Sterile Insect Technique (SIT) field trial targeting the suppression of Aedes albopictus in Greece.

The sterile insect technique (SIT) involves releasing large numbers of sterile males to outcompete wild males in mating with females, leading to a decline in pest populations. In the current study, we conducted a suppression trial in Greece against the invasive dengue vector mosquito Aedes albopictus (Skuse) through the weekly release of sterile males for 22 weeks from June to September 2019. Our approach included the long-distance transport of sterile mosquitoes, and their release at a density of 2,547 ± 159 sterile males per hectare per week as part of an area-wide integrated pest management strategy (AW-IPM). The repeated releases of sterile males resulted in a gradual reduction in egg density, reaching 78% from mid-June to early September. This reduction remained between 70% and 78% for four weeks after the end of the releases. Additionally, in the SIT intervention area, the ovitrap index, representing the percentage of traps containing eggs, remained lower throughout the trial than in the control area. This trial represents a significant advance in the field of mosquito control, as it explores the viability and efficacy of producing and transporting sterile males from a distant facility to the release area. Our results provide valuable insights for future SIT programmes targeting Ae. Albopictus, and the methodology we employed can serve as a starting point for developing more refined and effective release protocols, including the transportation of sterile males over long distances from production units to intervention areas.

Title: Essai sur le terrain de la Technique de l'Insecte Stérile (TIS) ciblant la suppression d'Aedes albopictus en Grèce. Abstract: La technique de l'insecte stérile (TIS) consiste à libérer un grand nombre de mâles stériles pour supplanter les mâles sauvages lors de l'accouplement avec les femelles, entraînant ainsi un déclin des populations de nuisibles. Dans la présente étude, nous avons mené un essai de suppression en Grèce contre le moustique vecteur invasif de la dengue, Aedes albopictus (Skuse), par le biais de la libération hebdomadaire de mâles stériles pendant 22 semaines de juin à septembre 2019. Notre approche comprenait le transport sur de longues distances de moustiques stériles, et leur lâcher à une densité de 2 547 ± 159 mâles stériles par hectare et par semaine dans le cadre d'une stratégie de lutte intégrée contre les nuisibles à l'échelle de la zone (AW-IPM). Les lâchers répétés de mâles stériles ont entraîné une réduction progressive de la densité des œufs, atteignant 78 % de la mi-juin au début septembre. Cette réduction est restée entre 70 % et 78 % pendant quatre semaines après la fin des lâchers. De plus, dans la zone d'intervention de la TIS, l'indice d'oviposition, représentant le pourcentage de pièges contenant des œufs, est resté plus faible que dans la zone témoin tout au long de l'essai. Cet essai représente une avancée significative dans le domaine de la lutte contre les moustiques, car il explore la viabilité et l'efficacité de la production et du transport de mâles stériles depuis une installation éloignée vers la zone de lâcher. Nos résultats fournissent des informations précieuses pour les futurs programmes de TIS ciblant Ae. albopictus et la méthodologie que nous avons utilisée pourra servir de point de départ pour développer des protocoles de libération plus raffinés et plus efficaces, y compris le transport de mâles stériles sur de longues distances depuis les unités de production jusqu'aux zones d'intervention.

The Roles of Mating, Age, and Diet in Starvation Resistance in Bactrocera oleae (Olive Fruit Fly).

The olive fruit fly (Bactrocera oleae (Rossi) (Diptera: Tephritidae)), although a pest of major economic importance for the olive industry, has not been sufficiently studied with respect to the factors affecting its survival resistance to food deprivation. In the present study, we examined the effect of the interaction between mating status (virgin/mated), age class (11-20/21-30/31-40/41-50), and diet quality (protein plus sugar or only sugar) on starvation resistance in B. oleae under constant laboratory conditions. We conducted a total of 16 treatments (2 × 4 × 2 = 16) for each gender. Our results showed that starvation resistance in B. oleae did not differ significantly between females and males. The main conclusions of our study regarding mating status, age, and diet indicated that mated adults showed much less starvation resistance compared to virgins, younger adults endured longer, and the adults fed a restricted diet endured longer than those fed a full diet. A three-way interaction between mating status, diet, and age class was also identified and was the same for both genders. The interaction between mating status, age class, and diet also had a significant influence on starvation resistance in both sexes.

Plant viruses induce plant volatiles that are detected by aphid parasitoids.

Aphis gossypii (Sternorrhyncha: Aphididae) aphids are vectors of important plant viruses among which cucumber mosaic virus (CMV) and potato virus Y (PVY). Virus-infected plants attract aphid vectors and affect their behavior and growth performance either positively or negatively depending on mode of transmission. Viruses cause changes in the composition and the amount of volatile organic compounds (VOCs) released by the plant that attract aphids. The aphid parasitoid Aphidius colemani (Hymenoptera: Aphelinidae) has been shown to have higher parasitism and survival rates on aphids fed on virus-infected than aphids fed on non-infected plants. We hypothesized that parasitoids distinguish virus-infected plants and are attracted to them regardless of the presence of their aphid hosts. Herein, we examined the attraction of the A. colemani parasitoid to infected pepper plants with each of CMV or PVY without the presence of aphids. The dynamic headspace technique was used to collect VOCs from non-infected and CMV or PVY-infected pepper plants. Identification was performed with gas chromatography-mass spectrometry (GC-MS). The response of the parasitoids on virus-infected vs non-infected pepper plants was tested by Y-tube olfactometer assays. The results revealed that parasitoids displayed a preference to CMV and PVY infected plants compared to those that were not infected.

Laboratory study on larval competition between two related mosquito species: Aedes (Stegomyia) albopictus and Aedes (Stegomyia) cretinus.

Aedes albopictus (Asian tiger mosquito) and Aedes cretinus are closely related mosquito species with similar morphological and bio-ecological characteristics. These species have been detected in specific areas of Athens, Greece, with Ae. albopictus developing significantly higher population densities than the native mosquito Ae. cretinus. In a laboratory factorial experimental design, we investigated the potential of interspecific and intraspecific competition between larvae of these species under various food and population density conditions. Duration of larval development, survival rate from the first larval instar until adulthood and the wing length of females were measured. When these two species developed on their own, larvae developed faster and the females were larger at high food provision, indicating intraspecific competition. When the two species developed in the same environment and food provision was low, Ae. albopictus outcompeted Ae. cretinus. In particular, the larval developmental time when these species competed with each other was 1.3 to 2.4 days shorter for Ae. albopictus and 0.9 to 1.4 days longer for Ae. cretinus, compared with single species development. Interspecific competition resulted to larger Ae. albopictus females at limited food availability and low density of individuals. Our findings indicate that Ae. albopictus is a superior competitor to Ae. cretinus, primarily at limited larval food resources, and this may account for the expansion of Ae. albopictus and the limited presence of Ae. cretinus in areas of Athens, Greece, where these related species co-exist.

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.

Seasonal Appearance, Abundance, and Host Preference of Philaenus spumarius and Neophilaenus campestris (Hemiptera: Aphrophoridae) in Olive Groves in Greece.

Xylella fastidiosa Wells et al. (Xanthomonadales: Xanthomonadaceae) is a xylem inhabiting bacterium which is exclusively transmitted by xylem sap feeding insects. Among them, Philaenus spumarius Linnaeus and Neophilaenus campestris Fallén are the most abundant species in Europe. During 2018 and 2019 a survey was conducted in olive groves in Greece aimed to improve the knowledge about the biology and ecology of those spittlebugs in areas with Mediterranean climate. Moreover, the host preference of the nymphs was studied. The nymphs of P. spumarius and N. campestris were observed between early March and middle May depending on geographic location and year. The spittlebug adults were present during two periods every year, one in spring and another one in autumn and early winter. During summer months the spittlebugs were totally absent from olive groves. Our observation on host plant selection revealed that there was a strong preference of N. campestris nymphs for plants belonging to the family Poaceae. On the contrary, P. spumarius nymphs were polyphagous. The most preferable plants for the nymphs of this species belonged to the families Asteraceae and Fabaceae. The importance of these findings for control measures for these spittlebugs is discussed.

Knowledge, Attitude, and Practices Survey in Greece before the Implementation of Sterile Insect Technique against Aedes albopictus.

Community involvement in Aedes albopictus management can be very efficient and result in raising awareness among citizens. Toward this end, a door-to-door campaign can encourage active community participation in vector control. The current study describes the results of an intervention where a KAP (knowledge, attitude, practices) survey tool was paired with a door-to-door campaign and was implemented as an intervention method in Vravrona area (Attica, Greece) before the release of sterile males (sterile insect technique, SIT) against Aedes albopictus. The KAP tool was used to shed light on the knowledge, practices, and attitudes of local community members in order to better prepare and motivate participation in household mosquito control and to assess current understanding of SIT. Each household also received specific information about mosquito source habitat in their own yards at the time of the initial KAP survey. These household data were complemented by standardized mosquito trapping in the municipality. Our findings indicate that citizens' attitude toward SIT ranged from indecisive to fully supportive, while 77.5% of the respondents agreed that the SIT has many advantages over chemical control methods. Furthermore, the results demonstrate that using the door-to-door campaign as an intervention and prerelease method before SIT can suppress the initial mosquito population and potentially improve its efficacy. Lastly, we show that the presence of local municipality officials during door-to-door visits was associated with increased willingness from the residents to participate in the intervention.

Efficacy and residual activity of commercially available entomopathogenic nematode strains for Mediterranean fruit fly control and their ability to infect infested fruits.

BACKGROUND: Entomopathogenic nematodes (EPN) show potential in controlling larvae of the Mediterranean fruit fly (medfly) Ceratitis capitate, but previous studies mainly concern species and strains that are not commercially available. The use of EPN for control of Mediterranean fruit fly is further hampered by the cost of using nematodes. In this study, the efficacy and residual activity of commercial strains of three EPN species, Steinernema carpοcapsae, S. feltiae and Heterοrhabditis bacteriοphοra medfly) C. capitata, in the soil substrate and inside fruits were evaluated. RESULTS: Suspensions of these species were applied at a dose of 1.5 mi m-2 on a soil substrate wherein medfly larvae were added sequentially for a period of 4 weeks post application at 20 °C. S. feltiae provided the highest suppression up to 50% as assessed by adult medfly emergence because it had the highest immediate activity and long residual activity. Furthermore, S. feltiae, and to a lesser degree S. carpocapsae, were able to move and infect medfly larvae inside infested apples and oranges left in the surface of the substrate wherein EPN were applied, reducing significantly adult medfly emergence (60-78%). CONCLUSION: These results support the efficacy and feasibility of applying a single, relatively low dose of S. feltiae in autumn, off-season, targeting overwintering medfly larvae with the scope of reducing the number of adult medflies emerging later in the new season.

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.

The Effect of Weather Variables on Mosquito Activity: A Snapshot of the Main Point of Entry of Cyprus.

Mosquitoes are vectors of pathogens, causing human and animal diseases. Their ability to adapt and expand worldwide increases spread of mosquito-borne diseases. Climate changes contribute in enhancing these "epidemic conditions". Understanding the effect of weather variables on mosquito seasonality and host searching activity contributes towards risk control of the mosquito-borne disease outbreaks. To enable early detection of Aedes invasive species we developed a surveillance network for both invasive and native mosquitoes at the main point of entry for the first time in Cyprus. Mosquito sampling was carried out for one year (May 2017-June 2018), at bimonthly intervals around Limassol port. Morphological and molecular identification confirmed the presence of 5 species in the study region: Culex. pipiens, Aedes detritus, Ae. caspius, Culiseta longiareolata and Cs. annulata. No invasive Aedes mosquito species were detected. The Pearson's correlation and multiple linear regression were used to compare number of sampled mosquitoes and weather variables for three most numerous species (Cx. pipiens, Ae. detritus and Ae. caspius). The population densities of the most numerous species were highest from February to April. Number of Cx. pipiens (-0.48), Ae. detritus (-0.40) and Ae. caspius (-0.38) specimens sampled was negatively correlated with average daily temperature. Monthly relative humidity showed positive correlation with the numbers of the species sampled, Cx. pipiens (0.66) Ae. detritus (0.68), and Ae. caspius (0.71). Mosquito abundance of Cx. pipiens (0.97) and Ae. detritus (0.98) was strongly correlated to seasonal precipitation as well. Our work is a stepping stone to further stimulate implementation of International Health Regulations and implementation of early warning surveillance system for detection of invasive Aedes mosquitoes, native mosquitoes and arboviruses they may transmit. A network for the surveillance of both invasive and native mosquito species at the main point of entry for the first time in Cyprus was developed. Number of mosquitoes sampled was correlated with weather factors to identify parameters that might predict mosquito activity and species distribution to the prevention of international spread of vector mosquitoes and vector-borne diseases.

The influence of organic matter content and media compaction on the dispersal of entomopathogenic nematodes with different foraging strategies.

In laboratory experiments, we investigated how media with varying ratio of peat:sand and two levels of compaction influence dispersal success of entomopathogenic nematode (EPN) species with different foraging strategies: Steinernema carpocapsae (ambusher), Heterorhabditis downesi (cruiser) and Steinernema feltiae (intermediate). Success was measured by the numbers of nematodes moving through a 4 cm column and invading a wax moth larva. We found that both compaction and increasing peat content generally decreased EPN infective juvenile (IJ) success for all three species. Of the three species, H. downesi was the least affected by peat content, and S. carpocapsae was the most adversely influenced by compaction. In addition, sex ratios of the invading IJs of the two Steinernema species were differentially influenced by peat content, and in the case of S. feltiae, sex ratio was also affected by compaction. This indicates that dispersal of male and female IJs is differentially affected by soil parameters and that this differentiation is species-specific. In conclusion, our study shows that organic matter: sand ratio and soil compaction have a marked influence on EPN foraging behaviour with implications for harnessing them as biological pest control agents.

Metabolomics of aging assessed in individual parasitoid wasps.

Metabolomics studies of low-biomass organisms, such as small insects, have previously relied on the pooling of biological samples to overcome detection limits, particularly using NMR. We show that the differentiation of metabolite profiles of individual 1 mg parasitoid wasps of different ages is possible when using a modified sample preparation and a combination of untargeted NMR and LC-MS based metabolomics. Changes were observed between newly emerged and older wasps in glycerolipids, amino acids and circulatory sugars. This advance in chemical profiling has important implications for the study of the behaviour and ecology of parasitoids and many other species of small organisms because predictions and observations are typically made at the level of the individual. Thus, the metabolomic state of low-biomass individuals can now be related to their behaviour and ecological performance. We discuss specifically the utility of age-related metabolomic profiling but our new approach can be applied to a wide range of biological research.

Detecting non-binomial sex allocation when developmental mortality operates.

Optimal sex allocation theory is one of the most intricately developed areas of evolutionary ecology. Under a range of conditions, particularly under population sub-division, selection favours sex being allocated to offspring non-randomly, generating non-binomial variances of offspring group sex ratios. Detecting non-binomial sex allocation is complicated by stochastic developmental mortality, as offspring sex can often only be identified on maturity with the sex of non-maturing offspring remaining unknown. We show that current approaches for detecting non-binomiality have limited ability to detect non-binomial sex allocation when developmental mortality has occurred. We present a new procedure using an explicit model of sex allocation and mortality and develop a Bayesian model selection approach (available as an R package). We use the double and multiplicative binomial distributions to model over- and under-dispersed sex allocation and show how to calculate Bayes factors for comparing these alternative models to the null hypothesis of binomial sex allocation. The ability to detect non-binomial sex allocation is greatly increased, particularly in cases where mortality is common. The use of Bayesian methods allows for the quantification of the evidence in favour of each hypothesis, and our modelling approach provides an improved descriptive capability over existing approaches. We use a simulation study to demonstrate substantial improvements in power for detecting non-binomial sex allocation in situations where current methods fail, and we illustrate the approach in real scenarios using empirically obtained datasets on the sexual composition of groups of gregarious parasitoid wasps.

Encyrtid parasitoids of soft scale insects: biology, behavior, and their use in biological control.

Parasitoids of the hymenopterous family Encyrtidae are one of the most important groups of natural enemies of soft scale insects and have been used extensively in biological control. We summarize existing knowledge of the biology, ecology, and behavior of these parasitoids and how it relates to biological control. Soft scale stage/size and phenology are important determinants of host range and host utilization, which are key aspects in understanding how control by these parasitoids is exerted. Furthermore, the nutritional ecology of encyrtids and their physiological interactions with their hosts affect soft scale insect population dynamics. Lastly, the interactions among encyrtids, heteronomous parasitoids, and ants shape parasitoid species complexes and consequently have a direct impact on the biological control of soft scale insects.

Mutually beneficial host exploitation and ultra-biased sex ratios in quasisocial parasitoids.

Selfish interests usually preclude resource sharing, but under some conditions collective actions enhance per capita gains. Such Allee effects underlay early explanations of social evolution but current understanding focusses on kin selection (inclusive fitness). We find an Allee effect that explains unusual quasisociality (cooperative brood care) among parasitoid wasps without invoking or precluding kin selection effects. In Sclerodermus harmandi, individual females produce most offspring when exploiting small hosts alone. However, larger hosts are more successfully exploited by larger groups of females, with the per-female benefits outweighing the costs of host sharing. Further, the extremely biased sex ratios (97% female) are better explained by mutually beneficial female-female interactions that increase the reproductive value of daughters (local resource enhancement), rather than by the usually invoked local mate competition between males. Thus, atypical quasisocial behaviour in a parasitoid wasp directly enhances reproductive success and selects for very extremely female-biased sex ratios.

Parasitoid developmental mortality in the field: patterns, causes and consequences for sex ratio and virginity.

1. Sex ratio theory predicts that developmental mortality can affect sex ratio optima under Local Mate Competition and also lead to 'virgin' broods containing only females with no sibling-mating opportunities on maturity. 2. Estimates of developmental mortality and its sex ratio effects have been laboratory based, and both models and laboratory studies have treated mortality as a phenomenon without identifying its biological causes. 3. We contribute a large set of field data on Metaphycus luteolus Timberlake (Hymenoptera: Encyrtidae), an endoparasitoid of soft scale insects (Hemiptera: Coccidae), which has sex allocation conditional on host quality and female-biased brood sex ratios. Developmental mortality within broods can be both assessed and attributed to distinct causes, including encapsulation by the host and larval-larval competition. 4. Thirty per cent of M. luteolus offspring die during development with 65% of this mortality because of encapsulation and 28% because of larval competition. The distributions of mortality overall and for each cause of mortality separately were overdispersed. 5. The probability of an individual being encapsulated increased with clutch size, while the probability of being killed by a brood mate declined with increasing clutch size and with increasing per capita availability of resources. 6. The sexual compositions of broods at emergence were influenced by both the degree and the type of mortality operating. At higher levels of mortality, single sex broods were more common and sex ratios were less precise. Overall, virginity was more prevalent than predicted and was more greatly affected by the occurrence of competition than by other sources of mortality, almost certainly because competition tended to eliminate males. 7. The reproductive and developmental biology of M. luteolus appears to be influenced by a complex interplay of maternal clutch size and sex allocation strategies, offspring-offspring developmental interactions, host defence mechanisms and postemergence mating behaviour. Despite the great sophistication of sex ratio theory, it has not yet evolved to the point where it is capable of considering all of these influences simultaneously.