Larvicidal efficacies and chemical composition of essential oils of Pinus sylvestris and Syzygium aromaticum against mosquitoes

Objective:To assess the chemical composition and mosquito larvicidal potentials of essential oils of locally sourced Pinus sylvestris(P.sylvestris)and Syzygium aromatkum(S.aromaticum)against Aedes aegypti(A.aegypti)and Culex quinquefasciatus(C.quinquefasciatus).Method:The chemical composition of the essential oils of both plants was determined using GCMS while the larvicidal bioassay was carried out using different concentrations of the oils against the larvae of A.aegypti and C.quinquefasciatus in accordance with the standard protocol.Results:The results as determined by GC-MS showed that oil of S.aromaticum has eugenol(80.5%)as its principal constituent while P.sylvestris has 3-Cyclohexene-1-methanol,.alpha.,.alpha.4-trimethyl(27.1%)as its dominant constituent.Both oils achieved over 85%larval mortality within24 h.The larvae of A.aegypti were more susceptible to the oils[LC_(50)(S.aromaticum)=92.56 mg/L,LC_(50)(P.sylvestris)=100.39 mg/L]than C.quinquefasciatus[LC_(50)(S.aromaticum)=124.42 mg/L;LC_(50)(P.sylvestris)=128.00 mg/L].S.aromaticum oil was more toxic to the mosquito larvae than oil of P.sylvatris but the difference in lethal concentrations was insignificant(P>0.05).Condusioii:The results justify the larvicidal potentials of both essential oils and the need to incorporate them in vector management and control.

Studies on effects of Andrographis paniculata(Burm.f.) and Andrographis lineata nees(Family:Acanthaceae) extracts against two mosquitoes Culex quinquefasciatus(Say.) and Aedes aegypti(Linn.)

Objective:To investigate the studies on effects of Andrographis paniculata(A.paniculata) (Burm.f.) and Andrographis lineata(A.lineata) nees(Family:Acanthaeeae)extracts against two mosquitoes Culex quinquefasciatus(Cx.quinquefasciatus)(Say.) and Aedes aegypti(Ae.aegypti) (Linn.).Methods:The aqueous and petroleum ether extracts of two plant species.A.paniculata and A.lineate were examined against the larvae of,4.aegypti(L.) and Cx.quinquefasciatus with gradually increasing concenlration ie.from 50 to 200 ppm of solvent extracts and to test their activity in combination with each other.Results:In a 24 h bioassay experiment with plant extracts,highest mortalities were recorded at 200 ppm of concentrations for leaves of 4.lineta and A.paniculata individually.For combination effect,only 150 ppm of the mixture of solvent extracts of petroleum ether:aqueous(1:1) extracts showed 100%mortality after 24 h of exposure. Conclusions:The results show that,insecticides of plant combination is ecofriend and has better larvicidal activity compared to individual extracts.

Biochemistry of Melanization in Mosquitoes

Melanization/pigmentation is an important physiological phenomenon in insects,which is involved incuticular tanning,parasite encapsulation,and egg shell hardening.During the past few years,we have beenusing mosquito eggs as a model to study the detailed biochemistry of melanization reactions in mosquitoes.Chorion of newly oviposited eggs is white and soft,but the chorion becomes black and hard within 3～4 hfollowing oviposition. Once the chorion turns black,the eggs become resistant to desiccation and solubiliza-tion.Floodwater mosquitoes,such as Aedes aegypti,oviposit on substrates at the edge of water and theeggs hatch only after being flooded with water following adequate rainfall.Consequently,the ability towithstand the desiccation and other environmental adversities is critical for egg survival.Our research deal-ing with chorion melanization in mosquitoes is aimed at understanding the biochemical processes and mech-

Efficacy of diurnal BG-Sentinel traps to capture nocturnal adult Armigeres subalbatus mosquitoes and impact of altitudinal variations in forests

Objective:To establish an efficacious and efficient surveillance method of Armigeres(Ar.)subalbatus,a known filarial vector,surviving in forest habitats to estimate realistic population density and assess the impact of altitudinal variations on the efficacy of the trap.Methods:In the study,12 locations in areas with an altitudinal range from 82 m to 920 m were selected in three reserve forests for night sampling of adult Ar.subalbatus mosquito using standard chemical lure based BG-Sentinel traps and CDC-light traps in pairs.Effects of locations and time were estimated on the efficacy of traps as mosquito density using multifactor analysis of variance for significant differences.Impact of altitudinal variations on the efficacy of traps was assessed using multiple regression with slope comparison.Results:BG-Sentinel trap collected significantly more adult Ar.subalbatus,4.43 folds in Gorumara NP,5.19 folds in Neora Valley NP and 12.10 folds in Mahananda WLS than the CDC-light trap irrespective to locations.BG-Sentinel traps were tolerant of altitudinal variations(80 m-170 m)and showed no significant relationship between density and altitudes in contrast to CDC-light traps which showed a significant negative impact on capturing efficacy with increase in altitudes(P<0.001).Conclusions:The study suggests that BG-Sentinel traps can be used effectively and efficiently to collect more Ar.subalbatus mosquitoes during night time in comparison to CDC-light trap under complex climatic conditions of forest and variable altitudes.

Accelerated evolution of constraint elements for hematophagic adaptation in mosquitoes

Comparative genomics is a powerful approach that comprehensively interprets the genome. Herein, we performed whole genome comparative analysis of 16 Diptera genomes, including four mosquitoes and 12 Drosophilae. We found more than 540 000 constraint elements （CEs） in the Diptera genome, with the majority found in the intergenic, coding and intronic regions. Accelerated elements （AEs） identified in mosquitoes were mostly in the proteincoding regions （〉93%）, which differs from vertebrates in genomic distribution. Some genes functionally enriched in blood digestion, body temperature regulation and insecticide resistance showed rapid evolution not only in the lineage of the recent common ancestor of mosquitoes （RCAM）, but also in some mosquito lineages. This may be associated with lineage-specific traits and/or adaptations in comparison with other insects. Our findings revealed that although universally fast evolution acted on biological systems in RCAM, such as hematophagy, same adaptations also appear to have occurred through distinct degrees of evolution in different mosquito species, enabling them to be successful blood feeders in different environments.

Wolbachia-infected mosquitoes:The answer to the dengue endemic in Pakistan?

Dengue is an infectious disease of viral etiology characterized by acute flu-like symptoms with potentially lethal complications.The virus is transmitted by the mosquitoes of the Aedes genus^([1]).These mosquitoes dwell around small bodies of stagnant water,road puddles and open sewers.

Stage-structured models for interacting wild and sterile mosquitoes

Using the sterile insect technique,in which sterile mosquitoes are released to reduce or eradicate the wild mosquito population,is an effective weapon to prevent transmission of mosquito-borne diseases. To study the impact of the sterile insect technique on the disease transmissions,we formulate stage-structured discrete-time mathematical models,based on difference equations,for the interactive dynamics of the wild and sterile mosquitoes. We incorporate different strategies for releasing sterile mosquitoes,investigate the model dynamics,and compare the impact of the different release strategies.Numerical examples are also provided to demonstrate dynamical features of the models.

Bio-Control of Fresh and Dried Portion of <i>Hyptis suaveolens</i>Plant on Mosquitoes Population during Raining Season in Nigeria Police Academy, Kano

Comparative study was conducted on fresh and dried portions of Hyptis suaveolens plant, to control mosquito population, and determine which portion has the highest efficacy in killing the vector. The study was conducted in Nigeria Police Academy, Wudil, Kano State Nigeria. The result revealed that both portions of the test plant (Hyptis suaveolens) have toxic effect on mosquitoes. During the study, three (3) experimental units were established and nine (9) sub units were made, three within each of the major units at different locations within the study area. Mosquitoes were reared at all units and various quantities test plant (both fresh and dried portions) were introduced at different quantities and observed at different times of the day (Morning, Afternoon and Evening) within 24 hours in order to test their efficacy on mosquitoes. A total of two hundred and fifty three (253) death rate (DR) with a total % mortality rate (% M.R) of 217.9 mosquitoes were recorded from the three different experimental units with the highest % MR from the cadets hostels. There was a gradual overall mortality rate increase in every experimental unit as the quantity (concentration) of the test plant (Hyptis suaveolens) introduced into the units increased. More so, a resultant highest cumulative death rate of 119 was recorded in the evening. Comparatively, fresh portion of the test plant was more effective for killing the adult mosquitoes during the study. Statistically, it also shows that, there was a strong correlative relationship (r = 0.9832) between the quantity (concentration) of the test plant (H. suaveolens) and the death rate of mosquito vectors.

Citrus-based essential oils could be used for dengue vector mosquitoes control

Dear Editor The mosquitoes Aedes aegypli(I,) and Aedes albopictus (Skuse)(Diptera:Culicidae) are important vectors of dengue fever in tropical and subtropical parts of the world. Chemical control has been considered as an effective tool for their management in different areas of the world.However, injudicious use of the chemicals result in environmental pollution,ill effects to humans and other animals,and the development of insecticide resistance,which ultimately limits the efficacy of many insecticides!’].Essential oils can be used for mosquito control with no harmful effects to the ecosystem and non-target organisms.Essentials oils from different plants have been found very

Topical Preparation of Newer and Safer Analogs of N,N-diethyl-2-phenylacetamide (DEPA) against <i>Aedes aegypti</i>Mosquitoes

Cosmetic acceptability and primary skin irritation are the two main parameters for assessing the suitability of any topical formulation meant for protection against the painful bites of mosquitoes. In the present study four newer analogs of N,N-diethyl-2-phenylacetamide (DEPA), were synthesized and formulated for topical application as insect repellent. They were assessed for their irritant behavior on rabbit’s skin for erythema and edema. The topical formulations of the analogs were also assessed for their protection time at varying concentrations against Aedes aegypti mosquitoes.

Larvicidal and repellent activities of Sida acuta Burm.F.(Family:Malvaceae) against three important vector mosquitoes

Objective:To determine the larvicidal and repellent activities of Sida acuta Burm.F.（Family: Malvaceae）extract against Culex quinquefasciatus,Aedes aegypti and Anopheles stephensi. Methods:Twenty five late III instar larve of three mosquito species were exposed to various concentrations（15-90 mg/L） and were assayed in the laboratory by using the protocol of WHO 2005:the 24 h LC50 values of the Sida acuta leaf extract was determined following Probit analysis. The repellent efficacy was determined against three mosquito species at three concentrations viz.,1.0,2.5 and 5.0 mg/cm2 under the laboratory conditions.Results:Results showed varying degree of larvicidal activity of crude extract of Sida acuta against three important mosquitoes with LC50 values ranging between 38 to 48 mg/L.The crude extract had strong repellent action against three species of mosquitoes as it provided 100%protection against Anopheles stephensi for 180 min followed by Aedes aegypti（150 min） and Culex quinquefasciatus（120 min）.Conclusions: From the results it can be concluded the crude extract of Sida acuta was an excellent potential for controlling Culex quinquefasciatus,Aedes aegypti and Anopleles stephensi mosquitoes.

Seasonal abundance and potential of Japanese encephalitis virus infection in mosquitoes at the nesting colony of ardeid birds,Thailand

Objective:To investigate the abundance and seasonal dynamics of mosquitoes,and to detect Japanese encephalitis virus(JEV)in these mosquitoes at the nesting colony of ardeid birds.Methods:Mosquitoes were collected bimonthly from July 2009 to May 2010 by Centers for Disease Control.Light traps and dry ice,as a source of CO_2,were employed to attract mosquitoes.Mosquitoes were first identified,pooled into groups of upto 50 mosquitoes by species,and tested for JEV infection by viral isolation and reverse transcriptase polymerase chain reaction.Results:A total of 20370 mosquitoes comprising 14 species in five genera were collected.The five most abundant mosquito species collected were Culex tritaeniorhynchus(95.46%),Culex vishnui(2.68%),Culex gelidus(0.72%),Anopheles peditaeniatus(0.58%)and Culex quinquefasciatus(0.22%).Mosquito peak densities were observed in July.All of 416 mosquito pools were negative for JEV.Conclusions:This study provides new information about mosquito species and status of JEV infection in mosquitoes in Thailand.Further study should be done to continue a close survey for the presence of this virus in the ardeid birds.

Evaluation of biological control agents for mosquitoes control in artificial breeding places

Objective:To evaluate the entomological impact of chlorpyrifos-methyl,Bacillus thuringiensis, and Gambusia affinis on mosquitoes control in artificial breeding places.Methods:A Latin square design with 4 replicates was performed in order to evaluate the efficacy of chlorpyrifosmethyl, Bacillus thuringiensis,and Gambusia affinis on larva.The larvicide was applied at the dosage of 100 mg a.h/ha,Bacillus thuringiensis at the recommended dosage and 10 fishes per m^2 were applied at 1×1 m^2 artificial breeding sites.The larval densities for both anopheline and culicine were counted according to larvae /10 dippers prior and 24 h after application.Results: All three control agents are effective for mosquito density reduction,and the difference between the three agents and the control is significant(P【0.05).There is also significant difference among chlorpyrifos-methyl,Bacillus thuringiensis and Gambusia affinis.Bacillus thuringiensis exhibited more reduction on mosquito larval density than fish and larvicide(P【0.05).Conclusions: Bacillus thuringiensis in comparison with two other agents is the appropriate method for larviciding in the breeding places.Although long term assessing for biological activities as well as monitoring and mapping of resistance is required.

Immatures of Lutzia fuscana(Wiedemann,1820)(Diptera:Culicidae) in ricefields:implications for biological control of vector mosquitoes

Objective:Rice fields are dynamic mosquito larval habitats with assemblage of different predator taxa,including the larva of the mosquitoes Lutzia.Entomological surveillance in the ricefields is essential to evaluate the potential of these predators as biological resource to regulate vector mosquito population.In view of this,a survey of ricefields for immatures of different mosquito species including Lutzia was conducted.Methods:Survey of selected ricefields was carried out to evaluate the species composition of mosquitoes.Laboratory evaluation of the immatures of Lutzia mosquitoes was carried out to assess its predation potential using mosquitoes and chironomid as preys.Results:The survey revealed the presence of five mosquito species belonging to the genera Anopheles and Culex and the predatory immatures of the mosquito Lutzia Juscana(Wiedemann,1820).The ratio of prey and predatory larva ranged between 1.46 and 4.78 during the study period,with a significant correlation on the relative abundance of the larval stages of Lt.Juscana and Anopheles and Culex larvae.Under laboratory conditions,a single IV instar larvae of Lt.fuscana was found to consume on an average 5 to 15 equivalent instars of Anopheles sp.and Culex sp.larvae per day depending on its age.The prey consumption reduced with the larval stage approaching pupation.When provided with equal numbers of chironomid and Anopheles or Culex larvae,larva of Lt.fuscana consumed mosquito larvae significantly more compared to chironomids.Conclusion: The survey results and the preliminary study on predation are suggestive of the role of Lt.Juscana in the regulation of vector mosquito populations naturally in the ricefields.Since Lt.Juscana is common in many Asian countries,further studies on bioecology will be helpful to justify their use in mosquito control programme.

Modelling of the Abundance of Malaria Mosquitoes Using Poisson Mixed Model

Anopheles funestus and Anopheles gambiae are malaria vector mosquitoes. Knowing their resting behavior is important for implementing control methods. The aim of this study was to investigate the resting behaviour of the two malaria mosquitoes. The study was conducted in Kilombero River Valley and mosquitoes were collected using indoor and outdoor traps from 2012-2015. Poisson mixed models were used to quantify the impact of environment variables on resting behaviour. A log ratio rate between the type of trap and its interaction with environmental variables was used to determine if there was a change over time in the resting behaviour. A total of 4696 mosquitoes were resting indoors of which 57% were A. funestus and 43% were A. gambiae. Similarly, a total of 12,028 mosquitoes were resting outdoor of which 13% were A. funestus and 87% were A. gambiae. Temperature was significant and affected the resting behaviour of A. funestus. Humidity, saturation deficit and temperature were significant variables influencing the resting behaviour of A. gambiae. A. funestus was resting indoor while A. gambiae was resting outdoor over time generally. The findings of this study on the effects of environmental variables and the variations in the resting behaviour of A. gambiae and A. funestus could be used as a guide to implementing appropriate intervention measures such as indoor residential spraying (IRS), insecticide treated nets (ITNs) and mosquito repellents.

Mitogenome-based phylogeny of mosquitoes(Diptera:Culicidae)

Mosquitoes are of great medical significance as vectors of many deadly diseases.Mitogenomes have been widely used in phylogenetic studies,but mitogenome knowledge within the family Culicidae is limited,and Culicidae phylogeny is far from resolved.In this study,we surveyed the mitogenomes of 149 Culicidae species,including 7 newly sequenced species.Comparative analysis of 149 mosquito mitogenomes shows gene composition and order to be identical to that of an ancestral insect,and the AT bias,length variation,and codon usage are all consistent with that of other reported Dipteran mitogenomes.Phylogenetic analyses based on the DNA sequences of the 13 protein-coding genes from the 149 species robustly support the monophyly of the subfamily Anophelinae and the tribes Aedini,Culicini,Mansonini,Sabethini,and Toxorhynchitini.To resolve ambiguous relationships between clades within the subfamily Culicinae,we performed topological tests and show that Aedini is a sister to Culicini and that Uranotaenini is a sister to(Mansonini)+(Toxorhynchitini+Sabethini).In addition,we estimated divergence times using a Bayesian relaxation clock based on the sequence data and 3 fossil calibration points.The results show mosquitoes diverged during the Early Jurassic with massive Culicinae radiations during the Cretaceous,coincident with the emergence of angiosperms and the burst of mammals and birds.Overall,this study,which uses the largest number of Culicidae mitogenomes sequenced to date,comprehensively reveals the mitogenome characteristics and mitogenome-based phylogeny and divergence times of Culicidae,providing information for further studies on the mitogenome,phylogeny,evolution,and taxonomic revision of Culicidae.

Evolutionary genomics analysis reveals gene expansion and functional diversity of arylalkylamine N-acetyltransferases in the Culicinae subfamily of mosquitoes

Arylalkylamine N-acetyltransferase(aaNAT),considered a potential new insecticide target,catalyzes the acetylation of arylalkylamine substrates such as serotonin and dopamine and,hence,mediates diverse functions in insects.However,the origin of insect aaNATs(iaaNATs)and the evolutionary process that generates multiple aaNATs in mosquitoes remain largely unknown.Here,we have analyzed the genomes of 33 species to explore and expand our understanding of the molecular evolution of this gene family in detail.We show that aaNAT orthologs are present in Bacteria,Cephalochordata,Chondrichthyes,Cnidaria,Crustacea,Mammalia,Placozoa,and Teleoste,as well as those from a number of insects,but are absent in some species of Annelida,Echinozoa,and Mollusca as well as Arachnida.Particularly,more than 10 aaNATs were detected in the Culicinae subfamily of mosquitoes.Molecular evolutionary analysis of aaNAT/aaNAT-like genes in mosquitoes reveals that tandem duplication events led to gene expansion in the Culicinae subfamily of mosquitoes more than 190 million years ago.Further selection analysis demonstrates that mosquito aaNATs evolved under strongly positive pressures that generated functional diversity following gene duplication events.Overall,this study may provide novel insights into the molecular evolution of the aaNAT family in mosquitoes.

Mosquito densovirus signifcantly reduces the vector susceptibility to dengue virus serotype 2 in Aedes albopictus mosquitoes(Diptera:Culicidae)

Background Dengue virus(DENV)is a major public health threat,with Aedes albopictus being the confrmed vector responsible for dengue epidemics in Guangzhou,China.Mosquito densoviruses(MDVs)are pathogenic mosquitospecifc viruses,and a novel MDV was previously isolated from Ae.albopictus in Guangzhou.This study aims to determine the prevalence of MDVs in wild Ae.albopictus populations and investigate their potential interactions with DENV and impact on vector susceptibility for DENV.Methods The prevalence of MDV in wild mosquitoes in China was investigated using open access sequencing data and PCR detection in Ae.albopictus in Guangzhou.The viral infection rate and titers in MDV-persistent C6/36 cells were evaluated at 12,24,48,72,96,and 120 h post infection(hpi)by indirect immunofuorescence assay(IFA)and real time quantitative PCR(RT-qPCR).The midgut infection rate(MIR),dissemination rate(DR),and salivary gland infection rate(SGIR)in various tissues of MDV-infected mosquitoes were detected and quantifed at 0,5,10,and 15 days post infection(dpi)by RT-PCR and RT-qPCR.The chi-square test evaluated dengue virus serotype 2(DENV-2)and Aedes aegypti densovirus(AaeDV)infection rates and related indices in mosquitoes,while Tukey’s LSD and t-tests compared viral titers in C6/36 cells and tissues over time.Results The results revealed a relatively wide distribution of MDVs in Aedes,Culex,and Anopheles mosquitoes in China and an over 68%positive rate.In vitro,signifcant reductions in DENV-2 titers in supernatant at 120 hpi,and an appar‑ent decrease in DENV-2-positive cells at 96 and 120 hpi were observed.In vivo,DENV-2 in the ovaries and salivary glands was frst detected at 10 dpi in both monoinfected and superinfected Ae.albopictus females,while MDV super‑infection with DENV-2 suppressed the salivary gland infection rate at 15 dpi.DENV-2 titer in the ovary and salivary glands of Ae.albopictus was reduced in superinfected mosquitoes at 15 dpi.Conclusions MDVs is widespread in natural mosquito populations,and replication of DENV-2 is suppressed in MDVinfected Ae.albopictus,thus reducing vector susceptibility to DENV-2.Our study supports the hypothesis that MDVs may contribute to reducing transmission of DENV and provides an alternative strategy for mosquito-transmitted disease control.

Mosquito Control, Killing off the Females

In addition to causing discomfort, female mosquitoes introduce disease-carrying viruses and bacteria into the bloodstream of their victims. There are numerous publications describing the uses of sugary mosquito baits with promising results. Without temperature control measures however, these methods are mainly useful for only nectar-feeding insects, including male mosquitoes, because the warmth of the blood is a condition for the females to locate their meals. The efforts required to keep the baits fresh against the natural spoiling process make them less attractive or impractical to implement. These experiments address these issues by using warm baits of water, sugar, boric acid, and antibiotics. Overnight, the general areas became clear of blood-sucking female mosquitoes while in numbers, the harmless males concentrated into the immediate vicinities. Control vs. experiment protocol established no other logical explanation for this phenomenon other than that females were attracted and killed by the bait. As expected, there was no female mosquito’s activity in these areas. There weren’t many left to do the work.

Pyrethroid resistance in mosquitoes

Repeated blood feedings throughout their life span have made mosquitoes ideal transmitters of a wide variety of disease agents. Vector control is a very important part of the current global strategy for the control of mosquito-associated diseases and insecticide application is the most important component in this effort. Pyrethroids, which account for 25% of the world insecticide market, are currently the most widely used insecticides for the indoor control of mosquitoes and are the only chemical recommended for the treatment of mosquito nets, the main tool for preventing malaria in Africa. However, mosquito-borne diseases are now resurgent, largely because of insecticide resistance that has developed in mosquito vectors and the anti-parasite drug resistance of parasites. This paper reviews our current knowledge of the molecular mechanisms governing metabolic detoxification and the development of target site insensitivity that leads to pyrethroid resistance in mosquitoes.