New contained semi-field cages are being developed and used to test novel vector control strategies of dengue and malaria vectors. We herein describe a new Quarantine Insectary Level-2 (QIC-2) laboratory and field cages (James Cook University Mosquito Research Facility Semi-Field System; MRF SFS) that are being used to measure the impact of the endosymbiont Wolbachia pipientis on populations of Aedes aegypti in Cairns Australia. The MRF consists of a single QIC-2 laboratory/insectary that connects through a central corridor to two identical QIC-2 semi-field cages. The semi-field cages are constructed of two layers of 0. 25 mm stainless steel wire mesh to prevent escape of mosquitoes and ingress of other insects. The cages are covered by an aluminum security mesh to prevent penetration of the cages by branches and other missiles in the advent of a tropical cyclone. Parts of the cage are protected from UV light and rainfall by 90% shade cloth and a vinyl cover. A wooden structure simulating the understory of a Queenslander-style house is also situated at one end of each cage. The remainder of the internal aspect of the cage is covered with mulch and potted plants to emulate a typical yard. An air conditioning system comprised of two external ACs that feed cooled, moistened air into the cage units. The air is released from the central ceiling beam from a long cloth tube that disperses the airflow and also prevents mosquitoes from escaping the cage via the AC system. Sensors located inside and outside the cage monitor ambient temperature and relative humidity, with AC controlled to match ambient conditions. Data loggers set in the cages and outside found a <2°C temperature difference. Additional security features include air curtains over exit doors, sticky traps to monitor for escaping mosquitoes between layers of the mesh, a lockable vestibule leading from the connecting corridor to the cage and from inside to outside of the insectary, and screened (0. 25 mm mesh) drains within the insectary and the cage. A set of standard operating procedures (SOP) has been developed to ensure that security is maintained and for enhanced surveillance for escaping mosquitoes on the JCU campus where the MRF is located. A cohort of male and female Aedes aegypti mosquitoes were released in the cage and sampled every 3–4 days to determine daily survival within the cage; log linear regression from BG-sentinel trapping collections produced an estimated daily survival of 0. 93 and 0. 78 for females and males, respectively. The MRF SFS allows us to test novel control strategies within a secure, contained environment. The air-conditioning system maintains conditions within the MRF cages comparable to outside ambient conditions. This cage provides a realistic transitional platform between the laboratory and the field in which to test novel control measures on quarantine level insects.
Dengue is the most abundant arboviral infection in the tropics, with 50–100 million cases and 5 million people at risk annually [1]. Currently, there is no available human vaccine, thus dengue prevention is limited to control strategies attacking the mosquito vectors Aedes aegypti and Aedes albopictus. Outside of community education programs and source reduction campaigns that seek to remove artificial containers that produce the vectors, most government programs rely upon insecticides to reduce vector populations. These methods are often inefficient, costly and ineffective. Furthermore, many populations of A. aegypti have developed physiological resistance to many pesticides, rendering them ineffective [2]. Thus, novel population control strategies are being developed to control vectors of dengue and other mosquito-borne diseases. Releases of genetically modified (GM) A. aegypti that are refractory to dengue infection and transmission, and the inundative release of sterile males are currently being developed to reduce populations of A. aegypti [3], [4]. Our research group is investigating the use of strains of the endosymbiotic bacteria Wolbachia pipientis to induce life-shortening and dengue virus interference in populations of A. aegypti. The Wolbachia infection is driven to fixation in populations of A. aegypti via a cytoplasmic incompatibility mechanism. The wMelPop strain is known to shorten the life span of A. aegypti reducing the number of individuals that survive long enough to transmit dengue [5], and can also interfere with the replication and transmission of several viruses in A. aegypti, including DENV-2 and chikungunya virus [6]. Entomopathogenic fungi are also being studied for their life-shortening impact on A. aegypti [7]. Novel control strategies require confirmation under field conditions before they can be deployed operationally. Furthermore, experiments involving population replacement methods involving GM and novel agents such as Wolbachia and fungi that are conducted out of the laboratory must be under tight containment to avoid accidental release. A cross-disciplinary scientific working group developed guidelines for testing of gene drive systems within secure flight cages [8]. These facilities, termed “semi-field system” (SFS) [9], typically consist of secure biocontainment laboratory for insect rearing, secure field cage for experimental release, and associated security features such as fencing, moats and pass-coded gates. Within the cages, experimental houses or huts simulating domestic premises to be tested are featured. This is especially important for A. aegypti, a mosquito that typically feeds on humans and harbours within houses and other human premises. Natural substrates of soil, grass and native vegetation are included. Natural larval habitat such as puddles for Anopheles malaria vectors and artificial containers for Aedes are included. Biocontainment structures typically include double-door atriums, air curtains, mosquito surveillance traps, double layers of insect-proof screening, screened water drains, etc. In the tropics, facilities must often be built to withstand heavy rain and strong winds, often to tropical storm, cyclone or hurricane strength. We describe a SFS that features a biocontainment level 2 laboratory/insectary that connects directly to 2 identical Quarantine Insectary Containment level 2 (QIC-2) semi-field cages. This new facility, the James Cook University Mosquito Research Facility (MRF), is currently being used to investigate the impact of wMelPop and wMel strains of Wolbachia infection on survival of A. aegypti, and the dynamics of its spread within a population of wild type A. aegypti. Each of the SFS cages contains the ground floor of a simulated Queenslander house and associated yard. Queenslander houses are typically timber houses set on concrete or wooden pillars, and are common throughout much of Queensland, Australia (http: //en. wikipedia. org/wiki/Queenslander_%28architecture%29). They are generally unscreened to maximise ventilation, and the ground floor is often not fully enclosed, allowing free access to mosquitoes. Dengue transmission is often most intense in suburbs dominated by these older types of housing [10]. We describe the security and containment features of the MRF, measure the environmental conditions inside and outside the cage and the impact of its climate control system, and also examine the survival of wild type A. aegypti within the cage. Finally, we provide standard operating procedures (SOP; File S1) designed to prevent escape of released mosquitoes.
Human ethics approval for use of human volunteers to blood feed colony (dengue free) A. aegypti was obtained (JCU Human Ethics H2250). Volunteers were examined for fever before each blood feeding, excluded if feverish, and could withdraw at anytime. Written consent was obtained from all staff involved in blood feeding. The MRF was constructed to provide a simulated Cairns urban environment, under QIC-2 containment levels (http: //www. daff. gov. au/aqis/import/general-info/qap/class7/quarantine_approved_criteria_qap_class_7. 2_quarantine_insectary_containment_level_2_qic2_facilities), for testing novel control strategies on A. aegypti. The MRF is built on 133 m2 of land on the Smithfield campus of James Cook University (16°48′58”S, 145°41′15”E) located ca. 15 km northwest of the city of Cairns, Queensland Australia. Cairns is located in the wet tropics of northern Queensland, and has a pronounced wet and dry monsoonal climate; the mean daily temperature ranges from 21°C in winter to 27°C in summer, and an average of 1992 mm of rain falls annually (Australian Bureau of Meteorology; http: //www. bom. gov. au/index. shtml). Cairns has a history of dengue outbreaks [10], [11], and A. aegypti are present in most urban areas. The campus building site was chosen as it is practical for researchers but, more importantly, it is situated within tropical rainforest and is isolated from urban areas of Cairns where A. aegypti is common. Thus, we think that any escaping A. aegypti are highly unlikely to breed with existing populations in the Cairns region. Construction on the MRF began in March 2008 and finished in January 2009. The cost of the facility in $AUS was $469,000 for the cage, $888,000 for the laboratory and $364,000 for the air conditioning system including controller. Total cost was $1,721,000; with Goods and Services Tax (10%) this was $1,893,000. Of this total, 55% was material costs, and 45% labor. The MRF design (Figure 1) allowed us to provide direct and secure access between the rearing laboratory and the SFS cages. Two cages were built so that treatment and control experiments could be conducted simultaneously. A service road connects to a loading bay located near the entry to the MRF laboratory. The screening of the cages reduced incoming light and thus potential solar gain. We measured light passing through the cage layers into the SFS cages using a Extech EasyView EA30 light meter (Extech Instruments Corporation, Waltham, MA 02451 U. S. A.) during mid day on clear conditions. We measured temperature and RH inside and outside each cage to test the ability of the shade cloth awning and AC system to maintain ambient conditions. Data loggers (Esis Hygrocon DS1923, Esis Pty Ltd, PO Box 450, Pennant Hills NSW 1715 AUSTRALIA) were set 24 cm above ground on a 8 L plastic bucket located within the Queenslander house and in the yard in the center of each cage, and run while the AC was on and off to investigate the impact of solar gain and the AC unit on conditions within the cages. Outside, 2 data loggers were set, one under a shaded tree ca. 1. 5 m off the ground (equivalent to 1. 5 m Stevenson screen height used by Bureau of Meteorology) and the other set on a upturned bucket in a shaded area adjacent to cage A. None of the data loggers were exposed to direct sunlight that could heat the unit and provide inaccurate temperature readings. Several systems are deployed at the MRF SFS to provide security against vandalism and to minimise the accidental release of insects. The cages are surrounded by 2 m high fencing topped with barbed wire to prevent access by animals and humans. Each cage has an auto-locking door that could only be opened once the entry door into the vestibule was closed. Before being opened, the entry door activated an air curtain above the cage side door that blew air downward over the entryway. The interior also had overlapping screens composed of fine polyester Tentex 72007 cloth (located on the vestibule side) that had a metal chain weight sewn into the bottom to ensure the screens securely overlapped. All doors entering the laboratory are auto-locking, and keys are only available to JCU staff working on the project. The doors have all been fitted with rubber seals. In total, there are 6 doors (3 from cage to insectary, and 3 from insectary to external) between each cage and the external exit of the MRF SFS. Within each vestibule entry into the cage, a BG-Sentinel trap (BGS, Biogents GmbH, Regensburg, Germany) [12] runs continuously and a sweepnet is provided for staff to capture any escaped mosquitoes. All drains within the cages have stainless steel basket screens (0. 25 ml) covered with fine mesh socks that are regularly inspected and cleaned. The external and internal walls of the cage are inspected for damage weekly. All supply air and return air grilles are fitted with 0. 25 mm stainless steel mesh within the MRF facility. Fire extinguishers are located within the cages and laboratory, and fire detectors are located in the laboratory, air-conditioning system and plant rooms. The building is fitted with a “Notifier” system that automatically dials out to the fire brigade and campus security personnel in the event of a fire alarm. A set of SOPs (File S1) are used to maintain surveillance and security within the MRF SFS. Extensive monitoring is conducted on the JCU campus to detect mosquitoes that may have escaped the SFS. Sticky ovitraps [13] and 4 BGS traps are also situated in buildings near the MRF, and are serviced weekly. Sticky traps consisting of 700 BGS ml red plastic cups containing a sticky panel insert are placed within the containment space between the mesh layers of each portal frame (Figure S1). If required, breaches of the cage sections can be rectified by replacement of the independently fitted double layers of 0. 25 BGS mm stainless steel mesh. Any mosquitoes collected are identified in the laboratory, and A. aegypti are sent to University of Queensland for identification of Wolbachia infection. The presence of Wolbachia was detected by polymerase chain reaction analysis using primers specific to the wMelPop IS5 insertion sequence as described in [14]. Several times a year container surveys are conducted on the JCU campus, and potential A. aegypti larval habitat is removed or treated with S-methoprene pellets. We sampled cohorts of male and female A. aegypti released within the MRF cage to determine their preferred resting sites. Three cohorts of 120 female and 60 male pupae were allowed to emerge in the cages at two day intervals. Mosquitoes were provided with daily human blood meals and access to oviposition sites as per regular experiment procedures. Separate areas in the cages were surveyed with a Prokopack aspirator [15] 3–7 days post-emergence. The cages were divided into five sections; (facing into the cage) left garden, inside Queenslander, right garden, behind Queenslander and front entrance of cage, and were surveyed in that order. Three surveys were performed at around dusk, when mosquitoes were less active, and three surveys were performed in mid-morning prior to blood-feeding. The dusk collections were performed 7,8, 9 days after the first release of pupae and the day collections were performed 9,10 and 11 days after the first release of pupae. One person (PHJ) performed all aspirator surveys and followed a specified route around objects (eg, plants, light fittings, furniture, sweaty towels) in each section. Mosquitoes were released back into the cages at the end of each survey. Data for all survey times were combined. For each cage, Fisher’s Exact Test was used to compare the total number of female and male mosquitoes captured within the Queenslander structure compared with those captured elsewhere in the cage. A cohort of known numbers of equally aged male and female Ae. aegypti were allowed to synchronously emerge in each cage to estimate daily survival within each MRF cage. Mosquitoes (F1 obtained from populations collected from over 280 ovitraps set in suburbs across Cairns) were reared in the MRF insectary as a single large cohort. Larvae were reared in 3. 4 L white buckets with approximately 2 L of water (ca. 100–150/bucket) and fed a diet of fish food (Tetramin). Temperature was maintained at 26°C with a 12∶12 photoperiod. Pupae were sexed using size as an indicator and 2500 female and 2500 male 0–24 hr old pupae were placed in buckets and allowed to emerge in each cage (total 5000 mosquitoes per cage). Mosquitoes within the SFS cages were blood fed on 1–2 human volunteers for 10 min. at around 10 AM each day. Two BGS traps were set in the Queenslanders in each cage and run for 30 min. and the mean number of male and female captured Ae aegypti calculated. Samples were not returned to the cages. After 22 days, all remaining mosquitoes were captured using BGS traps and human-bait sweepnet collections. Mosquito oviposition took place in 8 ovibuckets placed in the yard area of each cage. The ovibucket consisted of a 4 litre plastic bucket filled with 2 BGS L of a 20% hay infusion; a 10×15 cm red flannel cloth strip was attached inside the bucket as an oviposition substrate. Half of the ovibuckets in each cage were changed every three days so each ovibucket was in the cage for 6 days. Daily survival rates (DSR) were estimated using BGS trap sample and final trap-out data. Both methods of analysis assume that mortality is independent of age and are potentially biased as BGS trap samples were not returned to the cages [16], [17]. However, linear analyses were used for both estimates as the recapture rate was low (overall 10–12% of the total initial population), survival was high and data from the first collection period was very low. Mean BGS trap collections (+1) for females and males in each cage were loge transformed and fitted by linear regression against time (day of sample day 0 to day 15 for females and day 11 for males). The DSR were calculated from the resulting slopes [18]. For the DSR estimate for females using log-linear regression, the first sample point on day 3 was excluded from the regression as fewer mosquitoes were collected in the BGS-trap on day 3 than on the next sample day, day 7 (Figure 4A), likely due to a poor collection of teneral adult females by the BGS trap [19] on day 3. Male DSR were estimated based on samples from day 3 to day 11. The DSR based on the remaining number of female mosquitoes collected in each cage on day 22 was estimated by solving for p in the exponential decay equation where n is days and y is the number of mosquitoes on that day. As no males were collected in the final trap-out or in the BGS traps after day 7 (Figure 4B), day 11 and 0 males were used to estimate DSR.
Ambient light entering the cage was reduced by 98–99% (Table 1), and was reduced by well over 99% within the Queenslander. Temperature and relative humidity within the cages accurately tracked ambient conditions outside the cage during the Sept 2009 period (Figure 5, Figure S3). Indeed, the AC system appeared to reduce daily peak temperatures by about 2–3°C, suggesting that the shade cloth awning above the cage helped prevent significant solar gain within each cage. Temperature and RH were comparable between the two cages. The mean absolute difference in hourly temperature inside and outside the cage was 0. 92 and 1. 02°C, respectively, for cage A and B with the AC turned off; and 0. 71 and 0. 99°C, respectively, with the AC turned on. For RH, the mean absolute difference was 5. 6% and 5. 5%, respectively, for cage A and B with the AC turned off; and 2. 9% and 4. 8%, respectively, with the AC turned on. Temperature and RH within the Queenslander were comparable to both outside ambient and yard conditions within each SFS cage (Table 2). The level of solar gain was not high, and reflects the 99% reduction in light entering the cage. Thus, temperature did not become extreme when the AC system was off, although the AC did appear to reduce highest temperatures in the afternoon. Aberrant RH peaks within both cages during the day was caused by water from the automated sprinkler system. Long term temperatures in both cages remained comparable (Figure S3), with cage A ca. 0. 5°C warmer than cage B. Especially hot afternoon temperatures in early February 2010 exceeded 35°C, but were nearly identical inside and outside both cages. The exterior drainage system prevented overrunning and flooding within the MRF cage due to heavy tropical rains. Indeed, no evidence of flooding within the MRF cage has been observed despite extreme rain events in excess of 300 ml within 24 hr. Overflow of interior drains from rain penetrating the cage screens has also not been observed. The soil base of the cage allows much of the storm water to percolate out of the cage rather than being flushed through the floor drains. There is no evidence of A. aegypti escaping from the MRF-SFS. Aedes aegypti were occasionally captured in the BGS traps and sticky ovitraps located on the JCU campus. From February to June, 2009 a total of 47 (30 female and 17 male) A. aegypti were collected in 4 BGS traps from February to May 2009. During this time 14,800 non-infected and 48,000 Wolbachia-infected A. aegypti had been released in the cages. But none of the A. aegypti collected from the external traps was positive for Wolbachia by PCR assay. Whilst the absence of Wolbachia does not preclude the possibility that these mosquitoes escaped from the SFS cages, an alternative source of the mosquitoes was usually located. For example, the majority (25/47) of the A. aegypti were captured in one fortnight in a BGS trap located near an A. aegypti field bioassay from which adult mosquitoes had inadvertently escaped. Also, A. aegypti had been detected on campus before the cages were operational. Mosquito trapping and inspections detected larvae in potted plant bases, drain sumps and tyres. Although these sites were treated, breeding may have persisted. Unwanted arthropods, such as millipedes, phorid flies, ants and some spiders, were observed in the SFS cages. These were probably introduced before screening of the cages was completed, and may also have been entered the cages from contaminated mulch or ornamental plants. Many remained in the cages despite the steam-cleaning of the mulch. Most arthropod populations were self-limiting while spiders and their webs were removed by hand. Ants may been present in the site soil or tunnelled beneath fencing and ratwalls into the cages. These were subsequently controlled by placing ant baits containing AmdroTM (0. 73% hydramethylnon) within protective plastic petri dishes inside and outside each cage. A few geckoes (the exotic Hemidactylus frenatus (Dumeril and Bibron) ) that probably invaded the Queenslander before the cages were screened were also found in each cage. These were removed by hand or by spraying with DettolTM (active ingredient Chloroxylenol (4-chloro-3,5-dimethylphenol) ). Whilst the use of Dettol is not an approved method for removing geckos, it was the only effective one available. Spraying Dettol at the gecko would cause it to jump off the wall onto the floor rather than running to a crevice in the Queenslander wall or ceiling. Once on the floor the gecko could be quickly caught and killed by freezing. For cage A, significantly more A. aegypti females and males were collected inside the Queenslander structure compared with all the other areas of the cage (Fisher’s exact test, p = 0. 02). This was less apparent in Cage B where similar number of females were captured in the Queenslander, (Fisher’s exact test, p = 0. 32), but fewer mosquitoes were collected overall (Figure 6). The mean numbers of female and male A. aegypti collected in the BGS traps was consistent between the two cages (Figure 4; Figure S2). The day 22 trap-out collected 1,073 and 880 females from Cage A and Cage B, respectively; no males were collected from either cage. Estimated daily survival rate of females was similar for both cages across both estimation methods, ranging from 0. 92–0. 96 (Table 3). The DSRs for males were much lower, but there was nearly a 30% difference between estimates from the two methods, perhaps owing to different termination days.
The MRF provides a secure insectary for the production of mosquitoes and replicate quarantine level 2 SFS cages for conducting of experimental releases. The temperature and relative humidity within both SFS cages closely tracks ambient conditions outside the cages. We had fears that solar gain within the cages would result in high daytime temperatures that could be lethal to mosquitoes. Temperatures over 50°C were reported within the SFS in Tanzania, Africa [9]. However, the Tanzanian SFS had no AC system, and no protective awning to reduce solar gain. Our AC system was able to help maintain mean daily maximum temperatures within 1–2°C of external ambient (Figure 5, Figure S3). The multiple layers of shade cloth and screening reduced incident light within the cage by 98–99% (Table 1). This, coupled with ventilation facilitated by the void of 20–30 cm void beneath the shade cloth, ensured that the cage did not heat up appreciably when the AC was turned off (Figure 5). Temperature and relative humidity within the SFS Queenslander were similar to those recorded in the SFS yard, but light incidence was considerably reduced. Temperature and RH with in the SFS Queenslander are comparable to those occurring within a typical well-ventilated Queenslander house. Data loggers set from 1–8 Dec. 2007 in three rooms within a Queenslander house in Cairns demonstrated that average temperature was within 1°C of external Stevenson screen height temperature (S. Ritchie, unpublished data). However, cooler, high-humidity microclimates did exist in sheltered, moist areas such bathroom and laundry. The moist towels placed in the Queenslander within our SFS would have also provided a cooler, high humidity microclimate. A simple awning system also minimised solar gain and excessive temperature within in two smaller cages (7 m×6 m×4 m high) near the MRF-SFS. These cages were built of 0. 25 ml Tentex polyester covered with a 0. 2 m elevated 90% shade cloth awning. The mean maximum daily temperature (from 10 Feb. – 1 Mar 2010 using data loggers set 0. 24 above ground) in these cages was only 0. 44°C and 0. 17°C higher than ambient (J. Darbro, unpublished data). Thus, data from both the MRF-SFS and the adjacent small cages indicate that a simple elevated awning of shade cloth will provide shade and ventilation, preventing high solar gain and extreme temperatures within the cage. This would be a cheaper alternative to air conditioning units. Aspirator collections within the SFS cages indicated that most mosquitoes harboured within the Queenslander structure. Furthermore, we do not observe large numbers of mosquitoes resting on the cage walls. These observations indicate that the MRF SFS simulates a typical north Queensland urban environment for A. aegypti. Daily survival rate of female A. aegypti was quite high within the MRF SFS. Estimated daily survival rate of 0. 92–0. 96 was obtained from a released cohort of females A aegypti. Male DSR was much lower, (ca. 0. 5–0. 78), suggested that they died from starvation due to a lack of food or feeding. Either the flowering plants available in the cage were not suitable, or the males spent less time feeding compared with other behaviours such as mating. However, the high DSR estimates for females may be unrealistic high. Certainly mortality from predation, insecticide exposure and desiccation during prolonged flights are minimised within the cage. Female mosquitoes also had ready and easy access to a blood source (volunteer blood feeders were available every day) and oviposition sites, and thus were likely to expend less energy in searching for hosts or oviposition sites than wild mosquitoes. We acknowledge that the MRF SFS has limitations. Due to the high construction costs, we were limited to only two SFS cages. Thus, experimental replication will be minimal, requiring multiple sequential experiments in some instances. These experiments could be further complicated by seasonal differences between sequential runs. Environmental conditions within the SFS cage are also different from the natural urban environment. While temperature and RH were comparable to external ambient conditions (Table 2, Figure 5), the screening and shade cloth greatly reduced light and wind within the cage, and the limited space within the cage would may have greatly restricted flight activity. These could impact mosquito survival and the potential infection by agents such as Wolbachia. Thus, results from SFS experiments must be interpreted with caution, especially regarding extrapolation to field conditions. The MRF SFS is a highly secure environment. No Wolbachia-infected A. aegypti have been detected outside the SFS. Adult mosquitoes would have to escape through a double layer of 0. 25 mm stainless steel, limiting these events to a breech of the containment by damage to the structure by flying tree branches, sabotage or vehicular collision. Barring a breech of the cage screening, a mosquito would have to fly through 6 secure locked doors to escape. Both are highly unlikely events. Adult mosquitoes could oviposit in free water or even mulch within the cage. However, all drains have secure 0. 25 mm mesh baskets that would contain larvae. The oviposition buckets are the only source of free-standing water in the SFS cage. Thus, larvae hatched from eggs laid on mulch and other wet areas would not develop into adults. Nonetheless, care must be taken to eliminate free standing water in areas like plant axils and drains. In some instances regulatory bodies may require that genetic material not leave the SFS. Water from drainage and direct contact with the soil could allow for transfer of genetic material in our cage without the escape of living mosquito eggs, larvae or adults. A sealed concrete foundation, together with collection of waste water, would have to be used to prevent this. Finally, insects and other animals entered the cage in some instances. Most invaded the cage before it was screened, entered in mulch and plants brought into the cage or may have burrowed from the soil. Care must be taken to ensure contamination is minimal, and harmful mosquito predators, such as ants and geckoes, are eliminated. Nonetheless, contained SFS cages offer excellent opportunity to conduct research on insects. The secure environment prevents release of quarantine insects; to date, no Wolbachia-infected A. aegypti have been detected in surveillance traps on the JCU campus. The cage allows for the release of cohorts of known numbers. Thus, the direct impact of a control measure can be estimated by comparing changes in population between control and treatment cages. This approach as been used to study the impact of pesticides, repellents and parasite-vector interactions (for a review see Ferguson et al. [9]). Cohort cage studies can also be used to study the behaviour of mosquitoes [9], and to estimate the relative efficacy of traps [20]. The MRF SFS could also be used to conduct insecticide and repellent evaluations under controlled semi-field conditions without the ethical dilemma of disease risk. We hope to investigate the impact of competing oviposition containers on efficacy of ovitraps such as sticky ovitraps and lethal ovitraps. Furthermore, detailed studies on A. aegypti behaviour, such as the microclimate of preferred harbourage sites, can be conducted on released cohorts within the Queenslander structure. While we have not established populations within the cage, we believe it would be relatively easy to do so as has been done with Anopheles [9]. For our studies with Wolbachia, we will be able to observe the rate of Wolbachia invasion within a population of wild A. aegypti. These studies will measure the penetration of Wolbachia within wild A. aegypti after simultaneous release of known ratios of Wolbachia-infected and uninfected A. aegypti. This will occur over several generations and be used to estimate the time to fixation We are currently conducting invasion experiments using the wMelPop and wMel strains.
Novel vector control strategies require validation in the field before they can be widely accepted. Semi-field system (SFS) containment facilities are an intermediate step between laboratory and field trials that offer a safe, controlled environment that replicates field conditions. We developed a SFS laboratory and cage complex that simulates an urban house and yard, which is the primary habitat for Aedes aegypti, the mosquito vector of dengue in Cairns Australia. The SFS consists of a Quarantine Insectary Level-2 (QIC-2) laboratory, containing 3 constant temperature rooms, that is connected to two QIS-2 cages for housing released mosquitoes. Each cage contains the understory of a “Queenslander” timber house and associated yard. An automated air conditioning system keeps temperature and humidity to within 1°C and 5% RH of ambient conditions, respectively. Survival of released A. aegypti was high, especially for females. We are currently using the SFS to investigate the invasion of strains of Wolbachia within populations of A. aegypti.
public health and epidemiology/infectious diseases
A Secure Semi-Field System for the Study of Aedes aegypti