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.     

Genome-wide identification of major protein families of cyanobacteria and genomic insight into the circadian rhythm

Cyanobacteria are among the most abundant organisms present on earth and are considered to be one of the oldest known clades. Cyanobacteria are oxygenic photosynthetic bacteria and are well known as promising renewable sources of energy; therefore, it is important to understand aspects of their genomes in detail across species. Advances in sequencing technology and the availability of several cyanobacterial genomes have provided an excellent opportunity to understand the diversity and evolution of the cyanobacterial genome. Here, we compared the genomes of 62 different phototrophic cyanobacteria. Evaluation of genetic diversity of all the cyanobacteria species studied revealed that evolution from their common ancestors was polyphyletic. In addition, the genomes were very diverse and varied among species, and significant genomic diversity was observed at the species and strain level. Overall, we identified 56 different protein families of cyanobacteria species/strains and found that they varied significantly among strains of a species. The circadian clock proteins KaiA, KaiB and KaiC (KaiABC complex proteins) of cyanobacteria were found to be present and consistent in the majority of cyanobacterial species while absent in a few others. Evolutionary analysis of the KaiABC protein complex showed that the KaiA protein has a high frequency of polymorphism, and multiple alleles were found to be present at high frequency. These results demonstrated that evolution of phosphorylation events occurred via KaiA in the KaiABC complex. Furthermore, multiple sequence alignment showed that KaiA, KaiB and KaiC proteins are highly conserved in nature. Our results provide direct information regarding the presence of different protein or protein families in cyanobacteria. The information presented here will serve as a starting point to explore the genetic diversity of cyanobacteria with the potential to play important roles in biotechnological applications.     

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.     

Chromosomal evidence of incipient speciation in the Afrotropical malaria mosquito Anopheles funestus

The analysis of chromosomal polymorphism of paracentric inversions in anopheline mosquitoes has often been instrumental to the discovery of sibling species complexes and intraspecific genetic heterogeneities associated with incipient speciation processes. To investigate the population structure of Anopheles funestus Giles (Diptera: Culicidae), one of the three most important vectors of human malaria in sub-Saharan Africa, a three-year survey of chromosomal polymorphism was carried out on 4,638 karyotyped females collected indoors and outdoors from two villages of central Burkina Faso. Large and temporally stable departures from Hardy-Weinberg equilibrium due to significant deficits of heterokaryotypes were found irrespective of the place of capture, and of the spatial and temporal units chosen for the analysis. Significant linkage disequilibrium was observed among inversion systems on independently assorting chromosomal arms, indicating the existence of assortative mating phenomena. Results were consistent with the existence of two chromosomal forms characterized by contrasting degrees of inversion polymorphism maintained by limitations to gene flow. This hypothesis was supported by the reestablishment of Hardy-Weinberg and linkage equilibria when individual specimens were assigned to each chromosomal form according to two different algorithms. This pattern of chromosomal variability is suggestive of an incipient speciation process in An. funestus populations from Burkina Faso.     

Non-coding RNA gene families in the genomes of anopheline mosquitoes

Background

Only a small fraction of the mosquito species of the genus Anopheles are able to transmit malaria, one of the biggest killer diseases of poverty, which is mostly prevalent in the tropics. This diversity has genetic, yet unknown, causes. In a further attempt to contribute to the elucidation of these variances, the international “Anopheles Genomes Cluster Consortium” project (a.k.a. “16 Anopheles genomes project”) was established, aiming at a comprehensive genomic analysis of several anopheline species, most of which are malaria vectors. In the frame of the international consortium carrying out this project our team studied the genes encoding families of non-coding RNAs (ncRNAs), concentrating on four classes: microRNA (miRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), and in particular small nucleolar RNA (snoRNA) and, finally, transfer RNA (tRNA).

Results

Our analysis was carried out using, exclusively, computational approaches, and evaluating both the primary NGS reads as well as the respective genome assemblies produced by the consortium and stored in VectorBase; moreover, the results of RNAseq surveys in cases in which these were available and meaningful were also accessed in order to obtain supplementary data, as were “pre-genomic era” sequence data stored in nucleic acid databases. The investigation included the identification and analysis, in most species studied, of ncRNA genes belonging to several families, as well as the analysis of the evolutionary relations of some of those genes in cross-comparisons to other members of the genus Anopheles.

Conclusions

Our study led to the identification of members of these gene families in the majority of twenty different anopheline taxa. A set of tools for the study of the evolution and molecular biology of important disease vectors has, thus, been obtained.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1038) contains supplementary material, which is available to authorized users.     

Isolation and characterization of microsatellite DNA markers in the malaria vector Anopheles funestus

Screening of the Anopheles funestus genomic DNA library detected 18 new sequences with dinucleotide tandem repeats. Primers were designed to amplify the loci and 14 out of 18 gave a repeatable and scorable amplification. Deviations from Hardy–Weinberg expectations were tested for each locus in a sample of 30 wild Anopheles funestus females. No heterozygote deficiency was detected for 11 loci of 14, thus revealing the absence of null alleles. The number of alleles per locus ranged from 5 to 15, and observed heterozygosity from 0.13 to 0.85.     

Laboratory selection for and characteristics of pyrethroid resistance in the malaria vector Anopheles funestus

A laboratory colony of Anopheles funestus Giles (Diptera: Culicidae) was established in 2000 from material collected from southern Mozambique where pyrethroid resistance had been demonstrated in the wild population. A subsample of the colony was selected for pyrethroid resistance using 0.1% lambda-cyhalothrin. Bioassay susceptibility tests in subsequent generations F(2) to F(4) showed increased resistance with each successive generation. Survival of individual mosquitoes fed only on 10% sugar solution, increased with age up to 4 days, but by day 10 had decreased significantly. However, females that had been mated and given bloodmeals showed no such increase in mortality with age. Biochemical analysis of resistant and susceptible individuals showed increased monooxygenase and glutathione S-transferase activity but no significant correlation with age of the mosquitoes.     

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.     

曲霉高效基因编辑技术研究进展

曲霉Aspergillus spp.是自然界中分布极为广泛的一大类真菌,在工业上如黑曲霉等已经被人们广泛利用于食品添加剂等生产。因此,曲霉在工业、农业、医药领域均发挥着重要的作用。然而,有些曲霉如黄曲霉能够分泌致癌物黄曲霉毒素从而污染农产品;在临床上还有一类以烟曲霉为主的引起侵袭性真菌感染的条件致病曲霉菌。因此,曲霉就像一把双刃剑影响着人们的生活。曲霉菌丝具有发达的隔膜形成多细胞菌丝体并能在分生孢子梗上产生大量的分生孢子进行无性繁殖。研究曲霉的基因编辑技术对于控制医学和农业上有害曲霉的增殖和促进工业上有益曲霉的生长和代谢都具有非常重要的意义。长期以来,同源重组和随机整合一直是被用于研究曲霉基因功能的传统基因编辑方法,然而其操作费时费力且效率低、难以达到预期的要求。随着第三代基因编辑技术CRISPR/Cas9即成簇的规律间隔的短回文重复序列及其相关系统CRISPR/Cas9（Clustered regulatory interspaced short palindromic repeats-associated protein 9）建立以来,CRISPR/Cas9以编辑高效、操作简便等优势被广泛应用到不同物种中。目前,世界上多个研究团队已经建立了有效地用于不同曲霉物种的CRISPR-Cas9 基因编辑体系。本文概述了有关曲霉基因编辑的历史和进展,以期为尚未建立完整遗传编辑体系的其他曲霉物种或者丝状真菌引入高效CRISPR-Cas9体系提供帮助。     

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.     

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.