RNAi Trigger Delivery into Anopheles gambiae Pupae

Citation: Regna, K., Harrison, R. M., Heyse, S. A., Chiles, T. C., Michel, K., & Muskavitch, M. A. T. (2016). RNAi Trigger Delivery into Anopheles gambiae Pupae. Jove-Journal of Visualized Experiments(109), 9. doi:10.3791/53738RNA interference (RNAi), a naturally occurring phenomenon in eukaryotic organisms, is an extremely valuable tool that can be utilized in the laboratory for functional genomic studies. The ability to knockdown individual genes selectively via this reverse genetic technique has allowed many researchers to rapidly uncover the biological roles of numerous genes within many organisms, by evaluation of loss-of-function phenotypes. In the major human malaria vector Anopheles gambiae, the predominant method used to reduce the function of targeted genes involves injection of double-stranded (dsRNA) into the hemocoel of the adult mosquito. While this method has been successful, gene knockdown in adults excludes the functional assessment of genes that are expressed and potentially play roles during pre-adult stages, as well as genes that are expressed in limited numbers of cells in adult mosquitoes. We describe a method for the injection of Serine Protease Inhibitor 2 (SRPN2) dsRNA during the early pupal stage and validate SRPN2 protein knockdown by observing decreased target protein levels and the formation of melanotic pseudo-tumors in SRPN2 knockdown adult mosquitoes. This evident phenotype has been described previously for adult stage knockdown of SRPN2 function, and we have recapitulated this adult phenotype by SRPN2 knockdown initiated during pupal development. When used in conjunction with a dye-labeled dsRNA solution, this technique enables easy visualization by simple light microscopy of injection quality and distribution of dsRNA in the hemocoel

Plasmodium falciparum: linkage disequilibrium between loci in chromosomes 7 and 5 and chloroquine selective pressure in Northern Nigeria.

In view of the recent discovery (Molecular Cell 6, 861-871) of a (Lys76Thr) codon change in gene pfcrt on chromosome 7 which determines in vitro chloroquine resistance in Plasmodium falciparum, we have re-examined samples taken before treatment in our study in Zaria, Northern Nigeria (Parasitology, 119, 343-348). Drug resistance was present in 5/5 cases where the pfcrt 76Thr codon change was seen (100% positive predictive value). Drug sensitivity was found in 26/28 cases where the change was absent (93% negative predictive value). Allele pfcrt 76Thr showed strong linkage disequilibrium with pfmdr1 Tyr86 on chromosome 5, more complete than that between pfcrt and cg2 alleles situated between recombination cross-over points on chromosome 7. Physical linkage of cg2 with pfcrt may account for linkage disequilibrium between their alleles but in the case of genes pfmdr1 and pfcrt, on different chromosomes, it is likely that this is maintained epistatically through the selective pressure of chloroquine

The Evolution of the Anopheles 16 Genomes Project

We report the imminent completion of a set of reference genome assemblies for 16 species of Anopheles mosquitoes. In addition to providing a generally useful resource for comparative genomic analyses, these genome sequences will greatly facilitate exploration of the capacity exhibited by some Anopheline mosquito species to serve as vectors for malaria parasites. A community analysis project will commence soon to perform a thorough comparative genomic investigation of these newly sequenced genomes. Completion of this project via the use of short next-generation sequence reads required innovation in both the bioinformatic and laboratory realms, and the resulting knowledge gained could prove useful for genome sequencing projects targeting other unconventional genomes

Evolution of response dynamics underlying bacterial chemotaxis

© 2011 Soyer and Goldstein; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background: The ability to predict the function and structure of complex molecular mechanisms underlying cellular behaviour is one of the main aims of systems biology. To achieve it, we need to understand the evolutionary routes leading to a specific response dynamics that can underlie a given function and how biophysical and environmental factors affect which route is taken. Here, we apply such an evolutionary approach to the bacterial chemotaxis pathway, which is documented to display considerable complexity and diversity.Results: We construct evolutionarily accessible response dynamics starting from a linear response to absolute levels of attractant, to those observed in current-day Escherichia coli. We explicitly consider bacterial movement as a two-state process composed of non-instantaneous tumbling and swimming modes. We find that a linear response to attractant results in significant chemotaxis when sensitivity to attractant is low and when time spent tumbling is large. More importantly, such linear response is optimal in a regime where signalling has low sensitivity. As sensitivity increases, an adaptive response as seen in Escherichia coli becomes optimal and leads to 'perfect' chemotaxis with a low tumbling time. We find that as tumbling time decreases and sensitivity increases, there exist a parameter regime where the chemotaxis performance of the linear and adaptive responses overlap, suggesting that evolution of chemotaxis responses might provide an example for the principle of functional change in structural continuity.Conclusions: Our findings explain several results from diverse bacteria and lead to testable predictions regarding chemotaxis responses evolved in bacteria living under different biophysical constraints and with specific motility machinery. Further, they shed light on the potential evolutionary paths for the evolution of complex behaviours from simpler ones in incremental fashion

Sequencing of Culex quinquefasciatus establishes a platform for mosquito comparative genomics

Culex quinquefasciatus (the southern house mosquito) is an important mosquito vector of viruses such as West Nile virus and St. Louis encephalitis virus, as well as of nematodes that cause lymphatic filariasis. C. quinquefasciatus is one species within the Culex pipiens species complex and can be found throughout tropical and temperate climates of the world. The ability of C. quinquefasciatus to take blood meals from birds, livestock, and humans contributes to its ability to vector pathogens between species. Here, we describe the genomic sequence of C. quinquefasciatus: Its repertoire of 18,883 protein-coding genes is 22% larger than that of Aedes aegypti and 52% larger than that of Anopheles gambiae with multiple gene-family expansions, including olfactory and gustatory receptors, salivary gland genes, and genes associated with xenobiotic detoxification

RecG directs DNA synthesis during double-strand break repair

Homologous recombination provides a mechanism of DNA double-strand break repair (DSBR) that requires an intact, homologous template for DNA synthesis. When DNA synthesis associated with DSBR is convergent, the broken DNA strands are replaced and repair is accurate. However, if divergent DNA synthesis is established, over-replication of flanking DNA may occur with deleterious consequences. The RecG protein of Escherichia coli is a helicase and translocase that can re-model 3-way and 4-way DNA structures such as replication forks and Holliday junctions. However, the primary role of RecG in live cells has remained elusive. Here we show that, in the absence of RecG, attempted DSBR is accompanied by divergent DNA replication at the site of an induced chromosomal DNA double-strand break. Furthermore, DNA double-stand ends are generated in a recG mutant at sites known to block replication forks. These double-strand ends, also trigger DSBR and the divergent DNA replication characteristic of this mutant, which can explain over-replication of the terminus region of the chromosome. The loss of DNA associated with unwinding joint molecules previously observed in the absence of RuvAB and RecG, is suppressed by a helicase deficient PriA mutation (priA300), arguing that the action of RecG ensures that PriA is bound correctly on D-loops to direct DNA replication rather than to unwind joint molecules. This has led us to put forward a revised model of homologous recombination in which the re-modelling of branched intermediates by RecG plays a fundamental role in directing DNA synthesis and thus maintaining genomic stability

Long non-coding RNA discovery across the genus anopheles reveals conserved secondary structures within and beyond the Gambiae complex

Long non-coding RNAs (lncRNAs) have been defined as mRNA-like transcripts longer than 200 nucleotides that lack significant protein-coding potential, and many of them constitute scaffolds for ribonucleoprotein complexes with critical roles in epigenetic regulation. Various lncRNAs have been implicated in the modulation of chromatin structure, transcriptional and post-transcriptional gene regulation, and regulation of genomic stability in mammals, Caenorhabditis elegans, and Drosophila melanogaster. The purpose of this study is to identify the lncRNA landscape in the malaria vector An. gambiae and assess the evolutionary conservation of lncRNAs and their secondary structures across the Anopheles genus

Breakdown in the process of incipient speciation in Anopheles gambiae.

Understanding genetic causes and effects of speciation in sympatric populations of sexually reproducing eukaryotes is challenging, controversial, and of practical importance for controlling rapidly evolving pests and pathogens. The major African malaria vector mosquito Anopheles gambiae sensu stricto (s.s.) is considered to contain two incipient species with strong reproductive isolation, hybrids between the M and S molecular forms being very rare. Following recent observations of higher proportions of hybrid forms at a few sites in West Africa, we conducted new surveys of 12 sites in four contiguous countries (The Gambia, Senegal, Guinea-Bissau, and Republic of Guinea). Identification and genotyping of 3499 A. gambiae s.s. revealed high frequencies of M/S hybrid forms at each site, ranging from 5 to 42%, and a large spectrum of inbreeding coefficient values from 0.11 to 0.76, spanning most of the range expected between the alternative extremes of panmixia and assortative mating. Year-round sampling over 2 years at one of the sites in The Gambia showed that M/S hybrid forms had similar relative frequencies throughout periods of marked seasonal variation in mosquito breeding and abundance. Genome-wide scans with an Affymetrix high-density single-nucleotide polymorphism (SNP) microarray enabled replicate comparisons of pools of different molecular forms, in three separate populations. These showed strong differentiation between M and S forms only in the pericentromeric region of the X chromosome that contains the molecular form-specific marker locus, with only a few other loci showing minor differences. In the X chromosome, the M/S hybrid forms were more differentiated from M than from S forms, supporting a hypothesis of asymmetric introgression and backcrossing