Waseem Akram

Spatial mapping of gene expression in the salivary glands of the dengue vector mosquito, Aedes aegypti

BackgroundAedes aegypti mosquitoes are the main vectors of dengue viruses to humans. Understanding their biology and interactions with the pathogen are prerequisites for development of dengue transmission control strategies. Mosquito salivary glands are organs involved directly in pathogen transmission to vertebrate hosts. Information on the spatial distribution of gene expression in these organs is expected to assist in the development of novel disease control strategies, including those that entail the release of transgenic mosquitoes with impaired vector competence.ResultsWe report here the hybridization in situ patterns of 30 transcripts expressed in the salivary glands of adult Ae. aegypti females. Distinct spatial accumulation patterns were identified. The products of twelve genes are localized exclusively in the proximal-lateral lobes. Among these, three accumulate preferentially in the most anterior portion of the proximal-lateral lobe. This pattern revealed a salivary gland cell type previously undescribed in Ae. aegypti, which was validated by transmission electron microscopy. Five distinct gene products accumulate in the distal-lateral lobes and another five localize in the medial lobe. Seven transcripts are found in the distal-lateral and medial lobes. The transcriptional product of one gene accumulates in proximal- and distal-lateral lobes. Seven genes analyzed by quantitative PCR are expressed constitutively. The most abundant salivary gland transcripts are those localized within the proximal-lateral lobes, while previous work has shown that the distal-lateral lobes are the most active in protein synthesis. This incongruity suggests a role for translational regulation in mosquito saliva production.ConclusionsTransgenic mosquitoes with reduced vector competence have been proposed as tools for the control of dengue virus transmission. Expression of anti-dengue effector molecules in the distal-lateral lobes of Ae. aegypti salivary glands has been shown to reduce prevalence and mean intensities of viral infection. We anticipate greater efficiency of viral suppression if effector genes are expressed in all lobes of the salivary glands. Based on our data, a minimum of two promoters is necessary to drive the expression of one or more anti-dengue genes in all cells of the female salivary glands.

Predatory Potential of Chrysoperla carnea and Cryptolaemus montrouzieri Larvae on Different Stages of the Mealybug, Phenacoccus solenopsis: A Threat to Cotton in South Asia

Abstract The outbreaks of mealybug, Phenacoccus solenopsis Tinsley (Homoptera: Pseudococcidae), have created problems to cotton crops in South Asia in the recent years. To control this menace, predatory potential of Chrysoperla carnea and Cryptolaemus montrouzieri larvae were investigated under laboratory conditions (27 ± 5° C and 65 ± 5% RH). The experiments were conducted in no choice (only first, second, or third instar larvae of mealybug were offered at a time) and choice (first, second, and third instar larvae were offered simultaneously) feeding tests. Both predators had high consumption rates, with C. montrouzeiri being the most voracious feeder. In the no choice feeding tests, third instar larvae of C. montrouzeiri devoured the highest mean number of first instar P. solenopsis (439.38) In the choice feeding tests, a similar number of first instar nymphs (410) were consumed. In both feeding tests, C. carnea devoured relatively fewer numbers of P. solenopsis than C. montrouzeiri. Manlys preference index suggested that the both predators preferred first instar nymphs of P. solenopsis over second or third instar nymphs. Furthermore, studies on developmental rate and fecundity revealed that first instar nymphs of P. solenopsis significantly reduced development time but increased the fecundity of both predators.

First report of field evolved resistance to agrochemicals in dengue mosquito, Aedes albopictus (Diptera: Culicidae), from Pakistan

BackgroundAgrochemicals have been widely used in Pakistan for several years. This exposes mosquito populations, particularly those present around agricultural settings, to an intense selection pressure for insecticide resistance. The aim of the present study was to investigate the toxicity of representative agrochemicals against various populations of Aedes albopictus (Skuse) collected from three different regions from 2008-2010.ResultsFor organophosphates and pyrethroids, the resistance ratios compared with susceptible Lab-PK were in the range of 157-266 fold for chlorpyrifos, 24-52 fold for profenofos, 41-71 fold for triazofos, and 15-26 fold for cypermethrin, 15-53 fold for deltamethrin and 21-58 fold for lambdacyhalothrin. The resistance ratios for carbamates and new insecticides were in the range of 13-22 fold for methomyl, 24-30 fold for thiodicarb, and 41-101 fold for indoxacarb, 14-27 fold for emamectin benzoate and 23-50 fold for spinosad. Pair wise comparisons of the log LC50s of insecticides revealed correlation among several insecticides, suggesting a possible cross resistance mechanism. Moreover, resistance remained stable across 3 years, suggesting field selection for general fitness had also taken place for various populations of Ae. albopictus.ConclusionModerate to high level of resistance to agrochemicals in Pakistani field populations of Ae. albopictus is reported here first time. The geographic extent of resistance is unknown but, if widespread, may lead to problems in future vector control.

Genetics, cross-resistance and mechanism of resistance to spinosad in a field strain of Musca domestica L. (Diptera: Muscidae)

The house fly, Musca domestica L., is a cosmopolitan insect with the ability to develop resistance to insecticides used for their management. In the present study, we investigated the genetics of spinosad resistance, and cross-resistance potential to other insecticides by selecting a field strain with a commercial spinosad formulation. Bioassays with the field strain, before selection with spinosad, gave resistance ratios (RRs) of 4, 5, 66, 21 and 5 fold for spinosad, indoxacarb, abamectin, imidacloprid and deltamethrin, respectively, in comparison to a laboratory susceptible (Lab-susceptible) strain. After continuous selection of the field strain (Spin-SEL) with spinosad, the RR was increased up to 155 fold; however, the resistance was unstable (RR decreased 1.43 fold) when this strain was not exposed to spinosad for five generations. The Spin-SEL strain did not show cross-resistance to abamectin, indoxacarb or deltamethrin, but showed negative cross-resistance to imidacloprid. Crosses between the Spin-SEL and Lab-susceptible strains revealed an autosomal and incomplete dominant mode of resistance to spinosad. A direct test using a monogenic inheritance model based on Chi-square analysis revealed that the resistance was governed by more than one gene. Moreover, the resistance was neither overcome with the insecticide synergist piperonyl butoxide nor with S,S,S-tributylphosphorotrithioate. Lack of cross-resistance and instability of resistance suggest that rotation with spinosad could be an effective resistance management strategy.

Resistance to new chemical insecticides in the house fly, Musca domestica L., from dairies in Punjab, Pakistan

The house fly, Musca domestica L., is one of the major pests in dairy operations that has developed resistance to a number of insecticides with different modes of action. Adult house fly populations from six dairies in Punjab, Pakistan were evaluated for resistance to insecticides with novel modes of action (abamectin, emamectin benzoate, fipronil, imidacloprid, indoxacarb, and spinosad). Significant levels of resistance to most of the insecticides tested were observed in the present study. For avermectins at LC50 level, the resistance ratios were in the range of 38.40 to 94.44-fold for abamectin and 13.16 to 36.30-fold for emamectin benzoate. Fipronil LC50 resistance ratios exceeded 10-fold in three house fly populations, while all the populations had >10-fold resistance ratios for imidacloprid. Indoxacarb and spinosad had the lowest resistance ratios that ranged from 3.02 to 7.12-fold for indoxacarb and 2.91 to 9.0-fold for spinosad. As the resistance to fipronil, indoxacarb, and spinosad are emerging, therefore these chemicals should be used cautiously in management programs to retain the efficacy for longer times.

Resistance to conventional insecticides in Pakistani populations of Musca domestica L. (Diptera: Muscidae): a potential ectoparasite of dairy animals.

The house fly, Musca domestica L., is an important hygienic pest of humans and dairy animals with the potential to develop resistance to most chemical classes of insecticides. Six adult house fly strains from dairy farms in Punjab, Pakistan were evaluated for resistance to selected insecticides from organochlorine, organophosphate, carbamate and pyrethroid classes. For a chlorocyclodiene and two organophosphates tested, the resistance ratios (RR) at LC50 were in the range of 5.60–22.02 fold for endosulfan, 7.66–23.24 fold for profenofos and 2.47–7.44 fold for chlorpyrifos. For two pyrethroids and one carbamate, the RR values at LC50 were 30.22–70.02 for cypermethrin, 5.73–18.31 for deltamethrin, and 4.39–15.50 for methomyl. This is the first report of resistance to different classes of insecticides in Pakistani dairy populations of house flies. Regular insecticide resistance monitoring programs on dairy farms are needed to prevent field control failures. Moreover, integrated approaches including the judicious use of insecticides are needed to delay the development of insecticide resistance in house flies.

The Effect of Temperature on the Toxicity of Insecticides against Musca domestica L.: Implications for the Effective Management of Diarrhea

Background Diarrhea is an important cause of childhood mortality in developing countries like Pakistan because of unhygienic conditions, lack of awareness, and unwise use of preventive measures. Mechanical transmission of diarrheal pathogens by house flies, Musca domestica, is believed as the most effective route of diarrhea transmission. Although the use of insecticides as a preventive measure is common worldwide for the management of house flies, success of the measure could be compromised by the prevailing environmental temperature since it significantly affects toxicity of insecticides and thus their efficacy. Peaks of the house fly density and diarrheal cases are usually coincided and season specific, yet little is known about the season specific use of insecticides. Methodology/Principal Findings To determine the temperature-toxicity relationship in house flies, the effect of post-bioassays temperature (range, 20–34°C) on the toxicity of seven insecticides from organophosphate (chlorpyrifos, profenofos), pyrethroid (cypermethrin, deltamethrin) and new chemical (emamectin benzoate, fipronil, spinosad) classes was evaluated by using a feeding bioassay method. From 20–34°C, the toxicities of chlorpyrifos, profenofos, emamectin and fipronil increased 2.10, 2.93, 2.40 and 3.82 fold (i.e. positive temperature coefficient), respectively. Whereas, the toxicities of cypermethrin, deltamethrin and spinosad decreased 2.21, 2.42 and 3.16 fold (i.e. negative temperature coefficient), respectively. Conclusion/Significance These findings suggest that for the reduction in diarrheal cases, house flies should be controlled with insecticides according to the prevailing environmental temperature. Insecticides with a positive temperature coefficient may serve as potential candidates in controlling house flies and diarrhea epidemics in hot season and vice versa.

A cross sectional survey of knowledge, attitude and practices related to house flies among dairy farmers in Punjab, Pakistan

BackgroundHouse flies are of major public health concerns in areas with poor sanitation and hygienic conditions. Unfortunately, sanitation and hygiene have always been ignored in dairy farms particularly in the developing or low-income countries. Management of these flies mostly depends on the awareness regarding associated hazards and protective measures taken by the people to minimize risks associated with flies. The present study therefore explores the knowledge, attitude and practices taken by dairy farmers in Punjab, Pakistan against house flies.MethodsThe present study was based on a cross sectional self administered survey to a convenience sample of 173 small scale dairy farmers in four localities – Multan, Lahore, Shorkot and Faisalabad – of Pakistan. The relationships between socio-demographics, knowledge and preventive practices were investigated through logistic regression analysis and chi-square test of association.ResultsConsiderable number of dairy farmers 71/173 (41.04%) had no idea about the problems associated with house flies. Although 77/173 (44.51%) dairy farmers reported house flies as disease transmitters, only 23 (29.87%) farmers were familiar with diseases and 22 (28.57%) had somewhat idea of the mode of disease transmission. We found a positive association between dairy farmer’s education level and overall knowledge of house flies in multivariate analysis. Farmer’s education level and knowledge of the house flies breeding sites had a positive association with the adoption of house fly prevention practices by the respondents. However, knowledge of the problems associated with house flies and preventive measures had no association with house fly prevention practices.ConclusionThe present ethnoentomological survey provides information about knowledge, attitude and practices of dairy farmers related to house flies in Punjab, Pakistan. We conclude that the farmers’ education level and knowledge of the breeding sites had a positive association with the adoption of prevention practices against house flies. The study also highlights the need of targeting the lack of knowledge of dairy farmers for the successful management of house flies.

Insecticide Mixtures Could Enhance the Toxicity of Insecticides in a Resistant Dairy Population of Musca domestica L

House flies, Musca domestica L., are important pests of dairy operations worldwide, with the ability to adapt wide range of environmental conditions. There are a number of insecticides used for their management, but development of resistance is a serious problem. Insecticide mixtures could enhance the toxicity of insecticides in resistant insect pests, thus resulting as a potential resistance management tool. The toxicity of bifenthrin, cypermethrin, deltamethrin, chlorpyrifos, profenofos, emamectin benzoate and fipronil were assessed separately, and in mixtures against house flies. A field-collected population was significantly resistant to all the insecticides under investigation when compared with a laboratory susceptible strain. Most of the insecticide mixtures like one pyrethroid with other compounds evaluated under two conditions (1∶1-“A” and LC50: LC50-“B”) significantly increased the toxicity of pyrethroids in the field population. Under both conditions, the combination indices of pyrethroids with other compounds, in most of the cases, were significantly below 1, suggesting synergism. The enzyme inhibitors, PBO and DEF, when used in combination with insecticides against the resistant population, toxicities of bifenthrin, cypermethrin, deltamethrin and emamectin were significantly increased, suggesting esterase and monooxygenase based resistance mechanism. The toxicities of bifenthrin, cypermethrin and deltamethrin in the resistant population of house flies could be enhanced by the combination with chlorpyrifos, profenofos, emamectin and fipronil. The findings of the present study might have practical significance for resistance management in house flies.

Thiamethoxam Resistance in the House Fly, Musca domestica L.: Current Status, Resistance Selection, Cross-Resistance Potential and Possible Biochemical Mechanisms

The house fly, Musca domestica L., is an important ectoparasite with the ability to develop resistance to insecticides used for their control. Thiamethoxam, a neonicotinoid, is a relatively new insecticide and effectively used against house flies with a few reports of resistance around the globe. To understand the status of resistance to thiamethoxam, eight adult house fly strains were evaluated under laboratory conditions. In addition, to assess the risks of resistance development, cross-resistance potential and possible biochemical mechanisms, a field strain of house flies was selected with thiamethoxam in the laboratory. The results revealed that the field strains showed varying level of resistance to thiamethoxam with resistance ratios (RR) at LC50 ranged from 7.66-20.13 folds. Continuous selection of the field strain (Thia-SEL) for five generations increased the RR from initial 7.66 fold to 33.59 fold. However, resistance declined significantly when the Thia-SEL strain reared for the next five generations without exposure to thiamethoxam. Compared to the laboratory susceptible reference strain (Lab-susceptible), the Thia-SEL strain showed cross-resistance to imidacloprid. Synergism tests revealed that S,S,S-tributylphosphorotrithioate (DEF) and piperonyl butoxide (PBO) produced synergism of thiamethoxam effects in the Thia-SEL strain (2.94 and 5.00 fold, respectively). In addition, biochemical analyses revealed that the activities of carboxylesterase (CarE) and mixed function oxidase (MFO) in the Thia-SEL strain were significantly higher than the Lab-susceptible strain. It seems that metabolic detoxification by CarE and MFO was a major mechanism for thiamethoxam resistance in the Thia-SEL strain of house flies. The results could be helpful in the future to develop an improved control strategy against house flies.