Prevention of a dengue outbreak via the large-scale deployment of Sterile Insect Technology in a Brazilian city: a prospective study.

Background: Dengue is a global problem that seems to be worsening, as hyper-urbanization associated with climate change has led to a significant increase in the abundance and geographical spread of its principal vector, the Aedes aegypti mosquito. Currently available solutions have not been able to stop the spread of dengue which shows the urgent need to implement alternative technologies as practical solutions. In a previous pilot trial, we demonstrated the efficacy and safety of the method 'Natural Vector Control' (NVC) in suppressing the Ae. aegypti vector population and in blocking the occurrence of an outbreak of dengue in the treated areas. Here, we expand the use of the NVC program in a large-scale 20 months intervention period in an entire city in southern Brazil. Methods: Sterile male mosquitoes were produced from locally sourced Ae. aegypti mosquitoes by using a treatment that includes double-stranded RNA and thiotepa. Weekly massive releases of sterile male mosquitoes were performed in predefined areas of Ortigueira city from November 2020 to July 2022. Mosquito monitoring was performed by using ovitraps during the entire intervention period. Dengue incidence data was obtained from the Brazilian National Disease Surveillance System. Findings: During the two epidemiological seasons, the intervention in Ortigueira resulted in up to 98.7% suppression of live progeny of field Ae. aegypti mosquitoes recorded over time. More importantly, when comparing the 2020 and 2022 dengue outbreaks that occurred in the region, the post-intervention dengue incidence in Ortigueira was 97% lower compared to the control cities. Interpretation: The NVC method was confirmed to be a safe and efficient way to suppress Ae. aegypti field populations and prevent the occurrence of a dengue outbreak. Importantly, it has been shown to be applicable in large-scale, real-world conditions. Funding: This study was funded by Klabin S/A and Forrest Innovations Ltd.

Novel Sterile Insect Technology Program Results in Suppression of a Field Mosquito Population and Subsequently to Reduced Incidence of Dengue.

BACKGROUND: There is a steady rise in the global incidence of Aedes-borne arbovirus disease. It has become urgent to develop alternative solutions for mosquito vector control. We developed a new method of sterilization of male mosquitoes with the goal to suppress a local Aedes aegypti population and to prevent the spread of dengue. METHODS: Sterile male mosquitoes were produced from a locally acquired Ae. aegypti colony by using a treatment that includes double-stranded RNA and thiotepa. A field study was conducted with sterile mosquito releases being performed on a weekly basis in predefined areas. There were 2 intervention periods (INT1 and INT2), with treatment and control areas reversed between INT1 and INT2. RESULTS: During INT1, releases in the treated area resulted in up to 91.4% reduction of live progeny of field Ae. aegypti mosquitoes recorded over time, while the control neighborhoods (no releases of sterile male mosquitoes) remained highly infested. The successful implementations of the program during INT1 and INT2 were associated with 15.9-fold and 13.7-fold lower incidences of dengue in the treated area compared to the control areas, respectively. CONCLUSIONS: Our data show the success of this new sterile insect technology-based program in preventing the spread of dengue.

Larval application of sodium channel homologous dsRNA restores pyrethroid insecticide susceptibility in a resistant adult mosquito population.

BACKGROUND: Mosquitoes host and pass on to humans a variety of disease-causing pathogens such as infectious viruses and other parasitic microorganisms. The emergence and spread of insecticide resistance is threatening the effectiveness of current control measures for common mosquito vector borne diseases, such as malaria, dengue and Zika. Therefore, the emerging resistance to the widely used pyrethroid insecticides is an alarming problem for public health. Herein we demonstrated the use of RNA interference (RNAi) to increase susceptibility of adult mosquitoes to a widely used pyrethroid insecticide. METHODS: Experiments were performed on a field-collected pyrethroid resistant strain of Ae. aegypti (Rio de Janeiro; RJ). Larvae from the resistant Ae. aegypti population were soaked with double-stranded RNAs (dsRNAs) that correspond either to voltage-gate sodium channel (VGSC), P-glycoprotein, or P450 detoxification genes and reared to adulthood. Adult mortality rates in the presence of various Deltamethrin pyrethroid concentrations were used to assess mosquito insecticide susceptibility. RESULTS: We characterized the RJ Ae. aegypti strain with regard to its level of resistance to a pyrethroid insecticide and found that it was approximately 6 times more resistant to Deltamethrin compared to the laboratory Rockefeller strain. The RJ strain displayed a higher frequency of Val1016Ile and Phe1534Cys substitutions of the VGSC gene. The resistant strain also displayed a higher basal expression level of VGSC compared to the Rockefeller strain. When dsRNA-treated mosquitoes were subjected to a standard pyrethroid contact bioassay, only dsRNA targeting VGSC increased the adult mortality of the pyrethroid resistant strain. The dsRNA treatment proved effective in increasing adult mosquito susceptibility over a range of pyrethroid concentrations and these results were associated with dsRNA-specific small interfering RNAs in treated adults, and the corresponding specific down regulation of VGSC gene expression level. Finally, we demonstrated that the efficiency of our approach was further improved by 'tiling' along the VGSC gene in order to identify the most potent dsRNA sequences. CONCLUSIONS: These results demonstrate that dsRNA applied to mosquito larvae retains its biological activity into adulthood. Thus, the RNAi system reported here could be a useful approach to control the widespread insecticide resistance in mosquitoes and other insect vectors of human diseases.

Effective gene silencing in a microsporidian parasite associated with honeybee (Apis mellifera) colony declines.

Honeybee colonies are vulnerable to parasites and pathogens ranging from viruses to vertebrates. An increasingly prevalent disease of managed honeybees is caused by the microsporidian Nosema ceranae. Microsporidia are basal fungi and obligate parasites with much-reduced genomic and cellular components. A recent genome-sequencing effort for N. ceranae indicated the presence of machinery for RNA silencing in this species, suggesting that RNA interference (RNAi) might be exploited to regulate Nosema gene expression within bee hosts. Here we used controlled laboratory experiments to show that double-stranded RNA homologous to specific N. ceranae ADP/ATP transporter genes can specifically and differentially silence transcripts encoding these proteins. This inhibition also affects Nosema levels and host physiology. Gene silencing could be mediated solely by Nosema or in concert with known systemic RNAi mechanisms in their bee hosts. These results are novel for the microsporidia and provide a possible avenue for controlling a disease agent implicated in severe honeybee colony losses. Moreover, since microsporidia are pathogenic in several known veterinary and human diseases, this advance may have broader applications in the future for disease control.

Large-scale field application of RNAi technology reducing Israeli acute paralysis virus disease in honey bees (Apis mellifera, Hymenoptera: Apidae).

The importance of honey bees to the world economy far surpasses their contribution in terms of honey production; they are responsible for up to 30% of the world's food production through pollination of crops. Since fall 2006, honey bees in the U.S. have faced a serious population decline, due in part to a phenomenon called Colony Collapse Disorder (CCD), which is a disease syndrome that is likely caused by several factors. Data from an initial study in which investigators compared pathogens in honey bees affected by CCD suggested a putative role for Israeli Acute Paralysis Virus, IAPV. This is a single stranded RNA virus with no DNA stage placed taxonomically within the family Dicistroviridae. Although subsequent studies have failed to find IAPV in all CCD diagnosed colonies, IAPV has been shown to cause honey bee mortality. RNA interference technology (RNAi) has been used successfully to silence endogenous insect (including honey bee) genes both by injection and feeding. Moreover, RNAi was shown to prevent bees from succumbing to infection from IAPV under laboratory conditions. In the current study IAPV specific homologous dsRNA was used in the field, under natural beekeeping conditions in order to prevent mortality and improve the overall health of bees infected with IAPV. This controlled study included a total of 160 honey bee hives in two discrete climates, seasons and geographical locations (Florida and Pennsylvania). To our knowledge, this is the first successful large-scale real world use of RNAi for disease control.

Associative olfactory learning of honeybees to differential rewards in multiple contexts--effect of odor component and mixture similarity.

Discrimination among differentially rewarding flowers allows honeybees to maximize their foraging efficiency. We studied how honeybees are able to relate to differential positive rewards when the odor representations are either structurally dissimilar, structurally similar (or form a substrate-product duo in planta), or form a binary mixture sharing a common constituent. Bees were tested both in conditioning of the proboscis-extension response (PER) and in a free-flying context. Our results point to honeybees using olfactory associative learning to differentiate between two positively rewarded odors. In PER, subjects discriminated best between dissimilar odors; they initially generalized between similar odors, but eventually learned to discriminate between them. The discrimination between mixtures sharing a common constituent remained poor. Likewise, the difference in visits to low- and high-rewarding flowers of free-flying bees was greater for dissimilar odors than for binary mixtures sharing a common constituent. Consequences of the operant conditioning nature of the free-flying context are discussed.