The sex pheromone heptacosane enhances the mating competitiveness of sterile Aedes aegypti males.

BACKGROUND: Aedes aegypti is a vector that transmits various viral diseases, including dengue and Zika. The radiation-based sterile insect technique (SIT) has a limited effect on mosquito control because of the difficulty in irradiating males without reducing their mating competitiveness. In this study, the insect sex pheromone heptacosane was applied to Ae. aegypti males to investigate whether it could enhance the mating competitiveness of irradiated males. METHODS: Heptacosane was smeared on the abdomens of Ae. aegypti males that were allowed to mate with untreated virgin females. The insemination rate was used to assess the attractiveness of heptacosane-treated males to females. The pupae were irradiated with different doses of X-rays and γ-rays, and the emergence, survival time, egg number, and hatch rate were detected to find the optimal dose of X-ray and γ-ray radiation. The males irradiated at the optimal dose were smeared with heptacosane, released in different ratios with untreated males, and mated with females. The effect of heptacosane on the mating competitiveness of irradiated mosquitoes was then evaluated by the hatch rate, induced sterility, and mating competitiveness index. RESULTS: Applying heptacosane to Ae. aegypti males significantly increased the insemination rate of females by 20%. Pupal radiation did not affect egg number but significantly reduced survival time and hatch rate. The emergence of the pupae was not affected by X-ray radiation but was affected by γ-ray radiation. Pupae exposed to 60 Gy X-rays and 40 Gy γ-rays were selected for subsequent experiments. After 60 Gy X-ray irradiation or 40 Gy γ-ray irradiation, the average hatch rate was less than 0.1%, and the average survival time was more than 15 days. Moreover, at the same release ratio, the hatch rate of the irradiated group perfumed with heptacosane was lower than that of the group without heptacosane. Conversely, the male sterility and male mating competitiveness index were significantly increased due to the use of heptacosane. CONCLUSIONS: The sex pheromone heptacosane enhanced the interaction between Ae. aegypti males and females. Perfuming males irradiated by X-rays or γ-rays with heptacosane led to a significant increase in mating competitiveness. This study provided a new idea for improving the application effect of SIT.

Radiation dose-fractionation in adult Aedes aegypti mosquitoes.

Balancing process efficiency and adult sterile male biological quality is one of the challenges in the success of the sterile insect technique (SIT) against insect pest populations. For the SIT against mosquitoes, many stress factors need to be taken into consideration when producing sterile males that require high biological quality to remain competitive once released in the field. Pressures of mass rearing, sex sorting, irradiation treatments, packing, transport and release including handling procedures for each step, add to the overall stress budget of the sterile male post-release. Optimizing the irradiation step to achieve maximum sterility while keeping off-target somatic damage to a minimum can significantly improve male mating competitiveness. It is therefore worth examining various protocols that have been found to be effective in other insect species, such as dose fractionation. A fully sterilizing dose of 70 Gy was administered to Aedes aegypti males as one acute dose or fractionated into either two equal doses of 35 Gy, or one low dose of 10 Gy followed by a second dose of 60 Gy. The two doses were separated by either 1- or 2-day intervals. Longevity, flight ability, and mating competitiveness tests were performed to identify beneficial effects of the various treatments. Positive effects of fractionating dose were seen in terms of male longevity and mating competitiveness. Although applying split doses generally improved male quality parameters, the benefits may not outweigh the added labor in SIT programmes for the management of mosquito vectors.

Title: Fractionnement de la dose d'irradiation chez les moustiques Aedes aegypti adultes. Abstract: Équilibrer l'efficacité du processus et la qualité biologique des mâles adultes stériles est l'un des défis du succès de la technique des insectes stériles (TIS) contre les populations d'insectes nuisibles. Pour la TIS contre les moustiques, de nombreux facteurs de stress sont à prendre en compte lors de la production de mâles stériles qui nécessitent une haute qualité biologique pour rester compétitifs une fois relâchés au champ. Les pressions de l'élevage en masse, du triage par sexe, des traitements d'irradiation, de l'emballage, du transport et de la libération, y compris les procédures de manipulation pour chaque étape, s'ajoutent au budget de stress global du mâle stérile après la libération. L'optimisation de l'étape d'irradiation pour atteindre une stérilité maximale tout en minimisant les dommages somatiques hors cible peut améliorer considérablement la compétitivité de l'accouplement des mâles et il est donc important d'examiner divers protocoles qui se sont révélés efficaces chez d'autres espèces d'insectes, comme le fractionnement de dose. Une dose entièrement stérilisante de 70 Gy a été administrée aux mâles Aedes aegypti en une dose unique ou fractionnée en deux doses égales de 35 Gy, ou une faible dose de 10 Gy suivie d'une seconde dose de 60 Gy. Les deux doses étaient séparées par des intervalles de 1 ou 2 jours. Des tests de longévité, d'aptitude au vol et de compétitivité à l'accouplement ont été réalisés pour identifier les effets bénéfiques des différents traitements. Des effets positifs de la dose de fractionnement ont été observés en termes de longévité des mâles et de compétitivité à l'accouplement. Bien que l'application de doses fractionnées améliore généralement les paramètres de qualité des mâles, les avantages peuvent ne pas compenser le travail supplémentaire dans les programmes TIS pour la gestion des moustiques vecteurs.

Safety, regulatory and environmental issues with sterile insect technique-based mosquito vector control in European countries.

The globalisation of trade is opening the way to the spread of species in new regions where they may cause negative impacts. Invasive mosquito species such as Aedes aegypti and Aedes albopictus are raising concern for their capacity to transmit several arboviruses of public health and veterinary importance. Currently available integrated vector management measures do not achieve satisfactory results when deployed against these urban mosquitoes. Moreover, insecticides are losing their effectiveness owing to the resistance developed by the target species. Policies regulating the use of insecticides are progressively restricting their market availability and this trend is expected to continue. Genetic control methods, such as the sterile insect technique (SIT), based on the use of irradiation to sterilise male mosquitoes, are showing good efficacy in pilot trials at local scales in some Aedes albopictus-colonised urban areas in Europe, without any negative effects. The main limitation is the cost, which may be significantly reduced through the introduction of automation in the mass rearing and drone technology in the field release. These technological advancements require substantial investments at a scale that can only be achieved with centralised production and extensive distribution, which in turn may be granted only if the authorisation frameworks, including the regulation of international transportation and aerial release in an urban setting, are clarified and matured.

La mondialisation des échanges ouvre une voie d'accès à la propagation d'espèces exotiques dans de nouvelles régions, avec parfois des effets négatifs. Les moustiques appartenant à des espèces envahissantes comme Aedes aegypti et Aedes albopictus suscitent à cet égard une certaine inquiétude en raison de leur capacité à transmettre plusieurs arbovirus importants pour la santé publique et vétérinaire. Les mesures disponibles de gestion intégrée des vecteurs ne donnent pas de résultats satisfaisants contre ces moustiques, à l'habitat principalement urbain. En outre, les insecticides perdent en efficacité à mesure que les espèces cibles acquièrent une résistance. La réglementation actuelle sur l'utilisation des insecticides vise à restreindre graduellement la disponibilité de ces produits sur le marché, tendance qui devrait se poursuivre. Les méthodes de contrôle génétique telles que la technique de l'insecte stérile (TIS), qui repose sur l'utilisation de moustiques mâles rendus stériles par irradiation, ont présenté une efficacité satisfaisante lors d'essais pilotes effectués à l'échelle locale dans certaines zones urbaines d'Europe colonisées par Aedes albopictus, sans induire d'effets indésirables. La principale limitation de cette méthode est son coüt, mais celui-ci peut être significativement réduit en automatisant les procédures d'élevage de masse et en utilisant des drones pour les lâchers sur le terrain. Ces avancées technologiques nécessitent d'importants investissements à très grande échelle, qui ne sont réalisables qu'en centralisant la production et en élargissant la distribution ; à son tour, cette perspective ne peut être garantie qu'à travers une clarification et rationalisation des cadres réglementaires régissant les autorisations nécessaires, en particulier concernant les transports internationaux et les lâchers aériens au-dessus des zones urbaines.

La mundialización del comercio está abriendo las puertas a la diseminación de especies en nuevas regiones, donde su presencia puede tener efectos negativos. Hay especies invasoras de mosquito, como Aedes aegypti o Aedes albopictus, que suscitan inquietud por su capacidad de transmitir varios arbovirus de importancia para la salud pública y veterinaria. Las medidas de lucha integrada contra vectores que existen actualmente no ofrecen resultados satisfactorios contra estos mosquitos urbanos. Además, los insecticidas están perdiendo eficacia debido a las resistencias adquiridas por las especies destinatarias. Las políticas que regulan el uso de insecticidas están restringiendo progresivamente su disponibilidad comercial, tendencia que presumiblemente va a perdurar. La aplicación experimental a escala local de métodos de control genético como la técnica del insecto estéril (sterile insect technique, SIT), basada en el uso de radiación para esterilizar a mosquitos macho, está resultando eficaz en ciertas zonas urbanas de Europa colonizadas por Aedes albopictus, sin que se observe ningún efecto negativo. La principal limitación es el costo, que cabe reducir sustancialmente introduciendo la automatización en la cría masiva y empleando drones para las sueltas sobre el terreno. Estos avances tecnológicos exigen inversiones cuantiosas, de tal magnitud que solo son factibles si se plantea una producción centralizada y una distribución a gran escala, procesos que a su vez solo serán aprobados si previamente se aclaran y perfeccionan los regímenes de autorización, incluida la regulación del transporte internacional y de la suelta aérea en medios urbanos.

Development of the Sterile Insect Technique to control the dengue vector Aedes aegypti (Linnaeus) in Sri Lanka.

BACKGROUND: The Sterile Insect Technique (SIT) is presently being tested to control dengue in several countries. SIT aims to cause the decline of the target insect population through the release of a sufficient number of sterilized male insects. This induces sterility in the female population, as females that mate with sterilized males produce no offspring. Male insects are sterilized through the use of ionizing irradiation. This study aimed to evaluate variable parameters that may affect irradiation in mosquito pupae. METHODS: An Ae. aegypti colony was maintained under standard laboratory conditions. Male and female Ae. aegypti pupae were separated using a Fay and Morlan glass sorter and exposed to different doses of gamma radiation (40, 50, 60, 70 and 80 Gy) using a Co60 source. The effects of radiation on survival, flight ability and the reproductive capacity of Ae. aegypti were evaluated under laboratory conditions. In addition, mating competitiveness was evaluated for irradiated male Ae. aegypti mosquitoes to be used for future SIT programmes in Sri Lanka. RESULTS: Survival of irradiated pupae was reduced by irradiation in a dose-dependent manner but it was invariably greater than 90% in control, 40, 50, 60, 70 Gy in both male and female Ae. aegypti. Irradiation didn't show any significant adverse effects on flight ability of male and female mosquitoes, which consistently exceeded 90%. A similar number of eggs per female was observed between the non-irradiated groups and the irradiated groups for both irradiated males and females. Egg hatch rates were significantly lower when an irradiation dose above 50 Gy was used as compared to 40 Gy in both males and females. Irradiation at higher doses significantly reduced male and female survival when compared to the non-irradiated Ae. aegypti mosquitoes. Competitiveness index (C) scores of sterile and non-sterile males compared with non-irradiated male mosquitoes under laboratory and semi-field conditions were 0.56 and 0.51 respectively at 50 Gy. SIGNIFICATION: Based on the results obtained from the current study, a 50 Gy dose was selected as the optimal radiation dose for the production of sterile Ae. aegypti males for future SIT-based dengue control programmes aiming at the suppression of Ae. aegypti populations in Sri Lanka.

Integrated control of Aedes albopictus in Southwest Germany supported by the Sterile Insect Technique.

BACKGROUND: The invasive species Aedes albopictus, commonly known as the Asian tiger mosquito, has undergone extreme range expansion by means of steady introductions as blind passengers in vehicles traveling from the Mediterranean to south-west Germany. The more than 25 established populations in the State of Baden-Württemberg, Palatine and Hesse (south-west Germany) have become a major nuisance and public health threat. Aedes albopictus deserves special attention as a vector of arboviruses, including dengue, chikungunya and Zika viruses. In Germany, Ae. albopictus control programs are implemented by local communities under the auspices of health departments and regulatory offices. METHODS: The control strategy comprised three pillars: (i) community participation (CP) based on the elimination of breeding sites or improved environmental sanitation, using fizzy tablets based on Bacillus thuringiensis israelensis (fizzy Bti tablets; Culinex® Tab plus); (ii) door-to-door (DtD) control by trained staff through the application of high doses of a water-dispersible Bti granular formulation (Vectobac® WG) aimed at achieving a long-lasting killing effect; and (iii) implementation of the sterile insect technique (SIT) to eliminate remaining Ae. albopictus populations. Prior to initiating large-scale city-wide treatments on a routine basis, the efficacy of the three elements was evaluated in laboratory and semi-field trials. Special emphasis was given to the mass release of sterile Ae. albopictus males. RESULTS: More than 60% of the local residents actively participated in the first pillar (CP) of the large-scale control program. The most effective element of the program was found to be the DtD intervention, including the application of Vectobac® WG (3000 ITU/mg) to potential breeding sites (10 g per rainwater container, maximum of 200 l = maximum of approx. 150,000 ITU/l, and 2.5 g per container < 50 l) with a persistence of at least 3 weeks. In Ludwigshafen, larval source management resulted in a Container Index for Ae. albopictus of < 1% in 2020 compared to 10.9% in 2019. The mean number of Aedes eggs per ovitrap per 2 weeks was 4.4 in Ludwigshafen, 18.2 in Metzgergrün (Freiburg) (SIT area) and 22.4 in the control area in Gartenstadt (Freiburg). The strong reduction of the Ae. albopictus population by Bti application was followed by weekly releases of 1013 (Ludwigshafen) and 2320 (Freiburg) sterile Ae. albopictus males per hectare from May until October, resulting in a high percentage of sterile eggs. In the trial areas of Ludwigshafen and Frieburg, egg sterility reached 84.7 ± 12.5% and 62.7 ± 25.8%, respectively; in comparison, the natural sterility in the control area was 14.6 ± 7.3%. The field results were in line with data obtained in cage tests under laboratory conditions where sterility rates were 87.5 ± 9.2% after wild females mated with sterile males; in comparison, the sterility of eggs laid by females mated with unirradiated males was only 3.3 ± 2.8%. The overall egg sterility of about 84% in Ludwigshafen indicates that our goal to almost eradicate the Ae. albopictus population could be achieved. The time for inspection and treatment of a single property ranged from 19 to 26 min depending on the experience of the team and costs 6-8 euros per property. CONCLUSIONS: It is shown that an integrated control program based on a strict monitoring scheme can be most effective when it comprises three components, namely CP, DtD intervention that includes long-lasting Bti-larviciding to strongly reduce Ae. albopictus populations and SIT to reduce the remaining Ae. albopictus population to a minimum or even to eradicate it. The combined use of Bti and SIT is the most effective and selective tool against Ae. albopictus, one of the most dangerous mosquito vector species.

Effects of gamma radiation on the reproductive viability of Aedes aegypti and its descendants (Diptera: Culicidae).

This work evaluated the genetic damage in descendants of male pupae of Aedes (Stegomyia) aegypti (Diptera: Culicidae) separately exposed to 20, 30, and 40 Gy of gamma radiation in the context of Sterile Insect Technique (SIT). Despite the transmission of the dominant lethal mutation, the employed dose levels did not promote a marked reduction in adult mosquito emergence and fertility. This study emphasized that semi-sterilizing doses < 50 Gy for SIT of Aedes aegypti are not recommended.

Investigation of Developmental Stage/Age, Gamma Irradiation Dose, and Temperature in Sterilization of Male Aedes aegypti (Diptera: Culicidae) in a Sterile Insect Technique Program.

The sterilization process using gamma irradiation is a crucial component in a program using sterile insect technique (SIT) to control Aedes aegypti. Unfortunately, there is no efficient standard protocol for sterilizing mosquitoes that can produce a high level of sterility while maintaining mating ability and longevity. Therefore, we conducted a study of the critical factors necessary to develop such a standard protocol. In this study, male Ae. aegypti pupae, as well as adults aged 1 d and 3 d, were irradiated using a Gamma-cell 220 irradiator doses of 0, 20, 40, 60, 70, 80, and 100 Gray (Gy). In addition, male Ae. aegypti in the pupal and adult stage aged 1 d were irradiated at a dose of 70 Gy at various temperatures. Changes in emergence rates, longevity, sterility, and mating competitiveness were recorded for each combination of parameters. Results showed that an increase of irradiation dose leads to a rise of induced sterility at all developmental stages, while simultaneously reducing emergence rate, survival, and mating competitiveness. Higher temperatures resulted in increased levels of sterility, reduced longevity, and did not affect the ability to mate. This study found that an irradiation dose of 70 Gy at a temperature between 20.00 and 22.30°C administered in the pupal stage induced a high level of sterility (around 98%), while maintaining mating competitiveness and longevity.

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.

Radio-frequency exposure of the yellow fever mosquito (A. aegypti) from 2 to 240 GHz.

Fifth generation networks (5G) will be associated with a partial shift to higher carrier frequencies, including wavelengths comparable in size to insects. This may lead to higher absorption of radio frequency (RF) electromagnetic fields (EMF) by insects and could cause dielectric heating. The yellow fever mosquito (Aedes aegypti), a vector for diseases such as yellow and dengue fever, favors warm climates. Being exposed to higher frequency RF EMFs causing possible dielectric heating, could have an influence on behavior, physiology and morphology, and could be a possible factor for introduction of the species in regions where the yellow fever mosquito normally does not appear. In this study, the influence of far field RF exposure on A. aegypti was examined between 2 and 240 GHz. Using Finite Difference Time Domain (FDTD) simulations, the distribution of the electric field in and around the insect and the absorbed RF power were found for six different mosquito models (three male, three female). The 3D models were created from micro-CT scans of real mosquitoes. The dielectric properties used in the simulation were measured from a mixture of homogenized A. aegypti. For a given incident RF power, the absorption increases with increasing frequency between 2 and 90 GHz with a maximum between 90 and 240 GHz. The absorption was maximal in the region where the wavelength matches the size of the mosquito. For a same incident field strength, the power absorption by the mosquito is 16 times higher at 60 GHz than at 6 GHz. The higher absorption of RF power by future technologies can result in dielectric heating and potentially influence the biology of this mosquito.

Preparing Irradiated and Marked Male Aedes aegypti Mosquitoes for Release in an Operational Sterile Insect Technique Program.

The control of such human diseases as dengue, Zika, and chikungunya relies on the control of their vector, the Aedes aegypti mosquito, because there is no prevention. Control of mosquito vectors can rely on chemicals applied to the immature and adult stages, which can contribute to the mortality of non-targets and more importantly, lead to insecticide resistance in the vector. The sterile insect technique (SIT) is a method of controlling populations of pests through the release of sterilized adult males that mate with wild females to produce non-viable offspring. This paper describes the process of producing sterile males for use in an operational SIT program for the control of Aedes aegypti mosquitoes. Outlined here are the steps used in the program including rearing and maintaining a colony, separating male and female pupae, irradiating and marking adult males, and shipping Aedes aegypti males to the release site. Also discussed are procedural caveats, program limitations, and future objectives.

Irradiation induced inversions suppress recombination between the M locus and morphological markers in Aedes aegypti.

BACKGROUND: Aedes aegypti is the primary vector of arthropod-borne viruses and one of the most widespread and invasive mosquito species. Due to the lack of efficient specific drugs or vaccination strategies, vector population control methods, such as the sterile insect technique, are receiving renewed interest. However, availability of a reliable genetic sexing strategy is crucial, since there is almost zero tolerance for accidentally released females. Development of genetic sexing strains through classical genetics is hindered by genetic recombination that is not suppressed in males as is the case in many Diptera. Isolation of naturally-occurring or irradiation-induced inversions can enhance the genetic stability of genetic sexing strains developed through genetically linking desirable phenotypes with the male determining region. RESULTS: For the induction and isolation of inversions through irradiation, 200 male pupae of the 'BRA' wild type strain were irradiated at 30 Gy and 100 isomale lines were set up by crossing with homozygous 'red-eye' (re) mutant females. Recombination between re and the M locus and the white (w) gene (causing a recessive white eye phenotype when mutated) and the M locus was tested in 45 and 32 lines, respectively. One inversion (Inv35) reduced recombination between both re and the M locus, and wand the M locus, consistent with the presence of a rather extended inversion between the two morphological mutations, that includes the M locus. Another inversion (Inv5) reduced recombination only between w and the M locus. In search of naturally-occurring, recombination-suppressing inversions, homozygous females from the red eye and the white eye strains were crossed with seventeen and fourteen wild type strains collected worldwide, representing either recently colonized or long-established laboratory populations. Despite evidence of varying frequencies of recombination, no combination led to the elimination or substantial reduction of recombination. CONCLUSION: Inducing inversions through irradiation is a feasible strategy to isolate recombination suppressors either on the M or the m chromosome for Aedes aegypti. Such inversions can be incorporated in genetic sexing strains developed through classical genetics to enhance their genetic stability and support SIT or other approaches that aim to population suppression through male-delivered sterility.

Artificial Light at Night Increases Aedes aegypti Mosquito Biting Behavior with Implications for Arboviral Disease Transmission.

Aedes aegypti mosquito is a major vector of arboviral disease. Here, we report that the biting behavior of normally daytime active anthropophilic Ae. aegypti mosquitoes on human hosts is abnormally increased at night following exposure to artificial light at night (ALAN). Biting was examined using a human host assay where caged mosquitoes were exposed to a human arm and blood-feeding measured. Mosquitoes were tested during the daytime, nighttime, or challenged with ALAN. As predicted from the Ae. aegypti diel/circadian biting cycle, maximal biting occurred during daytime and lowest level occurred at night. Biting in the ALAN group was increased compared with time-matched nighttime controls. These data reveal that exposure to ALAN increases nocturnal blood-feeding behavior. This finding highlights the concern that globally increasing levels of light pollution could be impacting arboviral disease transmission, such as dengue fever and Zika, and has implications for application of countermeasures for mosquito vector control.

Cold Nights, City Lights: Artificial Light at Night Reduces Photoperiodically Induced Diapause in Urban and Rural Populations of Aedes albopictus (Diptera: Culicidae).

As the planet becomes increasingly urbanized, it is imperative that we understand the ecological and evolutionary consequences of urbanization on species. One common attribute of urbanization that differs from rural areas is the prevalence of artificial light at night (ALAN). For many species, light is one of the most important and reliable environmental cues, largely governing the timing of daily and seasonal activity patterns. Recently, it has been shown that ALAN can alter behavioral, phenological, and physiological traits in diverse taxa. For temperate insects, diapause is an essential trait for winter survival and commences in response to declining daylight hours in the fall. Diapause is under strong selection pressure in the mosquito, Aedes albopictus (Skuse); local adaptation and rapid evolution has been observed along a latitudinal cline. It is unknown how ALAN affects this photosensitive trait or if local adaptation has occurred along an urbanization gradient. Using a common garden experiment, we experimentally demonstrated that simulated ALAN reduces diapause incidence in this species by as much as 40%. There was no difference, however, between urban and rural demes. We also calculated diapause incidence from wild demes in urban areas to determine whether wild populations exhibited lower than predicted incidence compared to estimates from total nocturnal darkness. In early fall, lower than predicted diapause incidence was recorded, but all demes reached nearly 100% diapause before terminating egg laying. It is possible that nocturnal resting behavior in vegetation limits the amount of ALAN exposure this species experiences potentially limiting local adaptation.

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.

Demonstration of resistance to satyrization behavior in Aedes aegypti from La Réunion island.

Aedes aegypti and Aedes albopictus are competent vectors of arboviruses such as dengue and chikungunya viruses which co-exist in some areas, including La Réunion island, Indian Ocean. A type of reproductive interference called satyrization has been described in sympatric species where dominant species mating fails to produce hybrids and thus reduces the fitness and tends to control the spread of the other species. Here, we investigated satyrization in laboratory experiments to provide insights on the potential impact on Ae. aegypti of a control campaign including a sterile insect technique component against Ae. albopictus. Different mating crosses were used to test sympatric, conspecific-interspecific and allopatric effects of irradiated and non-irradiated male Ae. albopictus on female Ae. aegypti, including in a situation of skewed male ratio. Our results suggest that there was only a low level of satyrization between sympatric populations of Ae. aegypti and Ae. albopictus colonized from La Réunion island. A male Ae. albopictus to female Ae. aegypti ratio of 3:1 did not increase the level of satyrization. Female Ae. aegypti previously mated to male Ae. albopictus were not prevented from being inseminated by conspecific males. A satyrization effect was not seen between allopatric Ae. albopictus and Ae. aegypti strains from La Réunion Island either. The tested Ae. aegypti strain from La Réunion island has therefore developed full resistance to satyrization and so releasing sterile male Ae. albopictus may not suppress Ae. aegypti populations if an overflooding of irradiated male Ae. albopictus leads to similar results. The management strategy of two competent species in a sympatric area is discussed.

TITLE: Démonstration de la résistance au comportement de satyrisation chez Aedes aegypti de l'île de La Réunion. ABSTRACT: Aedes aegypti et Aedes albopictus sont des vecteurs compétents d'arbovirus, tels que les virus de la dengue et du chikungunya, qui coexistent dans certaines régions, y compris à La Réunion, dans l'Océan Indien. Une interférence sur la reproduction appelée satyrisation a été décrite chez les espèces sympatriques où l'accouplement d'une espèce dominante ne produit pas d'hybrides et donc réduit le succès reproducteur et tend à contrôler la propagation des autres espèces. Ici, nous avons étudié la satyrisation dans des expériences de laboratoire pour fournir des informations sur l'impact potentiel sur Ae. aegypti d'une campagne de lutte contre Ae. albopictus basée sur la technique de l'insecte stérile. Différents croisements d'accouplements ont été utilisés pour tester les effets sympatriques, conspécifiques-interspécifiques et allopatriques de mâles Ae. albopictus irradiés et non irradiés sur les femelles Ae. aegypti, y compris dans une situation de ratio masculin asymétrique. Nos résultats suggèrent qu'il n'y avait qu'un faible niveau de satyrisation entre les populations sympatriques d'Ae. aegypti et Ae. albopictus colonisées à La Réunion. Un rapport mâles Ae. albopictus sur femelles Ae. aegypti de 3 contre 1 n'a pas augmenté le niveau de satyrisation. Les femelles Ae. aegypti déjà accouplées à un mâle Ae. albopictus n'ont pas été empêchées d'être inséminées par des mâles conspécifiques. Aucun effet de satyrisation n'a été observé entre les souches allopatriques d'Ae. albopictus et d'Ae. aegypti de La Réunion. La souche testée d'Ae. aegypti de La Réunion a donc développé une résistance totale à la satyrisation et en conséquence la libération de mâles stériles d'Ae. albopictus peut ne pas supprimer les populations d'Ae. aegypti si des lâchers massifs de mâles irradiés d'Ae. albopictus conduisent à des résultats similaires. La stratégie de gestion de deux espèces compétentes dans une zone sympatrique est discutée.

Impact of temperature on the extrinsic incubation period of Zika virus in Aedes aegypti.

Since Zika virus (ZIKV) emerged as a global human health threat, numerous studies have pointed to Aedes aegypti as the primary vector due to its high competence and propensity to feed on humans. The majority of vector competence studies have been conducted between 26-28°C, but arboviral extrinsic incubation periods (EIPs), and therefore transmission efficiency, are known to be affected strongly by temperature. To better understand the relationship between ZIKV EIPs and temperature, we evaluated the effect of adult mosquito exposure temperature on ZIKV infection, dissemination, and transmission in Ae. aegypti at four temperatures: 18°C, 21°C, 26°C, and 30°C. Mosquitoes were exposed to viremic mice infected with a 2015 Puerto Rican ZIKV strain, and engorged mosquitoes were sorted into the four temperatures with 80% RH and constant access to 10% sucrose. ZIKV infection, dissemination, and transmission rates were assessed via RT-qPCR from individual mosquito bodies, legs and wings, and saliva, respectively, at three to five time points per temperature from three to 31 days, based on expectations from other flavivirus EIPs. The median time from ZIKV ingestion to transmission (median EIP, EIP50) at each temperature was estimated by fitting a generalized linear mixed model for each temperature. EIP50 ranged from 5.1 days at 30°C to 24.2 days at 21°C. At 26°C, EIP50 was 9.6 days. At 18°C, only 15% transmitted by day 31 so EIP50 could not be estimated. This is among the first studies to characterize the effects of temperature on ZIKV EIP in Ae. aegypti, and the first to do so based on feeding of mosquitoes on a live, viremic host. This information is critical for modeling ZIKV transmission dynamics to understand geographic and seasonal limits of ZIKV risk; it is especially relevant for determining risk in subtropical regions with established Ae. aegypti populations and relatively high rates of return travel from the tropics (e.g. California or Florida), as these regions typically experience cooler temperature ranges than tropical regions.

Acoustic tracheal rupture provides insights into larval mosquito respiration.

Acoustic larviciding (AL) occurs by exposing mosquito larvae to acoustic energy that ruptures their dorsal tracheal trunks (DTTs) by the expulsion of gas bubbles into the body. In studying this technique, we serendipitously identified undescribed anatomical and physiological respiratory features. The classical theory of respiration is that the siphon and DTTs play obligate roles in respiration. Our results contradict the accepted theory that culicine larvae respire via atmospheric gas exchange. We identified an undescribed tracheal occlusion (TO) at the posterior extremities the DTTs. The TOs appear necessary for the acoustic rupture of DTTs; this constriction prevents the escape of energized gas from the siphon and allows the tracheal system to be pressurized. With a pressurized isolated tracheal system, metabolic gas exchange directly with the atmosphere is unlikely and could mostly occur through the chitin and setae. Future studies are needed to explore respiration and elucidate the mechanisms of oxygen absorption and carbon dioxide elimination.

Gamma-Irradiation Reduces Survivorship, Feeding Behavior, and Oviposition of Female Aedes aegypti.

Aedes aegypti is a prominent disease vector that is difficult to control through traditional integrated vector management due to its cryptic peridomestic immature-stage habitat and adult resting behavior, increasing resistance to pesticide formulations approved by the US Environmental Protection Agency, escalating deregistration of approved pesticides, and slow development of new effective chemical control measures. One novel method to control Ae. aegypti is the sterile insect technique (SIT) that leverages the mass release of irradiated (sterilized) males to overwhelm mate choice of natural populations of females. However, one potential liability of SIT is sex sorting errors prior to irradiation, resulting in accidental release of females. Our goal in this study was to test the extent to which irradiation affects female life-history parameters to assess the potential impacts of releasing irradiated females accidentally sorted with males. In this study, we determined that a radiation dose ≥30 Gy-a dose sufficient to sterilize males while preserving their mating competitiveness-may substantially impact longevity, bloodfeeding, oviposition, and egg hatch rate of female Ae. aegypti after being irradiated as pupae. These findings could reduce public concern for accidental release of females alongside irradiated males in an operational Ae. aegypti SIT control program.

Effects of larval diets and temperature regimes on life history traits, energy reserves and temperature tolerance of male Aedes aegypti (Diptera: Culicidae): optimizing rearing techniques for the sterile insect programmes.

BACKGROUND: Producing high quality sterile males is vital in Aedes aegypti rear-and-release birth control strategies. Larval diets, rearing temperatures, and their interactions determine the accumulation rates of essential nutrients in larvae, but these factors have been understudied in relation to mass-rearing techniques for producing eminent males. METHODS: We compared the effects of two larval diets, a cereal-legume-based diet (Khan's diet) and a standard larval diet developed in the FAO/IAEA Insect Pest Control Laboratory (IAEA 2 diet). Diets were tested at selected temperatures for both larval and male adult life history traits, adult extreme temperature tolerance, and mating capacity relative to energy reserves of reared male adult Ae. aegypti. RESULTS: Khan's diet resulted in shorter immature development time at each test temperature (except for 25 °C) than an IAEA 2 diet. Larvae reared at 28 °C and 32 °C with Khan's diet demonstrated low pupation rates (c.80%). We accounted for these phenomena as secondary sex ratio manipulation, because a higher proportion of male adults emerged at 28 °C and 32 °C than that for the IAEA 2 diet. In general, the pupal development time shortened as temperature increased, resulting in higher teneral energy reserves in male mosquitoes. High energy reserves allowed male mosquitoes reared with Khan's diet to have higher adult longevity (5-6 days longer when sugar-fed and 2-3 days longer when water-fed) and tolerance of heat stress than those fed on the IAEA 2 diet. The IAEA 2 diet produced larger male mosquitoes than Khan's diet did: mosquitoes fed on Khan's diet were 1.03-1.05 times smaller than those fed on the IAEA 2 diet at 28 °C and 32 °C. No evidence indicated reduced mating capacity for small mosquitoes fed on Khan's diet. CONCLUSIONS: Larvae reared at 28 °C and 32 °C with Khan's diet were characterized by shorter immature development time compared with those fed on the IAEA 2 diet. Adult mosquitoes produced from that larval rearing condition exhibited a significant male bias, long lifespan, and better endurance against extreme temperatures relative to energy reserves. Thus, the larval diet at rearing temperature of 28 °C and 32 °C optimized rearing techniques for the sterile insect programmes. However, mating competitiveness and flight performance of adult males require further investigation.

The effects of temperature and shading on mortality and development rates of Aedes aegypti (Diptera: Culicidae).

Urbanization has caused an increase in favorable habitats for Aedes aegypti (Diptera: Culicidae), given their ability to reproduce in small and often non-degradable artificial water-containers. While much work has been done on Ae. aegypti biology and ecology in urban landscapes, the role of shading on immature stages as an independent factor from temperature, and any possible interactions between these factors, remains unexamined. We assessed how temperature and shading affected egg hatch-rate, larval/pupal mortality, and larval development to adult stage under different factorial temperature (28; 31; 34; 37; 40° C) and shade (0%, 3,100 lux; 40%, 1,860 lux; 75%, 775 lux; 100%, 0 lux) regimes. Hatch-rate was significantly lower at 37° C (57 %), and no eggs hatched at 40° C. There was no significant effect caused by shading on hatchability. Larval and pupal mortality at 37° C was significantly higher (35%) compared to lower temperature groups, while the effects of shading were emergent at low temperatures. Developmental times from hatching to adult emergence were significantly reduced with increasing temperatures and with greater light exposures. The eco-physiological response of Ae. aegypti larvae to temperature and light regimes suggest a photosensitivity previously unstudied in this species.