1 Introduction

Mosquito (class-Insecta, order-Diptera) vectors are mainly responsible for transmitting certain diseases like malaria, dengue, chikungunya, Japanese encephalitis, yellow fever, lymphatic filariasis (Rozendal, 1997; Barik et al., 2012). It has been estimated that 3,900 million people in 128 countries are at risk of infection with dengue viruses (WHO, 2015). In India, transmission of dengue and chikungunya is increasing day by day throughout the country. About 97,740 dengue cases and 26,912 chikungunya cases are reported in India during 2015 (NVBDCP, 2015). Aedes aegypti is the principal vector of dengue and chikungunya for which there is no approved vaccine or medication (Shelly and McInnis, 2011). Although the biological and chemical methods show some notable success in mosquito control but in some cases it has not been sustainable in the long-term due to their insecticide resistance, re-invasion, and environmental damage (Wilke et al., 2009). Potential applications for reducing transmission of mosquito-borne diseases by releasing genetically modified mosquitoes have undergone extensive research (Boete and Koella, 2003; Cattevuccia et al., 2003).

Use of various radiation techniques in entomology have considerably contributed to insect pest management since the last four decades. Nuclear techniques (Sterile Insect Technique and F₁ sterility) have been used successfully against a number of pest species such as Mediterranean fruit fly Ceratitis capitata (Wiedemann), melon fly Bactrocera cucurbitae (Coquilett), pink bollworm Pectinophora gossypiella (Saunders), codling moth Cydia pomonella (L.) and tsetse fly Glossina austeni Newstead (Tan, 2000; Wyss, 2000; Hendrichs et al., 2005; Klassen and Curtis, 2005).

In the present investigation, we tried to investigate the effect of gamma radiation at higher doses on pupal viability, adult emergence and malformation if any on F₁ progeny of field collected Aedes aegypti.

2 Materials and Methods

2.1 Mosquito collection

Larvae of Aedes aegypti were collected through dropper from different localities of Berhampur city (17^o 48¢ and 23^o 34¢ N latitudes and meridians of 80^o 24¢ and 87^o 29¢ E longitudes) of Odisha state, India and transported to the laboratory for rearing. Identification of field collected Aedes mosquito is done as per the morphological identification keys. All the colonies were maintained in an insectary at a temperature of 25 ± 1^oC and relative humidity of 65 ± 5% with 12 hour light and dark photoperiods. The adults were fed on cotton pads soaked in 10% glucose solution and soaked raisins. The larvae were reared in white enamel trays provided with yeast powder and dog biscuit at a ratio of 2:3 as food (Helinski et al., 2006). The amount of food depends upon the number and stage of larvae (Thomas and James, 1997). Blood meals to females were given from swiss mice. The F₁ progeny of field collected mosquitoes were taken for the current investigation.

2.2 Experimental set-up

The experimental mosquitoes were obtained from the same culture batch and were randomly distributed for different doses of radiation exposure. Mosquitoes in the control group underwent exactly the same handling stages as the experimental mosquitoes (Helinski et al., 2006). F₁ pupae of wild Aedes aegypti were taken the day before irradiation from the culture tray for the experiment. The irradiation of pupae is preferably performed on one day old pupae since irradiation of young pupae results in a reduced emergence (Abdel-Malek et al., 1967; Curtis, 1976) and shorter survival (Davis et al., 1959). Pupae were irradiated in a small plastic lid filled with water as previously described by Helinski et al. (2006). Pupae were exposed to gamma radiation generated by a cobalt-60 source (Gamma 5000 irradiator) available in the P.G. Department of Botany, Berhampur University, Berhampur, India. The dose rate of radiation was 7.619 KGy/h at the time of experiment. F₁ pupae were irradiated with various doses like 0Gy (control), 100Gy, 250Gy, 400Gy, 550Gy, 700Gy, 850Gy and 1000Gy. After irradiation of pupae, different parameters such as pupal viability, malformed pupae, malformed adults and adult emergence were observed at 24hr interval up to 72hr for each radiation dose. For malformation study, only mosquitoes that successfully emerged from pupae were positively scored while semi-emerged adults and dead adults were scored as non-emerged as earlier described by Helinski et al. (2006).

2.3 Data analysis

The data were usually obtained in different replicates. Data were computed for means, standard error and analysis of variance (ANOVA). Means were separated at the 5% significance level by least significant difference (LSD) test.

3 Results

Irradiation of Aedes aegypti is performed in the present study to investigate the effect of different radiation doses on pupal viability, adult emergence and malformation of pupae and adults if any.

3.1 Effect of γ-radiation on pupal viability of Aedes aegypti

To determine the radio sensitivity, one day old F₁ pupae of field collected Aedes aegypti were irradiated to a range of gamma radiation as mentioned above in the materials and methods. A set of pupae without radiation exposure served as control. Our investigation demonstrated that the pupal viability decreased with an increase in radiation dose in a dose dependant manner. It was observed that 99.2%, 61.4%, 42.5%, 13.4%, 7.5%, 6.7% and 3.9% viable pupae found after exposure to gamma radiation at 100Gy, 250Gy, 400Gy, 550Gy, 700Gy, 850Gy and 1000Gy respectively (d.f. = 7, F = 44155.85). In addition, 100%, 96.0%, 85.0%, 92.3%, 91.1%, 87% and 84.2% survival after 24hr and 99.2%, 65.4%, 54.3%, 37.4%, 27.9%, 35.0% and 13.9% survival after 48hr exposure were found at above gamma radiation doses (d.f. = 7, F= 30577.30). Further, irradiation at higher doses like 550Gy, 700Gy, 850Gy and 1000Gy survivability of pupae were 13.4%, 7.5%, 6.7% and 3.9% after 72hr of exposure to gamma radiation (Figure 1).

3.2 Effect of γ-radiation on adult emergence of Aedes aegypti

Impact of gamma radiation at higher doses on emergence of adult from irradiated pupae of Aedes aegypti was also carried out in the present study after demonstrating the pupal viability at different radiation doses. Hundred percent adult emergences was noticed in non-irradiated pupae (control) while the percent of adult emergence from irradiated pupae at different gamma radiation doses decreased with an increase in the radiation doses. The percent adult emergence from irradiated pupae were about 99.2%, 61.3%, 42.4%, 13.4%, 7.2%, 6.4%, and 3.0% at 100Gy, 250Gy, 400Gy, 550Gy, 700Gy, 850Gy and 1000Gy respectively (Table 1). It was also found that adult emergence from irradiated pupae started immediately after irradiation and completed after 48hr exposure to gamma radiation in all most all radiation doses.

3.3 Formation of malformed pupa and adult of Aedes aegypti after γ-radiation exposure

In the present investigation, morphological deformities due to the effect of γ-radiation at higher doses on both pupae and adults of Aedes aegypti were noticed after irradiation that indicates the somatic damage of pupae and adults.

Exposure of one day old pupae of Aedes aegypti with different radiation doses gave rise to noticeable pupal abnormalities. It was observed that malformed pupae were obtained after 24hr irradiation of pupae at 250 Gy and more doses of gamma radiation. Pupal-adults intermediates were induced as a result of this radiation exposure.

Deformed pupae failed to complete their metamorphosis properly which could not emerge completely and remain concealed in the puparia until they die. In some cases, it was observed that head, thorax and part of the abdomen with fore wings were released but the rest of the body still attached to the puparia (Figure 2a). Further, it was also observed that few malformed adults with curled legs, crumpled wings and with curved abdomen (Figure 2b) were obtained due to the effects of high dose of gamma radiation.

4 Discussion

The sensitivity of organisms to ionizing radiation is well documented and exposures to ionizing radiation have been shown to cause adverse effect to different organisms (Gomberg and Gould, 1953). There are a number of applications of ionizing radiation in entomology (Bakri et al., 2005), including disinfestations of commodities for quarantine and phytosanitary purposes, and reproductive sterilization of insects for pest management programmes using the Sterile Insect Technique (SIT) (Dyck et al., 2005; Meheta, 2009). Radiation can also apply in various ways to facilitate the use of biological agents for control of arthropod pests and weeds (Carpenter, 1997, 2000; Greany and Carpenter, 2000). Radio sensitivity varies with the stage of embryologic development (Ayvaz et al., 2008). Insects exhibit a pronounced inherent resistance to ionizing radiation. In general, adult insects are considered to be at least 100 times less sensitive to such radiation than other vertebrate (O’Brien and Wolfe, 1964). The range of lethal exposures to ionizing radiation is generally 2 and 9Gy for most mammals (Bond et al., 1965) and 1 and 1000Gy for most adult insects (Ducoff, 1972; Casarett, 1968). The eggs of Plodia interpunctella are quite radiosensitive and become 25 times more radio resistant at 72 h than at 2 h post-oviposition (Brower, 1974). Review of literature indicated that Anopheles arabiensis is more radiation resistant than other anopheline mosquitoes (Helinski et al., 2006). Pupae of An. gambiae were more radiation-resistant than adults (Curtis, 1976). For the irradiation of insects, gamma rays are usually used due to their high energy and penetration (Helinski et al., 2009). The pupal stage is irradiated in the present study due to ease of handling compared to the adult stage as earlier reported by Helinski et al. (2006). A series of irradiation doses up to 1000Gy were applied to pupae to determine the effect of gamma radiation on pupal viability of F₁ progeny of field collected Aedes mosquitoes. Results achieved here are similar to previous studies by Curtis (1976) with An. gambiae, and Patterson et al.(1975) in Cx. quinquefasciatus, found increased mortality of that species at 24hr post-irradiation of pupae with an increase in radiation dose.

Adult emergence from irradiated pupae of Aedes mosquitoes were also carried out. It was noticed that the percent adult emergence decreased with increase in radiation doses which was almost similar to the findings of Helinski and co-workers (2006) where the adult emergence of An. arabiensis mosquitoes was similar or reduced as compared to un-irradiated control mosquitoes of same species. Further, it has been reported that, when the pupae of Plodia interpunctella were irradiated, the percentage of adult emergence was decreased by doses up to 650 Gy (Brower, 1974). The percentage adult emergence was decreased in accordance with increasing in gamma radiation doses of 200 to 800 Gy to pupae of Ephestia calidella (Guenee) (Boshra and Mikhaiel, 2006).

Andresen and Curtis (2005) reported that significant somatic damage occurs due to the effect of γ-radiation on Anopheles mosquitoes. Exposure of one day old pupae of Aedes aegypti with different radiation doses in the present investigation gave rise to noticeable pupal and adult deformities indicating somatic damage. In contrary, Helinski et al.(2006) and Abdel–Malek et al.(1967) reported that irradiation had no effect in the emergence of adult from the pupae even when the dose applied was high. Further, Helinski et al. (2009) reported that pupae are more somatically damaged by the irradiation than adults which are similar to our present investigation.

5 Conclusion

Mosquito is posing a threat in all most all part of the world. Although great efforts have been made, currently no effective vaccine and drug available for dengue and chikungunya. Therefore, mosquito vector control is the only option for prevention of mosquito borne diseases. Conventional larval control methods gained only partial results due to the peculiar ecobiology of the species. Radiation has been used since the 1950s to sterilize the insects, for sterile insect technique to control insect population. This information can be utilized in planning radiation induced control programme for the control of wild Aedes mosquitoes.

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