Effects of Gamma Irradiation Against Mating Competitiveness of Male Culex quinquefasciatus (Diptera:Culicidae) in Laboratory

Culex quinquefasciatus is the main vector of lymphatic filariasis in Pekalongan City. Sterile Insect Tehnique (SIT) could be complementary vector control for filariasis. The key success of the technique depend on the ability of laboratory-reared sterile males with the wild-type females.The aim of the research was to determine the mating competitiveness, the fecundity and the fertility of sterile male Culex quinquefasciatus. The pupae of Cx. quinquefasciatus were gamma irradiated at the doses of 60Gy, 70Gy, and 80Gy, while unirradiated pupae were prepared as control. The mosquitoes emerging from the irradiated pupae could mate with a normal female in the cages. It were observed for the mean female laying eggs, the fecundity, the fertility and the mating competitiveness. The data were analyzed by one way ANOVA. The result showed that the irradiated Cx. quinquefasciatus at the doses tested did not affect on the fecundity and the mating competitiveness, but the fertility was disturbed (sterile). A dose of 70 Gy was the optimum dose or a fertility rate of 1.8% (98.2% sterile), and the value of competitiveness (C index) was 0.568. Based on the result, the irradiated Cx. quinquefasciatus can be recommended for semifield application.


INTRODUCTION
Lymphatic filariasis is a communicable disease caused by filarial worms and transmitted through the bite of various kinds of mosquitoes. There are three species of worms causing lymphatic filariasis in Indonesia, namely: Wuchereria bancrofti, Brugia malayi and Brugia timori 1 . The three species exist in Indonesia, but more than 70 percent of cases of filariasis in Indonesia were caused by Brugia malayi 2 .
Pekalongan City is one of the endemic regions of lymphatic filariasis caused by Wuchereria bancrofti with Culex quinquefasciatus being the vector 3

. Well-preserved places for
Culex quinquefasciatus breeding, small coverage of Mass Drugs Administration (60.3 percent), people's lack of awareness of environmental sanitation, the finding of filarial larvae in mosquitoes and vector's resistence to insecticides are the factors that keeps filarial transmission going in Pekalongan City 4,5,6 . This also proves that the mass drug administration in the last five years has not effectively cut the chain of filarial transmission. An important effort that should be made is conducting surveilance of vector of filariasis as the basis for vector control in cutting the transmission chain. Vector control of filariasis in Indonesia has not been done in a dedicated way due to extremely complex issues. Fertile Insect Technique (SIT) is one of vector control efforts that can be made. SIT is a highly specific biological vector control technique which only affects target species. This technique only reduces the population size in the field, and not kills off the population, and graduated release may reduce the mosquito population 7 . SIT pricinple is that male sterile insects (Cx. quinquefasciatus) are released in a considerable number to a target area.
The released sterile male Cx. quinquefasciatus will compete with wild type males to mate wild type females. The mating between sterile male Cx. quinquefasciatus and wild type females is expected to produce no offspring, which eventually will reduce the population size of Cx quinquefasciatus in that area 8 .
SIT is an environment-friendly, effective and potential vector control technique. This technique is also called species-specific control, by which vector is killed by the vector itself (autocidal technique) 9 . The technical procedure of this technique is relatively easy. Male mosquitoes are irradiated in the laboratory and then released to their habitat periodically.
Irradiation may cause male mosquitoes to be sterile as it causes damages to spermatogenesis and stops the production of sperms (aspermia). Sperm inactivation also causes sterilization as sperms are not able to move to fertilize egg cell. The inability to mate serves as another sterilization factor as irradiation causes damages to somatic cells of internal genital organs and prevents egg cell fertilization 8  SIT application for Cx. quinquefasciatus control has never been done in Indonesia, thus a preliminary test should be conducted for supporting the success of SIT application in the nature.
Some preliminary tests are needed to support SIT application in the field, one of which is mating competitiveness of sterile male Cx. quinquefasciatus. This research was intended to evaluate the effect of gamma irradiation on fecundity, fertility and mating competitiveness of sterile male Cx. quinquefasciatus at the laboratory scale. It is expected that this research supports the feasibility of SIT application in lymphatic filarasis vector control.

Ethics statement
The present study was performed with the approval of the Ethics in Research National Institute

Sample Mosquitoes
The research sample consisted of Culex quinquefasciatus pupae of second to third generation from the field isolate in Pabean Subdistrict, Pekalongan City kept at the Laboratory of Entomology of the Center for Research and Development of Animal-Borne Disease Control of Banjarnegara. The colony was from the Culex spp larvae in Pabean Subdistrict, Pekalongan City, and then kept at the laboratory, and the larvae were fed with uncrushed dog feed (Pedigree®). The administration of larva feed was adjusted to the age, after instar 3 was moved to cultivation tray sized 27 cm x 35 cm x 5 cm with a density of 400-600 mosquitoes/tray. The larvae were raised until they grew into pupae. The pupae raised every day were taken and put into mosquito cage. The emerging mosquitoes in the cage were identified and separated by Cx. quinquefasciatus and given 10% sugar solution and marmot blood to enable them to lay eggs.
The eggs produced were incubated and raised until they turned into pupae. The male and female pupae derived from the colonization at the laboratory were separated (male pupae are smaller than female pupae) using a sieving device (90-95 percent small pupae are males) according to the method of Nurhayati et al (2010) 13 . The identified male pupae were inserted into a bottle, kept in a cool box and carried to PAIR BATAN Jakarta to be irradiated. Female pupae were inserted into a 40 cm x 40 cm x 40 cm sized mosquito cage and then kept at the Laboratory of Entomology of the Center for Research and Development of Animal-Borne Disease Control of Banjarnegara before test.

Gamma irradiation
Irradiation was conducted at the Center for Technology of Isotope and Radiation BATAN, Jakarta. Irradiation was carried out when Cx. qinquefasciatus pupae were ≥15 hours old using Gamma Cell-220 irradiator and based on the the position of radioactive sources around the material, so more uniform absorption doses were obtained. The sample were placed in plastic pots with lower diameter of 4.5 cm, upper diameter of 6.5 cm and height of 6.5 cm, filled with 20 ml of water. Every plastic pot was filled with 200 pupae to which irradiation doses of 60, 70 and 80 Gy were administered separately and alternately. The doses were determined based on the results of dose test of previous research. The control group consisted of non-irradiated male Cx. quinquefasciatus having been prepared at the Laboratory of Entomology of the Center for Research and Development of Animal-Borne Disease Control of Banjarnegara at the same age of that of the irradiated pupae. After the gamma irradiation process, male Cx. quinquefasciatus pupae were raised until they became mature, and observation was conducted on their fertility, fecundity and mating competitiveness.

Fecundity and fertility
Mature male Cx. quinquefasciatus (n=25) on which gamma irradiation was administered were mated with normal female mosquitoes (n=25) in a cubic cage sized 30 cm x 30 cm x 30 cm. The control group consisted of normal male Cx. quinquefasciatus (n=25) that were mated with normal female mosquitoes (n=25). After three days of mating period, the male mosquitoes were released from the cage using aspirator. Female mosquitoes were fed with blood (marmot) until they were full, and then individually moved to a transparent plastic vial tubbe for oviposition process 14 . This treatment was repeated four times. The observation of the number of eggs, eggs that hatched and eggs that did not hatch was conducted manually using stereo microscope with a 4x magnification. Eggs with open operculum were eggs that hatched, while eggs that did not hatch were marked with closed operculum 15 .

Mating competitiveness
The observation of mating competitiveness was conducted at the Laboratory of  Table 1.
The mosquitoes in the mating cage were given 10% sugar solution. For five days starting from the fourth day-the mosquitoes were given marmot blood for one hour each day. Gravid female mosquitoes were taken individually and inserted into 150 ml sized paper glass filled with 50 ml of 10% straw water as a place to lay eggs. The observation of the number of eggs and hatching eggs was conducted every day for five days. The observation was conducted for one gonotrophic cycle.

Data analysis
The parameters measured in this research were the number of gravid female mosquitoes,

Effects of irradiation on fecundity
The data of fecundity and number of normal female Cx. quinquefasciatis mosquitoes successfully laying eggs by mating with irradiated males and normal males (non-irradiated) as control group are presented in Table 2. The percentage of female mosquitoes successfully laying eggs was 40 percent in the control group and 39-43 percent in the treatment group, and there was no difference statistically (F = 2.201; p = 0,099).

Effects of irradiation on Fertility
Fertility rate is the percentage of the number of hatching eggs out of the number of eggs produced by female mosquitoes mating with sterile or normal male mosquitoes. The fertility rates of female mosquitoes mating with irradiated and control male mosquitoes are shown in Table 3. Normal female mosquitoes mating with normal males had the highest fertility at 94.1 percent, while those mating with irradiated males had fertility of around 1.3-4.8 percent. The higher the irradiation dose, the lower the fertility rate. The results of the statistical test show a significant difference in the fertility rate of treatment group and control group (F = 896.614; p = 0.00).

Effects of irradiation on mating competitiveness
The mating competitiveness values of sterile Cx. quinquefasciatus at various irradiation doses are presented in Table 4. The highest C index value was obtained at the irradiation dose of 70 Gy (C index = 0.53), while the lowest was at the irradiation dose of 80 Gy (C index = 0.36).
The statistical test results show no significant difference in the mating competitiveness of sterile male Cx. quinquefasciatus at different irradiation doses (F = 0.526; p > 0.05).

DISCUSSION
The ability of mosquitoes to lay eggs is related to the structures and functions of the reproductive organs. Mosquitoes with healthy condition of reproductive organs and ability to function normally tend to have more successful oviposition than the otherwise. Mosquitoes may be unable to lay eggs because of their ovaries' inability to produce eggs or to have oviposition.
The two factors are related to the biological aspect, especially the factor that is related to abnormalities of the organ function due to genetic factors 17 . Table 2  The number of eggs produced by female mosquitoes is affected by the amount of blood sucked, biological factor and ecological factor 17 . According to Clements, to produce 85.5 eggs, on average, a mosquito needs 3-5 mg of blood. Eggs will not be produced if the amount of blood sucked is less than 0.5 mg 20 . The size of the mosquito's body is a biological factor, which affects the size of its abdomen and ovary. Larger ovary, which functions as a place in which eggs are produced, has bigger capacity and productivity. Large mosquitoes tend to have larger abdomens. In addition, the bigger the amount of blood is sucked, the bigger the number of the eggs is produced 21 .
The eggs produced by female Cx. quinquefasciatus show that the insemination process went on, but the embryo formed could not survive due to the dominant lethal mutation properties carried by irradiated male mosquito sperm cells. Dominant lethal mutation does not inhibit the process of male nor female gametogenesis, and there is no inhibition in the zygot formation either, but the embryo will experience death 9 . The dominant lethal mutation can be seen from the decreasing fertility rate with the increase in the gamma irradiation doses Some eggs do not hatch because there is no fertilization between sterile sperm cells and egg cells because male Cx. quiquefasciatus are unable to perfectly copulate with normal females as their genital organs change, preventing the sperm cells to be delivered perfectly . Decreased egg fertility also shows the ability of sterile male Cx. quiquefasciatus in competing with normal male Cx. quiquefasciatus in the nature in finding mates for mating.
High mating competitiveness shows that the gamma irradiation dose of Co-60 administered in the pupal stadium did not have any effect on the mating competitiveness. Every species needs a certain optimum gamma irradiation dose for the sterilization of eggs without affecting its mating competitiveness 22,26 In this research the irradiation dose did not have any effect on the mating competitiveness statistically as it was possibly caused by relatively small range of the doses tested. Some research has proven that higher doses will cause negative effect on mating competitiveness 26 .
This pattern is also found in Ae. Aegypti 15 and An arabiensis 23 . Higher irradiation doses (> 120 Gy) needed for sterilizing males may also cause such males to be unable to transfer sperm cells into female mosquitoes 13 .
Sterile males' success in mating may be caused by their ability to compete with normal males in getting mates. The main factor of the decrease in mating competitivenss in SIT is gamma irradiation sterilization process. Ionizing gamma irradiation may cause damages to somatic cells, which may also deteriorate male Ae. aegypti's form 16,25 . Low physical form may also worsen males' ability to mate with females. The results of some previous research show that the mating competitiveness of male mosquitoes has a negative correlation with the increase in gamma irradiation dose 16,18 .
A dose of 70 Gy is an optimum dose to be used at a fertility rate of 1.8 percent, with competitiveness value of 0.53. This shows that the irradiation dose of 70 Gy can decrease the egg hatching rate by 98.2 percent. Mating competitiveness value or C Index is useful for determining the number of sterile male mosquitoes spread in the nature following the method of Sasmita and Ernawan 18 . In this research the mating competitiveness of Cx. quinquefasciatus was 0.53. This means that the number of sterile males released should be at least twice of the population of males in the nature in order to increase mating possibility with normal females.
Our results that wild Cx. quinquefasciatus males irradiated at the optimum dose (70 Gy) can compete successfully with unirradiated male for wild females in laboratory. The results of this research can be used as the baseline for further testing at semi-field and limited-field scale and for assessing SIT feasibility in the vector control of lymphatic filariasis in Pekalongan City.