Tick strains
An acaricide susceptible (Su) R. microplus tick strain, previously reported (21,22) and a multiple acaricide-resistant tick strain dubbed San Alfonso (SA)(23) were used in this study (Table 2). This strain has been maintained by continuous infestation of restrained bovines under controlled conditions without exposure to pyrethroids for the last 17 years and designated for records as the San Alfonso (SA) strain for the name of the ranch where it was originally isolated; notably, in spite of the absence of pyrethroid pressure, the ticks are still exhibiting a pyrethroid resistance phenotype according to the larval package bioassays. The SA strain is used as reference for the tick acaricide resistance monitoring programs of the Mexican Federal Government. The SA strain was reared and maintained at Departamento de Ectoparásitos y Dípteros del Servicio Nacional de Sanidad, Inocuidad y Calidad Agroalimentaria (SENASICA- SADER) and the susceptible strain was maintained at Centro Nacional de Investigación Disciplinaria en Salud Animal e Inocuidad (CENID-SAI-INIFAP). Two one-year-old Bos taurus (Holstein Friesian) bovines were used for tick infestations. Animal care and use was according to the mexican norm NOM-062-ZOO-1999, and its technical specifications for production, care and use of laboratory animals can be found at http://www.fmvz.unam.mx/fmvz/principal/archivos/062ZOO.PDF
Table 2. Evolution of the San Alfonso strain resistances over the years without selection pressure.
Percentage of larval mortality
|
Organophosphorous
|
Pyrethroids
|
Amidines
|
Year
|
Coumaphos
|
Diazinon
|
Chlorpiriphos
|
Cypermethrin
|
Deltamethrin
|
Flumethrin
|
Amitraz
|
2001*
|
98.6
|
58.6
|
99.7
|
37.5
|
30.8
|
14.5
|
25.2
|
2006*
|
20
|
0.0
|
0.0
|
0.0
|
0.0
|
0.0
|
0.0
|
2012
|
100
|
90.58
|
99.10
|
0.0
|
0.0
|
0.0
|
0.0
|
2013
|
91.88
|
91.56
|
99.57
|
0.0
|
0.0
|
0.0
|
0.62
|
2016
|
73.24
|
100
|
100
|
0.0
|
0.0
|
0.0
|
10.76
|
* Data from 2001 and 2006 has already been reported in (23,38).
Each reference strain was obtained by infesting a bovine with 1x 104 fifteen days old larvae, engorged tick females were collected 19 to 20 days after infestation, maintained in an incubator with 80% relative humidity at 28°C. Females are deposited on a plastic petri dish and allowed to lay embryos, that are then collected and aliquot in 2mg aliquots, embryos are mantained in the same incubation conditions until eclosion. Eclosed larvae are used to infest a new bovine.
Ovary dissection
Engorged female ticks were collected 19-20 days after infestation, during this period they grow and mature over a stabulated bovine at ambient temperature and natural illumination, feeding themselves with blood to grow and mature. After 20 days of development, they become adults whose ovaries that are full of immature eggs; ticks in this condition are considered preengorged, in order to have enough protein to allow their eggs to mature they begin to consume copious amounts of blood and become “engorged”. We previously found (20) that the ovaries of the preengorged females are the most responsive to physiological treatments.
Ovaries were dissected as described by Cossio et. al., 2015 (20). Briefly: pre-engorged female ticks were washed with distilled water previous to dissection. A transversal cut between the first and second leg pairs separating the whole anterior area. Organs, were extruded into Jan & Jan solution (NaCl 128mM, KCl 2mM, 4mM MgCl2, Sucrose 36mM, HEPES 5mM pH= 7.3) without Ca2+ to avoid neurotransmitter depletion (24), ovaries were separated from the rest of the organs and washed Jan & Jan solutions without Ca2+, to be further processed.
DNA extraction
Ten dissected ovaries were frozen with liquid nitrogen in a ceramic mortar and finely grounded. The resulting frozen powder was resuspended in about 500µl in 0.1 M sodium citrate, pH 8.5, 50mM EDTA, 0.1% SDS and 1mg/ml PCR grade proteinase K (Thermo Fisher, Waltham, Massachusetts, United States). The tissue suspension was incubated for 2h at 60°C, extracted three times with 50% phenol pH 7.0, 50% chloroform; and then extracted once with 100% chloroform. The resulting extract was supplemented with a 10th of its volume with Sodium acetate 5M pH 5 and precipitated with 3 times its volume of absolute ethanol. Centrifuged for 5 minutes at 12,000g, washed once with 70% ethanol, air dried and resuspended in 100µl of molecular biology grade distilled water.
PCR amplification and SNPs determination
Oligonucleotides for the para-sodium channel relevant domains were synthetized accordingly to the ones reported in (13). PCR was performed using hotstart procedure as follows: 5 minutes at 95°C before adding Taq DNApolymerase (Thermo Fisher Scientific, Waltham, Massachusetts, United States) using the following amplification conditions, 30 seconds denaturation at 95°C, 30 seconds annealing at 59°C and 30 seconds polymerization at 72°C for 35 cycles and a final extension cycle at 72°C for 7 minutes.
The oligonucleotide sequences used were the following:
RmNaDomainIIF1; TACGTGTGTTCAAGCTAGCCAA
RmNaDomainIIR1; ACTTTCTTCGTAGTTCTTGCCAA
RmNaDomainIIIF1; AAGAGGACCAACCGGAATACG
RmNaDomainIIIR1; TCTTCTTTTGTTCATTGAAATTGT
PCR products were sequenced at the “Unidad de Síntesis y secuenciación de ADN del Instituto de Bitecnología” using the same oligonucleotides used for amplification.
All mutations reported in this Table 1 and the rest of this work follow the naming convention for R. microplus based on nucleotide position within the mRNA sequence [GenBank:AF134216.2]. Twenty individuals of the susceptible and the resistant population, were sampled, the corresponding amplicons were sequenced bidirectionally and analized for the presence of SNP using as reference GenBank:AF134216.2. We found no evidence of heterozigocity.
Acaricide discriminant doses Bioassays (DD)
Ticks were assayed and its toxicological profile was verified by acaricide discriminant doses Bioassays (18) (Table 2). Bioassays were performed with acaricides diluted in trichloro ethylene/ olive oil (2:1) at the following pesticide concentration: coumaphos 0.2%, diazinon 0.08%, chlorpiriphos 0.2%, cypermethrin 0.05%, deltamethrin 0.09%, flumethrin 0.01%. 670µl of each dilution was applied evenly to a 7.7 by 8.5 cm piece of filter paper; these concentrations were experimentally determined using susceptible populations as the lowest concentrations that would kill 100% of the larvae in the packet when the SENASICA-SADER program was established. Since then the concentrations have been fixed for methodological reasons. The trichloroethylene was evaporated from the filter paper in an extraction hood for 2 h. The treated papers were then folded in half and sealed on the sides with clips, forming a packet into which 100 larvae were placed; the packet was then sealed with another clip. Packets were kept at 28˚C (± 2.0°C) 80-90% relative humidity for 24 hours, live (motile), immobile larvae were considered death, both populations were counted and the data was reported as the percentage of mortality for each tick group each acaricide concentration.
Whole-mount preparation of contractile ovaries
Ovaries were dissected as described above in Jan & Jan solution (NaCl 128mM, KCl 2mM, 4mM MgCl2, Sucrose 36 mM, HEPES 5mM pH = 7.3) without Ca2+ to avoid neurotransmitter depletion during ovary dissection and preparation. Ovaries were loosely immobilized on a Sylgard plate in a perfusion chamber using stainless steel pins (Austerliz Insect Pins, minutiens 0.1mm Fine Science tools); organs that may be involved in xenobiotic detoxification such as the fat body, the Malpighian tubules and the intestine were discarded. Mounted ovaries had the Jan & Jan Ca2+ free solution and were substituted with complete Jan & Jan solution (NaCl 128mM, KCl 2mM, 4mM MgCl2, Sucrose 36 mM, 2mM Ca2+, HEPES 5mM pH = 7.3).
Videometric analysis of ovary response to cypermethrin
Ovary muscle contraction was videometrically recorded exactly as reported in (20). For each ovary, the ovary area average of the first 15 frames before cypermethrin addition was used as A0. Normalized ovary contraction index (NCI) is defined as the ovary area value of each frame Af normalized with A0 using the following formula:
Normalized contraction index = Af/A0 * 100. The addition of Ca2+ increased in ovary muscle tone reflecting tissue integrity and defining ovary initial area (A0). NCI is defined as the area of the ovary at any moment divided by A0 and multiplied by 100. A NCI smaller than 100 means that the tissue has an increase in tone or contraction. A NCI bigger than 100 means that the tissue relaxes or loses tone. The effect of cypermethrin tested on muscle tone (A1) was measured for 70s of exposure to treatment and averaged. Muscle contraction was induced by depolarization with 15mM KCl. Final or maximal ovary contraction (A2) is defined as the average of the last 70 seconds after the addition of KCl. The samples were exposed to cypermethrin by perfusion with Jan & Jan solution supplemented with the corresponding cypermethrin concentrations tested (3, 5, 9 and 15µM). Cypermethrin was dissolved in DMSO and its final concentration was 0.05% in all experiments. In all cases n = 9 contractile ovaries.
Time series statistical analysis
For each data point (frame) the normalized contraction index was averaged between samples of the same treatment (n = 9) and its standard deviation was calculated. Control time series were obtained with susceptible contractile ovaries in (0.05% DMSO in complete Jan & Jan solution without cypermethrin). Experimental time series were compared to controls using ANOVA followed by Dunnett´s multiple comparison test accordingly to Shumway(25). P values ≤ 0.01 were considered significative.