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Digital holographic reconstruction of diffraction patterns recorded using large aperture parabolic mirrors in a Z-configuration provide accurate, highly detailed, video-rate 3D tracking of mosquito flight in a 250×250×1200mm chamber.

Genetic strategies for large scale pest or vector control using modified insects are not yet operational in Africa, and currently rely on import of the modified strains to begin preliminary, contained studies. Early involvement of research teams from participating countries is crucial to evaluate candidate field interventions. Following the recommended phased approach for novel strategies, evaluation should begin with studies in containment facilities. Experiences to prepare facilities and build international teams for research on transgenic mosquitoes revealed some important organizing themes underlying the concept of “facilities readiness,” or the point at which studies in containment may proceed, in sub-Saharan African settings. First, “compliance” for research with novel or non-native living organisms was defined as the fulfillment of all legislative and regulatory requirements. This is not limited to regulations regarding use of transgenic organisms. Second, the concept of “colony utility” was related to the characteristics of laboratory colonies being produced so that results of studies may be validated across time, sites, and strains or technologies; so that the appropriate candidate strains are moved forward toward field studies. Third, the importance of achieving “defensible science” was recognized, including that study conclusions can be traced back to evidence, covering the concerns of various stakeholders over the long term. This, combined with good stewardship of resources and appropriate funding, covers a diverse set of criteria for declaring when “facilities readiness” has been attained. It is proposed that, despite the additional demands on time and resources, only with the balance of and rigorous achievement of each of these organizing themes can collaborative research into novel strategies in vector or pest control reliably progress past initial containment studies.

Transgenic mosquitoes are being developed as novel components of area-wide approaches to vector-borne disease control. Best practice is to develop these in phases, beginning with laboratory studies, before moving to field testing and inclusion in control programs, to ensure safety and prevent costly field testing of unsuitable strains. The process of identifying and developing good candidate strains requires maintenance of transgenic colonies over many generations in containment facilities. By working in disease endemic countries with target vector populations, laboratory strains may be developed and selected for properties that will enhance intended control efficacy in the next phase, while avoiding traits that introduce unnecessary risks. Candidate strains aiming toward field use must consistently achieve established performance criteria, throughout the process of scaling up from small study colonies to production of sufficient numbers for field testing and possible open release. Maintenance of a consistent quality can be demonstrated by a set of insect quality and insectary operating indicators, measured over time at predetermined intervals. These indicators: inform comparability of studies using various candidate strains at different times and locations; provide evidence of conformity relevant to compliance with terms of approval for regulated use; and can be used to validate some assumptions related to risk assessments covering the contained phase and for release into the environment.

Background: Malaria still remains a serious health burden in developing countries, causing more than 1 million deaths annually. Given the lack of an effective vaccine against its major etiological agent, Plasmodium falciparum, and the growing resistance of this parasite to the currently available drugs repertoire and of Anopheles mosquitoes to insecticides, the development of innovative control measures is an imperative to reduce malaria transmission. Paratransgenesis, the modification of symbiotic organisms to deliver anti-pathogen effector molecules, represents a novel strategy against Plasmodium development in mosquito vectors, showing the potential to reduce parasite development. However, the field application of laboratory-based evidence of paratransgenesis imposes the use of more realistic confined semi-field environments.Methods: Large cages were used to evaluate the ability of bacteria of the genus Asaia expressing green fluorescent protein (Asaia gfp), to diffuse in Anopheles stephensi and Anopheles gambiae target mosquito populations. Asaia gfp was introduced in large cages through the release of paratransgenic males or by sugar feeding stations. Recombinant bacteria transmission was directly detected by fluorescent microscopy, and further assessed by molecular analysis.Results: Here we show the first known trial in semi-field condition on paratransgenic anophelines. Modified bacteria were able to spread at high rate in different populations of An. stephensi and An. gambiae, dominant malaria vectors, exploring horizontal ways and successfully colonising mosquito midguts. Moreover, in An. gambiae, vertical and trans-stadial diffusion mechanisms were demonstrated.Conclusions: Our results demonstrate the considerable ability of modified Asaia to colonise different populations of malaria vectors, including pecies where its association is not primary, in large environments. The data support the potential to employ transgenic Asaia as a tool for malaria control, disclosing promising perspective for its field application with suitable effector molecules.

The persistence of transgenes in the environment is a consideration in risk assessments of transgenic organisms. Combining mathematical models that predict the frequency of transgenes and experimental demonstrations can validate the model predictions, or can detect significant biological deviations that were neither apparent nor included as model parameters. In order to assess the correlation between predictions and observations, models were constructed to estimate the frequency of a transgene causing male sexual sterility in simulated populations of a malaria mosquito Anopheles gambiae that were seeded with transgenic females at various proportions. Concurrently, overlapping-generation laboratory populations similar to those being modeled were initialized with various starting transgene proportions, and the subsequent proportions of transgenic individuals in populations were determined weekly until the transgene disappeared. The specific transgene being tested contained a homing endonuclease gene expressed in testes, I-PpoI, that cleaves the ribosomal DNA and results in complete male sexual sterility with no effect on female fertility. The transgene was observed to disappear more rapidly than the model predicted in all cases. The period before ovipositions that contained no transgenic progeny ranged from as little as three weeks after cage initiation to as long as 11 weeks.

Development of new genetic approaches to either interfere with the ability of mosquitoes to transmit dengue virus or to reduce vector population density requires progressive evaluation from the laboratory to contained field trials, before open field release. Trials in contained outdoor facilities are an important part of this process because they can be used to evaluate the effectiveness and reliability of modified strains in settings that include natural environmental variations without releasing mosquitoes into the open field. We describe a simple and cost-effective semi-field system designed to study Aedes aegypti carrying a dominant lethal gene (fsRIDL) in semi-field conditions. We provide a protocol for establishing, maintaining, and monitoring stable Ae. aegypti population densities inside field cages.

Knowledge of the frequency of contact between a mosquito species and its different hosts is essential to understand the role of each vector species in the transmission of diseases to humans and/or animals. However, no data are so far available on the feeding habits of Aedes albopictus in Italy or in other recently colonized temperate regions of Europe, due to difficulties in collecting blood-fed females of this diurnal and exophilic species. We analyzed Ae. albopictus host-feeding patterns in two urban and two rural sites within the area of Rome (Italy). Ae. albopictus was collected using sticky-traps and the blood-meal origin of 303 females was determined by direct dot-ELISA. The blood-fed sample, although representing only 4% of the total Ae. albopictus collected, demonstrates the useful application of sticky-trap in studying the feeding behavior of the species. The human blood index was significantly different among sites, ranging from 79-96% in urban sites to 23-55% in rural sites, where horses and bovines represented the most bitten hosts. The results obtained confirm the plastic feeding behavior shown by Ae. albopictus in its original range of distribution and highlights the high potential of this species as a vector of human pathogens in urban areas of Italy, where both humans and the mosquito itself may reach very high densities.

The patchy geographical distributions of classic Kaposi's sarcoma (KS) and human herpesvirus type 8 (HHV-8), better known as Kaposi's sarcoma-associated herpesvirus (KSHV) remain unexplained. It has been proposed that certain species of bloodsucking insects (‘promoter arthropods') promote the reactivation of HHV-8/KSHV and facilitate both HHV-8/KSHV transmission and KS development. This hypothesis was tested by sampling the presence and density of human-biting Diptera with CDC light traps in two areas of Sardinia with contrasting incidence rates of classic KS. In total, 11 030 specimens (99.9% sandflies and 0.1% mosquitoes) belonging to 10 species were collected from 40 rural sites. Five of these species are considered to be possible promoter arthropods because of the irritation their bites cause: Phlebotomus perniciosus Newstead; Phlebotomus perfiliewi Parrot (Diptera: Psychodidae); Aedes berlandi Seguy; Culiseta annulata (Schrank) and Culex theileri Theobald (Diptera: Culicidae). Five species are probable ‘non-promoters' because their bites are not particularly irritating: Culiseta longiareolata (Macquart); Culex pipiens s.l.; Anopheles algeriensis Theobald; Anopheles maculipennis s.l., and Anopheles plumbeus Stephens. A significant correlation was found between the geographical distribution of promoter arthropods and incidence rates of KS (Spearman's r = 0.59,P < 0.01). Promoter arthropods were more likely to be caught in areas with cutaneous leishmaniasis and a past high prevalence of malaria, and in areas of limestone, acid volcanic soil and cereal cultivation. The study supports the association between promoter arthropods and classic KS, which may explain the geographic variability of KS and HHV-8/KSHV, and highlights the links with a number of variables previously associated with the incidence of KS.

Development of new operational techniques for collection and monitoring of adult Stegomyia mosquitoes is considered a pressing need for surveillance and prevention of arboviruses. Here we report the results from a trial carried out in 2 dengue-endemic villages in Thailand to compare the ability to collect Aedes adults of a sticky trap versus a CDC backpack aspirator, which has been used routinely at the study area for entomological/epidemiological surveys. Our comparison was based on a comparable sampling effort required to carry out collections with 2 approaches. Over 19,000 specimens were collected, approximately 90% of which were Culex spp. Sticky traps collected significantly more Aedes aegypti and Aedes albopictus females than did backpack aspirators when located outdoors. The percentage of positive sticky-trap catches was double for Ae. aegypti and almost 20 times higher for Ae. albopictus. Operational benefits of the sticky trap are discussed within the context of the results obtained.

Lysozymes are important regulators of the immune system in most organisms. These enzymes can show a direct bactericidal activity or upregulate the signaling cascades that result in the production of antimicrobial peptides. In mosquitoes and particularly in the malaria-vector species of the genus Anopheles, lysozymes are present in different tissues and developmental stage expression profiles and are involved in the innate immunity and digestion of bacteria. In An. gambiae, lysozyme C1 protects the oocysts from melanization, thus contributing to an increased prevalence and intensity of Plasmodium berghei infection. The role and importance of lysozymes in the mosquito and in the regulation of oocysts development and the mechanism of action are still unclear.

Policy Platform A Regulatory Structure for Working with Genetically Modified Mosquitoes: Lessons from Mexico Janine M. Ramsey 1 , J. Guillermo Bond 1 , Maria Elena Macotela 1 , Luca Facchinelli 2,3 , Laura Valerio 2,4 , David M. Brown 5 , Thomas W. Scott 2 , Anthony A. James 5,6 * 1 Centro Regional de Investigacio´n en Salud Pu´blica, Instituto Nacional de Salud Pu´blica, Tapachula, Chiapas, Me´xico, 2 Department of Entomology, University of California, Davis, California, United States of America, 3 Department of Experimental Medicine, Functional Genomics Center, University of Perugia, Perugia, Italy, 4 Pasteur Institute–Cenci Bolognetti Foundation, University of Rome Sapienza, Rome, Italy, 5 Department of Microbiology and Molecular Genetics, University of California, Irvine, California, United States of America, 6 Department of Molecular Biology and Biochemistry, University of California, Irvine, California, United States of America Introduction Sustainable and effective control of dengue is hampered due to a number of factors, including the lack of evidence- based, locally relevant interventions; insuf- ficient information regarding key compo- nents of virus transmission and vector ecology; failure to implement precise and efficient surveillance systems; inefficient healthcare systems; ineffective health pro- motion and outreach resulting in lack of community dialogue and participation; and a paucity of efficient diagnostic strategies and clinical attention [1]. In- creased research efforts in response to the complexity of this problem have focused on the development of novel technologies that would enhance existing tools for vector-borne disease prevention [2–4]. Genetic strategies to reduce or replace mosquito populations and thereby inter- rupt transmission of dengue viruses are among the new approaches being consid- ered [5–7]. Many of these approaches take advantage of molecular genetic tools to engineer traits that cause lethal pheno- types or confer resistance to the pathogen in the mosquito. Genetic strategies are being advanced through a series of overlapping domains that inform the decision making on feasibility, safety, efficacy, and acceptabil- ity. Although the need to focus on science- based regulation using a risk-assessment framework is gaining support [8], there has been a relative lack of attention on broader community regulations that are explicitly or indirectly required to bring a genetics-based product to the field [3,9– 11]. An evidence-based approach would facilitate the integration, efficacy, and acceptability of policy for an intervention strategy. We addressed the regulatory challenges associated with testing a strain of Aedes aegypti engineered to result in population suppression in contained field trials in southwestern Mexico [12]. This large research effort (designated hereafter as the ‘‘Project’’) combined elements of scientific and social discovery and devel- opment as the basis for moving a new technology from the laboratory to the field. Unlike the rollout of other public- health products such as drugs, vaccines, and insecticides, no pipelines exist to move candidate genetically modified mosquitoes (GMMs) from the laboratory through safety and efficacy trials to field deploy- ment. This lack of a preexisting structure made it necessary for the scientists in the Project to play critical, unbiased roles in formulating the product development pathway. The challenge offered a unique opportunity for potential end users and beneficiaries of the technology to be involved from the beginning in product discovery and development. This ap- proach ensures that requirements for safety and efficacy are included as design features engineered into the modified mosquito strains [13]. It is incumbent on the researchers to identify gaps and assist in development of regulatory norms that should be applied to the products they create. These norms include not only statutory regulations but also a broader regulatory environment that addresses the needs and concerns of all communities in which the product will be applied. We describe here the regulatory and social structures used for obtaining approvals in Mexico. This review of our approach is intended to stimulate analysis and dia- logue that will help refine regulatory practices of genetic-based strategies for vector-borne disease control. Regulatory Domains for the Discovery and Development of Genetically Modified Mosquitoes An initial challenge of the Project was identifying relevant communities [14]. No consensus existed as to what comprises a relevant community to engage for a GMMs research project, nor were there any widely accepted methods for identify- ing their members. We adopted a defini- tion (modified from [15]) in which the community consists of all those individuals who share the identified risks and/or will benefit from the outcome of the proposed research project. In this context, the community coalesces as a result of the project and evolves continuously as it progresses through conceptualization, dis- cussion, and implementation [16]. The community is formed ultimately by those individuals, groups, organizations, and agencies that have legitimate interest in the research, and therefore they must be engaged in an effective and timely man- ner. Successfully moving a novel technology from the laboratory to practical application Citation: Ramsey JM, Bond JG, Macotela ME, Facchinelli L, Valerio L, et al. (2014) A Regulatory Structure for Working with Genetically Modified Mosquitoes: Lessons from Mexico. PLoS Negl Trop Dis 8(3): e2623. doi:10.1371/journal.pntd.0002623 Editor: Jesus G. Valenzuela, National Institute of Allergy and Infectious Diseases, United States of America Published March 13, 2014 Copyright: s 2014 Ramsey et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Research and Policy for Infectious Disease Dynamics program of the Science and Technology Directory, Department of Homeland Security; Fogarty International Center, National Institutes of Health; Pasteur Institute – Cenci Bolognetti Foundation; Foundation for the National Institutes of Health through the Grand Challenges in Global Health (GCGH) initiative. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors declare no conflict of interest. * E-mail: aajames@uci.edu PLOS Neglected Tropical Diseases | www.plosntds.org March 2014 | Volume 8 | Issue 3 | e2623

Transgenic strains of Aedes aegypti have been engineered to help control transmission of dengue virus. Although resources have been invested in developing the strains, we lack data on the ecology of mosquitoes that could impact the success of this approach. Although studies of intra-specific competition have been conducted using Ae. aegypti larvae, none of these studies examine mixed age cohorts at densities that occur in the field, with natural nutrient levels. Experiments were conducted in Mexico to determine the impact of direct and delayed density dependence on Ae. aegypti populations. Natural water, food, and larval densities were used to estimate the impacts of density dependence on larval survival, development, and adult body size. Direct and delayed density-dependent factors had a significant impact on larval survival, larval development, and adult body size. These results indicate that control methods attempting to reduce mosquito populations may be counteracted by density-dependent population regulation.

We demonstrate the utility of models as aids in the design and development of experiments aimed at measuring the effects of proposed vector population control strategies. We describe the exploration of a stochastic, age-structured model that simulates field cage experiments that test the ability of a female-killing strain of the mosquito Aedes aegypti (L.) to suppress a wild-type population. Model output predicts that choices of release ratio and population size can impact mean extinction time and variability in extinction time among experiments. We find that unless fitness costs are >80% they will not be detectable in experiments with high release ratios. At lower release ratios, the predicted length of the experiment increases significantly for fitness costs >20%. Experiments with small populations may more accurately reflect field conditions, but extinction can occur even in the absence of a functional female-killing construct because of stochastic effects. We illustrate how the model can be used to explore experimental designs that aim to study the impact of density dependence and immigration; predictions indicate that cage population eradication may not always be obtainable in an operationally realistic time frame. We propose a method to predict the extinction time of a cage population based on the rate of population reduction with the goal of shortening the duration of the experiment. We discuss the model as a tool for exploring and assessing the utility of a wider range of scenarios than would be feasible to test experimentally because of financial and temporal restraints.

Background A genetically-engineered strain of the dengue mosquito vector Aedes aegypti, designated OX3604C, was evaluated in large outdoor cage trials for its potential to improve dengue prevention efforts by inducing population suppression. OX3604C is engineered with a repressible genetic construct that causes a female-specific flightless phenotype. Wild-type females that mate with homozygous OX3604C males will not produce reproductive female offspring. Weekly introductions of OX3604C males eliminated all three targeted Ae. aegypti populations after 10–20 weeks in a previous laboratory cage experiment. As part of the phased, progressive evaluation of this technology, we carried out an assessment in large outdoor field enclosures in dengue endemic southern Mexico. Methodology/Principal Findings OX3604C males were introduced weekly into field cages containing stable target populations, initially at 10∶1 ratios. Statistically significant target population decreases were detected in 4 of 5 treatment cages after 17 weeks, but none of the treatment populations were eliminated. Mating competitiveness experiments, carried out to explore the discrepancy between lab and field cage results revealed a maximum mating disadvantage of up 59.1% for OX3604C males, which accounted for a significant part of the 97% fitness cost predicted by a mathematical model to be necessary to produce the field cage results. Conclusions/Significance Our results indicate that OX3604C may not be effective in large-scale releases. A strain with the same transgene that is not encumbered by a large mating disadvantage, however, could have improved prospects for dengue prevention. Insights from large outdoor cage experiments may provide an important part of the progressive, stepwise evaluation of genetically-engineered mosquitoes., Author Summary The absence of a commercially-available dengue vaccine or anti-viral drug makes control of Aedes aegypti, the principal dengue mosquito vector, the only available method to prevent this disease. Sustained, effective application of vector control, however, has been difficult and this led to the call for innovative strategies, including genetic approaches. Here, the authors investigated the ability of a genetically-engineered strain of Ae. aegypti to eliminate wild mosquito populations in large outdoor cages. Females of the engineered strain cannot fly and, therefore, cannot mate or take blood meals necessary to lay viable eggs. Wild females that mate with genetically-engineered males, therefore, will not produce reproductive female offspring. In this study, although population reductions were detected in 4 of 5 field cages, none of the wild mosquito populations were eliminated. A mating disadvantage for genetically-engineered males appeared to account for a significant part of their fitness disadvantage. Results suggest that this specific strain may not be effective in a large-scale release and that new strains with the same or similar transgene, but improved mating performance, may be more effective for preventing dengue. Results also indicate that large outdoor cage experiments may provide valuable insights into the progressive, stepwise assessment of genetically-engineered mosquitoes.

Most Aedes aegypti dispersal studies have focused on females because of their central role in dengue virus transmission. Only a few mark-release-recapture (MRR) studies provided insights into male Ae. aegypti dispersal. To fill this knowledge gap, we conducted five male Ae. aegypti MRR experiments in a coastal village in southern Mexico. Small and large male cohorts were marked with fluorescent dusts, released outside buildings, and recaptures were carried out by using backpack aspirators. Recapture rates ranged between 0.35% and 6.55% and median distance traveled was 12-166 meters. A statistically significant difference in median distance traveled with large males dispersing farther than small ones was detected only in one experiment (MRR5: U = 3.5, P < 0.01). Male dispersal data will be useful for constructing and estimating parameter values and validating models that will be used to plan the most effective release strategies for genetically modified male Ae. aegypti.

Female Aedes aegypti are assumed to be primarily monandrous (i.e., mate only once in their lifetime), but true estimates of mating frequency have not been determined outside the laboratory. To assess polyandry in Ae. aegypti with first-generation progeny from wild mosquitoes, stable isotope semen-labeled males ((15)N or (13)C) were allowed to mate with unlabeled females in semifield enclosures (22.5 m(3)) in a dengue-endemic area in southern Mexico. On average, 14% of females were positive for both labels, indicating that they received semen from more than one male. Our results provide evidence of a small but potentially significant rate of multiple mating within a 48-hour period and provide an approach for future open-field studies of polyandry in this species. Polyandry has implications for understanding mosquito ecology, evolution, and reproductive behavior as well as genetic strategies for mosquito control.

Collection methods currently used for large-scale sampling of adult Stegomyia mosquitoes (Diptera: Culicidae) present several operational limitations, which constitute major drawbacks to the epidemiological surveillance of arboviruses, the evaluation of the impact of control strategies, and the surveillance of the spreading of allochthonous species into non-endemic regions. Here, we describe a new sticky trap designed to capture adult container-breeding mosquitoes and to monitor their population dynamics. We tested the sampling properties of the sticky trap in Rome, Italy, where Aedes (Stegomyia) albopictus is common. The results of our observations, and the comparison between sticky trap catches and catches made with the standard oviposition trap, are presented. The sticky trap collected significantly larger numbers of Ae. albopictus females than any other Culicidae species representing > 90% of the total catches. A maximum of 83 An. albopictus females was collected in a single week. A high correlation (Pearson correlation coefficient r = 0.96) was found between the number of females and the number of eggs collected by the traps. The functional relationship between the number of eggs and the number of adult females was assessed by major axis regression fitted to log(1 + x)-transformed trap counts as y = 0.065 + 1.695x. Trap samples significantly departed from a random distribution; Taylor's power law was fitted to the trap samples to quantify the degree of aggregation in the catches, returning the equations s(2) = 2.401 m(1.325) for the sticky trap and s(2) = 13.068 m(1.441) for the ovitrap, with s(2) and m denoting the weekly catch variance and mean, respectively, indicating that eggs were significantly more aggregated than mosquitoes (P < 0.0001). Taylor's power law parameters were used to estimate the minimum number of sample units necessary to obtain sample estimates with a fixed degree of precision and sensitivity. For the range of densities encountered in our study area during the Ae. albopictus breeding season, the sticky trap was more precise and sensitive than the ovitrap. At low population densities (c. < 0.1 mosquito/trap), however, the ovitrap was more sensitive at detecting the presence of this species. Overall, our results indicate that our new model of sticky trap can be used to sample Ae. albopictus females in urban environments, and, possibly, other container-breeding Stegomyia mosquitoes (e.g. Aedes aegypti). The technical properties of the new trap are discussed with respect to its possible application in monitoring the population dynamics of container-breeding mosquitoes, in studying their bionomics, and in vector surveillance and, possibly, control.

The 'promoter-arthropod' hypothesis, which postulates that exposure to the bites of certain species of haematophagous arthropods is an environmental risk cofactor linked to human herpes virus 8 (HHV-8) and Kaposi's sarcoma, was investigated in the Po River valley, northern Italy. The presence and density of adult female mosquitoes (Diptera: Culicidae) was determined by CDC light trap catches in two adjacent districts, at variance with respect to Kaposi's sarcoma incidence and HHV-8 seroprevalence. A total of 3910 specimens belonging to 11 species was collected in 34 rural sites (six municipalities) representative of the two districts. Five of these species are considered to be possible 'promoters' because of the irritation their bites cause humans: Aedes vexans (Meigen) and Ae. caspius (Pallas) (87% of sampled promoters), Culex modestus Ficalbi, Culiseta annulata (Schrank) and Coquillettidia richiardii (Ficalbi). Six are probable 'non-promoters': Cx. pipiens s.l., Cx. martinii Medschid, Anopheles claviger (Meigen), An. maculipennis s.l., An. plumbeus Stephens and Uranotaenia unguiculata Edwards. The density of promoters by site was correlated with the incidence rates of Kaposi's sarcoma at the district level (Pearson's r = 0.33, P = 0.06) and at the municipal level (r = 0.50, P< 0.01). Similar correlations emerged for non-promoters (r = 0.48, P< 0.01 and r = 0.42, P = 0.01, respectively). The density of promoters was higher than that of non-promoters in sites with livestock (odds ratio, OR = 2.8, 95% CI 2.2-3.6) and in municipalities with Kaposi's sarcoma cases (OR = 2.5, 95% CI 1.7-3.5). The study provides additional evidence of the association between the density of some mosquito species and Kaposi's sarcoma.

Background Male Anopheles mosquitoes that swarm rely in part on features of the environment including visual stimuli to locate swarms. Swarming is believed to be the primary behaviour during which mating occurs in the field, but is not a common behaviour in the laboratory. Features that stimulate male Anopheles gambiae G3 strain swarming were created in novel large indoor cages. Methods The following visual features were tested in all combinations to determine which were important for swarm formation. Large cages and fading ceiling lights at dusk alone did not stimulate swarming while a dark foreground and contrasting illuminated background with a contrasting landmark stimulated and localized swarm formation during artificial twilight. Given the need to test transgenic strains in as natural a setting as possible, in this study it was investigated whether induced swarm behaviour and cage size would affect relative mating performance of wild-type and transgenic β2Ppo1 and β2Ppo2 A. gambiae sexually sterile males. Results Even using a mosquito colony that has been in laboratory culture for 39 years, swarming behaviour was induced by this novel arrangement. The presence of swarming stimuli was associated with an increase in insemination frequency from 74.3 to 97.7% in large cages. Transgenic males showed a lower competitiveness in large cages compared to small cages regardless of the presence of swarming stimuli. Conclusions The results of the present study are discussed in view of the progressive evaluation of genetically modified A. gambiae strains and the potential applications of reproducing swarms in controlled conditions to dissect the mating behaviour of this species and the mechanisms controlling it.