Adult female S. sichuanensis were reared on T. molitor pupa (0.1–0.15 g), which were reared on wheat bran and pollution-free vegetables, at the Provincial Key Laboratory of Forest Protection (College of Forestry, Sichuan Agricultural University, Sichuan Province, China). Wasps that had emerged for 6 days and completed mating were used in the experiments.
Before the experiment, mated female wasps and host pupae (1:1 ratio) were placed in finger-shaped glass tubes (6 cm × 1 cm). This procedure was designed to ensure that the wasps were familiar with and efficient at injecting and paralysing hosts. If hosts could be paralysed within 3 h, we considered experienced to be successful. This familiarization helped to maintain equal-instars pupa in the treatment and control groups. Experienced wasps and pupae (1:1 ratio) were placed in finger-shaped glass tubes. The tube orifices were sealed by cotton wool, and the tubes were kept under natural light. When wasps successfully paralysed the host for 24 h, we defined it as 1 day.
Preparation of venom and calculation of protein content
Venom sacs were carefully removed from the abdomens of female S. sichuanensis under a dissecting microscope and placed in 20 μL Dulbecco’s phosphate buffered saline (PBS: 0.80 % NaCl, 0.01 mol L−1 phosphate, pH 7.0–7.2) in the cap of an 0.5-mL centrifuge tube. The sac was then torn open to release the venom into the solution, and the empty sac was removed. After replacing the cap on the centrifuge tube, the tube was briefly centrifuged at 10,000×g and 4 °C (10 s), and the venom was collected and placed on ice. Venom from up to 20 wasps was collected in this way and pooled in 1 tube. The venom was then passed through a pipette tip to ensure thorough mixing, and centrifuged for 30 s (10,000×g, 4 °C) to pelletize; the supernatant was collected and stored at −20 °C. A standard curve was prepared using bovine serum albumin, and venom protein (0–100 μg) was quantified spectrophotometrically according to Bradford (1976). The calculation of protein content test was repeated five times by using 5 samples.
Natural injection and venom injection
Parasitism (natural injection)
Wasps and pupae (n = 40 each) were placed in finger-shaped glass tubes, and the tube orifices were sealed with cotton wool. The tubes were kept under natural light. After pupae were naturally injected and paralysed by wasps, they were removed from the tubes and kept at 27 ± 1 °C and 85 % relative humidity (RH).
The prepared venom was diluted with PBS to a concentration of 0.3 venom reservoir equivalents [VRE, one VRE was defined as the supernatant from one torn venom reservoir in 1 µL PBS; pupae could be complete paralytic when injected with 0.3 VRE (Zhuo et al. 2013a)], and the diluent (1 μL) was injected into the abdomen of each pupa. Pupae were injected in groups of 40 individuals, and the same numbers of pupae were injected with PBS as a control. The injected pupae were kept at 27 ± 1 °C and 85 % RH.
Pupae that had been injected with venom and PBS and kept for 1–6 and 10 days (some of the injected pupae would die after 10 days) were used as treatment groups in the subsequent experiments respectively. Pupae that had been parasitized (naturally injected) and non-parasitized and kept for 1–6 and 10 days were used as controls respectively. All pupae from 1 to 6 days were used for biochemical analyses and pupae at 10 days were used for observation of the fat body. All treatments were measured 3 times by using 40 pupae for each treatment and each nutrient, except analysis of fat-body protein and observation of fat body.
Sample pre-treatment followed methods described by Tan et al. (2003). We added 4 mL 0.15 mol L−1 perchloric acid to 1 g T. molitor pupae, and the pupae were ground to a homogenate. Then, 6 mL 0.15 mol L−1 perchloric acid was used to wash the mortar four times, and this solution was transferred into a centrifuge tube and centrifuged at 5000×g for 20 min. The supernatant was collected and filtered into a 25-mL volumetric flask. Distilled water was added to constant volume, and this mixture was used for sugar extraction.
Trehalose content followed methods described by Tan et al. (2003). Sugar extract (1 mL) was added to 2 mL 0.075 mol L−1 sulfuric acid and the mixture was heated in a water bath for 10 min, 60 °C. While the mixture was cooling, 2 mL 30 % KOH was added, and the solution was boiled for 20 min to destroy reducing sugars. One milliliter of this solution was then added to 4 mL anthrone reagent (1.0 g anthrone dissolved in 1 L 95 % concentrated sulfuric acid) and mixed well. The mixed solution was boiled in a water bath for 10 min. While the solution cooled, the OD620 value was determined to calculate trehalose content.
A trehalose standard curve was prepared using standard (200 µg mL−1) glucose solution (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, and 0.7 mL). Water was added to each tube to bring the volume to 1 mL. Anthrone reagent (4 mL) was then added to each tube and mixed well, and the tubes were heated for 10 min. The OD620 value was measured while the solution was cooling. A standard curve was drawn with sugar content as the abscissa and OD620 value as the ordinate.
Glycogen content was measured by adding 2 mL sugar extract, 6 mL 95 % ethanol, and 3 drops saturated Na2SO4 solution to a centrifuge tube. The solution was mixed well, left to stand for 3 days at 4 °C, and then centrifuged at 5000×g for 15 min. The supernatant was discarded and the residual liquid was briefly heated at 70 °C to remove ethanol. The precipitate was removed and dissolved in water; the dissolved solution was transferred into a 10-mL volumetric flask, and distilled water was added to constant volume and mixed well. One milliliter of this solution was added to 2 mL 0.075 mol L−1 sulfuric acid, and the mixture was heated in a water bath for 10 min. While the mixture cooled, 2 mL 30 % KOH was added, and this solution was boiled for 20 min to destroy reducing sugars. One milliliter of the boiled solution was added to 4 mL anthrone reagent and mixed well, and the mixed solution was boiled in a water bath for 10 min. The OD620 value was determined while the solution was cooling, for calculation of glycogen content.
A standard glycogen curve was prepared as described for trehalose in “Glycogen content” section.
Reducing sugar content
Reducing sugar content was determined by adding 1 mL sugar extract, 0.5 mL 3,5-dinitrosalicylic acid reagent, 6.3 g 3,5-dinitrosalicylic acid, and 262 mL 2 mol L−1 NaOH to a hot solution of 182 g potassium sodium tartrate dissolved in 500 mL water. Then, 5 g recrystallized phenol and 5 g Na2SO3 were dissolved in the solution and mixed well. As the solution was cooling, it was transferred to a 1000-mL volumetric flask, and distilled water was added to constant volume and mixed well. The yellow solution was stored in a brown bottle for 7 days and then heated in boiling water for 5 min. While it cooled, 4 mL double-distilled water (DDW) was added to the solution and mixed well, and the OD540 value was measured to determine the content of reducing sugar.
A standard was prepared using 0, 0.2, 0.3, 0.4, 0.5, and 0.6 mL standard glucose solution (200 µg mL−1). Water was added to each tube to bring the volume to 1 mL. Then, 0.5 mL 3,5-dinitrosalicylic acid reagent was added to each tube and mixed well, and the solution was heated for 5 min. While the solution cooled, 4 mL water was added to each tube and mixed well, and the OD540 value was measured. A standard curve was drawn with glucose content as the abscissa and OD540 value as the ordinate.
Analysis of hemolymph protein
Hemolymph collection (piercing method) followed methods described by Gäde et al. (2007). Hemolymph protein was analyzed by centrifuging 5 µL pupal hemolymph at 1000×g for 10 min to remove blood cells. The supernatant was collected to determine protein contents by electrophoresis according to Bradford (1976). The treatment was measured 3 times by using different pupae. We used sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The concentrations of spacer and separating gels were 5 and 12 % respectively. Before appearance of the indicator in the separating gel, the electrical current was set at 5 mA; when the indicator appeared, the electrical current was set at 10 mA. Electrophoresis was terminated when the indicator was 1 cm from the bottom of the gel. The gel was then stained by Coomassie brilliant blue R250, scanned, and analyzed.
Analysis of fat-body protein
The pupal fat body was cleaned three times by PBS and dried using filter paper. The fat was weighed, ground, and centrifuged at 10,000×g for 10 min. The supernatant consisted of soluble extract of fat-body protein, which was determined as described for hemolymph protein (“Analysis of hemolymph protein” section).
Analysis of hemolymph lipid droplets
Hemolymph lipid droplets were analyzed according to Nakamatsu and Tanaka (2003). Pupal hemolymph (5 µL) was centrifuged at 1000×g for 10 min, and the supernatant was extracted by chloroform for 5 min. Then, 0.5 mL concentrated sulfuric acid was added and the solution was heated in boiling water for 10 min. After cooling to room temperature, 1 mL glutaraldehyde solution was added and the solution was stained for 30 min. Finally, the OD547 value was determined to calculate the content of hemolymph lipid droplets. A standard curve was prepared using cholesterol (2.5 mg mL−1 carbinol, concentration range 0–50 mg mL−1).
Analysis of fat-body lipid droplets
The pupal fat body was cleaned by PBS three times, dried using filter paper, freeze-dried, and weighed. The fat body was then homogenized by 0.5 mL chloroform and centrifuged at 1000×g for 10 min, and the supernatant was collected to determine the content of fat-body lipid droplets as described for hemolymph (“Analysis of hemolymph lipid droplets” section).
Observation of fat body
The complete or broken fat body was carefully removed under a dissecting microscope, stained by 0.1 % Sudan IV solution, and photographed with an camera system (Gel Doc XR, Bio-Rad, USA) for observation.
All data were analyzed by one-way analysis of variance (ANOVA) and Duncan’s multiple-range tests, and all analyses were performed in SPSS 18.0 (SPSS Inc., Chicago, IL).