Field comparison of broad-spectrum white LED-baited traps with narrow-spectrum green LED-baited traps in the capture of Anopheles mosquitoes (Diptera: Culicidae).

Light-Emitting Diodes (LEDs) have been effective light sources in attracting Anopheles mosquitoes, but the broad-spectrum white light, even with a wide-ranging application in lighting, have not been evaluated yet. In this study, the white light was field evaluated against the green one in the light trapping of anopheline mosquitoes by using two non-suction Silva traps and two CDC-type suction light traps. Anopheline mosquitoes were captured for two 21-night periods of collecting (2022 and 2023). In the first period, two LEDs were used per Silva trap, but three were used in the second one to increase the luminance/illuminance at traps. A CDC-type suction light trap equipped with an incandescent lamp was used in 2022 and a CDC-type suction light trap equipped with a 6 V-white light (higher luminance/illuminance) in 2023. A total of eight species and 3,289 specimens were captured in both periods. The most frequent species were Anopheles triannulatus s.l., An. goeldii, An. evansae and An. argyritarsis. In 2022, white LEDs were less attractive to anopheline mosquitoes than the other light sources, but without statistical difference among treatments (F = 2.703; P = 0.0752; df = 2). In 2023, even with an increased luminance/illuminance at traps, no statistical difference was found between the two LED-baited Silva traps (F = 6.690; P = 0.0024; df = 2), but rather between the 6 V-white-baited CDC-type suction light trap and green-baited Silva traps. Due to some drawbacks and the lower abundance of individuals caught by using white LEDs, the narrow-banded green LEDs is preferable to white ones for attracting anophelines.

Semi-field evaluation of novel chemical lures for Aedes aegypti, Culex quinquefasciatus, and Anopheles minimus (Diptera: Culicidae) in Thailand.

BACKGROUND: Entomological surveillance is an important means of assessing the efficacy of insect vector management programs and estimating disease transmission thresholds. Among baited traps, Biogents' BG-Sentinel (BGS) trap baited with BG-Lure is considered to have the most similar outcome to, and be a possible replacement for, human-landing catches for the epidemiologically relevant monitoring of adult Aedes aegypti and Culex quinquefasciatus. In contrast to the BGS trap, the Black Hole ultraviolet (UV) light trap, which is widely used to catch nocturnal flying insects, is not baited with synthetic human odor-mimicking lures. METHODS: We evaluated the L-lactic acid-based Kasetsart University (KU)-lures nos. 1-6 as novel candidate chemical lures for the diurnal species Ae. aegypti and the nocturnal species Cx. quinquefasciatus using two commercial traps (the BGS trap and the Black Hole UV light trap) in a semi-field screen (SFS) house. Firstly, we optimized the dose of each KU-lure in an SFS house (140 m3). Secondly, six different candidate KU-lures were screened by comparing their percent attraction using a single discriminating dose (0.5 g). Finally, we evaluated the synergism of the KU-lures selected in this way with commercially available traps. RESULTS: BGS traps baited with KU-lure no. 1 exhibited the greatest percent attraction for Ae. aegypti (29.5% ± 14.3%), whereas those baited with KU-lure no. 6 most strongly attracted Cx. quinquefasciatus (33.3% ± 10.7%). Interestingly, BGS traps treated with 10 g BG-Lure did not significantly attract more Ae. aegypti or Cx. quinquefasciatus than the untreated BGS traps. CO2 at a flow rate of 250 ml/min most strongly attracted both Ae. aegypti and Cx. quinquefasciatus (42.2% ± 14.2% and 75.1% ± 16.9%, respectively). BGS and Black Hole UV light traps with KU-lure no. 6 exhibited a stronger attraction for Cx. quinquefasciatus than untreated traps, and the percent attraction did not differ between the treated traps. CONCLUSIONS: Synergistic effects of KU-lures nos. 1 and 6 with the mosquito traps were demonstrated for both the diurnal and nocturnal species in the SFS house assays. However, further studies are urgently needed for the development of species-specific lures to increase trap efficacy in the field for local vector mosquitoes in Thailand.

Effect of irradiation on the survival and susceptibility of female Anopheles arabiensis to natural isolates of Plasmodium falciparum.

BACKGROUND: The sterile insect technique (SIT) is a vector control strategy relying on the mass release of sterile males into wild vector populations. Current sex separation techniques are not fully efficient and could lead to the release of a small proportion of females. It is therefore important to evaluate the effect of irradiation on the ability of released females to transmit pathogens. This study aimed to assess the effect of irradiation on the survival and competence of Anopheles arabiensis females for Plasmodium falciparum in laboratory conditions. METHODS: Pupae were irradiated at 95 Gy of gamma-rays, and emerging females were challenged with one of 14 natural isolates of P. falciparum. Seven days post-blood meal (dpbm), irradiated and unirradiated-control females were dissected to assess the presence of oocysts, using 8 parasite isolates. On 14 dpbm, sporozoite dissemination in the head/thorax was also examined, using 10 parasites isolates including 4 in common with the 7 dpbm dissection (oocyst data). The survivorship of irradiated and unirradiated-control mosquitoes was monitored. RESULTS: Overall, irradiation reduced the proportion of mosquitoes infected with the oocyst stages by 17% but this effect was highly inconsistent among parasite isolates. Secondly, there was no significant effect of irradiation on the number of developing oocysts. Thirdly, there was no significant difference in both the sporozoite infection rate and load between the irradiated and unirradiated-control mosquitoes. Fourthly, irradiation had varying effects on female survival with either a negative effect or no effect. CONCLUSIONS: The effect of irradiation on mosquito competence strongly varied among parasite isolates. Because of such isolate variability and, the fact that different parasite isolates were used to collect oocyst and sporozoite data, the irradiation-mediated reduction of oocyst prevalence was not confirmed for the sporozoite stages. Our data indicate that irradiated female An. arabiensis could contribute to malaria transmission, and highlight the need for perfect sexing tools, which would prevent the release of females as part of SIT programmes.

The role of oxygen depletion and subsequent radioprotective effects during irradiation of mosquito pupae in water.

BACKGROUND: Radiation induced sterility is the basis of the Sterile Insect Technique, by which a target insect pest population is suppressed by releasing artificially reared sterile males of the pest species in overflooding numbers over a target site. In order for the sterile males to be of high biological quality, effective standard irradiation protocols are required. Following studies investigating the effects of mosquito pupae irradiation in water versus in air, there is a need to investigate the oxy-regulatory behavior of mosquito pupae in water to better understand the consequences of irradiation in hypoxic versus normoxic conditions. METHODS: Pupae of Aedes aegypti, Ae. albopictus, and Anopheles arabiensis were submerged in water inside air-tight 2 ml glass vials at a density of 100 pupae/ml and the dissolved oxygen (DO) levels in the water were measured and plotted over time. In addition, male pupae of Ae. aegypti (aged 40-44 h), Ae. albopictus (aged 40-44 h) and An. arabiensis (aged 20-24 h) were irradiated in a gammacell220 at increasing doses in either hypoxic (water with < 0.5% O2 content) or normoxic (in air) conditions. The males were then mated to virgin females and resulting eggs were checked for induced sterility. RESULTS: All three species depleted the water of DO to levels under 0.5% within 30 minutes, with An. arabiensis consuming oxygen the fastest at under 10 minutes. Following irradiation, the protective effect of hypoxia was observed across species and doses (P < 0.0001), increasing at higher doses. This effect was most pronounced in An. arabiensis. CONCLUSIONS: The consumption of dissolved oxygen by pupae submerged in water was significantly different between species, indicating that their oxy-regulatory capacity seems to have possibly evolved according to their preferred breeding site characteristics. This needs to be considered when sterilizing male mosquitoes at pupal stage in water. Depending on species, their DO consumption rates and their density, irradiation doses needed to achieve full sterility may vary significantly. Further assessments are required to ascertain optimal conditions in terms of ambient atmosphere during pupal irradiation to produce competitive sterile males, and temperature and density dependent effects are expected.

A rapid quality control test to foster the development of the sterile insect technique against Anopheles arabiensis.

BACKGROUND: With the fight against malaria reportedly stalling there is an urgent demand for alternative and sustainable control measures. As the sterile insect technique (SIT) edges closer to becoming a viable complementary tool in mosquito control, it will be necessary to find standardized techniques of assessing male quality throughout the production system and post-irradiation handling. Flight ability is known to be a direct marker of insect quality. A new version of the reference International Atomic Energy Agency/Food and Agricultural Organization (IAEA/FAO) flight test device (FTD), modified to measure the flight ability and in turn quality of male Anopheles arabiensis within a 2-h period via a series of verification experiments is presented. METHODS: Anopheles arabiensis juveniles were mass reared in a rack and tray system. 7500 male pupae were sexed under a stereomicroscope (2500 per treatment). Stress treatments included irradiation (with 50, 90, 120 or 160 Gy, using a Gammacell 220), chilling (at 0, 4, 8 and 10 °C) and compaction weight (5, 15, 25, and 50 g). Controls did not undergo any stress treatment. Three days post-emergence, adult males were subjected to either chilling or compaction (or were previously irradiated at pupal stage), after which two repeats (100 males) from each treatment and control group were placed in a FTD to measure flight ability. Additionally, one male was caged with 10 virgin females for 4 days to assess mating capacity (five repeats). Survival was monitored daily for a period of 15 days on remaining adults (two repeats). RESULTS: Flight ability results accurately predicted male quality following irradiation, with the first significant difference occurring at an irradiation dose of 90 Gy, a result which was reflected in both survival and insemination rates. A weight of 5 g or more significantly reduced flight ability and insemination rate, with survival appearing less sensitive and not significantly impacted until a weight of 15 g was imposed. Flight ability was significantly reduced after treatments at 4 °C with the insemination rate more sensitive to chilling with survival again less sensitive (8 and 0 °C, respectively). CONCLUSIONS: The reported results conclude that the output of a short flight ability test, adapted from the previously tested Aedes FTD, is an accurate indicator of male mosquito quality and could be a useful tool for the development of the SIT against An. arabiensis.

Identification of critical factors that significantly affect the dose-response in mosquitoes irradiated as pupae.

BACKGROUND: The sterile insect technique (SIT) for use against mosquitoes consists of several steps including the production of the target species in large numbers, the separation of males and females, the sterilization of the males, and the packing, transport and release of the sterile males at the target site. The sterility of the males is the basis of the technique; for this, efficient and standardized irradiation methods are needed to ensure that the required level of sterility is reliably and reproducibly achieved. While several reports have found that certain biological factors, handling methods and varying irradiation procedures can alter the level of induced sterility in insects, few studies exist in which the methodologies are adequately described and discussed for the reproductive sterilization of mosquitoes. Numerous irradiation studies on mosquito pupae have resulted in varying levels of sterility. Therefore, we initiated a series of small-scale experiments to first investigate variable parameters that may influence dose-response in mosquito pupae, and secondly, identify those factors that potentially have a significantly large effect and need further attention. METHODS: In this study, we compiled the results of a series of experiments investigating variable parameters such as pupal age (Aedes aegypti), pupal size (Ae. aegypti), geographical origin of mosquito strains (Ae. aegypti and Ae. albopictus), exposure methods (in wet versus dry conditions, Ae. albopictus) and subsequently in low versus high oxygen environments [submerged in water (low O2 (< 5 %)] and in air [high O2 (~ 21 %)] on the radiosensitivity of male pupae (Ae. aegypti, Ae. albopictus and Anopheles arabiensis). RESULTS: Results indicate that radiosensitvity of Ae. aegypti decreases with increasing pupal age (99% induced sterility in youngest pupae, compared to 93% in oldest pupae), but does not change with differences in pupal size (P = 0.94). Differing geographical origin of the same mosquito species did not result in variations in radiosensitivity in Ae. aegypti pupae [Brazil, Indonesia, France (La Reunion), Thailand] or Ae. albopictus [Italy, France (La Reunion)]. Differences in induced sterility were seen following irradiation of pupae that were in wet versus dry conditions, which led to further tests showing significant radioprotective effects of oxygen depletion during irradiation procedures in three tested mosquito species, as seen in other insects. CONCLUSIONS: These findings infer the necessity to further evaluate significant factors and reassess dose-response for mosquitoes with controlled variables to be able to formulate protocols to achieve reliable and reproducible levels of sterility for application in the frame of the SIT.

Metabolic rate does not vary with seasonal change in Anopheles arabiensis adults in South Africa.

An important component of South Africa's malaria elimination agenda is identifying the entomological drivers of residual transmission, especially those that present opportunities for enhanced vector control. Seasonal mosquito density correlates directly with malaria transmission in South Africa. Transmission is highest during the warm rainy season and lowest but not entirely absent during the cooler dry season. The factors that sustain dry-season mosquito survival remain unknown. The aim of this project was therefore to investigate seasonal change in metabolic rate to determine the presence or absence of winter dormancy in malaria vector mosquitoes. Metabolic rate, determined by CO2 production during closed-system respirometry, was measured from wild anophelines collected from KwaZulu-Natal Province, South Africa. Monthly sampling spanned all four seasons (summer, autumn, winter, and spring) in 2017. Anopheles arabiensis and An. parensis specimens formed the majority of the total 437 identified specimens (n = 216 and n = 162, respectively). Metabolic rate data from wild-caught mosquitoes showed no significant seasonal disparities for An. arabiensis and An. parensis males and females. Further laboratory experiments assessed the effect of manipulated photoperiod, representing seasonal day-length changes, on the metabolic rate of colonized An. arabiensis mosquitoes. Simulations of midwinter (10 h:14 h light dark) and midsummer (14 h:10 h) day-length showed no significant effect on the metabolic rate of these mosquitoes. Age (in days) had a significant effect on the metabolic rate of both male and female colonized adult An. arabiensis mosquitoes which may be linked to developmental factors during maturation of adults. These data suggest that the South African populations of the malaria vector species An. arabiensis and An. parensis do not curtail their breeding and foraging activities during the colder and drier winter months. Overwintering by diapause does not appear to be triggered in the adult mosquito stage in An. arabiensis. However, their respective population densities do decrease considerably during winter leading to reduced malaria transmission and the opportunity for control by winter larviciding of known breeding sites.

Evaluation of an ultraviolet LED trap for catching Anopheles and Culex mosquitoes in south-eastern Tanzania.

BACKGROUND: Improved surveillance techniques are required to accelerate efforts against major arthropod-borne diseases such as malaria, dengue, filariasis, Zika and yellow-fever. Light-emitting diodes (LEDs) are increasingly used in mosquito traps because they improve energy efficiency and battery longevity relative to incandescent bulbs. This study evaluated the efficacy of a new ultraviolet LED trap (Mosclean) against standard mosquito collection methods. METHODS: The study was conducted in controlled semi-field settings and in field conditions in rural south-eastern Tanzania. The Mosclean trap was compared to commonly used techniques, namely CDC-light traps, human landing catches (HLCs), BG-Sentinel traps and Suna traps. RESULTS: When simultaneously placed inside the same semi-field chamber, the Mosclean trap caught twice as many Anopheles arabiensis as the CDC-light trap, and equal numbers to HLCs. Similar results were obtained when traps were tested individually in the chambers. Under field settings, Mosclean traps caught equal numbers of An. arabiensis and twice as many Culex mosquitoes as CDC-light traps. It was also better at trapping malaria vectors compared to both Suna and BG-Sentinel traps, and was more efficient in collecting mosquitoes indoors than outdoors. The majority of An. arabiensis females caught by Mosclean traps were parous (63.6%) and inseminated (89.8%). In comparison, the females caught by CDC-light traps were 43.9% parous and 92.8% inseminated. CONCLUSIONS: The UV LED trap (Mosclean trap) was efficacious for sampling Anopheles and Culex mosquitoes. Its efficacy was comparable to and in some instances better than traps commonly used for vector surveillance. The Mosclean trap was more productive in sampling mosquitoes indoors compared to outdoors. The trap can be used indoors near human-occupied nets, or outdoors, in which case additional CO2 improves catches. We conclude that this trap may have potential for mosquito surveillance. However, we recommend additional field tests to validate these findings in multiple settings and to assess the potential of LEDs to attract non-target organisms, especially outdoors.

The impact of Anopheles gambiae egg storage for mass rearing and production success.

BACKGROUND: Mass rearing requires a large colony from which male individuals can be harvested for sterilization and release. Attention is needed when monitoring life parameters of the reared population, knowing that any variations within the target population would lead to mismatching between two populations. The aim of this study was to assess the impact of Anopheles gambiae sensu stricto (s.s.) egg storage on hatchability and life history traits. For each parameter, comparison was made between freshly laid and stored eggs in three densities (40, 80, 120 eggs). METHODS: Anopheles gambiae s.s. freshly laid eggs were collected from the Tropical Pesticide Research Institute (TPRI) insectary. Eggs to be stored were kept at - 20 °C for 10 min and then transferred to refrigerators at 4 °C for intervals of 5, 10, 15, 20, and 25 days. After respective storage days, the eggs were transferred from refrigerators to ambient temperature of (25 ± 2) °C for 24 h and then placed in incubators for 24 h. Thereafter eggs were hatched. The egg hatchability, emerged larvae development, larvae survival and emerged adult sex ratios were monitored. RESULTS: This study found that hatching rates decreased with increase in storage time. The difference was significant in eggs stored for 10 and 15 days (P < 0.05). There were no significant differences in hatching rates between An. gambiae eggs stored for 5 days and freshly hatched eggs (P > 0.05). Anopheles larvae development (L1 to pupae) was not significantly affected by storage time across all hatching densities. The study also found that larvae survival decreased with increase in egg storage time. However, there was no significant difference between larvae from freshly hatched eggs and those from eggs at 5 and 10 storage days (P > 0.05) but not for eggs stored for 15 days. Furthermore, there was a decrease in emerged adult males and increase in females relative to increased time of egg storage. The difference was significant (P < 0.05) at 15 storage days but not for eggs stored for 5 and 10 days (in triplicate densities). CONCLUSION: From this study it was concluded that storing An. gambiae eggs at 4 °C and 48 ± 2% relative humidity (RH) for 5 days is the optimal condition and time that did not affect egg hatching rates, larval development and survivorship and emerged adult mosquito sex ratio.

Impact of irradiation on the reproductive traits of field and laboratory An. arabiensis mosquitoes.

BACKGROUND: The sterile insect technique (SIT) aims at suppressing or decreasing insect pest populations by introducing sterile insects into wild populations. SIT requires the mass-production of insects and their sterilization through, for example, radiation. However, both mass-rearing and radiation can affect the life history traits of insects making them less competitive than their wild counterparts. In the malaria mosquito Anopheles arabiensis, some progress has been made to improve the mating competitiveness of mass-reared irradiated males. However, to date, no study has explored the relative effects of colonization and irradiation on important reproductive traits in this species. Such data may help to focus research efforts more precisely to improve current techniques. METHODS: Two strains of An. arabiensis originating from the same locality were used: one reared in the laboratory for five generations and the second collected as late larval instars in the field prior to experimentation. Pupae were irradiated with 95 Gy and some adult reproductive traits, including insemination rate, fecundity, oviposition behavior, fertility and male survivorship, were assessed in different mating combinations. RESULTS: Our study revealed the different effects of mosquito strain and irradiation on reproductive processes. The insemination rate was higher in field (67.3%) than in laboratory (54.9%) females and was negatively affected by both female and male irradiation (un-irradiated vs irradiated: 70.2 vs 51.3% in females; 67.7 vs 53.7% in males). Irradiated females did not produce eggs and egg prevalence was lower in the field strain (75.4%) than in the laboratory strain (83.9%). The hatching rate was higher in the field strain (88.7%) than in the laboratory strain (70.6%) as well as in un-irradiated mosquitoes (96.5%) than in irradiated ones (49%). Larval viability was higher in the field strain (96.2%) than in the laboratory strain (78.5%) and in un-irradiated mosquitoes (97.6%) than irradiated ones (52%). Finally, field males lived longer than laboratory males (25.1 vs 20.5 days, respectively). CONCLUSIONS: Our results revealed that both irradiation and colonization alter reproductive traits. However, different developmental stages are not equally affected. It is necessary to consider as many fitness traits as possible to evaluate the efficacy of the sterile insect technique.

Longevity of mass-reared, irradiated and packed male Anopheles arabiensis and Aedes aegypti under simulated environmental field conditions.

BACKGROUND: To ensure the success of a mosquito control programme that integrates the sterile insect technique (SIT), it is highly desirable to release sterile males with a maximal lifespan to increase release effectiveness. Understanding sterile male survival under field conditions is thus critical for determining the number of males to be released. Our study aimed to investigate the effect of mass rearing, irradiation, chilling, packing and release time on irradiated male mosquito longevity. METHODS: Anopheles arabiensis and Aedes aegypti immature stages were mass-reared using a rack and tray system. Batches of 50 males irradiated at the pupal stage were immobilised, packed into canisters and chilled for 6 hours at 6 °C. Mosquitoes were then transferred either in the early morning or early evening into climate chambers set to simulate the weather conditions, typical of the beginning of the rainy season in Khartoum, Sudan and Juazeiro, Brazil for An. arabiensis and Ae. aegypti, respectively. The longevity of experimental males was assessed and compared to mass-reared control males subjected either to simulated field or laboratory conditions. RESULTS: The combined irradiation, chilling and packing treatments significantly reduced the longevity of both An. arabiensis and Ae. aegypti under simulated field conditions (P < 0.001). However, packing alone did not significantly reduce longevity of Ae. aegypti (P = 0.38) but did in An. arabiensis (P < 0.001). Overall, the longevity of mass reared, irradiated and packed males was significantly reduced, with the median survival time (days) lower following an early morning introduction (4.62 ± 0.20) compared to an evening (7.34 ± 0.35) in An. arabiensis (P < 0.001). However, there was no significant difference in longevity between morning (9.07 ± 0.54) and evening (7.76 ± 0.50) in Ae. aegypti (P = 0.14). CONCLUSIONS: Our study showed that sterile mass-reared males have a reduced lifespan in comparison to laboratory-maintained controls under simulated field conditions, and that An. arabiensis appeared to be more sensitive to the handling process and release time than Ae. aegypti. Longevity and release time are important parameters to be considered for a successful area-wide integrated vector control programme with a SIT component.

Photoperiodic responses of Sahelian malaria mosquitoes Anopheles coluzzii and An. arabiensis.

BACKGROUND: Throughout large parts of sub-Saharan Africa, seasonal malaria transmission follows mosquito density, approaching zero during the dry season and peaking during the wet season. The mechanisms by which malaria mosquitoes survive the long dry season, when no larval sites are available remain largely unknown, despite being long recognized as a critical target for vector control. Previous work in the West African Sahel has led to the hypothesis that Anopheles coluzzii (formerly M-form Anopheles gambiae) undergoes aestivation (dry-season diapause), while Anopheles gambiae (s.s.) (formerly S-form An. gambiae) and Anopheles arabiensis repopulate each wet season via long-distance migration. The environmental cues used by these species to signal the oncoming dry season have not been determined; however, studies, mostly addressing mosquitoes from temperate zones, have highlighted photoperiod and temperature as the most common token stimuli for diapause initiation. We subjected newly established colonies of An. coluzzii and An. arabiensis from the Sahel to changes in photoperiod to assess and compare their responses in terms of longevity and other relevant phenotypes. RESULTS: Our results showed that short photoperiod alone and to a lesser extent, lower nightly temperature (representing the early dry season), significantly increased longevity of An. coluzzii (by ~30%, P < 0.001) but not of An. arabiensis. Further, dry season conditions increased body size but not relative lipid content of An. coluzzii, whereas body size of An. arabiensis decreased under these conditions. CONCLUSIONS: These species-specific responses underscore the capacity of tropical anophelines to detect mild changes (~1 h) in photoperiod and thus support the role of photoperiod as a token stimulus for An. coluzzii in induction of aestivation, although, these responses fall short of a complete recapitulation of aestivation under laboratory conditions.

Light manipulation of mosquito behaviour: acute and sustained photic suppression of biting activity in the Anopheles gambiae malaria mosquito.

BACKGROUND: Host-seeking behaviours in anopheline mosquitoes are time-of-day specific, with a greater propensity for nocturnal biting. We investigated how a short exposure to light presented during the night or late day can inhibit biting activity and modulate flight activity behaviour. RESULTS: Anopheles gambiae (s.s.), maintained on a 12:12 LD cycle, were exposed transiently to white light for 10-min at the onset of night and the proportion taking a blood meal in a human biting assay was recorded every 2 h over an 8-h duration. The pulse significantly reduced biting propensity in mosquitoes 2 h following administration, in some trials for 4 h, and with no differences detected after 6 h. Conversely, biting levels were significantly elevated when mosquitoes were exposed to a dark treatment during the late day, suggesting that light suppresses biting behaviour even during the late daytime. These data reveal a potent effect of a discrete light pulse on biting behaviour that is both immediate and sustained. We expanded this approach to develop a method to reduce biting propensity throughout the night by exposing mosquitoes to a series of 6- or 10-min pulses presented every 2 h. We reveal both an immediate suppressive effect of light during the exposure period and 2 h after the pulse. This response was found to be effective during most times of the night: however, differential responses that were time-of-day specific suggest an underlying circadian property of the mosquito physiology that results in an altered treatment efficacy. Finally, we examined the immediate and sustained effects of light on mosquito flight activity behaviour following exposure to a 30-min pulse, and observed activity suppression during early night, and elevated activity during the late night. CONCLUSIONS: As mosquitoes and malaria parasites are becoming increasingly resistant to insecticide and drug treatment respectively, there is a necessity for the development of innovative control strategies beyond insecticide-treated nets (ITNs) and residual spraying. These data reveal the potent inhibitory effects of light exposure and the utility of multiple photic pulses presented at intervals during the night/late daytime, may prove to be an effective tool that complements established control methods.

Effect of ionising (gamma) radiation on female Anopheles arabiensis.

Background: In a mosquito sterile insect technique programme the ideal scenario is to release male mosquitoes only. However, because there are currently no sex separation strategies which guarantee total female elimination, this study investigated the effect of irradiation on physiological and reproductive fitness of females of an Anopheles arabiensis genetic sexing strain. Methods: Female pupae were irradiated at 70 Gy and the effects of irradiation on adult emergence, longevity, blood-feeding capability, mating ability, fecundity and fertility were assessed. Results and conclusion: Irradiation reduced adult emergence and fecundity but did not affect adult survivorship, mating and blood feeding ability, which suggests that irradiated female mosquitoes can transmit disease pathogens.

Comparative physiological plasticity to desiccation in distinct populations of the malarial mosquito Anopheles coluzzii.

BACKGROUND: In West Africa, populations of the malaria vector mosquito, Anopheles coluzzii, are seasonally exposed to strong desiccating conditions during the dry season. Their dynamics strictly follows the pace of the availability of suitable larval development sites (water collections). Accordingly, mosquitoes can reproduce all year long where permanent breeding is possible, or stop reproduction and virtually disappear at the onset of the dry season when surface water dries up, like observed in temporary habitats of dry savannah areas. This highlights the strong adaptive abilities of this mosquito species, which relies at least in part, upon physiological and molecular mechanisms of specific signatures. METHODS: Here, we analysed a range of physiological and molecular responses expressed by geographically different populations of An. coluzzii inhabiting permanent and temporary breeding sites from the north and the south-west of Burkina Faso. Four mosquito colonies, namely (i) Oursi, built from females breeding in permanent habitats of the north; (ii) Déou, from temporary northern habitats; (iii) Soumousso from south-western temporary breeding sites; and (iv) Bama, from permanent habitats of the same south-western zone, were reared in climatic chambers under contrasted environmental conditions, mimicking temperature, relative humidity and light regimen occurring in northern Burkina Faso. Female mosquitoes were analysed for the seasonal variation in their amounts of proteins, triglycerides and free-circulating metabolites. The expression level of genes coding for the adipokinetic (AKH-I) and the AKH/corazonin-related peptides (ACP) were also assessed and compared among populations and environmental conditions. RESULTS: Our analysis did not reveal an apparent pattern of physiological and molecular variations strictly correlated with either the larval ecotype or the geographical origin of the mosquitoes. However, specific distinct responses were observed among populations, suggesting that dry season survival may rely on more complex ecological parameters at a micro-habitat scale. Interestingly, the physiological and molecular data support the hypothesis that different aestivation abilities exist among populations of An. coluzzii inhabiting contrasted ecological settings. In particular, the striking metabotypes differentiation and the AKH mRNA expression level observed in females from temporary northern populations may suggest the existence of a "strong" aestivation strategy in these specimens. CONCLUSION: Our work provides insights into the physiological and molecular basis of dry and rainy season responses in An. coluzzii, and highlights the important diversity of the mechanisms involved. Such results represent key data for understanding the ecophysiological mechanisms underpinning the strong adaptive potential of this malaria vector species, which undoubtedly contributes to the spreading of mosquito distribution areas in space and time.

Mating competitiveness of sterile genetic sexing strain males (GAMA) under laboratory and semi-field conditions: Steps towards the use of the Sterile Insect Technique to control the major malaria vector Anopheles arabiensis in South Africa.

BACKGROUND: Anopheles arabiensis Patton is primarily responsible for malaria transmission in South Africa after successful suppression of other major vector species using indoor spraying of residual insecticides. Control of An. arabiensis using current insecticide based approaches is proving difficult owing to the development of insecticide resistance, and variable feeding and resting behaviours. The use of the sterile insect technique as an area-wide integrated pest management system to supplement the control of An. arabiensis was proposed for South Africa and is currently under investigation. The success of this technique is dependent on the ability of laboratory-reared sterile males to compete with wild males for mates. As part of the research and development of the SIT technique for use against An. arabiensis in South Africa, radio-sensitivity and mating competitiveness of a local An. arabiensis sexing strain were assessed. METHODS: The optimal irradiation dose inducing male sterility without compromising mating vigour was tested using Cobalt 60 irradiation doses ranging from 70-100 Gy. Relative mating competitiveness of sterile laboratory-reared males (GAMA strain) compared to fertile wild-type males (AMAL strain) for virgin wild-type females (AMAL) was investigated under laboratory and semi-field conditions using large outdoor cages. Three different sterile male to fertile male to wild-type female ratios were evaluated [1:1:1, 5:1:1 and 10:1:1 (sterile males: fertile, wild-type males: fertile, wild-type females)]. RESULTS: Irradiation at the doses tested did not affect adult emergence but had a moderate effect on adult survivorship and mating vigour. A dose of 75 Gy was selected for the competitiveness assays. Mating competitiveness experiments showed that irradiated GAMA male mosquitoes are a third as competitive as their fertile AMAL counterparts under semi-field conditions. However, they were not as competitive under laboratory conditions. An inundative ratio of 10:1 induced the highest sterility in the representative wild-type population, with potential to effectively suppress reproduction. CONCLUSION: Laboratory-reared and sterilised GAMA male An. arabiensis at a release ratio of 3:1 (3 sterile males to 1 wild, fertile male) can successfully compete for insemination of wild-type females. These results will be used to inform subsequent small-scale pilot field releases in South Africa.

Participation of irradiated Anopheles arabiensis males in swarms following field release in Sudan.

BACKGROUND: The success of the sterile insect technique (SIT) depends the release of large numbers of sterile males, which are able to compete for mates with the wild male population within the target area. Unfortunately, the processes of colonisation, mass production and irradiation may reduce the competitiveness of sterile males through genetic selection, loss of natural traits and somatic damage. In this context, the capacity of released sterile Anopheles arabiensis males to survive, disperse and participate in swarms at occurring at varying distances from the release site was studied using mark-release-recapture (MRR) techniques. METHODS: In order to assess their participation in swarms, irradiated and marked laboratory-reared male mosquitoes were released 50, 100 or 200 m from the known site of a large swarm on three consecutive nights. Males were collected from this large swarm on subsequent nights. Over the three days a total of 8,100 males were released. Mean distance travelled (MDT), daily probability of survival and estimated population size were calculated from the recapture data. An effect of male age at the time of release on these parameters was observed. RESULTS: Five per cent of the males released over three days were recaptured. In two-, three- and four-day-old males, MDT was 118, 178 and 170 m, and the daily survival probability 0.95, 0.90 and 0.75, respectively. From the recapture data on the first day following each release, the Lincoln index gives an estimation of 32,546 males in the natural population. DISCUSSION: Sterile An. arabiensis males released into the field were able to find and participate in existing swarms, and possibly even initiate swarms. The survival probability decreased with the age of male on release but the swarm participation and the distance travelled by older males seemed higher than for younger males. The inclusion of a pre-release period may thus be beneficial to male competitiveness and increase the attractiveness of adult sexing techniques, such as blood spiking.

Mating competitiveness of sterile male Anopheles coluzzii in large cages.

BACKGROUND: Understanding the factors that account for male mating competitiveness is critical to the development of the sterile insect technique (SIT). Here, the effects of partial sterilization with 90 Gy of radiation on sexual competitiveness of Anopheles coluzzii allowed to mate in different ratios of sterile to untreated males have been assessed. Moreover, competitiveness was compared between males allowed one versus two days of contact with females. METHODS: Sterile and untreated males four to six days of age were released in large cages (~1.75 sq m) with females of similar age at the following ratios of sterile males: untreated males: untreated virgin females: 100:100:100, 300:100:100, 500:100:100 (three replicates of each) and left for two days. Competitiveness was determined by assessing the egg hatch rate and the insemination rate, determined by dissecting recaptured females. An additional experiment was conducted with a ratio of 500:100:100 and a mating period of either one or two days. Two controls of 0:100:100 (untreated control) and 100:0:100 (sterile control) were used in each experiment. RESULTS: When males and females consort for two days with different ratios, a significant difference in insemination rate was observed between ratio treatments. The competitiveness index (C) of sterile males compared to controls was 0.53. The number of days of exposure to mates significantly increased the insemination rate, as did the increased number of males present in the untreated: sterile male ratio treatments, but the number of days of exposure did not have any effect on the hatch rate. DISCUSSION: The comparability of the hatch rates between experiments suggest that An. coluzzii mating competitiveness experiments in large cages could be run for one instead of two days, shortening the required length of the experiment. Sterilized males were half as competitive as untreated males, but an effective release ratio of at least five sterile for one untreated male has the potential to impact the fertility of a wild female population. However, further trials in field conditions with wild males and females should be undertaken to estimate the ratio of sterile males to wild males required to produce an effect on wild populations.

Environmental temperatures significantly change the impact of insecticides measured using WHOPES protocols.

BACKGROUND: Insecticides are critical components of malaria control programmes. In a variety of insect species, temperature plays a fundamental role in determining the outcome of insecticide exposure. However, surprisingly little is known about how temperature affects the efficacy of chemical interventions against malaria vectors. METHODS: Anopheles stephensi, with no recent history of insecticide exposure, were exposed to the organophosphate malathion or the pyrethroid permethrin at 12, 18, 22, or 26°C, using the WHO tube resistance-monitoring assay. To evaluate the effect of pre-exposure temperature on susceptibility, adult mosquitoes were kept at 18 or 26°C until just before exposure, and then moved to the opposite temperature. Twenty-four hours after exposure, mosquitoes exposed at <26°C were moved to 26°C and recovery was observed. Susceptibility was assessed in terms of survival 24 hours after exposure; data were analysed as generalized linear models using a binomial error distribution and logit link function. RESULTS: Lowering the exposure temperature from the laboratory standard 26°C can strongly reduce the susceptibility of female An. stephensi to the WHO resistance-discriminating concentration of malathion (χ2(df=3) = 29.0, p < 0.001). While the susceptibility of these mosquitoes to the resistance-discriminating concentration of permethrin was not as strongly temperature-dependent, recovery was observed in mosquitoes moved from 12, 18 or 22°C to 26°C 24 hours after exposure. For permethrin especially, the thermal history of the mosquito was important in determining the ultimate outcome of insecticide exposure for survival (permethrin: pre-exposure temperature: F1,29 = 14.2, p < 0.001; exposure temp: F1,29 = 1.1, p = 0.3; concentration: F1,29 = 85.2, p < 0.001; exposure temp x conc: F1,29 = 5.8, p = 0.02). The effect of acclimation temperature on malathion susceptibility depended on the exposure temperature (exposure temp: F1,79 = 98.4, p < 0.001; pre-exposure temp: F1,79 = 0.03, p = 0.9; pre-exp temp x exp temp F1,79 = 6.0, p = 0.02). CONCLUSIONS: A single population of An. stephensi could be classified by WHO criteria as susceptible or resistant to a given chemical, depending on the temperature at which the mosquitoes were exposed. Investigating the performance of vector control tools under different temperature conditions will augment the ability to better understand the epidemiological significance of insecticide resistance and select the most effective products for a given environment.

The effects of genetic manipulation, dieldrin treatment and irradiation on the mating competitiveness of male Anopheles arabiensis in field cages.

BACKGROUND: To enable the release of only sterile male Anopheles arabiensis mosquitoes for the sterile insect technique, the genetic background of a wild-type strain was modified to create a genetic sexing strain ANO IPCL1 that was based on a dieldrin resistance mutation. Secondly, the eggs of ANO IPCL1 require treatment with dieldrin to allow complete elimination of female L1 larvae from the production line. Finally, male mosquito pupae need to be treated with an irradiation dose of 75 Gy for sterilization. The effects of these treatments on the competitiveness of male An. arabiensis were studied. METHODS: The competitiveness of ANO IPCL1 males that were treated either with irradiation or both dieldrin and irradiation, was compared with that of the wild-type strain (An. arabiensis Dongola) at a 1:1 ratio in 5.36 m3 semi-field cages located in a climate-controlled greenhouse. In addition, three irradiated: untreated male ratios were tested in semi-field cages (1:1, 5:1 and 10:1) and their competition for virgin wild-type females was assessed. RESULTS: The ANO IPCL1 males were equally competitive as the wild-type males in this semi-field setting. The ANO IPCL1 males irradiated at 75 Gy were approximately half as competitive as the unirradiated wild-type males. ANO IPCL1 males that had been treated with dieldrin as eggs, and irradiated with 75 Gy as pupae were slightly more competitive than males that were only irradiated. Ratios of 1:1, 5:1 and 10:1 irradiated ANO IPCL1 males: untreated wild-type males resulted in 31, 66 and 81% induced sterility in the female cage population, respectively. CONCLUSIONS: An irradiation dose of 75 Gy reduced the competitiveness of male ANO IPCL1 significantly and will need to be compensated by releasing higher numbers of sterile males in the field. However, the dieldrin treatment used to eliminate females appears to have an unexpected radioprotectant effect, however the mechanism is not understood. A sterile to wild-type ratio of 10:1 effectively reduced the population's fertility under the experimental field cage conditions, but further studies in the field will be needed to confirm the efficiency of sterile ANO IPCL1 males when competing against wild males for wild females.