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| iB_02827 | iB fragment
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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.
Note: The adult morphology was not analyzed systematically at 3/9/11 dpi. Only obvious phenotypes, visible without magnification, have been annotated.
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
- larval pantagmatic defects
- At least two tagmata (head, thorax, abdomen or terminus) show similar/ comparable defects.
- empty eggs
- Just empty shells are visible, no cuticle has been developed.
- strong defects
- L1 cuticle larvae which show severe defects. These strong defects are subdivided into three categories
- strongly fragmented (cuticle crumbs)
- No segments or appendages are identifiable, only residues of cuticle and bristles can be identified.
- cuticle remnants (with unidentifiable segments)
- Segments or parts of appendages are recognizable, but identity is not clear.
- cuticle remnants (with some identifiable segments)
- At least one segment or appendage is clearly regonizable.
- number of eggs on slide
- affected embryos usually (not always) do not hatch. Only the not hatched were used for cuticle analysis.
For the embryonic muscle analysis the hatched as well as the not hatched were analysed. - total number of affected eggs/embryos/larvae
- Summary of all different phenotypes/ phenotypic series which were annotated.
- inside-out
- The L1 larva shows a partial or complete inversion., e.g. bristles, appendages or parts of the abdominal segments are inverted into the interior of the cuticle.
- starved
- Dissected females show a strong resorption of fatbody predominately in the abdomen and the oogenesis is blocked. These are considered as eventually lethal phenotypes and the blocked oogenesis probably a secondary defect due to starvation.
- eclosion not fulfilled
- The emergence of the adult from the pupa stage is interrupted. This phenotype shows pupal as well as adult features.
Lethality 11 days after pupal injection: 10.0% (includes death as pupa, adult)
eclosion not fulfilled (with pupal and adult features) - Number of animals showing the phenotype: 1 (3 dpi)Larval injection procedure
Day 0: 10 female L5-6 larvae of the D17Xhom strain (females express RFP in the eyes and in the CNS; pupae express GFP in the thorax) were injected with dsRNA.
11 days post injection (dpi): Pupal morphology Documentation of larval/ pupal death and defects of metamorphosis (eclosion, delayed development). Screening for alterations of pupal head and appendages, pupal thorax, pupal thoracic musculature, pupal legs, pupal wings, pupal abdomen and pupal genital lobes.
16 dpi: Adult morphology Documentation of larval/ pupal and adult death and defects of metamorphosis (eclosion, delayed development). Screening for alterations of adult head and appendages, adult thorax, adult elytra and hindwings, adult legs and cuticle properties. For mating 4 males of the black strain were added.
19 dpi: Sieving and egg-lay Documentation of adult lethality and enduring metamorphosis defects.
22 dpi: Ovary analysis (Fertility) In case of a reduction of egg production 4 females were dissected and the gross morphology of the ovaries were analysed.
41 dpi: Fertility and stink gland analysis Documentation of defects in abdominal and thoracic stink glands development (colour, size, content) of the injected females. Check for fertility of injected larvae (no or decreased offspring).
Terms used in the larval injection procedure
- pupal/ adult pantagmatic defects
- At least two tagmata (head, thorax, abdomen or terminus) show similar/ comparable defects.
- pupal molt not fulfilled
- The hatching from the larva to the pupa is interrupted. Either it dies as prepupa or the phenotype shows larval as well as pupal features.
- eclosion not fulfilled
- The emergence of the adult from the pupa stage is interrupted. This phenotype shows pupal as well as adult features.
- starved
- Dissected females show a strong resorption of fatbody predominately in the abdomen and the oogenesis is blocked. These are considered as eventually lethal phenotypes and the blocked oogenesis probably a secondary defect due to starvation.
Lethalities 11 days after larval injection: -1.0% (includes death as larva, prepupa, pupa)
Lethalities 22 days after larval injection: -1.0% (includes death as larva, prepupa, pupa, adult)
- >TC015779 RA
MRFLNLIFI VCFTLTPII WAKAPGPRI INGKTAEKG QFPWQVAIH VTQPGVSTL CGGALLNEK WILTAGHCV KDATNFKIA VGSNHFNGD DPSRVVFQT SDYILHEDY NKYTLANDI GLIPLPQAV SFNDDIQPI ALPSQGLTD GSTVTVSGW GLTSDDGEE ASPELMYVD LVTISNSEC STAYDGLDI NNGVVCAKG PGTIVQSTC EGDSGGPLV TRDSNPTHV GIVSFGHPD GCESGKPAG FTRTYNYID WIKGKTGI
- >TC015779 RA
atgcgtttt ctcaattta atttttatt gtgtgtttt actcttacc ccaataatt tgggcaaaa gcgccaggt ccacgaatt ataaatggc aaaacggcc gaaaaaggt caatttccg tggcaagtg gcgattcat gtcacccaa cctggagtt tcaactttg tgtggaggg gctctcttg aacgaaaaa tggattcta acggccggt cattgcgtc aaagacgcc actaatttt aaaattgct gtgggctca aaccacttc aatggagat gatccctca cgggttgta ttccagaca agtgattac attttgcac gaagattac aacaagtat actctagct aacgatatt ggacttata ccattacca caagctgta tcattcaac gacgacatt caaccaatt gccctgcca tcacaggga ctaactgat ggtagtacg gtcaccgtc agtggctgg ggcctaacc agtgatgac ggcgaagaa gccagtcct gaactaatg tacgttgat ttagtcaca atttccaac tcggaatgt tccacggcc tatgacgga cttgacata aataacgga gttgtttgt gccaaaggg cccggaact atcgtccag agcacttgc gagggtgac agtggcggt ccgctcgtt acacgcgat tcaaacccc actcatgtt ggtattgtt agttttgga caccctgac ggttgtgag agtggcaaa cctgccggt ttcacgcgc acttataac tacattgat tggattaaa ggaaaaact ggtatttga
- >TC015779 RA
cttattttg agataaaat gcgttttct caatttaat ttttattgt gtgttttac tcttacccc aataatttg ggcaaaagc gccaggtcc acgaattat aaatggcaa aacggccga aaaaggtca atttccgtg gcaagtggc gattcatgt cacccaacc tggagtttc aactttgtg tggaggggc tctcttgaa cgaaaaatg gattctaac ggccggtca ttgcgtcaa agacgccac taattttaa aattgctgt gggctcaaa ccacttcaa tggagatga tccctcacg ggttgtatt ccagacaag tgattacat tttgcacga agattacaa caagtatac tctagctaa cgatattgg acttatacc attaccaca agctgtatc attcaacga cgacattca accaattgc cctgccatc acagggact aactgatgg tagtacggt caccgtcag tggctgggg cctaaccag tgatgacgg cgaagaagc cagtcctga actaatgta cgttgattt agtcacaat ttccaactc ggaatgttc cacggccta tgacggact tgacataaa taacggagt tgtttgtgc caaagggcc cggaactat cgtccagag cacttgcga gggtgacag tggcggtcc gctcgttac acgcgattc aaaccccac tcatgttgg tattgttag ttttggaca ccctgacgg ttgtgagag tggcaaacc tgccggttt cacgcgcac ttataacta cattgattg gattaaagg aaaaactgg tatttgaaa acaataaac aagtcgtat ttta