Complete Mitochondrial Genome of the Eggplant Fruit and Shoot Borer, Leucinodes orbonalis Guenée (Lepidoptera: Crambidae), and Comparison with Other Pyraloid Moths.

The eggplant fruit and shoot borer (EFSB) (Leucinodes orbonalis Guenée) is a devastating lepidopteran pest of eggplant (Solanum melongena L.) in the Philippines. Management of an insect pest like the EFSB requires an understanding of its biology, evolution, and adaptations. Genomic resources provide a starting point for understanding EFSB biology, as the resources can be used for phylogenetics and population structure studies. To date, genomic resources are scarce for EFSB; thus, this study generated its complete mitochondrial genome (mitogenome). The circular mitogenome is 15,244 bp-long. It contains 37 genes, namely 13 protein-coding, 22 tRNA, and 2 rRNA genes, and has conserved noncoding regions, motifs, and gene syntenies characteristic of lepidopteran mitogenomes. Some protein-coding genes start and end with non-canonical codons. The tRNA genes exhibit a conserved cloverleaf structure, with the exception in trnS1. Partitioned phylogenetic analysis using 72 pyraloids generated highly supported maximum likelihood and Bayesian inference trees revealing expected basal splits between Crambidae and Pyralidae, and Spilomelinae and Pyraustinae. Spilomelinae was recovered to be paraphyletic, with the EFSB robustly placed before the split of Spilomelinae and Pyraustinae. Overall, the EFSB mitogenome resource will be useful for delineations within Spilomelinae and population structure analysis.

Bacterial Survey in the Guts of Domestic Silkworms, Bombyx mori L.

Silkworm, Bombyx mori L., research involves studies on improving strains for enhanced sustainability of high-quality silk production. Several of these have investigated the factors affecting growth and development of silkworm larvae and cocoon characteristics that subsequently affect the yield and quality of silk. The gut microbiota has been reported to impact growth and development of silkworms and has been linked, in particular, with absorption and utilization of nutrients and immunity to diseases. The silkworm strains maintained in the Philippines lack sufficient biological data for use in strain improvement. This prompted efforts to augment the data by profiling bacterial communities through high-throughput 16S rRNA gene amplicon sequencing and analysis in four of the local silkworm strains that are bred and maintained in the country. Results of the study showed that the four silkworm strains are abundant in bacteria that belong to the genera Pseudomonas, Sphingomonas, Delftia, Methylobacterium and Acinetobacter. Results also showed that bacterial diversity and evenness increase as larvae mature, which can be correlated to larval development and shifts in the amount and age of mulberry leaves the larvae consume.

Global Profiling of Genes Expressed in the Silk Glands of Philippine-Reared Mulberry Silkworms (Bombyx mori).

RNA sequencing was used to assemble transcriptome data for Philippine-reared silkworm and compare gene expression profiles of strains reared in high- and low-temperature environments. RNA was isolated from the silk glands of fifth instar larvae and mRNA-enriched libraries were sequenced using Illumina NextSeq 500. Transcriptome reads were assembled using reference-based and de novo assemblers, and assemblies were evaluated using different metrics for transcriptome quality, including the read mapping rate, E90N50, RSEM-eval, and the presence of single-copy orthologs. All transcriptome assemblies were able to reconstruct >40,000 transcripts. Differential expression analysis found 476 differentially expressed genes (DEGs; 222 upregulated, 254 downregulated) in strains reared in different temperatures. Among the top DEGs were myrosinase, heat shock proteins, serine protease inhibitors, dehydrogenases, and regulators of the juvenile hormone. Validation of some of the top DEGs using qPCR supported the findings of the in silico analysis. GO term enrichment analysis reveals an overrepresentation of GO terms related to nucleotide metabolism and biosynthesis, lipid and carbohydrate metabolic processes, regulation of transcription, nucleotide binding, protein binding, metal binding, catalytic activity, oxidoreductase activity, and hydrolase activity. The data provided here will serve as a resource for improving local strains and increasing silk production of Philippine-reared B. mori strains.

Draft Genome Sequences of Two Streptomyces Isolates Obtained from Volcanic Soils in the Philippines.

We report here the draft genome sequences of two Streptomyces isolates, namely, UNOB3_S3 and UNOC14_S4, obtained from volcanic soils in Albay, Philippines. The genome assemblies comprised 7.63 Mb and 8.24 Mb, respectively. Genome mining revealed genes and biosynthetic gene clusters that encode putative insecticidal products.

Expansion of time window for mass spectrometric measurement of amide hydrogen/deuterium exchange reactions.

Backbone amide hydrogen exchange rates can be used to describe the dynamic properties of a protein. Amide hydrogen exchange rates in a native protein may vary from milliseconds (ms) to several years. Ideally, the rates of all amide hydrogens of the analyte protein can be determined individually. To achieve this goal, monitoring of a wider time window is critical, in addition to high sequence coverage and high sequence resolution. Significant improvements have been made to hydrogen/deuterium exchange mass spectrometry methods in the past decade for better sequence coverage and higher sequence resolution. On the other hand, little effort has been made to expand the experimental time window to accurately determine exchange rates of amide hydrogens. Many fast exchanging amide hydrogens are completely exchanged before completion of a typical short exchange time point (10-30 s) and many slow exchanging amide hydrogens do not start exchanging before a typical long exchanging time point (1-3 h). Here various experimental conditions, as well as a quenched-flow apparatus, are utilized to monitor cytochrome c amide hydrogen exchange behaviors over more than eight orders of magnitude (0.0044-1 000 000 s), when converted into the standard exchange condition (pH 7 and 23°C).

Evidence for trade-offs in detoxification and chemosensation gene signatures in Plutella xylostella.

BACKGROUND: Detoxification genes have been associated with insecticide adaptation in the diamondback moth, Plutella xylostella. The link between chemosensation genes and adaptation, however, remains unexplored. To gain a better understanding of the involvement of these genes in insecticide adaptation, the authors exposed lines of P. xylostella to either high uniform (HU) or low heterogeneous (LH) concentrations of permethrin, expecting primarily physiological or behavioral selection respectively. Initially, 454 pyrosequencing was applied, followed by an examination of expression profiles of candidate genes that responded to selection [cytochrome P450 (CYP), glutathione S-transferase (GST), carboxylesterase (CarE), chemosensory protein (CSP) and odorant-binding protein (OBP)] by quantitative PCR in the larvae. Toxicity and behavioral assays were also conducted to document the effects of the two forms of exposure. RESULTS: Pyrosequencing of the P. xylostella transcriptome from adult heads and third instars produced 198,753 reads with 52,752,486 bases. Quantitative PCR revealed overexpression of CYP4M14, CYP305B1 and CSP8 in HU larvae. OBP13, however, was highest in LH. Larvae from LH and HU lines had up to five- and 752-fold resistance levels respectively, which could be due to overexpression of P450s. However, the behavioral responses of all lines to a series of permethrin concentrations did not vary significantly in any of the generations examined, in spite of the observed upregulation of CSP8 and OBP13. CONCLUSION: Expression patterns from the target genes provide insights into behavioral and physiological responses to permethrin and suggest a new avenue of research on the role of chemosensation genes in insect adaptation to toxins.

Permanent genetic resources added to Molecular Ecology Resources Database 1 August 2012 - 30 September 2012.

This article documents the addition of 83 microsatellite marker loci and 96 pairs of single-nucleotide polymorphism (SNP) sequencing primers to the Molecular Ecology Resources Database. Loci were developed for the following species: Bembidion lampros, Inimicus japonicus, Lymnaea stagnalis, Panopea abbreviata, Pentadesma butyracea, Sycoscapter hirticola and Thanatephorus cucumeris (anamorph: Rhizoctonia solani). These loci were cross-tested on the following species: Pentadesma grandifolia and Pentadesma reyndersii. This article also documents the addition of 96 sequencing primer pairs and 88 allele-specific primers or probes for Plutella xylostella.

RNA interference-mediated knockdown of a cytochrome P450, CYP6BG1, from the diamondback moth, Plutella xylostella, reduces larval resistance to permethrin.

We have previously reported that a cytochrome P450, CYP6BG1, from Plutella xylostella was found to be overexpressed in 4th instars of a permethrin resistant strain and inducible in the susceptible counterpart. The findings suggested potential involvement of CYP6BG1 in permethrin resistance, hence warranted a functional analysis. To assess the functional link of the gene to permethrin resistance, we adopted RNA interference-mediated gene silencing (RNAi) by dsRNA droplet feeding. Here, real time PCR analyses show that oral delivery of dsRNA can efficiently reduce the expression of CYP6BG1. Knockdown of CYP6BG1 transcript was evident in midgut and larval tissues enclosed in carcass. As a consequence of knockdown, a significant reduction in resistance of larvae fed CYP6BG1 dsRNA was observed after 24 and 48h of exposure to permethrin. In addition, CYP6BG1 dsRNA feeding to larvae led to reduced total P450 activities of microsomal preparations toward model substrates p-nitroanisole and benzyloxyresorufin. These results indicate that the overexpressed CYP6BG1 participate in enhanced metabolism of permethrin, thereby, resistance. The knockdown of a non-overexpressed P450, CYP6BF1v4, from the same resistant P. xylostella strain did not lead to changes in the level of resistance to permethrin, supporting further the specific involvement of CYP6BG1 in the resistance.