Behavioral responses of Aedes aegypti,Aedes albopictus,Culex quinquefasciatus,and Anopheles minimus against various synthetic and natural repellent compounds

The behavioral responses of colony populations of Aedes aegypti, Aedes albopictus, Culex quinquefasciatus, and Anopheles minimus to four essential oils (citronella, hairy basil, catnip, and vetiver), two standard repellents (DEET and picaridin), and two synthetic pyrethroids (deltamethrin and permethrin) were conducted in the laboratory using an excito‐repellency test system. Results revealed that Cx. quinquefasciatus and An. minimus exhibited much stronger behavioral responses to all test compounds (65–98% escape for contact, 21.4–94.4% escape for non‐contact) compared to Ae. aegypti (3.7–72.2% escape (contact), 0–31.7% (non‐contact)) and Ae. albopictus (3.5–94.4% escape (contact), 11.2–63.7% (non‐contact)). In brief, essential oil from vetiver elicited the greatest irritant responses in Cx. quinquefasciatus (96.6%) and An. minimus (96.5%) compared to the other compounds tested. The synthetic pyrethroids caused a stronger contact irritant response (65–97.8% escape) than non‐contact repellents (0–50.8% escape for non‐contact) across all four mosquito species. Picaridin had the least effect on all mosquito species. Findings from the current study continue to support the screening of essential oils from various plant sources for protective properties against field mosquitoes.     

Behavioral responses of Anopheles species (Culicidae: Diptera) with varying surface exposure to pyrethroid‐treated netting in an excito‐repellency test system

Indoor Residual Spray (IRS) with insecticides has been a procedure used for decades to protect humans from biting mosquitoes and potential vectors of pathogens. The objective of this study was to determine the responses of three wild‐caught species of malaria vectors exposed to pyrethroids of three different surface coverage percents using an excito‐repellency test box. Each species was exposed to three insecticide‐treated surfaces at varying exposure levels (full coverage, 50%, and 25% of the maximum allowable by the test system) to a single standard field dose of either lambda‐cyhalothrin or alpha‐cypermethrin. Larger numbers of mosquitoes escaped the treated chambers in the direct contact test compared to the spatial repellent chambers in all three different treated surface exposures. No significant differences in the percent of escaped mosquitoes were detected in the 50% and full coverage surface coverage exposures, whereas the 25% coverage produced significantly lower avoidance responses for both compounds. This study found that varying levels of surface exposure with synthetic pyrethroids can impact the behavioral avoidance responses of Anopheles; however, it may also be possible to reduce the amount of coverage to achieve similar avoidance actions. This information may assist policy makers in designing more cost effective strategies involving residual insecticides to control mosquito vectors.     

Frequency of pyrethroid resistance in Aedes aegypti and Aedes albopictus (Diptera: Culicidae) in Thailand

Thirty‐two Aedes aegypti populations collected throughout Thailand and five populations of Aedes albopictus from southern Thailand were subjected to standard WHO contact bioassays to assess susceptibility to three commonly used synthetic pyrethroids: permethrin, deltamethrin, and lambda‐cyhalothrin. A wide degree of physiological response to permethrin was detected in Ae. aegypti, ranging from 56.5% survival (Lampang, northern Thailand) to only 4% (Kalasin in northeastern and Phuket in southern Thailand). All 32 populations of Ae. aegypti were found to have evidence of incipient resistance (62.5%) or levels of survival deemed resistant (37.5%) to permethrin. Four populations of Ae. albopictus were found with incipient resistance (97 – 80% mortality) and one with resistance (< 80%) to permethrin. The majority of Ae. aegypti populations (68.7%) was susceptible (> 98% mortality) to deltamethrin, with incipient resistance (observed 97–82% mortality) in other localities. In contrast, all populations of Ae. aegypti were completely susceptible (100% mortality) to the recommended operational dosage of lambda‐cyhalothrin. All five populations of Ae. albopictus were found completely susceptible to both deltamethrin and lambda‐cyhalothrin. Evidence of defined incipient or resistance to synthetic pyrethroids mandates appropriate response and countermeasures to mitigate further development and spread of resistance. In light of these findings, we conclude that routine and comprehensive susceptibility monitoring of dengue mosquito vectors to synthetic pyrethroids should be a required component of resistance management policies and disease control activities.     

Glutathione S-transferase detoxification as a potential pyrethroid resistance mechanism in the maize weevil, Sitophilus zeamais

Insecticide resistance patterns among 16 Brazilian populations of the maize weevil, Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae), were recognized by surveying resistance to three organophosphates (chlorpyrifos‐methyl, malathion, and pirimiphos‐methyl) and three pyrethroids (cypermethrin, deltamethrin, and permethrin). Two population clusters were obtained: one with three populations (Bragança Paulista, Cristalina, and Nova Andradina) showing low frequency of cypermethrin resistance (13–36%) and negligible frequency of deltamethrin resistance (2–9%); and another with six populations (Campos dos Goytacazes, Ivinhema, Patos de Minas, Penápolis, Uberlândia, and Venda Nova) showing low to negligible levels of pyrethroid resistance (0–23%). The remaining seven populations, including a susceptible, and a DDT‐ and pyrethroid‐resistant reference populations (Sete Lagoas and Jacarezinho, respectively), were significantly different from each other and from the two recognized clusters. In contrast with pyrethroid resistance, organophosphate resistance was negligible except for chlorpyrifos‐methyl in two populations (Fátima do Sul and Penápolis). There was no correlation between geographic distance and the Mahalanobis distance estimated from the resistance pattern ordination of the populations by canonical variate analysis, suggesting local selection and/or broad dispersal of resistant populations by grain trade. The results of biochemical in vitro studies measuring the activity of detoxification enzymes (esterases and glutathion S‐transferases) in conjunction with canonical correlation analysis suggest a major involvement of enhanced conjugation by glutathione S‐transferases (> 2‐fold increase) in pyrethroid resistance and, in the case of cypermethrin resistance, enhanced phosphotriesterase activity.     

Irritability and repellency of synthetic pyrethroids on an Aedes aegypti population from Thailand

The main objective of this study was to find the optimal dosage of deltamethrin, cyphenothrin, d‐tetramethrin, and tetramethrin that would elicit repellency and irritability responses of Aedes aegypti. The F1‐F3 generations of field mosquitoes collected from Pu Teuy Village, Sai‐Yok District, Kanchanaburi Province, Thailand, were tested with four pyrethroids to determine the LC₂₅, LC₅₀, and LC₉₉. These concentrations were 0.010%, 0.020%, and 0.055%, respectively, for deltamethrin; 0.113%, 0.167%, and 0.353%, respectively, for cyphenothrin; 2.091%, 2.770%, and 5.114%, respectively, for d‐tetramethrin; and 2.377%, 4.251%, and 10.715%, respectively, for tetramethrin. All dosages were tested in the excito‐repellency system. Survival analysis was used to compare each chamber of the test. It was found that cyphenothrin had a stronger repellent effect than the other pyrethroids, while the contact irritant effect was similar among compounds tested. The LC₅₀ of each pyrethroid was found to be the optimal dose for repelling Ae. aegypti. There was no significant difference in LC₉₉ values for either non‐contact or contact trials for each pyrethroid.     

Excito-Repellent Responses between <Emphasis Type="Italic">Culex quinquefasciatus</Emphasis> Permethrin Susceptible and Resistant Mosquitoes

The behavioral responses exhibited between four permethrin-resistant field-derived populations and a long-established, permethrin susceptible colony population of Culex quinquefasciatus mosquitoes was assessed for behavioral responses to permethrin in an excito-repellency (ER) test system. After a 30-min exposure to two different insecticide concentrations (200 and 500 mg/m²) in paired contact and noncontact test designs, the initial knockdown and escape (exit) responses were recorded followed by mortality after 24-h. All five test populations rapidly escaped from the chambers treated with permethrin regardless of background insecticide susceptibility status or chemical concentration. The greatest contact escape response was seen from the colonized population with 88 % of specimens escaping the chamber within 30-min. Contact irritancy was the predominant response compared to relatively weak noncontact spatial repellency in all test populations at both low and high concentrations. At the lower concentration, the contact escape responses did not appear influenced by background susceptibility status, whereas at the higher concentration the permethrin-resistant field populations exhibited significantly reduced avoidance behavior compared to the laboratory susceptible strain.     

A sodium channel mutation identified in Aedes aegypti selectively reduces cockroach sodium channel sensitivity to type I, but not type II pyrethroids

Voltage-gated sodium channels are the primary target of pyrethroid insecticides. Numerous point mutations in sodium channel genes have been identified in pyrethroid-resistant insect species, and many have been confirmed to reduce or abolish sensitivity of channels expressed in Xenopus oocytes to pyrethroids. Recently, several novel mutations were reported in sodium channel genes of pyrethroid-resistant Aedes mosquito populations. One of the mutations is a phenylalanine (F) to cysteine (C) change in segment 6 of domain III (IIIS6) of the Aedes mosquito sodium channel. Curiously, a previous study showed that alanine substitution of this F did not alter the action of deltamethrin, a type II pyrethroid, on a cockroach sodium channel. In this study, we changed this F to C in a pyrethroid-sensitive cockroach sodium channel and examined mutant channel sensitivity to permethrin as well as five other type I or type II pyrethroids in Xenopus oocytes. Interestingly, the F to C mutation drastically reduced channel sensitivity to three type I pyrethroids, permethrin, NRDC 157 (a deltamethrin analogue lacking the ??-cyano group) and bioresemthrin, but not to three type II pyrethroids, cypermethrin, deltamethrin and cyhalothrin. These results confirm the involvement of the F to C mutation in permethrin resistance, and raise the possibility that rotation of type I and type II pyrethroids might be considered in the control of insect pest populations where this particular mutation is present.     

Organophosphate and pyrethroid resistances in the tomato leaf miner Tuta absoluta (Lepidoptera: Gelechiidae) from Iran

The tomato leafminer, Tuta absoluta (Meyrich) (Lepidoptera: Gelechiidae), is a serious pest of tomato crops worldwide. The intensive use of chemical pesticides to control it has led to the selection of resistant populations. This study investigated the resistance of T. absoluta populations to pyrethroid and the organophosphate insecticides from ten regions of Iran. The resistance ratios at LC₅₀ for chlorpyrifos and diazinon varied among populations from 4.3 to 12 and from 1.4 to 9.0, respectively. The resistance ratios of the pyrethroids cypermethrin, deltamethrin and permethrin varied from 1.3 to 3.7, 2.7 to 13 and 1.2 to 4.3, respectively. Inclusion of synergists in toxicological bioassays and the variation observed in the activity of esterases, glutathione S‐transferase and cytochrome P450‐dependent monooxygenase suggest the existence of metabolically based resistance. Esterase and P450 biochemical assays were positively correlated with deltamethrin, and cypermethrin tolerance and diazinon tolerance correlated with esterase activity. The genes encoding the organophosphate and pyrethroid target sites acetylcholinesterase (ace1) and sodium channel (kdr) were partly sequenced. The genotyping revealed mutations in high frequencies in all populations leading to an A201S substitution in ace1 and three substitutions in the sodium channel gene L1014F, M918T, T929I. In summary, our results indicate the presence of organophosphate and pyrethroid resistance in Iranian T. absoluta populations with involvement of both detoxification enzymes and target site alterations. Most likely the populations of T. absoluta imported to Iran were resistant upon arrival.     

Host feeding responses of Aedes aegypti (L.) exposed to deltamethrin

Escape responses of mated and unmated nulliparous Aedes aegypti mosquitoes were compared using three different concentrations of deltamethrin in the presence or absence of a live animal host using an excito-repellency (ER) test system. Both insecticide contact (excitation) and non-contact (repellency) test configurations were compared. For contact trials, mated mosquitoes showed similar escape movements among the three concentrations when host stimuli were absent. Significant differences in responses were seen between the lower concentrations of (LC(50) and LC(75) ) deltamethrin with and without hosts present (P<0.05). Presence or absence of host stimuli produced no significant differences in escape response for unmated females when exposed to the highest concentration (LC(90) ) of deltamethrin. Our findings indicate that as deltamethrin concentrations decrease to sublethal levels, mating status and host cues play a more significant role in escape behavior. Therefore, insemination can influence the outcome of feeding success and flight movement of nulliparous female Ae. aegypti in contact with deltamethrin and in the presence of live host stimuli. The ER assay system serves as a useful tool for observing excitation and repellency responses of Ae. aegypti to insecticides in the presence or absence of other environmental and biological cues that can affect mosquito behavior.     

Novel Kdr mutations (K964R and A943V) in pyrethroid‐resistant populations of Triatoma mazzottii and Triatoma longipennis from Mexico and detoxifying enzymes

Although having five different ways of transmission the vector-borne is the principal way of transmission of Chagas disease, which involves insects of the subfamily Triatominae (Hemiptera: Reduviidae). Nineteen of the 31 species that occur in Mexico are associated with humans, and all are capable of transmitting the disease. Pyrethroids are the insecticides recommended for the control of these vectors in Mexico. We determined the susceptibility to the pyrethroids dcltamethrin and permethrin of peridomestic populations of Triatoma mazzottii Usinger and two populations of Triatoma longipetmis Usinger in comparison with a reference strain for each species. Bioassays were performed for the determination of the LD50 for both field populations and reference strains. A maximum of 27 fold resistance to deltamethrin was observed in T. mazzottii, meanwhile, for permethrin, T. longipennis from Jalisco show the highest value of 3.19 fold. There was significantly increased activity of esterases in field populations in comparison with their corresponding reference strain. The results of the search of kdr mutations related to the resistance to deltamethrin and permethrin in the evaluated species show the presence of mutations in the field populations, as is the case with individuals of T. mazzottii, for which the mutation was found A943V, and for the two populations of T. longipennis included in this study, we report the presence of the kdr mutation K964R. Evaluation of the various mechanisms involved in resistance to pyrethroids in triatomines from Mexico could guide us to the real justification for insecticide resistance monitoring.     

Carbon-based quantitation of pyrethrins by supercritical-fluid chromatography

All six insecticide active ingredients in pyrethrum extract were quantified by supercritical fluid chromatography and carbon calibration. Allethrin is a suitable reference compound for carbon calibration and pyrethrins calibrations. Carbon quantification in SFC is also applied to pyrethroids (phenothrin, permethrin, cypermethrin, fenvalerate and deltamethrin) and alkanes. Halogen substitution on pyrethroids requires halogens on the reference calibration compound. The method was applied to commercial extracts.     

拟除虫菊酯的杀虫活性和温度的关系

除了戊酸醚酯对苜蓿蚜呈弱的正温度系数外,杀灭菊酯、二氯苯醚菊酯、氯氰菊酯、溴氰菊酯、氟氰菊酯及百树菊酯,都呈负温度系数。杀灭菊酯、戊酸醚酯、二氯苯醚菊酯、氯氰菊酯、溴氰菊酯和氟氰菊酯,对梨网蝽全部呈正温度系数。氯氰菊酯对粘虫呈负温度系数,杀灭菊酯、戊酸醚酯、二氯苯醚菊酯和溴氰菊酯都呈正温度系数。杀灭菊酯和溴氰菊酯对粘虫卵也呈正温度系数。杀灭菊酯对小菜蛾的杀虫活性,受温度的影响不明显,而戊酸醚酯则呈负温度系数。杀灭菊酯和戊酸醚酯对蚊幼呈正温度系数,而二氯苯醚菊酯则呈负温度系数。     

Contact irritancy and spatial repellency behaviors in Anopheles albimanus Wiedemann (Diptera: Culicidae) collected in Orange Walk,Belize, C.A.

Anopheles albimanus Wiedemann is a vector of malaria in northern Belize. Previous studies have identified behavioral responses in this species when exposed to certain chemicals. We used a high throughput screening system to evaluate the contact irritant and spatial repellency responses exhibited by An. albimanus collected from Orange Walk, Belize, Central America. Anopheles albimanus behavior was significantly altered by a number of key public health insecticides. Significant irritant responses to alphacypermethrin, deltamethrin, permethrin, DDT, and propoxur were recorded, while spatial repellency responses were only elicited by DDT. Malathion resulted in neither a contact irritant or spatial repellency response. These results are in congruence with behavioral patterns previously observed for An. albimanus and for Aedes aegypti in the same system. This study produced baseline data on the behaviors of An. albimanus and confirms that the majority of compounds used as indoor residual sprays may induce premature exiting by An. albimanus in addition to killing the mosquito. This effect would decrease contact with humans and thus disrupt malaria transmission.     

Influence of nutritional and physiological status on behavioral responses of Aedes aegypti (Diptera: Culicidae) to deltamethrin and cypermethrin.

Excito-repellency responses of Aedes aegypti (L.) exposed to deltamethrin and cypermethrin were assessed using an excito-repellency test system. Contact irritancy and non-contact repellency assays compared non-bloodfed (unfed) parous (post-gravid), nulliparous, early blood-fed, late blood-fed, sugar-fed, and unmated female mosquitoes for behavioral responses based on nutritional and physiological conditions at the time of testing. Rates of escape during contact exposure with either compound were most pronounced in parous mosquitoes, followed by unmated mosquitoes, when compared to other conditional states. Significantly higher numbers of parous females also escaped from control chambers compared to other cohorts (P <0.05). Irritability of blood- and sugar-fed mosquitoes was noticeably suppressed. We conclude that nutritional and physiological conditions (including age) of mosquitoes at the time of testing can significantly influence behavioral responses (excito-repellency) to insecticides. The findings indicate that whether due to chronological age, nutrition, physiological state, or innate (circadian) activity patterns, careful consideration must be given to the selection of appropriate conditioned mosquitoes for testing.     

Isolation of broad-specificity domain antibody from phage library for development of pyrethroid immunoassay

An antibody to phenoxybenzoic acid (PBA), the conserved chemical region of pyrethroids, was developed using a domain antibody (DAB) library to enable pyrethroid detection in agricultural products. The DAB library, constructed without animal immunization and based on a human VH framework, displayed repertoires on filamentous bacteriophage. After four rounds of panning, we obtained five domain antibodies that are capable of binding to PBA. Antibody A3 has strong identification capability to cypermethrin, β-cypermethrin, and fenvalerate. The antibody A3 was used to develop an enzyme-linked immunosorbent assay (ELISA). The IC₅₀ values were 2.586, 1.814, and 2.251 μg/ml for cypermethrin, β-cypermethrin, and fenvalerate, respectively. The assay shows weak competition with flucythrinate but shows no competition with fenpropathrin, deltamethrin, and permethrin. The developed ELISA process was successfully applied to fortified Chinese cabbage samples, with the recoveries of cypermethrin, β-cypermethrin, and fenvalerate ranging from 84.4 to 112.3%. We developed an immunoassay to detect pyrethroids depending on the domain antibody library, which overcomes the limitation of requiring protein antigen to immunize animals raising antibody.     

Characterization of mosquito CYP6P7 and CYP6AA3: differences in substrate preference and kinetic properties

Cytochrome P450 monooxygenases are involved in insecticide resistance in insects. We previously observed an increase in CYP6P7 and CYP6AA3 mRNA expression in Anopheles minimus mosquitoes during the selection for deltamethrin resistance in the laboratory. CYP6AA3 has been shown to metabolize deltamethrin, while no information is known for CYP6P7. In this study, CYP6P7 was heterologously expressed in the Spodoptera frugiperda (Sf9) insect cells via baculovirus‐mediated expression system. The expressed CYP6P7 protein was used for exploitation of its enzymatic activity against insecticides after reconstitution with the An. minimus NADPH‐cytochrome P450 reductase enzyme in vitro. The ability of CYP6P7 to metabolize pyrethroids and insecticides in the organophosphate and carbamate groups was compared with CYP6AA3. The results revealed that both CYP6P7 and CYP6AA3 proteins could metabolize permethrin, cypermethrin, and deltamethrin pyrethroid insecticides, but showed the absence of activity against bioallethrin (pyrethroid), chlorpyrifos (organophosphate), and propoxur (carbamate). CYP6P7 had limited capacity in metabolizing λ‐cyhalothrin (pyrethroid), while CYP6AA3 displayed activity toward λ‐cyhalothrin. Kinetic properties suggested that CYP6AA3 had higher efficiency in metabolizing type I than type II pyrethroids, while catalytic efficiency of CYP6P7 toward both types was not significantly different. Their kinetic parameters in insecticide metabolism and preliminary inhibition studies by test compounds in the flavonoid, furanocoumarin, and methylenedioxyphenyl groups elucidated that CYP6P7 had different enzyme properties compared with CYP6AA3. © 2011 Wiley Periodicals, Inc.     

Lack of mutagenicity of synthetic pyrethroids in Salmonella typhimurium strains and in V79 Chinese hamster cells

Seven pyrethroids, i.e., cypermethrin, permethrin, deltamethrin, bioresmethrin, resmethrin, cismethrin and fenvalerate, were not found to be mutagenic in (a) Salmonella typhimurium strains TA100 or TA98 in the presence or absence of a rat liver activation system using the plate incorporation assay and fluctuation tests, or (b) V79 Chinese hamster cells in the presence or absence of hepatocytes.     

Potentiation/Antagonism of pyrethroids with organophosphate insecticides in Bemisia tabaci (Homoptera: Aleyrodidae)

The binary mixtures of pyrethroids cypermethrin, alpha-cypermethrin, zeta-cypermethrin, bifenthrin, fenpropathrin, lambda-cyhalothrin, and deltamethrin plus organophosphates ethion, profenofos, chlorpyrifos, quinalphos, acephate, methamidophos, methyl parathion, and triazophos were evaluated on putatively resistant field populations of Bemisia tabaci (Gennadius) (Homoptera: Aleyrodidae) from Pakistan using a leaf-dip bioassay. Ethion exhibited good potentiation with all the pyrethroids. Quinalphos potentiated cypermethrin, fenpropathrin, and lambda-cyhalothrin but not bifenthrin. Acephate was potentiating with bifenthrin and fenpropathrin but antagonistic with zeta-cypermethrin. A potentiation effect was also found when methamidophos was mixed with bifenthrin and fenpropathrin. However, profenofos was antagonistic with cypermethrin, bifenthrin, and lambda-cyhalothrin. Similarly, bifenthrin + methyl parathion and deltamethrin + triazophos mixtures were antagonistic when tested on several populations of B. tabaci. Chlorpyrifos was antagonistic with cypermethrin but had an additive effect with fenpropathrin.     

Chemically induced behavioral responses in Anopheles minimus and Anopheles harrisoni in Thailand

Behavioral responses of female mosquitoes representing two species in the Minimus Complex exposed to an operational field dose of bifenthrin or DEET (N,N-diethyl-m-toluamide) were described using an excito-repellency test system. Two test populations of An. minimus, one from the field (Tak Province, western Thailand), the other from a long-established laboratory colony, and Anopheles harrisoni collected from Kanchanaburi Province, western Thailand, were used. Results showed that all test populations rapidly escaped after direct contact with surfaces treated with either bifenthrin or DEET compared to match-paired untreated controls. Greater escape response by exposed females to bifenthrin and DEET were observed in the An. minimus colony compared to the two field populations. Field-collected An. minimus demonstrated a more rapid escape response to DEET than to bifenthrin, whereas An. harrisoni showed a converse response. Although fewer females escaped from test chambers without direct contact with treated surfaces compared to contact tests, the spatial repellency response was significantly pronounced in all test populations compared to match-paired controls (P < 0.05). DEET was found to perform as both a contact stimulant and moderate spatial repellent.     

Pyrethroid induced behavioral responses of Anopheles dirus, a vector of malaria in Thailand

Contact and noncontact behavioral actions of wild-caught Anopheles dirus in response to the operational field dose of three synthetic pyrethroids (bifenthrin, α-cypermethrin and λ-cyhalothrin) were evaluated using an exito-repellency test chamber. DEET was used as the repellency standard for comparison with the other three synthetic pyrethroids. Results showed that test specimens rapidly escaped from the test chamber when exposed to direct contact with a surface treated with each of the three synthetic pyrethroids and DEET. Alpha-cypermethrin demonstrated the strongest irritant action (84.9% escape), followed by DEET (77.0%), λ-cyhalothrin (68.6%) and bifenthrin (68.3%). In the noncontact configuration, fewer mosquitoes escaped from the test chambers as compared to contact trials, although a significant escape response was still observed as compared to the controls (P<0.05). We conclude that An. dirus exhibits both irritant and repellent actions in response the three pyrethroids testing in this study. The information obtained will allow us to better understand the behavioral responses of vectors to various chemicals and provide guidance when designing control strategies for targeting specific disease vectors.