An insight into the sialome of the blood-sucking bug Triatoma infestans, a vector of Chagas' disease

Triatoma infestans is a hemiptera, vector of Chagas' disease that feeds exclusively on vertebrate blood in all life stages. Hematophagous insects' salivary glands (SG) produce potent pharmacological compounds that counteract host hemostasis, including anticlotting, antiplatelet, and vasodilatory molecules. To obtain a further insight into the salivary biochemical and pharmacological complexity of this insect, a cDNA library from its SG was randomly sequenced. Also, salivary proteins were submitted to two-dimensional gel (2D-gel) electrophoresis followed by MS analysis. We present the analysis of a set of 1534 (SG) cDNA sequences, 645 of which coded for proteins of a putative secretory nature. Most salivary proteins described as lipocalins matched peptide sequences obtained from proteomic results.     

High Level of Pyrethroid Resistance in an Anopheles funestus Population of the Chokwe District in Mozambique

Background

Although Anopheles funestus is difficult to rear, it is crucial to analyse field populations of this malaria vector in order to successfully characterise mechanisms of insecticide resistance observed in this species in Africa. In this study we carried out a large-scale field collection and rearing of An. funestus from Mozambique in order to analyse its susceptibility status to insecticides and to broadly characterise the main resistance mechanisms involved in natural populations.

Methodology/Principal Findings

3,000 F₁ adults were obtained through larval rearing. WHO susceptibility assays indicated a very high resistance to pyrethroids with no mortality recorded after 1h30min exposure and less than 50% mortality at 3h30min. Resistance to the carbamate, bendiocarb was also noted, with 70% mortality after 1h exposure. In contrast, no DDT resistance was observed, indicating that no kdr-type resistance was involved. The sequencing of the acetylcholinesterase gene indicated the absence of the G119S and F455W mutations associated with carbamate and organophosphate resistance. This could explain the absence of malathion resistance in this population. Both biochemical assays and quantitative PCR implicated up-regulated P450 genes in pyrethroid resistance, with GSTs playing a secondary role. The carbamate resistance observed in this population is probably conferred by the observed altered AChE with esterases also involved.

Conclusion/Significance

The high level of pyrethroid resistance in this population despite the cessation of pyrethroid use for IRS in 1999 is a serious concern for resistance management strategies such as rotational use of insecticides. As DDT has now been re-introduced for IRS, susceptibility to DDT needs to be closely monitored to prevent the appearance and spread of resistance to this insecticide.     

Proteomics analysis reveals insight into the mechanism of H-Ras-mediated transformation

We implemented a proteomics approach to the systematical analysis of the alterations in the proteome of NIH3T3 cells transformed by oncogenic H-RasV12. Forty-four proteins associated with Ras-mediated transformation have been identified, and 28 proteins were not previously reported. RT-PCR analysis showed that approximately 44% of target proteins identified showed concomitant changes in mRNA abundance. A principal finding was the up-regulation of gankyrin, which was the first evidence to show that gankyrin pathway was implicated in Ras-activated transformation.     

Spatial and temporal variations of the chromosomal inversion polymorphism of Anopheles funestus in Senegal

The polymorphism of paracentric inversions of An. funestus polytene chromosomes was studied along a transect in Senegal in order to assess their variations at the spatial and temporal level. There was an increase in the degree of chromosomal polymorphism from the West to South-East. At the geographical level the variations in inversion frequencies were highly significant whatever the chromosomal arm considered. However, the variations in the chromosomal inversion frequencies did not change significantly over either seasons or years, except for inversion 3b in the village of Dielmo. Such geographical variability within a relatively limited area, associated to temporal stability, suggest a restricted gene flow between the populations studied, probably due to discontinuities in the An. funestus distribution and to its bioecology.     

Advances in the study of Anopheles funestus, a major vector of malaria in Africa

The recent literature on cytogenetic and molecular studies of Anopheles funestus, a major vector of malaria in Africa, is reviewed. Molecular data from West and Central Africa suggest a new species in the group closely allied to Anopheles rivulorum. Cytogenetic and molecular studies of populations from West, Central, East and southern Africa indicate considerable genetic structuring within An. funestus itself, which may well restrict the spread of pyrethroid resistance that has been demonstrated in southern Africa.     

Cryptic species within Anopheles longipalpis from southern Africa and phylogenetic comparison with members of the An. funestus group

House-resting Anopheles mosquitoes are targeted for vector control interventions; however, without proper species identification, the importance of these Anopheles to malaria transmission is unknown. Anopheles longipalpis, a non-vector species, has been found in significant numbers resting indoors in houses in southern Zambia, potentially impacting on the utilization of scarce resources for vector control. The identification of An. longipalpis is currently based on classical morphology using minor characteristics in the adult stage and major ones in the larval stage. The close similarity to the major malaria vector An. funestus led to investigations into the development of a molecular assay for identification of An. longipalpis. Molecular analysis of An. longipalpis from South Africa and Zambia revealed marked differences in size and nucleotide sequence in the second internal transcribed spacer (ITS2) region of ribosomal DNA between these two populations, leading to the conclusion that more than one species was being analysed. Phylogenetic analysis showed the Zambian samples aligned with An. funestus, An. vaneedeni and An. parensis, whereas the South African sample aligned with An. leesoni, a species that is considered to be more closely related to the Asian An. minimus subgroup than to the African An. funestus subgroup. Species-specific primers were designed to be used in a multiplex PCR assay to distinguish between these two cryptic species and members of the An. funestus subgroup for which there is already a multiplex PCR assay.     

Immunogold determination of Plasmodium falciparum circumsporozoite protein in Anopheles stephensi salivary gland cells

The distribution of circumsporozoite (CS) proteins of Plasmodium falciparum sporozoites was observed during the passage of mature sporozoites in the hemocoel of Anopheles stephensi and during their entrance and sojourn in the salivary gland cells (SGC). The CS protein was visualized using a monoclonal antibody (3SP2) and immunogold labeling on ultrathin cryosections. In the hemocoel the sporozoites cease synthesizing CS protein, and some of it is shedded resulting in a patchy labeling pattern on the outer pellicular membrane. No internal labeling was observed. The sporozoites enter the SGC by puncturing the basal or lateral membrane. Inside the SGC, CS protein synthesis is turned on again; the Golgi system, nuclear envelope and all 3 pellicular membranes contain CS immunoreactivity. In the last phase of maturation, micronemes display abundant CS immunoreactivity. Rhoptries also show some immunogold labeling, but not as much as the micronemes.     

An insight into the lignin peroxidase of Macrophomina phaseolina

Macrophomina phaseolina is one of the deadliest necrotrophic fungal pathogens that infect more than 500 plant species including major food, fiber, and oil crops all throughout the globe. It secretes a cocktail of ligninolytic enzymes along with other hydrolytic enzymes for degrading the woody lignocellulosic plant cell wall and penetrating into the host tissue. Among them, lignin peroxidase has been reported only in Phanerochaete chrysosporium so far. But interestingly, a recent study has revealed a second occurrence of lignin peroxidase in M. phaseolina. However, lignin peroxidases are of much significance biotechnologically because of their potential applications in bio-remedial waste treatment and in catalyzing difficult chemical transformations. Besides, this enzyme also possesses agricultural and environmental importance on account of their role in lignin biodegradation. In the present work, different properties of the lignin peroxidase of M. phaseolina along with predicting the 3-D structure and its active sites were investigated by the use of various computational tools. The data from this study will pave the way for more detailed exploration of this enzyme in wet lab and thereby facilitating the strategies to be designed against such deadly weapons of Macrophomina phaseolina. Furthermore, the insight of such a ligninolytic enzyme will contribute to the assessment of its potentiality as a bioremediation tool.     

The Role of Oxidative Stress in the Longevity and Insecticide Resistance Phenotype of the Major Malaria Vectors Anopheles arabiensis and Anopheles funestus

Oxidative stress plays numerous biological roles, both functional and pathological. The role of oxidative stress in various epidemiologically relevant biological traits in Anopheles mosquitoes is not well established. In this study, the effects of oxidative stress on the longevity and insecticide resistance phenotype in the major malaria vector species An. arabiensis and An. funestus were examined. Responses to dietary copper sulphate and hydrogen peroxide were used as proxies for the oxidative stress phenotype by determining the effect of copper on longevity and hydrogen peroxide lethal dose. Glutathione peroxidase and catalase activities were determined colorimetrically. Oxidative burden was quantified as protein carbonyl content. Changes in insecticide resistance phenotype were monitored by WHO bioassay. Insecticide resistant individuals showed an increased capacity for coping with oxidative stress, mediated by increased glutathione peroxidase and catalase activity. This effect was observed in both species, as well as in laboratory strains and F₁ individuals derived from wild-caught An. funestus mothers. Phenotypic capacity for coping with oxidative stress was greatest in strains with elevated Cytochrome P450 activity. Synergism of oxidative stress defence enzymes by dietary supplementation with haematin, 3-Amino-1, 2, 4-triazole and Sodium diethyldithiocarbamate significantly increased pyrethroid-induced mortality in An. arabiensis and An. funestus. It is therefore concluded that defence against oxidative stress underlies the augmentation of the insecticide resistance phenotype associated with multiple blood-feeding. This is because multiple blood-feeding ultimately leads to a reduction of oxidative stress in insecticide resistant females, and also reduces the oxidative burden induced by DDT and pyrethroids, by inducing increased glutathione peroxidase activity. This study highlights the importance of oxidative stress in the longevity and insecticide resistance phenotype in malaria vectors.     

Cryo-EM structure of the DNA-dependent protein kinase catalytic subunit at subnanometer resolution reveals alpha helices and insight into DNA binding

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) regulates the nonhomologous end joining pathway for repair of double-stranded DNA (dsDNA) breaks. Here, we present a 7A resolution structure of DNA-PKcs determined by cryo-electron microscopy single-particle reconstruction. This structure is composed of density rods throughout the molecule that are indicative of alpha helices and reveals structural features not observed in lower resolution EM structures. Docking of homology models into the DNA-PKcs structure demonstrates that up to eight helical HEAT repeat motifs fit well within the density. Surprisingly, models for the kinase domain can be docked into either the crown or base of the molecule at this resolution, although real space refinement suggests that the base location is the best fit. We propose a model for the interaction of DNA with DNA-PKcs in which one turn of dsDNA enters the central channel and interacts with a resolved alpha-helical protrusion.     

Exploring mechanisms of multiple insecticide resistance in a population of the malaria vector Anopheles funestus in Benin

Background

The insecticide resistance status of the malaria vector Anopheles funestus and the underlying resistance mechanisms remain uncharacterised in many parts of Africa, notably in Benin, West Africa. To fill this gap in our knowledge, we assessed the susceptibility status of a population of this species in Pahou, Southern Benin and investigated the potential resistance mechanisms.

Methodology/Principal Findings

WHO bioassays revealed a multiple resistance profile for An. funestus in Pahou. This population is highly resistant to DDT with no mortality in females after 1h exposure to 4%DDT. Resistance was observed against the Type I pyrethroid permethrin and the carbamate bendiocarb. A moderate resistance was detected against deltamethrin (type II pyrethroids). A total susceptibility was observed against malathion, an organophosphate. Pre-exposure to PBO did not change the mortality rates for DDT indicating that cytochrome P450s play no role in DDT resistance in Pahou. No L1014F kdr mutation was detected but a correlation between haplotypes of two fragments of the Voltage-Gated Sodium Channel gene and resistance was observed suggesting that mutations in other exons may confer the knockdown resistance in this species. Biochemical assays revealed elevated levels of GSTs and cytochrome mono-oxygenases in Pahou. No G119S mutation and no altered acetylcholinesterase gene were detected in the Pahou population. qPCR analysis of five detoxification genes revealed that the GSTe2 is associated to the DDT resistance in this population with a significantly higher expression in DDT resistant samples. A significant over-expression of CYP6P9a and CYP6P9b previously associated with pyrethroid resistance was also seen but at a lower fold change than in southern Africa.

Conclusion

The multiple insecticide resistance profile of this An. funestus population in Benin shows that more attention should be paid to this important malaria vector for the implementation and management of current and future malaria vector control programs in this country.