An overview of malaria transmission from the perspective of Amazon Anopheles vectors

In the Americas, areas with a high risk of malaria transmission are mainly located in the Amazon Forest, which extends across nine countries. One keystone step to understanding the Plasmodium life cycle in Anopheles species from the Amazon Region is to obtain experimentally infected mosquito vectors. Several attempts to colonise Ano- pheles species have been conducted, but with only short-lived success or no success at all. In this review, we review the literature on malaria transmission from the perspective of its Amazon vectors. Currently, it is possible to develop experimental Plasmodium vivax infection of the colonised and field-captured vectors in laboratories located close to Amazonian endemic areas. We are also reviewing studies related to the immune response to P. vivax infection of Anopheles aquasalis, a coastal mosquito species. Finally, we discuss the importance of the modulation of Plasmodium infection by the vector microbiota and also consider the anopheline genomes. The establishment of experimental mosquito infections with Plasmodium falciparum, Plasmodium yoelii and Plasmodium berghei parasites that could provide interesting models for studying malaria in the Amazonian scenario is important. Understanding the molecular mechanisms involved in the development of the parasites in New World vectors is crucial in order to better determine the interaction process and vectorial competence.     

The remarkable journey of adaptation of the Plasmodium falciparum malaria parasite to New World anopheline mosquitoes

Plasmodium falciparum originated in Africa, dispersed around the world as a result of human migration and had to adapt to several different indigenous anopheline mosquitoes. Anophelines from the New World are evolutionary distant form African ones and this probably resulted in a more stringent selection of Plasmodium as it adapted to these vectors. It is thought that Plasmodium has been genetically selected by some anopheline species through unknown mechanisms. The mosquito immune system can greatly limit infection and P. falciparum evolved a strategy to evade these responses, at least in part mediated by Pfs47, a highly polymorphic gene. We propose that adaptation of P. falciparum to new vectors may require evasion of their immune system. Parasites with a Pfs47 haplotype compatible with the indigenous mosquito vector would be able to survive and be transmitted. The mosquito antiplasmodial response could be an important determinant of P. falciparum population structure and could affect malaria transmission in the Americas.     

Confirmation that Plasmodium falciparum has aperiodic infectivity to Anopheles gambiae

Abstract. In preparation for field studies of transmission-blocking malaria vaccines, a study was carried out to determine whether P. falciparum infections obtained in An. gambiae blood-fed at 16.00 hours were quantitatively similar to infections obtained at 23.00 hours. Using a group of children aged 5-12 years from villages at Ahero, near Kisumu in Kenya, 71/74 (96%) of whom were found to be positive for P. falciparum parasitaemia, one batch of fifty colony-bred An. gambiae females were fed on volunteers at 16.00 hours and another batch at 23.00 hours. No statistically significant differences were found in the proportions of mosquitoes becoming infected, the numbers of children infecting mosquitoes or the mean numbers of malaria oocysts developing in mosquitoes blood-fed at the different times. Because mosquito infections obtained by day (16.00 hours) are equivalent in quantity to those obtained at night (23.00 hours), experimental infections can be carried out in the afternoon, when it is most convenient, rather than during the night.     

Effect of anti-mosquito antibodies on the infectivity of the rodent malaria parasite Plasmodium berghei to Anopheles farauti

The effect of mouse anti-mosquito antibodies, present in the bloodmeal, on the infectivity of Plasmodium berghei Vincke to Anopheles farauti Laveran was investigated. Significantly fewer oocysts developed in mosquitoes feeding on mice immunized with sugar-fed mosquito midgut antigens than in mosquitoes feeding on control mice. Mosquitoes feeding on mice immunized with the midgut antigens derived from sugar-fed mosquitoes also showed reduced mortality and had lower infection rates than those fed on unimmunized mice. Blood-fed midgut antigen was less effective in producing these effects than sugar-fed midgut antigen.     

Diversion of Anopheles gambiae from children to other hosts following exposure to permethrin-treated bednets

Permethrin-treated bednets reduce mortality and morbidity from malaria in Gambian children. However, it is not certain how this effect is achieved, as neither mosquito numbers nor the human blood index of indoor-resting female Anopheles gambiae Giles sensu lato (Diptera: Culicidae) mosquitoes have been reduced when treated bednets were introduced into a community. One possibility is that insecticide-treated bednets divert mosquitoes from children to adults. To investigate this hypothesis, a cross-over trial with insecticide-treated bednets was undertaken in two small Gambian villages. To differentiate mosquitoes that had fed on children from those that had fed on adults, all children in the study villages were immunized with rabies vaccine before the trial. Using the detection of rabies antibody in a bloodmeal as an indicator that a mosquito had bitten a child, it was found that the percentage of blood-fed mosquitoes caught indoors that had bitten a child fell significantly from 30.8% to 9.2% and from 28.0% to 6.9% in each village after insecticide-treated bednets were introduced. To investigate the possibility that some diversion to animals had occurred, a PCR analysis for human beta-globin DNA was undertaken on selected samples. The results of this investigation were confusing, as some rabies-antibody positive bloodmeals were negative for human DNA. This may have been due to cross-reacting antibodies in animal sera and/or DNA degradation by digestion in the mosquito. Although good evidence for diversion of mosquitoes away from children was obtained, it remains uncertain whether diversion was mainly to adult humans, to animals or to both.     

Behaviour and fitness of γHCH/dieldrin resistant and susceptible female Anopheles gambiae and An.stephensi mosquitoes in the absence of insecticide

The effects of gamma HCH/dieldrin resistance genes on various fitness components of mosquito larvae and adult females in the absence of insecticide were investigated in backcrossed strains of Anopheles gambiae Giles and An.stephensi Liston. Among larvae, heterozygotes (RS) developed slightly but significantly faster than homozygotes for resistance (RR) or susceptibility (SS). The lifetime fecundity of RR females in population cages was only half to two-thirds that of SS and RS females despite similar longevities; several reasons were identified: RR gravid females were less responsive to oviposition-site stimuli, their spontaneous activity--as measured in an acoustic actograph--was only half that of SS or RS females, and RR females produced fewer eggs per unit bloodmeal. When inseminated females were recorded in LD 12:12, RR were again less active than SS or RS. When the lighting was switched to a regime simulating full-moonlight, the activity pattern of SS and RS changed and they flew for longer periods. In contrast, the activity of RR females was the same in LD 12:12 as in 'moonlight'. In a test simulation of potential predation, RR mosquitoes took to flight least readily. All component tests on adult females therefore point to RR as being the least fit of the three genotypes. The behavioural tests suggest that resistance has raised the response threshold of RR females to diverse stimuli. A possible physiological mechanism underlying RR behaviour is that a change in the cyclodiene receptor on the chloride channels has increased their permeability to chloride ions, causing hyper-inhibition of the nervous system.     

Plasmodium berghei: plasmodium perforin-like protein 5 is required for mosquito midgut invasion in Anopheles stephensi

During its life cycle the malarial parasite Plasmodium forms three invasive stages which have to invade different and specific cells for replication to ensue. Invasion is vital to parasite survival and consequently proteins responsible for invasion are considered to be candidate vaccine/drug targets. Plasmodium perforin-like proteins (PPLPs) have been implicated in invasion because they contain a predicted pore-forming domain. Ookinetes express three PPLPs, and one of them (PPLP3) has previously been shown to be essential for mosquito midgut invasion. In this study we show through phenotypic analysis of loss-of-function mutants that PPLP5 is equally essential for mosquito infection. Deltapplp5 ookinetes cannot invade midgut epithelial cells, but subsequent parasite development is rescued if the midgut is bypassed by injection of ookinetes into the hemocoel. The indistinguishable phenotypes of Deltapplp5 and Deltapplp3 ookinetes strongly suggest that these two proteins contribute to a common process.     

Plasmodium simium/Plasmodium vivax infections in southern brown howler monkeys from the Atlantic Forest

Blood infection by the simian parasite, Plasmodium simium, wasidentified in captive (n = 45, 4.4%) and in wild Alouatta clamitansmonkeys (n = 20, 35%) from the Atlantic Forest of southern Brazil. A single malariainfection was symptomatic and the monkey presented clinical and haematologicalalterations. A high frequency of Plasmodium vivax-specificantibodies was detected among these monkeys, with 87% of the monkeys testing positiveagainst P. vivax antigens. These findings highlight the possibilityof malaria as a zoonosis in the remaining Atlantic Forest and its impact on theepidemiology of the disease.     

The Duffy binding protein as a key target for a Plasmodium vivax vaccine: lessons from the Brazilian Amazon

Plasmodium vivax infects human erythrocytes through a major pathwaythat requires interaction between an apical parasite protein, the Duffy bindingprotein (PvDBP) and its receptor on reticulocytes, the Duffy antigen/receptor forchemokines (DARC). The importance of the interaction between PvDBP (region II, DBPII)and DARC to P. vivax infection has motivated our malaria researchgroup at Oswaldo Cruz Foundation (state of Minas Gerais, Brazil) to conduct a numberof immunoepidemiological studies to characterise the naturally acquired immunity toPvDBP in populations living in the Amazon rainforest. In this review, we provide anupdate on the immunology and molecular epidemiology of PvDBP in the BrazilianAmazon - an area of markedly unstable malaria transmission - andcompare it with data from other parts of Latin America, as well as Asia andOceania.     

The behaviour of mosquitoes in relation to humans under holed bednets: the evidence from experimental huts

The physical integrity of bednets is a concern of national malaria control programs, as it is a key factor in determining the rate of replacement of bednets. It is largely assumed that increased numbers of holes will result in a loss of protection of sleepers from potentially infective bites. Experimental hut studies are valuable in understanding mosquito behaviour indoors, particularly as it relates to blood feeding and mortality. This review summarises findings from experimental hut studies, focusing on two issues: (i) the effect of different numbers or sizes of holes in bednets and (ii) feeding behaviour and mortality with holed nets as compared with unholed nets. As might be expected, increasing numbers and area of holes resulted in increased blood feeding by mosquitoes on sleepers. However, the presence of holes did not generally have a large effect on the mortality of mosquitoes. Successfully entering a holed mosquito net does not necessarily mean that mosquitoes spend less time in contact with the net, which could explain the lack in differences in mortality. Further behavioural studies are necessary to understand mosquito behaviour around nets and the importance of holed nets on malaria transmission.     

Activity and mating competitiveness of γHCH/dieldrin resistant and susceptible male and virgin female Anopheles gambiae and An.stephensi mosquitoes, with assessment of an insecticide-rotation strategy

The effects of gamma HCH/dieldrin resistance genes on flight activity and mating competitiveness were investigated in males from backcrossed strains of Anopheles gambiae Giles and An.stephensi Liston. Activity of males and virgin females of both species, as recorded in an acoustic actograph, occurred mainly at dusk (the E peak). The activity pattern of An.gambiae males was not affected by resistance genes; in mating competition and predator avoidance experiments, however, RR males were less successful than RS males which were less successful than SS males. The activity pattern of An.stephensi differed from An.gambiae in that the E peaks of RR males and females in a gradual dusk regime were out of synchrony with those of SS and RS, the E peaks of RR occurring slightly later. Thus, RR males and females tended to mate assortatively in mate competition experiments. When a sudden dusk regime was substituted for the gradual dusk regime, activity of RR An.stephensi became synchronized with SS and RS activity, but in mating competition experiments RR still tended to mate assortatively. Estimates of male competitiveness, together with previously-obtained estimates of female fitness, were included in population genetics models. Computer simulations showed that the frequency of resistance in populations of An.gambiae and An.stephensi should decrease in the absence of insecticide at a rate comparable with known field reversions.     

Molecular cloning and expression pattern of 14-3-3ζ from the malaria vector, Anopheles sinensis

14-3-3 proteins are known to play a pivotal role in cell survival, apoptosis and signal transduction. The 14-3-3ζ isoform has been cloned and characterized from many eukaryotic organisms, including the fruit fly and silkworm. However, no study on mosquito 14-3-3 has been reported to date. In an attempt to investigate the function of 14-3-3 in midgut epithelial cells undergoing apoptosis, a cDNA library was generated from the malaria vector, Anopheles sinensis , which was treated with apoptosis-inducing Actinomycin-D. We were able to identify and obtain A. sinensis 14-3-3ζ cDNA ( Ansi14-3-3ζ ) from expressed sequence tags (EST) analysis after conducting massive sequencing of the A. sinensis cDNA library. Ansi14-3-3ζ has very high homology to 14-3-3 homologs of various insects, such as Anopheles gambiae (100%), Aedes aegypti (100%), Drosophila melanogaster (96%), Bombyx mori (93%), Apis mellifera (93%) and Mus musculus (81%), indicating that mosquito 14-3-3ζ is a highly conserved gene in diverse organisms. Analysis of temporal expression patterns showed that Ansi14-3-3ζ mRNA is highly expressed in egg, early pupae and adult stages and is also expressed, although at low levels, in fourth instar larvae and late pupae. In response to two immune elicitors (lipopolysaccharide and laminarin), no striking induction of 14-3-3ζ mRNA was observed in A. sinensis . Further studies of the precise biological function, inducibility and subcellular distribution of 14-3-3ζ are required in Plasmodium invasion-induced apoptotic midgut cells in A. sinensis in the context of the Time Bomb model.     

Positional cloning of rp2 QTL associates the P450 genes CYP6Z1, CYP6Z3 and CYP6M7 with pyrethroid resistance in the malaria vector Anopheles funestus

Pyrethroid resistance in Anopheles funestus is threatening malaria control inAfrica. Elucidation of underlying resistance mechanisms is crucial to improve the successof future control programs. A positional cloning approach was used to identify genesconferring resistance in the uncharacterised rp2 quantitative trait locus (QTL)previously detected in this vector using F6 advanced intercross lines (AIL). A113 kb BAC clone spanning rp2 was identified and sequenced revealing acluster of 15 P450 genes and one salivary protein gene (SG7-2). Contrary toA. gambiae, AfCYP6M1 is triplicated in A. funestus, whileAgCYP6Z2 orthologue is absent. Five hundred and sixty-five new singlenucleotide polymorphisms (SNPs) were identified for genetic mapping from rp2P450s and other genes revealing high genetic polymorphisms with one SNP every36 bp. A significant genotype/phenotype association was detected forrp2 P450s but not for a cluster of cuticular protein genes previouslyassociated with resistance in A. gambiae. QTL mapping using F6 AIL confirms therp2 QTL with an increase logarithm of odds score of 5. Multiplex geneexpression profiling of 15 P450s and other genes around rp2 followed byindividual validation using qRT–PCR indicated a significant overexpression in theresistant FUMOZ-R strain of the P450s AfCYP6Z1, AfCYP6Z3,AfCYP6M7 and the glutathione-s-transferase GSTe2 with respective foldchange of 11.2, 6.3, 5.5 and 2.8. Polymorphisms analysis of AfCYP6Z1 andAfCYP6Z3 identified amino acid changes potentially associated with resistancefurther indicating that these genes are controlling the pyrethroid resistance explained bythe rp2 QTL. The characterisation of this rp2 QTL significantly improvesour understanding of resistance mechanisms in A. funestus.     

Aspidosperma (Apocynaceae) plant cytotoxicity and activity towards malaria parasites. Part I: Aspidosperma nitidum (Benth) used as a remedy to treat fever and malaria in the Amazon

Infusions of Aspidosperma nitidum (Apocynaceae) wood bark are usedto treat fever and malaria in the Amazon Region. Several species of this family areknown to possess indole alkaloids and other classes of secondary metabolites, whereasterpenoids, an inositol and the indole alkaloids harmane-3 acid and braznitiduminehave been described in A. nitidum . In the present study, extractsfrom the wood bark, leaves and branches of this species were prepared for assaysagainst malaria parasites and cytotoxicity testing using human hepatoma and normalmonkey kidney cells. The wood bark extracts were active against Plasmodiumfalciparum and showed a low cytotoxicity in vitro, whereas the leaf andbranch extracts and the pure alkaloid braznitidumine were inactive. A crude methanolextract was subjected to acid-base fractionation aimed at obtaining alkaloid-richfractions, which were active at low concentrations against P.falciparum and in mice infected with and sensitive Plasmodiumberghei parasites. Our data validate the antimalarial usefulness ofA. nitidum wood bark, a remedy that can most likely help tocontrol malaria. However, the molecules responsible for this antimalarial activityhave not yet been identified. Considering their high selectivity index, thealkaloid-rich fractions from the plant bark might be useful in the development of newantimalarials.     

Larvicidal and insect growth regulator effect of α-amyrin acetate from Catharanthus Roseus Linn against the malaria vector Anopheles Stephensi Liston (Diptera: Culicidae)

Vector control is a serious concern in developing countries. Over the past two decades, phytochemicals have received progressively more attention as insecticide alternatives, and they have recently become the focus in the concept of integrated vector control. α-Amyrin acetate, the n-hexane fraction of acetone extract from the leaves of Catharanthus roseus , was evaluated for its larvicidal, pupicidal and fecundity effects as well as insect growth regulator activity against the malaria vector Anopheles stephensi Liston. The highest concentration of 1 p.p.m. produced 100% mortality in first to second instars and 94% mortality in third and fourth instars. In addition, the duration of larval instars and the total developmental time were prolonged, while female longevity and fecundity were markedly decreased. The suppression of pupation and adult emergence was probably due to its action similar to juvenile hormone analogs in combination with growth regulator activity and toxicity, which reduced the overall performance of the malaria vector An. stephensi .     

Dystroglycan binding to laminin α1LG4 module influences epithelial morphogenesis of salivary gland and lung in vitro

Dystroglycan is a receptor for the basement membrane components laminin-1, -2, perlecan, and agrin. Genetic studies have revealed a role for dystroglycan in basement membrane formation of the early embryo. Dystroglycan binding to the E3 fragment of laminin-1 is involved in kidney epithelial cell development, as revealed by antibody perturbation experiments. E3 is the most distal part of the carboxyterminus of laminin alpha1 chain, and is composed of two laminin globular (LG) domains (LG4 and LG5). Dystroglycan-E3 interactions are mediated solely by discrete domains within LG4. Here we examined the role of this interaction for the development of mouse embryonic salivary gland and lung. Dystroglycan mRNA was expressed in epithelium of developing salivary gland and lung. Immunofluorescence demonstrated dystroglycan on the basal side of epithelial cells in these tissues. Antibodies against dystroglycan that block binding of alpha-dystroglycan to laminin-1 perturbed epithelial branching morphogenesis in salivary gland and lung organ cultures. Inhibition of branching morphogenesis was also seen in cultures treated with polyclonal anti-E3 antibodies. One monoclonal antibody (mAb 200) against LG4 blocked interactions between a-dystroglycan and recombinant laminin alpha1LG4-5, and also inhibited salivary gland and lung branching morphogenesis. Three other mAbs, also specific for the alpha1 carboxyterminus and known not to block branching morphogenesis, failed to block binding of alpha-dystroglycan to recombinant laminin alpha1LG4-5. These findings clarify why mAbs against the carboxyterminus of laminin alpha1 differ in their capacity to block epithelial morphogenesis and suggest that dystroglycan binding to alpha1LG4 is important for epithelial morphogenesis of several organs.     

Corynebacterium diphtheriae putative tellurite-resistance protein (CDCE8392_0813) contributes to the intracellular survival in human epithelial cells and lethality of Caenorhabditis elegans

Corynebacterium diphtheriae, the aetiologic agent of diphtheria, also represents a global medical challenge because of the existence of invasive strains as causative agents of systemic infections. Although tellurite (TeO3^2-) is toxic to most microorganisms, TeO3^2--resistant bacteria, including C. diphtheriae, exist in nature. The presence of TeO3^2--resistance (Te^R) determinants in pathogenic bacteria might provide selective advantages in the natural environment. In the present study, we investigated the role of the putative Te^R determinant (CDCE8392_813gene) in the virulence attributes of diphtheria bacilli. The disruption of CDCE8392_0813 gene expression in the LDCIC-L1 mutant increased susceptibility to TeO3^2- and reactive oxygen species (hydrogen peroxide), but not to other antimicrobial agents. The LDCIC-L1 mutant also showed a decrease in both the lethality of Caenorhabditis elegans and the survival inside of human epithelial cells compared to wild-type strain. Conversely, the haemagglutinating activity and adherence to and formation of biofilms on different abiotic surfaces were not regulated through the CDCE8392_0813 gene. In conclusion, the CDCE8392_813 gene contributes to the Te^R and pathogenic potential of C. diphtheriae.     

A new species of Bembidion ( Ecuadion) from Ecuador (Coleoptera,Carabidae, Bembidiini), with a key to members of the georgeballi species group

A new species of ground beetle, Bembidion ricei, is described from the Andes mountains of Ecuador east of Quito. It belongs to the georgeballi species group of subgenus Ecuadion, and is most similar to Bembidion georgeballi. A key to the species of the group is provided.     

The shift from low to high non-structural protein 1 expression in rotavirus-infected MA-104 cells

A hallmark of group/species A rotavirus (RVA) replication in MA-104 cells is the logarithmic increase in viral mRNAs that occurs four-12 h post-infection. Viral protein synthesis typically lags closely behind mRNA synthesis but continues after mRNA levels plateau. However, RVA non-structural protein 1 (NSP1) is present at very low levels throughout viral replication despite showing robust protein synthesis. NSP1 has the contrasting properties of being susceptible to proteasomal degradation, but being stabilised against proteasomal degradation by viral proteins and/or viral mRNAs. We aimed to determine the kinetics of the accumulation and intracellular distribution of NSP1 in MA-104 cells infected with rhesus rotavirus (RRV). NSP1 preferentially localises to the perinuclear region of the cytoplasm of infected cells, forming abundant granules that are heterogeneous in size. Late in infection, large NSP1 granules predominate, coincident with a shift from low to high NSP1 expression levels. Our results indicate that rotavirus NSP1 is a late viral protein in MA-104 cells infected with RRV, presumably as a result of altered protein turnover.     

Six new species of Agrilus Curtis, 1825 (Coleoptera,Buprestidae, Agrilinae) from the Oriental Region related to the emerald ash borer, A. planipennis Fairmaire, 1888 and synonymy of Sarawakita Obenberger, 1924

Six new species of Agrilus Curtis, 1825 with affinities to the emerald ash borer, Agrilus planipennis Fairmaire, 1888, are described from the Oriental Region: Agrilus crepuscularis sp. n. (Malaysia); Agrilus pseudolubopetri sp. n. (Laos); Agrilus sapphirinus sp. n.(Laos); Agrilus seramensis sp. n.(Indonesia); Agrilus spineus sp. n. (Malaysia); and Agrilus tomentipennis sp. n. (Laos). The genus Sarawakita Obenberger, 1924 syn. nov. is considered a junior synonym of Agrilus.