Sample sizes for identifying the key types of container occupied by dengue-vector pupae: the use of entropy in analyses of compositional data.

A method has been developed for estimating the sample sizes needed to identify categories that comprise a large proportion of a compositional data-set. The method is to be used in the design of surveys of mosquito pupae, for identifying the key container types from which the majority of adult dengue vectors emerge. Although a finite-population correction was devised for estimating the mean of a negative binomial distribution, other complications of parametric approaches make them unlikely to yield methods simple enough to be practically applicable. The Shannon-Wiener index was therefore investigated as a more useful alternative, at the cost of theoretical generalizability, in an approach based on re-sampling methods in conjunction with the use of entropy. This index can be used to summarize the degree to which pupae are either concentrated in a few container types, or dispersed among many. An empirical relationship between the index and the repeatability of surveys of differing sample sizes was observed. A step-wise rule, based on the entropy of the cumulative data, was devised for determining the sample size, in terms of the number of houses positive for pupae, at which a pupal survey might reasonably be stopped.

Aedes aegypti pupal/demographic surveys in southern Mexico: consistency and practicality.

In interventions aimed at the control of the immature stages of Aedes aegypti (L.), the principal vector of the dengue viruses, attempts are often made to treat or manage all larval habitats in households. When there are resource-constraints, however, a concentration of effort on the types of container that produce the most pupae may be required. Identification of these 'key' container types requires surveys of the immature stages and particularly - since these give the best estimates of the numbers of adults produced - of the numbers of pupae in local containers. Although there has been no clearly defined or standardized protocol for the sampling of Ae. aegypti pupae for many years, a methodology for 'pupal/demographic' surveys, which may allow the risk of dengue outbreaks in a given setting to be estimated, has been recently described. The consistency and practicality of using such surveys has now been investigated in three cities in the Mexican state of Chiapas, Mexico. Using a combination of 'quadrat'- and transect-sampling methods, 600 houses in each city were each sampled twice. Containers within each study household were searched for pupae and larvae. Although 107,297 containers, belonging to 26 categories, were observed, only 16,032 were found to contain water and 96% and 92% of these 'wet' containers contained no pupae and no third- or fourth-instar larvae, respectively. Although the random 'quadrat' sampling gave similar results to sampling along transects, there were statistically significant differences in the numbers of pupae according to container type and locality. The most important containers for pupal production were found to be large cement wash basins, which were present in almost every household investigated and from which 84% (10,257/12,271) of all pupae were collected. A focus on this class of container could serve as the basis of a targeted intervention strategy. When traditional Stegomyia indices were calculated they appeared to be correlated with the assessments of pupal abundance. The methodology for pupal/demographic surveys appears to be practical and to give consistent results, although it remains to be seen if monitoring of pupal productivity can adequately reflect the impact of vector-control interventions.

Effect of Novaluron (Rimon 10 EC) on the mosquitoes Anopheles albimanus, Anopheles pseudopunctipennis, Aedes aegypti, Aedes albopictus and Culex quinquefasciatus from Chiapas, Mexico.

Dengue fever is a serious problem in Mexico and vector control has not been effective enough at preventing outbreaks. Malaria is largely under control, but it is important that new control measures continue to be developed. Novaluron, a novel host-specific insect growth regulator and chitin synthesis inhibitor, has proved to be effective against agricultural pests, but its efficacy against larval mosquito vectors under field conditions remains unknown. In accordance with the World Health Organization Pesticide Evaluation Scheme, phase I, II and III studies were conducted to evaluate the efficacy and residual effect of Novaluron (Rimon 10 EC, Makhteshim, Beer-Sheva, Israel) on the malaria vectors Anopheles albimanus Wiedemann (Diptera: Culicidae) and Anopheles pseudopunctipennis Theobald, the dengue vectors Aedes aegypti (L) and Aedes albopictus Skuse and the nuisance mosquito Culex quinquefasciatus Say. Laboratory susceptibility tests yielded diagnostic concentrations for all five target species. Field trials to identify the optimum field dosage of Novaluron against Anopheles mosquitoes were carried out under semi-natural conditions in artificial plots and in vessels with wild mosquitoes. Efficacy was measured by monitoring mortality of larvae and pupae and the percentage of inhibition of emergence from floating cages. Dosages of Novaluron for field tests were based on pupal LC(99) (lethal concentration 99%) of An. pseudopunctipennis (0.166 mg/L) in plots and average pupal LC(99) of Ae. aegypti and Ae. albopictus (0.55 mg/L). At all dosages tested, Novaluron significantly reduced larval populations of An. albimanus, Culex coronator Dyar & Knab, Ae. albopictus and Cx. quinquefasciatus by approximately 90%, inhibited adult emergence of An. albimanus and An. pseudopunctipennis by approximately 97% for almost 4 months in experimental plots, and inhibited adult emergence of Ae. aegypti and Ae. albopictus by approximately 97% for up to 14 weeks. Recommended dosages of Novaluron for non-container breeding and container breeding mosquitoes are 0.166 mg/L and 0.55 mg/L, respectively. Overall, the residual effect was more sustained than that of temephos. The lowest dosage of Novaluron had less of an impact on non-target organisms than did temephos. Small-scale field trials in natural breeding sites treated with Novaluron at 0.6 L/ha eliminated adult emergence of An. albimanus and Cx. coronator for 8 weeks. For phase III studies, Novaluron was tested at the local and village levels, applying the optimum field rate to all natural breeding habitats within 1 km of a pair of neighbouring villages. Village-scale trials of Novaluron at 0.6 L/ha reduced An. albimanus larval populations for at least 8 weeks and, more importantly, sharply reduced the densities of adult host-seeking mosquitoes approaching houses. We conclude that Novaluron is effective and environmentally safer than temephos.

Sublethal iridovirus disease of the mosquito Aedes aegypti is due to viral replication not cytotoxicity.

Invertebrate iridescent viruses (Iridoviridae) possess a highly cytotoxic protein. In mosquitoes (Diptera: Culicidae), invertebrate iridescent virus 6 (IIV-6) usually causes covert (inapparent) infection that reduces fitness. To determine whether sublethal effects of IIV-6 are principally due to cytotoxicity of the viral inoculum (which inhibits macromolecular synthesis in the host), or caused by replication of the virus larvae of the mosquito Aedes aegypti (L) were exposed to untreated IIV-6 virus that had previously been deactivated by heat or ultraviolet light. Control larvae were not exposed to virus. Larval development time was shortest in control larvae and extended in larvae exposed to untreated virus. Covertly infected mosquitoes laid significantly fewer eggs, produced between 20 and 35% fewer progeny and had reduced longevity compared to other treatments. Wing length was shortest in mosquitoes exposed to heat-deactivated virus. Multivariate analysis of the same data identified fecundity and progeny production as the most influential variables in defining differences among treatments. Overall, viral infection resulted in a 34% decrease in the net reproductive rate (R0) of covertly infected mosquitoes, vs. only 5-17% decrease of R0 following treatments with deactivated virus, compared to controls. Sublethal effects of IIV-6 in Ae. aegypti appear to be mainly due to virus replication, rather than cytotoxic effects of the viral inoculum.

Selective oviposition by Aedes aegypti (Diptera: culicidae) in response to Mesocyclops longisetus (Copepoda: Cyclopoidea) under laboratory and field conditions.

The influence of predacious Mesocyclops longisetus Thiebaud on the selection of oviposition sites by prey Aedes aegypti (L.) was studied under laboratory and field conditions. In both cases, gravid Ae. aegypti females were significantly more attracted to ovitraps containing copepods or to ovitraps with water in which copepods were held previously than to distilled water. Monoterpene and sesquiterpene compounds including 3-carene, alpha-terpinene, alpha-copaene, alpha-longipinene, alpha-cedrene, and delta-cadinene were found in hexane extracts of copepods by gas chromatography and mass spectrometry analyses. These compounds may be responsible for attracting gravid Ae. aegypti females and may increase the number of potential prey for the copepod.

Update on the biology of Triatoma dimidiata Latreille (Hemiptera: Reduviidae) under laboratory conditions.

Feeding time, postfeeding defecation delay, and life cycle for each stage of a cohort of recently colonized Mexican Triatoma dimidiata were evaluated, and results were compared to existing published information on this species. Seventy-five nymphs (41.7%) completed a cycle with an average time from N-I to adult of 142 +/- 64 days. The average span in days for each stage was 20.2 for N-I, 17.9 for N-II, 10.1 for N-III, 43.6 for N-IV, and 55.1 for N-V. First-stage nymphs had the highest mean feeding time (25 min) and the longest postfeeding defecation delay (45 min). Differences among biological data from previous studies and the present study confirm the importance of conducting research on the behavior of the indigenous triatomine species from various countries.

Description and morphometric analysis of the eggs of Anopheles (Anopheles) vestitipennis (Diptera: Culcidae) from southern Mexico.

Light and scanning electron microscopy were used to compare the eggs of Anopheles vestitipennis Dyar & Knab females collected from human and animal baits in 9 villages of southern Mexico. An. vestitipennis eggs are boat-shaped, with lateral floats extending the length of the egg. Both the deck and dorsal surface are covered with hexagonal and pentagonal chorionic cells that contain round tubercles in the cell field. Crowns that enclose 3-5 lobed tubercles are present at both egg poles. By light microscopy, the mean length/width ratio of eggs of females caught at human bait were statistically different from those of females caught in horse-baited animal traps. In a regression tree model that included 19 egg attributes, the same egg groups could be separated by their posterior crown length/width ratio and mean anterior cell deck form factor (an index of their roundness). These findings support of the possible existence of 2 An. vestitipennis subpopulations with different host preferences in southern Mexico.

Gonotrophic cycle and survivorship of Anopheles vestitipennis (Diptera: Culicidae) in two different ecological areas of southern Mexico.

The duration of the gonotrophic cycle and survivorship of Anopheles vestitipennis Dyar & Knab was estimated in 2 malarious areas of Chiapas, Mexico: the Lacandon Forest and the Pacific Ocean Coastal Plain. Blood-engorged females held in an outdoor cage required 2.75 d for egg maturation, and 3.75 d for the duration of the gonotrophic cycle. Duration of the gonotrophic cycle also was estimated by parous-nulliparous dynamics for 20 consecutive days and autocorrelation time-series analysis, and by mark-recapture techniques. These methods depicted differences between the Lacandon Forest (3-d cycle) and the Coastal Plain (2-3 d cycles). Daily survival rates were estimated vertically and were generally higher in the Lacandon Forest (0.68) than in the Coastal Plain (0.45-0.58). The probability of mosquitoes surviving the sporogonic cycle was 10-100 times greater in the Lacandon Forest. The pregravid rate was 8.2%, and 29.3% of females with primary follicles beyond Christophers' stage III had traces of red blood in the gut. The 1st statistic indicated that 8.2% of females required > 1 blood meal for initial egg development, the 2nd statistic indicated that 29.3% of females take > 1 blood meal during a gonotrophic cycle. In summary, the enhanced vectorial role of this species is explained partially by high longevity and multiple blood-feeding habits.

Resistance management strategies in malaria vector mosquito control. Baseline data for a large-scale field trial against Anopheles albimanus in Mexico.

A high level of DDT resistance and low levels of resistance to organophosphorus, carbamate and pyrethroid insecticides were detected by discriminating dose assays in field populations of Anopheles albimanus in Chiapas, southern Mexico, prior to a large-scale resistance management project described by Hemingway et al. (1997). Biochemical assays showed that the DDT resistance was caused by elevated levels of glutathione S-transferase (GST) activity leading to increased rates of metabolism of DDT to DDE. The numbers of individuals with elevated GST and DDT resistance were well correlated, suggesting that this is the only major DDT resistance mechanism in this population. The carbamate resistance in this population is conferred by an altered acetylcholinesterase (AChE)-based resistance mechanism. The level of resistance observed in the bioassays correlates with the frequency of individuals homozygous for the altered AChE allele. This suggests that the level of resistance conferred by this mechanism in its heterozygous state is below the level of detection by the WHO carbamate discriminating dosage bioassay. The low levels of organophosphate (OP) and pyrethroid resistance could be conferred by either the elevated esterase or monooxygenase enzymes. The esterases were elevated only with the substrate pNPA, and are unlikely to be causing broad spectrum OP resistance. The altered AChE mechanism may also be contributing to the OP but not the pyrethroid resistance. Significant differences in resistance gene frequencies were obtained from the F1 mosquitoes resulting from adults obtained by different collection methods. This may be caused by different insecticide selection pressures on the insects immediately prior to collection, or may be an indication that the indoor- and outdoor-resting A. albimanus collections are not from a randomly mating single population. The underlying genetic variability of the populations is currently being investigated by molecular methods.

Colonization of Anopheles pseudopunctipennis from Mexico.

Two colonies of Anopheles pseudopunctipennis, Tapachula and Abasolo strains, were established under laboratory conditions with a thermoperiod (29 degrees C during the day; 24 degrees C during the night) and artificial dusk. To stimulate mating, a light beam from a flashlight was shone on the cage shortly after lights off. This procedure was repeated for the first 6 mosquito generations (parental to F6) and thereafter light stimulation was unnecessary for mating. The Tapachula colony has been maintained for 24 generations in 24 months, with insemination rates in females > 80% since the F3, and a monthly production of 30,000 pupae since the F7. Using the same procedure, the Abasolo colony from northeastern Mexico has been maintained for 13 generations in 14 months, with insemination rates of 26-52%.

Selective and conventional house-spraying of DDT and bendiocarb against Anopheles pseudopunctipennis in southern Mexico.

Indoor feeding behaviors and mortalities of Anopheles pseudopunctipennis females were evaluated following contact with selective (bands covering mosquitoes' preferred resting areas) and full applications of DDT and bendiocarb on indoor sprayable surfaces. The DDT residues provoked strong avoidance behavior. To a lesser degree, mosquitoes were also repelled by bendiocarb-sprayed surfaces. Because of strong irritancy/repellency, unfed mosquitoes were driven outdoors in proportionally higher numbers. The resting time on selectively or fully DDT-sprayed huts was greatly reduced in comparison to bendiocarb-sprayed huts. Although unfed mosquitoes tended to rest on non-DDT-sprayed surfaces in the selectively treated hut, the man-biting rate was similar with both types of treatments. Unfed mosquitoes were repelled less from selectively bendiocarb-treated surfaces. Similar reductions in postfed resting times were observed on all surfaces suggesting that once fed, mosquitoes rested on sprayed surfaces for shorter intervals of time. Engorged mosquitoes had normal resting behavior (pre- and postspray) within the range of preferred resting heights in both DDT- and bendiocarb-sprayed huts, but the proportion of mosquitoes fed in the DDT-treated huts was lower. Selective spraying of walls was as effective as spraying the complete walls with both insecticides, but DDT was more effective in reducing mosquito-human contact. These studies show that by more effectively targeting vector behavior, a cost-effective alternative to traditional control techniques can be achieved.

Behaviour of Anopheles albimanus in relation to pyrethroid-treated bednets.

Responses of the malaria vector Anopheles albimanus to pyrethroid impregnated bednets made of cotton or nylon, compared with untreated nets, were investigated in houses occupied by two people inside and/or outside two bednets, in coastal Chiapas, México. The pyrethroid used was lambdacyhalothrin 30 mg a.i./m2. Bioassay mortality rates of An.albimanus exposed to treated nets for 3 or 15 min, rose from 40-55% to 90-100% for nylon nets 3-19 weeks post-treatment, but were consistently lower for treated cotton nets. An.albimanus females (collected unfed on human bait) were released in houses surrounded by curtains for trapping mosquitoes that exited from the house. Floor sheets were used in and around each experimental house for retrieving any mosquitoes knocked-down and/or killed. During post-treatment assessment for 17 weeks, An.albimanus blood-feeding success rates were 23-24% with untreated nets, 14-18% with treated cotton nets and 8-15% with treated nylon nets, significantly reduced when both human baits were inside the treated bednets, but not when one or more baits were outside the treated bednet(s) within the house. Proportions of mosquitoes leaving houses < 3 h post-release were 53-59% from houses with untreated bednets versus 65-78% with treated bednets. Except in one case (when both humans were outside treated cotton nets), these increased early exit rates were significant, whether or not the human baits were inside the treated bednets indoors. Mortality rates of An.albimanus females exiting overnight (22.00-06.00 hours) averaged 15-39% from houses with treated cotton and 16-46% with treated nylon nets, very significantly greater than the control mean rates of 6-8% mortality with untreated nets. Observations on wild-caught An.albimanus females marked with fluorescent powder and released indoors revealed that few mosquitoes (3-11%) actually contacted the bednets unless both human baits remained under them - when contact rates were 22% on treated nylon, 23% on treated cotton and 42% on untreated nets (P approximately 0.05). The mean resting time was significantly longer on untreated (14.4 min) than on treated nylon (5.8 min) or cotton (9.5 min) bednets, whereas mean resting times on other surfaces indoors were 16.5-19.8 min. Proportions exiting within 2h of release were significantly more from houses with treated houses (33-35%) than with untreated nets (8%). However, mortality rates of mosquitoes that landed on treated nets were very significantly greater (90-100%) than after landing on untreated nets (10%). Thus, despite some excito-repellency, lambdacyhalothrin-impregnated bednets (especially made of nylon) proved to be effective as an alternative to house-spraying against An.albimanus.

Influence of plant abundance on nectar feeding by Aedes aegypti (Diptera: Culicidae) in southern Mexico.

The availability of flowering plants affected the sugar feeding rates of female Aedes aegypti (L.) in 4 areas of a small city in southern Mexico. The proportion of mosquitoes containing sugar varied from 8 to 21% in 4 areas in direct relation to blooming plant abundance. Human density was similar in the 4 areas (range, 3.9-5.4 per house), whereas the number of flowering plants per house increased on the outskirts (range, 3.1-5.4 plants per house). Equal proportions of sugar positive females were nulliparous or parous, indicating similar sugar feeding at any age. In addition, nearly 60% of positive females were at the Christophers stage II, indicating a greater need for flight fuel during the early stages of egg development. We conclude that Ae. aegypti feeds frequently on nectar and that this activity is modulated by nectar availability.

Genetic differences among Anopheles vestitipennis subpopulations collected using different methods in Chiapas state, southern México.

Biting activity and population genetic studies of the malaria vector Anopheles vestitipennis were conducted in southern México. Three subpopulations were collected from 2 villages; 2 subpopulations were from the same village, one on human bait and one with an animal-baited trap; the third was collected from a cattle corral in the 2nd village (280 km away SSE). The anthropophilic subpopulation had steady activity with 61% of bites occurring before midnight, significantly different from those of the 2 zoophilic subpopulations, which had 78-82% of bites before midnight and 2 biting peaks, one at 1900-2100 h and the other at 0400-0500 h. Isozyme analysis (13 enzymes) of these subpopulations indicated that differences between the 2 sympatric subpopulations (D = 0.07), collected using 2 different methods, were greater than that between the 2 allopatric ones (D = 0.03). These studies suggest the existence of 2 genetically different subpopulations of An. vestitipennis with specific host preferences.

Low-volume application by mist-blower compared with conventional compression sprayer treatment of houses with residual pyrethroid to control the malaria vector Anopheles albimanus in Mexico.

Village-scale trials were carried out in southern Mexico to compare the efficacy of indoor-spraying of the pyrethroid insecticide lambda-cyhalothrin applied either as low-volume (LV) aqueous emulsion or as wettable-powder (WP) aqueous suspension for residual control of the principal coastal malaria vector Anopheles albimanus. Three indoor spray rounds were conducted at 3-month intervals using back-pack mist-blowers to apply lambda-cyhalothrin 12.5 mg a.i./m2 by LV, whereas the WP was applied by conventional compression sprayer at a mean rate of 26.5 mg a.i./m2. Both treatments caused mosquito mortality indoors and outdoors (collected inside house curtains) as a result of contact with treated surfaces before and after feeding, but had no significant impact on overall population density of An. albimanus resting indoors or assessed by human bait collections. Contact bioassays showed that WP and LV treatments with lambda-cyhalothrin were effective for 12-20 weeks (> 75% mortality) without causing excito-repellency. Compared to the WP treatment (8 houses/man/day), LV treatment (25 houses/man/day) was more than 3 times quicker per house, potentially saving 68% of labour costs. This is offset, however, by the much lower unit price of a compression sprayer (e.g. Hudson 'X-pert' at US$120) than a mist-blower (e.g. 'Super Jolly' at US$350), and higher running costs for LV applications. It was calculated, therefore, that LV becomes more economical than WP after 18.8 treatments/100 houses/10 men at equivalent rates of application, or after 7.6 spray rounds with half-rate LV applications.

Control of Anopheles albimanus mosquitos in southern Mexico by spraying their preferred indoor resting sites.

The traditional indoor spraying technique to control Anopheles albimanus mosquitos was compared with a selective method that targets their preferred resting sites in coastal villages of southern Mexico. We also determined whether mosquitos changed their preferred resting sites following insecticide applications. In the selective spraying approach, two 1-m wide horizontal swaths of bendiocarb insecticide were applied 0.75-1.75 m from the base of the walls and at 1-m upwards from the base of the inner roof at a target dose of 0.4 g/m2. A 2-year follow-up (1988-90), which included four insecticide treatments, showed that the selective spraying was as effective as full spraying for controlling A. albimanus. Also the mosquitos preferred resting sites that were not modified by the insecticide applications. Finally, the use of this techniques required 46% less time and cost 67% less than conventional spraying.

Remote sensing of tropical wetlands for malaria control in Chiapas, Mexico.

Malaria, transmitted by anopheline mosquitoes, remains a serious health problem in the tropics. Most malaria eradication efforts focus on control of anopheline vectors. These efforts include the NASA Di-Mod project, whose current goal is to integrate remote sensing, geographic information systems (GIS), and field research to predict anopheline mosquito population dynamics in the Pacific coastal plain of Chiapas, Mexico. Field studies demonstrate that high larval production of Anopheles albimanus, the principal malaria vector in the plain, can be linked to a small number of larval habitat-types, determined by larval sampling and cluster analysis of wetlands in the coastal plain. Analysis of wet and dry season Landsat Thematic Mapper (TM) satellite imagery identified 16 land cover units within an 185-km2 study area in the coastal zone. A hierarchical approach was used to link the larval habitat-types with the larger land cover units and make predictions of potential and actual low, medium, and high anopheline production. The TM-based map and GIS techniques were then used to predict differences in anopheline production at two villages, La Victoria and Efrain Gutierrez. La Victoria was predicted to have much higher Anopheles albimanus production, based upon a 2-10 times greater extent of medium- and high-producing land cover units in its vicinity. This difference between villages was independently supported by sampling (with light traps) of adults, which were 5-10 times more abundant in La Victoria.

Intradomiciliary behavior of Anopheles albimanus on the coastal plain of southern Mexico: implications for malaria control.

The postfeeding indoor resting behavior of Anopheles albimanus in experimental houses in southern México was investigated by using a mark-recapture procedure. The majority of mosquitoes rested inside houses after taking a blood meal indoors. There was a higher landing frequency on interior surfaces other than walls and roofs; however, mosquitoes rested for longer periods on these 2 surfaces. Successive landings on walls after short flights showed that mosquitoes gradually increased their mean landing height from 1.0 to 1.4 m. Similarly, mosquitoes resting at the base of inner roofs had a successive landing height range of about 0.5 m. Based on these observations and the potential for reduction of nearly 50% in the quantity of insecticide used and the time needed to apply it, village-scale studies involving the selective spraying of a 1-m-wide swath of insecticide on walls and on roofs are recommended in this area.