Hemolymph

A. gambiae Keele strain mosquitoes were maintained on sugar solution at 27 °C and 70% humidity with a 12-h light/dark cycle according to standard rearing procedures [50 ]. For microarray assays, the carcasses and midguts from approximately 40 mosquitoes were dissected on ice 24 h after ingestion of blood infected with the wt Anka 2.34 or CTRP⁻ P. berghei strain, or the wt NF54 or CTRP⁻P. falciparum strain, or noninfected human blood. P. falciparum gametocyte cultures were prepared as previously described, and mosquitoes were fed on cultures through a membrane feeder at 27 °C and then maintained at 24 °C [51 (link)]. P. berghei infections were done at 21 °C as previously described [7 (link)]. Mosquito midguts were dissected at 7–8 d after feeding and stained with 0.2% mercurochrome. Oocyst numbers per midgut were determined using a light-contrast microscope (Olympus, Tokyo, Japan). P. berghei infections with gene-silenced (RNAi) mosquitoes were performed with a transgenic GFP P. berghei strain and infection phenotypes were determined as previously described [7 (link)]. For preparation of bacterially challenged samples for microarray analyses, 4-d-old female mosquitoes were first injected with approximately 20,000 heat-inactivated E. coli or S. aureus and approximately 20 whole mosquitoes were collected 4 h after challenge [52 (link)]. For bacterial challenge of gene-silenced (RNAi) mosquitoes, E. coli and S. aureus were cultured in LB broth overnight, then washed three times with phosphate-buffered saline (PBS) before being resuspended in PBS. Approximately 27,000 live E. coli or 55,000 S. aureus in a 50-nl PBS suspension were injected into the mosquito hemolymph 4 d after the dsRNA injections. RNA was extracted from dissected tissues or whole mosquitoes by using the RNeasy kit (Qiagen, Valencia, California, United States). Quantification of RNA was performed using a Biophotometer (Eppendorf, Hamburg, Germany) spectrophotometer, and quality assessment was determined by RNA Nano LabChip analysis on an Agilent Bioanalyzer 2100.

Normality and homogeneity of variance were tested for residuals and predicted values for all data. Average values from the last time points (130–150 min) were calculated for TER, TEP, and SCC to obtain stable values of the parameters. The effect of salinity on TER, TEP and the transfer of radiolabelled Ca²⁺ as well as on the hemolymph ion concentration was assessed with an Independent Samples t-test. The effect of salinity on SCC was assessed with a non-parametric Mann–Whitney U test. The statistical analyses for electrophysiology data were performed using IBM SPSS 22 (SPSS Inc., Chicago, IL, United States). A P-value of ≤0.05 was regarded as significant. Statistical differences in mRNA expression between salinities (14 vs. 28) were analyzed using an unpaired t-test (two-tailed). P values < 0.001 (^∗∗∗), 0.01 (^∗∗), and 0.05 (^∗) were considered as significant. Statistical analysis was performed using GraphPad Prism version 7.0a for MacOSX (GraphPad Software, La Jolla, CA, United States)⁵. All data are presented as mean values ± standard error of the mean.

Phenoloxidase Assay. The phenoloxidase (PO) assay is used to infer the strength of the immune response by measuring the degradation of the substrate by the enzyme PO over time in hemolymph (Gonzalez-Santoyo and Cordoba-Aguila, 2012 (link)). A subset of each host plant treatment group (n = 30) were chosen to have hemolymph collected for the PO assay. On the fourth day of fifth instar, 10.0 µl of hemolymph was collected using a micropipette and sterile insect pin to puncture the integument at the base of the third proleg. The hemolymph was immersed into 500 µl of phosphate-buffered saline (PBS) (Sigma-Aldrich) solution. Next, 100 µl of the PBS-hemolymph mixture was divided between two wells of a 96-well polystyrene microplate (Fisher-Scientific): 1) PO activated at the time the samples were taken (standing PO) and 2) all available PO including the stored, non-activated enzymes (total PO). To activate stored PO, 10.0 µl of 10% cetylpyridinium chloride (CPC) (Sigma-Aldrich) was added to each total PO well, followed by a 20-min incubation period. To correct for this additional volume, 10 µl of water was added to each of the standing PO well. Afterwards, 200 µl of 5 mM dopamine solution (Sigma-Aldrich) was placed in every well to act as the substrate for phenoloxidase. The plate was then immediately placed in a microplate reader (Bio-Rad iMark Microplate Absorbance Reader) for 45 min with readings made every 30 s at a wavelength of 490 nm. The linear phase of the reaction (determined to be between 0 and 20 min) was used for all analyses. Data were extracted from the spectrophotometer using Microplate Manager (MPM) software (Bio-Rad v.6.3).
Sephadex Bead Assay. Melanization were measured by injecting Sephadex beads into the hemocoel of the larvae (n = 15 for each host plant treatment). Following bead preparation methods in Smilanich et al. (2009a) (link), DEAE Sephadex-A25 chromatography beads (40–120 µm diam) (Sigma-Aldrich) were dyed with a 0.1% solution Congo Red Dye (Sigma-Aldrich) and left to dry in the hood before use. A 30-gauge needle (Sigma-Aldrich) fastened onto a syringe was used to administer 10 beads immersed in PBS. After hemolymph collection for the PO assay, larvae were injected with beads in the same wound site created during hemolymph collection or within a 5 mm radius of the wound site. Larvae were returned to their individual 2.0 oz cups and given 24 h to mount an immune response before being freeze-killed. Beads were recovered from dissected larvae and photographed using a dissecting microscope connected to a digital camera (Carl Ziess Discovery V.8, AXIOCAM Software, Oberkochen, Baden-Wurttenburg, Germany). Beads were photographed at ×80 magnification, and their red value was scored in Adobe Photoshop (v6.0; Adobe System Inc., San Jose, California, United States). The red value of the bead ranges from 0–255 with 0 = pure gray and 255 = pure red. The mean red value was obtained for each bead within a caterpillar and these values averaged to provide a red value score for each individual caterpillar. The mean red value was transformed into a percentage of melanization [1—(red value/maximum red value)] for ease of interpretation. With the transformed red value, the higher the value the darker the bead, which means more melanization and a stronger immune response (Rantala and Roff, 2007 (link); Smilanich et al., 2009b (link)).

For SDS-PAGE and Western blotting, a 100 µL aliquot per sample was diluted with 100 µL 2 × reducing Laemmli sample buffer (containing 5% [v/v] β-mercaptoethanol), boiled for 5 min at 100°C and a 5 µL aliquot per sample was applied to 4–20% Tris-glycine (TGX^TM) gels (BioRad, UK). Electrophoresis was carried out at 165 V for 52 min; gels were then transferred for Western blotting analysis using semi-dry transfer (1 h at 15 V), even protein transfer was assessed by PonceauS red stain (Sigma, UK). The membranes were blocked in 5% [w/v] bovine serum albumin (BSA, Sigma, UK) in TBS-T for 1 h at room temperature and incubated in primary antibodies overnight at 4°C on a shaking platform. Primary antibodies used for haemolymph were: anti-PAD2 (ab50257), as this is the most phylogenetically conserved PAD isozyme, and anti-citrullinated histone H3 (citH3, ab5103) as a marker for ETosis assessment; both diluted 1/1000 in TBS-T. For assessment of EV surface markers, CD63 and Flotillin 1 were used as described above. Following primary antibody incubation, the blots were washed with TBS-T (3 × 10 min), incubated in secondary antibody for 1 h at room temperature (using HRP-labelled anti-rabbit IgG; BioRad, diluted 1/3000 in TBS-T). Following washing (5 × 10 min in TBS-T), visualization was carried out using ECL (Amersham Biosciences) and the UVP BioDoc-ITTM System (ThermoFisher Scientific, Dartford, UK). Protein densitometry analysis was carried out in ImageJ [46 (link)].

For identification of putative deiminated/citrullinated proteins in crab haemolymph, enrichment was carried out using the F95 pan-citrulline antibody (MABN328, Merck) in conjunction with the Catch-and-Release Immunoprecipitation Kit (Merck). Immunoprecipitation was carried out on mini-agarose columns together with the F95 antibody and the affinity ligand, overnight at 4°C on a rotating platform and proteins thereafter eluted according to the manufacturer’s instructions (Merck, UK). F95-enriched proteins from the parasitized and control haemolymph were then subjected either to SDS-PAGE and silver staining, or to liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis for identification of protein hits.