Characterisation of the Small RNAs in the Biomedically Important Green-Bottle Blowfly Lucilia sericata

Abstract

BackgroundThe 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.ResultsWe 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.ConclusionsWe 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.

The transcriptional responses of cultured wound cells to the excretions and secretions of medicinal Lucilia sericata larvae

“…The eggs were initially laid onto bovine liver, then removed and sent by overnight courier to our laboratory with an ice‐pack to delay hatching. Eggs were surface sterilized as previously reported . Briefly this consisted of a 5 minute immersion in 1% sodium hypochlorite followed by 5 minutes in 70% ethanol followed by a final rinse in ddH 2 0.…”

“…Eggs were surface sterilized as previously reported. 18 Briefly this consisted of a 5 minute immersion in 1% sodium hypochlorite followed by 5 minutes in 70% ethanol followed by a final rinse in ddH 2 0. The eggs were hatched overnight in the absence of food and then the larvae used for collection of ES.…”

Biogenesis and Function of Small Non-Coding RNAs Derived from Eukaryotic Ribosomal RNA

“…Small rRF detection attests of their relative stability and implies that they do not result from normal cellular ribosome turnover. The caveat has to be taken into account that the study of rRFs has been hampered, and is still hampered, by the long-set bioinformatics pipelines that consider rRFs as mere degradation products and systematically remove small RNA sequencing reads mapping to rRNAs from the data 56,86 .…”

Genome Sequence of a Proteus mirabilis Strain Isolated from the Salivary Glands of Larval Lucilia sericata

“…Concurrently, the flies do not appear to effectively control P. mirabilis ( 19 ). Further, Proteus species have been identified in salivary gland samples of L. sericata ( 14 , 20 ), a relatively clean tissue and a major source of molecules contributing to molecular antibacterial activities important to maggot therapy ( 21 – 24 ). Finally, swarming signals associated with P. mirabilis have been linked to fly attraction and oviposition, making the species a model for interkingdom signaling between bacteria and insects ( 7 ), which might have implications for medical, forensic, and agricultural research with decomposer flies and for microbial ecology.…”