The four other mutations S T V I
The four other mutations (S281T, V317I, T328A and A329S) were not detected in the absence of the A286S. This could suggest that when all of them are present, there would be a tri-dimensional structural modification that would interfere with the binding of the insecticide and produce different levels of resistance. It should be noted that the RFSan population, with a very high percentage of survival at the discriminating concentration, always presented the four mutations and that the Jaguar and Juarez strains, with a survival rate between 30 and 49%, respectively, presented the two mutation patterns, the A286 L and the A286S, plus the four mutations. The U-MOR population, with a lower survival percentage than these last two populations, presented only the pattern of four mutations with A286S. Perhaps this situation is similar to that described previously by Zhang et al. (2016) for Brown planthopper, but it is not possible to elucidate the synergistic or compensatory effects of the different mutations.
Fipronil insensitivity could be due to multiple mutations in the Rdl as demonstrated in D. melanogaster by Remnant et al. (2014). The authors identified three fipronil-resistant strains (A301S + T350S, A301S + T360I and A/S301 + M/I360). In the present study, the mutation A286S detected in R. microplus could be analogous to 302 in D. melanogaster and to 296 in Anopheles funestus resistance to fipronil, and the V317I to 327 in both strains. In some fipronil-resistant populations, no mutations in the rdl loci were detected. Of the 16 fipronil resistant populations, seven had amino AG957 pathway alterations. This may be due to the low number of individuals analysed, which may coincide with a low allelic resistance frequency in the population. In the present study, it is difficult to correlate mutations to different levels of resistance and we cannot discard the presence of a different mechanism of resistance, such as metabolic detoxification (Punyawattoe et al., 2013; Elzaki et al., 2015) or gene duplication as observed in resistant individuals of Myzus persicae and D. melanogaster (Anthony et al., 1998; Remnant et al., 2013). Further studies on modelling, gene expression and cloning should be carried out in order to elucidate these hypotheses. Expression of recombinant RDL receptors could help to elucidate the synergistic or compensatory effects of the different point mutations.
Conclusions The mutation T290L present in the Gaba-Cl of dieldrin-resistant R. australis tick strains from Australia is not present in the tick populations from Brazil and Uruguay analysed in this study. Other mutations in the transmembrane 2 domain of Gaba-Cl were detected in fipronil- and lindane-resistant ticks. The amino acid change A286S/L could be related to the same alanine residue replaced by serine (A302S) described in insects resistant to cyclodienes and phenylpyrazoles.
Conflicts of interest
Introduction Investigations of intracellular channels present unique technical challenges. Perhaps most importantly, channels expressed exclusively on intracellular membranes are largely inaccessible to the direct application of the powerful patch-clamp techniques that led to rapid characterization of plasma membrane ion channels. As an alternative, intracellular channels can be studied in isolated vesicle fractions, but membrane fractionation techniques are always imperfect, with unavoidable contamination of membranes prepared from one organelle with those from other compartments. While low level contamination may not be critical to typical biochemical studies, contamination can be a fatal confounder in single molecule assays such as single channel recordings. These and other technical obstacles have impeded progress. Nonetheless, anion conductances have been demonstrated in numerous intracellular compartments and a host of discreet chloride channel activities have been described , , . Perhaps counter-intuitively, the regulation of concentration or amount of chloride itself within compartments has not been seen as the major role of these intracellular chloride channels. Instead, the key role of chloride permeability has been thought to be to function as a short-circuiting conductance to allow transport by electrogenic cation transport mechanisms. For example, acidification of intracellular organelles by the electrogenic proton ATPase is recognized as a process that requires a short-circuiting conductance to allow transmembrane cation transport , . Other processes which may require a chloride short-circuiting conductance across intracellular membranes include calcium transport across the sarcoplasmic reticulum (SR) ,  and potassium influx into secretory vesicles .