Mosquito protein microassay. II. Modification for potential field use

A simple microassay is described for determining protein levels in individual mosquitoes; this method should be useful on a routine basis in the field. This method requires only a small portion of a homogenate of a single mosquito. Sugar-fed adult female Anopheles albimanus showed a slow decline in protein level for 14 days post-emergence. Blood volumes and blood-meal protein levels were microassayed, and changes in protein level were monitored throughout the reproductive cycle. Blood-fed Anopheles albimanus protein levels return only slowly to pre-feeding levels following egg deposition.     

Characterization of aquatic mosquito habitat, natural enemies, and immature mosquitoes in the Artibonite Valley, Haiti

This paper characterizes water body types harboring immature mosquitoes in a low-lying area of Haiti and investigates the relationship between immature Anopheles albimanus abundance and aquatic predator presence. Larval An. albimanus were found in permanent and semi-permanent groundwater habitats including (in order of greatest abundance) hoof/footprints, ditches, rice fields, and ground pools. High levels of species co-occurrence were observed in habitats. Among water bodies positive for immature Anopheles, 42.9% also contained immature Culex species. Significant association between An. albimanus abundance and the absence of fish predators was detected. Results from the multivariate negative binomial regression suggest that the interactive effect of increasing distance from the Artibonite River and elevation are positively associated with the abundance of immature An. albimanus. The presence of fish predators was not associated with the abundance of An. albimanus larvae in habitats while controlling for habitat distance and elevation. The results of this study provide baseline entomological information to inform vector control programs in the country.     

Infectivity of two strains of Plasmodium vivax to Anopheles albitarsis mosquitoes from Colombia

Anopheles albitarsis obtained from Villavicencio, Colombia, were colonized in the laboratory using force-mating techniques. Laboratory reared mosquitoes were allowed to feed on Aotus monkeys infected with the Salvador II or the Rio Meta strains of Plasmodium vivax from El Salvador and Colombia, respectively. In comparison with other species, the An. albitarsis were less susceptible than Anopheles freeborni, Anopheles culicifacies and strains of Anopheles albimanus from El Salvador, Panama and Colombia and more susceptible than a strain of An. albimanus from Haiti.     

Ookinete destruction within the mosquito midgut lumen explains Anopheles albimanus refractoriness to Plasmodium falciparum (3D7A) oocyst infection

Previous studies have shown that the central American mosquito vector, Anopheles albimanus, is generally refractory to oocyst infection with allopatric isolates of the human malaria parasite Plasmodium falciparum. However, the reasons for the refractoriness of A. albimanus to infection with such isolates of P. falciparum are unknown. In the current study, we investigated the infectivity of the P. falciparum clone 3D7A to laboratory-reared A. albimanus and another natural vector of human malaria, Anopheles stephensi. Plasmodium falciparum gametocytes grown in vitro were simultaneously fed to both mosquito species and the progress of malaria infection compared. In 22 independent paired experimental feeds, no mature oocysts were observed on the midguts of A. albimanus 10days after bloodfeeding. In contrast, high levels of oocyst infection were found on the midguts of simultaneously fed A. stephensi. Direct immunofluorescence microscopy and light microscopical examination of Giemsa-stained histological sections were used to identify when the P. falciparum clone 3D7A failed to establish mature oocyst infections in A. albimanus. Similar densities of macrogametes/zygotes, and immature retort-form and mature ookinetes were found within the bloodmeals of both mosquito species. However, in A. albimanus, ookinetes were seldom associated with the peritrophic matrix, and were neither observed in the ectoperitrophic space nor the midgut epithelium. In contrast, ookinetes were frequently observed in these midgut compartments in A. stephensi. Additionally, young oocysts were observed on the midguts of A. stephensi but not A. albimanus 2days after bloodfeeding. Vital staining of the immature retort-form and mature ookinetes found within the luminal bloodmeal, demonstrated that a significantly greater proportion of these malaria parasite stages were non-viable in A. albimanus compared with A. stephensi. Overall, our observations indicate that ookinetes of the P. falciparum clone 3D7A are destroyed within the bloodmeal of A. albimanus and that the midgut lumen, rather than the midgut epithelium, is the site of mosquito refractoriness in this particular malaria parasite-mosquito vector combination.     

Habitat suitability for three species of Anopheles mosquitoes: larval growth and survival in reciprocal placement experiments

Larval habitats of the main malaria vectors in Belize are associated with three distinctly different aquatic environments: marshes with sparse macrophytes and cyanobacterial mats (Anopheles albimanus), tall dense macrophyte marshes (An. vestitipennis), and floating detritus assemblages within freshwater rivers (An. darlingi). We assessed species-specific habitat suitability based upon nutrient characteristics using larval survival rates (SR) and wing lengths (WL) from floating habitat enclosures. Anopheles albimanus showed a high SR (81%) in all three habitats, while An. vestitipennis had a similarly high SR in its own habitat (82%) and An. darlingi's habitat (81%). Anopheles darlingi only showed high SR (85%) in its own habitat. Both An. vestitipennis and An. darlingi showed very low SR in the An. albimanus habitat. There were no significant WL differences among field-caught, laboratory-reared, and experimental populations of An. vestitipennis and An. albimanus, with the exception of An. vestitipennis experimental populations and An. vestitipennis field populations placed in the An. albimanus habitat. Habitat quality indicators, particulate organic carbon (POC), dissolved organic carbon (DOC), and particulate organic nitrogen (PON), were consistently higher in An. vestitipennis habitats than in the habitats of the other two species. Correspondingly, An. vestitipennis adults were larger when measured both as dry mass and from WL. There were no differences in dry mass, lipids, or protein content among the same species reared at different locations. We compared SR and WL among mosquitoes from shaded and unshaded containers to test whether the high mortality rates for An. vestitipennis and An. darlingi in the An. albimanus habitat were due to intense sun exposure. There were no significant differences among developmental times, survivorship, or adult size for shaded versus sun-exposed populations. This indicates that other factors such as larval toxins, predator avoidance, interspecific species competition, etc. may be responsible for the higher mortality rates in those species not adapted to this particular habitat.     

Studies on infections with the Berok strain of Plasmodium cynomolgi in monkeys and mosquitoes

Infections with the Berok strain of Plasmodium cynomolgi were induced in Macaca mulatta, Macaca fascicularis, Macaca nemestrina, Aotus lemurinus griseimembra, Aotus azarae boliviensis, and Saimiri boliviensis monkeys. Transmission was obtained with sporozoites developing in Anopheles peditaeniatus, Anopheles maculatus, Anopheles quadrimaculatus, Anopheles culicifacies, and Anopheles dirus mosquitoes. This strain of P. cynomolgi offers significant potential for a number of experimental studies. The parasite induces high-density parasite counts in both Old World and New World monkeys; rhesus monkeys readily support the development of gametocytes infectious to different anopheline mosquitoes routinely maintained in the laboratory; the gametocytes are infective to laboratory-maintained Anopheles albimanus, a vector rarely susceptible to plasmodia of Old World monkeys; encapsulated oocysts are produced in An. culicifacies as well as in Anopheles gambiae; and the parasite has been adapted to long-term in vitro culture.     

Independent selection of multiple mechanisms for pyrethroid resistance in Guatemalan Anopheles albimanus (Diptera: Culicidae).

Isofemale lines were established containing either, both, or neither of the elevated esterase and oxidase resistance mechanisms conferring pyrethroid resistance in a Guatemalan strain of Anopheles albimanus (Wiedemann). Plots of esterase and oxidase levels for individual mosquitoes from these single families correlated with data obtained using oxidase and esterase synergists in bioassays run in the bottle format. Mixed populations of pyrethroid-resistant A. albimanus adult females were selected using DDT, permethrin, or malathion; and the esterase and oxidase levels of the individual progeny were plotted. These data showed that the 3 classes of insecticide selected the 2 mechanisms differently. These results are discussed in terms of the problem of multiresistance surveillance in the field, especially concerning pyrethroid insecticides and the interaction of agricultural and public health insecticide application.     

Determination of the resistance to organophosphate, carbamate, and pyrethroid insecticides in Panamanian Anopheles albimanus (Diptera: Culicidae) mosquitoes

Introduction. The susceptibility of Anopheles albimanus to organophosphates, carbamates and pyrethroid insecticides was unknown in the Panama communities of Aguas Claras, Pintupo and Puente Bayano, located in the Amerindian Reservation of Madungandi. This region is considered a malaria transmission area, where An. albimanus is the main vector. Objective. The resistance to organophosphate insecticides, carbamates and pyrethroids was evaluated in field populations of the Anopheles albimanus in Panama. Materials and methods. Progeny of An. albimanus collected in three localities in the indigenous Madugandi region were exposed to bioassays of susceptibility to organophosphate insecticides (fenitrothion, malathion and chlorpyrifos), the carbamate (propoxur) and pyrethroids (deltamethrin, lambdacyhalothrin, cyfluthrin and cypermethrin). The protocols were in accordance with those established for adult mosquitoes by World Health Organization. Results. The three strains of the An. albimanus were resistant to the pyrethroid insecticides deltamethrin, lambdacyhalothrin, cyfluthrin and cypermethrin. Susceptibility remained for the organophosphate insecticides fenitrothion, malathion, chlorpyrifos, and the carbamate insecticide propoxur. Conclusion. The results provided important information to the vector control program, contributing to the application of new strategies on the use of insecticides, and thereby lengthening the life of the insecticide in use.     

The Anopheles albimanus white gene: molecular characterization of the gene and a spontaneous white gene mutation

We have cloned and characterized the white gene of Anopheles albimanus. Comparison of the deduced amino acid sequence of this white gene with its homologs from six species of Diptera show that the An. albimanus gene is most similar to the white gene of An. gambiae (92% identity). A spontaneous white-eyed mutant An. albimanus was caused by an approximately 10 kb insertion into a CT dinucleotide repeat region of intron 2 of the white locus. The flanks of this insertion are long (at least 400 bp), nearly perfect inverted terminal repeat sequences. This cloned white gene should be useful as a marker for germ line transformation of An. albimanus. This revised version was published online in July 2006 with corrections to the Cover Date.     

Late Pleistocene environmental changes lead to unstable demography and population divergence of Anopheles albimanus in the northern Neotropics

We investigated the historical demography of Anopheles albimanus using mosquitoes from five countries and three different DNA regions, the mitochondrial cytochrome oxidase subunit I gene (COI), the single copy nuclear white gene and the ribosomal internal transcribed spacer two (ITS2). All the molecular markers supported the taxonomic status of a single species of An. albimanus. Furthermore, agreement between the COI and the white genes suggested a scenario of Pleistocene geographic fragmentation (i.e., population contraction) and subsequent range expansion across southern Central America.     

The sporogonic cycle of Plasmodium reichenowi

Plasmodium reichenowi, a malarial parasite of the chimpanzee, was infective to Anopheles freeborni, Anopheles quadrimaculatus, Anopheles stephensi, Anopheles maculatus, Anopheles dirus, and Anopheles culicifacies mosquitoes. Anopheles gambiae and Anopheles albimanus were not infected. Mean oocyst diameters of P. reichenowi were smaller than those of the other chimpanzee parasite, Plasmodium schwetzi. Sporozoites were present in the salivary glands of An. freeborni at 15 days when held at 25 to 26 C.     

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.     

Infectivity of the Santa Lucia (El Salvador) strain of Plasmodium falciparum to different anophelines.

Anopheles freeborni mosquitoes were much more heavily infected with the Santa Lucia strain of Plasmodium falciparum from coastal El Salvador than were any of the other species tested. Of 5 strains of A. albimanus examined, the most heavily infected was the CA-109A and the least was the Melara, both of which come from coastal El Salvador. Of the exotic anophelines, the A. maculatus was infected at a slightly higher level than was the A. balabacensis. The incidence of highly infected individual mosquitoes was greatest in the Panama-Escobal strain of A. albimanus from the Republic of Panama; the incidence was lowest in the Melara strain from El Salvador. All strains of A. albimanus developed infected salivary glands, but the A. freeborni and A. maculatus mosquitoes appeared to develop infected glands more effeciently. Infection rates in A. freeborni mosquitoes were highest if mosquitoes were fed on Aotus trivirgatus monkeys between the 19th and 25th days of patent gametocytemia.     

Plasmodium vivax: ookinete destruction and oocyst development arrest are responsible for Anopheles albimanus resistance to circumsporozoite phenotype VK247 parasites

Anopheles albimanus and An. pseudopunctipennis differ in their susceptibilities to Plasmodium vivax circumsporozoite phenotypes. An. pseudopunctipennis is susceptible to phenotype VK247 but almost refractory to VK210. In contrast, An. albimanus is almost refractory to VK247 but susceptible to VK210. To investigate the site in the mosquito and the parasite stage at which resistance mechanisms affect VK247 development in An. albimanus, parasite development was followed in a series of experiments in which both mosquitoes species were simultaneously infected with blood from patients. Parasite phenotype was determined in mature oocysts and salivary gland sporozoites by use of immunofluorescence and Western blot assays and/or gene identification. Ookinete maturation and their densities within the bloodmeal bolus were similar in both mosquito species. Ookinete densities on the internal midgut surface of An. albimanus were 4.7 times higher than those in An. pseudopunctipennis; however, the densities of developing oocysts on the external midgut surface were 6.12 times higher in the latter species. Electron microscopy observation of ookinetes in An. albimanus midgut epithelium indicated severe parasite damage. These results indicate that P. vivax VK247 parasites are destroyed at different parasite stages during migration in An. albimanus midguts. A portion, accumulated on the internal midgut surface, is probably destroyed by the mosquito's digestive enzymes and another portion is most likely destroyed by mosquito defense molecules within the midgut epithelium. A third group, reaching the external midgut surface, initiates oocyst development, but over 90% of them interrupt their development and die. The identification of mechanisms that participate in parasite destruction could provide new elements to construct transgenic mosquitoes resistant to malaria parasites.     

Studies on a newly isolated strain of Plasmodium brasilianum in Aotus and Saimiri monkeys and different anophelines

A strain of Plasmodium brasilianum was isolated from an Aotus vociferans monkey from Peru. The parasite readily infected Aotus monkeys from Bolivia and Columbia and Saimiri sciureus monkeys from Peru and Bolivia. Highest level mosquito infections were obtained by feeding on the Saimiri monkeys. The most susceptible mosquito was Anopheles freeborni, followed by Anopheles dirus, Anopheles stephensi, Anopheles gambiae, Anopheles culicifacies, Anopheles maculatus and Anopheles albimanus. Anopheles quadrimaculatus were also susceptible to infection. Degenerating oocysts were observed in An. dirus mosquitoes infected with this parasite. Transmission via the bites of infected An. maculatus mosquitoes was obtained to 3 Bolivian Saimiri monkeys; prepatent periods were 27, 27, and 29 days.     

P elements are found in the genomes of nematoceran insects of the genus Anopheles

We report the identification of genomic sequences in various anopheline mosquitoes (family Culicidae: suborder Nematocera: order Diptera) showing homology to the class II, short inverted-terminal-repeat (ITR) transposable element P from Drosophila melanogaster (family Drosophilidae; suborder Brachycera: order Diptera). Anopheles gambiae appears to have at least six distinct P elements. Other anopheline species, including four additional members of the An. gambiae species complex (An. arabiensis, An. merus, An. melas and An. quadriannulatus), Anopheles stephensi (all subgenus Cellia), An. quadrimaculatus (subgenus Anopheles) and Anopheles albimanus (subgenus Nyssorhynchus) also possess P elements similar to those found in An. gambiae. Ten distinct P element types were identified in the genus Anopheles. At least two of the An. gambiae elements appears to be intact and potentially functional. Phylogenetic analysis of the anopheline P elements reveals them to belong to a distinctly different clade from the brachyceran P elements.     

Maintenance of chromosome arm integrity between two Anopheles mosquito subgenera

Low-resolution chromosomal homology between Anopheles gambiae and A. albimanus was determined by polytene chromosome in situ cross hybridization of 17 recombinant DNA and PCR products hybridizing to 23 loci. Hybridization results reflect that the chromosomes have rearranged in the form of autosomal whole-arm translocations and numerous paracentric inversions and not by large detectable pericentric inversions or partial arm translocations. An. gambiae and An. albimanus chromosomes hence differ from each other by possessing alternative autosomal arm associations and rearranged internal structure of each arm, but the integrity of the whole arms has remained conserved. In addition, a photomap of the larval salivary gland polytene chromosomes of An. albimanus that we used to identify sites of hybridization in this species is presented that delineates further banding details than maps published in the past.     

Comparative genomics of insect juvenile hormone biosynthesis

The biosynthesis of insect juvenile hormone (JH) and its neuroendocrine control are attractive targets for chemical control of insect pests and vectors of disease. To facilitate the molecular study of JH biosynthesis, we analyzed ESTs from the glands producing JH, the corpora allata (CA) in the cockroach Diploptera punctata, an insect long used as a physiological model species and compared them with ESTs from the CA of the mosquitoes Aedes aegypti and Anopheles albimanus. The predicted genes were analyzed according to their probable functions with the Gene Ontology classification, and compared to Drosophila and Anopheles gambiae genes. A large number of reciprocal matches in the cDNA libraries of cockroach and mosquito CA were found. These matches defined known and suspected enzymes of the JH biosynthetic pathway, but also several proteins associated with signal transduction that might play a role in the modulation of JH synthesis by neuropeptides. The identification in both cockroach and mosquito CA of homologs of the small ligand binding proteins from insects, Takeout/JH binding protein and retinol-binding protein highlights a hitherto unsuspected complexity of metabolite trafficking, perhaps JH precursor trafficking, in these endocrine glands. Furthermore, many reciprocal matches for genes of unknown function may provide a fertile ground for an in-depth study of allatal-specific cell physiology. ESTs are deposited in GenBank under the accession numbers DV 017592-DV 018447 (Diploptera punctata); DR 746432-DV 747949 (Aedes aegypti); and DR 747950-DR 748310 (Anopheles albimanus).     

Plasmodium berghei ookinete densities in three anopheline species

Plasmodium berghei ookinete kinetics and densities were examined in the blood meals of 3 species of Anopheles mosquitoes fed simultaneously from a gametocytemic mouse. Simple techniques were developed for estimating relative and absolute ookinete densities within individual mosquito blood meals. The kinetics of ookinete formation were similar in all 3 species, with peak ookinete densities occurring from 12 to 24 hr postingestion. Ookinete densities consistently were lower in Anopheles stephensi than in Anopheles albimanus or Anopheles freeborni and could not be accounted for by species differences in blood meal volumes or gametocyte densities. Likely explanations involve species differences in blood meal hemolysis or sampling error as the result of ookinete emigration from the blood meal.