Exploration of microbial communities in the guts and casts of Eudrilus eugeniae, Perionyx excavatus, and Eisenia fetida.

Earthworms and their casts have been widely used for organic waste degradation and plant growth promotion. The microbial communities that reside in the guts and casts of earthworms markedly influence both applications. In the present study, next-generation sequencing was applied to identify the microbial communities in the guts and casts of three epigeic earthworm species, Eudrilus eugeniae, Perionyx excavatus, and Eisenia fetida, reared under two different feeding conditions. A total of 580 genera belonging to 43 phyla were identified. By comparing bacterial diversity among samples divided into groups based on the earthworm species, sample types, and conditions, the beta diversity analysis supported the impact of the sample type and suggested that there was significant dissimilarity of the bacterial diversity between the gut and cast. Besides, bacterial Phylum compositions within the group were compared. The result showed that the top three high relative frequency phyla found in the casts were the same regardless of earthworm species, while those found in the gut depended on both the condition and earthworm species. Focusing on the cellulolytic and plant growth-promoting bacteria, certain cellulolytic bacteria, Paenibacillus, Comamonas, and Cytophaga, were found only in the cast. Citrobacter and Streptomyces aculeolatus were detected only in the guts of earthworms reared in the bedding containing vegetables and bedding alone, respectively. Besides, Actinomadura and Burkholderia were detected only in the gut of E. eugeniae and E. fetida, respectively. The results proved that the microbial composition was affected by sample type, condition, and earthworm species. In addition, the proportion of these beneficial bacteria was also influenced by these factors. Hence, the information from this study can be used as a guide for selecting earthworm species or their casts for more efficient organic waste decomposition and plant growth promotion.

Identification of potential candidate genes involved in the sex determination cascade in an aquatic firefly, Sclerotia aquatilis (Coleoptera, Lampyridae).

Sexual differentiation, dimorphism, and courtship behavior are the downstream developmental programs of the sex determination cascade. The sex determination cascade in arthropods often involves key genes, transformer (tra), doublesex (dsx), transformer-2 (tra2), and fruitless (fru). These genes are conserved among insect taxa; however, they have never been reported in fireflies. In this study, the candidate genes for these key genes were identified for the first time in an aquatic firefly, Sclerotia aquatilis using transcriptome analysis. A comparative protein-protein interaction (PPI) network of sex determination cascade was reconstructed for S. aquatilis based on a network of a model insect, Drosophila melanogaster. Subsequently, a sex determination cascade in S. aquatilis was proposed based on the amino acid sequence structures and expression profiles of these candidates. This study describes the first efforts toward understanding the molecular control of sex determination cascade in fireflies.

Reconstruction of insect hormone pathways in an aquatic firefly, Sclerotia aquatilis (Coleoptera: Lampyridae), using RNA-seq.

Insect hormones: ecdysteroids and juvenile hormones have crucial functions during the regulation of different developmental pathways in insects. Insect metamorphosis is one of the primary pathways regulated by these hormones. The insect hormone biosynthetic pathway is conserved among arthropods, including insects, with some variations in the form of hormones used among each group of insects. In this study, the candidate genes involved in the insect hormone pathways and their functional roles were assessed in an aquatic firefly, Sclerotia aquatilis using a high-throughput RNA sequencing technique. Illumina next-generation sequencing (NGS) was used to generate transcriptome data for the different developmental stages (i.e., larva, pupa, and adult) of S. aquatilis. A total of 82,022 unigenes were generated across all different developmental stages. Functional annotation was performed for each gene, based on multiple biological databases, generating 46,230 unigenes. These unigenes were subsequently mapped using KEGG pathways. Accordingly, 221 protein-encoding genes involved in the insect hormone pathways were identified, including, JHAMT, CYP15A1, JHE, and Halloween family genes. Twenty potential gene candidates associated with the biosynthetic and degradation pathways for insect hormones were subjected to real-time PCR, reverse transcriptase PCR (RT-PCR) and sequencing analyses. The real-time PCR results showed similar expression patterns as those observed for transcriptome expression profiles for most of the examined genes. RT-PCR and Sanger sequencing confirmed the expressed coding sequences of these gene candidates. This study is the first to examine firefly insect hormone pathways, facilitating a better understanding of firefly growth and development.

Heterologous biosynthesis of a fungal macrocyclic polylactone requires only two iterative polyketide synthases.

Menisporopsin A is a bioactive macrocyclic polylactone produced by the fungus Menisporopsis theobromae BCC 4162. A scheme for the biosynthesis of this compound has been proposed, in which reducing (R) and non-reducing (NR) polyketide synthases (PKSs) would catalyze the formation of each menisporopsin A subunit, while an additional non-ribosomal peptide synthetase (NRPS)-like enzyme would be required to perform multiple esterification and cyclolactonization reactions. Transcriptome analysis of M. theobromae identified an R-PKS gene, men1, and an NR-PKS gene, men2, which both exhibited highest expression levels during the menisporopsin A production phase. These were cloned into separate vectors for heterologous expression in Aspergillus oryzae NSAR1. Unexpectedly, coexpression of the two PKSs alone was sufficient to catalyze the formation of the macrocyclic polylactone, ascotrichalactone A, a structural derivative of menisporopsin A. The unanticipated esterification and cyclolactonization activities could reside in the unusual thioesterase domain of the NR-PKS, which is similar to that of the NRPS catalyzing elongation and cyclization of trilactone in enterobactin biosynthesis and that of modular PKSs catalyzing macrodiolide formation in elaiophylin and conglobatin biosyntheses.

Comparative mitochondrial genome analysis of the firefly, Inflata indica (Coleoptera: Lampyridae) and the first evidence of heteroplasmy in fireflies.

The first mitochondrial genome of the firefly genus Inflata Boontop, Inflata indica (Lampyridae, Luciolinae) is reported here. Thirteen protein coding genes, 22 tRNA, 2 rRNA genes and a 1570 bp non-coding region were annotated. The comparative analysis showed that the size, GC content, gene content and gene arrangement of this mitochondrial genome are similar to those of other Lampyridae. The mitochondrial phylogenomic analysis showed that I. indica is clustered with other fireflies belonging to the subfamily Luciolinae and most closely related to Pteroptyx maipo. The evolutionary relationship within group of Luciolinae fireflies presented by complete COI phylogeny differs from the relationship based on the mitochondrial phylogenomic tree. This individual firefly also carries mitochondrial DNA (mtDNA) heteroplasmy; however, no potential effect of heteroplasmic mutations could be observed in this study. Hence, this study is reporting not only the first mitochondrial genome of the genus Inflata and the comparative mitochondrial genome analysis of fireflies but also the first mitochondrial DNA heteroplasmy in Lampyridae.

Transcriptome analysis reveals candidate genes involved in luciferin metabolism in Luciola aquatilis (Coleoptera: Lampyridae).

Bioluminescence, which living organisms such as fireflies emit light, has been studied extensively for over half a century. This intriguing reaction, having its origins in nature where glowing insects can signal things such as attraction or defense, is now widely used in biotechnology with applications of bioluminescence and chemiluminescence. Luciferase, a key enzyme in this reaction, has been well characterized; however, the enzymes involved in the biosynthetic pathway of its substrate, luciferin, remains unsolved at present. To elucidate the luciferin metabolism, we performed a de novo transcriptome analysis using larvae of the firefly species, Luciola aquatilis. Here, a comparative analysis is performed with the model coleopteran insect Tribolium casteneum to elucidate the metabolic pathways in L. aquatilis. Based on a template luciferin biosynthetic pathway, combined with a range of protein and pathway databases, and various prediction tools for functional annotation, the candidate genes, enzymes, and biochemical reactions involved in luciferin metabolism are proposed for L. aquatilis. The candidate gene expression is validated in the adult L. aquatilis using reverse transcription PCR (RT-PCR). This study provides useful information on the bio-production of luciferin in the firefly and will benefit to future applications of the valuable firefly bioluminescence system.

Biosynthetic origins of menisporopsin A.

Menisporopsin A, produced by Menisporopsis theobromae, shows antimalarial, antimycobacterial, and cytotoxic activities. Here, we report the first (13)C incorporations at individual carbons of menisporopsin A using sodium [1-(13)C] and [2-(13)C] acetate. This result indicates that each of the subunits of the pentalactone menisporopsin A is assembled by a polyketide synthase.

Prior infection of Manduca sexta with non-pathogenic Escherichia coli elicits immunity to pathogenic Photorhabdus luminescens: roles of immune-related proteins shown by RNA interference.

Prior infection of Manduca sexta caterpillars with the non-pathogenic bacterium Escherichia coli elicits effective immunity against subsequent infection by the usually lethal and highly virulent insect pathogen Photorhabdus luminescens TT01. Induction of this protective effect is associated with up-regulation of both microbial pattern recognition protein genes (hemolin, immulectin-2 and peptidoglycan recognition protein) and anti-bacterial effector genes (attacin, cecropin, lebocin, lysozyme and moricin). We used RNA interference to knock down over-transcription of members of both these sets of genes one at a time. Interfering with expression of individual recognition proteins had a drastic adverse effect on the E. coli elicited immunity. RNAi knock-down of immulectin-2 caused the greatest reduction in immunity, followed by hemolin and peptidoglycan recognition protein (PGRP) in that order, to the extent that knock-down of any one of these three proteins left the insects more susceptible to P. luminescens infection than insects that had not experienced prior infection with E. coli. Interfering with the expression of individual antibacterial effector proteins and peptides had a much less marked effect on immunity. Knock-down of attacin, cecropin or moricin caused treated insects to be more susceptible to P. luminescens infection than controls that had been pre-infected with E. coli but which had not received the specific RNAi reagents, but they were still less susceptible than insects that had not been pre-infected with E. coli. RNAi knock-down with expression of lebocin or lysozyme had no effect on E. coli-induced immunity to P. luminescens, indicating that these effectors are not involved in the response. By bleeding pre-infected caterpillars and growing the pathogen directly within cell-free insect haemolymph, we showed that at least part of the protection elicited by previous exposure to E. coli is due to the presence of factors within the blood plasma that inhibit the growth of P. luminescens. The production of these factors is inhibited by RNAi treatment with ds-RNA reagents that knock down hemolin, immulectin-2, and PGRP. These results demonstrate that the insect immune system can be effectively primed by prior infection with non-pathogenic bacteria against subsequent infection by a highly virulent pathogen. Given the continuous normal exposure of insects to environmental and symbiotic bacteria, we suggest that prior infection is likely to play a significant and underestimated role in determining the level of insect immunity found in nature.