Plasmodium vivax polymorphs and Plasmodium falciparum circumsporozoite proteins in Anopheles (Diptera: Culicidae) from Belize, Central America.

Eight species of Anopheles mosquitoes from indoor/outdoor human landing collections in Belize, Central America, were examined for human Plasmodium circumsporozoite protein (CSP) using an enzyme-linked immunosorbent assay (ELISA). A total of 14 of 9,104 females tested were positive from general surveys throughout Belize and three of 11,966 were positive from a longitudinal study in Caledonia, northern Belize. ELISA results, using pooled head-thorax preparations and species-specific monoclonal antibodies directed against the circumsporozoite proteins of Plasmodium falciparum and two Plasmodium vivax polymorphs (210 and VK247), found four species reactive: Anopheles vestitipennis (3 pools), Anopheles darlingi (2 pools), Anopheles albimanus (10 pools), and Anopheles gabaldoni (2 pools). The minimum field infection rates (MFIR) for combined Plasmodium species from the general survey were 0.282% for An. vestitipennis, 0.271% for An. darlingi, 0.126% for An. albimanus, and 0.395% for An. gabaldoni. MFIRs for combined Plasmodium species from the longitudinal study in the village of Caledonia were 0.018% for both An. vestitipennis and An. albimanus and 1.66% for An. gabaldoni. Positive CSP pools were collected from the Cayo, Corozal, Orange Walk, Stann Creek, and Toledo political districts. No CSP positive pools were detected from collections in the Belize District. The study provides valuable information on the spatial distribution and species type of Plasmodium positive mosquitoes. This information, in combination with other vector data, suggest that An. vestitipennis and An. darlingi are commonly involved in malaria transmission. Additionally, these species appear to be much more efficient vectors than An. albimanus in Belize.

Bacterial abundance in larval habitats of four species of Anopheles (Diptera: Culicidae) in Belize, Central America.

Numbers of free-living and attached bacteria were counted in surface waters from larval habitats of four species of Anopheles mosquitoes: Anopheles albimanus Wiedemann, An. darlingi Root, An. vestitipennis Dyar and Knab, and An. pseudopunctiopennis Theobald, using a direct count method and DAPI staining technique. Bacterial counts from larval habitats were compared to those from adjacent open water. Several additional variables such as total suspended solids (TSS), particulate organic carbon (POC), and dissolved organic carbon (DOC) were also recorded in order to establish possible relationships with bacterial characteristics. Our results showed that the waters from larval habitats were enriched with bacteria as well as POC and DOC compared to open water. The major component of all samples consisted of cocci, the proportion of rods was similar and there were significantly more attached rods in habitat samples than in open water samples. Anopheles vestitipennis habitats had the highest values of each of the categories of bacteria as well as of POC and DOC.

Larval habitats of anopheline mosquitoes in the Upper Orinoco, Venezuela.

Survey of larval habitats of anopheline mosquitoes was conducted in Ocamo in the State of Amazonas, southern Venezuela. The sampled habitats belonged to three different hydrological types: lagoons (26 habitats), forest pools including flooded forest (16 habitats), and forest streams (4 habitats). Out of 46 habitats surveyed, 31 contained anopheline larvae. Six species were found: Anopheles darlingi, Anopheles triannulatus, Anopheles oswaldoi, Anopheles peryassui, Anopheles punctimacula, and Anopheles mediopunctatus. Anopheles triannulatus was the most abundant species. Significantly higher numbers of anopheline larvae, in general, and of An. triannulatus specifically were found in lagoons with submersed macrophytes and sparse emergent graminoids than in forest pools with detritus.

Characterization and detection of Anopheles vestitipennis and Anopheles punctimacula (Diptera: Culicidae) larval habitats in Belize with field survey and SPOT satellite imagery.

Surveys of larval habitats of Anopheles vestitipennis and Anopheles punctimacula were conducted in Belize, Central America. Habitat analysis and classification resulted in delineation of eight habitat types defined by dominant life forms and hydrology. Percent cover of tall dense macrophytes, shrubs, open water, and pH were significantly different between sites with and without An. vestitipennis. For An. punctimacula, percent cover of tall dense macrophytes, trees, detritus, open water, and water depth were significantly different between larvae positive and negative sites. The discriminant function for An. vestitipennis correctly predicted the presence of larvae in 65% of sites and correctly predicted the absence of larvae in 88% of sites. The discriminant function for An. punctimacula correctly predicted 81% of sites for the presence of larvae and 45% for the absence of larvae. Canonical discriminant analysis of the three groups of habitats (An. vestitipennis positive; An. punctimacula positive; all negative) confirmed that while larval habitats of An. punctimacula are clustered in the tree dominated area, larval habitats of An. vestitipennis were found in both tree dominated and tall dense macrophyte dominated environments. The forest larval habitats of An. vestitipennis and An. punctimacula seem to be randomly distributed among different forest types. Both species tend to occur in denser forests with more detritus, shallower water, and slightly higher pH. Classification of dry season (February) SPOT multispectral satellite imagery produced 10 land cover types with the swamp forest and tall dense marsh classes being of particular interest. The accuracy assessment showed that commission errors for the tall, dense marsh and swamp forest appeared to be minor; but omission errors were significant, especially for the swamp forest (perhaps because no swamp forests are flooded in February). This means that where the classification indicates there are An. vestitipennis breeding sites, they probably do exist; but breeding sites in many locations are not identified and could be more abundant than indicated.

Use of generalized regression tree models to characterize vegetation favoring Anopheles albimanus breeding.

We propose the use of generalized tree models (GTMs) to analyze data from entomological field studies. Generalized tree models can be used to characterize environments with different mosquito breeding capacity. A GTM simultaneously analyzes a set of predictor variables (e.g., vegetation coverage) in relation to a response variable (e.g., counts of Anopheles albimanus larvae), and how it varies with respect to a set of criterion variables (e.g., presence of predators). The algorithm produces a treelike graphical display with its root at the top and 2 branches stemming down from each node. At each node, conditions on the value of predictors partition the observations into subgroups (environments) in which the relation between response and criterion variables is most homogeneous.

Characterization of Anopheles pseudopunctipennis larval habitats.

A survey of Anopheles pseudopunctipennis larval habitats was performed throughout most of its known geographic range. Eleven key environment variables characterized most larval habitats of this important vector of malaria in the Americas. Larval habitats occurred mainly in valley and foothill areas which were often situated in arid regions. Immatures were found primarily during the dry season in sun-exposed freshwater stream pools with clear, shallow, stagnant water containing abundant filamentous green algae and/or aquatic vegetation.

Anopheles albimanus (Diptera: Culicidae) and cyanobacteria: an example of larval habitat selection.

Northern Belize has extensive herbaceous wetlands. Those dominated by sparse emergent macrophytes, rushes (Eleocharis spp.) and sawgrass (Cladium jamaicense Crantz), often develop floating mats of cyanobacteria (blue-green algae). These mats provide suitable habitat for larvae of the malaria transmitting mosquito Anopheles albimanus Wiedemann. Presence/absence of A. albimanus larvae and cyanobacterial mats was assessed in marshes located throughout northern Belize. Of the 21 marshes examined during the 1993 wet and 1994 dry seasons, cyanobacterial mats were found in 11 and A. albimanus larvae were detected in 9 of these 11 marshes. No A. albimanus larvae were found in marshes without cyanobacterial mats. Mosquito larvae were collected along two 1,000 m long transects in both the wet season (August 1993) and the dry season (March 1994) to delineate larval distribution in marshes with cyanobacterial mats. A. albimanus larval densities in cyanobacterial mats were relatively high in both seasons: 2.8 and 2.3 larvae per dip in the wet and dry seasons, respectively, in Chan Chen marsh; and 0.8 and 1.02 larvae per dip in Buena Vista marsh. Numbers of larvae per dip did not significantly change with increasing distance from houses/pastures or margins of the marsh. A field experiment showed a strong preference of ovipositing A. albimanus for cyanobacterial mats. Higher temperatures and higher CO2 emissions from cyanobacterial mats are possible ovipositional cues.

Characterization of Anopheles darlingi (Diptera: Culicidae) larval habitats in Belize, Central America.

Surveys for larvae of Anopheles darlingi Root were conducted in April, May, and August 1994 in riverine habitats of central Belize (Cayo and Belize districts). An. darlingi was present during both the dry and wet seasons. Larvae were encountered most frequently in patches of floating debris along river margins. The floating mats were often formed by bamboo hanging over the banks and dense submersed bamboo roots. Larvae were found less frequently in lake margins, small lagoons, and ground pools with submersed roots and patches of floating leaves or vegetation. In addition to their association with floating debris, larvae of An. darlingi were associated positively with shade and submersed plants in riverine environments. Samples from river habitats showed the larvae of Anopheles albimanus Wiedemann to be strongly associated with sun-exposed sites containing green or blue-green algae. Unlike An. darlingi, An. albimanus was an ubiquitous mosquito, the immatures of which occurred in a wide variety of riverine and nonriverine aquatic habitats. Based on published reports and our experience, the association of An. darlingi with river systems was verified, and its distribution in Central America and Mexico was mapped.

Predictions of malaria vector distribution in Belize based on multispectral satellite data.

Use of multispectral satellite data to predict arthropod-borne disease trouble spots is dependent on clear understandings of environmental factors that determine the presence of disease vectors. A blind test of remote sensing-based predictions for the spatial distribution of a malaria vector, Anopheles pseudopunctipennis, was conducted as a follow-up to two years of studies on vector-environmental relationships in Belize. Four of eight sites that were predicted to be high probability locations for presence of An. pseudopunctipennis were positive and all low probability sites (0 of 12) were negative. The absence of An. pseudopunctipennis at four high probability locations probably reflects the low densities that seem to characterize field populations of this species, i.e., the population densities were below the threshold of our sampling effort. Another important malaria vector, An. darlingi, was also present at all high probability sites and absent at all low probability sites. Anopheles darlingi, like An. pseudopunctipennis, is a riverine species. Prior to these collections at ecologically defined locations, this species was last detected in Belize in 1946.

Landscape surrounding human settlements and Anopheles albimanus (Diptera: Culicidae) abundance in Southern Chiapas, Mexico.

Landscape characteristics that may influence important components of the Anopheles albimanus Wiedemann life cycle, including potential breeding sites, suitable diurnal resting sites, and possible sources of blood meals, were analyzed at 14 villages in a malarious area of southern Mexico. An. albimanus adults were collected weekly in each village using UV-light traps between July 1991 and August 1992. Based on rainfall, the study was divided into 6 seasonal periods. Villages were considered to have high mosquito abundance when >5 mosquitoes per trap per night were collected during any 1 of the 6 seasonal periods. The extension and frequency of 11 land cover types surrounding villages were determined using aerial photographs and subsequently verified through field surveys. Elevation was the main landscape feature that separated villages with low and high mosquito abundance. All villages with high mosquito abundance were below 25 m. Transitional and mangrove land cover types were found only in the high mosquito abundance group. Flooded areas as potential breeding sites and potential adult resting sites in unmanaged pastures were significantly more frequent in areas surrounding villages with high mosquito abundance. No significant differences in density of cattle and horses were found among village groups. Overall, surrounding breeding sites located at low elevations in flooded unmanaged pastures seemed to be the most important determinants of An. albimanus adult abundance in the villages.

Predictions of adult Anopheles albimanus densities in villages based on distances to remotely sensed larval habitats.

Remote sensing is particularly helpful for assessing the location and extent of vegetation formations, such as herbaceous wetlands, that are difficult to examine on the ground. Marshes that are sparsely populated with emergent macrophytes and dense cyanobacterial mats have previously been identified as very productive Anopheles albimanus larval habitats. This type of habitat was detectable on a classified multispectral System Probatoire d'Observation de la Terre image of northern Belize as a mixture of two isoclasses. A similar spectral signature is characteristic for vegetation of river margins consisting of aquatic grasses and water hyacinth, which constitutes another productive larval habitat. Based on the distance between human settlements (sites) of various sizes and the nearest marsh/river exhibiting this particular class combination, we selected two groups of sites: those located closer than 500 m and those located more than 1,500 m from such habitats. Based on previous adult collections near larval habitats, we defined a landing rate of 0.5 mosquitoes/human/min from 6:30 PM to 8:00 PM as the threshold for high (> or = 0.5 mosquitoes/human/min) versus low (< 0.5 mosquitoes/human/min) densities of An. albimanus. Sites located less than 500 m from the habitat were predicted as having values higher than this threshold, while lower values were predicted for sites located greater than 1,500 m from the habitat. Predictions were verified by collections of mosquitoes landing on humans. The predictions were 100% accurate for sites in the > 1,500-m category and 89% accurate for sites in the < 500-m category.

Remote sensing as a landscape epidemiologic tool to identify villages at high risk for malaria transmission.

A landscape approach using remote sensing and geographic information system (GIS) technologies was developed to discriminate between villages at high and low risk for malaria transmission, as defined by adult Anopheles albimanus abundance. Satellite data for an area in southern Chiapas, Mexico were digitally processed to generate a map of landscape elements. The GIS processes were used to determine the proportion of mapped landscape elements surrounding 40 villages where An. albimanus abundance data had been collected. The relationships between vector abundance and landscape element proportions were investigated using stepwise discriminant analysis and stepwise linear regression. Both analyses indicated that the most important landscape elements in terms of explaining vector abundance were transitional swamp and unmanaged pasture. Discriminant functions generated for these two elements were able to correctly distinguish between villages with high and low vector abundance, with an overall accuracy of 90%. Regression results found both transitional swamp and unmanaged pasture proportions to be predictive of vector abundance during the mid-to-late wet season. This approach, which integrates remotely sensed data and GIS capabilities to identify villages with high vector-human contact risk, provides a promising tool for malaria surveillance programs that depend on labor-intensive field techniques. This is particularly relevant in areas where the lack of accurate surveillance capabilities may result in no malaria control action when, in fact, directed action is necessary. In general, this landscape approach could be applied to other vector-borne diseases in areas where 1) the landscape elements critical to vector survival are known and 2) these elements can be detected at remote sensing scales.

Spatial patterns of Anopheles freeborni and Culex tarsalis (Diptera: Culicidae) larvae in California rice fields.

Spatial patterns of Anopheles freeborni Aitken and Culex tarsalis Coquillett larvae were studied during summer by sampling with a standard mosquito dipper in 104 rice fields in northern California. Culex tarsalis larval abundance was highest initially, then decreased and remained low through late summer. An. freeborni larval abundance was low initially, increased steadily, and peaked in mid-August. The degree of aggregation for both species as measured using Taylor's power law and Iwao's Patchiness Regression was highest among the first instars and then decreased as the larvae aged. Seasonal peaks in the degree of aggregation were observed. Analysis of covariance showed that for Taylor's model both instar and time effects were statistically significant, with instar showing the largest effect. In comparison, all slopes resulting from Iwao's model were significantly different, indicating that this model was affected by specific combinations of instar, week, and location and, thus, was less useful in developing an area-wide sampling plan. Optimal sample size was estimated using two methods. One method calculated the number of dips needed to estimate population abundance at three fixed-precision levels. The second calculated the minimum number of dips needed to collect at least one larva. The latter requires a substantially smaller sample size and may provide an effective method for monitoring larval mosquito abundance for control purposes.

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.

Preliminary observations on the changing roles of malaria vectors in southern Belize.

A survey for larval and adult Anopheles mosquitoes was conducted in Toledo District of southern Belize during August-September 1992. We surveyed for larvae in 145 habitats and conducted paired indoor-outdoor collections of adult mosquitoes landing on humans at 6 houses. In 1940-41, Kumm and Ram reported Anopheles darlingi females to be the most common Anopheles mosquitoes inside houses and reported no specimens of Anopheles vestitipennis in southern Belize. In our 1992 survey we found no An. darlingi mosquitoes either as adults or larvae. More An. vestitipennis females were captured indoors than outdoors, whereas most Anopheles albimanus and Anopheles apicimacula females were captured outdoors. All 3 species were represented occasionally in 145 collections of larvae from diverse habitats. Anopheles vestitipennis now appears to be a potentially important vector of malaria during the wet season in Toledo District.

PIP: Kumm and Ram surveyed for the presence of larval and adult Anopheles mosquitoes in Belize in 1940-41. They found An. darlingi to be the most common of Anopheles species inside houses and reported observing no An. vestitipennis in southern Belize. That study was conducted before the DDT malaria control program was implemented in the country and offers the most recent publication of such findings for the area up to the publication of this more recent report by the authors. The authors report findings from a recent survey of larval and adult Anopheles mosquitoes in the Toledo District during August-September 1992. The presence of larvae was surveyed in 145 habitats, while paired indoor-outdoor collections of adult mosquitoes landing on humans were taken at six houses. The authors, however, report finding no An. darlingi mosquitoes, either as adults or larvae. More An. vestitipennis females were captured indoors than outdoors and most An. albimanus and An. apicimacula females were captured outdoors. All three species were represented occasionally in the collections of larvae from diverse habitats. These findings clearly suggest that An. vestitipennis is a potentially important vector of malaria in the wet season in Toledo District.

Dynamics of population densities and vegetation associations of Anopheles albimanus larvae in a coastal area of southern Chiapas, Mexico.

Spatial and seasonal variations on Anopheles albimanus larval densities and their plant associations were investigated in larval habitats in southern Mexico between April 1989 and May 1990. Thirty-four plant groups were dominant in larval habitats. Dense larval populations were associated with 3 genera of plants, Cynodon, Echinocloa and Fimbristylis and no larvae were found in habitats with Salvinia and Rhizophora. Low significant positive or negative associations were documented with the other 12 plant genera. Larval habitats were classified according to the morphology of their dominant plants. Higher larval densities were observed in the groups characterized by relatively short emergent vegetation. The distribution of habitat-types within 5 identified vegetation units showed a significantly dependent relationship. For the entire study period, highest larval densities were detected in flooded pasture/grassland vegetation units. For all vegetation units, higher larval densities were found when the dominant plant type covered between 25-50% of the breeding site. The integration of data from habitat-types into vegetation units did not result in loss of information.

Limnological and botanical characterization of larval habitats for two primary malarial vectors, Anopheles albimanus and Anopheles pseudopunctipennis, in coastal areas of Chiapas State, Mexico.

Field surveys of mosquito breeding sites on the Pacific coastal plain and foothill regions of southern Chiapas, Mexico, were carried out in the dry and wet seasons of 1988. At each site, selected environmental variables were measured or estimated, presence and percent cover of aquatic plants recorded, a water sample collected for subsequent analyses, and 10-30 dips made for mosquito larvae. Logistic regression and discriminant analyses revealed that the occurrence of Anopheles albimanus larvae in both the wet and dry seasons was positively associated with planktonic algae and negatively associated with altitude. In the dry season, An. albimanus larvae were largely restricted to the margins of permanent water bodies and were associated with the presence of floating plants, particularly Eichhornia crassipes. During the wet season An. albimanus larvae were positively associated with emergent plants, particularly seasonally flooded Cyperaceae, and phosphorus (PO4) concentrations, and were negatively associated with abundant filamentous algae, high levels of total suspended solids (TSS) and Salvinia. In the dry season, An. pseudopunctipennis larvae were positively associated with filamentous algae, altitude and the presence of Heteranthera if encountered in a riverine setting, and were negatively associated with water depth. During the wet season, flooding eliminated typical flood plain An. pseudopunctipennis habitats, and larvae were rarely encountered.