Kayvan Etebari

A study on interaspecific biodiversity of eight groups of silkworm (Bombyx mori) by biochemical markers

Abstract The recognition of biodiversity in different races and lines of silkworm (Bombyx mori) is very useful for breeding programs and production of high efficiency hybrids. In this study eight groups of silkworm were selected including 103, 107, Xihang 1 and 2 of Japanese origin and 104, 110, Koming 1 and 2 of Chinese origin. The activity levels of three enzymes including alkaline phosphatase, alanine aminotransferase and aspartate aminotransferase in haemolymph of fifth instar larva were measured. Moreover, the quantitative amount of total protein, cholesterol and glucose of haemolymph was evaluated. The data reveal that the activity level of measured macromolecules except for alkaline phosphatase were significantly different in all the groups. Hierarchical agglomerative clustering under UPGMA model separated line 104 from other groups. Two groups of Koming 1 and Xihang 1 had the most intergroup similarities.

Wolbachia Infection Modifies the Profile, Shuttling and Structure of MicroRNAs in a Mosquito Cell Line

MicroRNAs (miRNAs) are small non-coding RNAs that play important roles in many biological processes such as development, cell signaling and immune response. Small RNA deep sequencing technology provided an opportunity for a thorough survey of the miRNA profile of a mosquito cell line from Aedes aegypti. We characterized the miRNA composition of the nucleus and the cytoplasm of uninfected cells and compared it with the one of cells infected with the endosymbiotic bacterium Wolbachia strain wMelPop-CLA. We found an overall increase of small RNAs between 18 and 28 nucleotides in both cellular compartments in Wolbachia-infected cells and identified specific miRNAs induced and/or suppressed by the Wolbachia infection. We discuss the mechanisms that the cell may use to shuttle miRNAs between the cytoplasm and the nucleus. In addition, we identified piRNAs that changed their abundance in response to Wolbachia infection. The miRNAs and piRNAs identified in this study provide promising leads for investigations into the host-endosymbiont interactions and for better understanding of how Wolbachia manipulates the host miRNA machinery in order to facilitate its persistent replication in infected cells.

Diazinon resistance in different selected strains of Chilo suppressalis (Lepidoptera: Crambidae) in northern Iran.

ABSTRACT Asiatic rice borer, Chilo suppressalis (Walker) (Lepidoptera: Crambidae), is a cosmopolitan and destructive pest in rice fields of the world. This pest was reported in 1973 in Iran, and it has since spread widely in rice, Oryza sativa L., fields throughout the country. In this study, we tried to evaluate comparative toxicity of diazinon in five colonies of C. suppressalis, collected from Babol (Ba), Amol (Am) of Mazandaran Province and Rasht (Ra), Sheikhmahale (Sh), and Gourabzarmikh (Go) of Guilan Province, northern Iran. The LD50 values were compared. We also evaluated the general esterases, alkaline phosphatase (ALP), glutathione transferase (GST), and acetylcholinesterase (AChE) activities from the five populations. The LD50 values of Ra, Ba, Am, and Sh (12.64, 11.4, 7.17, and 3.71 µg/ mg larva-1) were 13.67-, 12.33-, 7.75-, and 4.02-fold higher than Go population (0.924 µg/mg larva-1). Using &agr;-naphthyl acetate as substrate, the general esterase activities in Ra, Ba, Am, and Sh colonies were, respectively, 1.81-, 1.68-, 1.75-, and 1.35-fold more than those in Go population. When &bgr;-naphthyl acetate was used as the substrate, activity ratio was measured 1.98-, 2.58-, 1.25-, and 1.24-fold compared with the Go population. Glutathione transferase activities in Ra, Ba, Am, and Sh populations were 1.27-, 1.68-, 0.98-, and 1.7-fold more than those in Go, when 1-chloro-2,4-dinitrobenzene was used as the substrate. When 1,2-dichloro-4-nitro-benzene was used as the substrate, activity ratio was measured 1.14-, 1.42-, 0.56-, and 0.95-fold compared with Go population. The ALP activity demonstrated a significant difference among these populations and in Ra, Ba, Am, and Sh larvae were 3.54-, 4.62-, 3.84-, and 2.18-fold more than Go. The AChE inhibition or I50 value was 0.19, 0.22, 0.31, 0.19, and 0.26 mM in Ra, Ba, Am, Sh and Go populations, respectively. However, the results showed no significant differences in studied colonies. These biochemical characterizations of general esterases ALP, GST, and AChE were consistent with diazinon bioassay in the five populations. It is inferred from increased esterase, alkaline phosphatase and glutathione transferase, activities that might play an important role in the increasing resistance in C. suppressalis to diazinon among these five populations.

Identification of microRNAs from Plutella xylostella larvae associated with parasitization by Diadegma semiclausum

MicroRNAs (miRNAs) as small non-coding RNAs play important roles in many biological processes such as development, cell signaling and immune response. Small RNA deep sequencing technology provided an opportunity for a thorough survey of miRNAs in a global key pest Plutella xylostella as well as comparative analysis of miRNA expression profile of the insect in association with parasitization by Diadegma semiclausum. Combining the deep sequencing data and bioinformatics, 235 miRNAs were identified from P. xylostella. Differential expression of host cellular miRNAs in response to parasitism was examined by making small RNA libraries from parasitized and naive second instar larvae of P. xylostella. Bantam, miR-276*, miR-10, miR-31 and miR-184 were detected as five most abundant miRNAs in both libraries and 96 miRNAs were identified that were differentially expressed after parasitization. Bantam*, miR-184 and miR-281* were significantly down-regulated and two miRNAs miR-279b and miR-2944b* were highly induced in parasitized larvae. Interestingly, high copy numbers and differential expression of several miRNA passenger strands (miRNA*) suggest their potential roles in host-parasitoid interaction. In conclusion, expression profiling of miRNAs provided insights into their possible involvement in insect immune response to parasitism and offer an important resource for further studies.

Conserved microRNA miR-8 blocks activation of the Toll pathway by upregulating Serpin 27 transcripts

microRNAs (miRNAs) play significant regulatory roles in gene expression at the post-transcriptional level. This includes modulating processes such as development, immunity, cancer, and host-pathogen interactions. It was recently shown that the phylogenetically deeply conserved miRNA, miR-8, plays a role in maintaining the homeostasis of immunity by suppressing the production of anti-microbial peptides. In this study, we show that miR-8 from the insect Plutella xylostella positively regulates the transcript levels of the serine protease inhibitor Serpin 27, which has been shown to regulate activation of the Toll pathway and prophenoloxidase involved in the melanization response in insects. Interestingly, miR-8 is downregulated following parasitization by Diadegma semiclausum leading to significant declines in Serpin 27 transcript levels. This allows upregulation of antimicrobial peptides, such as gloverin, that are controlled by the Toll pathway and activation of proteolytic cascades essential for humoral immune responses to foreign invasion.

Genome wide discovery of long intergenic non-coding RNAs in Diamondback moth ( Plutella xylostella ) and their expression in insecticide resistant strains

Long non-coding RNAs (lncRNAs) play important roles in genomic imprinting, cancer, differentiation and regulation of gene expression. Here, we identified 3844 long intergenic ncRNAs (lincRNA) in Plutella xylostella, which is a notorious pest of cruciferous plants that has developed field resistance to all classes of insecticides, including Bacillus thuringiensis (Bt) endotoxins. Further, we found that some of those lincRNAs may potentially serve as precursors for the production of small ncRNAs. We found 280 and 350 lincRNAs that are differentially expressed in Chlorpyrifos and Fipronil resistant larvae. A survey on P. xylostella midgut transcriptome data from Bt-resistant populations revealed 59 altered lincRNA in two resistant strains compared with the susceptible population. We validated the transcript levels of a number of putative lincRNAs in deltamethrin-resistant larvae that were exposed to deltamethrin, which indicated that this group of lincRNAs might be involved in the response to xenobiotics in this insect. To functionally characterize DBM lincRNAs, gene ontology (GO) enrichment of their associated protein-coding genes was extracted and showed over representation of protein, DNA and RNA binding GO terms. The data presented here will facilitate future studies to unravel the function of lincRNAs in insecticide resistance or the response to xenobiotics of eukaryotic cells.

Effects of hypervitaminosis of vitamin B3 on silkworm biology.

A high-dose of vitamin B3 in silkworm diet interrupts larval feeding and normal growth. High mortality of larvae occurs during molting and they cannot complete this process normally. Also the larvae exhibit nicotinamide hypervitaminosis symptoms such as immobility, dyspepsia, darkening of the skin, inability to excrete normally, exerting brownish fluid from anus and swelling of rectal muscles. Maximum larval weights in 1, 2 and 3 g/l treatments were 2.9, 1.6 and 1.2 g respectively, while maximum larval weight in the control was 5.6 g. Larval stage compared to control had increased 18, 26 and 31 days respectively. The concentration increase of uric acid in haemolymph demonstrates the hyperuricemia, while other measured biochemical compounds show significant decrease; sodium and potassium did not change significantly.

Zika virus alters the microRNA expression profile and elicits an RNAi response in Aedes aegypti mosquitoes

Zika virus (ZIKV), a flavivirus transmitted primarily by Aedes aegypti, has recently spread globally in an unprecedented fashion, yet we have a poor understanding of host-microbe interactions in this system. To gain insights into the interplay between ZIKV and the mosquito, we sequenced the small RNA profiles in ZIKV-infected and non-infected Ae. aegypti mosquitoes at 2, 7 and 14 days post-infection. ZIKA induced an RNAi response in the mosquito with virus-derived short interfering RNAs and PIWI-interacting RNAs dramatically increased in abundance post-infection. Further, we found 17 host microRNAs (miRNAs) that were modulated by ZIKV infection at all time points. Strikingly, many of these regulated miRNAs have been reported to have their expression altered by dengue and West Nile viruses, while the response was divergent from that induced by the alphavirus Chikungunya virus in mosquitoes. This suggests that conserved miRNA responses occur within mosquitoes in response to flavivirus infection. This study expands our understanding of ZIKV-vector interactions and provides potential avenues to be further investigated to target ZIKV in the mosquito host.

Accuracy of microRNA discovery pipelines in non-model organisms using closely related species genomes.

Mapping small reads to genome reference is an essential and more common approach to identify microRNAs (miRNAs) in an organism. Using closely related species genomes as proxy references can facilitate miRNA expression studies in non-model species that their genomes are not available. However, the level of error this introduces is mostly unknown, as this is the result of evolutionary distance between the proxy reference and the species of interest. To evaluate the accuracy of miRNA discovery pipelines in non-model organisms, small RNA library data from a mosquito, Aedes aegypti, were mapped to three well annotated insect genomes as proxy references using miRanalyzer with two strict and loose mapping criteria. In addition, another web-based miRNA discovery pipeline (DSAP) was used as a control for program performance. Using miRanalyzer, more than 80% reduction was observed in the number of mapped reads using strict criterion when proxy genome references were used; however, only 20% reduction was recorded for mapped reads to other species known mature miRNA datasets. Except a few changes in ranking, mapping criteria did not make any significant differences in the profile of the most abundant miRNAs in A. aegypti when its original or a proxy genome was used as reference. However, more variation was observed in miRNA ranking profile when DSAP was used as analysing tool. Overall, the results also suggested that using a proxy reference did not change the most abundant miRNAs’ differential expression profiles when infected or non-infected libraries were compared. However, usage of a proxy reference could provide about 67% of the original outcome from more extremely up- or down-regulated miRNA profiles. Although using closely related species genome incurred some losses in the number of miRNAs, the most abundant miRNAs along with their differential expression profile would be acceptable based on the sensitivity level of each project.

Dengue virus infection alters post-transcriptional modification of microRNAs in the mosquito vector Aedes aegypti.

Recent discoveries regarding the importance of isomiRs have increased our understanding of the regulatory complexities of the miRNAome. Observed changes in the miRNA profiles in mosquitoes infected with flaviviruses have implicated small RNAs in the interactions between viruses and their vectors. Here we analysed the isomiR profiles of both uninfected and infected Aedes aegypti mosquitoes with the major human pathogen dengue virus (DENV). We found that several specific isomiRs were significantly altered in their abundance patterns in response to DENV infection potentially affecting their target repertoire. Notable among these were isomiR variants which displayed arm-switching. We also demonstrate that modifications to the 3p end of miRNAs are vastly more prevalent than those at the 5p ends. We also observed that in only 45% of Ae. aegypti miRNAs the most abundant read matches the exact sequence reported in miRBase. Further, we found positive correlations between the number of mature miRNA reads, pre-miRNA length, GC content and secondary structure minimum free energy with the number of isomiRs. The findings presented here provide some evidence that isomiR production is not a random phenomenon and may be important in DENV replication in its vector.