Effect of water salinity on immature performance and lifespan of adult Asian tiger mosquito.

BACKGROUND: Aedes albopictus (Skuse, 1894) is a vector for pathogens like dengue, chikungunya, and Zika viruses. Its adaptive capacity enables reproduction in temperate climates and development mainly in artificial containers with fresh water in urbanized areas. Nevertheless, breeding in coastal areas may also occur along with its aggressive invasiveness. Global warming and the consequent rise in sea levels will increase saline (> 30 ppt) or brackish (0.5-30 ppt salt) water in coastal regions. To address whether Ae. albopictus can breed in brackish water, we initiated the current study that analyses the survival of immature stages at different salinity concentrations and explores whether carryover effects occur in the resulting adults. This possible adaptation is important when considering the potential for development in new habitats and expansion of one of the world's most invasive species. METHODS: We investigated the influence of salinity on the survival of Ae. albopictus larvae and adults under laboratory-controlled conditions. First instar larvae were exposed to different salinity concentrations (0 to 30 ppt) and their development time, pupation, adult emergence, and overall survival were monitored daily. We used Kaplan-Meier and Cox regression models to analyze the survival rates at different salinity levels. Furthermore, life tables were constructed under each salinity concentration. RESULTS: Increasing salt concentrations significantly increased the mortality risk during immature development, while no significant effect was observed on adult mortality risk. A comparison between distilled and bottled water revealed a notable increase in overall mortality risk for individuals developing in distilled water. However, no significant effects were found when analyzing survival from the first larval stage to adult emergence and adult lifespan. The life expectancy of immature stages decreased with increasing salt concentrations, although salinity concentration did not significantly impact adult life expectancy. CONCLUSIONS: Our findings suggest that Ae. albopictus, previously considered freshwater species, can successfully develop and survive in brackish waters, even in the absence of characteristic structures found in euryhaline species. These adaptations may enable Ae. albopictus to establish new breeding sites and colonize unexplored territories. Knowledge of these physiological adaptations of Ae. albopictus to salinity should be pursued to increase the range of control of the species, and to make more accurate predictions of its dispersal and vectoring ability.

Insecticide resistant mosquitoes remain thermal stress resistant, without loss of thermal plasticity.

A major component of mosquito's climate change response is their heat tolerance, and any ability to rapidly adjust to extreme environmental conditions through phenotypic plasticity. The excessive use of insecticides for the control of major mosquito species leads to resistant populations, however it is largely unclear if this concurrently impacts thermal stress resistance and their potential to adjust tolerance via phenotypic plasticity. Culex pipiens pipiens, Culex pipiens molestus and Aedes albopictus populations obtained from the same region were subjected for 12 generations to selection trials to larvicides Diflubenzuron (DFB) and Bacillus thuringiensis subsp. israelensis (Bti) to develop insecticide resistance. Adults emerging from the selected populations were acclimated at different temperatures and the upper and lower critical thermal limits (CTmax and CTmin) were estimated using dynamic thermal assays. In addition, the supercooling points (SCPs) of non-acclimated adults of resistant and control populations were determined. Our results revealed marked differences in thermal response among the three species, the different acclimation regimes and sexes. Aedes albopictus was more resistant in high than low temperatures compared to both Culex pipiens biotypes. Culex forms responded similarly to heat but differently to cold stress. In both forms, females responded better than males to all thermal stressors. Acclimation at higher and lower temperatures improves CTmax and CTmin values, respectively in both insecticide resistant and control populations of all three species. Overall, selection to insecticides did not affect the thermal performance of adults. Hence, insecticide-resistant mosquito populations perform similarly to untreated ones and are capable of readily adapting to new environmental changes rising concerns regarding their geographic range expansion and disease transmission globally.

Residual efficacy of selected larvicides against Culex pipiens pipiens (Diptera: Culicidae) under laboratory and semi-field conditions.

Mosquitoes are a threat worldwide since they are vectors of important pathogens and parasites such as malaria, dengue, yellow fever, and West Nile. The residual toxicity of several commercial mosquito larvicides was evaluated for the control of Culex pipiens pipiens under controlled laboratory and semi-field conditions during late spring and summer of 2013. The evaluation included six different active ingredient formulations, i.e., diflubenzuron Du-Dim), Bacillus thuringiensis var. israelensis (Bti) (Vectobac), spinosad (Mozkill), S-methoprene (Biopren), temephos (Abate), and polydimethylsiloxane (PDMS) (Aquatain), that are currently registered of and had been registered in the past for mosquito control. Under controlled laboratory conditions, the residual activity ranged from 1 week (S-methoprene) up to 2 months (spinosad, PDMS). Exposure of larvicides under semi-field conditions resulted in noticeable differences regarding their efficacy as compared to the laboratory bioassays. Exposure of S-methoprene, Bti, and spinosad, for up to 3 days, resulted in similar adult emergence to the controls. On the other hand, the residual efficacy of diflubenzuron, temephos, and PDMS ranged from 14 to 28 days, depending on the season of exposure. Longevity and fecundity of adults that had emerged from surviving larvae, in most of the cases tested, did not differ significantly from that of the controls. The results of the present study demonstrate the necessity of both field and laboratory studies to draw safe conclusions regarding the efficacy of larvicides against mosquitoes and the selection of the proper formulation for each application scenario. In addition, defining the seasonal variation in the residual toxicity of the tested formulations could be useful for improving mosquitos' management programs.

Quality Control Methods for Aedes albopictus Sterile Male Transportation.

Genetic based mosquito control methods have been gaining ground in recent years for their potential to achieve effective suppression or replacement of vector populations without hampering environments or causing any public health risk. These methods require the mass rearing of the target species in large facilities sized to produce millions of sterile males, as already well established for a number of insects of agricultural importance. Assessing the performance of released males in Sterile Insect Technique (SIT) control programs is of the utmost importance for the success of the operation. Besides the negative effects of mass rearing and sterilization, the handling of sterilized insects and shipment to distant areas may also negatively impact the quality of sterilized males. The aim of the current study was to design and executive quality control (QC) tests for sterilized Aedes albopictus (Asian tiger mosquito) males delivered by air shipment from the mass production facility located in Italy to Greece and Montenegro field release sites. Mass reared mosquito strains were based on biological materials received from Italy, Greece and Montenegro. Tests conducted at the mass rearing facility before transportation revealed a rather high residual female contamination following mechanical sex separation (approximately 1.5% females, regardless of the mosquito strain). Irradiated males of all three mosquito strains induced high levels of sterility to females. Shipment lasting approximately 24 h resulted in approximately 15% mortality, while when shipment lasted nearly two days this increased to almost 40%. The flight ability of sterilized males following one day transportation time was satisfactory (over 60%). The response of sterile males to food and water starvation was comparable and slightly lower than that of wild non-transported males. Longevity of sterile males was shorter than that of wild counterparts and it seems it was not affected by mating to wild females. Both mating propensity and mating competitiveness for wild virgin females was higher for the wild, control males compared to the sterile, transported ones. Overall, the performance of sterile male Ae. albopictus delivered from the mass rearing facility of Italy to Greece in approximately 24 h was satisfactory. Transportation lasting two days or longer incurred detrimental effects on males, which called into question the outcome of the SIT release programs. In conclusion, our results demonstrate the need of quality control procedures, especially when sterile male production facilities are not near to the releasing point. Transportation could be a serious drawback for the implementation of Sterile Insect Releases and, consequently, it is important to establish an efficient and fast transportation of sterilized males in advance.

Effects of Selection to Diflubenzuron and Bacillus thuringiensis Var. Israelensis on the Overwintering Successes of Aedes albopictus (Diptera: Culicidae).

Aedes albopictus is an invasive mosquito species responsible for local transmission of chikungunya and dengue viruses in Europe. In the absence of available treatments, insecticides-based control remains one of the most important viable strategies to prevent emerging problems. Diflubenzuron (DFB) and Bacillus thuringiensis var. israelensis (Bti) are among the most commonly used larvicides for Ae. albopictus control with consequent concerns for the potential development of resistance. Studies on the resistance emergence in Ae. albopictus and its persistence in the wild to both DFB and Bti are essential for the efficient and sustainable planning of the control programmes. In this context, larvae from a recently laboratory established population were subjected to increasing selective pressure for nine successive generations using both DFB and Bti. The resistance levels and the overwintering success of the selected populations relative to control (colonies that received no selection) were determined. Results revealed an 8.5- and 1.6-fold increase on the resistance levels following selection with DFB and Bti, respectively. The selection process to both larvicides had no apparent impacts on the overwintering capability relative to control, suggesting the successful persistence of the selected individuals in the wild on an annual base.

Short-Term Selection to Diflubenzuron and Bacillus thuringiensis Var. Israelensis Differentially Affects the Winter Survival of Culex pipiens f. Pipiens and Culex pipiens f. Molestus (Diptera: Culicidae).

The Culex pipiens (Diptera: Culicidae) mosquito is of high medical importance as it is considered the prime vector of West Nile virus. In Europe, this species consists of two forms, named pipiens and molestus, that exhibit substantial differences in their overwintering biology. Diflubenzuron (DFB) and Bacillus thuringiensis var. israelensis (Bti) are two of the most used larvicides in mosquito control, including that of Culex pipiens. The high dependency on these two larvicides poses major concerns for resistance development. The evolution and stability of resistance to insecticides has been associated with fitness costs that may be manifested under stressful conditions, such as the winter period. This study investigated how short-term selection of pipiens and molestus forms to both larvicides affect their overwintering success. Larvae from each form were subjected to the same selective pressure (80% mortality) for three successive generations with DFB and Bti. At the end of this process, the winter survival between the selected populations and the controls (colonies without selection) was determined for each form. Selection to both larvicides significantly reduced the winter survival rates of molestus but not of pipiens form, indicating potential differences in the persistence of the selected individuals from year to year between the two forms.

Efficacy of polydimethylsiloxane against Culex pipiens (Diptera: Culicidae).

We evaluated, under controlled laboratory conditions, the insecticidal activity of polydimethylsiloxane (PDMS) for the control of the house mosquito, Culex pipiens. In a first series of bioassays, we tested the residual effect of different PDMS doses to control 3rd instar larvae. The label dose caused high mortality rates (>80%) even after 50 days from the initial application. Mortality levels at half the label dose were significantly higher compared to control, during the entire experimental period following a gradual decrease over time. Similar trends were observed when the 1/4 and the 1/8 of the label dose were applied with a much steeper decrease at long time intervals since the initial application especially for the lowest dose. However, after the 10th day of the bioassays and until the end of the experiments, mortality rates in the case of the label dose were higher compared to all other doses after the initial 10 days post application. Conversely, the insecticidal efficacy of PDMS against pupae was generally lower in comparison with larvae. Indeed, 10 days after the application, mortality was notably reduced, regardless of the dose rate tested. In a second series of bioassays, we assessed the efficacy of short exposures of different instars of larvae and nymphs to registered formulation and label dose. Mortality was well over 95% for 2nd instar larvae at exposure intervals ranging from 1 to 4 days. Significant mortality was recorded to 3rd instar larvae even at 15 min of exposure. Exposures longer than 30 min caused high mortality rates to 3rd instar larvae. Similar results were also recorded in pupae. Finally, in a third series of bioassays, the efficacy of PDMS in egg hatch was tested. Egg hatch was completely suppressed in the treated substrate (all egg rafts were "sinking" at the bottom of the test cups). Moreover, there was no oviposition in the case of the application of the label dose, while some egg rafts were recorded at one-eighth of the label dose. In both cases, it was recorded that PDMS acts as an oviposition deterrent. Τhe results of this study show that PDMS is effective in a wide range of conditions and application scenarios.

Reduction in Egg Fertility of Aedes albopictus Mosquitoes in Greece Following Releases of Imported Sterile Males.

Aedes albopictus, an invasive mosquito species, is currently well established in many European countries, posing high risks to human health. A preliminary trial using repetitive releases of irradiated sterile male mosquitoes was designed, implemented and evaluated for the first time in Greece. The main scope of this trial was to investigate the efficacy of sterile insect technique (SIT) on wild population egg hatch rate in Greece using mass-produced sterile male mosquitoes from another country (Italy). The study was conducted in Vravrona area, close to Athens International Airport (Attica Region). The number of eggs laid in ovitraps was similar in all intervention and control plots. However, a significant reduction in egg hatch rate was recorded in the SIT plot in comparison with both control plots starting two weeks after the first release. This trial validates the logistics (transportation, releases handling and monitoring) as a major step towards implementing efficient, environmentally safe control approaches as an additional tool against the invasive Aedes species in Greece and more widely in Europe.

Oviposition-deterrent effect of linalool - a compound of citrus essential oils - on female Mediterranean fruit flies, Ceratitis capitata (Diptera: Tephritidae).

BACKGROUND: Linalool is a natural scent, found in essential oils (EOs) of several plants. It is widely used as a fragrant, also possessing toxic, oviposition-deterrent and repellent properties against many insect species. Ceratitis capitata (medfly) is one of the most notorious insect pests for international fresh fruit production. Despite the available information on the repellent effects of EOs on herbivorous insects, possible oviposition-deterrent effects of linalool against C. capitata remain unknown. OBJECTIVES AND RESULTS: We tested whether different linalool concentrations, application methods on fruit hosts and time since application elicited oviposition-deterrence on medfly, from bitter oranges, apples and nectarines, under constant laboratory conditions. We also tested possible repellent properties of linalool on adult medflies in field conditions. Females drilled fewer oviposition stings and deposited fewer eggs per fruit in all linalool-treated hosts. Spraying and topical-droplet application were the most efficient methods on bitter oranges, although spraying was the least effective method in apples. Bitter oranges and nectarines that were offered to females immediately post-exposure to linalool received more oviposition stings and eggs than those offered 3 days post-exposure to linalool solutions. Conversely, apples tested 3 days following linalool application received significantly more oviposition stings and eggs than those tested immediately and 1 day post-exposure. More adults in total, and more females, were captured in traps located on untreated-control than on linalool-treated trees, especially in citrus hosts. CONCLUSION: Linalool has a potential future use in environmentally friendly control strategies against C. capitata. © 2020 Society of Chemical Industry.

Differential response to larval crowding of a long- and a short-lived medfly biotype.

Response of endophytic fruit fly species (Tephritidae) to larval crowding is a form of scramble competition that may affect important life history traits of adults, such as survival and reproduction. Recent empirical evidence demonstrates large differences in adult life history traits, especially longevity, among Mediterranean fruit fly (Ceratitis capitata; "medfly") biotypes obtained from different regions of the world. However, whether the evolution of long lifespan is associated with response to stress induced by larval crowding has not been fully elucidated. We investigated, under constant laboratory conditions, the response of a short- and a long-lived medfly biotypes to stress induced by larval crowding. Survival and development of larvae and pupae and the size of resulting pupae were recorded. The lifespan and age-specific egg production patterns of the obtained adults were recorded. Our findings reveal that increased larval density reduced immature survival (larvae and pupae) in the short-lived biotype but had rather neutral effects on the longed-lived one. Only larvae of the long-lived biotype were capable of prolonging their developmental duration under the highest crowding regime to successfully pupate and emerge as adults. Response of emerging adults to larvae crowding conditions was similar in the two medfly biotypes. Those individuals emerging from high larval density regimes had reduced longevity and fecundity. Long-lived biotype individuals, however, appeared to suffer a higher cost in longevity compared with the short-lived one. The importance of our findings to understand the evolution of long lifespan is discussed.

Towards improving sterile insect technique: Exposure to orange oil compounds increases sexual signalling and longevity in Ceratitis capitata males of the Vienna 8 GSS.

The Mediterranean fruit fly (medfly), Ceratitis capitata, is a notorious insect pest causing huge economic losses worldwide. The sterile insect technique (SIT) is widely used for its control. Using sexually mature sterilized males of the Vienna 8 (tsl) strain in the laboratory, we explored whether exposure of males to citrus compounds (separately or in a mixture) affects their sexual behaviour and if nutritional conditions and age modulate those effects. Exposed males exhibited increased sexual signalling compared to unexposed ones, particularly when fed a rich adult diet. Interestingly, and for the first time reported in medfly, exposure of Vienna 8 males to a mixture of citrus compounds increases longevity under poor adult diet conditions. We discuss the possible associated mechanisms and provide some practical implications of our results towards improving the effectiveness of SIT.

Toxic and hormetic-like effects of three components of citrus essential oils on adult Mediterranean fruit flies (Ceratitis capitata).

Plant essential oils (EOs) and a wide range of their individual components are involved in a variety of biological interactions with insect pests including stimulatory, deterrent, toxic and even hormetic effects. Both the beneficial and toxic properties of citrus EOs on the Mediterranean fruit fly (medfly) have been experimentally evidenced over the last years. However, no information is available regarding the toxic or beneficial effects of the major components of citrus EOs via contact with the adults of the Mediterranean fruit fly. In the present study, we explored the toxicity of limonene, linalool and α-pinene (3 of the main compounds of citrus EOs) against adult medflies and identified the effects of sub-lethal doses of limonene on fitness traits in a relaxed [full diet (yeast and sugar)] and in a stressful (sugar only) feeding environment. Our results demonstrate that all three compounds inferred high toxicity to adult medflies regardless of the diet, with males being more sensitive than females. Sub-lethal doses of limonene (LD20) enhanced the lifespan of adult medflies when they were deprived of protein. Fecundity was positively affected when females were exposed to limonene sub-lethal doses. Therefore, limonene, a major constituent of citrus EOs, induces high mortality at increased doses and positive effects on life history traits of medfly adults through contact at low sub-lethal doses. A hormetic-like effect of limonene to adult medflies and its possible underlying mechanisms are discussed.

The odor of a plant metabolite affects life history traits in dietary restricted adult olive flies.

Food quality shapes life history traits either directly or through response of individuals to additional environmental factors, such as chemical cues. Plant extracts used as food additives modulate key life history traits; however little is known regarding such effects for olfactory chemical cues. Exploiting an interesting experimental system that involves the olive fly (Bactrocera oleae) and the plant metabolite α-pinene we asked whether exposure of adults to this compound modulates adult longevity and female reproduction in similar manner in a stressful - dietary (protein) restricted (DR) and in a relaxed- full diet (FD) feeding environment. Accordingly, we exposed males and females to the aroma of α-pinene and measured lifespan and age-specific fecundity in the above two dietary contexts. Our results demonstrate that exposure to α-pinene increased longevity in males and fecundity in females only under dietary restricted conditions. In relaxed food conditions, females exposed to α-pinene shifted high egg-laying towards younger ages compared to non-exposed ones. This is the first report demonstrating that a plant compound affects key life history traits of adult olive flies through olfaction. These effects are sex-specific and more pronounced in dietary restricted adults. Possible underlying mechanisms and the ecological significance are discussed.

Aromatized to find mates: α-pinene aroma boosts the mating success of adult olive fruit flies.

BACKGROUND: Contrary to other Tephritidae, female but also male olive flies, Bactrocera oleae release pheromones during their sexual communication. Alpha-pinene, a common plant volatile found in high amounts in unripe olive fruit and leaves has been detected as one of the major components of the female pheromone. However, possible effects of α-pinene and that of other host volatiles on the mating behavior of the olive fly have not been investigated. METHODOLOGY: Using wild olive flies, reared on olive fruit for 3 generations in the laboratory, we explored whether exposure of male and female olive flies to α-pinene affects their sexual performance. RESULTS: Exposure of sexually mature adult olive flies to the aroma of α-pinene significantly increases the mating performance over non-exposed individuals. Interestingly, exposure to α-pinene boosts the mating success of both males and female olive flies. CONCLUSIONS: This is the first report of such an effect on the olive fly, and the first time that a single plant volatile has been reported to induce such a phenomenon on both sexes of a single species. We discuss the possible associated mechanism and provide some practical implications.