Chemical Composition and Use of Momordica charantia L. and Hyptis spicigera Lam. Extracts as Mosquito Larvicides and Insect Growth Regulators against Malarial Vector, Anopheles gambiae Giles

Background: Mosquitoes are responsible of numerous vector-borne diseases and among these diseases is malaria which takes away lives of thousands of people, especially children of under five, particularly in Africa. To reduce the mortality and economic burdens of this deadly disease, synthetic insecticide has been of use to control its main vector, Anopheles gambiae. Due to adverse effects caused by these conventional products on non-target organisms and the environment, plants have been of first choice as they proved their effectiveness against mosquitoes and are said to be eco-friendly and relatively safer. Therefore, the aim of this study was to screen the phytochemicals and determine the larvicidal and adult emergence exhibitory activities of Momordica charantia and Hyptis spicigera leaves extracts against Anopheles gambiae larvae. Methods: The WHO standard protocol for larvicidal activity and IGRs was followed up and concentrations from 500 - 2000 ppm for larvicidal and 500 - 1500 ppm for IGRs were set up. Results: There was the presence of alkaloids, flavonoids, tannins, phenols, steroids, terpenoids and oils in Momordica charantia against alkaloids, taponins, tannins, phenols, steroids, terpenoids and oils in Hyptis spicigera. Methanol crude extract registered the lowest LC50 value of 270.6 ppm followed by ethyl acetate and hexane fractions recorded the LC50 of 742.1 and 756 ppm, respectively for larvicidal activity of Momordica charantia against LC50 of 760 and 867.5 ppm for hexane fraction and methanol crude extract, respectively for Hyptis spicigera. Methanol crude extract and hexane fraction of both plants as well as the ethyl acetate fraction of M. charantia registered greater emergence inhibition with IE50 values of 590.2, 842.3 and 982 ppm for methanol crude extract, hexane and ethyl acetate fractions of M. charantia, respectively against 901.7 and 873.2 for methanol crude extract and hexane fraction of H. spicigera, respectively. Conclusion: The toxicity and adult emergence inhibition may be associated with constituents in both plants that interfere with the normal neuronal, respiratory and endocrine systems functions.

Chemical Composition, Larvicidal and Adult Emergence Inhibition Activities of Balanites aegyptiaca Del. Seed and Aristolochia albida Duch. Root Extracts against Malaria Vector, Anopheles gambiae Giles

Background: Anopheles gambiae is enemy number one of mankind in Africa and particularly in Cameroon due to its ability of transmitting malaria which is the deadliest disease in this part of the world. Synthetic insecticides have been used to control malaria vectors but they have negative effects on non-target organisms and are environmentally unfriendly. Control of mosquitoes at larval stages using phytochemicals is currently the leading tool to reduce the mosquito population and so the reduction of malaria transmission rates. Therefore, the present study was to evaluate the phytochemical contents, larvicidal and adult emergence inhibition activities of Balanites aegyptiaca seed and Aristolochia albida root solvents extracts against Anopheles gambiae larvae. Methods: The World Health Organization standard protocols were followed for the different bioassays. Concentrations ranging from 500 - 2000 ppm for larvicidal and 500 - 1500 ppm for IGRs were used. Results: Both plants showed the presence of alkaloids, flavonoids, saponins, tannins, phenols, terpenoids and oils except the absence of phenols in Aristolochia albida. The larvicidal activity of Aristolochia albida extracts showed that hexane and methanol fractions were the most active killing relatively all exposed larvae with the LC50 values of 420.1 and 453 ppm, respectively. The same observation was made in hexane fraction from Balanites aegyptiaca (LC50 = 588 ppm). The insect growth inhibitory activity of Aristolochia albida extracts proved that hexane and methanol fractions relatively caused a 100% inhibition in the mosquito development, recording the EI50 values of 482.4 and 555.6 ppm, respectively. The same trend was observed with Balanites aegyptiaca hexane fraction registering better EI50 of 623.9 ppm. Conclusions: Our findings demonstrate that Balanites aegyptiaca seed and Aristolochia albida seed extracts are rich in phytochemicals capable of killing mosquito larvae and disrupting mosquito larval development. This could contribute to the control of mosquito populations and improved management of malaria.

Larvicidal efficacy of monoterpenes against the larvae of Anopheles gambiae

Objective:To evaluate the larvicidal efficacy of eight volatile components of essential oils against 3rd instar larvae of Anopheles gambiae s.s.Methods:Larvicidal effects of each compound were evaluated in both laboratory and semi-field trials.Stock solution was prepared and serial dilutions were made in six concentrations for each compound.A total of 20 larvae were exposed to larvicides for each replicate and monitored at intervals of 12,24,48 and 72 h.Larvae monitoring was done on basis of dead and live larvae in all intervals.Results:All assayed compounds were larvicides and presented varying degrees of larval toxicity,with LC50 values ranging from 1.28 to 1 938.92 mg/L depending on the treatment time(12,24,48 or 72 h).(-)-Perillyl alcohol presented the strongest larvicidal activity towards Anopheles gambiae larvae,with LC_(50) values of 73.60,18.36,1.72 and1.28 mg/L after 12,24,48 and 72 h of exposure,respectively.The next strongest were(-)-isopulegol(LC_(50)= 135.10,49.39,34.39 and 20.22 mg/L) and(-)-carvone epoxide(LC_(50)= 168.86,124.74,80.84 and 23.46 mg/L).After 12,24 and 48 h of treatment,hydroxydihydrocarvone was the least toxic compound,with LC_(50) values of 1 938.92,1 172.18 and 401.03 mg/L,respectively.Conclusions:The data obtained in this study suggest that all evaluated monoterpenes,especially(-)-perillyl alcohol,have remarkable larvicidal effects and may be considered as potential sources for the development of suitable natural larvicides for mosquito management programs.Further small-scale field trials should be conducted.

Anopheles gambiae larvicidal and adulticidal potential of Phyllanthus amarus(Schumach and Thonn,1827)obtained from different localities of Nigeria

Objective:To screen phytochemicals in ethanolic leaf extracts of Phyllanthus amarus collected from three different geographical zones in Nigeria and evaluate their effects on larva and adult of Anopheles gambiae.Methods:The sample extracts of Phyllanthus amarus prepared with ethanol solvent were tested against Anopheles gambiae at two important developmental stages of its life cycle using slightly modified WHO protocols.Results:Alkaloids,saponins,tannins,flavonoids,glycosides,phenols,and terpenes were detected in each extract.Among these samples,the extract from northwest exhibited the highest larvicidal activity(LC50=263.02 ppm),followed by southeast and southwest extracts(LC50=288.40 and 295.12 ppm,respectively after 48 h),while the extract from southwest exhibited the highest adulticidal activity(LC50=275.42 ppm),followed by northwest and southeast extract(LC50=301.99 and 316.22 ppm,respectively after 24 h).A 50%larva mortality was almost attained at 600 ppm after 48 h duration of exposure to the northwest extract.Conclusions:The tested samples possess strong larvicidal and adulticidal property against Anopheles gambiae which depends on their chemical composition and localities of collection.Further studies are needed to explore the insecticidal activity against a wider range of mosquito species,and to identify active ingredient(s)of the extract responsible for such activity.

Bioinformatics-Based Identification of Chemosensory Proteins in African Malaria Mosquito, Anopheles gambiae

Chemosensory proteins (CSPs) are identifiable by four spatially conserved Cys-teine residues in their primary structure or by two disulfide bridges in their tertiary structure according to the previously identified olfactory specific-D related proteins. A genomics- and bioinformatics-based approach is taken in the present study to identify the putative CSPs in the malaria-carrying mosquito, Anopheles gambiae. The results show that five out of the nine annotated candidates are the most possible Anopheles CSPs of A. gambiae. This study lays the foundation for further functional identification of Anopheles CSPs, though all of these candidates need additional experimental verification.

Anopheles gambiae strain(Ag55)cultured cells originated from Anopheles coluzzii and are phagocytic with hemocyte-like gene expression

Anopheles gambiae and Anopheles coluzzii are closely related species that are predominant vectors of malaria in Africa.Recently,A.gambiae form M was renamed A.coluzzii and we now conclude on the basis of a diagnostic PCR-restriction fragment length polymorphism assay that Ag55 cells were derived from A.coluzzii.We established an Ag55 cell transcriptome,and KEGG pathway analysis showed that Ag55 cells are enriched in phagosome pathway transcripts.The Ag55 transcriptome has an abundance of specific transcripts characteristic of mosquito hemocytes.Functional E.coli bioparticle uptake experiments visualized by fluorescence microscopy and confocal microscopy and quantified by flow cytometry establish the phagocytic competence of Ag55 cells.Results from this investigation of Ag55 cell properties will guide researchers in the use and engineering of the Ag55 cell line to better enable investigations of Plasmodium,other microbes,and insecticidal toxins.

Ecdysis triggering hormone peptide in the African malaria mosquito Anopheles gambiae:The peptide structure for receptor activation

Infections by mosquito-borne diseases represent one of the leading causes of death in third world countries.The rapid progression of resistance to conventional insecticide causes a significant threat to the highly efficient preventive methods currently in place.Insect neuropeptidergic system offers potential targets to control the insect vectors.The essential roles of the neuropeptide ecdysis triggering hormone(ETH)in insect development and reproduction led us to attempt understanding of the fundamentals of the biochemical interaction between ETH and its receptor in the African malaria mosquito Anopheles gambiae.One of two ETH peptides of the African malaria mosquito(AgETH1),a small peptide hormone with 17 amino acid residues(SESPGFFIKLSKSVPRI-NH2),was studied to elucidate its molecular structure.N-termini deletions and mutations of conserved amino acids in the ligand revealed the critical residues for the receptor activation.The solution structure of AgETH1 using 2D 1H-1H nuclear magnetic resonance(NMR)spectroscopy and nuclear overhauser effect(NOE)derived constraints revealed a short alpha helix between residues 3S and 11S.The NMR solution structure of AgETH1 will be of significant assistance for designing a new class of insecticidal compounds that acts on the AgETH receptor aiming for in silico docking studies.

Efficacy of PermaNet^(■)3.0 and PermaNet^(■)2.0 nets against laboratory-reared and wild Anopheles gambiae sensu lato populations in northern Tanzania

Background:Mosquitoes have developed resistance against pyrethroids,the only class of insecticides approved for use on long-lasting insecticidal nets(LLINs).The present study sought to evaluate the efficacy of the pyrethroid synergist PermaNet^(■)3.0 LLIN versus the pyrethroid-only PermaNet^(■)2.0 LLIN,in an East African hut design in Lower Moshi,northern Tanzania.In this setting,resistance to pyrethroid insecticides has been identified in Anopheles gambiae mosquitoes.Methods:Standard World Health Organization bioefficacy evaluations were conducted in both laboratory and experimental huts.Experimental hut evaluations were conducted in an area where there was presence of a population of highly pyrethroid-resistant An.arabiensis mosquitoes.All nets used were subjected to cone bioassays and then to experimental hut trials.Mosquito mortality,blood-feeding inhibition and personal protection rate were compared between untreated nets,unwashed LLINs and LLINs that were washed 20 times.Results:Both washed and unwashed PermaNet^(■)2.0 and PermaNet^(■)3.0 LLINs had knockdown and mortality rates of 100%against a susceptible strain of An.gambiae sensu stricto.The adjusted mortality rate of the wild mosquito population after use of the unwashed PermaNet^(■)3.0 and PermaNet^(■)2.0 nets was found to be higher than after use of the washed PermaNet^(■)2.0 and PermaNet^(■)3.0 nets.Conclusions:Given the increasing incidence of pyrethroid resistance in An.gambiae mosquitoes in Tanzania,we recommend that consideration is given to its distribution in areas with pyrethroid-resistant malaria vectors within the framework of a national insecticide-resistance management plan.

Alstonia boonei De Wild oil extract in the management of mosquito(Anopheles gambiae),a vector of malaria disease

Objective:To evaluate the insecticidal potential of Alstonia boonei(A.boonei)oils and derivatives against different life stages of a malaria vector,Anopheles gambiae.Methods:The leaf,stem bark and root bark of A.boonei were collected from an open field and air dried before being blended to fine powder.Oils from this plant were extracted by cold extraction and were prepared at different concentrations.Contact toxicity of A.boonei was tested against the larvae and pupae of the insect while smoke toxicity of the plant materials in form of mosquito coil was tested against the adult insect.Results:Alstodine recorded the highest insect mortality rate and the order of susceptibility of the life stages of the insect to the plant was pupae<adult<larvae.Alstodine recorded the highest repellant activity(100%)after 4-5 h of application.However,all the treatments achieved high repellency(above 70%)after 6-7 h of application compared with the control.The formulated mosquito coil(smoke)of A.boonei oil extracts and derivatives showed high rate of protectability as they achieved above 55%protection.Moreover,alstodine(83.22%)showed the greatest smoke toxicity effect on the insect as it recorded almost the same percentage protection as the positive control(Raid synthetic insecticide)which recorded 83.56%protection.Conclusions:This present study has proven A.boonei oil extracts and derivatives as a potential botanical insecticide which could serve as a new thoroughfare of mosquito control.Moreover,the order of effectiveness of the plant can be arranged thus:alstodine>alstonine>stem bark extract>leaf extract>root bark extract.

Observation of granulations in the basal body of ovarioles and follicular dilatations for the determination of physiological age of Anopheles gambiae s.s.

Objective:To explore ovariole basal body granulations and follicular dilatations for determining physiological age inAnopheles gambiaes.s.(An.gambiaes.s.).Methods:Mosquitoes were collected by using window trap catch and identified morphologically.For the first lot ofmosquitoes,they were dissected,and ovary was left in distilled water for reading ovarian tracheoles and the second was cut and transferred to another blade in a physiological liquid for verification of ovariole basal body granulations.The same approach was followed with the second lot of mosquitoes where follicular dilatations were found after classic dilaceration of ovaries were transferred into physiological liquid.The other body parts of mosquitoes were used to identify the species of theAn.gambiaes.s.complex by PCR.Results:Among the 123An.gambiae s.s.of the first lot,the method of Detinova determined the age of 89 mosquitoes versus 114 for the observation of granulations(P>0.05).Among the 112An.gambiae s.s.of the second lot,the method of Detinova determined the age of 84 mosquitoes versus 93 for the observation of follicular dilatations(P>0.05).Unlike the method of Detinova,observation of follicular dilatations and basal body granulations of ovarioles were possible beyond the stage II Christophers.Conclusions:Overall,the observation of follicular dilatations and ovariole basal body granulations are reliable for the determination of the physiological age inAn.gambiaes.s.Furthermore,these two methods can be used beyond the stage II.

Potential geographical distribution of Anopheles gambiae worldwide under climate change

Background:Anopheles gambiae(An.gambiae)is considered the most effective malaria vector worldwide and is widely distributed in Africa.The purpose of this study is to determine the potential impact of climate change on An.gambiae and predict the present and future potential suitable habitat globally.Methods:In this study,environmental variables,global occurrence data of An.gambiae,and the maximum entropy model(MaxEnt)were used to evaluate the contribution of environmental factors and predict the habitat suitability of An.gambiae under the current and future scenarios.Results:Among all environmental variables,isothermality(Bio3,34.5%)contributed the most to An.gambiae distribution.Under current climate conditions,the potential suitable areas for An.gambiae are mainly located near the equator(approximately 30°N-30°S),with a total area of 16.58 million km2,including central and northern South America,a fraction of areas near the equator of North America,central and southern Africa,some tropical regions of southern Asia,and small areas of Oceania.The areas of potential suitable habitats would be reduced to varying degrees in future climate scenarios.Conclusions:Potential suitable habitats for An.gambiae may not be limited to Africa.Necessary surveillance and preventive measures should be undertaken in high-risk regions,including those outside Africa,to monitor and control the spread of An.gambiae.

Biochemical characterization of chitin synthase activity and inhibition in the African malaria mosquito, Anopheles gambiae

Chitin synthase （CHS） is an important enzyme catalyzing the formation of chitin polymers in all chitin containing organisms and a potential target site for insect pest control. However, our understanding of biochemical properties of insect CHSs has been very limited. We here report enzymatic and inhibitory properties of CHS prepared from the African malaria mosquito, Anopheles gambiae. Our study, which represents the first time to use a nonradioactive method to assay CHS activity in an insect species, determined the optimal conditions for measuring the enzyme activity, including pH, temperature, and concentrations of the substrate uridine diphosphate N-acetyl-D-glucosamine （UDP- GlcNAc） and Mg＋＋. The optimal pH was about 6.5-7.0, and the highest activity was detected at temperatures between 37℃ and 44℃. Dithithreitol is required to prevent melanization of the enzyme extract. CHS activity was enhanced at low concentration of GIcNAc, but inhibited at high concentrations. Proteolytic activation of the activity is significant both in the 500×g supernatant and the 40 000×g pellet. Our study revealed only slight in vitro inhibition ofA. gambiae CHS activity by diflubenzuron and nikkomycin Z at the highest concentration （2.5μmol/L） examined. There was no in vitro inhibition by polyoxin D at any concentration examined. Furthermore, we did not observe any in vivo inhibition of CHS activity by any of these chemicals at any concentration examined. Our results suggest that the inhibition of chitin synthesis by these chemicals is not due to direct inhibition of CHS in A. gambiae.

Insecticidal Activity of Crude Extracts of Hyptis suaveolens(Bush Mint)on Anopheles Mosquitoes Collected from Lafia,Nasarawa State,Nigeria

Anopheles gambiae is a vector that is responsible for the transmission of malaria parasites which causes high morbidity and mortality in Nigeria and the world at large.Human-vector contact can be reduced by the use of conventional repellents being sold in the market,though some of these repellent are not environmentally friendly and An.gambiae have developed resistance to some of these repellents.To this end,the phytochemical constituents and insecticidal activity of crude extracts of Hyptis suaveolens(bush mint)was determined on adult An.gambiae mosquitoes collected from Lafia,Nasarawa State,Nigeria to evaluate its effect in controlling them.Here,70%ethanolic and diethyl ether fat crude extracts were made from H.suaveolens dried leaves and used to carry out the experiment.The phytochemical screening of the ethanol extract revealed the presence of alkaloids,flavonoid,saponins,tannins,steroids and reducing sugar.Glycoside was not detected in the ethanol extract.Only steroids was detected in diethyl ether extract the rest of the phytochemical tested were absent.An.gambiae larvae were collected from the field and were raised to F1 progeny adults that were used for the study.WHO protocol for carrying out human bait repellency cage test was used.Human hand treated with the extracts was exposed to a cage containing 30 female mosquitoes for each of the extracts respectively.The ethanolic crude extract treatment proved to be more effective in repelling mosquitoes with 0%(0/30)mosquito landing than diethyl ether extract which had 40%(12/30)mosquitoes landing and the control hand(untreated left hand)had the highest landing rate 63.3%(19/30)which showed very high significant difference(χ^(2)=27.2619,df=2,P<0.00001)in relation to the treatments.After 24 hours holding period,the mortality rate of exposed mosquitoes was observed to be highest in the ethanolic treatment 73.3%(22/30).In conclusion,H.suaveolens extracts have repellency potential in controlling adult An.gambiae.

Spatial distribution and habitat characterization of mosquito species during the dry season along the Mara River and its tributaries,in Kenya and Tanzania

Background:Vector-borne diseases are increasingly becoming a major health problem among communities living along the major rivers of Africa.Although larger water bodies such as lakes and dams have been extensively researched,rivers and their tributaries have largely been ignored.This study sought to establish the spatial distribution of mosquito species during the dry season and further characterize their habitats along the Mara River and its tributaries.Methods:In this cross-sectional survey,mosquito larvae were sampled along the Mara River,its two perennial tributaries(Amala and Nyangores),drying streams,and adjacent aquatic habitats(e.g.swamps,puddles that receive direct sunlight[open sunlit puddles],rock pools,hippo and livestock hoof prints,and vegetated pools).Each habitat was dipped 20 times using a standard dipper.Distance between breeding sites and human habitation was determined using global positioning system coordinates.The collected mosquito larvae were identified using standard taxonomic keys.Water physico-chemical parameters were measured in situ using a multiparameter meter.Mean mosquito larvae per habitat type were compared using analysis of variance and chi-square tests,while the relationship between mosquito larvae and physicochemical parameters was evaluated using a generalized linear mixed model.The Cox-Stuart test was used to detect trends of mosquito larvae distribution.The test allowed for verification of monotonic tendency(rejection of null hypothesis of trend absence)and its variability.Results:A total of 4001 mosquito larvae were collected,of which 2712(67.8%)were collected from river/stream edge habitats and 1289(32.2%)were sampled from aquatic habitats located in the terrestrial ecosystem about 50 m away from the main river/streams.Anopheles gambiae s.s,An.arabiensis,and An.funestus group,the three most potent vectors of malaria in Sub-Saharan Africa,together with other anopheline mosquitoes,were the most dominant mosquito species(70.3%),followed by Culex quinquefasciatus and Cx.pipiens complex combined(29.5%).Drying streams accounted for the highest number of larvae captured compared to the other habitat types.A stronger relationship between mosquito larvae abundance and dissolved oxygen(Z=7.37,P≤0.001),temperature(Z=7.65,P≤0.001),turbidity(Z=−5.25,P≤0.001),and distance to the nearest human habitation(Z=4.57,P≤0.001),was observed.Conclusions:Presence of malaria and non-malaria mosquito larvae within the Mara River basin calls for immediate action to curtail the insurgence of vector-borne diseases within the basin.A vector control program should be conducted during the dry period,targeting drying streams shown to produce the highest number of larval mosquitoes.

Bioefcacy and durability of Olyset® Plus, a permethrin and piperonyl butoxide-treated insecticidal net in a 3-year long trial in Kenya

Background:Long-lasting insecticide nets(LLINs)are a core malaria intervention.LLINs should retain efficacy against mosquito vectors for a minimum of three years.Efficacy and durability of Olyset®Plus,a permethrin and piperonyl butoxide(PBO)treated LLIN,was evaluated versus permethrin treated Olyset®Net.In the absence of WHO guidelines of how to evaluate PBO nets,and considering the manufacturer’s product claim,Olyset®Plus was evaluated as a pyrethroid LLIN.Methods:This was a household randomized controlled trial in a malaria endemic rice cultivation zone of Kirinyaga County,Kenya between 2014 and 2017.Cone bioassays and tunnel tests were done against Anopheles gambiae Kisumu.The chemical content,fabric integrity and LLIN survivorship were monitored.Comparisons between nets were tested for signifcance using the Chi-square test.Exact binomial distribution with 95%confdence intervals(95%CI)was used for percentages.The WHO efcacy criteria used were≥95%knockdown and/or≥80%mortality rate in cone bioassays and≥80%mortality and/or≥90%blood-feeding inhibition in tunnel tests.Results:At 36 months,Olyset®Plus lost 52%permethrin and 87%PBO content;Olyset®Net lost 24%permethrin.Over 80%of Olyset®Plus and Olyset®Net passed the WHO efcacy criteria for LLINs up to 18 and 12 months,respec‑tively.At month 36,91.2%Olyset®Plus and 86.4%Olyset®Net survived,while 72%and 63%developed at least one hole.The proportionate Hole Index(pHI)values representing nets in good,serviceable and torn condition were 49.6%,27.1%and 23.2%,respectively for Olyset®Plus,and 44.9%,32.8%and 22.2%,respectively for Olyset®Net but were not signifcantly diferent.Conclusions:Olyset®Plus retained efcacy above or close to the WHO efcacy criteria for about 2 years than Olyset®Net(1–1.5 years).Both nets did not meet the 3-year WHO efcacy criteria,and showed little attrition,comparable physical durability and survivorship,with 50%of Olyset®Plus having good and serviceable condition after 3 years.Better community education on appropriate use and upkeep of LLINs is essential to ensure efectiveness of LLIN based malaria interventions.

Accurate determination of DNA yield from individual mosquitoes for population genomic applications

Accurate estimates of DNA quantity are likely to become increasingly important for successful genomic screening of insect populations via recently developed, highly multiplexed genotyping assays and high-throughput sequencing methods. Here we show that genomic DNA extractions from single Anopheles gambiae Giles using a standard commercial kit-based methodology yield extracts with concentrations below the linear range of spectrophotometric absorbance at 260 nm. Concentrations determined by spectropho- tometry were not reproducible, and are therefore neither accurate nor reliable. However, DNA quantification using a fluorescent nucleic acid stain （PicoGreen） gave highly reproducible concentration estimates, and indicated that, on average, single mosquitoes yielded approximately 300 ng of DNA. Such a total yield is currently insufficient for many highthroughput genome screening applications, necessitating whole genome amplification of all or most individuals in a population prior to genotyping.