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Proteomics profiling of the honeybee parasite Tropilaelpas mercedesae across post-embryonic development

ABSTRACT: All T. mercedesae samples were collected from capped brood cells using a soft paintbrush and soft tweezers. Reproductive mites (Rep) were collected from brood cells containing white-eyed pupae. Protonymphs (Pro) and one deutonymphs (Deu) were sampled from brood cells containing purple-eyed pupae. Adult T. mercedesae (Adu) were collected from cells close to emerge (about one day before adult bees emerge). For each group (protonymphs, deutonymphs, adults, and reproductive mites), collected mites from all 12 colonies were randomly allocated to one of three replicates (50 mg for each replicate). All samples were rinsed with PBS and air-dried to remove many contaminates that may have been attached to the cuticle, flash-frozen using liquid nitrogen, and stored at -80°C for further processing.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Tropilaelaps Mercedesae

TISSUE(S): Whole Body

SUBMITTER: Bin Han

LAB HEAD: Shufa Xu

PROVIDER: PXD051347 | Pride | 2024-04-13

REPOSITORIES: Pride

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Project description:Background: Synaptic transmission is required for functional maturation of the CNS and to induce gene transcription involved in neuronal plasticity. Our aim is to identify genes that are transcriptionally regulated by synaptic transmission during development. cDNA microarrays will be used to compare gene expression in normal embryos and embryos with no synaptic transmission. Using the Gal4 UAS system, the tetanus toxin light fragment (TET) will be expressed throughout the nervous system. The effects of TET are well characterised: the synaptic protein n-Synaptobrevin is cleaved, blocking evokes vesicle fusion in TET expressing neurons and as a result synaptic transmission is blocked. Embryos expressing an inactive, mutant version of TET (iTET) show no detectable phenotype. Thus a comparison of gene expression in iTET expressing embryos and TET expressing embryos focuses our screen cleanly and specifically on genes that are regulated by synaptic transmission. At the end of larval life, during metamorphosis, there is a second phase of nervous system development with the same requirement for neural circuit differentiation and maturation as in the embryo. We will compare gene expression in TET expressing and iTET expressing pupae to identify genes which are regulated by synaptic transmission in this second phase of nervous system development. We will focus our analysis on genes which are similarly regulated in both systems. Plan: elav Gal4 (expressed in all the neurons) for the embryos and Heat Shock Gal4 (which allows a temporally controlled expression) for the pupae are used to drive UAS TET and UAS iTET throughout the nervous system. Crosses are set up with flies homozygous for Gal4 or UAS transgenes, so that all the progeny have the same genotype. Total RNA is extracted from 500 carefully staged embryos that have been collected at the time of hatching. Pupae are heat shocked at 24 hours after puparium formation, and heads are then collected 5 days after puparium formation. TET expressing pupae are paralysed but morphologically normal whereas iTET expressing pupae emerge as normal adults. Total RNA will be extracted from 250 heads of each genotype. Probes made from TET and iTET expressing embryos extracts will be hybridised together to the chip and so will be probes made from TET and iTET expressing pupae extracts. Genes regulated in both systems will be compared together.

Dataset Information

Proteomics profiling of the honeybee parasite Tropilaelpas mercedesae across post-embryonic development

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