Comparative analysis of the Potter Tower and a new Track Sprayer for the application of residual sprays in the laboratory.

BACKGROUND: Efforts to evaluate the residual efficacy of new indoor residual spraying (IRS) formulations have identified limitations with the industry standard laboratory sprayer, the Potter Spray Tower (PT). Calibrating the PT can be time-consuming, and the dosing of surfaces may not be as accurate or uniform as previously assumed. METHODS: To address these limitations, the Micron Horizontal Track Sprayer with Spray Cabinet (TS) was developed to provide higher efficiency, ease of operation and deposition uniformity equal to or better than the PT. A series of studies were performed using a fluorescent tracer and three IRS formulations (Actellic® 300CS, K-Othrine WG250 and Suspend PolyZone) sprayed onto surfaces using either the PT or the TS. RESULTS: Deposition volumes could be accurately calibrated for both spray systems. However, the uniformity of spray deposits was higher for the TS compared to the PT. Less than 12% of the volume sprayed using the PT reaches the target surface, with the remaining 88% unaccounted for, presumably vented out of the fume hood or coating the internal surfaces of the tower. In contrast, the TS deposits most of the spray on the floor of the spray chamber, with the rest contained therein. The total sprayed surface area in one run of the TS is 1.2 m2, and the operational zone for spray target placement is 0.7 m2, meaning that 58% of the applied volume deposits onto the targets. The TS can treat multiple surfaces (18 standard 15 × 15 cm tiles) in a single application, whereas the PT treats one surface at a time and a maximum area of around 0.0225 m2. An assessment of the time taken to perform spraying, including the setup, calibration and cleaning, showed that the cost of application using the TS was around 25-35 × less per tile sprayed. Standard operating procedures (SOPs) for calibration and use of both the Potter Tower and Track Sprayer have been developed. CONCLUSIONS: Overall, the TS represents a significant improvement over the PT in terms of the efficiency and accuracy of IRS formulation applications onto test substrates and offers a useful additional tool for researchers and manufacturers wanting to screen new active ingredients or evaluate the efficacy of IRS or other sprayable formulations for insect control.

Overcoming insecticide resistance in Anopheles mosquitoes by using faster-acting solid forms of deltamethrin.

BACKGROUND: Controlling malaria-transmitting Anopheles mosquitoes with pyrethroid insecticides is becoming increasingly challenging because of widespread resistance amongst vector populations. The development of new insecticides and insecticidal formulations is time consuming and costly, however. A more active crystalline form of deltamethrin, prepared by heating the commercial crystalline form, previously was reported to be 12-times faster acting against susceptible North American Anopheles quadrimaculatus mosquitoes. Herein the potential for heat-activated deltamethrin dispersed on chalk to overcome various resistance mechanisms amongst five West African Anopheles strains is investigated, and its long-term sustained lethality evaluated. METHODS: The more active deltamethrin form was generated in a commercial dust containing deltamethrin by heating the material as purchased. Tarsal contact bioassays were conducted to investigate its efficacy, potency, and speed of action against resistant Anopheles populations compared to the commercially available form of deltamethrin dust. RESULTS: In all cases, D-Fense Dust heated to generate the more active form of deltamethrin was substantially more effective than the commercially available formulation. 100% of both Banfora M and Kisumu populations were knocked down 10 min post-exposure with no recovery afterwards. Gaoua-ara and Tiefora strains exhibited 100% knockdown within 15 min, and the VK7 2014 strain exhibited 100% knockdown within 20 min. In all cases, 100% mortality was observed 24 h post-exposure. Conversely, the commercial formulation (unheated) resulted in less than 4% mortality amongst VK7 2014, Banfora, and Gaoua-ara populations by 24 h, and Tiefora and Kisumu mosquitoes experienced 14 and 47% mortality by 24 h, respectively. The heat-activated dust maintained comparable efficacy 13 months after heating. CONCLUSIONS: The heat-activated form of commercial deltamethrin D-Fense Dust outperformed the material as purchased, dramatically increasing efficacy against all tested pyrethroid-resistant strains. This increase in lethality was retained for 13 months of storage under ambient conditions in the laboratory. Higher energy forms of commonly used insecticides may be employed to overcome various resistance mechanisms seen in African Anopheles mosquitoes through more rapid uptake of insecticide molecules from their respective solid surfaces. That is, resistant mosquitoes can be killed with an insecticide to which they are resistant without altering the molecular composition of the insecticide.

Insecticides for Mosquito Control: Improving and Validating Methods to Strengthen the Evidence Base.

Efforts to eliminate vector-borne diseases, for example malaria which caused an estimated 619,000 deaths in 2021 [...].

Unmanned aerial vehicles for surveillance and control of vectors of malaria and other vector-borne diseases.

The use of Unmanned Aerial Vehicles (UAVs) has expanded rapidly in ecological conservation and agriculture, with a growing literature describing their potential applications in global health efforts including vector control. Vector-borne diseases carry severe public health and economic impacts to over half of the global population yet conventional approaches to the surveillance and treatment of vector habitats is typically laborious and slow. The high mobility of UAVs allows them to reach remote areas that might otherwise be inaccessible to ground-based teams. Given the rapidly expanding examples of these tools in vector control programmes, there is a need to establish the current knowledge base of applications for UAVs in this context and assess the strengths and challenges compared to conventional methodologies. This review aims to summarize the currently available knowledge on the capabilities of UAVs in both malaria control and in vector control more broadly in cases where the technology could be readily adapted to malaria vectors. This review will cover the current use of UAVs in vector habitat surveillance and deployment of control payloads, in comparison with their existing conventional approaches. Finally, this review will highlight the logistical and regulatory challenges in scaling up the use of UAVs in malaria control programmes and highlight potential future developments.

A new WHO bottle bioassay method to assess the susceptibility of mosquito vectors to public health insecticides: results from a WHO-coordinated multi-centre study.

BACKGROUND: The continued spread of insecticide resistance in mosquito vectors of malaria and arboviral diseases may lead to operational failure of insecticide-based interventions if resistance is not monitored and managed efficiently. This study aimed to develop and validate a new WHO glass bottle bioassay method as an alternative to the WHO standard insecticide tube test to monitor mosquito susceptibility to new public health insecticides with particular modes of action, physical properties or both. METHODS: A multi-centre study involving 21 laboratories worldwide generated data on the susceptibility of seven mosquito species (Aedes aegypti, Aedes albopictus, Anopheles gambiae sensu stricto [An. gambiae s.s.], Anopheles funestus, Anopheles stephensi, Anopheles minimus and Anopheles albimanus) to seven public health insecticides in five classes, including pyrethroids (metofluthrin, prallethrin and transfluthrin), neonicotinoids (clothianidin), pyrroles (chlorfenapyr), juvenile hormone mimics (pyriproxyfen) and butenolides (flupyradifurone), in glass bottle assays. The data were analysed using a Bayesian binomial model to determine the concentration-response curves for each insecticide-species combination and to assess the within-bioassay variability in the susceptibility endpoints, namely the concentration that kills 50% and 99% of the test population (LC50 and LC99, respectively) and the concentration that inhibits oviposition of the test population by 50% and 99% (OI50 and OI99), to measure mortality and the sterilizing effect, respectively. RESULTS: Overall, about 200,000 mosquitoes were tested with the new bottle bioassay, and LC50/LC99 or OI50/OI99 values were determined for all insecticides. Variation was seen between laboratories in estimates for some mosquito species-insecticide combinations, while other test results were consistent. The variation was generally greater with transfluthrin and flupyradifurone than with the other compounds tested, especially against Anopheles species. Overall, the mean within-bioassay variability in mortality and oviposition inhibition were < 10% for most mosquito species-insecticide combinations. CONCLUSION: Our findings, based on the largest susceptibility dataset ever produced on mosquitoes, showed that the new WHO bottle bioassay is adequate for evaluating mosquito susceptibility to new and promising public health insecticides currently deployed for vector control. The datasets presented in this study have been used recently by the WHO to establish 17 new insecticide discriminating concentrations (DCs) for either Aedes spp. or Anopheles spp. The bottle bioassay and DCs can now be widely used to monitor baseline insecticide susceptibility of wild populations of vectors of malaria and Aedes-borne diseases worldwide.

Reviewing the WHO Tube Bioassay Methodology: Accurate Method Reporting and Numbers of Mosquitoes Are Key to Producing Robust Results.

Accurately monitoring insecticide resistance in target mosquito populations is important for combating malaria and other vector-borne diseases, and robust methods are key. The "WHO susceptibility bioassay" has been available from the World Health Organization for 60+ years: mosquitoes of known physiological status are exposed to a discriminating concentration of insecticide. Several changes to the test procedures have been made historically, which may seem minor but could impact bioassay results. The published test procedures and literature for this method were reviewed for methodological details. Areas where there was room for interpretation in the test procedures or where the test procedures were not being followed were assessed experimentally for their impact on bioassay results: covering or uncovering of the tube end during exposure; the number of mosquitoes per test unit; and mosquito age. Many publications do not cite the most recent test procedures; methodological details are reported which contradict the test procedures referenced, or methodological details are not fully reported. As a result, the precise methodology is unclear. Experimental testing showed that using fewer than the recommended 15-30 mosquitoes per test unit significantly reduced mortality, covering the exposure tube had no significant effect, and using mosquitoes older than 2-5 days old increased mortality, particularly in the resistant strain. Recommendations are made for improved reporting of experimental parameters.

A Practical Insecticide Resistance Monitoring Bioassay for Orally Ingested Dinotefuran in Anopheles Malaria Vectors.

Attractive Toxic Sugar Baits (ATSB) deployed outdoors are likely to be particularly effective against outdoor biting mosquitoes and, if they contain insecticides with a different mode of action, mosquitoes resistant to pyrethroids. One such ATSB based on the neonicotinoid dinotefuran is currently under evaluation in Africa. As with any insecticide-based intervention, it will be important to monitor for the possible emergence of vector resistance. While methods for detecting resistance to insecticides via tarsal contact are recommended by the World Health Organization (WHO), these may not be applicable for orally ingested insecticides. Here, a new ingestion assay, appropriate for a controlled laboratory setting, is described using fluorescein sodium salt (uranine) as a feeding marker. Conventional topical application bioassays, more appropriate for routine deployment, have also been used to apply dinotefuran to the thorax of adult Anopheles mosquitoes with an organic carrier to bypass lipid cuticle barriers. The two methods were compared by establishing lethal doses (LD) in several Anopheles strains. The similarity of the ratios of susceptibility to dinotefuran between pairs of pyrethroid susceptible and resistant strains validates topical application as a suitable, more practical and field applicable method for monitoring for the emergence of resistance to orally ingested dinotefuran. A discriminating dose is proposed, which will be further validated against field populations and used to routinely monitor for the emergence of resistance alongside ATSB trials.

The Residual Efficacy of SumiShield™ 50WG and K-Othrine® WG250 IRS Formulations Applied to Different Building Materials against Anopheles and Aedes Mosquitoes.

Insecticides with novel modes of action are required to complement the pyrethroids currently relied upon for controlling malaria vectors. One example of this is the neonicotinoid clothianidin, the active ingredient in the indoor residual spray (IRS) SumiShield™ 50WG. In a preliminary experiment, the mortality of insecticide-susceptible and resistant An. gambiae adults exposed to filter papers treated with this IRS product reached 80% by 3 days post-exposure and 100% by 6 days post-exposure. Next, cement, wood, and mud tiles were treated with the clothianidin or a deltamethrin-based IRS formulation (K-Othrine WG250). Insecticide resistant and susceptible Anopheles and Aedes were exposed to these surfaces periodically for up to 18 months. Pyrethroid resistant Cx. quinquefasciatus was also exposed at 9 months. Between exposures, tiles were stored in heat and relative humidity conditions reflecting those found in the field. On these surfaces, the clothianidin IRS was effective at killing both susceptible and resistant An. gambiae for 18 months post-treatment, while mortality amongst the resistant strains when exposed to the deltamethrin IRS was not above that of the negative control. Greater efficacy of clothianidin was also demonstrated against insecticide resistant strains of An. funestus compared to deltamethrin, though the potency was lower when compared with An. gambiae. In general, higher efficacy of the clothianidin IRS was observed on cement and mud compared to wood, though it demonstrated poorer residual activity against Ae.aegypti and Cx. quinquefasciatus.

Tenebenal: a meta-diamide with potential for use as a novel mode of action insecticide for public health.

BACKGROUND: There is an urgent need for insecticides with novel modes of action against mosquito vectors. Broflanilide is a meta-diamide, discovered and named Tenebenal™ by Mitsui Chemicals Agro, Inc., which has been identified as a candidate insecticide for use in public health products. METHODS: To evaluate its potential for use in public health, Tenebenal™ was screened using an array of methodologies against Anopheles and Aedes strains. Initially it was assessed for intrinsic efficacy by topical application. Tarsal contact bioassays were then conducted to further investigate its efficacy, as well as its potency and speed of action. The potential of the compound for use in indoor residual spray (IRS) applications was investigated by testing the residual efficacy of a prototype IRS formulation on a range of typical house building substrates, and its potential for use in long-lasting insecticidal nets (LLIN) was tested using dipped net samples. Finally, bioassays using well-characterized insecticide-resistant mosquito strains and an in silico screen for mutations in the insecticide's target site were performed to assess the risk of cross-resistance to Tenebenal™. RESULTS: Tenebenal™ was effective as a tarsal contact insecticide against both Aedes and Anopheles mosquitoes, with no apparent cross-resistance caused by mechanisms that have evolved to insecticides currently used in vector control. Topical application showed potent intrinsic activity against a Kisumu reference strain and an insecticide-resistant strain of Anopheles gambiae. Applied to filter paper in a WHO tube bioassay, Tenebenal™ was effective in killing 100% of susceptible and resistant strains of An. gambiae and Aedes aegypti at a concentration of 0.01%. The discriminating concentration of 11.91 µg/bottle shows it to be very potent relative to chemistries previously identified as having potential for vector control. Mortality occurs within 24 h of exposure, 80% of this mortality occurring within the first 10 h, a speed of kill somewhat slower than seen with pyrethroids due to the mode of action. The potential of Tenebenal™ for development in LLIN and IRS products was demonstrated. At least 12 months residual efficacy of a prototype IRS formulation applied at concentrations up to 200 mg of AI/sq m was demonstrated on a range of representative wall substrates, and up to 18 months on more inert substrates. A dipped net with an application rate of around 2 g/sq m Tenebenal™ killed 100% of exposed mosquitoes within a 3-min exposure in a WHO cone test. CONCLUSIONS: Tenebenal™ is a potent insecticide against adult Aedes and Anopheles mosquitoes, including strains resistant to classes of insecticide currently used in vector control. The compound has shown great potential in laboratory assessment and warrants further investigation into development for the control of pyrethroid-resistant mosquitoes.

Characterisation of Anopheles strains used for laboratory screening of new vector control products.

BACKGROUND: Insecticides formulated into products that target Anopheles mosquitos have had an immense impact on reducing malaria cases in Africa. However, resistance to currently used insecticides is spreading rapidly and there is an urgent need for alternative public health insecticides. Potential new insecticides must be screened against a range of characterized mosquito strains to identify potential resistance liabilities. The Liverpool School of Tropical Medicine maintains three susceptible and four resistant Anopheles strains that are widely used for screening for new insecticides. The properties of these strains are described in this paper. METHODS: WHO tube susceptibility bioassays were used for colony selection and to screen for resistance to the major classes of public health insecticides. Topical and tarsal contact bioassays were used to produce dose response curves to assess resistance intensity. Bioassays with the synergist piperonyl butoxide were also performed. Taqman™ assays were used to screen for known target site resistance alleles (kdr and ace-1). RT-qPCR was used to quantify expression of genes associated with pyrethroid resistance. RESULTS: Pyrethroid selection pressure has maintained resistance to this class in all four resistant strains. Some carbamate and organophosphate resistance has been lost through lack of exposure to these insecticide classes. The Anopheles gambiae (sensu lato) strains, VK7 2014, Banfora M and Tiassalé 13 have higher levels of pyrethroid resistance than the An. funestus FUMOZ-R strain. Elevated expression of P450s is found in all four strains and the 1014F kdr mutation is present in all three An. gambiae strains at varying frequencies. Tarsal contact data and overexpression of CYP4G16 and SAP2 suggest penetration barriers and/or sequestration also confer resistance in Banfora M. CONCLUSIONS: Continual selection with deltamethrin has maintained a stable pyrethroid-resistant phenotype over many generations. In conjunction with a standardized rearing regime, this ensures quality control of strains over time allowing for robust product comparison and selection of optimal products for further development. The identification of multiple mechanisms underpinning insecticide resistance highlights the importance of screening new compounds against a range of mosquito strains.

Ecology of reproduction of Anopheles arabiensis in an urban area of Bobo-Dioulasso, Burkina Faso (West Africa): Monthly swarming and mating frequency and their relation to environmental factors.

Swarming is a key part of the natural system of reproduction of anopheline mosquito populations, and a better understanding of swarming and mating systems in a targeted species in its natural habitat would contribute to better design control strategies with a greater chance of success. Our study investigated the monthly occurrence of swarming and the mating frequency (within swarms) of Anopheles arabiensis in Dioulassoba, Burkina Faso and their relationship with local environmental factors. Mosquitoes collected from swarms were described in terms of body size, recent sugar meal intake, and female repletion, insemination, and Plasmodium falciparum infection status. Swarms of An. arabiensis were found in each month of the year. Both start and end times of swarming varied significantly between months, correlating with the time of sunset. Swarming mostly started after or coincided with sunset from late July to early October but occurred before sunset from late October to early July. Swarming duration, the number of mosquitoes and mating pairs per swarm, and time to first mating were significantly different between months in an inverse relationship with the monthly rainfall. The number of mating pairs was strongly and positively correlated with swarm size. Almost all the females caught in copula were inseminated but a very few were blood fed; no P. falciparum infection was observed. Males caught in copula and in solo were similar in body size and in the proportion which had taken a recent sugar meal. Our investigations showed that An. arabiensis reproductive activities are most frequent during the dry season, suggesting either the species' preference for dry climatic conditions or a lack of available breeding sites during the rainy season due to the seasonal flooding in this area. Targeting interventions to kill mosquitoes in swarms or to achieve an over-flooding ratio of sterile males during the rainy season would increase their efficiency in reducing the population density of this vector.

Assessment of the developmental success of Anopheles coluzzii larvae under different nutrient regimes: effects of diet quality, food amount and larval density.

BACKGROUND: In a context of increasing resistance of both vectors toward main classes of insecticides used in public health and parasites toward anti-malarial drugs, development of new and complementary molecules or control approaches is fundamental to achieve the objective of controlling or even eliminating malaria. Concerning vector control, the sterile insect technique and other genetic control approaches are among promising complementary tools in an integrated management strategy for malaria control. These approaches rely not only on a good understanding of vector biology (especially during larval stages), but also on the availability of adequate supplies and protocols for efficient mosquito rearing. The aim of this study was to assess the factors impacting the life history of Anopheles coluzzii mosquitoes at the larval stage, in the context of genetic and sterile insect approaches to control malaria vectors. METHODS: The effect of different larval diets and larval rearing densities on the development of An. coluzzii were evaluated in the laboratory. Emergence rate (ER), pre-imaginal developmental time (DT) and adult wing length (WL) were measured under different food regimes. Four diets were tested among which three were provided by the Insect Pest Control Laboratory (IPCL) of the FAO/IAEA Joint division. RESULTS: Data showed significant differences in the quality of the different diets and suggested a negative density dependence in all three life history parameters measured under tested rearing conditions. ER and WL increased with food availability, but decreased with increasing larval density. Conversely DT was shortened with increasing food availability but increased with larval density. These data demonstrates intraspecific larval competition modulated by food amount and space availability. Of the four diets tested, the one made of a mix of tuna meal, bovine liver powder, brewer's yeast, squid liver powder and vitamin mix (diet 2) yielded the best results as it produced a good balance between ER, DT and WL. Food availability for optimal development (highest survival at shortest time) was in the range of 180-400 µg/larvae/day for the three diets provided by the IPCL. CONCLUSION: There is an interaction between diet type, diet concentration and larval density. Best results in terms of optimal larvae development parameters happen when moderately high values of those three variables are observed.

Optimization of Mass-Rearing Methods for Anopheles arabiensis Larval Stages: Effects of Rearing Water Temperature and Larval Density on Mosquito Life-History Traits.

Insect mass-rearing is an essential requirement for the sterile insect technique. Production at a large scale requires the development of standardized rearing procedures to produce good quality males able to compete with wild males to mate with wild females. Three sets of experiments (using trays placed on the table, the whole tray-rack system, and climate-controlled chambers) have been conducted aiming to determine the optimal water temperature and number of eggs to aliquot into each larval rearing tray to achieve the highest production of pupae. No difference was found in time to pupation, sex ratio, or male body size as a result of altering larval density. However, higher larval densities resulted in decreased emergence rate and female body size. A constant water temperature of 22°C delayed hatching and did not allow Anopheles arabiensis to complete larval development. Hatching eggs in water at 22°C and then increasing water temperature to 27°C resulted in decreased pupae production compared to eggs hatched and larvae maintained at a water temperature of 27°C throughout. Water temperature and larval density affected the production parameters of An. arabiensis mosquitoes, which has implications for mass release programs. We conclude that 4,000 eggs per 4 liter and a water temperature of 27°C were the optimal conditions for mass-rearing this mosquito species which yielded 105,000 pupae/larval rearing unit. These results are valuable information in the development of standard operation procedures for the efficient large-scale rearing of An. arabiensis mosquitoes.

Reverse osmosis and ultrafiltration for recovery and reuse of larval rearing water in Anopheles arabiensis mass production: Effect of water quality on larval development and fitness of emerging adults.

BACKGROUND: Countries around the world are showing increased interest in applying the sterile insect technique against mosquito disease vectors. Many countries in which mosquitoes are endemic, and so where vector control using the sterile insect technique may be considered, are located in arid zones where water provision can be costly or unreliable. Water reuse provides an alternate form of water supply. In order to reduce the cost of mass rearing of Anopheles arabiensis mosquitoes, the possibility of recycling and reusing larval rearing water was explored. METHODS: The used rearing water ('dirty water') was collected after the tilting of rearing trays for collection of larvae/pupae, and larvae/pupae separation events and underwent treatment processes consisting of ultrafiltration and reverse osmosis. First-instar An. arabiensis larvae were randomly assigned to different water-type treatments, 500 larvae per laboratory rearing tray: 'clean' dechlorinated water, routinely used in rearing; dirty water; and 'recycled' dirty water treated using reverse osmosis and ultrafiltration. Several parameters of insect quality were then compared: larval development, pupation rate, adult emergence, body size and longevity. Water quality of the samples was analyzed in terms of ammonia, nitrite, nitrate, sulphate, dissolved oxygen, chloride, and phosphate concentrations after the larvae had all pupated or died. Surface water temperatures were also recorded continuously during larval development. RESULTS: Pupation rates and adult emergence were similar in all water treatments. Adult body sizes of larvae reared in recycled water were similar to those reared in clean water, but larger than those reared in the dirty larval water treatment, whereas the adult longevity of larvae reared in recycled water was significantly increased relative to both 'clean' and 'dirty' water. Dirty larval water contained significantly higher concentrations of ammonium, sulfate, phosphate and chloride and lower levels of dissolved oxygen than clean water. These parameters significantly varied during the period of larval development. After dirty water was recycled by ultrafiltration and reverse osmosis, all the parameters measured were the same as those in clean water. CONCLUSION: This study demonstrated the potential for using recycled larval rearing water to supplement clean dechlorinated water supplies. Recycling used water improved its quality and of the reared mosquitoes. As water demands and environmental pressures grow, recycling of larval rearing water will improve the sustainability and affordability of mosquito mass-rearing.

Optimization of mosquito egg production under mass rearing setting: effects of cage volume, blood meal source and adult population density for the malaria vector, Anopheles arabiensis.

BACKGROUND: Anopheles arabiensis is one of the major malaria vectors that put millions of people in endemic countries at risk. Mass-rearing of this mosquito is crucial for strategies that use sterile insect technique to suppress vector populations. The sterile insect technique (SIT) package for this mosquito species is being developed by the Insect Pest Control Subprogramme of the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture. To improve mass-rearing outcomes for An. arabiensis, the question of whether the egg production by females would be affected by the size of the adult holding cages, the source of the blood meal and the total number of pupae that could be loaded into the cages was addressed and finally the impact of adding additional pupae to the cage daily to maintain adult numbers on egg productivity assessed. METHODS: Mass production cages of two different volumes, two different sources of blood meal (bovine and porcine) and two different population densities (cages originally loaded with either 15,000 or 20,000 pupae) were tested and evaluated on the basis of eggs produced/cage or per female. Males and females pupae with a ratio of 1:1 were added to the cages at day 1 and 2 of pupation. The emerging adults had constant access to 5% sugar solution and blood fed via the Hemotek membrane feeding system. Eggs were collected either twice a week or daily. A generalized linear model was used to identify factors which gave significantly higher egg production. RESULTS: Neither cage volume nor blood meal source affected egg production per cage or per female. However, increasing population density to 20,000 pupae had a negative effect on eggs produced per cage and per female. Although high density negatively impacted egg production, adding 1000 daily additional pupae compensating for daily mortality resulted in a substantial increase in egg production. Moreover, in all tests the first and the third egg batches collected were significantly higher than others eggs batches. With the equipment and protocols described here and routinely used at the Insect Pest Control Laboratory (IPCL), it was possible to produce up to 120,000 eggs/cage/day. CONCLUSION: These results demonstrated that 15,000 is the optimal number of pupae to be loaded into the Anopheles Mass production cages. Under this condition, an average of 40 eggs per female was obtained for five gonotrophic cycles. However, an improvement in egg production can be achieved by daily addition, to the original 15,000 pupae, of one thousand pupae a day. Interestingly, feeding females with bovine or porcine blood using both large and small versions of the mass production cage did not affect egg productivity.

Benchmarking vector arthropod culture: an example using the African malaria mosquito, Anopheles gambiae (Diptera: Culicidae).

BACKGROUND: Numerous important characteristics of adult arthropods are related to their size; this is influenced by conditions experienced as immatures. Arthropods cultured in the laboratory for research, or mass-reared for novel control methods, must therefore be of a standard size range and known quality so that results are reproducible. METHODS: A simple two-step technique to assess laboratory culture methods was demonstrated using the mosquito Anopheles gambiae s.s. as a model. First, the ranges of key development outcomes were determined using various diet levels. The observed outcomes described the physiologically constrained limits. Secondly, the same outcomes were measured when using a standard operating procedure (SOP) for comparison with the determined ranges. RESULTS: The standard method resulted in similar development rates to those of high and medium diets, wing length between those resulting from the high and medium diets, and larval survival exceeding all benchmark diet level values. The SOP used to produce experimental material was shown to produces high-quality material, relative to the biologically constrained limits. CONCLUSIONS: The comparison between all possible phenotypic outcomes, as determined by biological constraints, with those outcomes obtained using a given rearing protocol is termed "benchmarking". A method is here demonstrated which could be easily adapted to other arthropods, to objectively assess important characters obtained, and methods used, during routine culture that may affect outcomes of research.

Reusing larval rearing water and its effect on development and quality of Anopheles arabiensis mosquitoes.

BACKGROUND: There is growing interest in applying the sterile insect technique (SIT) against mosquitoes. Mass production of mosquitoes for large-scale releases demands a huge amount of water. Yet, many arid and/or seasonally arid countries face the difficulties of acute water shortage, deterioration of water quality and environmental constraints. The re-use of water to rear successive generations of larvae is attractive as a way to reduce water usage and running costs, and help to make this control method viable. METHODS: To determine whether dirty larval water was a suitable rearing medium for Anopheles arabiensis, in place of the 'clean' dechlorinated water routinely used, a series of three experiments was carried out to evaluate the effect of dirty water or mixed clean and dirty water on several parameters of insect quality. Batches of 100 fresh eggs were distributed in dirty water or added to clean water to test the effect of dirty water on egg hatching, whereas first-instar larvae were used to determine the effect on immature development time, pupation, adult emergence, body size, and longevity. Moreover, to assess the effect of dirty water on larval mortality, pupation rate, adult emergence, and longevity, L4 larvae collected after the tilting or larvae/pupae separation events were returned either to the dirty water or added to clean water. RESULTS: Results indicated that reusing dirty water or using a 50:50 mix of clean and dirty water did not affect egg hatching. Moreover, no difference was found in time to pupation, larval mortality or sex ratio when first-instar larvae were added to clean water, dirty water, or a 75:25, 50:50 or 25:75 mix of clean and dirty water and reared until emergence. When late-instar larvae were put back into their own rearing water, there was no effect on pupation rate, emergence rate or female longevity, though male longevity was reduced. When reared from first-instar larvae, however, dirty water decreased pupation rate, emergence rate, body size, and adult longevity. CONCLUSIONS: Re-used larval-rearing water has no impact on egg hatching, development time or mortality of the immature stages of An. arabiensis. However, dirty water is not suitable for the production of high quality adult mosquitoes. Recycling processes to improve water quality and increase insect quality will be investigated, since it may have important implications for the implementation of the SIT in areas where clean water is a scarce or costly resource.

Combining the Sterile Insect Technique with the Incompatible Insect Technique: III-Robust Mating Competitiveness of Irradiated Triple Wolbachia-Infected Aedes albopictus Males under Semi-Field Conditions.

Combination of the sterile insect technique with the incompatible insect technique is considered to be a safe approach to control Aedes albopictus populations in the absence of an accurate and scalable sex separation system or genetic sexing strain. Our previous study has shown that the triple Wolbachia-infected Ae. albopictus strain (wAlbA, wAlbB and wPip) was suitable for mass rearing and females could be completely sterilized as pupae with a radiation dose of at least 28 Gy. However, whether this radiation dose can influence the mating competitiveness of the triple infected males was still unknown. In this study we aimed to evaluate the effects of irradiation on the male mating competitiveness of the triple infected strain under laboratory and semi-field conditions. The results herein indicate that irradiation with a lower, female-sterilizing dose has no negative impact on the longevity of triple infected males while a reduced lifespan was observed in the wild type males (wAlbA and wAlbB) irradiated with a higher male-sterilizing dose, in small cages. At different sterile: fertile release ratios in small cages, triple-infected males induced 39.8, 81.6 and 87.8% sterility in a wild type female population at 1:1, 5:1 and 10:1 release ratios, respectively, relative to a fertile control population. Similarly, irradiated triple infected males induced 31.3, 70.5 and 89.3% sterility at 1:1, 5:1 and 10:1 release ratios, respectively, again relative to the fertile control. Under semi-field conditions at a 5:1 release ratio, relative to wild type males, the mean male mating competitiveness index of 28 Gy irradiated triple-infected males was significantly higher than 35 Gy irradiated wild type males, while triple infected males showed no difference in mean mating competitiveness to either irradiated triple-infected or irradiated wild type males. An unexpected difference was also observed in the relative male mating competitiveness of the triple infected strain after irradiation at 28 Gy dose in small vs large cages, with a higher male mating competitiveness index calculated from results of experiments in the large cages. Based on these results, we consider that the male mating performance of the triple infected strain after irradiation at 28 Gy, a dose required for complete female sterility and the avoidance of population replacement, is approximately equal to that of the wild type males under semi-field conditions. Though field evaluation is required, this suggests that the triple infected strain is suitable for irradiation and release as part of a combined SIT-IIT approach to Ae. albopictus control.

More than one rabbit out of the hat: Radiation, transgenic and symbiont-based approaches for sustainable management of mosquito and tsetse fly populations.

Mosquitoes (Diptera: Culicidae) and tsetse flies (Diptera: Glossinidae) are bloodsucking vectors of human and animal pathogens. Mosquito-borne diseases (malaria, filariasis, dengue, zika, and chikungunya) cause severe mortality and morbidity annually, and tsetse fly-borne diseases (African trypanosomes causing sleeping sickness in humans and nagana in livestock) cost Sub-Saharan Africa an estimated US$ 4750 million annually. Current reliance on insecticides for vector control is unsustainable: due to increasing insecticide resistance and growing concerns about health and environmental impacts of chemical control there is a growing need for novel, effective and safe biologically-based methods that are more sustainable. The integration of the sterile insect technique has proven successful to manage crop pests and disease vectors, particularly tsetse flies, and is likely to prove effective against mosquito vectors, particularly once sex-separation methods are improved. Transgenic and symbiont-based approaches are in development, and more advanced in (particularly Aedes) mosquitoes than in tsetse flies; however, issues around stability, sustainability and biosecurity have to be addressed, especially when considering population replacement approaches. Regulatory issues and those relating to intellectual property and economic cost of application must also be overcome. Standardised methods to assess insect quality are required to compare and predict efficacy of the different approaches. Different combinations of these three approaches could be integrated to maximise their benefits, and all have the potential to be used in tsetse and mosquito area-wide integrated pest management programmes.

Combining the Sterile Insect Technique with Wolbachia-Based Approaches: II--A Safer Approach to Aedes albopictus Population Suppression Programmes, Designed to Minimize the Consequences of Inadvertent Female Release.

Due to the absence of a perfect method for mosquito sex separation, the combination of the sterile insect technique and the incompatible insect technique is now being considered as a potentially effective method to control Aedes albopictus. In this present study first we examine the minimum pupal irradiation dose required to induce complete sterility in Wolbachia triple-infected (HC), double-infected (GUA) and uninfected (GT) female Ae. albopictus. The HC line is a candidate for Ae. albopictus population suppression programmes, but due to the risk of population replacement which characterizes this triple infected line, the individuals to be released need to be additionally irradiated. After determining the minimum irradiation dose required for complete female sterility, we test whether sterilization is sufficient to prevent invasion of the triple infection from the HC females into double-infected (GUA) populations. Our results indicate that irradiated Ae. albopictus HC, GUA and GT strain females have decreased fecundity and egg hatch rate when irradiated, inversely proportional to the dose, and the complete sterilization of females can be acquired by pupal irradiation with doses above 28 Gy. PCR-based analysis of F1 and F2 progeny indicate that the irradiated HC females, cannot spread the new Wolbachia wPip strain into a small cage GUA population, released at a 1:5 ratio. Considering the above results, we conclude that irradiation can be used to reduce the risk of population replacement caused by an unintentional release of Wolbachia triple-infected Ae. albopictus HC strain females during male release for population suppression.