Characterisation of the small RNAs in the biomedically important green-bottle blowfly Lucilia sericata.

BACKGROUND: The green bottle fly maggot, Lucilia sericata, is a species with importance in medicine, agriculture and forensics. Improved understanding of this species' biology is of great potential benefit to many research communities. MicroRNAs (miRNA) are a short non-protein coding regulatory RNA, which directly regulate a host of protein coding genes at the translational level. They have been shown to have developmental and tissue specific distributions where they impact directly on gene regulation. In order to improve understanding of the biology of L. sericata maggots we have performed small RNA-sequencing of their secretions and tissue at different developmental stages. RESULTS: We have successfully isolated RNA from the secretions of L. sericata maggots. Illumina small RNA-sequencing of these secretions and the three tissues (crop, salivary gland, gut) revealed that the most common small RNA fragments were derived from ribosomal RNA and transfer RNAs of both insect and bacterial origins. These RNA fragments were highly specific, with the most common tRNAs, such as GlyGCC, predominantly represented by reads derived from the 5' end of the mature maggot tRNA. Each library also had a unique profile of miRNAs with a high abundance of miR-10-5p in the maggot secretions and gut and miR-8 in the food storage organ the crop and salivary glands. The pattern of small RNAs in the bioactive maggot secretions suggests they originate from a combination of saliva, foregut and hindgut tissues. Droplet digital RT-PCR validation of the RNA-sequencing data shows that not only are there differences in the tissue profiles for miRNAs and small RNA fragments but that these are also modulated through developmental stages of the insect. CONCLUSIONS: We have identified the small-RNAome of the medicinal maggots L. sericata and shown that there are distinct subsets of miRNAs expressed in specific tissues that also alter during the development of the insect. Furthermore there are very specific RNA fragments derived from other non-coding RNAs present in tissues and in the secretions. This new knowledge has applicability in diverse research fields including wound healing, agriculture and forensics.

Phylogenetic species identification in Rattus highlights rapid radiation and morphological similarity of New Guinean species.

The genus Rattus is highly speciose, the taxonomy is complex, and individuals are often difficult to identify to the species level. Previous studies have demonstrated the usefulness of phylogenetic approaches to identification in Rattus but some species, especially among the endemics of the New Guinean region, showed poor resolution. Possible reasons for this are simple misidentification, incomplete gene lineage sorting, hybridization, and phylogenetically distinct lineages that are unrecognised taxonomically. To assess these explanations we analysed 217 samples, representing nominally 25 Rattus species, collected in New Guinea, Asia, Australia and the Pacific. To reduce misidentification problems we sequenced museum specimens from earlier morphological studies and recently collected tissues from samples with associated voucher specimens. We also reassessed vouchers from previously sequenced specimens. We inferred combined and separate phylogenies from two mitochondrial DNA regions comprising 550 base pair D-loop sequences and both long (655 base pair) and short (150 base pair) cytochrome oxidase I sequences. Our phylogenetic species identification for 17 species was consistent with morphological designations and current taxonomy thus reinforcing the usefulness of this approach. We reduced misidentifications and consequently the number of polyphyletic species in our phylogenies but the New Guinean Rattus clades still exhibited considerable complexity. Only three of our eight New Guinean species were monophyletic. We found good evidence for either incomplete mitochondrial lineage sorting or hybridization between species within two pairs, R. leucopus/R. cf. verecundus and R. steini/R. praetor. Additionally, our results showed that R. praetor, R. niobe and R. verecundus each likely encompass more than one species. Our study clearly points to the need for a revised taxonomy of the rats of New Guinea, based on broader sampling and informed by both morphology and phylogenetics. The remaining taxonomic complexity highlights the recent and rapid radiation of Rattus in the Australo-Papuan region.