Morphological and proteomic characterization of midgut of the malaria vector Anopheles albimanus at early time after a blood feeding.

The midgut of anopheline mosquito is the entry of Plasmodium, the causative agent of malaria.When the mosquito feeds on parasite infected host, Plasmodium parasites reach the midgut and must confront digestive enzymes, the innate immune response and go across the peritrophic matrix (PM), a thick extracellular sheath secreted by the mosquito midgut epithelial cells. Then, to continue its development, the parasite must reach the salivary glands to achieve transmission to a vertebrate host. We report here the morphological and biochemical descriptions of the midgut changes after a blood meal in Anopheles albimanus. Before blood feeding, midgut epithelial cells contained numerous electrondense vesicles distributed in the central to apical side. These vesicles were secreted to the luminal side of the midgut after a blood meal. At early times after blood ingest, the PM is formed near microvilli as a granulous amorphous material and after it consolidates forming a highly organized fibrillar structure, constituted by layers of electrondense and electronlucent regions. Proteomic comparative analysis of sugar and blood fed midguts showed several molecules that modify their abundance after blood intake; these include innate immunity, cytoskeletal, stress response, signaling, and digestive, detoxifying and metabolism enzymes. Biological significance In the midgut of mosquitoes during bloodfeeding, many simultaneous processes occur, including digestion, innate immune activities, cytoskeleton modifications, construction of a peritrophic matrix and hormone production, between others. Mechanical forces are very intense during bloodfeeding and epithelial and muscular cells must resist the stress, modifying the actin cytoskeleton and coordinating intracellular responses by signaling. Microorganisms present in midgut contents reproduce and interact with epithelial cells triggering innate immune response. When infectious agents are present in the blood meal they must traverse the peritrophic matrix, an envelope formed from secretion products of epithelial cells, and evade the immune system in order to reach the epithelium and continue their journey towards salivary glands, in preparation for the transmission to the new hosts. During all these processes, proteins of mosquitoes are modified in order to deal with mechanical and biological challenges, and the aim of this work is to study these changes.

GP35 ANOAL, an abundant acidic glycoprotein of female Anopheles albimanus saliva.

Salivary glands of female mosquitoes produce proteins, not completely described yet, that participate in carbohydrate and blood feeding. Here, we report an acidic glycoprotein of 35 kDa (GP35 ANOAL) secreted in the saliva of the malaria vector mosquito Anopheles albimanus. GP35 ANOAL is produced exclusively in the distal lateral lobes of adult female salivary glands, it has a pI of 4.45 and is negatively stained by regular silver stain. An 888 bp cDNA clone encoding a predicted product of 240 amino acids has a signal peptide, potential post-translational modification sites, and a disintegrin signature RGD. The GP35 ANOAL sequence depicts high similarities with the 30 kDa saliva allergen of Aedes aegypti, 30 kDa allergen-like hypothetical proteins, and GE-rich proteins present in several Anopheles species, as well as in Ae. albopictus and Culex pipiens quinquefasciatus. The function of this protein family is still unknown.

Pteridine concentrations differ between insectary-reared and field-collected Anopheles albimanus mosquitoes of the same physiological age.

Biopterin, isoxanthopterin and 6-pterincarboxylic acid were identified in the head of the malaria vector mosquito Anopheles albimanus Weidemann (Diptera: Culicidae) by HPLC. Total pteridine concentrations (TPC) were estimated in heads, body parts (BP: abdomen, legs and wings) and whole bodies of insectary-reared and field-collected females, by spectrofluorometry, to investigate whether they could be used for age determination. Pteridine concentrations diminished with age in both mosquito groups. TPC correlated with chronological age in insectary-reared sugar-fed females (heads: r2 = 0.35, BP: r2 = 0.34, P < 0.001), but lower correlation occurred in blood-fed females (heads: r2 = 0.22, BP: r2 = 0.27). TPC differed among females of the same age fed with blood at different times (P < 0.05), indicating that bloodmeals modify the diminution rate of pteridines with age. Nevertheless, a polynomial significant correlation was documented for TPC and the number of ovipositions (heads: r2 = 0.24, BP: r2 = 0.27, whole body: r2 = 0.52, P < 0.001) in insectary-reared mosquitoes. This correlation was lower in field-collected mosquitoes (heads: r2 = 0.14, BP: r2 = 0.10, P < 0.05), which showed a remarkable pteridine increase in one-parous females. The correlation of TPC in whole body with physiological age was much less (r2 = 0.03). These observations indicate that TPC determination by spectrofluorometry is not a reliable method to estimate the age of An. albimanus females from the feral population.

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.

Different prevalences of Plasmodium vivax phenotypes VK210 and VK247 associated with the distribution of Anopheles albimanus and Anopheles pseudopunctipennis in Mexico.

The geographic distribution of Plasmodium vivax circumsporozoite protein phenotypes from patient blood used to infect colonized Anopheles albimanus and An. pseudopunctipennis was investigated in southern Mexico. Parasite phenotype types were determined in blood samples by a polymerase chain reaction and oligoprobe hybridization or by immunofluorescent assay of sporozoites. The proportion of infected mosquitoes and the number of oocysts per mosquito confirmed previous in vitro observations indicating that An. albimanus is more susceptible to VK210 and that An. pseudopunctipennis is more susceptible to VK247. All patients living on the coast were infected with VK210 and most patients living above 170 meters above sea level had VK247. Both phenotypes infected patients from intermediate altitudes. These results concur with the distribution of the anophelines, indicating that An. albimanus is the main vector of the phenotype VK210, but that An. pseudopunctipennis transmits both phenotypes. These conditions have direct implications on parasite transmission rates and malaria epidemiology in Mexico.

Scorpine, an anti-malaria and anti-bacterial agent purified from scorpion venom.

A novel peptide, scorpine, was isolated from the venom of the scorpion Pandinus imperator, with anti-bacterial activity and a potent inhibitory effect on the ookinete (ED(50) 0.7 microM) and gamete (ED(50) 10 microM) stages of Plasmodium berghei development. It has 75 amino acids, three disulfide bridges with a molecular mass of 8350 Da. Scorpine has a unique amino acid sequence, similar only to some cecropins in its N-terminal segment and to some defensins in its C-terminal region. Its gene was cloned from a cDNA library.

Susceptibility of three laboratory strains of Anopheles albimanus (Diptera: Culicidae) to coindigenous Plasmodium vivax circumsporozoite protein phenotypes in southern Mexico.

The susceptibility to two coindigenous Plasmodium vivax Grassi & Feletti phenotypes VK210 and VK247 of three colonized Anopheles albimanus Wiedemann strains (white-striped, green and brown) from southern Mexico was investigated. Mosquitoes of the three strains were simultaneously fed with P. vivax-infected patient blood and examined 1 wk later for the presence of oocysts. The circumsporozoite protein phenotype type (VK210 and VK247) was determined by immunoflorescence of salivary gland sporozoites using monoclonal antibodies. The proportions of specimens infected and the number of oocyst per mosquito indicated that all mosquito strains were more susceptible to the phenotype VK210 than to VK247, but the white-striped strain was more susceptible to both parasite phenotypes than the other two strains.

Morphological and cytochemical characterization of female Anopheles albimanus (Diptera: Culicidae) hemocytes.

Hemocytes of 2- to 3-d-old female Anopheles albimanus Wiedemann are described by morphology, cytochemistry, and functional criteria. Supplemented Grace's insect medium in a modified Foley's perfusion method was used to obtain hemolymph from An. albimanus. Morphological analysis indicated 3 types of hemocytes were present, prohemocytes, plasmatocytes, and granular cells. Prohemocytes were small round cells with a high nuclear/cytoplasmic ratio. Plasmatocytes were the most abundant cell types in the hemolymph, and appeared as small to large and spindle-shaped cells with round or elongate nucleus, variable number of vacuoles, small granules, and pseudopodia. Granular cells were small to large and round with a large number of cytoplasmic granules, vacuoles, and numerous filopodia. Ultrastructurally, prohemocytes were undifferentiated with abundant free ribosomes and with few small electron-dense granules. Plasmatocytes were rich in mitochondria, rough endoplasmic reticulum, free ribosomes, small electron-dense granules, numerous peripheral vacuoles and with an important organelle polarization. Granular cells contained numerous large electron-dense granular inclusions and vacuoles. Cytochemical studies showed that plasmatocytes and granular cells have cationic bactericidal proteins. Only granular cells showed phenoloxidase and probably lysosomal activities. In vitro functional studies demonstrated that both plasmatocytes and granular cells were able to attach to glass slides, and only plasmatocyte had phagocytic activity and motility. These results characterize the hemocytes of An. albimanus and suggest that plasmatocytes and granular cells may have a role in defense responses to foreign organisms.

Risk factors for Plasmodium vivax infection in the Lacandon forest, southern Mexico.

A study was conducted to characterize the risk of Plasmodium vivax infection in the Lacandon forest, southern Mexico. Blood samples and questionnaire data were collected in 1992. Malaria cases (n = 137) were identified by the presence of symptoms and a positive thick blood smear. The control group included individuals with negative antibody titres and no history of malaria (n = 4994). From 7628 individuals studied, 1006 had anti-P. vivax antibodies. Seroprevalence increased with age. Risk factors associated with infection included: place of birth outside the village of residence (odds ratio, OR 11.67; 95% CI 5.21-26.11); no use of medical services (OR 4.69, 95% CI 3.01-7.29), never using bed-nets (OR 3.98, 95 % CI 1.23-12.86) and poor knowledge of malaria transmission, prevention and treatment (OR 2.30, 95 % CI 1.30-4.07). Health education represents the best recommendation for controlling the disease in the area.

Comparison of serological and parasitological assessments of Onchocerca volvulus transmission after 7 years of mass ivermectin treatment in Mexico.

OBJECTIVE AND METHOD: To compare the utility of an ELISA using 3 recombinant antigens with that of the skin biopsy to estimate incidence of infections in a sentinel cohort of individuals living in an endemic community in southern Mexico during a set of 11 subsequent ivermectin treatments. RESULTS: The apparent community prevalence of infection and microfilarial skin infection before and after 11 treatments with ivermectin plus nodulectomy were 78% and 13%, and 0.68 mf/mg and 0.04 mf/mg, respectively, as measured by skin biopsy. Of a group of 286 individuals participating in all surveys, a sentinel cohort of 42 mf and serologically negative individuals had been followed since 1994. The annual percentage of individuals becoming positive in this cohort was 24% (10/42), 28% (9/33), 0%, and 4.3% (1/23) in 1995, 1996, 1997 and 1998, respectively. Likewise, the incidence in children 5 years and under (n = 13) within this sentinel cohort was 15% (2/13), 18% (2/11), 0% and 11% (1/9), respectively. All individuals became positive to both tests simultaneously, indicating that seroconversion assessed infection incidence as accurately as skin biopsy in the sentinel group. CONCLUSION: Incidence monitoring of a sentinel cohort provides an estimation of the parasite transmission in the community; it is less costly than massive sampling, and a finger prick blood test might be more acceptable in some communities.

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.

Detection of Onchocerca volvulus infection in Simulium ochraceum sensu lato: comparison of a PCR assay and fly dissection in a Mexican hypoendemic community.

Detection of Onchocerca volvulus larvae in vector populations is of prime importance in the assessment of the effectiveness of onchocerciasis control programmes. Traditionally, detection of larvae is attained by the dissection of flies, but this time-consuming method cannot easily discriminate between species of Onchocerca. The genome of all Onchocerca species has a unique 150 bp repeat, which can be amplified by PCR, and O. volvulus-specific DNA probes can detect these products by Southern blot (SB). This study optimizes a PCR/SB assay, and compares it with fly dissection to estimate the prevalence (p) and intensity of infection (m) in the local vector population of a Mexican community that has become hypoendemic as a result of 7 years of treatment with ivermectin and nodulectomy. The PCR detected 1 infected fly in a pool of 99 uninfected flies, but the optimal pool size was 50 flies. At the community level, 1 out of 10,550 flies was positive (p = 0.0095%, 95% confidence intervals CI = 0.00024-0.05280%; m = 0.00027 larvae/parous fly, CI = -0.00026-0.00081) by PCR, and 4 out of 10,772 flies (p = 0.0371%, CI = 0.01012-0.09505%; m = 0.00107 larvae/parous fly, 95% CI = 0.00002-0.00212) by dissection (observed m = 0.0005). Both methods produce statistically similar estimates of the prevalence and intensity, indicating that pool screening is a viable alternative for entomological surveillance in areas where the intensity of transmission is becoming extremely low as a result of control interventions.

Differential susceptibilities of Anopheles albimanus and Anopheles pseudopunctipennis to infections with coindigenous Plasmodium vivax variants VK210 and VK247 in southern Mexico.

The susceptibilities to coindigenous Plasmodium vivax of colonized Anopheles albimanus and Anopheles pseudopunctipennis from southern Mexico were investigated by simultaneous feeding with infected blood obtained from patients. The genes encoding circumsporozoite protein variant types (VK210 and VK247) in blood samples were determined by PCR and oligonucleotide probe hybridization. A. albimanus was more susceptible to VK210, and A. pseudopunctipennis was more susceptible to VK247.

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.

Plasmodium vivax: a monoclonal antibody recognizes a circumsporozoite protein precursor on the sporozoite surface.

The major surface circumsporozoite (CS) proteins are known to play a role in malaria sporozoite development and invasion of invertebrate and vertebrate host cells. Plasmodium vivax CS protein processing during mosquito midgut oocyst and salivary gland sporozoite development was studied using monoclonal antibodies which recognize different CS protein epitopes. Monoclonal antibodies which react with the CS amino acid repeat sequences by ELISA recognized a 50-kDa precursor protein in immature oocyst and additional 47- and 42-kDa proteins in older oocysts. A 42-kDa CS protein was detected after initial sporozoite invasion of mosquito salivary glands and an additional 50-kDa precursor CS protein observed later in infected salivary glands. These data confirm previous results with other Plasmodium species, in which more CS protein precursors were detected in oocysts than in salivary gland sporozoites. A monoclonal antibody (PvPCS) was characterized which reacts with an epitope found only in the 50-kDa precursor CS protein. PvPCS reacted with all P. vivax sporozoite strains tested by indirect immunofluorescent assay, homogeneously staining the sporozoite periphery with much lower intensity than that produced by anti-CS repeat antibodies. Immunoelectron microscopy using PvPCS showed that the CS protein precursor was associated with peripheral cytoplasmic vacuoles and membranes of sporoblast and budding sporozoites in development oocysts. In salivary gland sporozoites, the CS protein precursor was primarily associated with micronemes and sporozoite membranes. Our results suggest that the 50-kDa CS protein precursor is synthesized intracellularly and secreted on the membrane surface, where it is proteolytically processed to form the 42-kDa mature CS protein. These data indicate that differences in CS protein processing in oocyst and salivary gland sporozoites development may occur.

Determination of sample sizes for the estimation of Onchocerca volvulus (Filarioidea: Onchocercidae) infection rates in biting populations of Simulium ochraceum s.l. (Diptera: Simuliidae) and its application to ivermectin control programs.

Monthly samples of biting Simulium ochraceum s.l. Walker were collected before and after ivermectin treatment in southern Mexico and analyzed for Onchocerca volvulus Leuckart infection rates, infection intensity, and the characteristics of larval distribution among parous flies. The variance over mean ratio (VMR) indicated that in all cases this distribution departed from Poisson and was strongly aggregated (VMR > 1). The negative binomial was found to be an adequate model with a small value of the aggregation parameter k, but the degree of larval overdispersion increased as the mean larval load decreased, invalidating the use of a common kc value. A linear relationship between k and the mean (mu) was established, k(mu) = k1 mu, which permitted exploration of the relationship between the observed proportion of infected flies, p, and the estimated mean larval burden per fly, m (all larval stages in parous flies). This would allow mean numbers of larvae per parous fly to be predicted from presence-absence data (e.g., from infection rates provided by polymerase chain reaction methods applied to pools of flies), assuming that k1 is a known parameter. Given that both p and m are naturally low in S. ochraceum, their relationship was practically linear within the range of observed values. Predictions were tested with the Mexican data from which the clumping parameter was estimated as well as for Guatemalan data for which this information was not available. Results showed a highly satisfactory degree of agreement between predictions and observations. The sample sizes required to estimate mean larval loads from prevalence data for fixed levels of precision (defined as the ratio between SE[m] and m) were calculated for realistic S. ochraceum infection rates (those found in published pre- and postcontrol field surveys as well as in this work). For the special case in which the relationship between k and the mean is linear and goes through the origin, k(mu) = k1 mu, the number of flies to be examined for O. volvulus infections does not explicitly depend on the aggregation parameter, but rather on the unknown proportion of infected flies. Practical recommendations for the calculation of sample sizes are discussed. For infection levels < 0.2%, a minimum number between 6,000 and 13,000 parous flies would have to be examined to estimate the mean larval load with a precision between 0.20 and 0.30. The linearity between onchocercal infection rate and infection intensity in the fly population indicates that relationships between the former and onchocerciasis patterns in the human population should be further explored for the purposes of monitoring the impact of ivermectin control programs through entomological evaluations.

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.