Deep, Staged Transcriptomic Resources for the Novel Coleopteran Models Atrachya menetriesi and Callosobruchus maculatus

Abstract

Despite recent efforts to sample broadly across metazoan and insect diversity, current sequence resources in the Coleoptera do not adequately describe the diversity of the clade. Here we present deep, staged transcriptomic data for two coleopteran species, Atrachya menetriesi (Faldermann 1835) and Callosobruchus maculatus (Fabricius 1775). Our sampling covered key stages in ovary and early embryonic development in each species. We utilized this data to build combined assemblies for each species which were then analysed in detail. The combined A. menetriesi assembly consists of 228,096 contigs with an N50 of 1,598 bp, while the combined C. maculatus assembly consists of 128,837 contigs with an N50 of 2,263 bp. For these assemblies, 34.6% and 32.4% of contigs were identified using Blast2GO, and 97% and 98.3% of the BUSCO set of metazoan orthologs were present, respectively. We also carried out manual annotation of developmental signalling pathways and found that nearly all expected genes were present in each transcriptome. Our analyses show that both transcriptomes are of high quality. Lastly, we performed read mapping utilising our timed, stage specific RNA samples to identify differentially expressed contigs. The resources presented here will provide a firm basis for a variety of experimentation, both in developmental biology and in comparative genomic studies.

Fig 1. Phylogenetic positions of A. menetriesi and C. maculatus.

A) Cladogram of Coleoptera simplified from that determined by [16]. Black asterisk indicates the superfamily in which the Chrysomelidae are located. B) Cladogram of Chrysomelidae simplified from that determined by [21]. Black and grey asterisks indicate the sub-families in which A. menetriesi and C. maculatus are located (respectively). C) A. menetriesi and D) C. maculatus adults.

Fig 2. Summary of RNA sources, life stages sampled and sequencing results.

A.menetriesi data is presented at left and C. maculatus data at right.

Fig 3. Overview of results of differential expression analysis performed by RSEM within the Trinity framework.

A) and B) show the sample correlation matrix for A. menetriesi and C. maculatus respectively. C) and D) show relative expression of each differentially expressed contig, considered as isoforms, across time.

Fig 4. Blast2GO results.

A) Distribution of BLASTx best hits by species, showing metazoans only, for A. menetriesi in orange, C. maculatus in blue. B) Distribution of GO terms expressed as a percentage of annotated contigs which were assigned a term within each of the three (Molecular Function, Cellular Component, Biological Process) categories of GO ID.

Fig 5. Phylogenetic inter-relationships of ANTP HOXL class genes.

Phylogenetic relationships were reconstructed using MEGA 6 using the LG+Freqs model with 4 gamma categories and invariant sites, based on a 59 amino acid alignment spanning the homeodomain. Numbers at base of nodes represent bootstrap percentages of 1000 replicates. Scale bar at base of phylogeny gives substitutions per site at given unit distance. Red underline indicates A. menetriesi and C. maculatus sequences, coloured boxes are used to delineate known gene families (and in the case of Hox 6/7/8, a superfamily).

Fig 6. Maximum Likelihood Phylogeny of TGF-β ligands.

Phylogenetic relationships were determined using MEGA under the LG+Freqs model with 4 gamma categories and invariant sites, on the basis of a 72 amino acid alignment of mature peptide sequences. The given scale depicts the number of substitutions per site per unit length. Bootstrap percentages (of 1000 replicates) are given at base of nodes. A. menetriesi and C. maculatus sequences are underlined in red. Coloured boxes represent known gene families with representatives in our transcriptomic resources, while all gene families, including those not found in our datasets, are indicated at right.

Fig 7. KEGG style pathway maps showing recovery in our transcriptome resources.

A) the Wnt signalling pathway in canonical and non-canonical contexts, B) the Hedgehog signalling pathway and C) the Notch signalling pathway. Coloration of genes indicates presence, absence or ancestral absence from the Coleoptera as detailed in the key, which also gives other information as noted. All genes automatically annotated by KEGG KAAS server, with the exception of PAR-1, which was manually annotated.