Control Of Larvae Research Articles

Larvicidal Activity of Brugmansia candida against Aedes aegypti and Culex quinquefasciatus (Diptera: Culicidae)

Mosquitoes are well known as vectors of hazardous diseases for human. Plant extracts can be used as an alternative for larval control due to they are a rich source of bioactive chemicals and safe for the environment. The present study investigated the larvicidal activity of crude extracts derived from leaf and flower of Brugmansia candida against the second larval instar of Aedes aegypti and Culex quinquefasciatus . The larval mortality was observed at 24 and 48 h exposure of both leaf and flower extracts, at the concentration of 100, 250, 500, and 1000 ppm. The 24 h exposure of both extracts at the concentration of 500 and 1000 ppm resulted in larval mortality rates were significantly lower than those of 48 h exposure. However, the mortality rate was not significantly different at the lower concentrations of crude extracts. The results also suggested that there was no significant difference in the larvicidal effect between leaf and flower extracts at 24 and 48 h exposure for all concentrations. The LC 50 values at 48 h exposure for leaf extract were 789 and 791 ppm for Ae . aegypti and C . quinquefasciatus , respectively, whereas for flower extract were 772 and 780 ppm for Ae . aegypti and C . quinquefasciatus , respectively. Overall, B . candida showed larvicidal activity against Ae . aegypti and C . quinquefasciatus . This research contributes to new finding regarding the larvicidal activity of B . candida . This finding also supports the next study to develop B . candida as an alternative source for larval control agent.

Auto-dissemination strategy for larval control of the insecticide-resistant dengue vector Aedes aegypti in Lao PDR

Background: Dengue is reemerging in Laos and the most important recent outbreak was in 2013, with 44,098 cases. In 2019 (September), 27,904 dengue cases, including 54 deaths, were reported. Larviciding is one of the best options for larval control and in Laos, the organophosphate temephos has been the only larvicide used for vector control for decades. Recent studies showed that Aedes aegypti populations from Vientiane were resistant to this insecticide. In this context of dengue reemergence combined with high rates of urbanization, there was an urgent need to provide the public health authorities with new strategies, including new insecticides. We tested the efficacy of an innovative program using the auto-dissemination technique with the use of the In2Care® trap system in semi-field and large-scale field trials. Methods and materials: The auto-dissemination method using the pupacide insect growth regulator pyriproxyfen, was first investigated on the premises of the Institut Pasteur du Laos with 25 traps. This method was also implemented under large-scale field trials, in 2 dengue hotspots of Vientiane. In total, 600 traps were installed in 2 villages covering 1.2 hectares in each village. Aedes sp. densities were measured weekly with adult/larval traps and monthly with entomological indexes. Two other villages were selected as controls. Results: The small-field trial results showed a significant reduction of larvae and adult Aedes aegypti abundance during the treatment. The one year study in large-field trial has just finished and the results will allow us to test the hypothesis that Aedes densities measured weekly with adult/larval traps and monthly with entomological indexes, can decrease dramatically in the intervention zones vs. non-intervention zones. Conclusion: The results showed that the pyriproxyfen auto-dissemination strategy can represent an effective strategy to reduce mosquito densities in specific and determined areas and may represent a potential tool to be used in combination with other vector control strategies in Laos.

Effect of community-based intervention on awareness of dengue and its prevention among urban poor community in India: a systematic review approach

Owing to increased epidemic activity and difficulties in controlling the insect vector, dengue has become a major public health problem globally. The prevention and control of dengue rely mainly on vector control methods. A systematic review was conducted using four databases (PubMed, Cochrane, Google Scholar and Scopus) and a manual search of the reference lists of the identified studies. Data from included studies were extracted, analyzed. Applying all inclusion and exclusion criteria 23 articles was included. Further relevant articles using this keywords Aedes dengue breeding habits housing and community intervention were selected. Eight studies combined community participation programme with dengue control tools. Findings of the published literature indicate that at baseline, almost half the respondents did not know that dengue is serious but preventable, or that it is transmitted by mosquitoes. The analysis showed that dengue vector control is carried out by vertically structured programmes of national, state, and local administrative bodies through fogging and larval control, without any involvement of community-based organizations, and that vector control efforts were conducted in an isolated and irregular way. The most productive container types for Aedes pupae were cement tanks, drums, and discarded containers. Evidence that community-based dengue control programmes alone and in combination with other control activities can enhance the effectiveness of dengue control programmes is weak. This review demonstrates a paucity of reliable evidence for the effectiveness of any dengue vector control method. Standardized studies of higher quality to evaluate and compare methods must be prioritized to optimize cost-effective dengue prevention. Clear best practice guidelines for the methodology of entomological studies should be developed.

Larvicidal Activity of Bintaro (Cerbera odollam) against Culex quinquefasciatus

Mosquitoes are known for their bites which caused discomfort and its role as vector for various dangerous diseases that has been the bane of humanity since antiquity. One of them is Culex quinquefasciatus which facilitates the spread of arbovirus and nematodes that caused debilitating impact for human life. To eliminate mosquito, the study for evaluating plant-based mosquito larvicidal extract of Cerbera odollam extract against C. quinquefasciatus was conducted. Twenty-five second instar of C. quinquefasciatus were subjected to the crude and fractionated extracts of C. odollam, and the larval mortality was observed at 24, and 48 h of exposure. The results showed that the crude extracts of leaves, rinds, and stem bark exhibited low larvicidal activities. Interestingly, crude extract of the seed kernel exhibited high larvicidal activity. We found that the fractionated extract derived from the seed kernel extract using ethyl acetate and n-hexane exhibited high larvicidal activities, suggesting that C. odollam extract could be promoted as a material for larval control.

Toxicity of two groups of pesticides against the mosquito Aedes aegypti

The mosquito Aedes aegypti (Linnaeus) (Diptera: Culicidae) is a vector for several pathogens that affect human health worldwide. Therefore, mosquito control is the best approach to prevent disease outbreaks. In this milieu, it is preferable to evaluate the effectiveness of chemical pesticides at regular intervals to identify the most effective ones and use them during the outbreaks of diseases and spread of pests. Here, we aimed to study the toxicity of six pesticides, which are classified under two groups, namely pyrethroids and organophosphates, against A. aegypti mosquitoes to improve disease control in Saudi Arabia. Hortak was the most effective in larval mosquito control (LC50 = 0.0031 ppm), followed by Aquapal Super 20 EW (LC50 = 0.0389 ppm), whereas Solfac was the least effective (LC50 = 0.1119 ppm). In addition, the sensitivity of the tested larvae to Safrotin and Keen 600 EC was 8.1 and 58.9 times higher than that to Resfin-5, which was the least effective, respectively. Hortak and Safrotin exhibited the highest toxicity against the larvae of A. aegypti. Our findings confirm that the tested pesticides can be used in mosquito-control programs during epidemic outbreaks and emergency.

New distribution record of Anopheles rivulorum-like from Sadiola, Mali, with notes on malaria vector insecticide resistance.

The SEMOS gold mine in Sadiola, southwestern Mali, has been implementing a malaria vector control programme for 15y using indoor residual house spraying and sporadic larval control. Periodic screening of the vector populations have been carried out over the years to provide information to the control programme, mainly on vector species present and their insecticide resistance status. The data from five entomological surveys, carried out in 2006, 2011, 2014, 2016 and 2018, are presented. Adult mosquitoes were collected resting on walls inside houses and on verandas. Insecticide susceptibility assays were carried out and mosquitoes subsequently identified by species using molecular assays. The major malaria vector mosquitoes, Anopheles gambiae and Anopheles arabiensis were abundant at each sampling period with Anopheles coluzzii and Anopheles funestus being rare or absent. Anopheles rivulorum was identified in 2006 and Anopheles leesoni in 2016. The presence of Anopheles rivulorum-like, identified for the first time in 2018, was not screened for in previous surveys. Insecticide susceptibility bioassays showed resistance in both A. gambiae and A. arabiensis to pyrethroids, carbamates and dichlorodiphenyltrichloroethane over the 12y. This is the first record of A. rivulorum-like west of Côte d'Ivoire. Resistance levels to the three classes of insecticides were variable but appeared to decrease after pyrethroids were discontinued for house spraying.

COVID-19 lockdown: impact assessment on Aedes larval indices, breeding habitats, effects on vector control programme and prevention of dengue outbreaks

Aedes aegypti (Linn.) and Aedes albopictus (Skuse) are widespread vector mosquitoes responsible for the transmission of various disease-causing viruses to human including dengue virus (DENV). India is endemic for dengue disease and both of these vector mosquitoes are well established throughout India. Since, Aedes mosquitoes breeds in containers, WHO recommends to do a regular immature surveillance and implement appropriate control measures. Owing to the current COVID-19 pandemic, most of the countries have implemented continuous shutdown/lockdown, which affected the routine Aedes surveillance and vector control measures. In India, the first nation-wide lockdown was implemented on 24th, March 2020. As of now, Government of India has extended the lockdown till 30th, June 2020. In the present study, two rounds of Aedes surveillance was carried out in two localities of Bengaluru City (urban) of Karnataka State, India during the COVID-19 lockdown days and results were compared with pre-lockdown surveillance data to assess the impact of lockdown on Aedes larval indices, breeding habitats and dengue vector control programme. The recorded house index (HI) and Breteau index (BI) were 6.6 and 9.3 in K.P. Agrahara and 4.0 and 5.3 in Palace Guttahalli during pre-lockdown survey. The house index (HI) and Breteau index (BI) were found to be increased to 26.6 and 34.6 in K.P. Agrahara and 21.3 and 28.0 in Palace Guttahalli during the COVID-19 lockdown second survey. Aedes immature density has drastically increased in both the localities due to temporarily discontinued Aedes surveillance, larval control activities like source reduction and anti-larval measures during COVID-19 lockdown. The high indices show that the vector is increasing and this may result in higher dengue virus transmission. The results highly recommend to implement the Aedes vector control programme with limited health staffs following the physical distance and other protectives measures to prevent dengue outbreaks.

Spatial and temporal distribution of Anopheles mosquito's larvae and its determinants in two urban sites in Tanzania with different malaria transmission levels

BackgroundIn order to be able to design and implement control measures directed to the mosquito larva stages an understanding of the spatial and temporal distribution and its determinants in different malaria transmission settings is important. This study therefore, intended to determine the spatial and temporal distribution of Anopheles mosquito's larvae and its determinants in two urban sites with different transmission levels, in Tanzania. MethodologyThis study was conducted in Dodoma and Morogoro regions in Tanzania. The study was an ecological study of repeated cross-sectional type. Searching for water bodies in the selected wards was done by going around all streets. Potential breeding sites were given unique identification numbers and larval sampling was done using the standard dipping method with a 350 ml mosquito scoop and a calibrated pipette. Visual identification of presence of larvae and its abundance in each sampling were used to describe the larvae density. A sample of Anopheles mosquitoes which emerged from collected larvae, were processed for species identification using PCR. Descriptive statistics were arrived at by calculating different proportions for the variables. The overall impact of the variables on the density of Anopheles larvae was tested using multiple logistic regression. Variables with p-value less than 0.05 were regarded as significant. ResultsA total of 724 water bodies out of which, 576 (79.6%) potential breeding sites were analyzed. It was found that, most (96.2%) of the potential breeding sites were manmade and most (59.5%) were less than 5 m in diameter and 87.2% were within 100 m from human settlement. Out of all the potential breeding sites, 69.8% and 30.2% were in Morogoro and Dodoma respectively, out of which 72.2% and 68.4% respectively, were found during rainy season. Habitats with clean water, at a distance of 10–100 m from the house, in natural, shaded and partial sunlight habitats had higher odds of having high density of mosquito larvae than their counterparts (p < .05). The PCR analysis showed that 72.5% were An. arabiensis, 4.5% An. gambiaes.s, 0.5% An.coustaniand 20% An. quadrianulatuswhile 2.5% of the samples could not be identified because DNA was not amplified. Conclusion and recommendationType of water, distance from the breeding site to human settlement, light intensity and habitat origin were significant predictors of variation on the spatial and temporal distribution of Anopheles mosquito breeding sites. With increased global emphasis on control measures that targets mosquito immature stages; we recommend that larval control measures should be developed while considering the findings from this study.

Community acceptance of yeast interfering RNA larvicide technology for control of Aedes mosquitoes in Trinidad.

RNA interference (RNAi), a technique used to investigate gene function in insects and other organisms, is attracting attention as a potential new technology for mosquito control. Saccharomyces cerevisiae (baker’s yeast) was recently engineered to produce interfering RNA molecules that silence genes required for mosquito survival, but which do not correspond to genes in humans or other non-target organisms. The resulting yeast pesticides, which facilitate cost-effective production and delivery of interfering RNA to mosquito larvae that eat the yeast, effectively kill mosquitoes in laboratory and semi-field trials. In preparation for field evaluation of larvicides in Trinidad, a Caribbean island with endemic diseases resulting from pathogens transmitted by Aedes mosquitoes, adult residents living in the prospective trial site communities of Curepe, St. Augustine, and Tamana were engaged. Open community forums and paper surveys were used to assess the potential acceptability, societal desirability, and sustainability of yeast interfering RNA larvicides. These assessments revealed that Trinidadians have good working knowledge of mosquitoes and mosquito-borne illnesses. A majority of the respondents practiced some method of larval mosquito control and agreed that they would use a new larvicide if it were proven to be safe and effective. During the community engagement forums, participants were educated about mosquito biology, mosquito-borne diseases, and the new yeast larvicides. When invited to provide feedback, engagement forum attendees were strongly supportive of the new technology, raised few concerns, and provided helpful advice regarding optimal larvicide formulations, insecticide application, operational approaches for using the larvicides, and pricing. The results of these studies suggest that the participants are supportive of the potential use of yeast interfering RNA larvicides in Trinidad and that the communities assessed in this investigation represent viable field sites.

Assessment of selected larvicides for the control of Culex pipiens biotype pipiens and Culex pipiens biotype molestus under laboratory and semi-field conditions.

The insecticidal efficacy of selected mosquito larvicides [teflubenzuron, S-methoprene, diflubenzuron, temephos, Bacillus thuringiensis var. israelensis (Bti) and polydimethylsiloxane (PDMS)], applied alone or in combination, against two Culex pipiens biotypes, Cx. p. pipiens and Cx. p. molestus, was compared under laboratory and semi-field conditions. Registered larvicides were evaluated at the label dose, whereas those not registered for mosquito control were tested at the label dose per area, i.e. as plant protection products. Teflubenzuron and temephos were also evaluated at half the label dose, alone or in combination with a non-ionic surfactant. Larval mortality and adult emergence were assessed after 1, 3, 5, 7, 9, 11, 16 and 20 days of exposure. In all cases, PDMS showed complete (100%) larval control after 3 days of exposure against both biotypes. With temephos, all larvae were dead after 1 day of exposure, even at half the label dose. For teflubenzuron, mortality reached 100% at the end of the bioassays and adult emergence was avoided completely, whereas for diflubenzuron, mortality was close to 100% at the end of the trials. By contrast, in S-methoprene-treated water, control was below the 90% mortality threshold for both biotypes. In semi-field trails, in all treatments with Bti, all larvae of both biotypes were dead after 1 day of exposure. Based on our data, and with the exception of S-methoprene, the larvicides tested remained below the threshold for effective treatment against larvae of Cx. p. pipiens and Cx. p. molestus for the entire duration of the study. Most of the larvicides tested showed high levels of efficacy against Cx. p. pipiens and Cx. p. molestus larvae in laboratory and semi-field bioassays. © 2020 Society of Chemical Industry.

Coordination among neighbors improves the efficacy of Zika control despite economic costs.

Emerging mosquito-borne viruses like Zika, dengue, and chikungunya pose a major threat to public health, especially in low-income regions of Central and South America, southeast Asia, and the Caribbean. Outbreaks of these diseases are likely to have long-term social and economic consequences due to Zika-induced congenital microcephaly and other complications. Larval control of the container-inhabiting mosquitoes that transmit these infections is an important tool for mitigating outbreaks. However, metapopulation theory suggests that spatiotemporally uneven larvicide treatment can impede control effectiveness, as recolonization compensates for mortality within patches. Coordinating the timing of treatment among patches could therefore substantially improve epidemic control, but we must also consider economic constraints, since coordination may have costs that divert resources from treatment. To inform practical disease management strategies, we ask how coordination among neighbors in the timing of mosquito control efforts influences the size of a mosquito-borne infectious disease outbreak under the realistic assumption that coordination has costs. Using an SIR (Susceptible-Infectious-Recovered)/metapopulation model of mosquito and disease dynamics, we examine whether sharing surveillance information and coordinating larvicide treatment among neighboring patches reduces human infections when incorporating coordination costs. We examine how different types of coordination costs and different surveillance methods jointly influence the effectiveness of larval control. We find that the effect of coordination depends on both costs and the type of surveillance used to inform treatment. With epidemiological surveillance, coordination improves disease outcomes, even when costly. With demographic surveillance, coordination either improves or hampers disease control, depending on the type of costs and surveillance sensitivity. Our results suggest coordination among neighbors can improve management of mosquito-borne epidemics under many, but not all, assumptions about costs. Therefore, estimating coordination costs is an important step for most effectively applying metapopulation theory to strategies for managing outbreaks of mosquito-borne viral infections.

Regional Survey of Diamondback Moth (Lepidoptera: Plutellidae) Response to Maximum Dosages of Insecticides in Georgia and Florida.

We conducted maximum dose bioassays of insecticide for the control of diamondback moth (DBM), Plutella xylostella (Linnaeus), in cole crops, from 2016 to 2019 at several commercial locations in Georgia and Florida. The nominal maximum dose was defined as the highest labeled rate of an insecticide at the beginning of the survey in the equivalent of 935 liters/ha dilution. The results indicated low insecticide efficacy for high labeled rates of the following insecticides by common name (Insecticide Resistance Action Committee group number in parentheses). Our 4-yr survey identified very low levels of DBM larval control (<47%) by lambda-cyhalothrin (3), methoxyfenozide (18), pyriproxyfen (7C), novaluron (15), bifenthrin (3), chlorantraniliprole (28), indoxacarb (22A), and methomyl (1A). The best products for DBM control (>74%) listed in decreasing average levels of efficacy were naled (1B), cyclaniliprole (28), tolfenpyrad (21A), emamectin benzoate (6), and cyantraniliprole (28). Intermediate levels of control (61-71%) were obtained with Bacillus thuringiensis subspecies aizawai (11A), Bacillus thuringiensis, subsp. kurstaki, strain ABTS-351 (11A), and spinetoram (5). This rapid bioassay provided the grower with a ranking of insecticide efficacy for the control the DBM population for that farm site. These data allowed growers to make an informed decision on control quickly and plan for resistance management rotations for DBM that season.

Community factors affecting participation in larval source management for malaria control in Chikwawa District, Southern Malawi

BackgroundTo further reduce malaria, larval source management (LSM) is proposed as a complementary strategy to the existing strategies. LSM has potential to control insecticide resistant, outdoor biting and outdoor resting vectors. Concerns about costs and operational feasibility of implementation of LSM at large scale are among the reasons the strategy is not utilized in many African countries. Involving communities in LSM could increase intervention coverage, reduce costs of implementation and improve sustainability of operations. Community acceptance and participation in community-led LSM depends on a number of factors. These factors were explored under the Majete Malaria Project in Chikwawa district, southern Malawi.MethodsSeparate focus group discussions (FGDs) were conducted with members from the general community (n = 3); health animators (HAs) (n = 3); and LSM committee members (n = 3). In-depth interviews (IDIs) were conducted with community members. Framework analysis was employed to determine the factors contributing to community acceptance and participation in the locally-driven intervention.ResultsNine FGDs and 24 IDIs were held, involving 87 members of the community. Widespread knowledge of malaria as a health problem, its mode of transmission, mosquito larval habitats and mosquito control was recorded. High awareness of an association between creation of larval habitats and malaria transmission was reported. Perception of LSM as a tool for malaria control was high. The use of a microbial larvicide as a form of LSM was perceived as both safe and effective. However, actual participation in LSM by the different interviewee groups varied. Labour-intensiveness and time requirements of the LSM activities, lack of financial incentives, and concern about health risks when wading in water bodies contributed to lower participation.ConclusionCommunity involvement in LSM increased local awareness of malaria as a health problem, its risk factors and control strategies. However, community participation varied among the respondent groups, with labour and time demands of the activities, and lack of incentives, contributing to reduced participation. Innovative tools that can reduce the labour and time demands could improve community participation in the activities. Further studies are required to investigate the forms and modes of delivery of incentives in operational community-driven LSM interventions.

Optimizing the use of entomopathogenic nematodes for the management of Listronotus maculicollis (Coleoptera: Curculionidae): Split applications and combinations with imidacloprid

Entomopathogenic nematodes have shown potential as biocontrol agents targeting larvae of the annual bluegrass weevil, Listronotus maculicollis Kirby, a major golf course turf pest in eastern North America with widespread insecticide resistance. Control levels of single applications of nematodes have been too low and variable in the past to support application by golf course managers for weevil control. We examined if splitting nematode applications in two at half rates approximately 1 week apart as well as combinations of the nematodes with the neonicotinoid imidacloprid could improve nematode performance. In greenhouse experiments in pots with grass infested with ABW larvae, the entomopathogenic nematodes Steinernema carpocapsae, S. feltiae, and Heterorhabditis bacteriophora were similarly effective for larval control. Combination with imidacloprid resulted in additive weevil mortality. In a field experiment with natural infestations of larvae using 1.25 and 2.5 × 109 nematodes ha−1, respectively, of S. carpocapsae and H. bacteriophora, additive mortality in the combinations with imidacloprid amounted to 82% and 79% control, respectively, at the high nematode rate. In three additional field experiments using only S. carpocapsae, the low and high nematode rate provided on average 59% and 74% control, respectively, while their combinations with imidacloprid provided 75% and 84% control, respectively. Imidacloprid alone averaged 37% control. Mortality in all combination treatments was additive. Splitting S. carpocapsae applications provided a marginal increase in mortality that was significant only in one of three experiments. However, combining split applications with imidacloprid provided 88–95% control which could offer an effective alternative to synthetic insecticides.

Characterisation of anopheline larval habitats in southern Malawi

IntroductionIncreasing the knowledgebase of anopheline larval ecology could enable targeted deployment of malaria control efforts and consequently reduce costs of implementation. In Malawi, there exists a knowledge gap in anopheline larval ecology and, therefore, basis for targeted deployment of larval source management (LSM) for malaria control, specifically larvicides. We set out to characterize anopheline larval habitats in the Majete area of Malawi on the basis of habitat ecology and anopheline larval productivity to create a basis for larval control initiatives in the country. MethodsLongitudinal surveys were conducted in randomly selected larval habitats over a period of fifteen months in Chikwawa district, southern Malawi. Biotic and abiotic parameters of the habitats were modelled to determine their effect on the occurrence and densities of anopheline larvae. ResultsSeventy aquatic habitats were individually visited between 1-7 times over the study period. A total of 5,123 immature mosquitoes (3,359 anophelines, 1,497 culicines and 267 pupae) were collected. Anopheline and culicine larvae were observed in sympatry in aquatic habitats. Of the nine habitat types followed, dams, swamps, ponds, borehole runoffs and drainage channels were the five most productive habitat types for anopheline mosquitoes. Anopheline densities were higher in aquatic habitats with bare soil making up part of the surrounding land cover (p<0.01) and in aquatic habitats with culicine larvae (p<0.01) than in those surrounded by vegetation and not occupied by culicine larvae. Anopheline densities were significantly lower in highly turbid habitats than in clearer habitats (p<0.01). Presence of predators in the aquatic habitats significantly reduced the probability of anopheline larvae being present (p=0.04). ConclusionsAnopheline larval habitats are widespread in the study area. Presence of bare soil, culicine larvae, predators and the level of turbidity of water are the main determinants of anopheline larval densities in aquatic habitats in Majete, Malawi. While the most productive aquatic habitats should be prioritised, for the most effective control of vectors in the area all available aquatic habitats should be targeted, even those that are not characterized by the identified predictors. Further research is needed to determine whether targeted LSM would be cost-effective when habitat characterisation is included in cost analyses and to establish what methods would make the characterisation of habitats easier.

Biochemical and Molecular Characterization of Pichia pastoris Cells Expressing Multiple TMOF Genes (tmfA) for Mosquito Larval Control.

Trypsin modulating oostatic factor (TMOF), a decapeptide hormone synthesized by female mosquito ovaries, ganglia and the central nervous system of Aedes aegypti, terminates trypsin biosynthesis in larvae, and blood-fed female mosquitoes. Earlier, TMOF was cloned and expressed as a single copy in Chlorella dessicata and in Saccharomyces cerevisiae cells as a potential larvicide. Here we report the use of a methylotrophic yeast cells, Pichia pastoris, that efficiently express multi copies of heterologous proteins, that are readily ingested by mosquito larvae. P. pastoris was engineered using pPICZB (Invitrogen, CA, United States), and 2 genes: gfp-tmfA and tmfA inserted between KpnI and XbaI in the multiple cloning site. The plasmid carries a strong AOXI promoter and P. pastoris KM71 and KM71H cells were transformed by homologous recombination. The synthesis of GFP-TMOF was followed using UV and clones were analyzed using southern and Northern blot analyses. Cloning tmfA into KM71H and selection on high Zeocin concentration (2.0 mg/mL) identified a clone that carried 10 copies of tmfA. A comparison between a single and high copy (10 genes) insertions using Northern blot analyses showed that a tmfA transcript was highly expressed even after 120 h. SDS-PAGE analysis of KM71 cells transformed with gfp-tmfA identified a protein band that ran at the expected Mr of 31 kDa. Enzyme Linked Immunoadsorbant Assay (ELISA) analysis of the recombinant cells showed that 1.65 × 108 and 8.27 × 107 cells produce 229 and 114 μM of TMOF, respectively, and caused 100% larval mortality when fed to groups of 5 larvae in 25 mL water. These results indicate that the recombinant P. pastoris cells could be used in the future in the marsh to control mosquito populations.

Effectivity of the jackfruit leaves infusion (Artocarpus heterophyllus l.) as a larvacide to the mortality rate of Aedes aegypti larva

Larval stage control using temephos, a chemical larvicide powder, was not completely safe. WHO recommended to use biological controls. Jackfruit leaves which contain saponins, flavonoids, and tannins can be used as alternate. This study was conducted to determine the effectivity of jackfruit leaves (Artocarpus heterophyllus L.) infusion on the mortality of Aedes aegypti larvae using post test only control group design. 600 of Aedes aegypti larvae were divided into 1 control group and 5 experiment groups that consist jackfruit leaves infusion concentrations of 4%, 8%, 12%, 16%, 20% with four times replication. The mortality of larvae was observed every 8 hours to 48 hours. The LC50 and LT50 were obtained by probit analysis were 3.842% and 2.150 hours. In the repeated Kruskal-Wallis test and Mann-Whitney, there were significant differences (p&lt;0.05) in the mortality of Aedes aegypti larvae that died based on differences in the concentration of jackfruit leaf infusion. In the Spearman test, there were significant correlations (p&lt;0.05) between the concentration of jackfruit leaves infusion and the mortality of Aedes aegypti larvae and the death rate of Aedes aegypti larvae. The results of the study are expected to be alternatives to chemical larvicides commonly used by the community.

Identification and detection of a novel point mutation in the Chitin Synthase gene of Culex pipiens associated with diflubenzuron resistance.

BackgroundDiflubenzuron (DFB) is one of the most used insecticides in mosquito larval control including that of Culex pipiens, the proven vector of the recent West Nile Virus epidemics in Europe. Two mutations (I1043L and I1043M) in the chitin synthase (CHS) putative binding site of DFB have been previously reported in Cx. pipiens from Italy and associated with high levels of resistance against this larvicide.Methodology/Principal findingsHere we report the identification of a third mutation at the same I1043 position of the CHS gene resulting in the substitution of Isoleucine to Phenylalanine (I1043F). This mutation has also been found in agricultural pests and has been functionally validated with genome editing in Drosophila, showing to confer striking levels (>15,000 fold) of DFB resistance. The frequency of the I1043F mutation was found to be substantially higher in Cx. pipiens mosquitoes surviving DFB doses largely exceeding the recommended field dose, raising concerns about the future efficient use of this insecticide. We monitored the presence and frequency of DFB mutations in Cx. pipiens mosquitoes from several Mediterranean countries, including Italy, France, Greece, Portugal and Israel. Among the Cx. pipiens populations collected in Northern Italy all but one had at least one of the three DFB mutations at allele frequencies reaching 93.3% for the I1043M, 64.8% for the I1043L and 10% for the I1043F. The newly reported I1043F mutation was also identified in two heterozygote individuals from France (4.2% allelic frequency). In contrast to Italy and France, no DFB resistant mutations were identified in the Cx. pipiens mosquitoes sampled from Greece, Portugal and Israel.Conclusions/SignificanceThe findings of our study are of major concern for mosquito control programs in Europe, that rely on the use of a limited number of available larvicides, and highlight the necessity for the development of appropriate Insecticide Resistance Management (IRM) programs, to ensure the sustainable use of DFB.

Biological control of Aedes mosquito larvae with carnivorous aquatic plant, Utricularia macrorhiza

BackgroundBiological controls with predators of larval mosquito vectors have historically focused almost exclusively on insectivorous animals, with few studies examining predatory plants as potential larvacidal agents. In this study, we experimentally evaluate a generalist plant predator of North America, Utricularia macrorhiza, the common bladderwort, and evaluate its larvacidal efficiency for the mosquito vectors Aedes aegypti and Aedes albopictus in no-choice, laboratory experiments. We sought to determine first, whether U. macrorhiza is a competent predator of container-breeding mosquitoes, and secondly, its predation efficiency for early and late instar larvae of each mosquito species.MethodsNewly hatched, first-instar Ae. albopictus and Ae. aegypti larvae were separately exposed in cohorts of 10 to field-collected U. macrorhiza cuttings. Data on development time and larval survival were collected on a daily basis to ascertain the effectiveness of U. macrorhiza as a larval predator. Survival models were used to assess differences in larval survival between cohorts that were exposed to U. macrorhiza and those that were not. A permutation analysis was used to investigate whether storing U. macrorhiza in laboratory conditions for extended periods of time (1 month vs 6 months) affected its predation efficiency.ResultsOur results indicated a 100% and 95% reduction of survival of Ae. aegypti and Ae. albopictus larvae, respectively, in the presence of U. macrorhiza relative to controls within five days, with peak larvacidal efficiency in plant cuttings from ponds collected in August. Utricularia macrorhiza cuttings, which were prey-deprived, and maintained in laboratory conditions for 6 months were more effective larval predators than cuttings, which were maintained prey-free for 1 month.ConclusionsDue to the combination of high predation efficiency and the unique biological feature of facultative predation, we suggest that U. macrorhiza warrants further development as a method for larval mosquito control.

Abnormal Cone and Rod Photoreceptor Morphogenesis in gdf6a Mutant Zebrafish.

PurposeAnalysis of photoreceptor morphology and gene expression in mispatterned eyes of zebrafish growth differentiation factor 6a (gdf6a) mutants.MethodsRod and cone photoreceptors were compared between gdf6a mutant and control zebrafish from larval to late adult stages using transgenic labels, immunofluorescence, and confocal microscopy, as well as by transmission electron microscopy. To compare transcriptomes between larval gdf6a mutant and control zebrafish, RNA-Seq was performed on isolated eyes.ResultsAlthough rod and cone photoreceptors differentiate in gdf6a mutant zebrafish, the cells display aberrant growth and morphology. The cone outer segments, the light-detecting sensory endings, are reduced in size in the mutant larvae and fail to recover to control size at subsequent stages. In contrast, rods form temporarily expanded outer segments. The inner segments, which generate the required energy and proteins for the outer segments, are shortened in both rods and cones at all stages. RNA-Seq analysis provides a set of misregulated genes associated with the observed abnormal photoreceptor morphogenesis.Conclusions GDF6 mutations were previously identified in patients with Leber congenital amaurosis. Here, we reveal a unique photoreceptor phenotype in the gdf6a mutant zebrafish whereby rods and cones undergo abnormal maturation distinct for each cell type. Further, subsequent development shows partial recovery of cell morphology and maintenance of the photoreceptor layer. By conducting a transcriptomic analysis of the gdf6a larval eyes, we identified a collection of genes that are candidate regulators of photoreceptor size and morphology.