Identification and characterization of small non-coding RNA molecules transcribed in Heliothis virescens (Fabricius) (Lepidoptera: Noctuidae) larvae parasitized by Toxoneuron nigriceps (Viereck) (Hymenoptera: Braconidae).

Toxoneuron nigriceps (Hymenoptera, Braconidae) is an endophagous parasitoid of the tobacco budworm, Heliothis virescens (Lepidoptera, Noctuidae). Parasitized larvae show a complex array of pathological symptoms including immune response suppression, largely due to a symbiotic virus, named TnBV, belonging to the family Polydnaviridae (PDV). A striking characteristic of the TnBV, as well as the other PDV genomes sequenced to date, is the large preponderance of non-coding regions, which raises the question of their functional relevance. To start addressing this question, we decided to analyze the small RNA fraction present in haemocytes of parasitized larvae and search for TnBV-derived sequences that might interfere with the host immune response. To investigate this hypothesis in the case of TnBV, a specialized cDNA library, representative of the small RNA fraction present in haemocytes of parasitized larvae, was prepared. The generated cDNA library was validated by two types of quality control analyses. Real-time PCR Absolute Quantification was performed using a standard curve method. This analysis essentially allowed me to evaluate the concentration of the library and also to obtain indications on its purity, by assessing the presence and relative abundance of adapter dimers contaminants. A further quality test was carried out by cloning a small amount of the cDNA products in a plasmid vector and by sequencing through conventional technology few randomly selected clones. Two cloned sequences turned to be microRNA species already known in other organisms, miR-184 and miR-970, evolutionarily conserved from insects to mammals. In Drosophila melanogaster miR-184 is expressed in all developmental stages (Ping Li et al., 2011). Moreover, functional analysis identified a role for this miRNA in the female germline, where it is involved in multiple steps of oogenesis (Iovino et al., 2009). The presence of authentic miRNA species in our library, as well as the finding of a correct size range (21-27) in our cloned cDNA sequences represented valid quality control criteria, empowering further analysis of the cDNA library by high-throughput sequencing technologies. Specifically, cDNA sequencing was performed using the Illumina platform and resulted in about 6 million total reads. Sequence alignment with the TnBV genome was then performed using the FAST (Fast Aligning of Short Tags) software developed by the Institute of Applied Genomics of Udine. These preliminary analyses allowed the identification of 52 cDNA sequences, ranging in size between 20 and 27 nucleotides, that align with the viral genome. 46 are present in the viral genome in single copy, while 6 are located in two different positions and are therefore represented twice. These sequences were extensively analyzed by bioinformatical tools, in particular computational methods based on secondary structure prediction to assess the ability of these genomic regions to generate appropriate small RNA precursor molecules. Specific analyses were performed using a web tool, MirEval. 5 sequences were selected as potential miRNA species, deriving from viral genomic regions that show structural features typical of precursor-miRNA (pre-miRNA) molecules. RT-PCR experiments of these genomic regions, aimed at detecting presumptive primary-miRNA (pri-miRNA) species, indicated that these genomic regions are actively transcribed in haemocytes, 6 and 12 hours after parasitization. In parallel, transcriptional profiling of the TnBV genomic regions where the remaining presumptive TnBV small ncRNA sequences were mapped, as well as of randomly selected regions, that do not contain any protein coding genes or any putative small ncRNAs, indicated that the non-coding portion of the TnBV genome is extensively transcribed in host haemocytes. These analyses indicated that most, if not all, of the non-coding portion of the TnBV genome is transcribed in host haemocytes. This unexpected finding raises several questions concerning: 1) the functional meaning of generalized transcription and 2) the possible existance of specific mechanisms acting downstream of transcription to discriminate biologically relevant RNA molecules from “transcriptional noise”. Functional characterization of the sequences identified as potential microRNAs is expected to shed light on the role played by the non-coding regions of the TnBV genome during parasitization. Furthermore, future work aimed at target identification may disclose the possible involvement of these sequences in the alteration of host immune system. The generated information will be essential for defining a more comprehensive model of the immunosuppression phenomena occurring during parasitization.