Compound microscope

Puparia of the new species were collected on leaves of Schimasuperba from Zhejiang, Thousand Island Lake (hereafter TIL) and Gutianshan Nature Reserve, Shuangxikou village, China. No adult emergence was noticed during rearing of puparia for two weeks. Puparia were mounted following Dubey and David (2012) . The terminology for morphological structures follows Bink-Moenen (1983) , Martin (1985) and Gill (1990) . Habitus images were taken using a digital camera Canon IXUS 105 and a camera DFC 290 (Leica, Wetzlar, Germany) attached to a Leica stereomicroscope M 125 (Leica, Wetzlar, Germany). Puparial measurements and microphotographs were taken using a compound microscope (Carl Zeiss, Gottingen, Germany) from Zhejiang Agriculture and Forestry University (ZAFU). The scanning electron microscope images were taken by Hitachi TM-1000 Scanning Electron Microscope (Hitachi, Japan) from the Center of Electron Microscopy, Zhejiang University (Life Sciences Division). Adobe Photoshop 7 software was used for figure preparation. The holotype is deposited in the Insect Collections of Zhejiang Agriculture and Forestry University, Lin’an, China (ZAFU). One paratype will be deposited in the Shanghai Entomological Museum, Chinese Academy of Sciences (SEM-CAS) and the remainder in ZAFU.

For morphological description, specimens were removed from mounts and relaxed in a Sodium Dodecyl Sulphate-based buffer with Proteinase K, and then transferred into ethanol for observation with stereo microscopy, or into 1:1 ethanol : glycerol for imaging with a Zeiss compound microscope. Genitalia and terminal segments were dissected and mounted in temporary glycerol slide mounts, and are stored in glycerol microvials pinned with their respective specimens. Photographs of specimens were taken by using a Visionary Digital photomicrographic apparatus with Infinity optics and a Canon 60D camera, installed at the American Museum of Natural History, New York. Montage images were constructed from stacks using Helicon Focus. DNA from two paratype females was extracted according to a non-destructive protocol outlined previously (Parker and Maruyama 2013 (link)). The symbol “//” is used to separate different data labels attached to the specimens. The terminology used to describe the foveal system follows Park (1942) (link), as modified by Chandler (2001), except that the terms “mesoventral” and “metaventral” are used in place of “mesosternal” and “metasternal”, following the discussion of Herman (2013) (link).

Pole cells were detected in fixed 10- to 14-hour-old embryos using anti-Vas immunohistochemistry as previously described [60 (link)]. The following antibodies were used: 1:500 rabbit anti-Vas and 1:500 biotin goat anti-rabbit (Jackson Immuno Research Laboratories). Embryos were mounted in 80% glycerol and analyzed using a Zeiss compound microscope. Images of representative embryos were captured on a Nikon DsQi-2 microscope using a 20 × 0.75 NA air objective and DIC optics.

Recent collections were sampled via snorkelling from Macrocystis sp. in Wellington and collected from a large, drifting detached plant of Durvillaeaantarctica from Otago Harbour. They were immediately preserved in 95% ethanol. Specimens were examined and dissected using a Leica MZ12.5, in Wellington and drawn using a camera lucida attachment. Small appendages (mouthparts, uropods, telson) were temporarily mounted in glycerin and examined and drawn using a compound microscope (Zeiss, in Wellington) fitted with a camera lucida. The body lengths of specimens examined were measured by tracing individual’s mid-trunk lengths (tip of the rostrum to end of telson) using a camera lucida. For scanning electron microscope (SEM) imaging the specimens and appendages were dehydrated through a graduated ethanol series, acetone dried, mounted on studs, coated with gold-palladium and investigated via a SEM LEO1525.
Type material and other material examined is held at the "National Institute of Water and Atmospheric Research Invertebrate Collection at Wellington, New Zealand (NIWA) and the CeNak, Zoological Museum Hamburg.

A subset of sputum samples (500 μl) from ATB and NTB subjects (n = 8) were fixed with equal volume of 70% ethanol specifically for cytology analysis. Smear was prepared on a glass slide, dried for 15 mins at room temperature before heat fixing and Wright’s stain was applied for 3 mins. After washing with water, slides were incubated with diluted Wrights stain (stain/phosphate buffer saline; 1:4 (v/v)) for 15 mins. After second wash with water, slides were kept at room temperature for air drying and taken for observation of presence and absence of different types of cells at 400× magnification using a compound
microscope (Carl Zeiss). Epithelial cell, macrophage and neutrophil were counted from a total of 200 fields and presented in % with respect to total cell numbers.

Animals to be imaged were picked into a drop (5 to 7 μl) of M9 containing sodium azide (50 μm) on an agarose (1%) pad made on a glass slide. Coverslip was added once the animals were paralyzed, and the pad was almost dry. chil-27p::GFP/col-12p::mCherry expression was observed on Zeiss Axio Zoom V16 microscope and images were taken using the associated ZEN Microscopy software. For rpt-3p::GFP and sur-5::UbV-GFP, animals were treated with oomycete extract or 20 μm BTZ with DMSO control for 24 h at early L2 stage and expression was observed on Zeiss Compound microscope (AxioScope A1) at 40× magnification and images were taken using OCULAR. For chil-27p::GFP expression upon BTZ treatment, early L4 stage animals were treated with different doses of BTZ for 24 h and adults were imaged as described above.

For visual comparisons of Pnlp-29-GFP/Pcol-12-dsRed (frIs7) expression we took images on a Zeiss compound microscope using a GFP long-pass filter set. To quantify Pnlp-29-GFP (frIs7) expression levels we used the COPAS C. elegans BIOSORT (Union Biometrica, Holliston MA) at the Troemel lab (UCSD). We calculated the ratio of green fluorescence to the internal control Pcol-12-dsRed.