Upscaling irradiation protocols of Aedes albopictus pupae within an SIT program in Reunion Island.

The implementation of the sterile insect technique against Aedes albopictus relies on many parameters, in particular on the success of the sterilization of males to be released into the target area in overflooding numbers to mate with wild females. Achieving consistent sterility levels requires efficient and standardized irradiation protocols. Here, we assessed the effects of exposure environment, density of pupae, irradiation dose, quantity of water and location in the canister on the induced sterility of male pupae. We found that the irradiation of 2000 pupae in 130 ml of water and with a dose of 40 Gy was the best combination of factors to reliably sterilize male pupae with the specific irradiator used in our control program, allowing the sterilization of 14000 pupae per exposure cycle. The location in the canister had no effect on induced sterility. The results reported here allowed the standardization and optimization of irradiation protocols for a Sterile Insect Technique program to control Ae. albopictus on Reunion Island, which required the production of more than 300,000 sterile males per week.

Impact of substerilizing dose on histological changes in gonads and ovaries of Ephestia cautella (Lepidoptera: Pyralidae) by gamma radiation.

To investigate the effect of Gamma radiation on the reproductive systems of emerged female and male of Ephestia cautella (Walker) moths, mature pupae of both sexes were irradiated with doses of 50, 100 and 150 Gy. Histological study of the treated individuals showed in females that the ovaries appear sever damage in the follicular epithelium at all doses, which become thinness and separated from developing oocytes, moreover, some of the nurse cells were rupture. In males which treated with 150 Gy, it was noticed retardation in the stages of spermatogenesis and few numbers of sperm bundles and their dispersion in the testicular follicles' adults, on the other hand, the doses of 50 and 100 Gy showed little or moderate effects on the structure of the testis contents.

Radiation dose-rate is a neglected critical parameter in dose-response of insects.

Reproductive sterility is the basis of the sterile insect technique (SIT) and essential for its success in the field. Numerous factors that influence dose-response in insects have been identified. However, historically the radiation dose administered has been considered a constant. Efforts aiming to standardize protocols for mosquito irradiation found that, despite carefully controlling many variable factors, there was still an unknown element responsible for differences in expected sterility levels of insects irradiated with the same dose and handling protocols. Thus, together with previous inconclusive investigations, the question arose whether dose really equals dose in terms of biological response, no matter the rate at which the dose is administered. Interestingly, the dose rate effects studied in human nuclear medicine indicated that dose rate could alter dose-response in mammalian cells. Here, we conducted experiments to better understand the interaction of dose and dose rate to assess the effects in irradiated mosquitoes. Our findings suggest that not only does dose rate alter irradiation-induced effects, but that the interaction is not linear and may change with dose. We speculate that the recombination of reactive oxygen species (ROS) in treatments with moderate to high dose rates might minimize indirect radiation-induced effects in mosquitoes and decrease sterility levels, unless dose along with its direct effects is increased. Together with further studies to identify an optimum match of dose and dose rate, these results could assist in the development of improved methods for the production of high-quality sterile mosquitoes to enhance the efficiency of SIT programs.

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.

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.

60Co-γ Radiation Alters Developmental Stages of Zeugodacus cucurbitae (Diptera: Tephritidae) Through Apoptosis Pathways Gene Expression.

Radiation is considered as a promising insect pest control strategy for minimizing postharvest yield losses. Among various techniques, irradiation is a method of choice as it induces lethal biochemical or molecular changes that cause a downstream cascade of abrupt physiological abnormalities at the cellular level. In this study, we evaluated the effect of 60Co-γ radiation on various developmental stages of Zeugodacus cucurbitae Coquillett and subsequent carry-over effects on the progeny. For this purpose, we treated eggs with 30- and 50-Gy radiation doses of 60Co-γ. We found that radiation significantly affected cellular antioxidants, insect morphology, and gene expression profiles. Our results indicate that in response to various doses of irradiation reactive oxygen species, catalase, peroxidase, and superoxide dismutase activities were increased along with a significant increase in the malondialdehyde (MDA) content. We observed higher mortality rates during the pupal stage of the insects that hatched from irradiated eggs (50 Gy). Furthermore, the life span of the adults was reduced in response to 50 Gy radiation. The negative effects carried over to the next generation were marked by significantly lower fecundity in the F1 generation of the irradiation groups as compared to control. The radiation induced morphological abnormalities at the pupal, as well as the adult, stages. Furthermore, variations in the gene expression following irradiation are discussed. Taken together, our results signify the utility of 60Co-γ radiation for fruit fly postharvest management.

Comparison between pupal and adult X-ray radiation, designed for the sterile insect technique for Aedes albopictus control.

Aedes albopictus is a vector of several human viral diseases, including dengue, chikungunya, and Zika. New control method for Aedes albopictus is needed to replace traditional methods such as chemical insecticides which induce resistance, environmental contamination and toxicity to human. In sterile insect technique (SIT), male mosquitoes are sterilized by γ-ray or X-ray irradiation before released. In this study, the relative effectiveness of X-ray irradiation as a mosquito SIT was investigated. Both pupal and adult Aedes albopictus were subjected to different radiation doses and their emergence, survivorship, longevity, induced sterility, and male mating competitiveness were evaluated. Relative to controls, irradiation had no significant effect on emergence and survivorship but significantly reduce adult longevity. Induced sterility were essentially same for both irradiated pupal and adult. At a dose of 40 Gy, 97% and 100% sterility was respectively achieved for males and females. Mating competitiveness was reduced both in adult males and those derived from pupae exposed to 40 Gy. However, populations can be suppressed by increasing the release ratio (sterile: normal). When the release ratio was 7:1, 74% of the wild population could be suppressed. Overall, the results of the present study showed that SIT based on X-Ray irradiation is scientific and feasible to control Aedes albopictus.

Effect of gamma radiation as a post-harvest disinfestation treatment against life stages of the coffee berry borer, Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae).

Purpose: Gamma radiation is mainly used for disinfesting insect pests as an alternative for harmful fumigants. The specific dose of radiation is known to affect different developmental stages of insect pests. The study was conducted to determine the effective irradiation doses for inhibition of developmental stages and adult longevity of the coffee berry borer, Hypothenemus hampei (Ferrari). Materials and methods: Irradiation was carried with the following doses: five levels between 0.01 and 0.16 kGy for eggs, seven levels between 0.10 and 2.00 kGy for larva and prepupa, six levels between 0.10 and 1.60 kGy for pupa and ten levels between 0.10 and 3.20 kGy for adults. Results: Egg development was completely arrested at 0.160 kGy. A dose of 2.00 kGy caused 100% mortality in the first and second instar larva and 98.99% mortality in prepupa. The dose of 1.60 kGy prevented adult eclosion from the irradiated pupa. The adult mortality was 100% at 3.20 kGy. Conclusion: A dose of 3.20 kGy could successfully provide complete security from all developmental stages of H. hampei and prevent yield loss in green coffee as well as the spread of the pest.

UV-C radiation during the pupal stage affects morphological changes of wings in Tribolium castaneum (Col; Tenebrionidae).

Purpose: To reveal the effects of Ultraviolet-C (UV-C) on the elytra and hindwing morphology of Tribolium castaneum. Material and methods: Zero-day-old-pupae were irradiated with UV-C at a distance of 35 cm for 1, 2, 4, 8, 16, 32, or 64 min. Changes in wing morphologies were examined using light and scanning electron microscope. Results: UV-C radiation decreased the adult emergence rate and the insect body mass. Morphological changes of the elytra and hindwings in the adults were classified into nine grades. The treated insects had wrinkled and split elytra, and hindwings were not folded properly. Radiation altered the size of elytra, hindwings and wing shape. An analysis of the color intensity indicated that the irradiated beetles had darker elytra. The veins of hindwings became darker, while the membranous area had a lighter color than the control. UV-C radiation also affected the thickness of the elytra. Scanning electron microscopy revealed that UV-C caused deformity of elytra surface and decreased the number of hair sensilla. Conclusions: Results indicate that the elytra and hindwing morphology were altered by UV-C radiation. However, further analysis is required to evaluate the response of T. castaneum to UV-C radiation at the gene level.

Effect of Low-Oxygen Conditions Created by Modified Atmosphere Packaging on Radiation Tolerance in Drosophila suzukii (Diptera: Drosophilidae) in Sweet Cherries.

Modified atmosphere packaging (MAP) creates a low-oxygen (O2) environment that can increase the shelf life of fresh produce by decreasing respiration and the growth of pathogens. Low oxygen may also increase insect tolerance to irradiation (IR), and the use of MAP with products treated by IR to control quarantine pests before export may inadvertently compromise treatment efficacy. Spotted wing drosophila, Drosophila suzukii Matsumura (Diptera: Drosophilidae) is a quarantine pest of stone and small fruits and a potential target for postharvest IR treatment. The effect of low oxygen generated by MAP at ambient temperatures on the radiation tolerance of D. suzukii infesting sweet cherries was examined. Early pupal stage D. suzukii were inserted into ripe sweet cherries and treated by 1) MAP + IR, 2) IR alone, 3) MAP alone, or 4) no MAP and no IR and held for adult emergence. Three types of commercially available MAP products were tested that produced different oxygen concentrations between 3 and 15%, and a sublethal radiation dose (60 Gy) was used to allow comparisons between the treatments. Xtend PP61 bags (3.2-4.8% O2), Xtend PP71 bags (5.4-8.6% O2), and Xtend PP53 bags (13.6-15.4% O2) did not enhance survivorship to the adult stage in D. suzukii pupae irradiated at 60 Gy in sweet cherries. MAP use should not compromise phytosanitary IR treatment against D. suzukii in exported sweet cherries or other fruit.

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

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

Cloning and functional characterizations of an apoptogenic Hid gene in the Scuttle Fly, Megaselia scalaris (Diptera; Phoridae).

Although the mechanisms of apoptotic cell death have been well studied in the fruit fly, Drosophila melanogaster, it is unclear whether such mechanisms are conserved in other distantly related species. Using degenerate primers and PCR, we cloned a proapoptotic gene homologous to Head involution defective (Hid) from the Scuttle fly, Megaselia scalaris (MsHid). MsHid cDNA encodes a 197-amino acid-long polypeptide, which so far is the smallest HID protein. PCR analyses revealed that the MsHid gene consists of four exons and three introns. Ectopic expression of MsHid in various peptidergic neurons and non-neuronal tissues in Drosophila effectively induced apoptosis of these cells. However, deletion of either conserved domain, N-terminal IBM or C-terminal MTS, abolished the apoptogenic activity of MsHID, indicating that these two domains are indispensable. Expression of MsHid was found in all life stages, but more prominently in embryos and pupae. MsHid is actively expressed in the central nervous system (CNS), indicating its important role in CNS development. Together MsHID is likely to be an important cell death inducer during embryonic and post-embryonic development in this species. In addition, we found 2-fold induction of MsHid expression in UV-irradiated embryos, indicating a possible role for MsHid in UV-induced apoptosis.

UVB Radiation Delays Tribolium castaneum Metamorphosis by Influencing Ecdysteroid Metabolism.

Ultraviolet B (UVB) radiation is an important environmental factor. It is generally known that UVB exhibits high genotoxicity due to causing DNA damage, potentially leading to skin carcinogenesis and aging in mammals. However, little is known about the effects of UVB on the development and metamorphosis of insects, which are the most abundant terrestrial animals. In the present study, we performed dose-response analyses of the effects UVB irradiation on Tribolium castaneum metamorphosis, assessed the function of the T. castaneum prothoracicotropic hormone gene (Trcptth), and analyzed ecdysteroid pathway gene expression profile and ecdysterone titers post-UVB irradiation. The results showed that UVB not only caused death of T. castaneum larvae, but also delayed larval-pupal metamorphosis and reduced the size and emergence rate of pupae. In addition, we verified the function of Trcptth, which is responsible for regulating metamorphosis. It was also found that the expression profiles of Trcptth as well as ecdysteroidogenesis and response genes were influenced by UVB radiation. Therefore, a disturbance pulse of ecdysteroid may be involved in delaying development under exposure to irradiation. To our knowledge, this is the first report indicating that UVB can influence the metamorphosis of insects. This study will contribute to a better understanding of the impact of UVB on signaling mechanisms in insect metamorphosis.

The Effect of Ultraviolet-A Radiation Exposure on the Reproductive Ability, Longevity, and Development of the Dialeurodes citri (Homoptera: Aleyrodidae) F1 Generation.

Ultraviolet (UV) light has been used worldwide to monitor and trap insect pests. Whitefly adults show conspicuous positive phototactic behavior toward UV light stimuli; however, knowledge of the effect of UV light exposure on various life-history parameters of Dialeurodes citri remains limited. The present research aimed to investigate the effect of ultraviolet radiation (UV-A; long-wave) exposure on the reproduction and longevity of D. citri adults as well as the development of immature (eggs, larvae, and pupae) flies in the F1 generation. Paired D. citri adults were exposed to UV-A radiation for different periods (0, 1, 4, and 7 h/d) until the end of their life. The results of the experiment revealed that fecundity and oviposition rates increased when adults were irradiated for 1 and 4 h/d, but interestingly, both were significantly decreased compared with those of the controls after the longest exposure time (7 h/d). The longevity of adults of both sexes and the cumulative survival of F1 immatures were decreased with increased exposure time. Exposure to UV-A radiation prolonged the developmental time of immature stages, and a positive correlation was observed with exposure time. Exposure to UV light significantly inhibited egg hatching, larval development, pupation, and adult emergence. To the best of our knowledge, this is the first study describing the effect of UV radiation on a homopteran insect pest. This research may provide a foundation for the scientific community to use UV light in the field as an integrated pest management strategy to control this devastating agricultural pest.

Developmental inhibition and DNA damage of Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) by gamma radiation.

PURPOSE: Gamma radiation on Helicoverpa armigera Hübner was performed to assess developmental inhibition and to identify a potential quarantine treatment dose of the radiation. MATERIALS AND METHODS: Gamma radiation ((60)Co) treatment at different doses of 50, 100, 200, 300, and 400 Gy was carried out with egg, larvae, pupae and adults of H. armigera. RESULTS: Gamma radiation induced developmental inhibition of all stages of H. armigera. The effective dose values required for inhibition 99% (ED(99)) of hatching, pupation and adult emergence from the irradiated eggs were 550.7, 324.9 and 136.4 Gy, respectively. ED(99) values for inhibition of the larvae to adult emergence was 200.0 Gy. Irradiation on pupae could not completely inhibit adult emergence even at 400 Gy. ED(99) value for inhibition of F(1) egg hatchability from the irradiated adults was estimated to be 229.5 Gy. CONCLUSIONS: This study suggests that gamma radiation is a possible alternative to phytosanitary treatments. Irradiation treatment with minimum dose of 200 Gy can be suggested as optimum dose for larval treatment in quarantine.

Enhanced UV-B radiation during pupal stage reduce body mass and fat content, while increasing deformities, mortality and cell death in female adults of solitary bee Osmia bicornis.

The effects of enhanced UV-B radiation on the oogenesis and morpho-anatomical characteristics of the European solitary red mason bee Osmia bicornis L. (Hymenoptera: Megachilidae) were tested under laboratory conditions. Cocooned females in the pupal stage were exposed directly to different doses (0, 9.24, 12.32, and 24.64 kJ/m(2) /d) of artificial UV-B. Our experiments revealed that enhanced UV-B radiation can reduce body mass and fat body content, cause deformities and increase mortality. Following UV exposure at all 3 different doses, the body mass of bees was all significantly reduced compared to the control, with the highest UV dose causing the largest reduction. Similarly, following UV-B radiation, in treated groups the fat body index decreased and the fat body index was the lowest in the group receiving the highest dose of UV radiation. Mortality and morphological deformities, between untreated and exposed females varied considerably and increased with the dose of UV-B radiation. Morphological deformities were mainly manifested in the wings and mouthparts, and occurred more frequently with an increased dose of UV. Cell death was quantified by the Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (DNA fragmentation) during early stages of oogenesis of O. bicornis females. The bees, after UV-B exposure exhibited more germarium cells with fragmented DNA. The TUNEL test indicated that in germarium, low doses of UV-B poorly induced the cell death during early development. However, exposure to moderate UV-B dose increased programmed cell death. In females treated with the highest dose of UV-B the vast majority of germarium cells were TUNEL-positive.

Influence of modified atmosphere packaging on radiation tolerance in the phytosanitary pest melon fly (Diptera: Tephritidae).

Modified atmosphere packaging (MAP) produces a low-oxygen (O2) environment that can increase produce shelf life by decreasing product respiration and growth of pathogens. However, low O2 is known to increase insect tolerance to irradiation, and the use of MAP with products treated by irradiation before export to control quarantine pests may inadvertently compromise treatment efficacy. Melon fly, Bactrocera cucurbitae Coquillet (Diptera: Tephritidae), is an important economic and quarantine pest of tropical fruits and vegetables, and one of the most radiation-tolerant tephritid fruit flies known. The effect of low O2 generated by MAP on the radiation tolerance of B. cucurbitae was examined. Third-instar larval B. cucurbitae were inoculated into ripe papayas and treated by 1) MAP + irradiation, 2) irradiation alone, 3) MAP alone, or (4) no MAP and no irradiation, and held for adult emergence. Three types of commercially available MAP products were tested that produced O2 concentrations between 1 and 15%, and a sublethal radiation dose (50 Gy) was used to allow comparisons between treatments. Ziploc storage bags (1-4% O2) increased survivorship to adult from 14 to 25%, whereas Xtend PP61 bags (3-8% O2) and Xtend PP53 bags (11-15% O2) did not enhance survivorship to the adult stage in B. cucurbitae irradiated at 50 Gy. Radiation doses approved by the United States Department of Agriculture and the International Plant Protection Commission for B. cucurbitae and Ceratitis capitata (Wiedemann) (Mediterranean fruit fly) are 150 and 100 Gy, respectively. In large-scale tests, 9,000 B. cucurbitae and 3,800 C. capitata larvae infesting papayas in Ziploc bags were irradiated at 150 and 100 Gy, respectively, with no survivors to the adult stage. MAP can increase insect survivorship during irradiation treatment at certain doses and O2 concentrations, but should not compromise the efficacy of the 150-Gy generic radiation treatment for tephritid fruit flies or the 100-Gy radiation treatment for C. capitata.

Exposure of insect cells to ionising radiation in vivo induces persistent phosphorylation of a H2AX homologue (H2AvB).

The response of eukaryotic cells to ionising radiation (IR)-induced double-strand DNA breaks is highly conserved and involves a DNA repair mechanism characterised by the early phosphorylation of histone protein H2AX (producing the active form γH2AX). Although the expression of an induced γH2AX variant has been detected in Drosophila melanogaster, the expression and radiation response of a γH2AX homologue has not been reported in economically important fruit flies. We use Bactrocera tryoni (Diptera: Tephritidae, Queensland fruit fly or 'Q-fly') to investigate this response with a view to developing molecular assays to detect/quantify exposure of fruit flies to IR and consequent DNA damage. Deep sequencing confirmed the presence of a H2AX homologue that we have termed H2AvB (i.e. variant Bactrocera) and has an identical sequence to a histone reported from the human disease vector Glossina morsitans. A linear dose-response of γH2AvB (0-400 Gy IR) was observed in whole Q-fly pupal lysates 24h post-IR and was detected at doses as low as 20 Gy. γH2AvB signal peaked at ~20min after IR exposure and at 24h post-IR the signal remained elevated but declined significantly by 5 days. Persistent and dose-dependent γH2AvB signal could be detected and quantified either by western blot or by laser scanning cytometry up to 17 days post-IR exposure in histone extracts or isolated nuclei from adult Q-flies (irradiated as pupae). We conclude that IR exposure in Q-fly leads to persistent γH2AvB signals (over a period of days) that can easily be detected by western blot or quantitative immunofluorescence techniques. These approaches have potential as the basis for assays for detection and quantification of prior IR exposure in pest fruit flies.

High expression of PP2A-Aα is associated with diapause induction during the photoperiod-sensitive stage of the cotton bollworm, Helicoverpa armigera.

Protein phosphatase 2A (PP2A) is a major serine-threonine protein phosphatase which regulates metabolism, transcription, RNA splicing, translation, differentiation, cell cycle, oncogenic transformation and signal transduction. PP2A-Aα, an isoform of PP2A-A, is a structural subunit of the PP2A complex. We identified the photoperiod-sensitive stage for pupal diapause induction to be from the fifth instar to the early sixth instar larvae in the cotton bollworm, Helicoverpa armigera. PP2A-Aα cDNA from brains of diapause-destined fifth instar larvae was obtained by suppressive subtractive hybridization using nondiapause-destined larval brains as a control. Developmental expression of PP2A-Aα mRNA during the photoperiod-sensitive stage was higher in brains of diapause-destined larvae, and the PP2A-Aα protein showed a similar expression pattern as the mRNA. When larvae were transferred from diapause-inducing short days to long days during the diapause-sensitive stage, both PP2A-Aα mRNA and protein decreased significantly, and diapause incidence was also reduced. Thus, high PP2A-Aα expression during the diapause-sensitive stage may play a crucial role in photoperiodic induction of diapause, suggesting that it may be a new player involved in the molecular mechanism for diapause induction.

Mating competitiveness of Aedes albopictus radio-sterilized males in large enclosures exposed to natural conditions.

Mating competitiveness trials have been conducted in large net-screened enclosures (8 by 5 by 2.8 m) built in a natural shaded environment, in the summers of 2006, 2007, 2008, and 2009 in northern Italy. Aedes albopictus (Skuse) males were radio-sterilized by applying gamma radiations at doses in the range 30-60 Gy. Gamma radiation was administered to aged pupae at the rate of 2.3 Gy/min. Reared radiated males (originally collected in Rimini, Forli, Bologna, Matera, Pinerolo) and hybrid radiated males were tested against wild fertile males (originated from eggs collected in Rimini and Cesena) and reared fertile males, in multiple comparisons for mating competitiveness with reared or wild females. The ratio was kept constant at 100-100_100 (fertile males-radiated males_virgin females). Mating competitiveness was estimated through the calculation of the hatching rate of the eggs laid in oviposition traps positioned inside enclosures. No clear effect of the strains tested (reared, wild, or hybrid) was found. Results demonstrated that reducing the radiation dose from 60 to 30 Gy increases males' competitiveness. Laboratory investigations conducted after controversial results in the 2006 preliminary trials, showed that radiation induces precociousness in adult male emergence.