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7 protocols using mz 16a stereomicroscope

1

Microscopy and Measurement Protocols for Solenysa reflexlis

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Specimens were examined and measured by using a Leica MZ16A stereo microscope. Further details, such as epigynes, were studied with a Leica DM5500B compound microscope. Digital images were taken with a Leica DFC 500 camera and as a composite of multiple focus images assembled using the software package Leica Application Suite. Epigynes were cleared in methyl salicylate (Holm 1979 ) for examination under the microscope and temporarily mounted as described by Grandjean (1949) and Coddington (1983) . SEM images were taken by using a Hitachi S-3400N scanning electron microscope at China Agriculture University. For SEM examination, the PageBreakspecimens were prepared as described by Álvarez-Padilla and Hormiga (2008) . The non-chitinous abdominal tissue was digested with Sigma Pancreatin LP 1750 enzyme complex to expose the internal structures for examination. Due to the unavailability of specimen, no SEM image provided for the male palp of Solenysareflexlis.
All measurements are given in millimeters. The leg measurements are given in the following sequence: Total (femur, patella+tibia, metatarsus, tarsus). Terminology for the genital characters follows Tu and Hormiga (2011) . The specimens examined here have been deposited in the Department of Zoology, National Science Museum, Tokyo, Japan (NSMT) and in College of Life Sciences, Capital Normal University, Beijing (China).
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2

Measurement of Larval Size by Microscopy

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The size of the larvae was measured for all conditions from day 4 to day 7 of incubation. Larvae (n ≥ 60) were collected and washed in distiller water, killed with a short heat treatment (5s at 90°C) and transferred on a microscopy slide. Images of the larvae were captured using a digital microimaging Leica DMD108 and larval longitudinal size (length) was measured using ImageJ software (Schneider et al., 2012 (link)). The pictures of the larvae were captured with Leica MZ16A stereomicroscope with Leica Application Suite (LAS) microscope software.
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3

Feather Pigment Quantification Using HPLC

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We examined the patterns of pigment deposition in the feather rachi, barbs, and barbules using a Leica MZ16A stereomicroscope at a magnification of 100X, and photographed each feather using a Leica DFC550 camera. We also quantified melanin content using high performance liquid chromatography (HPLC) to measure degradation products of pheomelanin (4-amino-3hydroxyphenylalanine, 4-AHP, and thiazole-2,4,5-tricarboxylic acid, TTCA) and eumelanin (pyrrole-2,3,5-tricarboxylic acid, PTCA). Feather samples were homogenized with a Ten-Broeck homogenizer at a concentration of 1 mg/mL H 2 O. 100 µL (0.1 mg) aliquots were subjected to alkaline hydrogen peroxide oxidation (Ito et al., 2011) and hydroiodic acid hydrolysis (Wakamatsu et al., 2002) .
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4

Wasp Venom Gland Transcriptome Analysis

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Mated female wasps aged 1–3 days were anaesthetized at −70 °C for 5 min, swabbed with 75% ethanol (v/v), dried and then dissected in sterile Pringle’s phosphate-buffered saline (PBS) with 3 µL RNase inhibitor at 1 unit/µL (TOYOBO, Osaka, Japan) on an ice plate under a Leica MZ 16A stereomicroscope (Leica, Wetzlar, Germany). Venom glands and carcasses without venom apparatus were collected into TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Total RNA was extracted using TRIzol reagent according to the manufacture’s protocol. We used agarose gel electrophoresis, Qubit Fluorometer (Thermo Scientific, Wilmington, DE, USA), Agilent 2100 (Agilent Technologies, Santa Clara, CA USA), and nanodrop 2000 (Thermo Scientific, Wilmington, DE, USA) to determine the quality and quantity of the total RNA samples, respectively.
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5

Microcapsule Characterization by Microscopy

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Microcapsules size, morphology and topography were analyzed by microscopic techniques: (i) optical microscopy (OM) (Leica MZ16a stereo-microscope, Leica Microsystems Ltd., Switzerland) and (ii) scanning electron microscopy (SEM) (FE-SEM, model JSM-7000 F, Jeol Ltd., Tokyo, Japan). The average diameter of wet and dry microparticles was determined by optical microscopy using Olympus Soft Imaging Solutions GmbH, version E_LCmicro_09Okt2009. Sixty microparticles were randomly selected from batches produced in triplicate, to determine the size distribution.
Microcapsule formulations for SEM analysis were put on the high-conductive graphite tape. FE-SEM was linked to an EDS/INCA 350 (energy dispersive X-ray analyzer) manufactured by Oxford Instruments Ltd. (High Wycombe, UK). The ImageJ software was used for the determination of the size of pores on a microparticle surface.
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6

Wasp Venom Apparatus RNA Extraction

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Mated female wasps aged 2–7 days were chilled at 4 °C for 10 min and then rinsed in sterile phosphate-buffered saline (PBS, pH 7.2) followed by dissection in PBS with 1 unit/μL RNase inhibitor (Vazyme, Nanjing, China) on an ice plate under a Leica MZ 16A stereomicroscope (Leica, Wetzlar, Germany). The venom apparatus and carcass (minus the venom apparatus) were collected in 1 mL of TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Total RNA was extracted as per the manufacturer’s protocol. The quantity of the total RNA samples was determined by NanoDrop 2000 (Thermo Scientific, Wilmington, DE, USA) and stored at −80 °C for subsequent experiments.
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7

Transcriptome Analysis of Wasp Venom Glands

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Mated female wasps aged 2–5 days were anesthetized at 4°C for 10 min, rinsed in 75% ethanol (v/v) once, and then rinsed in sterile phosphate-buffered saline (PBS, pH 7.2) thrice. Subsequently, the females were dissected in PBS containing 1 unit/μL Murine RNase inhibitor (Vazyme, Nanjing, China) on an ice plate under a Leica MZ 16A stereomicroscope (Leica, Wetzlar, Germany), the venom apparatus (venom reservoirs and associated glands, henceforth, called the VG) and carcasses (the female body minus venom apparatus, henceforth, called the CA) were collected into 1 mL TRIzol reagent (Invitrogen, Carlsbad, CA, United States), respectively. Total RNA was extracted according to the manufacturer’s protocol. RNA degradation and contamination were monitored on 1% agarose gels. RNA purity was checked using the NanoPhotometer® spectrophotometer (IMPLEN, CA, United States). RNA concentration was measured using the Qubit® RNA Assay Kit in Qubit® 2.0 Flurometer (Life Technologies, CA, United States). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, United States).
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