The lack of data on mosquito susceptibility to candidate insecticides is a limiting factor for the success of control programs. These programs have often been implemented without information on the resistance selection risk posed by a given control agent in the field. For this reason, evaluations are often performed using the susceptibility of laboratory colonies as a reference, which do not necessarily reflect the natural variations that can be found among field populations, which has been demonstrated in other studies .
The results of this study provided a baseline dataset on the susceptibility of field populations of Ae. aegypti from Brazil to Bti. We also documented pre-existing exposure of the populations to control agents, in particular, to temephos, which has been largely used by the PNCD . Indeed, thirteen of fourteen populations investigated displayed high resistance to temephos, and all displayed high activity of detoxifying enzymes, including GST and esterases, which could be a consequence of prolonged and intensive use of temephos. The resistance ratios (RR) found for these samples were much higher than those observed in previous surveys from Brazil, in which RRs were lower than 20 in several populations, except for two populations from Ceará and Pernambuco States with RRs > 100 [27–33, 40, 48].
All Ae. aegypti populations in our study, regardless of their resistance to temephos and increased activity of DEs, displayed a level of susceptibility to Bti similar to two reference colonies. RR values at LC50 or LC90 were equal to or lower than 2, which is not considered biologically significant for resistance. These RRs are likely due to natural variations in toxicity ratios rather than to resistance selection, as observed in the studies described below. For instance, surveys carried out on around fifty Culex pipiens populations with no history of Bti exposure showed that variation in the susceptibility to Bti ranged from RRs of 2 to 12.5 [49, 50], while populations from three Bti-treated areas displayed RRs < 1 . In China, five populations of Anopheles sinensis that were exposed to chemical and microbial larvicides showed RRs to Bti between 1.7 and 5.9, although pre-treatment data were not available and it was not possible to estimate pre existing variation . Surveys based on thirty untreated and treated Aedes spp. populations showed a narrow range of RRs [35, 53–58]. Among these cases, the maximum recorded RR value at LC95 was 4, detected in a Bti-treated population of Ae. rusticus from France, a Bti-treated population of Ae. aegypti from Malaysia [35, 58], and an untreated population of Aedes albopictus from Malaysia . These data suggest that the RR values were not related to previous Bti exposure. The present study showed discrete variations in Bti RR values in Ae. aegypti populations, including two Bti-treated populations whose RRs were similar to the untreated populations. The data suggest that the variation of Ae. aegypti susceptibility to Bti could be lower than that detected for C. pipiens. To date, the only report of a high RR described in a field population previously exposed to Bti was in two C. pipiens populations from New York State (USA) that displayed RRs of 14 and 41 . However, data on the susceptibility of these populations before Bti treatment were not available, so there is no evidence that these high RRs were associated with exposure to Bti or if they were simply natural variations.
The analysis of Bti susceptibility in the populations in our study was performed in light of the disseminated temephos resistance reported for many areas of Brazil, which was confirmed by our data. In fact, Bti and temephos have distinct active principles, modes of action and target sites in insects, leading to the hypothesis that no cross-resistance is expected to occur. Data from the present study support this assertion since all populations showed a pattern of Bti susceptibility similar to the reference colonies, regardless of temephos resistance. Likewise, the RecR resistant colony, whose high resistance level (RR ≈ 180) was achieved under laboratory conditions , was still susceptible to Bti. As a consequence, the results from this work are not in agreement with previous reports whose findings suggest cross–effects of temephos and Bti, linked to DE alterations [34–36]. Boyer et al. (2007) observed an association between the exposure of Ochlerotatus cataphylla (Diptera: Culicidae) populations to Bti and temephos with a decrease in the sensitivity to these compounds and with an increase in the activity of GSTs and α-esterases. Those authors suggested that DEs may be involved in Bti detoxification . Another report showed that Ae. rusticus populations from a Bti-treated area in France had Bti RRs (LC50) of 7, which correlated with a 3-fold increase in GST activity. However, biochemical evidence that Bti toxins could be detoxified by these enzymes was not provided . In a general view, the association of DE in the resistance to insecticidal toxins from bacterial larvicides has not been related, while the alteration of target sites seems to play a major role in this process, besides others as the failure of protoxin processing and innate immune responses [12, 60–62]. The only report concerning the role of esterases in the metabolism of insecticidal bacterial toxins involved the resistance of the lepidopteran Helicoverpa armigera to Cry1Ac from Bacillus thuringiensis. In this case, esterases from a resistant strain were able to bind the Cry1Ac toxin, which could be the basis of resistance, although further investigation on this mechanism is needed . Our study showed that all Ae. aegypti populations exhibited increased or highly increased DE activity for at least three groups of enzymes that we investigated. In particular, esterases can be responsible for the detoxification of a wide range of xenobiotics, including insecticidal compounds. Nevertheless, the susceptibility of our sampled larvae to Bti was similar to the reference colonies (Rockefeller and RecL), which did not display alterations in any of the five enzymes investigated. We did not find an association between the increased activity of detoxifying enzymes and a decrease in susceptibility to Bti.