The range of the mosquito Aedes aegypti continues to expand, putting more than two billion people at risk of arboviral infection. The sterile insect technique (SIT) has been used to successfully combat agricultural pests at large scale, but not mosquitoes, mainly because of challenges with consistent production and distribution of high-quality male mosquitoes. We describe automated processes to rear and release millions of competitive, sterile male Wolbachia-infected mosquitoes, and use of these males in a large-scale suppression trial in Fresno County, California. In 2018, we released 14.4 million males across three replicate neighborhoods encompassing 293 hectares. At peak mosquito season, the number of female mosquitoes was 95.5% lower (95% CI, 93.6-96.9) in release areas compared to non-release areas, with the most geographically isolated neighborhood reaching a 99% reduction. This work demonstrates the high efficacy of mosquito SIT in an area ninefold larger than in previous similar trials, supporting the potential of this approach in public health and nuisance-mosquito eradication programs.

Efficient production of male Wolbachia-infected Aedes aegypti mosquitoes enables large-scale suppression of wild populations.

Living in the endosymbiotic world of Wolbachia: A centennial review.

Wolbachia is an endosymbiotic Alphaproteobacteria that can suppress insect-borne diseases through decreasing host virus transmission (population replacement) or through decreasing host population density (population suppression). We contrast natural Wolbachia infections in insect populations with Wolbachia transinfections in mosquitoes to gain insights into factors potentially affecting the long-term success of Wolbachia releases. Natural Wolbachia infections can spread rapidly, whereas the slow spread of transinfections is governed by deleterious effects on host fitness and demographic factors. Cytoplasmic incompatibility (CI) generated by Wolbachia is central to both population replacement and suppression programs, but CI in nature can be variable and evolve, as can Wolbachia fitness effects and virus blocking. Wolbachia spread is also influenced by environmental factors that decrease Wolbachia titer and reduce maternal Wolbachia transmission frequency. More information is needed on the interactions between Wolbachia and host nuclear/mitochondrial genomes, the interaction between invasion success and local ecological factors, and the long-term stability of Wolbachia-mediated virus blocking.

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Evolutionary Ecology of Wolbachia Releases for Disease Control

Background 
Important arboviral diseases, such as dengue, chikungunya, and Zika virus infections, are transmitted mainly by the Aedes aegypti vector. So far, controlling this vector species with current tools and strategies has not demonstrated sustainable and significant impacts. Our main objective was to evaluate whether open field release of sterile males, produced from combining the sterile insect technique using radiation with the insect incompatible technique through Wolbachia-induced incompatibility (SIT/IIT), could suppress natural populations of Ae. aegypti in semi-rural village settings in Thailand.


Methodology/Principal findings 
Irradiated Wolbachia-infected Aedes aegypti males produced by the SIT/IIT approach were completely sterile and were able to compete with the wild fertile ones. Open field release of these sterile males was conducted in an ecologically isolated village in Chachoengsao Province, eastern Thailand. House-to-house visit and media reports resulted in community acceptance and public awareness of the technology. During intervention, approximately 100–200 sterile males were released weekly in each household. After 6 months of sterile male release, a significant reduction (p<0.05) of the mean egg hatch rate (84%) and the mean number of females per household (97.30%) was achieved in the treatment areas when compared to the control ones.


Conclusions/Significance 
Our study represents the first open field release of sterile Ae. aegypti males developed from a combined SIT/IIT approach. Entomological assessment using ovitraps, adult sticky traps, and portable vacuum aspirators confirmed the success in reducing natural populations of Ae. aegypti females in treated areas. Public awareness through media resulted in positive support for practical use of this strategy in wider areas. Further study using a systematic randomized trial is needed to determine whether this approach could have a significant impact on the diseases transmitted by Ae. aegypti vector.

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Combined sterile insect technique and incompatible insect technique: The first proof-of-concept to suppress Aedes aegypti vector populations in semi-rural settings in Thailand.

Cytoplasmic incompatibility (CI) is the most common symbiont-induced reproductive manipulation. Specifically, symbiont-induced sperm modifications cause catastrophic mitotic defects in the fertilized embryo and ensuing lethality in crosses between symbiotic males and either aposymbiotic females or females harboring a different symbiont strain. However, if the female carries the same symbiont strain, then embryos develop properly, thereby imparting a relative fitness benefit to symbiont-transmitting mothers. Thus, CI drives maternally-transmitted bacteria to high frequencies in arthropods worldwide. In the past two decades, CI experienced a boom in interest due to its (i) deployment in worldwide efforts to curb mosquito-borne diseases, (ii) causation by bacteriophage genes, cifA and cifB, that modify sexual reproduction, and (iii) important impacts on arthropod speciation. This review serves as a gateway to experimental, conceptual, and quantitative themes of CI and outlines significant gaps in understanding CI's mechanism that are ripe for investigation from diverse subdisciplines in the life sciences.

Symbiont-mediated cytoplasmic incompatibility: what have we learned in 50 years?

The radiation-based sterile insect technique (SIT) has successfully suppressed field populations of several insect pest species, but its effect on mosquito vector control has been limited. The related incompatible insect technique (IIT)-which uses sterilization caused by the maternally inherited endosymbiotic bacteria Wolbachia-is a promising alternative, but can be undermined by accidental release of females infected with the same Wolbachia strain as the released males. Here we show that combining incompatible and sterile insect techniques (IIT-SIT) enables near elimination of field populations of the world's most invasive mosquito species, Aedes albopictus. Millions of factory-reared adult males with an artificial triple-Wolbachia infection were released, with prior pupal irradiation of the released mosquitoes to prevent unintentionally released triply infected females from successfully reproducing in the field. This successful field trial demonstrates the feasibility of area-wide application of combined IIT-SIT for mosquito vector control.

Incompatible and sterile insect techniques combined eliminate mosquitoes

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.

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

The invention discloses a preparation method of aedes albopictus stably carrying culex quinquefasciatus wolbachia and the application of the aedes albopictus stably carrying the culex quinquefasciatus wolbachia to prevention and control of the aedes albopictus The preparation method comprises the following steps that Guangzhou wild aedes albopictus treated with tetracycline are screened, so that aedes albopictus without wolbachia is obtained, and the aedes albopictus stably carrying the culex quinquefasciatus wolbachia is prepared The application of the aedes albopictus stably carrying the culex quinquefasciatus wolbachia to prevention and control of the aedes albopictus has the advantages that the cost is low, and sustainability and environment friendliness are achieved The aedes albopictus stably carrying the culex quinquefasciatus wolbachia is one of the most potential biological measures for prevention and control of mosquito-borne diseases, the problem that insects can easily establish drug resistance under the condition of using chemical insecticides is solved, and the problem that harm to the environment and the public health is caused is also solved

Preparation method of aedes albopictus stably carrying culex quinquefasciatus wolbachia and application of aedes albopictus stably carrying culex quinquefasciatus wolbachia to prevention and control of aedes albopictus

The utility model provides an adult mosquito raising device which comprises a mosquito cage and a feeding groove connected into the mosquito cage. An opening covered with an isolation net is formed in the top of the mosquito cage. The adult mosquito raising device further comprises blood feeding devices. Each blood feeding device is composed of a blood cavity and a water bath cavity, wherein the blood cavity contains blood, the water bath cavity contains hot water at constant temperature, the blood cavity is located above the opening, the water bath cavity is tightly attached to the peripheral side of the blood cavity and is divided through a partition plate for water isolation and heat conduction, and the bottom of the blood cavity is a casing bottom. According to the adult mosquito raising device, blood can be continuously heated through the external hot water at the constant temperature, operation is convenient, the temperature of the blood is easily kept constant, the blood feeding devices can be used for feeding blood at the same time, and the number of raised mosquitos is increased; due to the fact that a spawning groove is horizontally formed and is filled with cotton, damp is even, a piece of filter paper can be kept wet and cannot be excessively wet to be sunk, the adult mosquitos can evenly lay eggs on the filter paper, the phenomenon of egg overlapping is reduced, and follow-up counting and statistics are facilitated.

Liu Julian

Papers

Incompatible and sterile insect techniques combined eliminate mosquitoes

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

Preparation method of aedes albopictus stably carrying culex quinquefasciatus wolbachia and application of aedes albopictus stably carrying culex quinquefasciatus wolbachia to prevention and control of aedes albopictus

Adult mosquito raising device