Compound microscope

To measure compound toxicity, animals were grown to the L4 stage and exposed to compounds as described above. After 12 hours, animals were scored for viability by assessing locomotion and observing pharyngeal pumping. For compounds displaying significant toxicity over the negative control, serial dilution was used to identify the LD₅₀. LD₅₀ values were calculated using Graphpad Prism software.
To measure developmental delay, animals grown and exposed to compounds as described above were viewed under a compound microscope (Leica) and scored for tail development and linker cell survival. Tails were scored as adult if they had shed the L4 cuticle and showed tail ray and fan development.

Specimens were dissected and mounted on slides by using Canada balsam. All measurements were taken from slide-mounted specimens at 65×, 100×, 250×, or 400× magnification with a Leica compound microscope fitted with an eyepiece reticle. Body length, in micrometers (μm), was measured from the transverse trabecula to the metasomal apex, excluding the exserted part of the ovipositor. The remaining measurements are given either in micrometers or as ratios. Photographs were taken from slide-mounted specimens using a Nikon Ni-E system, and images were processed by Adobe Photoshop.
Abbreviations used are as follows: F1–6 = antennal funicular 1–6; POL = the shortest distance between posterior ocelli; OOL = the shortest distance between posterior ocellus and eye margin.
Terminology for morphological features follows Lin [6 (link)] and Rehmat and Anis [7 ].

We injected Evans Blue Dye as 1% solution by animal body weight (1 mg EBD/100 µl PBS/10 g) 24 hr prior to sample collection (Hamer et al., 2002 (link)). As a positive control for EBD uptake, we create an injured muscle area by inserting a 21-gauge needle 2–3 times into the jerboa gastrocnemius muscle. Samples were fresh frozen in OCT and cryosection at 12 µm thickness. Slides were processed for MF20 fluorescence with primary antibody incubation for 1 hr at RT before secondary antibody incubation. Slides were mounted for analysis: EBD signal is detected using the Cy5 filter and imaged using the Olympus compound microscope or imaged using the Leica SP5 confocal laser 633 nm.

The experiment was carried out with 400 fish specimens of cultured Lutjanus erythropterus fish species from Jerejak Island, Penang, Peninsular Malaysia (5.320097 longitude, 100.3189185 latitude). The length (cm) of each fish was measured prior to parasite examination. Fresh water medium was used as anesthetics to reduce the stress as well as for easy handling. After the fish has been anaesthetized, presence of ectoparasite was examined via external fish body examination and direct observation under light microscope [24 ]. The site specificity of parasite was obtained from head, body, and both sides of inner operculum.
First morphological identification of parasite was done by first staining the parasite with a few drops of lactophenol solutions (200 mL lactic acid, 200 g/L phenol, 400 mL glycerol, and 200 mL deionized water). Upon staining, slides were observed under the compound microscope (Leica, USA). Parasite found was taken out carefully from the infected area, and then the number of parasites obtained from each fish was recorded, preserved with 70% ethanol solution in universal bottle for further examination. After the pictures of parasites had been taken, identification of parasites collected was done by morphological observation using identification keys as suggested by Kua et al. [25 , 26 ].

A transgenic Drosophila model based on the Gal4/UAS system was used to target gene expression in fly tissues. For specific expression in border cells, we generated flies containing UAS-hE-cad WT or UAS-hE-cad R749W together with UAS-mCherry, which were all expressed using GAL4 driven from a border cell-specific promoter (slbo-Gal4, BDSC #58435). For the genetic interaction analysis in the adult eye, we established fly lines carrying simultaneously hE-cad and one RNAi targeting βPS integrin (RNAi #1 obtained from BDSC #27735 and RNAi #2 obtained from BDSC #33642) or UAS-mCherry (BDSC #35787), as control for titration of the number of UAS lines in the organism. These lines were then crossed with the GMR-Gal4 line and the appropriate progeny was selected. Eye phenotypes of at least 200 progeny flies (per condition) from three independent experiments were evaluated under a Leica compound microscope. Digital images were processed using Adobe Photoshop CS6. For further details, see Supplementary Materials and Methods.