Nd 100 spectrophotometer

Total RNA was extracted from the whole body (lice from Farms 1 and 2; bioassays) or only from the cephalotorax (lice from Farm 3; pre-post azamethiphos treatment) of adult C. rogercresseyi females and males. A Trizol protocol combined with the RNeasy Mini kit for animal tissues (Qiagen, Hilden, Germany) was used for the extraction. Lice tissues were homogenized in 1 ml Trizol using TissueLyser MM 301 (Qiagen Retsch, Hilden, Germany) and one stainless steel bead of 5 mm diameter (Qiagen). After mixing with 0.2 ml chloroform and a centrifugation step, the aqueous phase was transferred to a new vial and mixed with one volume of 70% ethanol. Total RNA was then isolated with RNeasy spin columns following the manufacturer’s protocol. The RNA was quantified with an ND-100 Spectrophotometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and the quality was checked with a 2100 Bioanalyzer instrument (Agilent Technologies, Santa Clara, California, USA) and an Agilent RNA 6000 Nano kit. First strand cDNA was synthesized with the RevertAid H Minus first strand cDNA synthesis kit (Thermo Fisher Scientific) using oligo (dT)₁₈ primers.

For Sample set II, DNA was isolated from all the salmon louse samples in 96-well format using DNeasy kit as per manufacturer’s instructions (Qiagen, Hilden, Germany). A spot-check of DNA quality and quantity was made for some of the samples using an ND-100 Spectrophotometer (Thermo Fisher Scientific, DE, USA). Prior to genotyping, all samples were re-organized onto 384 plate format.

Total RNA was extracted using RNeasy plus Mini kit (Qiagen, CA, USA), from female adult individuals, as per manufacturer’s protocol. The RNA was quantified and qualified on ND-100 Spectrophotometer (Thermo Fisher Scientific, DE, USA). First strand cDNA was synthesized from total RNA (1 μg) using qScript reverse transcriptase (Quanta Biosciences, MD, USA).

Five adult F2-females immobilized at 0.2 µg/L (S from family group 1) and five adult F2-females not visibly affected at 1 µg/L (R from family group 2) in addition to female parasites from the P0 and 2013 Ls A (n = 4 and n = 3) and Ls V (n = 5 and n = 7) groups were prepared for RNAseq analysis.
Total RNA was extracted from individual adult females using a Trizol protocol combined with RNeasy Mini kit for animal tissues (Qiagen, CA, USA). Lice tissues were disrupted and homogenized in 1 ml Trizol using TissueLyser MM 301 (Qiagen Retsch) and one stainless steel bead of 5 mm diameter (Qiagen). After mixing with 0.2 ml of chloroform and a centrifugation step, the aqueous phase was transferred to a new vial and mixed with one volume of 70% ethanol. Total RNA was then isolated with RNeasy spin columns following manufacturer’s protocol. Genomic DNA was removed from the extracted RNA with Turbo DNA-free TM kit (TURBO™ DNase Treatment and Removal Reagents, Ambion, Life Technologies). Subsequently, the RNA was cleaned and concentrated with RNA Clean & ConcentratorTM-5 (Zymo Research). The RNA was quantified with ND-100 Spectrophotometer (Thermo Fisher Scientific, DE, USA) and the quality was checked with a 2100 Bioanalyzer instrument (Agilent Technologies) and the Agilent RNA 6000 Nano kit.

Fibrinogen was purified from pooled mouse plasma by ammonium and ethanol precipitations, using a protocol adapted from Dietrich et al. (37 (link)). All steps were performed at 4 °C. A protease inhibitor mixture (55 mM ε-aminocaproic acid, 55 mM benzamidine, 11 μM pepstatin, 11 μM leupeptin, and 1.1 mM phenylmethylsulfonyl fluoride, in TBS) was added (1:10 vol/vol) to plasma and 1 vol saturated ammonium sulfate (760 g/L) was slowly (drop-by-drop) added to 3 vol plasma and incubated for 2 h. Following centrifugation at 12,000 g for 15 min, the pellet was resuspended in 2-(N-morpholino) ethanesulfonic acid (MES) buffer (55 mM ε-aminocaproic acid, 55 mM benzamidine, 1.1 μM pepstatin, 1.1 μM leupeptin, 110 μM phenylmethylsulfonyl fluoride, 22 mM 2-(N-morpholino)ethanesulfonic acid, in ddH₂O, pH 6.6), and the whole process was repeated a second time, followed by pellet resuspension and dialysis in TBS for 1 h. Then, 1 vol ice-cold 100% ethanol was added drop-by-drop to 13 vol ice-cold suspension and incubated on ice for 1 h. Following centrifugation at 12,000 g for 15 min, the pellet was resuspended and dialyzed against TBS overnight before the concentration was determined using a ND-100 Spectrophotometer (Thermo Fisher Scientific).