Quantifying flight aptitude variation in wild Anopheles gambiae in order to identify long-distance migrants.

BACKGROUND: In the West African Sahel, mosquito reproduction is halted during the 5-7 month-long dry season, due to the absence of surface waters required for larval development. However, recent studies have suggested that both Anopheles gambiae sensu stricto (s.s.) and Anopheles arabiensis repopulate this region via migration from distant locations where larval sites are perennial. Anopheles coluzzii engages in more regional migration, presumably within the Sahel, following shifting resources correlating with the ever-changing patterns of Sahelian rainfall. Understanding mosquito migration is key to controlling malaria-a disease that continues to claim more than 400,000 lives annually, especially those of African children. Using tethered flight data of wild mosquitoes, the distribution of flight parameters were evaluated as indicators of long-range migrants versus appetitive flyers, and the species specific seasonal differences and gonotrophic states compared between two flight activity modalities. Morphometrical differences were evaluated in the wings of mosquitoes exhibiting high flight activity (HFA) vs. low flight activity (LFA). METHODS: A novel tethered-flight assay was used to characterize flight in the three primary malaria vectors- An. arabiensis, An. coluzzii and An. gambiae s.s. The flights of tethered wild mosquitoes were audio-recorded from 21:00 h to 05:00 h in the following morning and three flight aptitude indices were examined: total flight duration, longest flight bout, and the number of flight bouts during the assay. RESULTS: The distributions of all flight indices were strongly skewed to the right, indicating that the population consisted of a majority of low-flight activity (LFA) mosquitoes and a minority of high-flight activity (HFA) mosquitoes. The median total flight was 586 s and the maximum value was 16,110 s (~ 4.5 h). In accordance with recent results, flight aptitude peaked in the wet season, and was higher in gravid females than in non-blood-fed females. Flight aptitude was also found to be higher in An. coluzzii compared to An. arabiensis, with intermediate values in An. gambiae s.s., but displaying no statistical difference. Evaluating differences in wing size and shape between LFA individuals and HFA ones, the wing size of HFA An. coluzzii was larger than that of LFAs during the wet season-its length was wider than predicted by allometry alone, indicating a change in wing shape. No statistically significant differences were found in the wing size/shape of An. gambiae s.s. or An. arabiensis. CONCLUSIONS: The partial agreement between the tethered flight results and recent results based on aerial sampling of these species suggest a degree of discrimination between appetitive flyers and long-distance migrants although identifying HFAs as long-distance migrants is not recommended without further investigation.

In vitro antimalarial activity of novel semisynthetic nocathiacin I antibiotics.

Presently, the arsenal of antimalarial drugs is limited and needs to be replenished. We evaluated the potential antimalarial activity of two water-soluble derivatives of nocathiacin (BMS461996 and BMS411886) against the asexual blood stages of Plasmodium falciparum. Nocathiacins are a thiazolyl peptide group of antibiotics, are structurally related to thiostrepton, have potent activity against a wide spectrum of multidrug-resistant Gram-positive bacteria, and inhibit protein synthesis. The in vitro growth inhibition assay was done using three laboratory strains of P. falciparum displaying various levels of chloroquine (CQ) susceptibility. Our results indicate that BMS461996 has potent antimalarial activity and inhibits parasite growth with mean 50% inhibitory concentrations (IC50s) of 51.55 nM for P. falciparum 3D7 (CQ susceptible), 85.67 nM for P. falciparum Dd2 (accelerated resistance to multiple drugs [ARMD]), and 99.44 nM for P. falciparum K1 (resistant to CQ, pyrimethamine, and sulfadoxine). Similar results at approximately 7-fold higher IC50s were obtained with BMS411886 than with BMS461996. We also tested the effect of BMS491996 on gametocytes; our results show that at a 20-fold excess of the mean IC50, gametocytes were deformed with a pyknotic nucleus and growth of stage I to IV gametocytes was arrested. This preliminary study shows a significant potential for nocathiacin analogues to be developed as antimalarial drug candidates and to warrant further investigation.

Induction of long-lived potential aestivation states in laboratory An. gambiae mosquitoes.

BACKGROUND: How anopheline mosquitoes persist through the long dry season in Africa remains a gap in our understanding of these malaria vectors. To span this period in locations such as the Sahelian zone of Mali, mosquitoes must either migrate to areas of permanent water, recolonize areas as they again become favorable, or survive in harsh conditions including high temperatures, low humidity, and an absence of surface water (required for breeding). Adult mosquitoes surviving through this season must dramatically extend their typical lifespan (averaging 2-3 weeks) to 7 months. Previous work has found evidence that the malaria mosquito An. coluzzii, survives over 200 days in the wild between rainy seasons in a presumed state of aestivation (hibernation), but this state has so far not been replicated in laboratory conditions. The inability to recapitulate aestivation in the lab hinders addressing key questions such as how this state is induced, how it affects malaria vector competence, and its impact on disease transmission. METHODS: In effort to induce aestivation, we held laboratory mosquitoes in climate-controlled incubators with a range of conditions that adjusted humidity (40-85% RH), temperature (18-27 °C), and light conditions (8-12 h of light) and evaluated their survivorship. These conditions were chosen to mimic the late rainy and dry seasons as well as relevant extremes these mosquitoes may experience during aestivation. RESULTS: We found that by priming mosquitoes in conditions simulating the late wet season in Mali, and maintaining mosquitoes in reduced light/temperature, mean mosquito survival increased from 18.34 ± 0.65 to 48.02 ± 2.87 days, median survival increased from 19 (95% CI 17-21) to 50 days (95% CI 40-58), and the maximum longevity increased from 38 to 109 days (P-adj < 0.001). While this increase falls short of the 200 + day survival seen in field mosquitoes, this extension is substantially higher than previously found through environmental or dietary modulation and is hard to reconcile with states other than aestivation. This finding will provide a platform for future characterization of this state, and allow for comparison to field collected samples.

Marking mosquitoes in their natural larval sites using 2H-enriched water: a promising approach for tracking over extended temporal and spatial scales.

BACKGROUND: 1.Tracking mosquitoes using current methods of mark-release-recapture are limited to small spatial and temporal scales exposing major gaps in understanding long-range movements and extended survival. Novel approaches to track mosquitoes may yield fresh insights into their biology which improves intervention activities to reduce disease transmission.Stable isotope enrichment of natural mosquito breeding sites allows large-scale marking of wild mosquitoes absent human handling. Mosquito larvae that develop in 2H-enriched water are expected to be detectable for over four months using tissue mass-fraction 2H measurements, providing opportunities for long-term mark-capture studies on a large scale. APPROACH: 2.A laboratory study followed by a field experiment of mosquito larval habitat 2H-enrichment was conducted in Mali, to evaluate potential labeling of wild mosquitoes. Twelve natural larval sites were enriched using [2H]-Deuterium-oxide (D2O, 99%). Enrichment level was maintained by supplementation following dilution by rains. Availability of 2H to mosquito larvae was enhanced by locally collected and cultured microorganisms (i.e. protozoa, algae and bacteria) reared in deuterated water, and provided as larval diet. Putative natural predators were removed from the larval sites and first instar larvae Anopheles gambiae s.l. larvae were added every other day. Emergence traps enabled collection of eclosing adults. Adult mosquitoes were kept at laboratory conditions for analysis of label attrition with age. RESULTS: 3.Deuterium enrichment of wild mosquitoes above background levels (maximum = 143.1 ppm) became apparent 5-6 days after initial exposure, after which 2H values increased steadily until ~24 days later (to a mean of approx. 220 ppm). Anopheles and Culex mosquitoes showed significantly different 2H values (211 and 194.2 ppm respectively). Both genera exhibited exponential label attrition (e (-x)) amounting to 21.6% by day 30 post emergence, after which attrition rate continuously decreased. Males of both taxa exhibited a higher mean 2H value compared to females. CONCLUSIONS: 4.Deuterium-oxide proved useful in marking mosquitoes in their natural larval sites and although costly, may prove valuable for studies of mosquitoes and other aquatic insects. Based on our field study, we provide a protocol for marking mosquito larval sites using deuterium-oxide.

Unidentified Plasmodium species in Australian black swans (Cygnus atratus) hatched and raised in North America.

A pair of Australian black swans (Cygnus atratus) with origins in Wakefield, Virginia, USA, was admitted to the quarantine area at the Baltimore Zoo for general health assessments before housing in the collections. During the quarantine period, no clinical signs of disease were manifest; however, upon examination of a blood smear, intraerythrocytic parasites were detected and initially determined to be Haemoproteus species. Diagnostic polymerase chain reaction (PCR) and sequencing results, however, indicated that the parasites were within the genus Plasmodium. Subclinical infections with Plasmodium species in birds may affect collection management, and transmission from refractory hosts to susceptible hosts should be considered when multispecies exhibits are used. In addition, changes in the dynamics of host-vector-parasite interactions might have significant impacts on wild or domesticated populations of birds.

Typing Plasmodium yoelii microsatellites using a simple and affordable fluorescent labeling method.

The rodent malaria parasite Plasmodium yoelii has been an important animal model for studying malaria pathology and host-parasite interactions. Compared with other rodent malaria parasites such as Plasmodium chabaudi, however, genetic mapping studies on P. yoelii have been limited, partly due to the absence of genetic markers and the lack of well characterized phenotypes. Taking advantage of the available genome sequence, we initiated a project to develop a high-resolution microsatellite (MS) map for P. yoelii to study malaria disease phenotypes. Here we report screening the P. yoelii genome for simple sequence repeats and development of an inexpensive method (modified from a previously reported procedure) for typing malaria parasite MS: instead of labeling individual polymerase chain reaction primers, a single fluorescently labeled primer was used to type the MS markers. We evaluated various polymerase chain reaction cycling conditions and M13-tailed/labeled M13 primer ratios to establish a simple and robust procedure for typing P. yoelii MS markers. We also compared typing efficiencies between individually labeled primers and the M13-tailed single labeled primer method and found that the two approaches were comparable. Preliminary analyses of seven P. yoelii isolates deposited at MR4 with 77 MS showed that the markers were highly polymorphic and that the isolates belonged to two groups, suggesting potential common ancestry or laboratory contaminations among the isolates. The MS markers and the typing method provide important tools for genetic studies of P. yoelii. There is a good possibility that this method can be applied to type MS from other malaria parasites including important human pathogens Plasmodium falciparum and Plasmodium vivax.

The contribution of dietary restriction to extended longevity in the malaria vector Anopheles coluzzii.

BACKGROUND: Variation in longevity has long been of interest in vector biology because of its implication in disease transmission through vectorial capacity. Recent studies suggest that Anopheles coluzzii adults persist during the ~7 month dry season via aestivation. Recently there has been a growing body of evidence linking dietary restriction and low ratio of dietary protein to carbohydrate with extended longevity of animals. Here, we evaluated the effects of dietary restriction and the protein : carbohydrate ratio on longevity of An. coluzzii. RESULTS: In our experiment, we combined dietary regimes with temperature and relative humidity to assess their effects on An. coluzzii longevity, in an attempt to simulate aestivation under laboratory conditions. Our results showed significant effects of both the physical and the dietary variables on longevity, but that diet regimen had a considerably greater effect than those of the physical conditions. Higher temperature and lower humidity reduced longevity. At 22 °C dietary protein (blood) shortened longevity when sugar was not restricted (RH = 85%), but extended longevity when sugar was restricted (RH = 50%). CONCLUSIONS: Dietary restriction extended longevity in accord with predictions, but protein : carbohydrate ratio had a negligible effect. We identified conditions that significantly extend longevity in malaria vectors, however, the extent of increase in longevity was insufficient to simulate aestivation.

Preliminary results of an anticircumsporozoite DNA vaccine trial for protection against avian malaria in captive African black-footed penguins (Spheniscus demersus).

Captive juvenile African black-footed penguins (Spheniscus demersus) housed in an outdoor enclosure at the Baltimore Zoo have an average 50% mortality from avian malarial (Plasmodium sp.) infection each year without intense monitoring for disease and chemotherapeutic intervention. During the 1996 malaria transmission season, the safety and efficacy of an anti-circumsporozoite (CSP) DNA vaccine encoding the Plasmodium gallinaceum CSP protein against P. relictum were studied. The goal was to reduce clinical disease and death without initiating sterile immunity after release into an area with stable, endemic avian malaria. The birds were monitored for adverse clinical signs associated with vaccination, the stimulation of an anti-CSP antibody response, and protection afforded by the vaccine. The presence of P. relictum in trapped culicine mosquitoes within the penguin enclosure was monitored to assess parasite pressure. Among the vaccinated penguins, the parasitemia rate dropped from approximately 50% to approximately 17% despite intense parasite pressure, as determined by mosquito infection rate. During the year of the vaccine trial, no mortalities due to malaria occurred and no undesirable vaccination side effects occurred. This is the first trial of an antimalarial vaccine in a captive penguin colony.

Plasmodium Juxtanucleare associated with mortality in black-footed penguins (Spheniscus demersus) admitted to a rehabilitation center.

Five black-footed penguins (Spheniscus demersus) admitted to the Southern African Foundation for the Conservation of Coastal Birds, in Cape Town, South Africa, died from malaria infection. Evidence for malaria as the cause of death included antemortem clinical signs, parasitemia, splenomegaly, pulmonary edema, and the presence of histologically visible schizonts in the reticuloendothelial system. A portion of the malarial small subunit ribosomal ribonucleic acid gene was detected by polymerase chain reaction from postmortem blood samples from all the birds. A species-specific variable region of this gene was compared with the same region on genes from other known avian malarial organisms, establishing that Plasmodium juxtanucleare was involved.

Hundreds of microsatellites for genotyping Plasmodium yoelii parasites.

Genetic crosses have been employed to study various traits of rodent malaria parasites and to locate loci that contribute to drug resistance, immune protection, and disease virulence. Compared with human malaria parasites, genetic crossing of rodent malaria parasites is more easily performed; however, genotyping methods using microsatellites (MSs) or large-scale single nucleotide polymorphisms (SNPs) that have been widely used in typing Plasmodium falciparum are not available for rodent malaria species. Here we report a genome-wide search of the Plasmodium yoelii yoelii (P. yoelii) genome for simple sequence repeats (SSRs) and the identification of nearly 600 polymorphic MS markers for typing the genomes of P. yoelii and Plasmodium berghei. The MS markers are randomly distributed across the 14 physical chromosomes assembled from genome sequences of three rodent malaria species, although some variations in the numbers of MS expected according to chromosome size exist. The majority of the MS markers are AT-rich repeats, similar to those found in the P. falciparum genome. The MS markers provide an important resource for genotyping, lay a foundation for developing linkage maps, and will greatly facilitate genetic studies of P. yoelii.

Disruption of a Plasmodium falciparum multidrug resistance-associated protein (PfMRP) alters its fitness and transport of antimalarial drugs and glutathione.

ATP-binding cassette transporters play an important role in drug resistance and nutrient transport. In the human malaria parasite Plasmodium falciparum, a homolog of the human p-glycoprotein (PfPgh-1) was shown to be involved in resistance to several drugs. More recently, many transporters were associated with higher IC(50) levels in responses to chloroquine (CQ) and quinine (QN) in field isolates. Subsequent studies, however, could not confirm the associations, although inaccuracy in drug tests in the later studies could contribute to the lack of associations. Here we disrupted a gene encoding a putative multidrug resistance-associated protein (PfMRP) that was previously shown to be associated with P. falciparum responses to CQ and QN. Parasites with disrupted PfMRP (W2/MRPDelta) could not grow to a parasitemia higher than 5% under normal culture conditions, possibly because of lower efficiency in removing toxic metabolites. The W2/MRPDelta parasite also accumulated more radioactive glutathione, CQ, and QN and became more sensitive to multiple antimalarial drugs, including CQ, QN, artemisinin, piperaquine, and primaquine. PfMRP was localized on the parasite surface membrane, within membrane-bound vesicles, and along the straight side of the D-shaped stage II gametocytes. The results suggest that PfMRP plays a role in the efflux of glutathione, CQ, and QN and contributes to parasite responses to multiple antimalarial drugs, possibly by pumping drugs outside the parasite.

Antimalarial drugs: current status and new developments.

Malaria continues to be a major threat in the developing world, with > 1 million clinical episodes and 3000 deaths every day. In the last century, malaria claimed between 150 and 300 million lives, accounting for 2 - 5% of all deaths. Currently approximately 40% of the world population resides in areas of active malaria transmission. The disease symptoms are most severe in young children and pregnant women. A total of 90% of the disease-associated mortality occurs in Subsaharan Africa, despite the fact that malaria is indigenous to most tropical regions. A licensed vaccine for malaria has not become a reality and antimalarial drugs are the only available method of treatment. Although chloroquine, the first synthetically developed antimalarial, proved to be an almost magical cure for > 30 years, the emergence and spread of chloroquine-resistant parasites has made it virtually ineffective in most parts of the world. Currently, artemisinin, a plant-derived antimalarial, is the only available drug that is globally effective against the parasite. Although several new drugs have been introduced in the past 30 years, widespread or isolated cases of resistance indicate that their window of effectiveness will be limited. Thus, there is an urgent need to develop new therapeutics and regimens for malaria control. This article presents an overview of the currently available antimalarial chemotherapy options and the efforts being undertaken to develop new drugs based on both the recent technological advances and modifications to the old remedies, and on combination therapies.

The effects of glucose concentration on the reciprocal regulation of rRNA promoters in Plasmodium falciparum.

The developmental progression of Plasmodium falciparum is remarkably sensitive to glucose concentration. We have investigated the effects of glucose concentration on the parasite development cycle as reflected by changes of ribosomal RNA (rRNA) transcription. We showed that glucose starvation differentially affects transcriptional control of the rRNA genes by sharply repressing transcription from those loci involved with asexual development of the parasite while up-regulating transcription at those loci involved with sexual development of the parasite. Temperature change also effects regulation of transcription. We found that the effects of temperature and glucose were synergistic. We identified and compared the upstream region of the transcription start sites of each gene. These putative promoter structures are considerably different from one another and contain structures remarkably similar to rRNA control elements in other organisms.

Measuring the effects of an ever-changing environment on malaria control.

The effectiveness of malaria control measures depends not only on the potency of the control measures themselves but also upon the influence of variables associated with the environment. Environmental variables have the capacity either to enhance or to impair the desired outcome. An optimal outcome in the field, which is ultimately the real goal of vaccine research, will result from prior knowledge of both the potency of the control measures and the role of environmental variables. Here we describe both the potential effectiveness of control measures and the problems associated with testing in an area of endemicity. We placed canaries with different immunologic backgrounds (e.g., naïve to malaria infection, vaccinated naïve, and immune) directly into an area where avian malaria, Plasmodium relictum, is endemic. In our study setting, canaries that are naïve to malaria infection routinely suffer approximately 50% mortality during their first period of exposure to the disease. In comparison, birds vaccinated and boosted with a DNA vaccine plasmid encoding the circumsporozoite protein of P. relictum exhibited a moderate degree of protection against natural infection (P < 0.01). In the second year we followed the fate of all surviving birds with no further manipulation. The vaccinated birds from the first year were no longer statistically distinguishable for protection against malaria from cages of naïve birds. During this period, 36% of vaccinated birds died of malaria. We postulate that the vaccine-induced protective immune responses prevented the acquisition of natural immunity similar to that concurrently acquired by birds in a neighboring cage. These results indicate that dominant environmental parameters associated with malaria deaths can be addressed before their application to a less malleable human system.

Description of a possible clonal expansion of Plasmodium vivax in Manaus, Amazonas, Brasil

Casos atípicos de P. vivax relatados no Município de Manaus nos levou a detectar um isolado genético de P. vivax. Regiöes variáveis do SSUrRNA foram examinadas no momento inicial da infecçäo e nas duas recrudescências de um mesmo paciente exibindo recrudescência/recidiva à cloroquina e primaquina. Um único isolado, encontrado em todos os estágios da infecçäo, sugere a presença de uma expansäo clonal