Genetic manipulations of insect populations for pest control have been advocated for some time, but there are few cases where manipulated individuals have been released in the field and no cases where they have successfully invaded target populations. Population transformation using the intracellular bacterium Wolbachia is particularly attractive because this maternally-inherited agent provides a powerful mechanism to invade natural populations through cytoplasmic incompatibility. When Wolbachia are introduced into mosquitoes, they interfere with pathogen transmission and influence key life history traits such as lifespan. Here we describe how the wMel Wolbachia infection, introduced into the dengue vector Aedes aegypti from Drosophila melanogaster, successfully invaded two natural A. aegypti populations in Australia, reaching near-fixation in a few months following releases of wMel-infected A. aegypti adults. Models with plausible parameter values indicate that Wolbachia-infected mosquitoes suffered relatively small fitness costs, leading to an unstable equilibrium frequency <30% that must be exceeded for invasion. These findings demonstrate that Wolbachia-based strategies can be deployed as a practical approach to dengue suppression with potential for area-wide implementation.

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Successful establishment of Wolbachia in Aedes populations to suppress dengue transmission

From the Oxford University Clinical Research Unit and Wellcome Trust Major Overseas Programme (C.P.S., J.J.F., B.W.), Hospital for Tropical Diseases (N.V.C.), Ho Chi Minh City, Vietnam; and the Centre for Tropical Medicine, University of Oxford, Churchill Hospital, Oxford, United Kingdom (C.P.S., J.J.F., B.W.). Address reprint requests to Dr. Farrar at the Hospital for Tropical Diseases, Oxford University Clinical Research Unit, 190 Ben Ham Tu, Quan 5, Ho Chi Minh City, Vietnam, or at jfarrar@oucru.org.

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Current concepts: Dengue

Dengue fever is the most important mosquito-borne viral disease of humans with more than 50 million cases estimated annually in more than 100 countries. Disturbingly, the geographic range of dengue is currently expanding and the severity of outbreaks is increasing. Control options for dengue are very limited and currently focus on reducing population abundance of the major mosquito vector, Aedes aegypti. These strategies are failing to reduce dengue incidence in tropical communities and there is an urgent need for effective alternatives. It has been proposed that endosymbiotic bacterial Wolbachia infections of insects might be used in novel strategies for dengue control. For example, the wMelPop-CLA Wolbachia strain reduces the lifespan of adult A. aegypti mosquitoes in stably transinfected lines. This life-shortening phenotype was predicted to reduce the potential for dengue transmission. The recent discovery that several Wolbachia infections, including wMelPop-CLA, can also directly influence the susceptibility of insects to infection with a range of insect and human pathogens has markedly changed the potential for Wolbachia infections to control human diseases. Here we describe the successful transinfection of A. aegypti with the avirulent wMel strain of Wolbachia, which induces the reproductive phenotype cytoplasmic incompatibility with minimal apparent fitness costs and high maternal transmission, providing optimal phenotypic effects for invasion. Under semi-field conditions, the wMel strain increased from an initial starting frequency of 0.65 to near fixation within a few generations, invading A. aegypti populations at an accelerated rate relative to trials with the wMelPop-CLA strain. We also show that wMel and wMelPop-CLA strains block transmission of dengue serotype 2 (DENV-2) in A. aegypti, forming the basis of a practical approach to dengue suppression.

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The wMel Wolbachia strain blocks dengue and invades caged Aedes aegypti populations.

The notion of the "biological individual" is crucial to studies of genetics, immunology, evolution, development, anatomy, and physiology. Each of these biological subdisciplines has a specific conception of individuality, which has historically provided conceptual contexts for integrating newly acquired data. During the past decade, nucleic acid analysis, especially genomic sequencing and high-throughput RNA techniques, has challenged each of these disciplinary definitions by finding significant interactions of animals and plants with symbiotic microorganisms that disrupt the boundaries that heretofore had characterized the biological individual. Animals cannot be considered individuals by anatomical or physiological criteria because a diversity of symbionts are both present and functional in completing metabolic pathways and serving other physiological functions. Similarly, these new studies have shown that animal development is incomplete without symbionts. Symbionts also constitute a second mode of genetic inheritance, providing selectable genetic variation for natural selection. The immune system also develops, in part, in dialogue with symbionts and thereby functions as a mechanism for integrating microbes into the animal-cell community. Recognizing the "holobiont"--the multicellular eukaryote plus its colonies of persistent symbionts--as a critically important unit of anatomy, development, physiology, immunology, and evolution opens up new investigative avenues and conceptually challenges the ways in which the biological subdisciplines have heretofore characterized living entities.

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A symbiotic view of life: we have never been individuals.

Chikungunya virus infection is a rapid-onset, febrile disease with intense asthenia, arthralgia, myalgia, headache, and rash. This mosquito-borne alphavirus has spread throughout the Caribbean and into much of Central America. Further spread in the Americas seems likely.

https://www.nejm.org/doi/suppl/10.1056/NEJMra1406035/suppl_file/nejmra1406035_appendix.pdf

Chikungunya Virus and the Global Spread of a Mosquito-Borne Disease

https://www.arca.fiocruz.br/bitstream/icict/13668/2/A%20Wolbachia%20Symbiont%20in%20Aedes%20aegypti.pdf

A Wolbachia Symbiont in Aedes aegypti Limits Infection with Dengue, Chikungunya, and Plasmodium

Wolbachia as an endosymbiont is widespread in insects and other arthropods and is best known for reproductive manipulations of the host. Recently, it has been shown that wMelpop and wMel strains of Wolbachia inhibit the replication of several RNA viruses, including dengue virus, and other vector-borne pathogens (e.g., Plasmodium and filarial nematodes) in mosquitoes, providing an alternative approach to limit the transmission of vector-borne pathogens. In this study, we tested the effect of Wolbachia on the replication of West Nile Virus (WNV). Surprisingly, accumulation of the genomic RNA of WNV for all three strains of WNV tested (New York 99, Kunjin, and New South Wales) was enhanced in Wolbachia-infected Aedes aegypti cells (Aag2). However, the amount of secreted virus was significantly reduced in the presence of Wolbachia. Intrathoracic injections showed that replication of WNV in A. aegypti mosquitoes infected with wMel strain of Wolbachia was not inhibited, whereas wMelPop strain of Wolbachia significantly reduced the replication of WNV in mosquitoes. Further, when wMelPop mosquitoes were orally fed with WNV, virus infection, transmission, and dissemination rates were very low in Wolbachia-free mosquitoes and were completely inhibited in the presence of Wolbachia. The results suggest that (i) despite the enhancement of viral genomic RNA replication in the Wolbachia-infected cell line the production of secreted virus was significantly inhibited, (ii) the antiviral effect in intrathoracically infected mosquitoes depends on the strain of Wolbachia, and (iii) replication of the virus in orally fed mosquitoes was completely inhibited in wMelPop strain of Wolbachia.

http://www.eliminatedengue.com/library/publication/document/scientifix_paper/JVirol-Jan2013v87n2p851-858.pdf

Effect of Wolbachia on Replication of West Nile Virus in a Mosquito Cell Line and Adult Mosquitoes

In order to assess the broad-scale applicability of field releases of Wolbachia for the biological control of insect-transmitted diseases, we determined the relationship between the larval diet of Aedes aegypti L. mosquitoes infected with Wolbachia strains and their susceptibility to dengue virus (DENV) infection via intrathoracic injection and oral inoculation. Larvae were reared on diets that varied in the quantity of food which had the effect of modifying development time and adult body size. Wolbachia wMel infection was associated with highly significant reductions in dengue serotype 2 (DENV-2) infection rates of between 80 and 97.5% following intrathoracic injection of adults emerging from three diet levels. Reductions were 100% in two diet level treatments following oral inoculation. Similarly, wMelPop infection was associated with highly significant reductions in DENV-2 infection rates of between 95 and 100% for intrathoracic injection and 97.5 and 100% for oral inoculation across diet level treatments. Larval diet level had no significant effect on DENV-2 infection rates in the presence of Wolbachia infection in mosquitoes that were intrathoracically injected with the virus. This indicates that the effectiveness of Wolbachia on vector competence disruption within Ae. aegypti is unlikely to be compromised by variable larval nutrition in field settings.

Effects of Larval Nutrition on Wolbachia -Based Dengue Virus Interference in Aedes aegypti (Diptera: Culicidae)

1 Australian Infectious Disease Research Centre, School of Biological Sciences, The University of 8 Queensland, St Lucia, QLD 4072, Australia 9 Australian Infectious Disease Research Centre, School of Chemistry and Molecular Biosciences, 10 The University of Queensland, St Lucia, QLD 4072, Australia 11 Mosquito Control Laboratory, Queensland Institute of Medical Research, Herston, QLD 4029, 12 Australia 13 14

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Guangjin Lu

Papers

A Wolbachia Symbiont in Aedes aegypti Limits Infection with Dengue, Chikungunya, and Plasmodium

Effect of Wolbachia on Replication of West Nile Virus in a Mosquito Cell Line and Adult Mosquitoes

Effects of Larval Nutrition on Wolbachia -Based Dengue Virus Interference in Aedes aegypti (Diptera: Culicidae)

Effect of Wolbachia on replication of West Nile Virus in mosquito cell line and adult