Danilo O. Carvalho

Suppression of a Field Population of Aedes aegypti in Brazil by Sustained Release of Transgenic Male Mosquitoes

The increasing burden of dengue, and the relative failure of traditional vector control programs highlight the need to develop new control methods. SIT using self-limiting genetic technology is one such promising method. A self-limiting strain of Aedes aegypti, OX513A, has already reached the stage of field evaluation. Sustained releases of OX513A Ae. aegypti males led to 80% suppression of a target wild Ae. aegypti population in the Cayman Islands in 2010. Here we describe sustained series of field releases of OX513A Ae. aegypti males in a suburb of Juazeiro, Bahia, Brazil. This study spanned over a year and reduced the local Ae. aegypti population by 95% (95% CI: 92.2%-97.5%) based on adult trap data and 81% (95% CI: 74.9-85.2%) based on ovitrap indices compared to the adjacent no-release control area. The mating competitiveness of the released males (0.031; 95% CI: 0.025-0.036) was similar to that estimated in the Cayman trials (0.059; 95% CI: 0.011 – 0.210), indicating that environmental and target-strain differences had little impact on the mating success of the OX513A males. We conclude that sustained release of OX513A males may be an effective and widely useful method for suppression of the key dengue vector Ae. aegypti. The observed level of suppression would likely be sufficient to prevent dengue epidemics in the locality tested and other areas with similar or lower transmission.

Mass production of genetically modified Aedes aegypti for field releases in Brazil.

New techniques and methods are being sought to try to win the battle against mosquitoes. Recent advances in molecular techniques have led to the development of new and innovative methods of mosquito control based around the Sterile Insect Technique (SIT)1-3. A control method known as RIDL (Release of Insects carrying a Dominant Lethal)4, is based around SIT, but uses genetic methods to remove the need for radiation-sterilization5-8. A RIDL strain of Ae. aegypti was successfully tested in the field in Grand Cayman9,10; further field use is planned or in progress in other countries around the world. Mass rearing of insects has been established in several insect species and to levels of billions a week. However, in mosquitoes, rearing has generally been performed on a much smaller scale, with most large scale rearing being performed in the 1970s and 80s. For a RIDL program it is desirable to release as few females as possible as they bite and transmit disease. In a mass rearing program there are several stages to produce the males to be released: egg production, rearing eggs until pupation, and then sorting males from females before release. These males are then used for a RIDL control program, released as either pupae or adults11,12. To suppress a mosquito population using RIDL a large number of high quality male adults need to be reared13,14. The following describes the methods for the mass rearing of OX513A, a RIDL strain of Ae. aegypti 8, for release and covers the techniques required for the production of eggs and mass rearing RIDL males for a control program.

Aedes aegypti Control Strategies in Brazil: Incorporation of New Technologies to Overcome the Persistence of Dengue Epidemics

Dengue is considered to be the most important mosquito-borne viral disease in the world. The Aedes aegypti mosquito, its vector, is highly anthropophilic and is very well adapted to urban environments. Although several vaccine candidates are in advanced stages of development no licensed dengue vaccine is yet available. As a result, controlling the spread of dengue still requires that mosquitoes be targeted directly. We review the current methods of dengue vector control focusing on recent technical advances. We first examine the history of Brazil’s National Dengue Control Plan in effect since 2002, and we describe its establishment and operation. With the persistent recurrence of dengue epidemics, current strategies should be reassessed to bring to the forefront a discussion of the possible implementation of new technologies in Brazil’s mosquito control program.

Two step male release strategy using transgenic mosquito lines to control transmission of vector-borne diseases.

Mosquitoes are responsible for the transmission of pathogens that cause devastating human diseases such as malaria and dengue. The current increase in mean global temperature and changing sea level interfere with precipitation frequency and some other climatic conditions which, in general, influence the rate of development of insects and etiologic agents causing acceleration as the temperature rises. The most common strategy employed to combat target mosquito species is the Integrated Vector Management (IVM), which comprises the use of multiple activities and various approaches to preventing the spread of a vector in infested areas. IVM programmes are becoming ineffective; and the global scenario is threatening, requiring new interventions for vector control and surveillance. Not surprisingly, there is a growing need to find alternative methods to combat the mosquito vectors. The possibility of using transgenic mosquitoes to fight against those diseases has been discussed over the last two decades and this use of transgenic lines to suppress populations or to replace them is still under investigation through field and laboratory trials. As an alternative, the available transgenic strategies could be improved by coupling suppression and substitution strategies. The idea is to first release a suppression line to significantly reduce the wild population, and once the first objective is reached a second release using a substitution line could be then performed. Examples of targeting this approach against vectors of malaria and dengue are discussed.

Dispersal of Engineered Male Aedes aegypti Mosquitoes

Background Aedes aegypti, the principal vector of dengue fever, have been genetically engineered for use in a sterile insect control programme. To improve our understanding of the dispersal ecology of mosquitoes and to inform appropriate release strategies of ‘genetically sterile’ male Aedes aegypti detailed knowledge of the dispersal ability of the released insects is needed. Methodology/Principal Findings The dispersal ability of released ‘genetically sterile’ male Aedes aegypti at a field site in Brazil has been estimated. Dispersal kernels embedded within a generalized linear model framework were used to analyse data collected from three large scale mark release recapture studies. The methodology has been applied to previously published dispersal data to compare the dispersal ability of ‘genetically sterile’ male Aedes aegypti in contrasting environments. We parameterised dispersal kernels and estimated the mean distance travelled for insects in Brazil: 52.8m (95% CI: 49.9m, 56.8m) and Malaysia: 58.0m (95% CI: 51.1m, 71.0m). Conclusions/Significance Our results provide specific, detailed estimates of the dispersal characteristics of released ‘genetically sterile’ male Aedes aegypti in the field. The comparative analysis indicates that despite differing environments and recapture rates, key features of the insects’ dispersal kernels are conserved across the two studies. The results can be used to inform both risk assessments and release programmes using ‘genetically sterile’ male Aedes aegypti.

Endogenously-expressed NH2-terminus of circumsporozoite protein interferes with sporozoite invasion of mosquito salivary glands

BackgroundThe circumsporozoite protein is the most abundant polypeptide expressed by sporozoites, the malaria parasite stage capable of infecting humans. Sporozoite invasion of mosquito salivary glands prior to transmission is likely mediated by a receptor/ligand-like interaction of the parasites with the target tissues, and the amino (NH2)-terminal portion of CSP is involved in this interaction but not the TSR region on the carboxyl (C)-terminus. Peptides based on the NH2-terminal domain could compete with the parasites for the salivary gland receptors and thus inhibit penetration.MethodsPeptides based on the NH2-terminus and TSR domains of the CSP from avian or human malaria parasites, Plasmodium gallinaceum and Plasmodium falciparum, respectively, were expressed endogenously in mosquito haemolymph using a transient (Sindbis virus-mediated) or stable (piggyBac-mediated transgenesis) system.ResultsTransient endogenous expression of partial NH2-terminus peptide from P. falciparum CSP in P. gallinaceum-infected Aedes aegypti resulted in a reduced number of sporozoites in the salivary glands. When a transgenic approach was used to express a partial CSP NH2-terminal domain from P. gallinaceum the number of sporozoites in the salivary glands did not show a difference when compared to controls. However, a significant difference could be observed when mosquitoes with a lower infection were analysed. The same result could not be observed with mosquitoes endogenously expressing peptides based on the TSR domain from either P. gallinaceum or P. falciparum.ConclusionThese results support the conclusion that CSP partial NH2-terminal domain can be endogenously expressed to promote a competition for the receptor used by sporozoites to invade salivary glands, and they could be used to block this interaction and reduce parasite transmission. The same effect cannot be obtained with peptides based on the TSR domain.

Implementation of the Artificial Feeders in Hematophagous Arthropod Research Cooperates to the Vertebrate Animal Use Replacement, Reduction and Refinement (3Rs) Principle

Vector-borne diseases are transmitted to humans by hematophagous arthropods and these blood-sucking organisms are target to researches worldwide. The laboratory colonization of these species is an important factor in the development of innovative strategies to control these vectors. However, this maintenance requires blood to make these invertebrates able to complete their life cycle. Although live vertebrate animals are frequently used for this feeding procedure, artificial feeders are available as potential alternatives to replace the use of live animals in some situations, especially in vector colony maintenance. The aim of this commentary is to discuss the use of artificial feeding methods concerning the 3Rs principle application. The scientific community focused on vector-borne diseases studies needs to strongly consider these artificial feeding options as a bioethical alternative to maintain blood-feeding arthropods in laboratory.

Effect of interruption of over-flooding releases of transgenic mosquitoes over wild population of Aedes aegypti: two case studies in Brazil

The number of mosquito populations resistant to insecticides is increasing along with the reemerging of vector‐borne diseases. New technologies are under evaluation to complement the strategies used against these mosquitoes. Transgenic mosquitoes are one approach that some countries are considering and they are being evaluated to control the wild population. Although they have achieved success in population suppression of Aedes aegypti (L.) (Diptera: Culicidae), these studies have not demonstrated what the outcomes are when releases are interrupted (ceased). In this study, after demonstrating suppression of Ae. aegypti using transgenic technology, changes in the spatial distribution of the infestation and the abundance of the vector Ae. aegypti were assessed in the post‐release period, along with fluctuation of transgenic mosquitoes in two areas of Brazil. In both pilot trials, there was an average suppression of ca. 70% of the wild population due to the release of transgenic males compared to the pre‐release period. In Juazeiro (Mandacaru), in the post‐release phase, the number of eggs per trap ranged between 0.06 and 14.41 (mean ± SE = 4.44 ± 0.44), and the ovitrap index (OI = number of ovitraps with eggs/total number of ovitraps recovered) ranged from 0.01 to 0.43 (0.13 ± 0.01). In Jacobina (Pedra Branca), during the post‐release phase, the number of eggs per trap ranged between 1 and 7.2 (1.72 ± 0.72), and the OI ranged from 1 to 0.83 (0.095 ± 0.032). The mosquito population in Juazeiro (Mandacaru) remained suppressed for 17 weeks after the release interruption, whereas in Jacobina (Pedra Branca) suppression lasted 32 weeks. In Juazeiro, transgenic larvae were detected up to 5 months after the interruption of the over‐flooding releases of transgenic males. In Jacobina, they were found up to 2 months after the release interruption. The number of eggs collected increased 4–5 months after the release interruption, which indicated that the Ae. aegypti population had been re‐established after the interruption of releases. The results demonstrate that the technique requires a continuous release in the treated areas, and after suppression, the release rate can be decreased and used as a barrier against external migration.

Mosquito pornoscopy: Observation and interruption of Aedes aegypti copulation to determine female polyandric event and mixed progeny

Ades aegypti is the most important arbovirus vector in the world, and new strategies are under evaluation. Biological studies mentioning the occurrence of a second mate in Aedes aegypti can interfere with vector control program planning, which involves male mosquito release technique. This study presents different experiments to show the occurrence of mixed progeny. Mixed male crosses (using a combination of different type of males in confinement with virgin females) showed no polyandric female. Individual crosses with male substitution in every gonotrophic cycle also did not show any polyandric female. Individual crosses with a 20 minutes interval, with subsequent male change, showed that only a few females presented mixed offspring. The copulation breach in three different moments, group A with full coitus length, group B the coitus was interrupted in 5–7 seconds after the start; and group C, which the copulation was interrupted 3 seconds after started. In summary, group A showed a majority of unique progeny from the first male; group B showed the higher frequency of mixed offspring and group C with the majority of the crosses belonging to the second male. To conclude, the occurrence of a viable second mate and mixed offspring is only possible when the copulation is interrupted; otherwise, the first mate is responsible for mixed progeny.

Oviplate: A Convenient and Space-Saving Method to Perform Individual Oviposition Assays in Aedes aegypti

Aedes aegypti is the principal vector of the urban arboviruses and the blood ingestion is important to produce the eggs in this species. To analyze the egg production in Ae. aegypti, researchers frequently use small cages or Drosophila vials to collect eggs from gravid females. Although it is affordable, the setup is time- and space-consuming, mainly when many mosquitoes need to be individually analyzed. This study presents an easy, cheap, and space-saving method to perform individual oviposition assays in Ae. aegypti using cell culture plates. This new method to access fecundity rate was named “oviplate”. The oviplates are setup with 12- or 24-well plates, distilled water and filter paper and they are 78 to 88% cheaper than the traditional Drosophila vial assay, respectively. Furthermore, to allocate 72 vitellogenic females in an insectary using Drosophila vial is necessary 4100 cm3 against 1400 cm3 and 700 cm3 when using 12- and 24-well plates, respectively. No statistical differences were found between the number of eggs laid in Drosophila vials and the oviplates, validating the method. The oviplate method is an affordable, and time- and space-efficient device, and it is simpler to perform individual fecundity analyses in Ae. aegypti.