Pupal Screen Example


Structure of the iBeetle-Base

The iBeetle-Base stores gene related information for all genes of the official gene set (red box). Among others, RNA and protein sequences can be downloaded and links lead to the respective annotation in the genome browser. Further, the Drosophila orthologs are displayed including links to FlyBase. Wherever available, the phenotypic data gathered in the iBeetle screen is displayed below the gene information in separate sections for the pupal and larval screening parts (yellow box).

Searching for gene IDs or phenotypes

The information can be searched from two sides: First, the “Gene search” uses gene IDs (TC number) or name of the ortholog or iB number to display the details page (gene search, left blue box). There, all information available for this gene is shown (blue and yellow boxes). If the entered search term is equivocal, a results list presents all hits for selection by the user.

Second, the “search for morphological defects” allows searching for affected structures and/or the nature of the defects. Here, the result list presents both the annotation and the pictures of the searched entity for further selection. Links to the respective details pages gives all information available for this gene.

Note that for all genes of the official gene set the gene information is s tored at iBeetle-Base while only a subset of the genes has been screened in the iBeetle screen.

Background information

iBeetle is a first pass screen

The iBeetle Screen is a first pass screen, i.e. the experiments were performed only once and off target controls were not included. Further, we aimed at keeping false negative results as low as possible with the trade-off of elevated false positive annotations. Finally, we deliberately used an intermediate dsRNA concentration (1ug/ul) in order to reveal the phenotypic series. Hence, the data presented here needs to be confirmed with the original fragment and non-overlapping fragments of dsRNA before being publishable.


Highly penetrant phenotypes with a direct phenotype-genotype relationship (e.g. lethality, wing blistering) were reproducible at a close to 100% rate in our hands. Regarding processes where the relation between gene function and phenotype is complex (e.g. embryonic development), the rate of reproducibility was between 70% and 80%. Reproducibility of low penetrance phenotypes (<50%) was significantly lower.

Search strategy

The search at iBeetle-Base allows for very specific searches (e.g. “larva, leg, size decreased”). However, in order not to miss interesting phenotypes, we recommend initiating your search broadly for defects of the structure and stage you are interested in (larva, leg). Further, a penetrance of >50% is recommended in order to increase the portion of reproducible datasets. Next, the list should be consolidated by browsing the pictures and detailed annotations displayed in the search results. Interesting results can be marked by checkboxes. Finally, the respective details pages should be browsed in order to learn more about additional phenotypes, the gene structure and sequences and its orthologs.

Rescreen strategy for the identification of morphological defects

Phenotypes need to be confirmed by both the original dsRNA fragment (see sequence on the details page) and a non-overlapping fragment for off target control. A higher concentration (e.g. 3ug/ul) than the one used in the screen (1ug/ul) is recommended because in some cases, this will reveal s tronger phenotypes. For mid scale re-screening it may be advantageous to buy dsRNAs commercially from Eupheria.com.

Screening procedure and terms

Pupal injection procedure

Day 0

10 female pupae of the pBA19 strain (muscle enhancer trap line) were injected with dsRNA.

3 days post injection (dpi)

Hatch control: Pupal and adult lethality as well as metamorphosis defects (molting, eclosion) were documented. For mating, 4 males of the black strain were added.

9 dpi

First egg-lay was collected and incubated for cuticle analysis. Adult lethality and egg production (reduced/ no egg-lay) was documented.

11 dpi

Second egg-lay was collected and incubated for embryonic muscle analysis. Adult lethality and egg production (reduced/ no egg-lay) were documented.

13 dpi: Egg productivity and Ovary analysis

The percentage of hatched larvae was documented and not hatched larvae/ eggs were embedded for cuticle analysis (15 dpi). In case of a reduction of egg production, 4 injected females were dissected to analyze the gross morphology of the ovaries.

14 dpi: Analysis of embryonic musculature and early embryonic development

Offspring of the injected females (hatched and not hatched larvae/ eggs) were analysed for embryonic lethality and muscle defects.

15 dpi: Analysis of larval instar 1 cuticle

Offspring of injected females were analysed and cuticle phenotypes were annotated.

22 dpi: Stink gland analysis

Documentation of defects in abdominal and thoracic stink glands (colour, size, content) of the injected femals.

Terms used in the pupal injection procedure