Laboratory and field strains.
One strain of Anopheles albitarsis s.s. species is kept as an autonomously mating colony in the Laboratório de Fisiologia e Controle de Artrópodes Vetores (Laficave)/ IOC/Fiocruz, for more than two decades uninterruptedly. This colony was original from collections performed around a rice plantation field in Massaranduba, Santa Catarina State, Brazil (Horosko III et al., 1997). We performed new collections of An. albitarsis in Massaranduba (26º34’55” S, 48º54’39” W), around the same rice fields that originated the laboratory colony over 20 years ago.
Local farmers informed us in Massaranduba that a variety of insecticides have been used in the rice fields at that time, such as Actara (thiamethoxam, Syngenta), EngeoTM Pleno (thiamethoxam + lambda-cyhalothrin, Syngenta), Incrivel (acetamiprid + alfa-cypermethrin, Iharabras), Karate Zeon (lambda-cyhalothrin, Syngenta), Mustang (zeta-cypermethrin, FMC) and Safety (etofenprox, Iharabras), in dosages varying from 160 to 200 mL/ha. Therefore, except for thiamethoxam, which is a neonicotinoid, all the other active ingredients were pyrethroids.
We performed the insect collections in corrals adjacent to flooded rice fields. We capture adult Anopheline females individually with a Castro aspirator [27] while resting on the walls after blood-feeding upon contained caws, 1-2h after twilight. We gently transferred the insects to small carton cages maintained in a humid chamber while shipped to the laboratory. Two field collections were carried out: in February 2015 and February 2016. These collections were authorized by Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio), SISBIO # 48127-2. In the laboratory, we morphologically classified the insects as An. albitarsis, following the Anopheles key of Consoli e Lourenço-de Oliveira (1994) and were induced to lay eggs to obtain the F1 generation, as previously described [38]. It was necessary to maintain a new colony of An. albitarsis s.s. to generate a sufficient quantity of insects for bioassays with insecticides. Therefore, subsequent generations were obtained through artificial mating, similarly as performed by Klein et al. (1990)[39] and Lima et al. (2004)[40]. Hereafter we refer to the colonies as ALBI-lab (An. albitarsis s.s. lab colony) and ALBI-field (An. albitarsis s.s. colony recently collected from the field).
Insectary conditions and mosquito rearing.
Laboratory colonies of Anophelines have been maintained accordingly to Lima et al. (2004)[40], with few improvements. Briefly, larvae were reared in plastic trays with 0.1% sea salt solution in dechlorinated tap water. For larvae feeding, we daily pulverized groundfish food ‘Marine Large Flakes’ (Tetra, Melle) was over the water surface and replaced the salt-water solution whenever we noticed a high volume of food in the bottom of the tray. We transferred the pupae daily to cylindrical carton cages (17 x 17 cm, diameter x height), with a 10% sugar solution offered to adults ad libitum. Anesthetized guineapigs were the source of blood-feeding for females (license CEUA – LW-20/14). We introduced in the cages plastic cups internally covered with filter paper and filled with salt-water four days after the blood-feeding. We transferred the eggs to a tray until larvae hatching, which were then replaced to a new tray and reared as above. Larvae and adults were maintained in distinct environments with 28 ± 1C and 26 ± 1C, respectively, in larvae and adult rooms. The relative humidity in adult rooms was maintained around 80% and a light/dark regimen of 12:12h. ALBI-field Colony was maintained similarly, except that we had to proceed with forced copulation and egg-laying induction, as aforementioned.
Insecticide bioassays.
Larvicide.
We performed the “Simplified knockdown bioassay” for Anopheline larvae, adapted from the Aedes protocol developed by Kawada et al. (2009)[41], as detailed by Silva et al. (2014)[42]. Briefly, we exposed 20 L4 larvae individually to a 20 mL pyrethroid deltamethrin (SIGMA-ALDRICH) solution in 50 mL plastic cups. Two concentrations were tested: 0.1 and 0.4 ppm in 20 cups each, in addition to two cups containing 0.4 ppm of the solvent acetone, as a negative control. The Larvae exposition lasted for 30 minutes, and the median knockdown time (KdT50), i.e., the time when half of the larvae (10) sank due to knockdown effect, were categorized in 1 (0-5 min), 2 (6-10 min), 3 (11-15 min), 4 (16-20 min), 5 (21-30 min) and 6 (> 30 min). The product of KdT50 of 0.1 and 0.4 ppm determined the susceptibility index (SI) of the strain, varying from 1 to 36. The larger the SI, the less susceptible the lineage was. In total, we have performed five independent assays in different days for ALBI-lab and ALBI-field (F5 generation of the 2015 field collection), totalizing 100 larvae of each strain tested per concentration. We obtained the average of the five assays as the final SI result.
Adulticides.
Bioassays with adult mosquitoes essentially followed World Health Organization-WHO tube test procedures [43] [44] with papers impregnated in our laboratory, instead. We performed a semi-quantitative time-response assay, where knockdown or mortality rates were monitored continuously until 120 minutes under exposure to the insecticide. The insecticides tested were the pyrethroids type I permethrin (0.75%), type II deltamethrin (0.05%), and the pseudo-pyrethroid etofenprox (0.5%), in addition to the organophosphate malathion (5%). These reagents were technical grade compounds (Pestanal, Sigma-Aldrich), dissolved in acetone, and diluted to their final concentration in silicone oil (Dow Corning). To impregnate the papers, we homogeneously distributed a total of 840 µL of the insecticide solution in a 12 X 14 cm filter paper (Whatman 1) and left it to air dry for two days. We used around 25 three to five days-old females per tube, in a total of three tubes/ insecticide/ strain. The negative control consisted of one tube containing paper impregnated only with the carrier silicone oil, in parallel to each test. We performed the whole assay twice, on different days for ALBI-lab e ALBI-field (F1 generation of the 2016 field collection). We submitted the recorded knockdown (pyrethroids) or mortality (organophosphate) data to Probit analysis, with the help of the software Statgraphics Centurion V. 16.103, in order to calculate the time of knockdown (KdT) or the lethal time (LT) to 50 and 90% of the lineages (KdT50 and KdT90, LT50 and LT90, respectively). We determined the ALBI-field knockdown or lethal time resistant ratios (RR) of each insecticide relative to ALBI-lab by the quotient between their respective KdT or LT values.
Molecular assays.
DNA extraction.
We tittered each insect (adult females) in 250 µL TNES lysis solution (250 mM Tris, 2 M NaCl, 100 mM EDTA and 2.5 % SDS) to obtain individual DNA [45], including a 56°C overnight incubation with 2 µl Proteinase K (20 mg/mL), and two alcoholic washes, first with 70% ethanol, followed by 100% isopropanol. We dissolved the final DNA pellet in 20 µL TE buffer (1 mM EDTA, 10mM Tris-HCl) and checked the DNA concentration with the kit Qubit® dsDNA BR Assay (ThermoFisher Scientific) in a Qubit 3 Fluorometer (ThermoFisher Scientific). We deployed an aliquot of 20 ng of each DNA sample to compose the ALBI-lab (200 samples) and ALBI-field (214 samples of the 2016 collection) DNA pools.
Amplification and sequencing of a COI mitochondrial gene fragment.
To confirm ALBI-field’s taxonomic status, we amplified and sequenced the COI (Cytochrome c oxidase I) mitochondrial gene. We randomly selected the DNA of 11 ALBI-field and 10 ALBI-lab individual females for this analysis. We deployed the primers LCO1490 (5’- GGTCAACAAATCATAAAGATATTGG-3’) and HCO2198 (5’-TAAACTTCAGGGTGACCAAAAAATCA-3’) [46] in a PCR amplification reaction as described elsewhere [47]. The purified amplicons were submitted to the Sanger sequencing method with the kit Big Dye 3.1 (Applied Biosystems) and followed to the DNA sequencing facility “Plataforma de Sequenciamento PDTIS/ Fiocruz”. We checked the quality of electropherograms and aligned the sequences with homologous COI sequences of the Albitarsis complex available in the GenBank (https://www.ncbi.nlm.nih.gov/genbank/): An. albitarsisH (DQ076222, DQ076223, DQ076224), An. deaneorum (DQ076226, DQ07 6227, DQ076229, DQ076230), An. albitarsis G (DQ076221, DQ076225) e An. oryzalimnetes (DQ076210, DQ076211, DQ076213, DQ0762105), with the software Geneious, v9.1.8 [48]. A Neighbor-joining (NJ) non-rooted tree was constructed, utilizing the Kimura-two-parameters model (K2P), with 1,000 permutations for Bootstrap, with the software Mega 7 [49].
Amplification, cloning, and sequencing of the IIS6 segment of the voltage-gated sodium channel gene (NaV).
We used the primers 42F (5’- TCGTGTTTTATGCGGAGAATGG-3’) and 422R (5’CACGGACGCAATTTGACTTGT-3’) designed for amplifying a fragment corresponding to An. darlingi IIS6 NaV segment (Loureiro et al., submitted) for amplifying a fragment corresponding to the IIS6 NaV segment. Amplification reactions made use of 1 µM of each primer, 1X of the GoTaq Polymerase 2x (Promega®), and 4 µL of the DNA pool in a 40 µL PCR reaction. Thermocycling conditions occurred as follow: 94°C/ 3 minutes, followed by 32 cycles of 94°C/30 seconds, 58°C/30 seconds, and 72°C/1 minute, for denaturation, primers annealing, and amplification, respectively, with an additional final step of 72°C/ 3 minutes. We submitted an aliquot of 4 µL of the products to a 1% agarose gel electrophoresis to check the amplification of an expected 400 bp amplicon. The remaining was purified with the kit Kit Agencourt AMPureXP (Beckman Coulter), according to the manufacture’s instructions.
We ligated the purified amplicons (4 µL) to the PJet plasmid with the CloneJET PCR Cloning Kit (Fermentas), submitted them to a transformation reaction with Escherichia coli DH5α competent cells in SOC medium (Invitrogen), and plated the transformed cells in an LB-agar solid medium with ampicillin (100 mg/mL) [45]. Around 96 colonies of each group were randomly selected and individually cultivated in 1 mL of CircleGrow (MP Biomedicals) liquid medium with ampicillin 0.075 mg/mL, in a 96 deep well plate. We obtained the plasmidial purified DNA by an alkaline-lysis procedure (Sambrooke e Russel, 2001) and filtration in a 0.22 µm filter microplate (Millipore). Purified plasmid DNAs (1 µL, around 400 ng) were submitted to the Sanger sequence method with the kit Big Dye 3.1 (Applied Biosystems) and followed to the DNA sequencing facility “Plataforma de Sequenciamento PDTIS/ Fiocruz”, as above. Sequences were evaluated and analyzed with Geneious, v9.1.8 (Kearse et al., 2012).
Anopheles albitarsis kdr genotyping.
Based on the obtained NaV IIS6 An. albitarsis haplotypes, we developed a customized TaqMan SNP Assay (ThemoFischer) for the variations observed in the 1014 NaV codon: TTA/ TTT (Leu+/Phekdr). We used plasmidial DNAs of known sequences as positive controls L/L and F/F. The heterozygote L/F consisted of an equimolar mixture of both homozygotes. Once we have characterized insects with distinct genotypes, their DNA replaced the plasmidial DNA as positive controls.
Before the TaqMan SNP genotyping reaction, the IIS6 NaV segment was amplified, as described above, with individual DNA, in a reaction with half of that total volume. These products were 10X diluted and used in the TaqMan Genotyping SNP reactions, as follow: 1 µL of 10X diluted IIS6 DNA, 1X TaqMan Genotyping Master Mix (Thermo Fischer), 1X CustomTaqMan SNP assay [primers: forward (5’-GCATACCTTTCTTCTTAGCGACTGT-3’) and reverse (5’-GCATGTTCGCAATGTTTGTATCAGT-3’), probes 1014 L (VIC- GAAACTTAGTCGTAAGTG) and 1014 F (FAM- GAAACTTTGTCGTAAGTG)], in a 10 µL reaction. We performed these qPCR reactions in a QuantStudio™ 6 FlexSystem (Thermo Fischer), according to standard TaqMan genotyping procedures.
Calculations.
We obtained genotypic frequencies (L/L, L/F, and F/F) by quoting the number of insects with the respective genotype and the total of evaluated samples. The formulas obtained the allelic frequencies:
f(L) = 2n L/L + n L/F / 2N
f(F) = 2n F/F + n L/F / 2N
where n is the number of insects with the respective genotype, and N is the total samples. We checked the Hardy-Weinberg Equilibrium by its classical equation [50].