scholarly journals CRISPR-mediated knock-in of transgenes into the malaria vector Anopheles funestus

2021 ◽  
Author(s):  
Charlotte Quinn ◽  
Amalia Anthousi ◽  
Charles Wondji ◽  
Tony Nolan
Keyword(s):  
Mosquito Species ◽  
Anopheles Funestus ◽  
Current Control ◽  
Sub Saharan Africa ◽  
Marker Genes ◽  
End Joining ◽  
Genetic Traits ◽  
Homology Directed Repair ◽  
Sub Saharan ◽  
Gene Drives

Abstract The ability to introduce mutations, or transgenes, of choice to precise genomic locations has revolutionized our ability to understand how genes and organisms work. In many mosquito species that are vectors of various human diseases, the advent of CRISPR genome editing tools has shed light on basic aspects of their biology that are relevant to their efficiency as disease vectors. This allows a better understanding of how current control tools work and opens up the possibility of novel genetic control approaches, such as gene drives, that deliberately introduce genetic traits into populations. Yet for the Anopheles funestus mosquito, a significant vector of malaria in sub-Saharan Africa and indeed the dominant vector species in many countries, transgenesis has yet to be achieved. We describe herein an optimized transformation system based on the germline delivery of CRISPR components that allows efficient cleavage of a previously validated genomic site and preferential repair of these cut sites via homology-directed repair (HDR), which allows the introduction of exogenous template sequence, rather than end-joining repair. The rates of transformation achieved are sufficiently high that it should be able to introduce alleles of choice to a target locus, and recover these, without the need to include additional dominant marker genes. Moreover, the high rates of HDR observed suggest that gene drives, which employ an HDR-type mechanism to ensure their proliferation in the genome, may be well suited to work in A. funestus.

scholarly journals CRISPR-mediated knock-in of transgenes into the malaria vector Anopheles funestus

2021 ◽  
Author(s):  
Charlotte Quinn ◽  
Amalia Anthousi ◽  
Charles Wondji ◽  
Tony Nolan
Keyword(s):  
Mosquito Species ◽  
Anopheles Funestus ◽  
Current Control ◽  
Sub Saharan Africa ◽  
Marker Genes ◽  
End Joining ◽  
Genetic Traits ◽  
Homology Directed Repair ◽  
Sub Saharan ◽  
Gene Drives

ABSTRACTThe ability to introduce mutations, or transgenes, of choice to precise genomic locations has revolutionised our ability to understand how genes and organisms work.In many mosquito species that are vectors of various human disease, the advent of CRISPR genome editing tools has shed light on basic aspects of their biology that are relevant to their efficiency as disease vectors. This allows a better understanding of how current control tools work and opens up the possibility of novel genetic control approaches, such as gene drives, that deliberately introduce genetic traits into populations. Yet for the Anopheles funestus mosquito, a significant vector of malaria in sub-Saharan Africa and indeed the dominant vector species in many countries, transgenesis has yet to be achieved.We describe herein an optimised transformation system based on the germline delivery of CRISPR components that allows efficient cleavage of a previously validated genomic site and preferential repair of these cut sites via homology-directed repair (HDR), which allows introduction of exogenous template sequence, rather than end-joining repair. The rates of transformation achieved are sufficiently high that it should be able to introduce alleles of choice to a target locus, and recover these, without the need to include additional dominant marker genes. Moreover, the high rates of HDR observed suggest that gene drives, which employ an HDR-type mechanism to ensure their proliferation in the genome, may be well suited to work in An. funestus.

scholarly journals The dominant malaria vector, Anopheles funestus from rural south-eastern Tanzania, is more strongly resistant to insecticides than Anopheles arabiensis

2020 ◽  
Author(s):  
Polius Gerazi Pinda ◽  
Claudia Eichenberger ◽  
Halfan S Ngowo ◽  
Dickson S Msaky ◽  
Said Abbasi ◽  
...  
Keyword(s):  
Malaria Transmission ◽  
Pyrethroid Resistance ◽  
Mosquito Species ◽  
Anopheles Funestus ◽  
Indoor Residual Spraying ◽  
Sub Saharan Africa ◽  
Malaria Vectors ◽  
Insecticide Treated Nets ◽  
South Eastern ◽  
Sub Saharan

Abstract Background: Long-lasting insecticide-treated nets (LLINs) and indoor residual spraying (IRS) have greatly reduced malaria transmission in sub-Saharan Africa, but are threatened by insecticide resistance in dominant malaria vectors. In south-eastern Tanzania, pyrethroid-resistant Anopheles funestus now transmit more than 80% of malaria infections even in villages where the species occurs at far lower densities than other vectors such as Anopheles arabiensis.Methods: To better understand the dominance of An. funestus in these settings and improve options for its control, this study compared intensities of resistance between females of this species and those of An. arabiensis , using WHO assays with 1×, 5× and 10× insecticide doses. Additional tests were done to assess the reversibility of such resistance using synergists. The mosquitoes were collected from villages across two districts in south-eastern Tanzania.Findings: Both species were resistant to the two pyrethroids (permethrin and deltamethrin) and the organochloride (DDT) but susceptible to the organophosphate (pirimiphos-methyl) at standard baseline doses (1×). However, An. funestus as opposed to An. arabiensis was also resistant to the carbamate (bendiocarb) at standard doses (1×). An. funestus showed strong resistance to pyrethroids, surviving the 5× doses and 10× doses except in one village. Pre-exposure to the synergist, piperonyl butoxide (PBO), reversed the pyrethroid-resistance in both An. arabiensis and An. funestus achieving mortalities >98%, except for An. funestus from two villages for which permethrin-associated mortalities exceeded 90% but not 98%.Conclusions : In these communities where An. funestus now dominates malaria transmission, the species also displays much stronger resistance to pyrethroids than its counterpart, An. arabiensis, and can readily survive more classes of insecticides, including carbamates. The resistance to pyrethroids in both mosquito species appears to be mostly metabolic and can be reversed significantly using synergists such as PBO. These findings may explain the continued persistence and dominance of An. funestus despite widespread use of pyrethroid-treated LLINs, and will also inform future choices of interventions to tackle malaria transmission in this area and other similar settings. Such interventions may include PBO-based LLINs or improved IRS with compounds such as organophosphates against which the vectors are still susceptible.

scholarly journals An increasing role of pyrethroid-resistant Anopheles funestus in malaria transmission in the Lake Zone, Tanzania

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Nancy S. Matowo ◽  
Jackline Martin ◽  
Manisha A. Kulkarni ◽  
Jacklin F. Mosha ◽  
Eliud Lukole ◽  
...  
Keyword(s):  
Malaria Transmission ◽  
Anopheles Funestus ◽  
Sub Saharan Africa ◽  
North West ◽  
Vector Population ◽  
Lake Zone ◽  
Sub Saharan ◽  
Long Lasting Insecticidal Nets ◽  
Study District

AbstractAnopheles funestus is playing an increasing role in malaria transmission in parts of sub-Saharan Africa, where An. gambiae s.s. has been effectively controlled by long-lasting insecticidal nets. We investigated vector population bionomics, insecticide resistance and malaria transmission dynamics in 86 study clusters in North-West Tanzania. An. funestus s.l. represented 94.5% (4740/5016) of all vectors and was responsible for the majority of malaria transmission (96.5%), with a sporozoite rate of 3.4% and average monthly entomological inoculation rate (EIR) of 4.57 per house. Micro-geographical heterogeneity in species composition, abundance and transmission was observed across the study district in relation to key ecological differences between northern and southern clusters, with significantly higher densities, proportions and EIR of An. funestus s.l. collected from the South. An. gambiae s.l. (5.5%) density, principally An. arabiensis (81.1%) and An. gambiae s.s. (18.9%), was much lower and closely correlated with seasonal rainfall. Both An. funestus s.l. and An. gambiae s.l. were similarly resistant to alpha-cypermethrin and permethrin. Overexpression of CYP9K1, CYP6P3, CYP6P4 and CYP6M2 and high L1014S-kdr mutation frequency were detected in An. gambiae s.s. populations. Study findings highlight the urgent need for novel vector control tools to tackle persistent malaria transmission in the Lake Region of Tanzania.

scholarly journals Molecular Diversity of Nematode Parasites in Afrotropical Reed Frogs (Hyperolius spp.)

Diversity ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 265
Author(s):  
Ulrich Sinsch ◽  
J. Maximilian Dehling ◽  
Patrick Scheid ◽  
Carsten Balczun
Keyword(s):  
Dna Sequences ◽  
Nematode Species ◽  
Sub Saharan Africa ◽  
Parasitic Nematodes ◽  
Marker Genes ◽  
Nematode Parasites ◽  
Diversity Assessment ◽  
Wide Range ◽  
Nematode Diversity ◽  
Sub Saharan

The diversity of nematodes infecting amphibians is understudied in tropical Africa and unknown in Rwanda. Diversity assessment is hampered by the fact that species descriptions refer mostly to morphological features that are unlinked to DNA sequences of marker genes available in public databases. In this paper, we explore the abundance and diversity of parasitic nematodes in reed frogs Hyperolius kivuensis (n = 115), H. parallelus (n = 45) and H. viridiflavus (n = 100) collected in Rwanda. Five nematode species were identified morphologically as Orneoascaris chrysanthemoides, O. schoutedeni, Gendria leberrei, Aplectana chamaeleonis and Rhabdias collaris. Corresponding DNA sequences of 18S and COI genes were determined and subsequently deposited in GenBank. Aplectana chamaeleonis showed the highest prevalence (8.7%), but O. chrysanthemoides the highest mean intensity of infection (6.0) and largest number (24) of individuals in H. kivuensis. To the best of our knowledge, all amphibian hosts are new records for these nematode species, which are known to infect a wide range of amphibian and reptile species. Our findings suggest that nematode diversity is probably lower than previously assumed due to low host specificity. As morphological species identification is often challenging, our data facilitate molecular identification of adult and specifically larval nematodes found in amphibians of Sub-Saharan Africa.

scholarly journals High-Throughput Genotyping of Common Chromosomal Inversions in the Afrotropical Malaria Mosquito Anopheles Funestus

Insects ◽  
2020 ◽  
Vol 11 (10) ◽  
pp. 693
Author(s):  
Martin Lukindu ◽  
R. Rebecca Love ◽  
Moussa W. Guelbeogo ◽  
Scott T. Small ◽  
Melissa T. Stephens ◽  
...  
Keyword(s):  
High Throughput ◽  
Anopheles Funestus ◽  
Sub Saharan Africa ◽  
Nucleotide Polymorphisms ◽  
Female Population ◽  
Ecotypic Differentiation ◽  
Chromosomal Inversions ◽  
Sub Saharan ◽  
Mechanistic Basis ◽  
Resting Behavior

Polymorphic chromosomal inversions have been implicated in local adaptation. In anopheline mosquitoes, inversions also contribute to epidemiologically relevant phenotypes such as resting behavior. Progress in understanding these phenotypes and their mechanistic basis has been hindered because the only available method for inversion genotyping relies on traditional cytogenetic karyotyping, a rate-limiting and technically difficult approach that is possible only for the fraction of the adult female population at the correct gonotrophic stage. Here, we focus on an understudied malaria vector of major importance in sub-Saharan Africa, Anopheles funestus. We ascertain and validate tag single nucleotide polymorphisms (SNPs) using high throughput molecular assays that allow rapid inversion genotyping of the three most common An. funestus inversions at scale, overcoming the cytogenetic karyotyping barrier. These same inversions are the only available markers for distinguishing two An. funestus ecotypes that differ in indoor resting behavior, Folonzo and Kiribina. Our new inversion genotyping tools will facilitate studies of ecotypic differentiation in An. funestus and provide a means to improve our understanding of the roles of Folonzo and Kiribina in malaria transmission.

scholarly journals Transmission potential of Rickettsia felis infection by Anopheles gambiae mosquitoes

2015 ◽  
Vol 112 (26) ◽  
pp. 8088-8093 ◽  
Author(s):  
Constentin Dieme ◽  
Yassina Bechah ◽  
Cristina Socolovschi ◽  
Gilles Audoly ◽  
Jean-Michel Berenger ◽  
...  
Keyword(s):  
Anopheles Gambiae ◽  
Mosquito Species ◽  
Unknown Origin ◽  
Sub Saharan Africa ◽  
Rickettsia Felis ◽  
Infected Blood ◽  
Transmission Potential ◽  
Membrane Feeding ◽  
High Prevalence ◽  
Sub Saharan

A growing number of recent reports have implicated Rickettsia felis as a human pathogen, paralleling the increasing detection of R. felis in arthropod hosts across the globe, primarily in fleas. Here Anopheles gambiae mosquitoes, the primary malarial vectors in sub-Saharan Africa, were fed with either blood meal infected with R. felis or infected cellular media administered in membrane feeding systems. In addition, a group of mosquitoes was fed on R. felis-infected BALB/c mice. The acquisition and persistence of R. felis in mosquitoes was demonstrated by quantitative PCR detection of the bacteria up to day 15 postinfection. R. felis was detected in mosquito feces up to day 14. Furthermore, R. felis was visualized by immunofluorescence in salivary glands, in and around the gut, and in the ovaries, although no vertical transmission was observed. R. felis was also found in the cotton used for sucrose feeding after the mosquitoes were fed infected blood. Natural bites from R. felis-infected An. gambiae were able to cause transient rickettsemias in mice, indicating that this mosquito species has the potential to be a vector of R. felis infection. This is particularly important given the recent report of high prevalence of R. felis infection in patients with “fever of unknown origin” in malaria-endemic areas.

scholarly journals Novel Wolbachia strains in Anopheles malaria vectors from Sub-Saharan Africa

2018 ◽  
Vol 3 ◽  
pp. 113 ◽  
Author(s):  
Claire L. Jeffries ◽  
Gena G. Lawrence ◽  
George Golovko ◽  
Mojca Kristan ◽  
James Orsborne ◽  
...  
Keyword(s):  
Control Strategies ◽  
Mosquito Species ◽  
Amplicon Sequencing ◽  
Pathogen Transmission ◽  
Sub Saharan Africa ◽  
Malaria Vectors ◽  
Endosymbiotic Bacterium ◽  
Prevalence Rates ◽  
Republic Of The Congo ◽  
Sub Saharan

Background: Wolbachia, a common insect endosymbiotic bacterium that can influence pathogen transmission and manipulate host reproduction, has historically been considered absent from the Anopheles (An.) genera, but has recently been found in An. gambiae s.l. populations.  As there are numerous Anopheles species that have the capacity to transmit malaria, we analysed a range of species to determine Wolbachia prevalence rates, characterise novel Wolbachia strains and determine any correlation between the presence of Plasmodium, Wolbachia and the competing endosymbiotic bacterium Asaia. Methods: Anopheles adult mosquitoes were collected from five malaria-endemic countries: Guinea, Democratic Republic of the Congo (DRC), Ghana, Uganda and Madagascar, between 2013 and 2017.  Molecular analysis of samples was undertaken using quantitative PCR, Sanger sequencing, Wolbachia multilocus sequence typing (MLST) and high-throughput amplicon sequencing of the bacterial 16S rRNA gene.  Results: Novel Wolbachia strains were discovered in five species: An. coluzzii, An. gambiae s.s., An. arabiensis, An. moucheti and An. species ‘A’, increasing the number of Anopheles species known to be naturally infected. Variable prevalence rates in different locations were observed and novel strains were phylogenetically diverse, clustering with Wolbachia supergroup B strains.  We also provide evidence for resident strain variants within An. species ‘A’.  Wolbachia is the dominant member of the microbiome in An. moucheti and An. species ‘A’, but present at lower densities in An. coluzzii.  Interestingly, no evidence of Wolbachia/Asaia co-infections was seen and Asaia infection densities were also shown to be variable and location dependent.  Conclusions: The important discovery of novel Wolbachia strains in Anopheles provides greater insight into the prevalence of resident Wolbachia strains in diverse malaria vectors.  Novel Wolbachia strains (particularly high-density strains) are ideal candidate strains for transinfection to create stable infections in other Anopheles mosquito species, which could be used for population replacement or suppression control strategies.

scholarly journals Increasing role of pyrethroid-resistant Anopheles funestus in malaria transmission in the Lake Zone, Tanzania: implications for the evaluation of novel vector control products

2021 ◽  
Author(s):  
Nancy S. Matowo ◽  
Jackline Martin ◽  
Manisha A. Kulkarni ◽  
Jacklin F. Mosha ◽  
Eliud Lukole ◽  
...  
Keyword(s):  
Vector Control ◽  
Malaria Transmission ◽  
Anopheles Funestus ◽  
Sub Saharan Africa ◽  
North West ◽  
Vector Population ◽  
Lake Zone ◽  
Sub Saharan ◽  
Study District

AbstractAnopheles funestus is playing an increasing role in malaria transmission in parts of sub-Saharan Africa, where An. gambiae s.s. has been effectively controlled by long-lasting insecticidal nets. We investigated vector population bionomics, insecticide resistance and malaria transmission dynamics in 86 study clusters in North-West Tanzania. An. funestus s.l. represented 94.5% (4740/5016) of all vectors and was responsible for the majority of malaria transmission (96.5%), with a sporozoite rate of 3.4% and average monthly entomological inoculation rate (EIR) of 4.57 per house. Micro-geographical heterogeneity in species composition, abundance and transmission was observed across the study district in relation to key ecological differences between northern and southern clusters, with significantly higher densities, proportions and EIR of An. funestus s.l. collected from the south. An. gambiae s.l. (5.5%) density, principally An. arabiensis (81.1%) and An. gambiae s.s. (18.9%), was much lower and closely correlated with seasonal rainfall. Both An. funestus s.l. and An. gambiae s.l. were similarly resistant to alpha-cypermethrin and permethrin. Overexpression of CYP9K1, CYP6P3, CYP6P4 and CYP6M2 and high L1014F-kdr mutation frequency were detected in An. gambiae s.s. populations. Study findings highlight the urgent need for novel vector control tools to tackle persistent malaria transmission in the Lake Region of Tanzania.

scholarly journals Novel Wolbachia strains in Anopheles malaria vectors from Sub-Saharan Africa

2018 ◽  
Vol 3 ◽  
pp. 113 ◽  
Author(s):  
Claire L. Jeffries ◽  
Gena G. Lawrence ◽  
George Golovko ◽  
Mojca Kristan ◽  
James Orsborne ◽  
...  
Keyword(s):  
Control Strategies ◽  
Mosquito Species ◽  
Amplicon Sequencing ◽  
Pathogen Transmission ◽  
Sub Saharan Africa ◽  
Malaria Vectors ◽  
Prevalence Rates ◽  
Population Replacement ◽  
Republic Of The Congo ◽  
Sub Saharan

Background: Wolbachia, a common insect endosymbiotic bacterium that can influence pathogen transmission and manipulate host reproduction, has historically been considered absent from the Anopheles (An.) genera, but has recently been found in An. gambiae s.l. populations in West Africa.  As there are numerous Anopheles species that have the capacity to transmit malaria, we analysed a range of species across five malaria endemic countries to determine Wolbachia prevalence rates, characterise novel Wolbachia strains and determine any correlation between the presence of Plasmodium, Wolbachia and the competing bacterium Asaia. Methods: Anopheles adult mosquitoes were collected from five malaria-endemic countries: Guinea, Democratic Republic of the Congo (DRC), Ghana, Uganda and Madagascar, between 2013 and 2017.  Molecular analysis was undertaken using quantitative PCR, Sanger sequencing, Wolbachia multilocus sequence typing (MLST) and high-throughput amplicon sequencing of the bacterial 16S rRNA gene.  Results: Novel Wolbachia strains were discovered in five species: An. coluzzii, An. gambiae s.s., An. arabiensis, An. moucheti and An. species A, increasing the number of Anopheles species known to be naturally infected. Variable prevalence rates in different locations were observed and novel strains were phylogenetically diverse, clustering with Wolbachia supergroup B strains.  We also provide evidence for resident strain variants within An. species A. Wolbachia is the dominant member of the microbiome in An. moucheti and An. species A but present at lower densities in An. coluzzii.  Interestingly, no evidence of Wolbachia/Asaia co-infections was seen and Asaia infection densities were shown to be variable and location dependent.  Conclusions: The important discovery of novel Wolbachia strains in Anopheles provides greater insight into the prevalence of resident Wolbachia strains in diverse malaria vectors.  Novel Wolbachia strains (particularly high-density strains) are ideal candidate strains for transinfection to create stable infections in other Anopheles mosquito species, which could be used for population replacement or suppression control strategies.

scholarly journals Evidence for natural hybridisation and novel Wolbachia strain superinfections in the Anopheles gambiae complex from Guinea

2019 ◽  
Author(s):  
Claire L Jeffries ◽  
Cintia Cansado-Utrilla ◽  
Abdoul H Beavogui ◽  
Caleb Stica ◽  
Eugene K Lama ◽  
...  
Keyword(s):  
Prevalence Rate ◽  
Mosquito Species ◽  
Wolbachia Strain ◽  
Pathogen Transmission ◽  
Sub Saharan Africa ◽  
Malaria Vectors ◽  
Prevalence Rates ◽  
Anopheles Gambiae Complex ◽  
Natural Hybridisation ◽  
Sub Saharan

1.SummaryWolbachia, a widespread bacterium which can influence mosquito-borne pathogen transmission, has recently been detected within Anopheles (An.) species that are malaria vectors in Sub-Saharan Africa. Although studies have reported Wolbachia strains in the An. gambiae complex, apparent low density and prevalence rates require confirmation. In this study, wild Anopheles mosquitoes collected from two regions of Guinea were investigated. In contrast to previous studies, RNA was extracted from adult females (n=516) to increase the chances for detection of actively expressed Wolbachia genes, determine Wolbachia prevalence rates and estimate relative strain densities. Molecular confirmation of mosquito species and Wolbachia Multilocus sequence typing (MLST) were carried out to analyse phylogenetic relationships of mosquito hosts and newly discovered Wolbachia strains. Strains were detected in An. gambiae s.s. (prevalence rates of 0.0-2.8%) from the Faranah region, An. melas (prevalence rate of 11.6% - 16/138) and hybrids between these two species (prevalence rate of 40.0% - 6/15) from Senguelen in the Maferinyah region. Furthermore, a novel high-density strain, termed wAnsX, was found in an unclassified Anopheles species. The discovery of novel Wolbachia strains (particularly in members, and hybrids, of the An. gambiae complex) provides further candidate strains that could be used for future Wolbachia-based malaria biocontrol strategies.

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