Progress 10/01/99 to 09/30/04
We developed linear regression models to age-grade female Aedes aegypti reared and maintained under controlled lab conditions. We analyzed by gas-liquid chromatography temporal differences between 2 cuticular hydrocarbons for predicting female age. We developed two new regression models, one for the cool-dry season and one for the rainy season. Both models resulted in better estimates of age than the original regression models and improved our ability to predict age of the Ae. Aegypti out to 15 calendar days. We adapted this method to age male Ae aegypti to investigate the potential of hydrocarbon analysis to the age of the Anopheles vectors of malaria. Using gas chromatography, we detected a significant relationship between the 2 cutilular hydrocarbons. We found the relative abundance of the ration of the cuticular hydrocarbons pentacosane and nonacosane decreased in early with the log of mosquito age and provide a model to predict if a mosquito is old enough to
transmit malaria. Since teneral females required about 2 days to mature and mate before taking their bloodmeal, only those more than 12 days old could transmit malaria. We found that a mosquito with a C29/C31 ration of 2.6 is only 10% probable to be old enough to transmit malaria whereas a mosquito with a ration of 1.8 is 90% probable.
Development of an automated system for accurately ageing adult mosquitoes.
- No publications reported this period
Progress 01/01/03 to 12/31/03
In previous studies, we developed linear regression models to age-grade female Aedes aegypti reared and maintained under controlled lab conditions. The models were based on temporal differences between 2 cuticular hydrocarbons, pentacosane (C25H52) and nonacosane (C29H60), which were extracted from Ae. aegypti legs and analyzed by gas-liquid chromatography. These initial models predicted female age up to 165 degree days (12-15 calendar days at 28 C). Older mosquitoes, however, could not be accurately aged. In the present study, original regression models were tested using age data obtained from mosquitoes maintained in a field lab and those that were marked, released, and recaptured in NE Thailand. Our field data lead to the development of 2 new regression models, one for the cool-dry season (Feb-Mar) and one for the rainy season (Jul-Aug). Both models resulted in better estimates of age than the original model and improve our ability to predict the age of Ae. aegypti
out to 15 calendar days. Females older than 15 days can be identified as such, but their exact age still cannot be estimated. Currently we are adapting this method to age male Ae aegypti and apply it to field studies of mating competitiveness. Success in accurately aging Ae aegypti in the field, led us to investigate the potential of hydrocarbon analysis to age the Anopheles vectors of malaria. Lab colony Anopheles stephensi were reared at 27C and females aged 3-21d were sampled to create 10 age groups provided 10% sucrose but no blood. Using previous methods, hydrocarbons extracts from each mosquito's legs were injected into a gas chromatograph with flame-ionization detection. This allowed quantification of hydrocarbons based on the integrated areas of specific peaks in the chromatograms; 6 peaks were distinct. With regression analysis, a significant relationship was detected between 2 of the peaks: nonacosane (C29) and hentriacontane (C31). Relative abundance of C29/C31 decreased
linearly with the log of mosquito age. We confirmed this relationship by repeating the experiment. We used the resulting C29/C31 values with the model to predict the age of each mosquito. The actual and predicted ages were highly correlated with a mean difference of 0.1d (SD = 4.2d). The relative abundance of these 2 hydrocarbons may therefore indicate if a mosquito is old enough to transmit malaria, e.g., in An. stephensi mosquitoes at 27 C, Plasmodium falciparum parasites require 10 d to develop into a human-infective form. Since teneral females require appox. 2 d for maturation and mating before taking their 1st bloodmeal, only those more than 12 d old would be able to transmit malaria. We modeled the probability that an individual mosquito has exceeded this transmission age based on its C29/C31 ratio. To do this, we created a 2nd regression model using the data from both replicates. We then generated prediction intervals for a 12-d-old mosquito, using confidence levels for
one-sided tests. The model indicates that a mosquito with a C29/C31 ratio of 2.6 is only 10% probable to be old enough to transmit malaria, whereas a mosquito with a ratio of 1.8 is 90% probable
Determining the age structure of mosquito populations is critical for evaluating disease risk and for evaluating impact of vector control strategies including replacement with genetically modified populations. We developed and continue to refine an automated system for accurately ageing adult mosquitoes.
- Buonaccursi, J., Harrington, L.C. and J. D. Edman. 2003. Estimation and comparison of mosquito survival rates with release-capture-removal data. J. Med. Entomol. 40: 6-17.
- De Benedictis, J., E. Chow-Shaffer, A. Costero, G.G. Clark, J.D. Edman and T.W. Scott. 2003. Identification of the people from whom engorged Aedes aegypti took blood meals in Florida, Puerto Rico, using polymerase chain reaction-based DNA profiling. Am. J. Trop. Med. Hyg. 68: 437-446.
- Edman, J.D. 2003. Fitness advantage in multiple blood feeding: the Aedes aegypti example, pp.63-74. IN: Ecological Aspects for Application of Genetically Modified Mosquitoes, W.Takken & T.Scott (eds). Wageningen UR Frontis Series (Kluwer) Dordrecht, The Netherlands.
- Gerade, B., S.H. Lee, J.D. Edman, T.W. Scott, L. Harrington and J.M. Clark. 2003. Field evaluation of Aedes aegypti (Diptera: Culicidae) age estimation by analysis of cuticular hydrocarbons. J. Med. Entomol. (in press)
- Brei, B., J.D. Edman, B. Gerade and J.M. Clark. Relative abundance of two culicular hydrocarbons indicates if a mosquito is old enough to transmit malaria parasites. J. Med. Entomol. (in press)2004
Progress 01/01/02 to 12/31/02
Age Determination by Cuticular Hydrocarbon Analysis. Field-collected, marked and released (FCMR) mosquitoes were recaptured during dry and wet seasons in Thailand to validate age-grading models. Statistical analyses were performed using Minitab 12 for linear regressions and analyses of covariance (ANCOVA). Linear regression data for females collected in the same season but different years were not statistically different. When the combined FCMR data from the wet and dry seasons were analyzed, however, they were statistically different (p = 0.035). Regression lines for predicted ages were compared to those for actual ages in both seasons. Data for the dry and wet season were statistically different from their respective actual ages indicating that separate models were needed for each season. Combined data were plotted as a function of degree day (DD) for dry and wet season females, respectively. Both data sets became noticeably curvelinear. A square root transformation
produced the best-fitted regression models. Models for each season were validated as before to determine performance using FCMR females. Regressions of predicted ages were compared to actual ages. Dry season model, slope and y-intercept values were the same; wet season, slope and y-intercept values were different. Wet season model appeared to fit data better but was less able to predict actual age compared to dry season model. Actual age structure of FCMR females from dry season was notice older than wet season. Still, both models performed similarly in the actual percent of correct predictions. Wild mosquitoes were collected in wet and dry seasons and their ages predicted by their seasonal square root model. Comparison of age distributions from wet and dry seasons revealed more young females during the wet season than during the dry, which correlates with our findings for FCMR females. In both distributions there are fewer individuals corresponding with the 1st oviposition (5-6 days)
of blood-fed females. This physiological age marker provides further evidence that the two new models may accurately predict female age. Age Determination by Mark-Recapture Analysis. It has been assumed that the probability of adult mosquitoes surviving does not change with age. A series of uniquely designed field studies tested this non-senescence hypothesis for Ae. aegypti in Thai villages. Synthetic age-cohorts were created with adults emerging over 2-day intervals from village pupae. Ten cohorts (0-2 days old to 18-20 days old) were marked and simultaneously released. Mosquitoes were recaptured over the next 2 days. A replicate release was conducted 4 days later. Data were analyzed using Proc GENMOD in SAS with age as a continuous variable. Significant age effects were detected (P <0.0001). The best-fit model for predicting age-cohort recapture over time was non-linear with age-squared. Results from this experiment refute the non-senescence hypothesis, challenge the notion that
mosquito biology does not change with age, and support more rigorous quantitative study of mosquito senescence affects on pathogen transmission.
The ability to age vector populations in the field will assist in predicting disease risk and in evaluating the efficacy of various vector control strategies.
- Harrington, L.C., J.P. Buonaccorsi, J.D. Edman, A. Costero, G.G. Clark, P. Kittayapong, and T.W. Scott. 2001. Analysis of survival rates for two age cohorts of Aedes aegypti (L.) (Diptera: Culicidae): Results from Puerto Rico and Thailand. J. Med. Entomol. 38: 537-547.
- Harrington, L.C. and J. D. Edman. 2001. Indirect evidence against "skip-oviposition" behavior by wild Aedes aegypti (Diptera: Culicidae) from Thailand. J. Med. Entomol. 38(5) 641-645.
- Harrington, L.C., J.D. Edman and T.W. Scott. 2001. Why do female Aedes aegypti (Diptera: Culicidae) feed preferentially and frequently on human blood? 2001. J. Med. Entomol. 38(3): 411-422.
- Buonaccorsi, J., Harrington, L. C. and J. D. Edman. 2002. Estimation and comparison of mosquito survival rates with release-recapture-removal data. J. Med. Entomol. In press
Progress 01/01/01 to 12/31/01
Analysis of cuticular hydrocarbons (CH) from lab-reared and field-collected Aedes aegypti females from Thailand, were conducted during the wet and dry season of 2000 and 2001. Using the relative abundance (RA) of C25/C25+C29 versus degree-day calculations for each collection, linear regression models were generated and compared. Age grading equations were not different from each other, indicating that the current age grading method based on the RA of specific CH peaks is independent of seasonal variation and consistent year to year. Of the 109 field-reared, marked and recaptured mosquitoes from Feb-Mar 2000, 75 have been analyzed and their ages in degree-days predicted using the age-grading equation. The model was able to predict the age (within the predictive interval of the model) of approximately 75 % of these recaptured mosquitoes up to 165 degree days (approx.15 calendar days). To determine if the model can accurately identify mosquitoes > 2 weeks old, lab-reared
females up to 88 days old were analyzed. Although the slope of the predicted degree-day versus actual age line was zero (indicating the specific ages of older mosquitoes could not be accurately estimated by this method), all old mosquitoes (15-88 days old) tested were estimated to be at least 15 days old. This indicates that once females attain this age, they maintain their CH complement over time. An additional CH peak (retention time 20.8 min) was observed and tentatively identified as a sterol molecule most closely related to 26-nor-5-cholesten-3-beta-one. A highly significant increase in the abundance of this sterol was seen in blood-fed vs sugar-fed females. Abundance does not increase significantly after multiple feedings, but this molecule appears to be a notable indicator that a mosquito has previously taken a blood meal. Various larval rearing methods were evaluated and compared to the original method use to lab-rear the Rockefeller strain. To date, no significant impact on
the CH complement of differentially reared mosquitoes has been observed.
The ability to age vector populations in the field will assist in predicting disease risk and in evaluating the efficacy of various vector control methods.
- Moncayo, J.D. Edman and J.T. Finn. 2000. Application of geographic information technology in determining risk of Eastern equine encephalomyelitis virus transmission. J. Amer. Mosq. Contr. Assoc. 16: 28-35.
- Moncayo, A., J.D. Edman and M.J. Turell. 2000. Effect of Eastern equine encephalomyelitis virus on the survival of Aedes albopictus, Anopheles quadrimaculatus and Coquillettidia perturbans (Diptera: Culicidae). J. Med.Entomol. 37: 701-706. Harrington, L.C., J.D. Edman and T.W. Scott. 2001. Why do female Aedes aegypti (Diptera: Culicidae) feed preferentially and frequently on human blood. J. Med. Entomol. 38: 411-422.
- Harrington, L.C., J.P. Buonaccorsi, J.D. Edman, A. Costero, P. Kittayapong, G.G. Clark and T.W. Scott. 2001. Analysis of survival of young and old Aedes aegypti (Diptera: Culicidae) from Puerto Rico and Thailand. J. Med. Entomol. 38: 537-547.
- Harrington, L.C. and J. D. Edman. 2001. Indirect evidence against delayed "skip-oviposition" behavior by Aedes aegypti (Diptera: Culicidae) in Thailand. J. Med. Entomol. 38: 641-645.
- Scott, T.W., A.C. Morrison, L.H. Lorenz, G.G. Clark, P.Reiter, D. Strickman, P. Kittayapong, H. Zhou and J.D. Edman. 2000. Longitudinal studies of Aedes aegypti (Diptera: Culicidae) in Thailand and Puerto Rico: population dynamics. J. Med. Entomol. 37: 77-88.
- Scott, T.W., P.H. Amerasinghe, A.C. Morrison, L.H. Lorenz, G.G. Clark, P. Reiter, D. Strickman, P. Kittayapong and J.D. Edman. 2000. Longitudinal studies of Aedes aegypti (Diptera: Culicidae) in Thailand and Puerto rico: blood feeding frequency. J. Med. Entomol. 37: 89-101.