Characterization of the pyrethroid resistance mechanisms in a Blattella germanica (Dictyoptera: Blattellidae) strain from Buenos Aires (Argentina).

The use of chemical insecticides is the main control method for Blattella germanica worldwide. The prolonged and frequent use of insecticides produced the selection of insecticide-resistant individuals. The German cockroach is one of the most widespread urban pests in Argentina. In the last decades, resistance monitoring studies in this country demonstrated that there is a high prevalence of pyrethroid-resistant populations of B. germanica in the field. In this work, we studied the resistance mechanisms of a field-collected strain of B. germanica at toxicological, enzymatic, and molecular levels. A resistance ratio of 100 was obtained for the resistant strain when it was exposed to ß-cypermethrin. The pretreatment with specific synergists (piperonyl butoxide and triphenyl phosphate) led to a significant increase in the toxicity of the pyrethroid, suggesting an involvement of oxidases and esterases in the detoxification of this insecticide. Moreover, esterase and oxidase activities in the resistant strain were 1.5-fold and 2-fold higher respectively, compared to the susceptible individuals. On the other hand, the voltage-gated sodium channel gene of the resistant cockroaches did not show nucleotidic substitutions in the domain II which are associated to knockdown resistance in this species. These results suggest that the main mechanism of resistance of the studied cockroaches' strain is metabolic, mainly due to an increase in the activity of oxidase and esterase enzymes. The results of this work in addition to other reports found in literature show that the extended use of a single active principle for cockroach control promotes the development of resistance leading to control failure in the field. In contrast, integrated pest management strategies include the use of different control tools in addition to chemical insecticides, which delay the appearance of resistance increasing the efficacy of pest control.

Repellence produced by monoterpenes on Rhodnius prolixus (Hemiptera: Reduviidae) decreases after continuous exposure to these compounds.

Botanical monoterpenes are secondary metabolites present in essential oils produced by plants. Some of them are insect repellents. The bloodsucking bug Rhodnius prolixus Ståhl (Hemiptera: Reduviidae) is one of the main vectors of Chagas disease in the north of South America and some countries in Central America. In this study, we studied the repellence produced by two monoterpenes, menthyl acetate and geraniol, on fifth instar nymphs of R. prolixus. In the absence of other stimuli, both menthyl acetate and geraniol produced a repellent effect from 740 µg/cm(2) and 74 µg/cm(2), respectively. Pre-exposure to each monoterpene reduced the repellent activity produced by the same substance. Additionally, pre-exposure to one monoterpene decreased the behavioral response of the nymphs to the other one. The repellent effect of both monoterpenes also decreased when nymphs' antennae were previously treated with the nitric oxide donor S-nitroso-N-acetyl-cysteine.

The Response of Susceptible and Pyrethroid-Resistant Blattella germanica (Dyctioptera: Blattellidae) to Shelter-Associated Cues.

In this work, it was studied the role of faeces in the location and permanence in a shelter in susceptible and pyrethroid-resistant individuals of Blattella germanica (Linnaeus 1767). Additionally, the effect of different concentrations of palmitic acid on the modulation of these behaviours was tested. A shelter constituted by a square cardboard structure was offered to susceptible as well as to resistant specimens. The shelter bases were treated with faecal extracts obtained from susceptible or resistant cockroaches, or with solutions of palmitic acid. The behaviour of susceptible as well as resistant specimens was analysed using infrared videography software. Susceptible's faecal extract attracted both specimens since the time spent by cockroaches to locate the treated shelters was lower, whereas the faecal extract from resistant insects did not elicit any effect on both strains. Faecal extracts showed an arrestant effect on both strains, suggested by the time spent inside the shelter that was significantly higher in their presence. On the other hand, treatment with palmitic acid produced an attractant or a repellent effect depending on the concentration and strain. The tested lower concentration was attractant to susceptible insects, but did not produce any effect on resistant ones. In addition, the higher concentrations did not produce any effect on susceptible individuals, but resulted repellent for resistant ones. Palmitic acid did not produce an arrestant effect on the strains as there was not an increase in time spent inside the shelter in the presence of this substance. An increase in the number of visits to the shelter and to the periphery was also observed in shelters treated with the faecal extract and with the lower concentration of palmitic acid. These results show that compounds of the susceptible faeces were attractant to cockroaches of both strains, while faecal extracts from resistant insects were not. Moreover, a dual effect of palmitic acid was observed, being attractant at low concentrations and repellent as concentration increased. Additionally, a difference in the concentration threshold at which the effect of this substance changes was observed between strains.

Feeding Behaviour of a Pyrethroid-Resistant Strain of the German Cockroach Blattella germanica (Linnaeus, 1767).

Many authors report biological differences among insecticide-resistant pest species, mainly associated with parameters of life history, but only a few studies approach differences at the behavioural level. Feeding behaviour in Blattella germanica (L.) is modulated by the detection of chemical volatiles emitted from food sources in order to be located a long-median distance, and also by the physicochemical properties of food. This work aimed to study the differences in the feeding behaviour of a susceptible and a pyrethroid-resistant strain of B. germanica, in the location and exploitation of a food source. Resistant males showed a lower performance in the feeding behaviour compared to susceptible males. Particularly, the time taken to locate the source was significantly higher in the resistant individuals, suggesting a lower capacity in the detection of food odours. In addition, although the Intake rate was negatively related to the stiffness of food for both strains, resistant individuals showed a lower intake rate compared to susceptible ones, when exploiting a food source of the same stiffness. In a control context, the phenotypic characteristics associated to feeding of pyrethroid-resistant individuals could induce a behavioural resistance mechanism caused by a reduction in the amount of toxic gel bait ingested. If that is the case, resistance of this strain would not be associated to physiological changes affecting the toxicity of the active principle of gel baits, but to a lower efficacy of these products against resistant individuals due to less amount of gel consumed, generating control problems of this pest.

Spatial Repellency Caused by Volatile Pyrethroids is Olfactory-Mediated in the German Cockroach Blattella germanica (Dictyoptera: Blattellidae).

Pyrethroids are synthetic insecticides that have a repellent action. This effect has been associated with an increase in the locomotor activity, which causes the avoidance of the insecticide-treated area (excito-repellency). In this work, we studied with behavior and electrophysiological recordings the occurrence of olfactory-mediated repellency caused by pyrethroids of different volatility in the German cockroach Blattella germanica (Linnaeus, 1767). Male cockroaches were spatially repelled when they were exposed to D-allethrin vapors and vapothrin vapors in a dose-dependent manner. No repellency was observed when insects were exposed to permethrin, a non-volatile pyrethroid. To confirm the role of olfaction in this phenomenon, we measured the electrical activity of the cockroaches' antennae in response to these insecticides. There was a significant increase in the electrical activity in response to D-allethrin and vapothrin, but no increase was observed in insects exposed to permethrin. Locomotor activity of cockroaches exposed to pyrethroids was measured in order to discard excito-repellency. No changes in locomotor activity were observed for any of the insecticides. Finally, we found that volatile pyrethroids in the vapor phase cause spatial repellency in cockroaches, being the first report of an olfactory-mediated repellency phenomenon caused by pyrethroids in cockroaches.

Synergism between cis-permethrin and benzoyl phenyl urea insect growth regulators against Aedes aegypti larvae.

Emulsifiable concentrate formulations of 2 insect growth regulator (IGR) benzoyl ureas (novaluron and diflubenzuron) and the pyrethroid cis-permethrin were individually bioassayed against late 3rd- or early 4th-stage larvae of susceptible Aedes aegypti. The emergence inhibition 50% were 0.038, 0.048, and 0.179 ppb for novaluron, diflubenzuron, and cis-permethrin, respectively. We also tested 1:1 (v:v) mixtures of cis-permethrin and benzoyl ureas. Emergence inhibition 50% for novaluron plus cis-permethrin and diflubenzuron plus cis-permethrin were 0.030 and 0.037, respectively, with combination indexes of 0.49 and 0.56, indicating a synergistic interaction between these insecticides. The percentage of larval mortality produced by 0.5 ppb of the mixtures of cis-permethrin and each IGR was compared to the addition of larval mortality produced in separated experiments by 0.25 ppb of each component, after 5, 9 and 13 days of continuous exposure. In all bioassays, the novaluron plus cis-permethrin mixture produced a significantly higher larvicidal effect (P < 0.05) compared with the individual contribution of each compound. The diflubenzuron plus cis-permethrin mixture showed significantly higher larval mortality (P < 0.05) compared with the contributive effect of each insecticide after 9 and 13 days of exposure, but there was no significant difference after 5 days of exposure.

Nitric oxide contributes to high-salt perception in a blood-sucking insect model.

In all organisms, salts produce either appetitive or aversive responses depending on the concentration. While low-salt concentration in food elicits positive responses to ingest, high-salt triggers aversion. Still the mechanisms involved in this dual behavior have just started to be uncovered in some organisms. In Rhodnius prolixus, using pharmacological and behavioral assays, we demonstrated that upon high-salt detection in food a nitric oxide (NO) dependent cascade is activated. This activation involves a soluble guanylate cyclase (sGC) and the production of cyclic guanosine monophosphate (cGMP). Thus, appetitive responses to low-salt diets turn to aversion whenever this cascade is activated. Conversely, insects feed over aversive high-salt solutions when it is blocked by reducing NO levels or by affecting the sGC activity. The activation of NO/sGC/cGMP cascade commands the avoidance feeding behavior in R. prolixus. Investigations in other insect species should examine the possibility that high-salt aversion is mediated by NO/sSG/cGMP signaling.

Modulation of the behavioral and electrical responses to the repellent DEET elicited by the pre-exposure to the same compound in Blattella germanica.

Insects under different stimuli from the environment modify behavioural responses due to changes in the sensitivity of neurons at the peripheral and/or at the central level of the nervous system. This phenomenon is called neuronal plasticity, and sensory adaptation is an example of it. An insect repellent is a chemical that produces oriented movements of the insects away from its source. In this work we studied the modulation of the behavioural and electrical response to the repellent N, N-diethyl-3-methylbenzamide (DEET) in males of the German cockroach B. germanica produced by previous exposure to the same repellent. Methods. We determined repellency using a circular arena, one half of which was treated with DEET. The time spent by insects in each half of the arena was measured, and a repellency coefficient (RC) was calculated. The RCs of pre-exposed and non-pre-exposed insects were compared. To determine a possible role of nitric oxide in the modulation of the response to DEET after pre-exposure, the nitric oxide donor S-nitroso-acetyl-cysteine (SNAC) was applied on cockroaches' antennae. The electrical activity of the cockroaches' antennae in response to DEET was recorded using electroantennogram (EAG) technique. The response to DEET was recorded also after a long stimulation with the same repellent, and after topical application of SNAC and dbcGMP (a cGMP analogue) on the antennae. Results. We found that previous exposure of B. germanica males to the repellent DEET produced an increase of the repellency at the behavioural level, measured as RC. A possible role of nitric oxide (NO) in the transduction pathway of this phenomenon is suggested, since treatment of the cockroaches with the NO donor SNAC also produced an increase of the repellency elicited by DEET. On the other hand, the response of the cockroaches' antennae exposed to DEET was determined electrophysiologically. The electrical activity in response to DEET decreased when the insects' antennae were stimulated with a long pulse of the repellent. The activity of the antennae was restored after 10 min. Treatment of the antennae either with SNAC or dbGMPc also produced a decrease in the response of the antennae to the repellent. Discussion.The previous exposure to a chemical stimulus can modify the behaviour associated to the same stimulus, increasing or decreasing the behavioural response. In the case of DEET we found that pre-exposure increased DEET repellency in male cockroaches. We also found NO involvement in a similar phenomenon. On the other hand, the test showed that DEET is perceived by insects' antennae as an odour. A long exposure of the antennae to DEET caused a transient decrease in the response of the antennae to the same compound. The same effect was achieved by treating the antennae with SNAC or dbcGMP, suggesting the involvement of the NO/cGMP system in the transduction pathway of the sensory adaptation phenomenon elicited by an odour in this species.

Locomotor behaviour of Blattella germanica modified by DEET.

N,N-diethyl-3-methylbenzamide (DEET) is the active principle of most insect repellents used worldwide. However, its toxicity on insects has not been widely studied. The aim of this work is to study the effects of DEET on the locomotor activity of Blattella germanica. DEET has a dose-dependent repellent activity on B. germanica. Locomotor activity was significantly lower when insects were pre-exposed to 700 µg/cm(2) of DEET for 20 or 30 minutes, but it did not change when pre-exposure was shorter. Locomotor activity of insects that were pre-exposed to 2.000 µg/cm(2) of DEET for 10 minutes was significantly lower than the movement registered in controls. No differences were observed when insects were pre-exposed to lower concentrations of DEET. A 30-minute pre-exposure to 700 µg/cm(2) of DEET caused a significant decrease in locomotor activity. Movement was totally recovered 24 h later. The locomotor activity measured during the exposure to different concentrations of DEET remained unchanged. Insects with decreased locomotor activity were repelled to the same extent than control insects by the same concentration of DEET. We demonstrated that the repellency and modification of locomotor activity elicited by DEET are non-associated phenomena. We also suggested that the reduction in locomotor activity indicates toxicity of DEET, probably to insect nervous system.

Adaptation of the repellency response to DEET in Rhodnius prolixus.

For many years it has been accepted that DEET interferes with the detection of odours from the host instead of having a repellent effect. However, recent work showed that DEET acts as an odorant molecule and elicits a behavioural response in the absence of other stimuli. Therefore, DEET must promote some phenomenon connected with the stimuli-sensory system interaction, such as a sensory adaptation, where the sensory system regulates its sensitivity to different stimuli intensities during continuous or repetitive exposure. In this work, we studied different aspects of the insect-DEET interaction through behavioural observations. Previous exposure of fifth instar Rhodnius prolixus nymphs to DEET decreased the behavioural response to this repellent. We observed a decrease in repellence after different times of continuous stimulation with DEET in a time-dependent manner. The response to DEET was recovered 10 min after exposure, when insects were continuously stimulated during 5 or 10 min; maximum repellence was recovered 20 min after exposure when insects were stimulated for 20 min. DEET produced a repellent effect when nymphs were exposed only to its vapours. These results suggest that exposure to DEET produces adaptation in R. prolixus nymphs, and that the behavioural response elicited by DEET occurs via olfaction when no other stimuli are present.

Decrease in DEET repellency caused by nitric oxide in Rhodnius prolixus.

N,N-diethyl-3-methylbenzamide (DEET) is widely used as an insect repellent; however, little is known about its mode of action. On the other hand, nitric oxide (NO) participates in the olfaction transduction pathway of insects. In this work, nitroso-acetyl-cysteine (SNAC), a nitric oxide donor, or dibutyril-cyclic-GMP (db-cGMP), the cyclic nucleotide analog, were applied on fifth instar nymphs of Rhodnius prolixus before exposing them to DEET, to obtain information about the possible role of NO/cGMP system in the olfaction process. In the first place, we exposed the nymphs to several DEET concentrations (70, 700, 1,750, and 3,500 microg/cm2). All these concentrations produced a repellent effect. A decrease in repellency during the course of the experiment was observed when the nymphs were exposed to high concentrations of DEET (700 and 1,750 microg/cm2), suggesting an adaptation phenomenon. The pre-treatment of the insects with 15 microg /insect of SNAC or 2 microg/insect of db-cGMP produced a reduction of the repellency. An increase in locomotor activity was observed in insects exposed to 350 or 700 microg/cm2 DEET. Although exposure to 70 microg/cm2 DEET produced a high repellency response, it did not modify the insects' locomotor activity. Insects treated with two doses of SNAC before being exposed to 350 microg/cm2 of DEET showed no differences in locomotor activity compared to controls.

Toxicity of pyrethroids and repellency of diethyltoluamide in two deltamethrin-resistant colonies of Triatoma infestans Klug, 1834 (Hemiptera: Reduviidae).

The aim of the currrent investigation was to evaluate (a) the toxicity of three pyrethroids (deltamethrin, lambda-cyhalothrin, and tetramethrin); (b) the effect of these insecticides on the locomotor activity; and (c) the repellent effect of N,N-diethyl-m-toluamide (DEET) on two deltamethrin-resistant strains of Triatoma infestans from Argentina (El Chorro and La Toma), and one susceptible strain. The resistance ratios (RRs) obtained for the La Toma strain were: > 10,769, 50.7, and > 5.2 for deltamethrin, lambda-cyhalothrin, and tetramethrin respectively. The RRs for the El Chorro strain were: > 10,769, 85.8, and > 5.2 for deltamethrin, lambda-cyhalothrin, and tetramethrin respectively. The hyperactivity usually caused by the three pyrethroids was in both the deltamethrin-resistant strains compared to the susceptible reference strain. No differences were observed in the repellent effect of DEET between the three groups. These results indicate that the deltamethrin-resistant insects have a cross resistance to lambda-cyhalothrin and tetramethrin, and are also resistant to the first symptom of pyrethroid poisoning (hyperactivity). However, the sensorial process related to DEET repellency does not appear to be altered.

Toxicity of pyrethroids and repellency of diethyltoluamide in two deltamethrin-resistant colonies of Triatoma infestans Klug, 1834 (Hemiptera: Reduviidae)

The aim of the currrent investigation was to evaluate (a) the toxicity of three pyrethroids (deltamethrin, lambda-cyhalothrin, and tetramethrin); (b) the effect of these insecticides on the locomotor activity; and (c) the repellent effect of N,N-diethyl-m-toluamide (DEET) on two deltamethrin-resistant strains of Triatoma infestans from Argentina (El Chorro and La Toma), and one susceptible strain. The resistance ratios (RRs) obtained for the La Toma strain were: > 10,769, 50.7, and > 5.2 for deltamethrin, lambda-cyhalothrin, and tetramethrin respectively. The RRs for the El Chorro strain were: > 10,769, 85.8, and > 5.2 for deltamethrin, lambda-cyhalothrin, and tetramethrin respectively. The hyperactivity usually caused by the three pyrethroids was in both the deltamethrin-resistant strains compared to the susceptible reference strain. No differences were observed in the repellent effect of DEET between the three groups. These results indicate that the deltamethrin-resistant insects have a cross resistance to lambda-cyhalothrin and tetramethrin, and are also resistant to the first symptom of pyrethroid poisoning (hyperactivity). However, the sensorial process related to DEET repellency does not appear to be altered.