For the iBeelte screen certain parameters such as pupal age at injection, in-cubation times, and annotations needed to be comparable and standardized be-tween the screeners. To this end a strict protocol was used by all screeners. Alt-hough some procedures were already described in former parts of the materials and methods chapter, I will give an overview of the detailed screening procedure for the pupal injection screen here.
All incubations were done at 31°C. Female pupae from the Pig-19 strain were injected and mated to males from the Black strain to allow easy discrimina-tion between injected females and non-injected males during later time points of the screening procedure. All observations, including important aspects of the ex-periments, were annotated in a common database (3.6.1) using a controlled vo-cabulary.
Injection was defined as day 0 in the protocol. Pupae were considered old enough if at least the base of the mandibles was darkened, which indicates about 75 % of pupal development. On each injection day 22 iBeetle dsRNAs (synthe-sized by Eupheria Biotech GmbH, Dresden, Germany) and two buffer controls were injected in 10 females each. The entity of the injections of one injection day was called a ‘repetition’. After injection the pupae were transferred to petri dishes containing approximately 10 g of flour.
After three days (day 3 of the protocol) the eclosure and lethality rate for each injection was documented and the now adult beetles were transferred to a 25-well block system. Male beetles from the Black strain were added. Since Tribolium beetles tend to cannibalize on pupae this was not done directly on the injection day.
On day 9 the first egg collection was done and the eggs were placed in 300 µm meshes above vials containing safflower oil. As soon as the animals hatched, they crawled through the mesh into the oil, so the hatch rate could be es-timated later. Differences in the amount of eggs in relation to the buffer controls were annotated in the database. Dead females were counted and documented.
A second egg collection was done on day 11. Again the amount of eggs in relation to the controls was checked and differences were annotated. The eggs were directly transferred to 180 µm meshes and put back to 31°C on a small amount of flour to allow hatching larvae to feed.
On day 13 the first egg collection (from day 9) was processed for cuticle analysis (3.5.6). If many non-hatched animals were detectable after dechorionization two preparations were done on one microscope slide, one of them was gently squeezed to allow stretching of the larvae to ease analysis of weaker cuticle phenotypes, the other one was embedded without squeezing as comparison. On day 13 of screening analysis also ovary preparations were per-formed for females showing affected egg deposition. The ovaries of usually four females of one iBeetle injection were dissected in PBS using fine forceps (Dumont
#5), shortly fixed in 3,5 % formaldehyde in PBS, and mounted in PBS on a cover slide. Dissected ovaries were subjected to a brief microscope analysis and all de-tectable phenotypes were annotated in the data base.
On day 14 fresh preparations from the second egg collection were done by de-chorionizing the eggs in two steps for three minutes in 50 % bleach each. Eggs and larvae were carefully mounted in Voltalef oil and covered with a cover slip.
The muscle pattern was analyzed under a Zeiss Axioplan 2 microscope using DIC and a mercury vapor lamp with FITC filter for detecting the GFP signal in the mus-cles. Any detectable phenotype was annotated in the database.
Cuticle analysis was done at protocol day 15 or later. Since cuticle prepara-tions are stable for several months this part of analysis was time wise more flexible and could be done later. Cuticles were analyzed and documented as described in 3.5.6. Only single plane images were taken (no Z-stacks).
3.6.1 Annotation of screening results
Important parameters during the screening as well as all observed pheno-types were annotated in a database using an online interface (ibeetle-base.uni-goettingen.de) and a controlled vocabulary. Free text fields allowed additional de-scriptions or comments. Pictures were uploaded for documentation. Part of every annotated phenotype was its penetrance. In case of ovary analysis, penetrance of phenotypes was annotated as the number of females showing an ovary phenotype compared to the total number (usually four) of dissected females. For muscle and
cuticle analysis the occurence of the phenotype in relation to the total number of counted eggs or animals on the slide was annotated. As an estimation, a subset of the animals on the slide was counted. This was done by counting the cuticles once or twice crosswise along the microscope slide. The occurence of the phenotype in relation to the total number of counted eggs or animals on the slide was annotat-ed. In case only few cuticles were on the slide, all animals or eggs were countannotat-ed.
Penetrances were annotated as: <30 %, 30-50 %, 50-80 % and >80 %. All pene-trances were referring to the total number of animals on the analysis slide. An ex-emplary screenshot of the iBeetle annotation database is shown below (Figure 3-1).
Figure 3-1 Database annotation
The picture shows an exemplary screenshot from the iBeetle annotation database. Depicted is the annotation of one of the positive controls on day 15 (cuticle analysis) of the pupal injection screen. Shown on the upper left is the affected animal tagma, in this case “larval head” (A). Directly below is the entity, the affected struc-ture (B). There are several dropdown menus to describe the phenotype in more detail (one dropdown menu is active). These are called ‘qualifiers’ (C). The last dropdown menu in the line describes how the entity is actual-ly altered in the annotated phenotype (modifier, D). The penetrance of the phenotype is also annotated (E) and additional comments can be entered (F). Every morphological phenotype is documented by taking and uploading pictures (G)