Browsing by Author "Premaratne, R.G."

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Effect of γ-radiation on fertility and survival of Aedes albopictus (skuse) males in the laboratory for Sterile Insect Technique
(Moleclar Medicine Unit, Faculty of Medicine, University of Kelaniya, Sri Lanka, 2015) Harishchandra, J.; Abeyewickreme, W.; Hapugoda, M.; Premaratne, R.G.; Gilles, J.R.S.
BACKGROUND: It is believed that Aedes albopictus, one of the dengue vectors in Sri Lanka can be controlled using Sterile Insect Technique (SIT) when integrated with other conventional control methods. The objective of this study was to determine the effective dose of gamma radiation for producing sterile males of Ae. albopictus mosquitoes. METHODS: A batch of male pupae (n=32) aged 24-48 hours in F1 was irradiated using a gamma-ray irradiator (Gamma 220, Atomic Energy of Canada Ltd., Co60) with 25 Gy in duplicates. Following the same procedure, different doses (30 Gy, 40 Gy, 50 Gy, 60 Gy and 70 Gy) were given to each pupal batch in duplicates. Then they were transferred to laboratory cages (30 cm X 30 cm X 30 cm) for emergence and supplied with 10% sucrose solution. Adult emergence rate and male longevity were recorded. Virgin females from the same cohort (F1) were introduced into each cage for mating and fed blood starting 5 days after emergence. Females were then isolated in to individual tubes and hatching rate of individual egg batches was determined after two weeks of egg maturation in hatching solution containing 0.25 g BNB, 0.05g BY in 700 ml distilled water. Spermachecae of female mosquitoes were dissected and insemination rates were calculated after egg laying. RESULTS: Male mosquito pupae in F1 showed low mortality (0-3.12%) immediately after exposing to radiation (0-48 hours). After 21 days of the irradiation, probability of survival of male mosquitoes in F1 were 0.578, 0.494, 0.453, 0.313, 0.328, 0.381 and 0.219 at 0Gy, 25 Gy, 30 Gy, 40 Gy, 50 Gy, 60 Gy and 70 Gy respectively (Kaplan Meier survival analysis). Log Rank test indicated significant differences of survival of control males with males irradiated at 40Gy, 50 Gy, 60 Gy and 70 Gy. The survival of males irradiated at 25 Gy and 30 Gy did not differ from each other and from the control. The survival of males irradiated at 40 Gy, 50 Gy, 60 Gy were not significantly different among each other. The mean hatching rate (Mean+SE) of the F2 progeny of Ae. albopictus males (F1 progeny) were 10.89+2.76%, 7.36+1.75%, 3.09+0.71%, 0.79+0.27%, 0.66+0.23% and 0% when irradiated at 25 Gy, 30 Gy, 40 Gy, 50 Gy, 60 Gy and 70 Gy respectively. The control group showed a hatching rate of 64.26 + 7.12%. It was found that insemination rate of the irradiated males among different doses and controls were above 90% in F1 based on spermatheca dissection of blood-fed females (F1). CONCLUSION: 50 Gy is recommended as the most suitable γ radiation dose to produce 99% sterility in Ae. albopictus males which has 0.328 survival probability after 21 days of irradiation. This dose can be used to produce sterile males of Ae. albopictus for population suppression.
IGamma- ray irradiation as a method for sterilization of males of Aedes albopictus (skuse) for its control through Sterile Insect Technique (SIT)
(Faculty of Graduate Studies, University of Kelaniya, 2015) Harishchandra, J.; Abeyewickreme, W.; Hapugoda, M.D.; Premaratne, R.G.; Gilles, J.R.L.
Sterile Insect Technique (SIT) can be integrated with other conventional control methods to suppress population of Aedes albopictus, a vector of dengue in Sri Lanka. A study was conducted to determine the effective dose of gamma radiation for producing sterile males of Ae. albopictus mosquitoes. A batch of male pupae (n=32) aged 24-48 hours in F1 was irradiated using Gamma 220, (Co60) irradiator with 25, 30, 40, 50, 60 and 70Gy in duplicates. Adult emergence rate and male longevity were recorded. Virgin females from the same cohort (F1) were introduced into each cage for mating and fed blood starting 5 days after emergence. Then, females were isolated in individual tubes and hatching rate of individual egg batches was calculated after two weeks of egg maturation in hatching solution. Spermacheca of female mosquitoes were dissected and insemination rates were calculated after egg laying. Male mosquito pupae in F1 showed low mortality (0-3.12%) immediately after exposing to radiation (0-48 hours). After 14 days of the irradiation, survival rate of male mosquitoes in F1 were 82.7%, 79.7%, 56.3%, 73.4%, 76.6%, 76.1% and 56.3% when irradiated at 0Gy, 25Gy, 30Gy, 40Gy, 50Gy, 60Gy and 70Gy respectively. No significant difference was observed among survival of males in different doses except in 30 Gy and 70 Gy (Kaplan-Meier survival analysis, Log Rank test). Low survival rate at 30 Gy could be due to mosquito handling errors and need to be retested. Based on the hatching rate of the F2 progeny, males (F1) showed 89%, 93%, 97%, 99%, 99% and 100% sterility when irradiated at 25Gy, 30Gy, 40Gy, 50Gy, 60Gy and 70Gy respectively. Insemination rate of the irradiated males among different doses and controls were above 90% in F1 based on spermatheca dissection of bloodfed females (F1). 50 Gy is recommended as the most suitable radiation dose to produce 99% sterility in Ae. albopictus males which has 76% survival after 14 days of irradiation. This dose can be used to produce sterile males of Ae. albopictus for a SIT programme.
Sterile Insect Technique for Controlling Aedes albopictus (Skuse); Sex Separation and Mating Competitiveness
(19th Conference on Postgraduate Research, International Postgraduate Research Conference 2018, Faculty of Graduate Studies,University of Kelaniya, Sri Lanka, 2018) Harishchandra, J.; Hapugoda, M.; Premaratne, R.G.; Abeyewickreme, W.
Sterile Insect Technique (SIT) introduced by Edward F. Knipling in 1950s has been shown as an effective and sustainable genetic approach to control populations of selected pest insects in area wide integrated pest management programmes. Successfulness of this approach depends on the availability of an efficient sexing method and production of sterile males competitive enough to compete with wild males. In the context of developing SIT for the control of dengue vector Aedes albopictus (Skuse) in Sri Lanka, a method for separation of male pupae was tested and mating competitiveness of irradiated males against wild males was evaluated under laboratory conditions. Male pupae of the F42 generation of Ae. albopictus (Gampaha strain) were separated from female pupae using mechanical sieving method. A set of three stainless steel sieves of mesh sizes 1.4, 1.25 and 1.12mm were placed one on the other in descending order of size towards the bottom, forming a column immersed in a water jar. All pupae were poured on to the top sieve and shaken gently for seven minutes. Pupae were collected from each sieve and the male and female counts were taken after emergence of adults. Male and female pupae used for the competitiveness assessment were separated by the above sieving method. Male pupae (F42, age 28-40hours) were exposed to Gamma radiation (Gamma 220, Atomic Energy of Canada Ltd., Co60) at a dose of 50Gy by placing 300 male pupae in each plastic vial with 10ml of water. Dosimetry was performed using Frickey method. Wild fertile males and females reared from field-collected eggs from Gampaha (F1) were used to compete and cross with irradiated males respectively in different ratios. Two days after the emergence, the adult mosquitoes were caged (Bug Dorm 30X30X30cm) with ratios of irradiated males: wild males: wild virgin females 1:1:1, 3:1:1 and 5:1:1 (females, n = 100). Fertile control was set with wild males and wild virgin females 1:1. and an irradiated control was set with irradiated males with wild virgin females 1:1. Mosquitoes were allowed to mate for 3 days with access to 10% sugar solution. Then the mosquitoes were fed with cattle blood and engorged females were isolated in oviposition tubes. After 4 days, females were returned into cages and the filter papers containing eggs were allowed to dry. Eggs were hatched after one week and the hatching rates were compared. From the pupae retained in the sieve of mesh size 1.12mm, 98.3% were males. The mean hatch rates of fertile and irradiated controls and with ratios of irradiated males: wild fertile males: wild virgin females 1:1:1, 3:1:1, 5:1:1 were 80.24%±1.63SEM, 2.42%±0.57SEM, 56.74%±5.79SEM, 37.33%±4.76SEM and 34.23%±4.73SEM respectively. Fried Competitive Index (C) of irradiated males compared to controls was 0.43. Results show sieving method can be used for separating male pupae for irradiation with 98% accuracy. Irradiated males were less than 50% competitive as wild males under laboratory conditions