Overnight access to sugar and response to DDT in Anopheles albimanus Wied

Bull. World Health Organ. 1974, 51, 311-313Bull. Organ. mond. Sante J Overnight access to sugar and response to DDT in Anopheles albimanus Wied. R. ELLIOTT1 & 0. OCHOA-AGUIRRE2 Abstract A study was made of the response of A. albimanus females to DDT after overnight access to sugar solu- tions. Tarsal contact with the solutions had a statisti- cally significant protective effect proportional to the strength of the solution. Prior access to sugar can produce considerable experimental error and thus invalidate DDT susceptibility tests. Reports of DDT resistance among anophelines should be treated with caution when the tests were conducted on insects that had access to sugar. In the WHO Region of the Americas, adult sus- ceptibility tests with the standard WHO equip- ment (1) are usually performed on female anophelines captured at animal bait on the evening before expo- sure to the insecticide. Instead of being freshly fed, as recommended in WHO's instructions, the mos- quitos have therefore had some 12-14 hours for digestion of the bloodmeal, and have been kept over- night with access to sugar solution supplied on cot- ton pads. Fleming & O'Connor have shown a that both these factors depress the susceptibility of A. aquasalis to DDT. They found that the reduction in 24-hour mortality below that observed in material exposed when freshly bloodfed without sugar was proportional to the concentration of the sugar solu- tion, and was a physical rather than a physiological effect, depending on tarsal contact with the cotton pads. As the number of tests performed on A. albi- manus over the last 14 years probably exceeds that for all other neotropical anophelines, the effect of sugar on the susceptibility of this species was investigated. 1 Formerly Scientist (Entomologist), project AMRO 0203, WHO/PAHO, Guatemala. Present address: 1 Dore Court, Ladies Spring Grove, Sheffield, S17 3LR, England. ' Chief, Entomology Section, National Malaria Eradi- cation Service, Guatemala. a FLEMING, G. A. & O'CONNOR, C. T. (1972) Some fac- tors affecting the results of susceptibility tests carried out on adults of Anopheles aquasalis (Curry) with DDT, Geneva, 1972 (WHO mimeographed document WHO/MAL/72.766; WHO/VBC/72.343). Materials and methods A colony of A. albimanus from Sanarate, El Pro- greso department, Guatemala, has beenmaintained by the Guatemalan National MalariaEradication Service since April 1960. It has retained complete resistance to dieldrin, but shows a balanced polymorphism with respect to the genetic factor for DDT resistance. The type of DDT resistance is unusual in that it depends on recovery from knockdown, and not the complete absence of response that is more common in Central American A. albimanus. The larvae are maintained on yeast in the early instars and Incapa- rina (a vegetable protein infant dietary supplement) later; the adults are fed on rabbits. Experimental material was obtained by placing pupae in a cage supplied with water only, instead of the usual sugar solution used for male nutrition. Female adults were given their first bloodmeal at 16 h 00 on the day before the DDT susceptibility test. They were then placed in groups of 25±4 in holding tubes of the WHO apparatus. Batches of the tubes were supplied with cane sugar solution in a range of different strengths (2.5%, 5%, 10%, 20%, 40%), or with water, absorbed on uniform cotton pads. At 08 h 00 the following day, when aged 2-3 days, the mosquitos were exposed to 4% DDT test papers for 1 hour or to 2% DDT test papers for 2 hours. For each batch of replicates exposed to DDT, there were 2 controls exposed for 2 hours to test papers without DDT, one with overnight access to water, and the other to the highest sugar concentration (initially 40%, later 20%). Mortality was counted after holding the tubes for a further 24 hours at 24.4C ±I.7C and a relative humidity of 80%±5%. Results As can be seen in Table 1, which shows the results for 66 replicates exposed to DDT, mortality showed a progressive decrease with increases in sugar con- centration up to 10%. There was no further decrease at 20 %, but at the highest concentration (40%) mor- 3271 - 311- 5 312 BRIEF COMMUNICATIONS Table 1. Response of A. albimanus to DDT after overnight access to sugar solutions. Concentration Number of Mean Standard of sugar (%) replicates a mortality (%) deviation (%) 0 12 70 12.8 2.5 12 59 10.4 5 12 53 14.9 10 12 43 11.2 20 12 43 15.3 40 6 67 15.0 a Each replicate contained 25 ± 4 bloodfed females. tality rose again to nearly the level observed with distilled water. Since the very thick and syrupy 40% solution regularly caused up to 8% mortality among controls not exposed to DDT, its use was discon- tinued; the effect appeared to be attributable to mechanical or physical trauma. Fig. 1 shows mean mortalities, standard deviations and ranges of mortality for the different sugar con- centrations. Compared with water, all sugar concen- trations up to 20% gave significantly reduced mor- tality (for the 2.5% concentration, P <0.05; for the 5%, 10% and 20% concentrations, P <0.01). Mor- talities with 10% and 20% sugar were significantly lower than with 2.5% sugar and significantly higher than with the 40% concentration (P<0.01). Mor- *%. MORTALITY 100 90 80 I 70 [ RANGE MAX. 60 tI\i5. I0S 50 i SD 40 wIRANGE MIN. 20- 20 10 0 2i5 10 20 40% CONCENTR. OF SUGAR Fig. 1. Mean mortalities (x), standard deviation (SD) and range of mortality in A. albimanus when exposed to DDT after overnight access to sugar solutions. talities caused by 2-hour exposure to 2% DDT (mean ()= 52 %; standard deviation (SD) = 24.6 %; 40 replicates) were quite close to those obtained with 1-hour exposure to 4% DDT (x= 54%; SD = 18.9 %; 20 replicates).a Discussion The protective effect against DDT of tarsal con- tact with sugar solutions during the night before testing was shown in this study to extend to A. albi- manus; the effect apparently reaches a maximum with sugar solutions between 10% and 20%, the range of concentration likely to be used in the field. In Central America, test material is usually kept overnight in a cage of 3 000 cm3 capacity or more (Austin, personal communication, 1973), but in South America paper cups of 220 cm3 or less are used, giving conditions of virtually forced contact with the sugar pad. However, the tendency of con- fined mosquitos to explore all parts of the receptacle before settling down probably means that even in the former case a good deal of contact takes place. Although, as frequently occurs with the WHO sus- ceptibility test for adult mosquitos, variance was high, a statistically significant effect of pre-test expo- sure to sugar solution has been shown. In the test series, the mean reduction of mortality brought about by 10% and 20% sugar, compared with water, was 27 %. As the grades of resistance to DDT in Central America are classified by 24-hour survival, after 1 hour of exposure to 4% DDT, of 0 %-4 %, 5%-24%, 25%/-49Y%, 50%/-74%, and 75%-100%, the use or non-use of sugar could alter the assessment of a vector population by 1 or even 2 classes. Since most tests in the Region of the Americas have been conducted with sugar-fed mosquitos, the results are probably comparable inter se, but not with those obtained elsewhere with the unmodified WHO method. The effect of sugar may, however, be the source of reports of survival at the standard dosage, and thus of resistance to DDT, in South American malaria vectors, such as A. nuneztovari and A. punctimacula, which are in fact still highly susceptible (3). The principal objection to the American modifi- cation of the WHO test is that the exposure to sugar is to some extent facultative, and thus adds an unnecessary source of variation to an experimental procedure that is already beset by sampling prob- a The calculation of SD (between batches) and the com- parison of the meanz by the t-test followed the methodology proposed by Swaroop (2). BRIEF COMMUNICATIONS 313 lems, uncontrollable meteorological conditions, and seasonal influences such as the aerial spraying of the cotton crop. REFERENCES 1. WHO Technical Report Series, No. 443, 1970 (Insec- ticide resistance and vector control: seventeenth report of the WHO Expert Committee on Insecticides), Annex IA and IB, pp. 47-65. 2. SwAROop, S. Statistical methods in malaria eradication, Geneva, World Health Organization, 1966 (Mono- graph Series, No. 51). 3. BROWN, A. W. A. & PAL, R. Insecticide resistance in anthropods, 2nd ed. Geneva, World Health Organiz- ation, 1971 (Monograph Series, No. 38).