Species: Psylliodes chrysocephala (Linnaeus) – Cabbage stem flea beetle
P. chrysocephala is found in Europe, North Africa and Asia. It is a pest of most brassica crops and is a serious pest of winter rape in Sweden, France, Switzerland, the Netherlands and the UK. In the UK, it is the most important pest of rape, leading to yield losses of up to 20%.
Adults spend the late summer feeding on wild or cultivated crucifer plants. They aestivate in the early part of the summer in sheltered areas and when adults emerge, they move to newly growing winter brassica crops, where they feed on the stems and leaves. The female lays her eggs in cracks in the soil near the base of the plant and the emerging larvae mine the stems and petioles. Larvae pupate in the soil. The adults chew holes in the leaves. The larvae usually mine the lower petioles, moving from ageing to healthy tissue, but will move to the stem and destroy the growing point if larval numbers are large or if the rosette is poorly developed. As well as causing direct damage, P. chrysocephala is associated with fungal and bacterial infection and it may transmit turnip crinkle virus. Infested plants are also more susceptible to frost damage.
Source: CABI Invasive Species Compendium https://www.cabi.org/isc
Single adult beetle (with parastoid wasp) from field sample collected at Rothamsted farm on 28 July 2017. GPS 51.8093°, -0.3548°.
Next Generation Sequencing
i) P. chrysocephala Illumina Hi-C sequencing 150 bp paired end:
901,426,372 reads and 114x coverage.
M. brassicae Illumina Hi-C sequencing 150 bp paired end:
367,204,982 reads and 399x coverage.
ii) unknown sex Illumina 10X sequencing 150 bp paired end, totalling 668,730,898 reads and 85x coverage. Individual male 150 bp paired end, totalling 264,428,764 reads and 33x coverage. Individual female 150 bp paired end, totalling 230,696,660 reads and 29x coverage.
iii) PacBio HiFi data, of mean read length 14,428, total reads 2,074,955, read length N50 15,228, and total bases 29,937,625,920. DNA was extracted using Circulomics kit and quantified using FEMTO Pulse (3000ng gDNA).
Non-sexed single CSFB individual (with parastoid wasp, Microctonus brassicae) used for PacBio HiFi (University of Delaware, USA) and multi-individual Hi-C Illumina sequencing (Arima Genomics USA) for both species. Hifiasm was used to assemble the PacBio HiFi, with Juicer then 3d-dna using Hi-C data for both specie chromosome level assembly. Haplotigs were removed (purge_haplotigs). Manual curation was done to bring the genomes together and check for miss-assemblies. Unmapped reads were mapped back to the original assembly to check for missing sequence and incorporated into the final assembly. Error correction was done with Illumina 10X library data for P. chrysocephala and Hi-C for M. brassicae using freebayes.
PGI RNA-seq from adult CSFB was assembled into a transcriptome (BUSCO: C:96.7%[S:95.2%,D:1.5%],F:0.7%,M:2.6%) and used in the Maker2 annotation pipeline with trained Augustus and Genemark gene predictors. PASA was used to update the gene models to add UTR, correct existing models and add isoforms. Non-coding RNA was annotated using Infernal v1.1.4.
A Pfam genomic track was created by converting to six reading frames and utilizing hmmer to identify loci of interest i.e. P450 pfam domains on the genome. Using this information, loci of interest including UDP, P450, ABC and IRAC gene models were found and curated using mapped RNA-seq and a Maker gene annotation.
A P. chrysocephala Wolbachia endosymbiont (1,438,854 bp) was assembled.
A complete annotated P. chrysocephala 23 chromosome assembly deposited at NCBI (PRJEB47896) (incl. raw data).
BUSCO (Insecta odb10): C:96.7%,F:0.5%,M:2.8%
15,768 gene models - BUSCO C:95.5%[S:91.5%,D:4.0%],F:0.8%,M:3.7%
Scaffold No. (incl Mt): 33
N bases (bp): 1,415,333
Total size (bp) (chr no.): 1,181,805,970 (23)
Curated: 109x P450, 93x ABC transporter, 31x UGT, and the majority of 117/130 IRAC gene models.
One Wolbachia endosymbiont was assembled (1,438,854 bp).
M. brassicae 12 chromosome assembly deposited at NCBI (PRJEB48227).
Lower coverage than I was expecting because of the parasitic wasp so coverage was quite low. Greater coverage would have produced a better assembly with less N's but still reasonable. Best example assembly of a parasitic wasp in NCBI though (M. brassicae).
These are files that were not submitted to NCBI but might be useful.
M. brassicae annotation not submitted to NCBI:
Gene annotation track (Protein information used as evidence so lots of isoforms that need removing but good BUSCO score for representation, needs cleaning up for submission to NCBI)