molecular and physiological analysis of Anopheles Funestus swarms in nchelenge zambia

Anopheles Funestus has been recognized as a major malaria vector in Africa for over 100 years, but knowledge on many aspects of the biology of this species is still lacking. Anopheles Funestus, as with most other anophelines, mate through swarming. A key event that is crucial for the An. Funestus male to mate is genitalia rotation. This involves the 135° to 180° rotation of claspers, which are tipped with claws. This physical change then enables the male to grasp the female during copulation. The aim of this investigation was to molecularly characterize wild An. Funestus swarms from Zambia and examine the degree of genitalia rotation within the swarm. Anopheles Funestus swarms were collected from Nchelenge, northern Zambia, during dusk periods in May 2016. All the adults from the swarm were analysed morphologically and identified to species level using a multiplex PCR assay. Anopheles Funestus s.s. specimens were molecularly characterized by restriction fragment length polymorphism type and Clade type assays. The different stages of genitalia rotation were examined in the adult males. A total of six swarms were observed during the study period and between 6 and 26 mosquitoes were caught from each swarm. Species analysis revealed that 90% of the males from the swarms were An. Funestus s.s. MW-type, with 84% belonging to clade I compared to 14% clade II and 2% failed to amplify. Very few specimens (3.4%) were identified as Anopheles gambiae s.s. Eighty percent of the males from the swarm had complete genitalia rotation. This is the first time that An. Funestus swarms have been molecularly identified to species level. Anopheles Funestus swarms appear to be species-specific with no evidence of clade-type differentiation within these swarms. The An. Funestus swarms consist mainly of males with fully rotated genitalia, which strongly suggests that swarming behaviour is triggered primarily when males have matured.

Application of hydrolysis probe analysis to identify clade types of the malaria vector mosquito Anopheles Funestus sensu stricto from Muheza, northeastern Tanzania

A hydrolysis probe analysis (TaqMan assay) was used to study clade types in Anopheles Funestus sensu stricto Giles, a major malaria vector in sub-Saharan Africa, with specimens collected from Muheza in Tanga, northeastern Tanzania. A total of 186 An. Funestus specimens were analysed, revealing that 176 (94.6%) were of clade I and 10 (5.4%) of clade II. These findings extend the distribution of clade type II from southern Mozambique and northern Zambia to northeastern Tanzania. The technique used can also be of great value in assessing the role and contribution of these clade types in malaria transmission and insecticide resistance frequencies for An. Funestus s.s.

characterization of the Anopheles Funestus group including Anopheles Funestus like from northern malawi

Background
Limited information is available on malaria vector composition, feeding habits and malaria transmission in northern Malawi. Evidence of mosquito species diversity in this area was established in 2009, when Anopheles Funestus-like, a new member of the An. Funestus group was described. Additional biological information is needed to identify this species and to understand its role in malaria transmission.

Methods
Anopheline mosquitoes were collected in northern Malawi and analyzed for Plasmodium species infection, blood meal source and susceptibility to insecticides. A new hydrolysis probe assay was designed to identify An. Funestus-like.

Results
Anopheles Funestus and An. rivulorum predominated in the indoor collections. Most An. Funestus-like were collected indoors, mainly fed on animals and were uninfected with P. falciparum. Anopheles Funestus showed insecticide resistance to deltamethrin and bendiocarb. A high-precision hydrolysis probe assay was successfully developed to identify An. Funestus-like.

Discussion
Four species in the An. Funestus group were collected in Karonga. Resistance to deltamethrin and bendiocarb was observed in An. Funestus and further investigation is needed on the insecticide resistance mechanisms. Anopheles Funestus-like, while collected indoors, is mainly zoophilic and most likely not a malaria vector.

Accession numbers
An. Funestus (GenBank accession no. KC771136), An. Funestus-like (GenBank accession no. KC771137), An. parensis GenBank accession no. KC771138) and An. vaneedeni GenBank accession no. KC771139).

molecular and physiological analysis of Anopheles Funestus swarms in nchelenge zambia

Anopheles Funestus has been recognized as a major malaria vector in Africa for over 100 years, but knowledge on many aspects of the biology of this species is still lacking. Anopheles Funestus, as with most other anophelines, mate through swarming. A key event that is crucial for the An. Funestus male to mate is genitalia rotation. This involves the 135° to 180° rotation of claspers, which are tipped with claws. This physical change then enables the male to grasp the female during copulation. The aim of this investigation was to molecularly characterize wild An. Funestus swarms from Zambia and examine the degree of genitalia rotation within the swarm. Anopheles Funestus swarms were collected from Nchelenge, northern Zambia, during dusk periods in May 2016. All the adults from the swarm were analysed morphologically and identified to species level using a multiplex PCR assay. Anopheles Funestus s.s. specimens were molecularly characterized by restriction fragment length polymorphism type and Clade type assays. The different stages of genitalia rotation were examined in the adult males. A total of six swarms were observed during the study period and between 6 and 26 mosquitoes were caught from each swarm. Species analysis revealed that 90% of the males from the swarms were An. Funestus s.s. MW-type, with 84% belonging to clade I compared to 14% clade II and 2% failed to amplify. Very few specimens (3.4%) were identified as Anopheles gambiae s.s. Eighty percent of the males from the swarm had complete genitalia rotation. This is the first time that An. Funestus swarms have been molecularly identified to species level. Anopheles Funestus swarms appear to be species-specific with no evidence of clade-type differentiation within these swarms. The An. Funestus swarms consist mainly of males with fully rotated genitalia, which strongly suggests that swarming behaviour is triggered primarily when males have matured.

Identification and characterization of microRNAs expressed in the African malaria vector Anopheles Funestus life stages using high throughput sequencing.

Background
Over the past several years, thousands of microRNAs (miRNAs) have been identified in the genomes of various insects through cloning and sequencing or even by computational prediction. However, the number of miRNAs identified in anopheline species is low and little is known about their role. The mosquito Anopheles Funestus is one of the dominant malaria vectors in Africa, which infects and kills millions of people every year. Therefore, small RNA molecules isolated from the four life stages (eggs, larvae, pupae and unfed adult females) of An. Funestus were sequenced using next generation sequencing technology.

Analysis of esterase enzyme activity in adults of the major malaria vector Anopheles Funestus.

Anopheles Funestus is a major vector of malaria in sub-Saharan Africa. In order to apply effective control measures against this vector, it is necessary to understand the underlying physiological factors that play a critical role in its development, reproduction, fertility and susceptibility to insecticides. One enzyme family involved in the above mentioned biological pathways is the esterases. The aim of this study was to analyse esterase activity levels at different ages during the life-span of adult Anopheles Funestus Giles in order to better understand the complex biological processes in this species. Isoenzyme electrophoresis (IEE) was used to examine the esterase activity in laboratory colonised An. Funestus adults aged between 2 h (h) and 30 days post eclosion as well as in wild An. Funestus adults aged between 2 h and 15 days post eclosion. Esterase activity was quantified by densitometry analysis of the IEE gels. Esterases were classified according to their activity inhibition by organic phosphates, eserine sulphate and sulphydryl reagents. Nine esterases IEE profiles were common to both the laboratory colonised and wild An. Funestus adults. These esterases were further divided into acetylesterases, arylesterases, carboxylesterases and acetylcholinesterase. The activity level of certain specific esterases was primarily influenced by age and/or gender. The information from this study contributes towards the general understanding of esterase enzyme activity variation in adults of a major malaria vector An. Funestus. This variation likely carries physiological and adaptive significance and may influence specific characteristics, such as reproductive fitness and insecticide resistance that are epidemiologically important.

Implication of Anopheles Funestus in malaria transmission in the city of Yaoundé, Cameroon.

The contribution of Anopheles Funestus to malaria transmission in the urban environment is still not well documented. The present study assesses the implication of An. Funestus in malaria transmission in two districts, Nsam and Mendong, in the city of Yaounde. Adult mosquitoes were collected using Centers for Disease Control and Prevention miniature light traps (CDC-LT) and human landing catches from April 2017 to March 2018 and were identified morphologically to the species level. Those belonging to the Anopheles gambiae complex and to the Anopheles Funestus group were further processed by PCR to identify members of each complex/group. Anopheline mosquitoes were analysed to determine their infection status using an enzyme-linked immunosorbent assay. Bioassays were conducted with 2–5-day-old female Anopheles Funestus and An. gambiae s.l. to determine their susceptibility to permethrin, deltamethrin and dichlorodiphenyltrichloroethane (DDT). Six anopheline species were collected in the peri-urban district of Mendong: Anopheles gambiae , An. coluzzii , An. Funestus , An. leesoni , An. ziemanni and An. marshallii ; only four out of the six were recorded in Nsam. Of the two members of the Anopheles gambiae complex collected, An. coluzzii was the most prevalent. Anopheles coluzzii was the most abundant species in Nsam, while An. Funestus was the most abundant in Mendong. Both Anopheles Funestus and An. gambiae s.l. were found to be infected with human Plasmodium at both sites, and both were found to be resistant to DDT, permethrin, and deltamethrin. This study confirms the participation of An. Funestus in malaria transmission in Yaounde and highlights the need to also target this species for sustainable control of malaria transmission.

Exploring the impact of glutathione S-transferase (GST)-based metabolic resistance to insecticide on vector competence of Anopheles Funestus for Plasmodium falciparum

Background: Malaria control heavily relies on insecticide-based interventions against mosquito vectors. However, the increasing spread of insecticide resistance is a major threat. The extent to which such resistance, notably metabolic resistance, influences the development of the Plasmodium parasite and its impact on overall malaria transmission remains poorly characterized. Here, we investigated whether glutathione S-transferase-based resistance could influence Plasmodium falciparum development in Anopheles Funestus . Methods: Anopheles Funestus females were infected with P. falciparum gametocytes and midguts were dissected at day 7 post infection for detection/quantification of oocysts. Infection parameters were compared between individuals with different L119F-GSTe2 genotypes, and the polymorphism of the GSTe2 gene was analyzed in infected and uninfected mosquito groups. Results: Overall, 403 An. Funestus  mosquitoes were dissected and genotyped. The frequency of the L119F-GSTe2 resistance allele was significantly higher in non-infected (55.88%) compared to infected (40.99%) mosquitoes (Fisher’s exact test, P

Molecular basis of permethrin and DDT resistance in an Anopheles Funestus population from Benin.

Insecticide resistance in Anopheles mosquitoes is threatening the success of malaria control programmes. In order to implement suitable insecticide resistance management strategies, it is necessary to understand the underlying mechanisms involved. To achieve this, the molecular basis of permethrin and DDT resistance in the principal malaria vector, Anopheles Funestus from inland Benin (Kpome), was investigated. Here, using a microarray-based genome-wide transcription and qRT-PCR analysis, we showed that metabolic resistance mechanisms through over-expression of cytochrome P450 and glutathione S-transferase genes (GSTs) are a major contributor to DDT and permethrin resistance in Anopheles Funestus from Kpome. The GSTe2 gene was the most upregulated detoxification gene in both DDT- [fold-change (FC: 16.0)] and permethrin-resistant (FC: 18.1) mosquitoes suggesting that upregulation of this gene could contribute to DDT resistance and cross-resistance to permethrin. CYP6P9a and CYP6P9b genes that have been previously associated with pyrethroid resistance were also significantly overexpressed with FC 5.4 and 4.8, respectively, in a permethrin resistant population. Noticeably, the GSTs, GSTd1-5 and GSTd3, were more upregulated in DDT-resistant than in permethrin-resistant Anopheles Funestus suggesting these genes are more implicated in DDT resistance. The absence of the L1014F or L1014S kdr mutations in the voltage-gated sodium channel gene coupled with the lack of directional selection at the gene further supported that knockdown resistance plays little role in this resistance. The major role played by metabolic resistance to pyrethroids in this An. Funestus population in Benin suggests that using novel control tools combining the P450 synergist piperonyl butoxide (PBO), such as PBO-based bednets, could help manage the growing pyrethroid resistance in this malaria vector in Benin.