessary to confirm that wAlbB-Tw mediated suppression is feasible in natural environments.

Recently introduced Wolbachia reduces bacterial species richness and reshapes bacterial community structure in Nilaparvata lugens.

BACKGROUND: Wolbachia has been developed as an effective tool to suppress insect pests and arbovirus transmission. Recently, the brown planthopper Nilaparvata lugens, a serious agricultural pest, has been successfully transinfected with Wolbachia wStri strain from Laodelphax striatellus. However, before conducting the field experiments, the impacts of wStri on the bacterial microbiota in N. lugens and how it differs from native Wolbachia wLug strain have not been clarified. RESULTS: Here, we found that wStri reduced bacterial diversity and shaped bacterial community structure more than wLug in both developmental stage and different adult tissues. Overall, the relative abundance of Wolbachia was negatively correlated with bacterial diversity, but the bacterial diversity gradually decreased only when the relative abundance of Wolbachia was higher than 60%. Further analysis found that wStri reduced species richness of other bacteria but not their evenness. wStri infection also affected many bacterial functions (e.g., amino acid metabolism & signaling and cellular processes) in the developmental stages, with a stronger effect than wLug in nymphs. Moreover, although Wolbachia occupied a high relative abundance in infected individuals, Acinetobacter was consistently a core part of microbiome. CONCLUSION: These results showed the significant impacts of recently introduced wStri on bacterial microbiota in N. lugens, with the effects differing from native wLug. This study will aid in understanding the relationship between Wolbachia, its host and the host's microbiota, and provide a reference for future field experiments.

Wolbachia wAlbB remains stable in Aedes aegypti over 15 years but exhibits genetic background-dependent variation in virus blocking.

The ability of the maternally transmitted endosymbiotic bacterium Wolbachia to induce cytoplasmic incompatibility (CI) and virus blocking makes it a promising weapon for combatting mosquito-borne diseases through either suppression or replacement of wild-type populations. Recent field trials show that both approaches significantly reduce the incidence of dengue fever in humans. However, new questions emerge about how Wolbachia-mosquito associations will co-evolve over time and whether Wolbachia-mediated virus blocking will be affected by the genetic diversity of mosquitoes and arboviruses in the real world. Here, we have compared the Wolbachia density and CI expression of two wAlbB-infected Aedes aegypti lines transinfected 15 years apart. We have also assessed wAlbB-mediated virus blocking against dengue (DENV), Zika (ZIKV), and Chikungunya (CHIKV) viruses and examined whether host genetic backgrounds modulate viral blocking effects by comparing ZIKV infection in mosquitoes with a Mexican genetic background to those with a Singaporean background. Our results show that over 15 years, wAlbB maintained the capacity to form a stable association with Ae. aegypti in terms of both density and CI expression. There were variations in wAlbB-induced virus blocking against CHIKV, DENV, and ZIKV, and higher inhibitory effects on ZIKV in mosquitoes on the Singaporean genetic background than on the Mexican background. These results provide important information concerning the robustness and long-term stability of Wolbachia as a biocontrol agent for arbovirus disease control.

Releasing incompatible males drives strong suppression across populations of wild and Wolbachia-carrying Aedes aegypti in Australia.

Releasing sterile or incompatible male insects is a proven method of population management in agricultural systems with the potential to revolutionize mosquito control. Through a collaborative venture with the "Debug" Verily Life Sciences team, we assessed the incompatible insect technique (IIT) with the mosquito vector Aedes aegypti in northern Australia in a replicated treatment control field trial. Backcrossing a US strain of Ae. aegypti carrying Wolbachia wAlbB from Aedes albopictus with a local strain, we generated a wAlbB2-F4 strain incompatible with both the wild-type (no Wolbachia) and wMel-Wolbachia Ae. aegypti now extant in North Queensland. The wAlbB2-F4 strain was manually mass reared with males separated from females using Verily sex-sorting technologies to obtain no detectable female contamination in the field. With community consent, we delivered a total of three million IIT males into three isolated landscapes of over 200 houses each, releasing ∼50 males per house three times a week over 20 wk. Detecting initial overflooding ratios of between 5:1 and 10:1, strong population declines well beyond 80% were detected across all treatment landscapes when compared to controls. Monitoring through the following season to observe the ongoing effect saw one treatment landscape devoid of adult Ae. aegypti early in the season. A second landscape showed reduced adults, and the third recovered fully. These encouraging results in suppressing both wild-type and wMel-Ae. aegypti confirms the utility of bidirectional incompatibility in the field setting, show the IIT to be robust, and indicate that the removal of this arbovirus vector from human-occupied landscapes may be achievable.

Microbes increase thermal sensitivity in the mosquito Aedes aegypti, with the potential to change disease distributions.

The mosquito Aedes aegypti is the primary vector of many disease-causing viruses, including dengue (DENV), Zika, chikungunya, and yellow fever. As consequences of climate change, we expect an increase in both global mean temperatures and extreme climatic events. When temperatures fluctuate, mosquito vectors will be increasingly exposed to temperatures beyond their upper thermal limits. Here, we examine how DENV infection alters Ae. aegypti thermotolerance by using a high-throughput physiological 'knockdown' assay modeled on studies in Drosophila. Such laboratory measures of thermal tolerance have previously been shown to accurately predict an insect's distribution in the field. We show that DENV infection increases thermal sensitivity, an effect that may ultimately limit the geographic range of the virus. We also show that the endosymbiotic bacterium Wolbachia pipientis, which is currently being released globally as a biological control agent, has a similar impact on thermal sensitivity in Ae. aegypti. Surprisingly, in the coinfected state, Wolbachia did not provide protection against DENV-associated effects on thermal tolerance, nor were the effects of the two infections additive. The latter suggests that the microbes may act by similar means, potentially through activation of shared immune pathways or energetic tradeoffs. Models predicting future ranges of both virus transmission and Wolbachia's efficacy following field release may wish to consider the effects these microbes have on host survival.

Abundance and Seasonality of Aedes aegypti (Diptera: Culicidae) in Two Suburban Localities of South Mexico, With Implications for Wolbachia (Rickettsiales: Rickettsiaceae)-Carrying Male Releases for Population Suppression.

We conducted a baseline characterization of the abundance and seasonality of Aedes aegypti (Linnaeus, 1762)-a vector of dengue, chikungunya, and Zika-in two suburban localities of Yucatan, Mexico, as the first step in the implementation of an integrated vector management (IVM) plan combining 'traditional Aedes control' (source reduction/truck-mounted ultra-low volume [ULV] spraying) and incompatible insect technique/sterile insect technique for population suppression in Yucatan, Mexico. Weekly entomological collections with ovitraps and BG-sentinel traps were performed in 1-ha quadrants of both localities for 1 yr. Three distinct periods/phases were identified, closely associated with precipitation: 1) a phase of low population abundance during the dry season (weekly average of Aedes eggs per ovitrap and adults per BG trap = 15.51 ± 0.71 and 10.07 ± 0.88, respectively); 2) a phase of population growth and greatest abundance of Aedes (49.03 ± 1.48 eggs and 25.69 ± 1.31 adults) during the rainy season; and finally 3) a phase of decline among populations (20.91 ± 0.97 eggs and 3.24 ± 0.21 adults) after the peak of the rainy season. Seasonal abundance and dynamics of Ae. aegypti populations suggest that it is feasible to develop and implement time-specific actions as part of an IVM approach incorporating integrating novel technologies (such as rear-and-release of Wolbachia-infected males) with classic (insecticide-based) approaches implemented routinely for vector control. In agreement with the local vector control program, we propose a pilot IVM strategy structured in a preparation phase, an attack phase with traditional vector control, and a suppression phase with inundative releases, which are described in this paper.

Stable Introduction of Plant-Virus-Inhibiting Wolbachia into Planthoppers for Rice Protection.

Progress has been made in developing the maternally inherited endosymbiotic bacterium Wolbachia as a tool for protecting humans from mosquito-borne diseases. In contrast, Wolbachia-based approaches have not yet been developed for the protection of plants from insect pests and their associated diseases, with a major challenge being the establishment of artificial Wolbachia infections expressing desired characteristics in the hemipterans that transmit the majority of plant viruses. Here, we report stable introduction of Wolbachia into the brown planthopper, Nilaparvata lugens, the most destructive rice pest that annually destroys millions of hectares of staple crops. The Wolbachia strain wStri from the small brown planthopper, Laodelphax striatellus, was transferred to this new host, where it showed high levels of cytoplasmic incompatibility, enabling rapid invasion of laboratory populations. Furthermore, wStri inhibited infection and transmission of Rice ragged stunt virus and mitigated virus-induced symptoms in rice plants, opening up the development of Wolbachia-based strategies against major agricultural pests and their transmitted pathogens. VIDEO ABSTRACT.

Newly introduced Cardinium endosymbiont reduces microbial diversity in the rice brown planthopper Nilaparvata lugens.

Symbiotic microorganisms in invertebrates play vital roles in host ecology and evolution. Cardinium, a common intracellular symbiont, is transinfected into the important agricultural pest Nilaparvata lugens (rice brown planthopper) to regulate its reproduction, but how this impacts its microbial community is unknown. Here, we characterized the bacterial microbiota from N. lugens, with or without Cardinium, at different developmental stages and in various adult tissues using 16S ribosomal ribonucleic acid (16S rRNA) gene sequencing. Upon infection with Cardinium, we found that microbial diversity in the different developmental stages of N. lugens (especially females), and in female midguts and male testes, was lower than that in the uninfected control. There was a negative correlation between Cardinium and most related genera and between Bacteroidetes and Proteobacteria. Although the microbial structure varied during Cardinium infection, Acinetobacter spp. were a core microbiome genus. The Cardinium infection enhanced the relative density of midgut-associated Acinetobacter spp., with both bacteria exhibiting tissue-specific tropism. In addition, this infection caused the changes of main microbial functions in N. lugens. These results offer insights into the effects of alien (i.e. newly introduced from other organism) Cardinium infection on N. lugens-associated microbiotas, aiding in the development of transinfected endosymbionts for pest control.

Toward implementation of combined incompatible and sterile insect techniques for mosquito control: Optimized chilling conditions for handling Aedes albopictus male adults prior to release.

Combined incompatible and sterile insect technique (IIT-SIT) has been considered to be an effective and safe approach to control mosquito populations. Immobilization of male adults by chilling is a crucial process required for the packing, transportation and release of the mosquitoes during the implementation of IIT-SIT for mosquito control. In this study, effects of chilling on the Aedes albopictus males with triple Wolbachia infections (HC line), a powerful weapon to fight against the wild type Ae. albopictus population via IIT-SIT, were evaluated under both laboratory and field conditions. Irradiated HC (IHC) males were exposed to 1, 5 and 10°C for 1, 2, 3, 6 and 24 h. The survival rate of the post-chilled IHC males was then monitored. Longevity of post-chilled IHC males was compared to non-chilled males under laboratory and semi-field conditions. Mating competitiveness of IHC/HC males after exposure to 5 or 10°C for 0, 3 and 24 h was then evaluated. Effects of compaction and transportation under chilled conditions on the survival rate of IHC males were also monitored. The optimal chilling conditions for handling IHC males were temperatures between 5 and 10°C for a duration of less than 3 h with no negative impacts on survival rate, longevity and mating competitiveness when compared to non-chilled males. However, the overall quality of post-chilled IHC/HC males decreased when exposed to low temperatures for 24 h. Reduced survival was observed when IHC males were stored at 5°C under a compaction height of 8 cm. Transportation with chilling temperatures fluctuating from 8 to 12°C has no negative impact on the survival of IHC males. This study identified the optimal chilling temperature and duration for the handling and transportation of Ae. albopictus IHC male adults without any detrimental effect on their survival, longevity and mating competitiveness. Further studies are required to develop drone release systems specific for chilled mosquitoes to improve release efficiency, as well as to compare the population suppression efficiency between release of post-chilled and non-chilled males in the field.

Wolbachia Inter-Strain Competition and Inhibition of Expression of Cytoplasmic Incompatibility in Mosquito.

Successful field trials have been reported as part of the effort to develop the maternally transmitted endosymbiontic bacteria Wolbachia as an intervention agent for controlling mosquito vectors and their transmitted diseases. In order to further improve this novel intervention, artificially transinfected mosquitoes must be optimized to display maximum pathogen blocking, the desired cytoplasmic incompatibility (CI) pattern, and the lowest possible fitness cost. Achieving such optimization, however, requires a better understanding of the interactions between the host and various Wolbabachia strains and their combinations. Here, we transferred the Wolbachia wMel strain by embryonic microinjection into Aedes albopictus, resulting in the successful establishment of a transinfected line, HM (wAlbAwAlbBwMel), with a triple-strain infection comprising wMel, wAlbA, and wAlbB. Surprisingly, no CI was induced when the triply infected males were crossed with the wild-type GUA females or with another triply infected HC females carrying wPip, wAlbA, and wAlbB, but specific removal of wAlbA from the HM (wAlbAwAlbBwMel) line resulted in the expression of CI after crosses with lines infected by either one, two, or three strains of Wolbachia. The transinfected line showed perfect maternal transmission of the triple infection, with fluctuating egg hatch rates that improved to normal levels after repeated outcrosses with GUA line. Strain-specific qPCR assays showed that wMel and wAlbB were present at the highest densities in the ovaries and midguts, respectively, of the HM (wAlbAwAlbBwMel) mosquitoes. These finding suggest that introducing a novel strain of Wolbachia into a Wolbachia-infected host may result in complicated interactions between Wolbachia and the host and between the various Wolbachia strains, with competition likely to occur between strains in the same supergroup.