Whole-Genome Shotgun Sequencing and Assembly of Anopheles gambiae G3, the Host of Malaria Parasite Plasmodium sp.

The recent advances in sequencing throughput and genome assembly algorithms have established whole-genome shotgun (WGS) assemblies as the cornerstone of the genomic infrastructure for many species. WGS assemblies can be constructed with comparative ease and give a comprehensive representation of the gene space even of large and complex genomes. One major obstacle in utilizing WGS assemblies for important research applications such as gene isolation or comparative genomics has been the lack of chromosomal positioning and contextualization of short sequence contigs. Assigning chromosomal locations to sequence contigs required the construction and integration of genome-wide physical maps and dense genetic linkage maps as well as synteny to model species. Recently, methods to rapidly construct ultra-dense linkage maps encompassing millions of genetic markers from WGS sequencing data of segregating populations have made possible the direct assignment of genetic positions to short sequence contigs. Here, we review recent developments in the integration of WGS assemblies and sequence-based linkage maps, discuss challenges for further improvement of the methodology and outline possible applications building on genetically anchored WGS assemblies.

Annotation of an improved whole-genome shotgun assembly of the Drosophila melanogaster genome predicted 297 protein-coding genes and six non-protein-coding genes, including known heterochromatic genes, and regions of similarity to known transposable elements. Fluorescence in situ hybridization was used to correlate the genomic sequence with the cytogenetic map; the annotated euchromatic sequence extends into the centric heterochromatin on each chromosome arm.

BackgroundMalaria remains one of the main causes of morbidity and mortality in Cameroon. To inform vector control intervention decision making, malaria vector surveillance was conducted monthly from October 2018 to September 2020 in five selected sentinel sites (Gounougou and Simatou in the North, and Bonabéri, Mangoum and Nyabessang in the South).MethodsHuman landing catches (HLCs), U.S. Centers for Disease Control and Prevention (CDC) light traps, and pyrethrum spray catches (PSCs) were used to assess vector density, species composition, human biting rate (HBR), endophagic index, indoor resting density (IRD), parity, sporozoite infection rates, entomological inoculation rate (EIR), and Anopheles vectorial capacity.ResultsA total of 139,322 Anopheles mosquitoes from 18 species (or 21 including identified sub-species) were collected across all sites. Out of the 18 species, 12 were malaria vectors including Anopheles gambiae sensu lato (s.l.), Anopheles funestus s.l.., Anopheles nili, Anopheles moucheti, Anopheles paludis, Anopheles demeilloni, Anopheles. pharoensis, Anopheles ziemanni, Anopheles multicinctus, Anopheles tenebrosus, Anopheles rufipes, and Anopheles marshallii. Anopheles gambiae s.l. remains the major malaria vector (71% of the total Anopheles) collected, though An. moucheti and An. paludis had the highest sporozoite rates in Nyabessang. The mean indoor HBR of Anopheles ranged from 11.0 bites/human/night (b/h/n) in Bonabéri to 104.0 b/h/n in Simatou, while outdoors, it varied from 24.2 b/h/n in Mangoum to 98.7 b/h/n in Simatou. Anopheles gambiae s.l. and An. moucheti were actively biting until at least 8:00 a.m. The mean Anopheles IRD was 17.1 females/room, and the parity rate was 68.9%. The mean EIRs for each site were 55.4 infective bites/human/month (ib/h/m) in Gounougou, 99.0 ib/h/m in Simatou, 51.2 ib/h/m in Mangoum, 24.4 ib/h/m in Nyabessang, and 18.1 ib/h/m in Bonabéri. Anopheles gambiae s.l. was confirmed as the main malaria vector with the highest vectorial capacity in all sites based on sporozoite rate, except in Nyabessang.ConclusionThese findings highlight the high malaria transmission occurring in Cameroon and will support the National Malaria Control Program to design evidence-based malaria vector control strategies, and deployment of effective and integrated vector control interventions to reduce malaria transmission and burden in Cameroon, where several Anopheles species could potentially maintain year-round transmission.

Insecticide resistance in the main malaria vectors in Africa is a major concern for malaria vector control program managers. The most common insecticides used for indoor residual spraying (IRS) and treating bed nets are becoming increasingly ineffective. The quest for safer and more effective insecticides for malaria vector control is urgent. This study sought to evaluate the efficacy of ACTELLIC 50 EC (pirimiphos methyl), an organophosphate, for IRS in Ghana, where there is high vector resistance to pyrethroids and organochlorines. Before the commencement of the study, standard World Health Organization (WHO) vector susceptibility tests against a common malaria vector, Anopheles gambiae s.l, were conducted using preparations of pyrethroids, organochlorines, carbamates, and organophosphates. The vector was found to be resistant to the pyrethroids, the organochlorines, and the carbamates, but susceptible to the organophosphates. The emulsifiable organophosphate concentrate formulation, ACTELLIC 50 EC, was then evaluated to determine the efficacy and the length of its residual effect. The wall bioassay test, using recommended cones from WHO, was conducted on sprayed surfaces with ACTELLIC 50 EC from 27 July 2009 to 16 October 2009. After 15 wk of trials on painted cement surface, it was found out that the main malaria vector, An. gambiae s.l, was susceptible to the insecticide even though the WHO Pesticide Evaluation recommends 2- to 3-mo duration of effective action. Therefore, it is recommended for use in IRS programs in this part of Ghana, where there is high vector resistance to most of the insecticides.

Conifers are the predominant gymnosperm. The size and complexity of their genomes has presented formidable technical challenges for whole-genome shotgun sequencing and assembly. We employed novel strategies that allowed us to determine the loblolly pine (Pinus taeda) reference genome sequence, the largest genome assembled to date. Most of the sequence data were derived from whole-genome shotgun sequencing of a single megagametophyte, the haploid tissue of a single pine seed. Although that constrained the quantity of available DNA, the resulting haploid sequence data were well-suited for assembly. The haploid sequence was augmented with multiple linking long-fragment mate pair libraries from the parental diploid DNA. For the longest fragments, we used novel fosmid DiTag libraries. Sequences from the linking libraries that did not match the megagametophyte were identified and removed. Assembly of the sequence data were aided by condensing the enormous number of paired-end reads into a much smaller set of longer “super-reads,” rendering subsequent assembly with an overlap-based assembly algorithm computationally feasible. To further improve the contiguity and biological utility of the genome sequence, additional scaffolding methods utilizing independent genome and transcriptome assemblies were implemented. The combination of these strategies resulted in a draft genome sequence of 20.15 billion bases, with an N50 scaffold size of 66.9 kbp.

Identification and evaluation expression level of arrestin 1 gene during the development stage of Anopheles stephensi

Malaria remains one of the most critical disease causing morbidity and mortality in Uganda. Indoor residual spraying (IRS) and the use of insecticide-treated bed nets are currently the predominant malaria vector control interventions. However, the emergence and spread of insecticide resistance among malaria vectors threaten the continued effectiveness of these interventions to control the disease, particularly in high transmission areas. To inform decisions on vector control, the current study evaluated the Anopheles malaria vector species and their susceptibility levels to 0.1% bendiocarb and 0.25% pirimiphos-methyl insecticides used in IRS intervention program in Namutumba district, Eastern Uganda. Anopheles larvae were collected between March and May 2017 from different breeding sites in the parishes of Nsinze and Nawaikona in Nsinze sub-county and reared to adults to assess the susceptibility status of populations in the study area. Mosquitoes were identified using morphological keys and species-specific polymerase chain reaction (PCR) assays. Susceptibility tests were conducted on 2- to 5-day-old non-blood-fed adult female Anopheles that emerged using insecticide-impregnated papers with 0.1% bendiocarb and 0.25% pirimiphos-methyl following standard World Health Organization (WHO) insecticide susceptibility bioassays. A Log-probit regression model was used to derive the knock-down rates for 50% and 95% of exposed mosquitoes. A total of 700 mosquito larvae were collected from different breeding sites. Morphological identification showed that 500 individuals that emerged belonged to Anopheles gambiae sensu lato (s.l.), the main malaria vector. The PCR results showed that the dominant sibling species under the A. gambiae complex was Anopheles arabiensis 99.5% (395/397). WHO bioassay tests revealed that the population of mosquitoes exhibited high levels of susceptibility (24-hour post-exposure mortality 98-100%) to both insecticides tested. The median knock-down time, KDT50, ranged from 6.6 to 81.4 minutes, while the KDT95 ranged from 21.6 to 118.9 minutes for 0.25% pirimiphos-methyl. The KDT50 for 0.1% bendiocarb ranged from 2.8 to 62.9 minutes, whereas the KDT95 ranged from 36.0 to 88.5 minutes. These findings indicate that bendiocarb and pirimiphos-methyl are still effective against the major malaria vector, A. arabiensis in Nsinze sub-county, Namutumba district, Uganda and can be effectively used for IRS. The study has provided baseline information on the insecticide susceptibility status on malaria vectors in the study area. However, routine continuous monitoring program of insecticide susceptibility and malaria vector composition is required so as to guide future decisions on insecticide use for IRS intervention toward malaria elimination and to track future changes in vector population.

Anopheles gambiae Giles is commonly called the African malaria mosquito because it is the most efficient vector of human malaria in the Afrotropical Region. They are considered to be one of the world’s most important human malaria vectors because of their susceptibility to the Plasmodium parasite, their preference for humans as a host, and their indoor-feeding behavior. Due to their short development time and their preference for developmental habitats near human dwellings, Anopheles gambiae are considered effective vectors of human malaria, as well as lymphatic filariasis (elephantiasis). This 6-page fact sheet was written by Sabrina A. White and Phillip E. Kaufman, and published by the UF Department of Entomology and Nematology, September 2014.
 EENY601/IN1048: African Malaria Mosquito Anopheles gambiae Giles (Insecta: Diptera: Culicidae) (ufl.edu)

BackgroundWith the rapid spread of pyrethroid resistance in the main malaria vectors from Benin and the various resistance mechanisms involved (metabolic resistance and knock-down resistance (kdr), it is important to foresee effective resistance management strategies. Thus, the knowledge of the insensitive acetylcholinesterase (ace-1R) effects on phenotypes of An. gambiae will help us to strengthen basic and operational research on thedevelopment of strategies that will use organophosphates or carbamates as alternatives against pyrethroids-resistant malaria vectors in the field.Methods Larvae and pupae of Anopheles gambiae s.l. mosquitoes were collected from the breeding sites in Ouemé, Atacora, and Alibori departments. CDC susceptibility tests were conducted on unfed female mosquitoes aged 2–5 days old. CDC bioassays were performed with stock solutions of fenitrothion (50 μg per bottle) and bendiocarb (12.5 μg per bottle). PCR techniques were used to detect species and Ace-1 mutations.Results Anopheles gambiae Seme and Kandi populations were susceptible to fenitrothion whereas Anopheles gambiae Tanguieta and Malanville populations were resistant. An. gambiae populations from Seme, Kandi and Malanville were fully susceptible to bendiocarb whereas those from Tanguieta have developed a strong resistance to the same insecticide. A slight decrease in mortality rate was observed with 97.91% in populations of mosquitoes from Malanville. PCR revealed that all specimens tested were Anopheles gambiae s.s..The presence of Ace-1R at very low frequency (0.01) was observed in Anopheles gambiae Malanville populations.ConclusionThis study demonstrated the need to monitor organophosphate (OPs) and Carbamates resistance among populations of the An. gambiae s.l. in Benin, to determine its spread and anticipate vector control failure where these insecticides are used. However, further studies are needed to understand the current distribution of the Ace-1R mutation in other localities in the south-north transect Benin.

Anopheles gambiae, which is the main malaria vector in Benin has developed high level of resistance to pyrethroid insecticides. This raises serious concerns to the future use of long- lasting insecticidal nets (LLIN) and indoor residual spraying (IRS). It is therefore important to seek better and effective resistance management strategies which will use organophosphates or carbamates as alternatives against pyrethroid resistant malaria vectors in the field. Larvae and pupae of A. gambiae s.l. mosquitoes were collected from the breeding sites in Dassa-Zoume and Zogbodomey districts. WHO susceptibility tests were conducted on unfed female mosquitoes aged 2-5 days old. WHO bioassays were performed with impregnated papers with fenitrothion 1%, pirimiphos-methyl 0.25%, and bendiocarb 0.1%. Polymerase chain reaction (PCR) techniques were used to detect species and Ace-1 mutations. A. gambiae Dassa-Zoume populations were susceptible to bendiocarb 0.1% with mortality rate of 99%. A. gambiae Zogbodomey populations were susceptible to pirimiphos-methyl 0.25% and fenitrothion 1% with mortality rates of 98.96 and 99%, respectively. PCR assay revealed that 100% of mosquitoes tested were A. gambiae s.s. The frequencies of Ace-1R mutation in A. gambiae Dassa-Zoume and Zogbodomey were 0%. Carbamates (bendiocarb) and organophosphates (fenitrothion and pirimiphos-methyl) have maintained their efficiency against A. gambiae Dassa-Zoume and Zogbodomey populations. Carbamates (bendiocarb) and organophosphates (fenitrothion and pirimiphos-methyl) have proven to be powerful alternatives against pyrethroid resistant malaria vectors such as A. gambiae Dassa-Zoume and Zogbodomey populations. The use of any of these three compounds in the centre Benin would be successful in malaria vector control.   Key   words:   Anopheles gambiae,   Ace-1,   resistance,   fenitrothion,   pirimiphos-methyl, bendiocarb, Benin.

Chitinase is an important enzyme responsible for chitin metabolism in a wide range of organisms including bacteria, yeasts and other fungi, nematodes and arthropods. However, current knowledge on chitinolytic enzymes, especially their structures, functions and regulation is very limited. In this study we have identified 20 chitinase and chitinase-like genes in the African malaria mosquito, Anopheles gambiae, through genome-wide searching and transcript profiling. We assigned these genes into eight different chitinase groupings (groups I–VIII). Domain analysis of their predicted proteins showed that all contained at least one catalytic domain. However, only seven (AgCht4, AgCht5-1, AgCht6, AgCht7, AgCht8, AgCht10 and AgCht23) displayed one or more chitin-binding domains. Analyses of stage- and tissue-specific gene expression revealed that most of these genes were expressed in larval stages. However, AgCht8 was mainly expressed in the pupal and adult stages. AgCht2 and AgCht12 were specifically expressed in the foregut, whereas AgCht13 was only expressed in the midgut. The high diversity and complexity of An. gambiae chitinase and chitinase-like genes suggest their diverse functions during different developmental stages and in different tissues of the insect. A comparative genomic analysis of these genes along with those present in Drosophila melanogaster, Tribolium castaneum and several other insect species led to a uniform classification and nomenclature of these genes. Our investigation also provided important information for conducting future studies on the functions of chitinase and chitinase-like genes in this important malaria vector and other species of arthropods.

The main malaria vectors in sub-Saharan Africa, the Anopheles gambiae (Giles; Diptera: Culicidae), normally breed in clean water sources. However, evidence suggests an on-going adaptation of Anopheline species to polluted breeding habitats in urban settings. This study aimed at understanding the adaptation to breeding in water bodies with different qualities, in five selected mosquito breeding sites in urban Accra, Ghana. The study sites were also evaluated for the WHO water-quality parameters as a measure of pollution, and insecticide residues. Field mosquitoes were evaluated for five genes; CYP6P3, CYP4H19, CYP4H24, GSTD1-4, and ABCC11-associated with insecticide detoxification-using quantitative RT-PCR, as well as Mono-oxygenase, Alpha Esterase, Glutathione S-transferase, and insensitive acetylcholinesterase (AChE) using biochemical enzyme assays. The lab-reared, insecticide susceptible An. gambiae Kisumu strain was bred in the most polluted water source for 10 generations and evaluated for the same genes and enzymes. The results revealed that the fold expression of the genes was higher in the larvae compared with the adults. The results also suggest that detoxification enzymes could be involved in the adaptation of An. gambiae to polluted breeding sites. Correlation analysis revealed a highly positive significant correlation between calcium levels and all five genes (P < 0.05). Stepwise linear regression to understand which of the variables predicted the expression of the genes revealed that sulphate was responsible for ABCC11 and CYP4H24, alkalinity for GSTD1-4, and calcium for CYP4H19 and CYP6P3. The detailed genetic basis of this adaptation need to be further investigated. A further understanding of this adaptation may provide outlooks for controlling malaria and other disease vectors adapted to polluted breeding water sources.

In America, of the 44 species of Anopheles, nine are main vectors of malaria and, of these, genetic information exists for seven. Hence, this study sought to know the gene flow and diversity of the seven principal vectors of malaria at the Americas level. For the seven species and the sequences of the mitochondrial cytochrome c oxidase I (COI) gene obtained from the GenBank and Bold System, genetic analyzes of populations and genetic structure were performed and haplotype networks and phylogenetic trees were obtained. For the seven species, 1440 sequences were analyzed and 519 haplotypes were detected. The Hd and π values were higher within a continental context than by countries. Neutrality tests indicated positive and negative values with most of these being significant (p < 0.05). Phylogenetic analyses for all the species recovered three clades with no geographic pattern among them. Studies suggest that native species of Anopheles from the Americas have greater haplotype diversity and low genetic differentiation due to the lack of physical barriers to impede gene flow among these populations. Moreover, all the species are interconnected by roadways. This scenario complicates the epidemiological picture of malaria in the Americas.

Complex eukaryotic genomes are now being sequenced at an accelerated pace primarily using whole-genome shotgun (WGS) sequence assembly approaches. WGS assembly was initially criticized because of its perceived inability to resolve repeat structures within genomes. Here, we quantify the effect of WGS sequence assembly on large, highly similar repeats by comparison of the segmental duplication content of two different human genome assemblies. Our analysis shows that large (> 15 kilobases) and highly identical (> 97%) duplications are not adequately resolved by WGS assembly. This leads to significant reduction in genome length and the loss of genes embedded within duplications. Comparable analyses of mouse genome assemblies confirm that strict WGS sequence assembly will oversimplify our understanding of mammalian genome structure and evolution; a hybrid strategy using a targeted clone-by-clone approach to resolve duplications is proposed.

The use of long lasting insecticide nets (LLINs) treated with pyrethroïd is known for its major contribution in malaria control. However, LLINs are suspected to induce behavioral changes in malaria vectors, which may in turn drastically affect their efficacy against Plasmodium sp. transmission. In sub Saharan Africa, where malaria imposes the heaviest burden, the main malaria vectors are widely resistant to pyrethroïds, the insecticide family used on LLINs, which also threatens LLIN efficiency. There is therefore a crucial need for deciphering how insecticide-impregnated materials might affect the host-seeking behavior of malaria vectors in regards to insecticide resistance. In this study, we explored the impact of permethrin-impregnated net on the host attractiveness for Anopheles gambiae mosquitoes, either susceptible to insecticides, or carrying the insecticide resistance conferring allele kdr. Groups of female mosquitoes were released in a dual-choice olfactometer and their movements towards an attractive odor source (a rabbit) protected by insecticide-treated (ITN) or untreated nets (UTN) were monitored. Kdr homozygous mosquitoes, resistant to insecticides, were more attracted by a host behind an ITN than an UTN, while the presence of insecticide on the net did not affect the choice of susceptible mosquitoes. These results suggest that permethrin-impregnated net is detectable by malaria vectors and that the kdr mutation impacts their response to a LLIN protected host. We discuss the implication of these results for malaria vector control.