Assessment of community support for Wolbachia-mediated population suppression as a control method for Aedes aegypti mosquitoes in a community cohort in Puerto Rico

Arboviral diseases transmitted by Aedes species mosquitoes pose an increasing public health challenge in tropical regions. Wolbachia-mediated population suppression (Wolbachia suppression) is a vector control method used to reduce Aedes mosquito populations by introducing male mosquitoes infected with Wolbachia, a naturally occurring endosymbiotic bacterium. When Wolbachia-infected male mosquitoes mate with female wild mosquitoes, the resulting eggs will not hatch. Public support is vital to the successful implementation and sustainability of vector control interventions. Communities Organized to Prevent Arboviruses (COPA) is a cohort study to determine the incidence of arboviral disease in Ponce, Puerto Rico and evaluate vector control methods. Focus groups were conducted with residents of COPA communities to gather their opinion on vector control methods; during 2018–2019, adult COPA participants were interviewed regarding their views on Wolbachia suppression; and a follow-up questionnaire was conducted among a subset of participants and non-participants residing in COPA communities. We analyzed factors associated with support for this method. Among 1,528 participants in the baseline survey, median age was 37 years and 63% were female. A total of 1,032 (68%) respondents supported Wolbachia suppression. Respondents with an income of $40,000 or more were 1.34 times as likely [95% CI: 1.03, 1.37] to support Wolbachia suppression than those who earned less than $40,000 annually. Respondents who reported repellant use were 1.19 times as likely to support Wolbachia suppression [95% CI: 1.03, 1.37]. A follow-up survey in 2020 showed that most COPA participants (86%) and non-participants living in COPA communities (84%) supported Wolbachia suppression during and after an educational campaign. The most frequent questions regarding this method were related to its impact on human and animal health, and the environment. Continuous community engagement and education efforts before and during the implementation of novel vector control interventions are necessary to increase and maintain community support.

Wolbachia cifB induces cytoplasmic incompatibility in the malaria mosquito vector

Wolbachia, a maternally inherited intracellular bacterial species, can manipulate host insect reproduction by cytoplasmic incompatibility (CI), which results in embryo lethality in crosses between infected males and uninfected females. CI is encoded by two prophage genes, cifA and cifB. Wolbachia, coupled with the sterile insect technique, has been used in field trials to control populations of the dengue vector Aedes albopictus, but CI-inducing strains are not known to infect the malaria vector Anopheles gambiae. Here we show that cifA and cifB can induce conditional sterility in the malaria vector An. gambiae. We used transgenic expression of these Wolbachia-derived genes in the An. gambiae germline to show that cifB is sufficient to cause embryonic lethality and that cifB-induced sterility is rescued by cifA expression in females. When we co-expressed cifA and cifB in male mosquitoes, the CI phenotype was attenuated. In female mosquitoes, cifB impaired fertility, which was overcome by co-expression of cifA. Our findings pave the way towards using CI to control malaria mosquito vectors.

Mosquito control, killing off the females.

In addition to causing discomfort, female mosquitoes introduce disease-carrying viruses and bacteria into the bloodstream of their victims. There are numerous publications describing the uses of sugary mosquito baits with promising results. Without temperature control measures however, these methods are mainly useful for only nectar-feeding insects, including male mosquitoes, because the warmth of the blood is a condition for the females to locate their meals. The efforts required to keep the baits fresh against the natural spoiling process make them less attractive or impractical to implement. These experiments address these issues by using warm baits of water, sugar, boric acid, and antibiotics. Overnight, the general areas became clear of blood-sucking female mosquitoes while in numbers, the harmless males concentrated into the immediate vicinities. Control vs. experiment protocol established no other logical explanation for this phenomenon other than that the females were attracted and killed by the bait. As expected, there was no mosquito egg-laying activity in these areas.

Mosquito control, killing off the females

In addition to causing discomfort, female mosquitoes introduce disease-carrying viruses and bacteria into the bloodstream of their victims. There are numerous publications describing the uses of sugary mosquito baits with promising results. However, without temperature control measures, these methods are mainly useful for only nectar feeding insects, including male mosquitoes, because the warmth of the blood is a condition for the females to locate their meals. The efforts required to keep the baits fresh against the natural spoiling process make them less attractive or impractical to implement. These experiments address these issues by using warm baits of water, sugar, boric acid and antibiotics. On the surface, the area became a sought-out island where mosquito activities flourished; nevertheless, the mosquitoes were exclusively males (almost). Control vs. experiment protocol established no other logical explanation for this phenomenon other than that the females were attracted and killed by the bait. As expected, there was no mosquito egg laying in these areas.

Mosquito control, killing off the females

In addition to causing discomfort, female mosquitoes introduce disease-carrying viruses and bacteria into the bloodstream of their victims. There are numerous publications describing the uses of sugary mosquito baits with promising results. However, without temperature control measures, these methods are mainly useful for only nectar feeding insects, including male mosquitoes, because the warmth of the blood is a condition for the females to locate their meals. The efforts required to keep the baits fresh against the natural spoiling process make them less attractive or impractical to implement. These experiments address these issues by using warm baits of water, sugar, boric acid and antibiotics. On the surface, the area became a sought-out island where mosquito activities flourished; nevertheless, the mosquitoes were exclusively males (almost). Control vs. experiment protocol established no other logical explanation for this phenomenon other than that the females were attracted and killed by the bait. As expected, there was no mosquito egg laying in these areas.

Predicting the spread and persistence of genetically modified dominant sterile male mosquitoes

Background Reproductive containment provides an opportunity to implement a staged-release strategy for genetic control of malaria vectors, in particular allowing predictions about the spread and persistence of (self-limiting) sterile and male-biased strains to be compared to outcomes before moving to (self-sustaining) gene-drive strains. Methods In this study, we: (i) describe a diffusion–advection–reaction model of the spread and persistence of a single cohort of male mosquitoes; (ii) elicit informative prior distributions for model parameters, for wild-type (WT) and genetically modified dominant sterile strains (DSM); (iii) estimate posterior distributions for WT strains using data from published mark-recapture-release (MRR) experiments, with inference performed through the Delayed-Rejection Adaptive Metropolis algorithm; and (iv) weight prior distributions, in order to make predictions about genetically modified strains using Bayes factors calculated for the WT strains. Results If a single cohort of 5000 genetically modified dominant sterile male mosquitoes are released at the same location as previous MRR experiments with their WT counterparts, there is a 90% probability that the expected number of released mosquitoes will fall to < 1 in 10 days, and that by 12 days there will be a 99% probability that no mosquitoes will be found more than 150 m from the release location. Conclusions Spread and persistence models should form a key component of risk assessments of novel genetic control strategies for malaria vectors. Our predictions, used in an independent risk assessment, suggest that genetically modified sterile male mosquitoes will remain within the locality of the release site, and that they will persist for a very limited amount of time. Data gathered following the release of these mosquitoes will enable us to test the accuracy of these predictions and also provide a means to update parameter distributions for genetic strains in a coherent (Bayesian) framework. We anticipate this will provide additional insights about how to conduct probabilistic risk assessments of stage-released genetically modified mosquitoes. Graphical abstract

A conserved female-specific larval requirement for MtnB function facilitates sex separation in multiple species of disease vector mosquitoes

Background Clusters of sex-specific loci are predicted to shape the boundaries of the M/m sex-determination locus of the dengue vector mosquito Aedes aegypti, but the identities of these genes are not known. Identification and characterization of these loci could promote a better understanding of mosquito sex chromosome evolution and lead to the elucidation of new strategies for male mosquito sex separation, a requirement for several emerging mosquito population control strategies that are dependent on the mass rearing and release of male mosquitoes. This investigation revealed that the methylthioribulose-1-phosphate dehydratase (MtnB) gene, which resides adjacent to the M/m locus and encodes an evolutionarily conserved component of the methionine salvage pathway, is required for survival of female larvae. Results Larval consumption of Saccharomyces cerevisiae (yeast) strains engineered to express interfering RNA corresponding to MtnB resulted in target gene silencing and significant female death, yet had no impact on A. aegypti male survival or fitness. Integration of the yeast larvicides into mass culturing protocols permitted scaled production of fit adult male mosquitoes. Moreover, silencing MtnB orthologs in Aedes albopictus, Anopheles gambiae, and Culex quinquefasciatus revealed a conserved female-specific larval requirement for MtnB among different species of mosquitoes. Conclusions The results of this investigation, which may have important implications for the study of mosquito sex chromosome evolution, indicate that silencing MtnB can facilitate sex separation in multiple species of disease vector insects. Graphical Abstract

Strong attraction of male mosquitoes (Aedes aegypti) to humans

Female Aedes aegypti mosquitoes integrate multiple sensory cues to locate human hosts for blood meals. While male mosquitoes do not blood feed, male Ae. aegypti swarm around and land on humans in nature. Basrur et al. (2020) generated male Aedes aegypti lacking the fruitless gene and discovered that they gained strong attraction to humans, similar to female mosquitoes. The authors assume that host-seeking is a female-specific trait, which they confirmed through experiments. However, all experiments were performed under confined laboratory conditions which appear to inhibit swarming behavior. We used semi-field experiments to demonstrate robust attraction of male Ae. aegypti to humans. Human-baited traps captured up to 25% of released males within 15 min, whereas control traps without humans as bait failed to capture males. Rapid attraction to humans was further demonstrated through videography. Males swarmed around and landed on human subjects, with no activity recorded in paired unbaited controls. The absence of female Ae. aegypti in these experiments rules out a hypothesis by Basrur et al. (2020) that males are attracted not to the human, but to host-seeking females near humans. Finally, we confirm the lack of male attraction to humans in small laboratory cages, even when using recently field-collected males. Our direct observations of male mosquito attraction to humans refute a key assumption of Basrur et al. (2020) and raise questions around conditions under which fruitless prevents male host-seeking. Male mosquito attraction to humans is likely to be important for mating success in wild populations and its basis should be further explored.

A functional requirement for sex-determination M/m locus region lncRNA genes in Aedes aegypti female larvae

Although many putative long non-coding RNA (lncRNA) genes have been identified in insect genomes, few of these genes have been functionally validated. A screen for female-specific larvicides that facilitate Aedes aegypti male sex separation uncovered multiple interfering RNAs with target sites in lncRNA genes located in the M/m locus region, including loci within or tightly linked to the sex determination locus. Larval consumption of a Saccharomyces cerevisiae (yeast) strain engineered to express interfering RNA corresponding to lncRNA transcripts resulted in significant female death, yet had no impact on male survival or fitness. Incorporation of the yeast larvicides into mass culturing protocols facilitated scaled production and separation of fit adult males, indicating that yeast larvicides could benefit mosquito population control strategies that rely on mass releases of male mosquitoes. These studies functionally verified a female-specific developmental requirement for M/m locus region lncRNA genes, suggesting that sexually antagonistic lncRNA genes found within this highly repetitive pericentromeric DNA sequence may be contributing to the evolution of A. aegypti sex chromosomes.

“Maskandi experience”: exploring the use of a cultural song for community engagement in preparation for a pilot Sterile Insect Technique release programme for malaria vector control in KwaZulu-Natal Province, South Africa 2019

Background An assessment of the Sterile Insect Technique (SIT) as a complementary malaria vector control tool, is at an advanced stage in South Africa. The technique involves the release of laboratory-reared sterilized male mosquitoes of the major malaria vector Anopheles arabiensis, raising social, ethical and regulatory concerns. Therefore, its implementation largely depends on community participation and acceptance. Against this background, it is critical that robust and effective community strategies are developed. This study describes the development of a cultural song to engage the community and increase awareness on SIT and malaria control in KwaZulu-Natal, South Africa. Methods An exploratory concurrent mixed-methods study was conducted to get opinions about the effectiveness of a cultural song developed to engage communities and increase acceptability of the SIT technology. Two self-administered surveys (expert and community) were conducted. Additionally, more in depth opinions of the song and its effectiveness in conveying the intended information were investigated through three community dialogue sessions with community members in the study area. Results A total of 40 experts and 54 community members participated in the survey. Four themes were identified in relation to the appropriateness and effectiveness of the song, with a fifth theme focused on recommendations for adaptations. Overall, the song was well received with the audience finding it entertaining and informative. Responses to unstructured questions posed after the song showed an increase in the knowledge on malaria transmission and SIT technology. In particular, the explanation that male mosquitoes do not bite allayed anxiety and fears about the SIT technology. Conclusion The song was deemed both culturally appropriate and informative in engaging community members about the SIT technology. It proved useful in promoting health messages and conveying SIT technology as a complementary malaria vector control tool. With minor adaptations, the song has potential as an area-wide community engagement tool in areas targeted for sterile male releases.