De novo Assembly of the Burying Beetle Nicrophorus orbicollis (Coleoptera: Silphidae) Transcriptome Across Developmental Stages with Identification of Key Immune Transcripts.

Burying beetles (Nicrophorus spp.) are among the relatively few insects that provide parental care while not belonging to the eusocial insects such as ants or bees. This behavior incurs energy costs as evidenced by immune deficits and shorter life-spans in reproducing beetles. In the absence of an assembled transcriptome, relatively little is known concerning the molecular biology of these beetles. This work details the assembly and analysis of the Nicrophorus orbicollis transcriptome at multiple developmental stages. RNA-Seq reads were obtained by next-generation sequencing and the transcriptome was assembled using the Trinity assembler. Validation of the assembly was performed by functional characterization using Gene Ontology (GO), Eukaryotic Orthologous Groups (KOG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Differential expression analysis highlights developmental stage-specific expression patterns, and immunity-related transcripts are discussed. The data presented provides a valuable molecular resource to aid further investigation into immunocompetence throughout this organism's sexual development.

ß-Glucuronidase activity in seedlings of the parasitic angiosperm Cusctua pentagona: developmental impact of the ß-glucuronidase inhibitor saccharic acid 1,4-lactone.

Endogenous plant ß-glucuronidase (ß-GUS) activity was detected in germinating seeds, seedlings, stems, flowers and haustoria of the parasitic angiosperm Cuscuta pentagona Engelm. In vitro characterisation of this activity showed it to have an acidic pH optimum, similar to previously characterised plant activities, and a sensitivity to the ß-GUS inhibitor saccharic acid 1,4-lactone (SAL). Application of SAL to seeds immediately after chemical scarification resulted in a significant developmental delay and, frequently, in the total arrest of seedling growth. In contrast, application of SAL subsequent to the emergence of the radicle did not produce a significant effect on the development of the seedling. Thus, the distribution of activity and the developmentally contingent potency of SAL in inhibiting growth suggest a role for ß-GUS at an early stage of seed germination or seedling growth. Further, the inability of the inhibitor to prevent subsequent shoot elongation suggests that at least some plant growth processes do not require this activity, or that it is required only at minimal levels and is unaffected by the application of SAL.

Surrogate splicing for functional analysis of sesquiterpene synthase genes.

A method for the recovery of full-length cDNAs from predicted terpene synthase genes containing introns is described. The approach utilizes Agrobacterium-mediated transient expression coupled with a reverse transcription-polydeoxyribonucleotide chain reaction assay to facilitate expression cloning of processed transcripts. Subsequent expression of intronless cDNAs in a suitable prokaryotic host provides for direct functional testing of the encoded gene product. The method was optimized by examining the expression of an intron-containing beta-glucuronidase gene agroinfiltrated into petunia (Petunia hybrida) leaves, and its utility was demonstrated by defining the function of two previously uncharacterized terpene synthases. A tobacco (Nicotiana tabacum) terpene synthase-like gene containing six predicted introns was characterized as having 5-epi-aristolochene synthase activity, while an Arabidopsis (Arabidopsis thaliana) gene previously annotated as a terpene synthase was shown to possess a novel sesquiterpene synthase activity for alpha-barbatene, thujopsene, and beta-chamigrene biosynthesis.