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-

Monitoring Report on Mosquitoes at Ports of Four Regions in East China during 2011—2014

[Objective] The aim was to understand the species composition, distribution, density, seasonal fluctuation and pathogens of mosquitoes in four regions of East China to provide basic data for prevention and control of mosquitoes at ports. [Method] The mosquitoes at 21 ports of four regions in East China were investigated during 2011-2014 by CO2 lamp method, egg collection method, scoop method and visual method. The species of mosquitoes was identified, and pathogens in mosquitoes were detected. [Result] From 2011 to 2014, 102 583 mosquitoes were captured at the ports, belonging to 18 species, 6 genera and 1 family. Culex pipi- ens quinquefasciatus or Culex pipiens pallens was the dominant species at most ports. Mosquitoes could be captured from May to October, and the captured mosquitoes were the most in June. The positive rate of egg collectors was 9.41%, and the positive rate of breeding places was 7.23%. Rare entry mosquitoes were captured, and most of them were C. pipiens pallens. The detection results of dengue fever, malaria and other pathogens were negative. [Conclusion] C. pipiens quinquefasciatus or C. pipiens pallens was dominant at most ports, and the peak period was June. There were breeding environments of mosquitoes at some ports, so the prevention and control of mosquitoes at these ports should be paid more at- tention to in future.

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.

Application of DNA Barcode Technology to the Identification of Exotic Mosquitoes at Ningbo Port

[Objective] The study aimed to solve the problem of morphological identi- fication difficulties and propose solutions for the identification of foreign mosquitoes that are difficult to identify. [Method] Based on the sequencing, alignment and anal- ysis of COl gene fragment, DNA barcode technology was used to identify 7 exotic mosquitoes, and the phylogenetic analysis was performed using MEGA6.0 and DNASTAR software. Then the morphological characteristics of the mosquitoes were reviewed. [Result[ These mosquitoes were Armigeres subalbatus, Culex gefidus, Anopheles gambiae, and Culiseta incidens. [Conclusion] DNA barcode technology is a useful supplement to the morphological classification method of 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.

Factors Affecting the Ovilarval Density of Aedes Spp. Mosquitoes in Selected Rice Fields of Mu^oz, Nueva Ecija

Variables among the macroclimate, microclimate and rice canopy categories and three other different farming systems were evaluated on their effects to the egg and larval density of Aedes spp. mosquitoes known as transmitters of animal and human diseases. No statistical difference in egg density （#eggs/mL） among farming systems （P = 0.345） were observed. However, there was significant difference in larval density （#1arvae/mL） among farming systems （P 〈 0.001） particularly between organic and conventional farms and between organic and mixed farms at （P 〈 0.05）. Among the variables in the macroclimate category, wind velocity and ambient temperature significantly influenced larval density in conventional farms. Among the variables in the microclimate category, water temperature significantly contributed to larval density in both the mixed and conventional farms whereas water turbidity, in conventional farms. Among the variables in the rice canopy category, the number of tillers per plant was a significant contributor to larval density in all farm types. No variable among the environmental exposure categories affected the larval density in organic farms.

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.

Our Experience in Collecting Tiger Mosquitoes Using Ovitraps in Strumica, Gevgelija and the Border Crossing to Greece

Collecting eggs of Aedes （Stegomyia） albopictus （Skuse, 1894） （Diptera, ram. Culicidae）, i.e. Asian tiger mosquito was carded out in August 2016. Fourteen （14） ovitraps were placed at 3 different places： the area Banja Bansko （near Strumica）, Mrzenci （Gevgelija） and at the border crossing between Macedonia-Greece （Bogorodica）. During one month the ovitraps were exposed for two weeks, near the vegetation that mosquitoes use as resting places, also in the places where people are present and close to potted flower arrangements. The collected eggs from the ovitraps were returned to the Entomological laboratory of the P.H.O. Center for public health-Skopje and put in an entomological （hand-made） cage. Under the optimum moisture and temperature, after a few days the adults emerged. The determination of species was performed using a binocular magnifier. A total of 50 mosquitoes were collected, of those 23 were male Ae. albopictus and 27 were female Ae. albopictus. These examples were verified by prof. Dusan Petric and the Medical entomology laboratory at the Faculty of Agficul^tre Novi Sad （Republic of Serbia）. The presence of Ae. albopictus in the Republic of Macedonia was for the first time determined. We are now even more motivated to investigate and prove the presence of tiger mosquitos in other locations in Macedonia.

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.