Wolbachia

B, Muhamad Azmi Mohammed, B, Ameyra Aman-Zuki, Wahida, Nurul Othman, Tagami, Yohsuke & Yaakop, Salmah, 2018, The role of a novel Wolbachia (Rickettsiales: Anaplasmataceae) synthetic peptide, WolFar, in regulating prostaglandin levels in the hemolymph of Acheta domesticus (Orthoptera: Gryllidae), Turkish Journal of Zoology 42 (4), pp. 422-431 : 423

2.1. Designation of Wolbachia synthesis peptide (WolFar) A total of 17 sequences of WSP were obtained from Mohammed et al. (2017). These sequences were amplified from an endoparasitoid species, Fopius arisanus , which was collected from five carambola cultivation sites in Peninsular Malaysia. The WSP sequences were aligned to search for the conserved region that comprises 29 nucleotides (5’ -AGC TAC TAC GTT CGT TTG CAA TAC AAC GG- 3’) translated into nine amino acids (SYY VRL QYN), and they were sent to Mimotopes Pty. Ltd. ( Australia) for peptide synthesis. The custom-synthesized peptide was made using mild 9-fluorenylmethoxycarbonyl (Fmoc) chemistry methods ( Sheppard, 2003). The peptide assembly and purification were computer-controlled to ensure the authenticity of the sequences. The synthesized peptides were then purified in an automated preparative HPLC system, and the best fractions were selected as the final product, with a final mass of 509.7 mg at 93% of purity by HPLC. The customized peptide was 1205.34 g / mol. The purified peptides were consistently estimated by analytical reverse-phase HPLC (RP-HPLC), and they were checked for correct identity using mass spectrometry (MS). The peptide treatments were prepared by diluting the peptide in solid form with deionized distilled water at a ratio of 1:1 (100%) to produce molarity of 0.83 mmol/L. The 100% peptide then underwent a series of dilutions to produce concentrations of 75% (0.63 mmol/L) and 50% (0.42 mmol/L).

2.2. Insect rearing

Fifth instar females of Acheta domesticus , weighing 200 ± 10 mg, were used as a model species. The cricket nymphs were bought from a pet shop and underwent a selective procedure for the fifth instar stage: the nymphs must not yet have formed the protruding ovipositor structure. According to Patton (1978), female A. domesticus reach their fifth instar stage when the ovipositor structure begins to show. Accordingly, all nymphs without ovipositors were brought to the lab, placed in transparent plastic containers (24.5 cm × 13.5 cm × 13.0 cm), and reared under lab conditions until the emergence of the ovipositor structure. The plastic containers were provided with pieces of cotton wool soaked in water as a drinking supply and a few dry twigs for the nymphs to jump on. Rabbit pellets (Harringtons, UK) and larvae of the mealworm Tenebrio molitor were supplied as food. The female crickets with newly formed ovipositor structures were selected and relocated into new plastic containers prior to the biochemical assay. All the rearing procedures were conducted at 28 °C with 68% relative humidity.

2.3. Peptide treatment assay

A total of 10 fifth instar nymphs of female A. domesticus , chilled to 2 °C for 10 min, were injected dorsoventrally between the third and fourth abdominal segments with 15 µL of Wolbachia synthetic peptide. As a negative control, 10 samples were injected with 15 µL of deionized distilled water. The synthetic peptide injection assays were set to 24 h, 48 h, and 72 h, in line with previous biochemical assay studies on insect immune response ( Bali and Kaur, 2013; Prisco et al., 2013), and the hemolymph was collected with micropipettes 24, 48, and 72 h after injection. The hemolymph was collected by piercing the prothoracic legs with a fine and sterile needle and pooled into Eppendorf tubes from all 10 samples. The average quantity of hemolymph collected was about 30.0 µL per insect. The tubes were then centrifuged (12,000 × g, 27 °C, 10 min) to remove cell wastes. Phosphate buffered saline (PBS), pH 7.4, was added to the hemolymph to achieve the different concentrations (50%, 75%, and 100%). The mixtures of hemolymph and PBS were transferred to new Eppendorf tubes, labeled with their treatment concentrations, in preparation for ELISA. Three replications were taken for each treatment as well as for the control, and the experiments were repeated twice.