Dataset Information

Exploration of RNA-Seq data to identify a potential pathogen of the leaf-mining moth, Stomphastis sp. (Lepidoptera: Gracillariidae)

ABSTRACT: The leaf-mining moth (Stomphastis sp.) was imported to Australia as a potential biological control agent of a weed, Bellyache bush (Jatropha gossypiifolia). The insect colony has been maintained in the quarantine facility for over eight years. Recently, significant mortality was observed in the population. The larvae demonstrated swollen intersegments with a fragile integument. The infected larvae are cloudy muted green or yellowish whereas a healthy late instar larva is a vivid green. They slowly dehydrate and eventually die and when death occurs, the larval body becomes rubbery and turns to black. We used next generation sequencing approach to identify the cause of mortality in the population. Total RNA was extracted from 20 larvae in two cohorts with and without apparent symptoms of disease for deep sequencing on NovaSeq platform after eukaryote ribosomal RNA depletion. We identified several non-insect sequences belonging to viruses, bacteria and fungi but none of those showed significant abundance or enrichment in the infected dataset. The sequences related to a unicellular yeast, Saccharomyces cerevisiae, are among the highly expressed non-insect contigs and more than 5% of reads in both libraries mapped to the genome of this opportunistic microorganism.

ORGANISM(S): unclassified Stomphastis

PROVIDER: GSE185938 | GEO | 2021/10/16

REPOSITORIES: GEO

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Project description:Arabidopsis thaliana plants (ecotype Landsberg erecta) were grown at 20ËC constant temperature, 8 hr/16 hr Light/Dark photoperiod at 50-60% ambient humidity, for 8 to 9 weeks. Short day conditions prevented the onset of flowering and the plants were thus maintained in growth stage 1 (leaf production) with 13 to 15 rosette leaves larger than 1mm (stage 1.13 to 1.14). Diamondback moth (DBM, Plutella xylostella) larvae were maintained on cabbage (Brassica oleracea) plants in a climate-controlled room at 25ÂºC, 12 hr photoperiod with 50%-60% relative humidity. Two days before exposing A. thaliana plants to herbivore treatment, plants were transferred to a climate-controlled room (22ÂºC, 50-60% humidity, 12 hr photoperiod). For insect treatment, seven Diamondback moth (DBM, Plutella xylostella) larvae (third to fifth instars) were placed on a group of four or five plants until time of harvest, for each time point separately. All rosette leaves were harvested at 1h, 4h, 12h, and 24h after onset of continuous herbivory and flash frozen in liquid nitrogen. Control plants were maintained under the same conditions and harvested in parallel. Keywords: time course, stress response, herbivory response For each time point (1h, 4h, 12h, and 24h) two biological replicates (DBM treatment) were performed. Control plants were harvested in parallel. Total RNA from each sample was used for two labelling reactions using dye-flips (Cy5 or Cy3) and labelled cDNA from treatment and control plants were co-hybridized. Thus, for each time-point four replicates were analyzed each comparing treatment with the corresponding control (rep1 and rep2 are dye-flips of biological replicate 1, and rep3 and rep4 are dye-flips of biological replicate 2). For background correction, we defined the mean of the lowest 10% of spot intensities from a particular subgrid as the background for that subgrid. This mean was subtracted from each spot in the subgrid. Signal intensities that did not exceed the background plus 3 standard deviations thereof were defined as not detectable and were excluded from further analyses. We normalized using loess curves thus generating log2 transformed expression ratios comparing DBM treatment with control. These ratios are given in the VALUE columns of each array.

Dataset Information

Exploration of RNA-Seq data to identify a potential pathogen of the leaf-mining moth, Stomphastis sp. (Lepidoptera: Gracillariidae)

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