Identification and Characteristics of microRNAs from Army Worm,Spodoptera frugiperda Cell Line Sf21

microRNAs play important regulatory role in all intrinsic cellular functions. Amongst lepidopteran insects, miRNAs from only Bombyx mori have been studied extensively with a little focus on Spodoptera sp. In the present study, we identified a total of 226 miRNAs from Spodoptera frugiperda cell line Sf21. Of the total, 116 miRNAs were well conserved within other insects, like B. mori, Drosophila melanogaster and Tribolium castenum while the remaining 110 miRNAs were identified as novel based on comparative analysis with the insect miRNA data set. Landscape distribution analysis based on Sf21 genome assembly revealed clustering of few novel miRNAs. A total of 5 miRNA clusters were identified and the largest one encodes 5 miRNA genes. In addition, 12 miRNAs were validated using northern blot analysis and putative functional role assignment for 6 Sf miRNAs was investigated by examining their relative abundance at different developmental stages of Spodoptera litura and body parts of 6^th instar larvae. Further, we identified a total of 809 potential target genes with GO terms for selected miRNAs, involved in different metabolic and signalling pathways of the insect. The newly identified miRNAs greatly enrich the repertoire of insect miRNAs and analysis of expression profiles reveal their involvement at various steps of biochemical pathways of the army worm.     

Biocontrol Potential of Steinernema thermophilum and Its Symbiont Xenorhabdus indica Against Lepidopteran Pests: Virulence to Egg and Larval Stages

Under laboratory conditions, the biocontrol potential of Steinernema thermophilum was tested against eggs and larval stages of two important lepidopteran insect pests, Helicoverpa armigera and Spodoptera litura (polyphagous pests), as well as Galleria mellonella (used as a model host). In terms of host susceptibility of lepidopteran larvae to S. thermophilum, based on the LC₅₀ 36 hr after treatment, G. mellonella (LC₅₀ = 16.28 IJ/larva) was found to be more susceptible than S. litura (LC₅₀ = 85 IJ/larva), whereas neither host was found to be significantly different from H. armigera (LC₅₀ = 54.68 IJ/larva). In addition to virulence to the larval stages, ovicidal activity up to 84% was observed at 200 IJ/50 and 100 eggs of H. armigera and S. litura, respectively. To our knowledge this is the first report of entomopathogenic nematode pathogenicity to lepidopteran eggs. Production of infective juvenile (IJ) nematodes/insect larva was also measured and found to be positively correlated with rate of IJ for H. armigera (r = 0.990), S. litura (r = 0.892), as well as G. mellonella (r = 0.834). Both Phase I and Phase II of symbiotic bacteria Xenorhabdus indica were tested separately against neonates of H. armigera and S. litura by feeding assays and found to be virulent to the target pests; phase variation did not affect the level of virulence. Thus S. thermophilum as well as the nematode’s symbiotic bacteria applied separately have the potential to be developed as biocontrol agents for key lepidopteran pests.     

The identification of microRNAs in the whitespotted bamboo shark (Chiloscyllium plagiosum) liver by Illumina sequencing

MicroRNAs are indispensable players in the regulation of a broad range of biological processes. Here, we report the first deep sequencing of the whitespotted bamboo shark (Chiloscyllium plagiosum) liver. We mapped 91 miRNAs in the Callorhinchus milii genome that have previously been described in the Danio rerio, Fugu rubripes, Oryzias latipes, Xenopus laevis, Xenopus tropicalis, Homo sapiens, and Mus musculus. In addition, 156 new putative candidate (PC) C. plagiosum miRNAs were identified. From these 247 miRNAs, 39 miRNA clusters were identified, and the expression of these clustered miRNAs was observed to vary significantly. A total of 7 candidate miRNAs were selected for expression confirmation by stem-loop RT-PCR. This study resulted in the addition of a significant number of novel miRNA sequences to GenBank and laid the foundation for further understanding of the function of miRNAs in the regulation of C. plagiosum liver development.     

Antifeedant and larvicidal activities of Rhein isolated from the flowers of Cassia fistula L.

Antifeedant and larvicidal activities of rhein (1,8-dihydroxyanthraquinone-3-carboxylic acid) isolated from the ethyl acetate extract of Cassia fistula flower were studied against lepidopteron pests Spodoptera litura and Helicoverpa armigera. Significant antifeedant activity was observed against H. armigera (76.13%) at 1000 ppm concentration. Rhein exhibited larvicidal activity against H. armigera (67.5), S. litura (36.25%) and the LC₅₀ values was 606.50 ppm for H. armigera and 1192.55 ppm for S.litura. The survived larvae produced malformed adults.     

Gene identification and proteomic analysis of the esterases of the cotton bollworm,Helicoverpa armigera

Some of the resistance of Helicoverpa armigera to conventional insecticides such as organophosphates and synthetic pyrethroids appears to be due to metabolic detoxification by carboxylesterases. To investigate the H. armigera carboxyl/cholinesterases, we created a data set of 39 putative paralogous H. armigera carboxyl/cholinesterase sequences from cDNA libraries and other sources. Phylogenetic analysis revealed a close relationship between these sequences and 70 carboxyl/cholinesterases from the recently sequenced genome of the silkworm, Bombyx mori, including several conserved clades of non-catalytic proteins. A juvenile hormone esterase candidate from H. armigera was identified, and B. mori orthologues were proposed for 31% of the sequences examined, however low similarity was found between lepidopteran sequences and esterases previously associated with insecticide resistance from other insect orders. A proteomic analysis of larval esterases then enabled us to match seven of the H. armigera carboxyl/cholinesterase sequences to specific esterase isozymes. All identified sequences were predicted to encode catalytically active carboxylesterases, including six proteins with N-terminal signal peptides and N-glycans, with two also containing C-terminal signals for glycosylphosphatidylinositol anchor attachment. Five of these sequences were matched to zones of activity on native PAGE at relative mobility values previously associated with insecticide resistance in this species.     

Novel regulation and functional interaction of polycistronic miRNAs

The importance of microRNAs in gene expression and disease is well recognized. However, what is less appreciated is that almost half of miRNA genes are organized in polycistronic clusters and are therefore coexpressed. The mir-11∼998 cluster consists of two miRNAs, miR-11 and miR-998. Here, we describe a novel layer of regulation that links the processing and expression of miR-998 to the presence of the mir-11 gene. We show that the presence of miR-11 in the pri-miRNA is required for processing by Drosha, and deletion of mir-11 prevents the expression of miR-998. Replacing mir-11 with an unrelated miRNA rescued miR-998 expression in vivo and in vitro, as did expressing miR-998 from a shorter, more canonical miRNA scaffold. The embedded regulation of miR-998 is functionally important because unchecked miR-998 expression in the absence of miR-11 resulted in pleiotropic developmental defects. This novel regulation of expression of miRNAs within a cluster is not limited to the mir-11∼998 cluster and, thus, likely reflects the more general cis-regulation of expression of individual miRNAs. Collectively, our results uncover a novel layer of regulation within miRNA clusters that tempers the functions of the individual miRNAs. Unlinking their expression has the potential to change the expression of multiple miRNA targets and shift a biological response.     

Combinatorial Action of MicroRNAs let-7 and miR-34 Effectively Synergizes with Erlotinib to Suppress Non-small Cell Lung Cancer Cell Proliferation

Lung cancer represents the leading cause of cancer-related deaths in men and women worldwide. Targeted therapeutics, including the epidermal growth factor receptor (EGFR) inhibitor erlotinib, have recently emerged as clinical alternatives for the treatment of non-small cell lung cancer (NSCLC). However, the development of therapeutic resistance is a major challenge, resulting in low 5-year survival rates. Due to their ability to act as tumor suppressors, microRNAs (miRNAs) are attractive candidates as adjuvant therapeutics for the treatment of NSCLC. In this study, we examine the ability of 2 tumor suppressor miRNAs, let-7b and miR-34a to sensitize KRAS;TP53 mutant non-small cell lung cancer cells to the action of erlotinib. Treatment with these miRNAs, individually or in combination, resulted in synergistic potentiation of the anti-proliferative effects of erlotinib. This effect was observed over a wide range of miRNA and erlotinib interactions, suggesting that let-7b and miR-34a target oncogenic pathways beyond those inhibited by EGFR. Combinatorial treatment with let-7b and miR-34a resulted in the strongest synergy with erlotinib, indicating that these miRNAs can effectively target multiple cellular pathways involved in cancer cell proliferation and resistance to erlotinib. Together, our findings indicate that NSCLC cells can be effectively sensitized to erlotinib by supplementation with tumor suppressor miRNAs, and suggest that the use of combinations of miRNAs as adjuvant therapeutics for the treatment of lung cancer is a viable clinical strategy.     

Identification,expression, and molecular evolution of microRNAs in the “living fossil” Triops cancriformis (tadpole shrimp)

MicroRNAs have been identified and analyzed in various model species, but an investigation of miRNAs in nonmodel species is required for a more complete understanding of miRNA evolution. In this study, we investigated the miRNAs of the nonmodel species Triops cancriformis (tadpole shrimp), a “living fossil,” whose morphological form has not changed in almost 200 million years. Dramatic ontogenetic changes occur during its development. To clarify the evolution of miRNAs, we comparatively analyzed its miRNAs and the components of its RNAi machinery. We used deep sequencing to analyze small RNA libraries from the six different developmental stages of T. cancriformis (egg, first–fourth instars, and adult), and also analyzed its genomic DNA with deep sequencing. We identified 180 miRNAs (87 conserved miRNAs and 93 novel candidate miRNAs), and deduced the components of its RNAi machinery: the DICER1, AGO1–3, PIWI, and AUB proteins. A comparative miRNA analysis of T. cancriformis and Drosophila melanogaster showed inconsistencies in the expression patterns of four conserved miRNAs. This suggests that although the miRNA sequences of the two species are very similar, their roles differ across the species. An miRNA conservation analysis revealed that most of the conserved T. cancriformis miRNAs share sequence similarities with those of arthropods, although T. cancriformis is called a “living fossil.” However, we found that let-7 and DICER1 of T. cancriformis are more similar to those of the vertebrates than to those of the arthropods. These results suggest that miRNA systems of T. cancriformis have evolved in a unique fashion.     

Identification of Wolbachia-responsive microRNAs in the two-spotted spider mite,Tetranychus urticae

Background

The two-spotted spider mite, Tetranychus urticae, is infected with Wolbachia, which have the ability to manipulate host reproduction and fitness. MicroRNAs (miRNAs) are small non-coding RNAs that are involved in many biological processes such as development, reproduction and host-pathogen interactions. Although miRNA was observed to involve in Wolbachia-host interactions in the other insect systems, its roles have not been fully deciphered in the two-spotted spider mite.

Results

Small RNA libraries of infected and uninfected T. urticae for both sexes (in total four libraries) were constructed. By integrating the mRNA data originated from the same samples, the target genes of the differentially expressed miRNAs were predicted. Then, GO and pathway analyses were performed for the target genes. Comparison of libraries showed that Wolbachia infection significantly regulated 91 miRNAs in females and 20 miRNAs in males, with an overall suppression of miRNAs in Wolbachia-infected libraries. A comparison of the miRNA and mRNA data predicted that the differentially expressed miRNAs negatively regulated 90 mRNAs in females and 9 mRNAs in males. An analysis of target genes showed that Wolbachia-responsive miRNAs regulated genes with function in sphingolipid metabolism, lysosome function, apoptosis and lipid transporting in both sexes, as well as reproduction in females.

Conclusion

Comparisons of the miRNA and mRNA data can help to identify miRNAs and miRNA target genes involving in Wolbachia-host interactions. The molecular targets identified in this study should be useful in further functional studies.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1122) contains supplementary material, which is available to authorized users.     

Enhancement of Larval RNAi Efficiency by Over-expressing Argonaute2 in Bombyx mori

RNA interference has been described as a powerful genetic tool for gene functional analysis and a promising approach for pest management. However, RNAi efficiency varies significantly among insect species due to distinct RNAi machineries. Lepidopteran insects include a large number of pests as well as model insects, such as the silkworm, Bombyx mori. However, only limited success of in vivo RNAi has been reported in lepidoptera, particularly during the larval stages when the worms feed the most and do the most harm to the host plant. Enhancing the efficiency of larval RNAi in lepidoptera is urgently needed to develop RNAi-based pest management strategies. In the present study, we investigate the function of the conserved RNAi core factor, Argonaute2 (Ago2), in mediating B. mori RNAi efficiency. We demonstrate that introducing BmAgo2 dsRNA inhibits the RNAi response in both BmN cells and embryos. Furthermore, we establish several transgenic silkworm lines to assess the roles of BmAgo2 in larval RNAi. Over-expressing BmAgo2 significantly facilitated both dsRNA-mediated larval RNAi when targeting DsRed using dsRNA injection and shRNA-mediated larval RNAi when targeting BmBlos2 using transgenic shRNA expression. Our results show that BmAgo2 is involved in RNAi in B. mori and provides a promising approach for improving larval RNAi efficiency in B. mori and in lepidopteran insects in general.     

Functional Characterization of Drosophila microRNAs by a Novel in Vivo Library

Animal microRNAs (miRNA) are implicated in the control of nearly all cellular functions. Due to high sequence redundancy within the miRNA gene pool, loss of most of these 21- to 24-bp long RNAs individually does not cause a phenotype. Thus, only very few miRNAs have been associated with clear functional roles. We constructed a transgenic UAS-miRNA library in Drosophila melanogaster that contains 180 fly miRNAs. This library circumvents the redundancy issues by facilitating the controlled misexpression of individual miRNAs and is a useful tool to complement loss-of-function approaches. Demonstrating the effectiveness of our library, 78 miRNAs induced clear phenotypes. Most of these miRNAs were previously unstudied. Furthermore, we present a simple system to create GFP sensors to monitor miRNA expression and test direct functional interactions in vivo. Finally, we focus on the miR-92 family and identify a direct target gene that is responsible for the specific wing phenotype induced by the misexpression of miR-92 family members.     

Comparisons of Mitochondrial DNA from the Sibling Species Heterodera glycines and H. schachtii

Restriction fragment patterns of mitochondrial DNA from sibling species of cyst nematodes Heterodera glycines and H. schachtii were examined. Fourteen restriction endonucleases recognizing four, five, and six base-pair sequences yielded a total of 90 scorable fragments of which 10% were shared by both species. Mitochondrial genome sizes for H. glycines and H. schachtii were estimated to be 22.5-23.5 kb and 23.0 kb, respectively. A single wild type mitochondrial genome was identified in all populations of H. glycines examined, although other mitochondrial genomes were present in some populations. The H. schachtii genome exhibited 57 scorable fragments, compared with 33 identified in the H. glycines wild type genome. The estimated nucleotide sequence divergence between the two species was p = 0.145. This estimate suggests these species diverged from a common ancestor 7.3-14.8 million years ago.