Infection of Aedes detritus hal. with mosquito iridescent virus.

268 NOTE Infection of Aedes detritus Hal. with Mosquito Iridescent Virus* by S. HASAN,1 C. VAGO 2 & G. KUHL 3 Mosquito iridescent virus (MIV) has been ob- served in various species of Culicidae such as Aedes annulipes Meigen (Weiser, 1965), Aedes taenior- hynchus Wiedemann (Clark et al., 1965), Aedes fulvus Wiedemann, Aedes vexans Meigen, Psoro- phora ferox Humboldt, and Aedes dorsalis Meigen (Chapman et al., 1966). Investigating the host spe- cificity of the MIV from Ae. taeniorhynchus in other mosquitos, Woodard & Chapman (1968) found that it was possible to transmit the virus in Aedes soli- citans. Linley & Nielsen (1968a, 1968b) have shown experimentally that healthy larvae could be infected orally from the macerated bodies of infected larvae and that subsequent transovarian transmission occurred. Recently Vago et al. (1969) found a spontaneous natural infection of MIV in Aedes detritus in Tunisia. Apart from ecological surveys, various studies have been made on the nature of infection in diseased larvae. Cadavers of diseased larvae collected from Tunisia were frozen and stored for further studies on the transmission of the virus. This study deals with the infection capacity of the virus to the larvae of Ae. detritus. Materials and methods All infection experiments were carried out using diseased larvae that had been stored at -20°C. The larvae were frozen just before or 2-3 hours after death. In all cases the larvae were examined for the characteristic symptoms caused by the virus. Larvae of Ae. detritus of mixed instars, collected from the marshy areas of the Camargue in southern France and reared at 18°C in pans with saline pond water, were used for infection purposes. Collections * This work was carried out under a Contractual Tech- nical Agreement with the World Health Organization. 1 Entomologist, Commonwealth Scientific and Industrial Research Organisation, Station de Recherches Cytopatho- logiques, INRA-CNRS, St. Christol, France. ' Director, Station de Recherches Cytopathologiques, INRA-CNRS, St. Christol, France. Professor, Laboratoire de Pathologie Comparee, Faculte des Sciences, Universite de Montpellier, Montpellier, France. ' Agent Technique, Institut National de la Recherche Agronomique, Station de Recherches Cytopathologiques, St. Christol, France. 2722H were made on several occasions throughout the winter to ensure a continuous supply of wild larvae. Because of the low temperature to which these larvae are exposed in nature, the infection experiments were carried out at 15°C. Two methods were used for infection, as described below. Oral infection. Five infected larvae were thorough- ly macerated in 5 ml of distilled water and the third- instar wild larvae were kept in this concentrated viral suspension for about 24 hours. At the end of this period the suspension was transferred to a rearing pan with 0.5% saline solution replacing the pond water normally used for rearing. An equal number of uninfected larvae was used as control under similar conditions. The larvae were fed with a mixture of yeast and dog buiscuits in 1: 3 proportions. Infection by injection. An infected larva was macerated and suspended in 1 ml of sterilized dis- tilled water. In order to obtain homogeniety the viral suspension was treated ultrasonically for 3-5 seconds using an MSE 100-watt ultrasonic disintegrator at an amplitude of 8 ,m. The suspension was then cen- trifuged at 650 g for 10 min to remove the fragments of body tissues. The supernatant was filtered through a 0.45-ju Millipore filter and the filtrate was inocul- tated into the haemocoel of the third-instar larvae at the level of 0.04 mg per individual larva. The larvae maintained as control were inoculated with an equal quantity of Ringer's solution for insects. The inoculation procedure was as follows: larvae were immobilized by placing them on moist filter paper and were viewed with a low power binocular microscope. A fine glass microneedle containing the inoculum fluid was then introduced into the body cavity of the abdomen of each larva at the level of the second segment. The inoculum was discharged by increasing the pressure in the microneedle by means of a small pressure pump. This was con- nected to the microneedle by means of rubber tubing and functioned only when an electric circuit was manually completed. The inoculated larvae were kept at 15°C and were observed regularly for the appearance of initial symptoms. As with the larvae infected orally, the diet was a mixture of yeast and dog biscuits. INFECTION OF AEDES DETRITUS WITH MOSQUITO IRIDESCENT VIRUS 269 Table 1. Infection rates of Aedes detritus with MIV administered by injection or orally NoNo. of oof Mraiy No. of }Ineto Group experimental No Mortalty infected Infection larvae idead larvae rate (%) larvae rate(% infected by injection 620 400 64.5 212 34.2 control 500 116 23.2 _ infected orally 200 19 9.5 16 8.0 control 200 7 3.5 Results and discussion In both types of infection experiment, only the fourth-instar larvae showed disease symptoms. The first signs of disease appeared after 10 days in larvae inoculated with MIV and after 20 days in those exposed to the virus in rearing pans. At this stage the infected larvae showed slight discoloration at both sides of the abdomen and the thorax with bluish iridescence when seen against a black back- ground. Later these larvae turned whitish in colour and their hypertrophied thoraxes developed a distinct blue iridescence. The infected larvae died within 2 weeks of the appearance of the initial symptoms, having become extremely sluggish just before their death. The formation of pupae and adults was observed only when the larvae did not show any apparent symptoms of MIV infection. Infection rates were estimated from the average results of 3 experiments that ran more or less simulta- neously with larvae of the same age group. The infection rate was much higher in the experimental larvae injected with MIV (34.2%) than in those infected orally (8.0%), as shown in Table 1. Some mortality occurred among the injected larvae within 48 hours of manipulation. The total mortality of both infected and non-infected larvae at this stage was about equal. In spite of this mortality the technique of inoculation has been found efficient for the multiplication of MIV in the laboratory and for detailed studies on the host-virus relationship. Although the possibility of multiplying MIV by injection was mentioned by Vago et al. (1969), this is the first time that the injection ofa viral suspension into the body cavity of a mosquito larva has been carried out successfully. Use of this technique may also facilitate the study of the specificity of the virus for different mosquito species. REFERENCES Chapman, H. C., Clark, T. B., Woodard, D. B. & Kellen, W. R. (1966) J. Inv. Path., 8, 545-546 Clark, T. B., Kellen, W. R., & Lum, T. M. (1965) J. Inv. Path., 7, 519-521 Linley, J. R. & Nielsen, H. T. (1968a) J. Inv. Path., 12, 7-16 Linley, J. R. & Nielsen, H. T. (1968b) J. Inv. Path., 12, 17-24 Vago, C., Rioux, J. A., Duthoit, J. L. & Dedet, J. P. (1969) Ann. Parasit. hum. comp., 44, 667-676 Weiser, J. (1965) Bull. Wld Hith Org., 33, 586-588 Woodard, D. B. & Chapman, H. C. (1968) J. Inv. Path. 11, 296-301