Meta-Analysis of Transcriptomes in Insects Showing Density-Dependent Polyphenism

With increasing public data, a statistical analysis approach called meta-analysis, which combines transcriptome results obtained from multiple studies, has succeeded in providing novel insights into targeted biological processes. Locusts and aphids are representative of insect groups that exhibit density-dependent plasticity. Although the physiological mechanisms underlying density-dependent polyphenism have been identified in aphids and locusts, the underlying molecular mechanisms remain largely unknown. In this study, we performed a meta-analysis of public transcriptomes to gain additional insights into the molecular underpinning of density-dependent plasticity. We collected RNA sequencing data of aphids and locusts from public databases and detected differentially expressed genes (DEGs) between crowded and isolated conditions. Gene set enrichment analysis was performed to reveal the characteristics of the DEGs. DNA replication (GO:0006260), DNA metabolic processes (GO:0006259), and mitotic cell cycle (GO:0000278) were enriched in response to crowded conditions. To date, these processes have scarcely been the focus of research. The importance of the oxidative stress response and neurological system modifications under isolated conditions has been highlighted. These biological processes, clarified by meta-analysis, are thought to play key roles in the regulation of density-dependent plasticity

Long-read genome sequencing of the Japanese parasitic wasp <i>Copidosoma floridanum</i> (Hymenoptera: Encyrtidae)

Supplementary Table S1: Statistics of the alternate contigs generated by Hifiasm Supplementary File 1:Match coordinate data between Cflo-US and Cflo-J Hifi assemblySupplementary File 2:Match coordinate data between Cflo-J subread assembly and Cflo-J Hifi assemblySupplementary Table S2: Statistics summary generated by k-mer assembly evaluation by Merqury Supplementary Figure S1: Quality assessment of Cflo-J Hifi assembly by Merqury and Blobtools. (A) The kmer spectrum assembly plot generated by Merqury. Different contigs are indicated by different colors.(B) BlobPlot for the primary assembly and (C) alternate contig. Each circle is scaled proportionally to the length of the contig and is colored by taxonomic annotations based on BLAST search results. Contigs are arranged based on GC content (x-axis) and read coverage (y-axis). Histograms show cumulative base pair length in coverage (y-axis) and GC content (x-axis).Supplementary Table S3 Summary of repetitive elements in the Hifi primary contigSupplementary Table S4: All results of functional annotation by Fanflow4InsectsSupplementary Table S5 Transcripts with no corresponding protein in the Cflo-US datasetSupplementaryTable S6: Genomic location of vasa geneSupplementary File 3: Annotation GTF file generated by BRAKER</table

Meta-Analysis of Transcriptomes in Insects Showing Density-Dependent Polyphenism

With increasing public data, a statistical analysis approach called meta-analysis, which combines transcriptome results obtained from multiple studies, has succeeded in providing novel insights into targeted biological processes. Locusts and aphids are representative of insect groups that exhibit density-dependent plasticity. Although the physiological mechanisms underlying density-dependent polyphenism have been identified in aphids and locusts, the underlying molecular mechanisms remain largely unknown. In this study, we performed a meta-analysis of public transcriptomes to gain additional insights into the molecular underpinning of density-dependent plasticity. We collected RNA sequencing data of aphids and locusts from public databases and detected differentially expressed genes (DEGs) between crowded and isolated conditions. Gene set enrichment analysis was performed to reveal the characteristics of the DEGs. DNA replication (GO:0006260), DNA metabolic processes (GO:0006259), and mitotic cell cycle (GO:0000278) were enriched in response to crowded conditions. To date, these processes have scarcely been the focus of research. The importance of the oxidative stress response and neurological system modifications under isolated conditions has been highlighted. These biological processes, clarified by meta-analysis, are thought to play key roles in the regulation of density-dependent plasticity

Ecdysteroid-induced programmed cell death is essential for sex-specific wing degeneration of the wingless-female winter moth.

The winter moth, Nyssiodes lefuarius, has a unique life history in that adults appear during early spring after a long pupal diapause from summer to winter. The moth exhibits striking sexual dimorphism in wing form; males have functional wings of normal size, whereas females lack wings. We previously found that cell death of the pupal epithelium of females appears to display condensed chromatin within phagocytes. To provide additional detailed data for interpreting the role of cell death, we performed light microscopy, transmission electron microscopy, and TUNEL assay. We consequently detected two modes of cell death, i.e., dying cells showed both DNA fragmentation derived from epithelial nuclei and autophagic vacuole formation. To elucidate the switching mechanism of sex-specific wing degeneration in females of N. lefuarius, we tested the effects of the steroid hormone 20-hydroxyecdysone (20E) on pupal diapause termination and wing morphogenesis in both sexes. When 20E (5.4 µg) was injected into both sexes within 2 days of pupation, wing degeneration started 4 days after 20E injection in females, whereas wing morphogenesis and scale formation started 6 days after 20E injection in males. We discuss two important findings: (1) degeneration of the pupal wing epithelium of females was not only due to apoptosis and phagocytotic activation but also to autophagy and epithelial cell shrinkage; and (2) 20E terminated the summer diapause of pupae, and triggered selective programmed cell death only of the female-pupal wing epithelium in the wingless female winter moth

Supplemental Material for Masuoka et al., 2018

Fig. S1-S4.pdf, Table S1-S2.pdf, Supplemental file caption.doc

TGF<i>β</i> signaling related genes are involved in hormonal mediation during termite soldier differentiation

<div><p>A working knowledge of the proximate factors intrinsic to sterile caste differentiation is necessary to understand the evolution of eusocial insects. Genomic and transcriptomic analyses in social hymenopteran insects have resulted in the hypothesis that sterile castes are generated by the novel function of co-opted or recruited universal gene networks found in solitary ancestors. However, transcriptome analysis during caste differentiation has not been tested in termites, and evolutionary processes associated with acquiring the caste are still unknown. Termites possess the soldier caste, which is regarded as the first acquired permanently sterile caste in the taxon. In this study, we performed a comparative transcriptome analysis in termite heads during 3 molting processes, i.e., worker, presoldier and soldier molts, under natural conditions in an incipient colony of the damp-wood termite <i>Zootermopsis nevadensis</i>. Although similar expression patterns were observed during each molting process, more than 50 genes were shown to be highly expressed before the presoldier (intermediate stage of soldier) molt. We then performed RNA interference (RNAi) of the candidate 13 genes, including transcription factors and uncharacterized protein genes, during presoldier differentiation induced by juvenile hormone (JH) analog treatment. Presoldiers induced after RNAi of two genes related to TGF<i>β</i> (Transforming growth factor beta) signaling were extremely unusual and possessed soldier-like phenotypes. These individuals also displayed aggressive behaviors similar to natural soldiers when confronted with <i>Formica</i> ants as hypothetical enemies. These presoldiers never molted into the next instar, presumably due to the decreased expression levels of the molting hormone (20-hydroxyecdysone; 20E) signaling genes. These results suggest that TGF<i>β</i> signaling was acquired for the novel function of regulating between JH and 20E signaling during soldier differentiation in termites.</p></div

RNAi effects and expression patterns of <i>ZnSox11</i> and <i>Znev_01548</i>.

<p>(A) Phenotypes of <i>GFP</i>, <i>ZnSox11</i> and <i>Znev_01548</i> dsRNA injected presoldiers. Each dsRNA was injected into the side of the thorax 24 hours after the JH analog (JHA) application. The emerged presoldiers were photographed 7 days after the molt. Approximately half of the <i>GFP</i> dsRNA injected presoldiers (14 out of 26, 53.8%) molted into soldiers with normal phenotypes (right). Scale bar indicates 5 mm. (B) Aggression levels of <i>GFP</i>, <i>ZnSox11</i> and <i>Znev_01548</i> dsRNA injected presoldiers (n = 6–9). The levels of natural soldiers are also shown on the right (n = 9). Numbers of individuals examined are shown in parentheses. The boxes and whiskers mean median, quartiles and range. Different letters over the bars indicate significant differences in each category (Kruskal-Wallis test: P = 6.35E-04, Steel-Dwass test: P < 0.05). (C) Expression patterns of <i>ZnSox11</i> and <i>Znev_01548</i> in the head provided by the qPCR analysis during each molting process in an incipient colony. Relative expression levels (mean ± S.E., biological triplicates) were calibrated by the expression levels in workers (GP0) as 1.0. The statistical results of three-way ANOVA are described in each box (**P < 0.01). The data is consistent with the use of parametric statistics by the Browne-Forsythe test (<i>ZnSox11</i>: P = 6.37E-01 (worker), 5.08E-02 (presoldier), 7.75E-01 (soldier); <i>Znev_01548</i>: P = 6.77E-01 (worker), 7.45E-01 (presoldier), 9.08E-01 (soldier)) prior to the use of the ANOVAs. (D) Expression patterns of <i>ZnSox11</i> and <i>Znev_01548</i> under the <i>ZnMet</i> RNAi treatment. Gray and red lines indicate the results under the <i>GFP</i> and <i>ZnMet</i> RNAi treatments, respectively. Relative expression levels (mean ± S.E., biological triplicates) were calibrated by the expression level of intact worker (day 0) as 1.0. The statistical results of two-way ANOVA are described in each box (*P < 0.05, **P < 0.01). The data is consistent with the use of parametric statistics by the Browne-Forsythe test (<i>ZnMet</i>: P = 7.91E-01 (<i>GFP</i>), 5.90E-01 (<i>ZnMet</i> RNAi); <i>ZnSox11</i>: P = 4.99E-01 (<i>GFP</i>), 8.43E-01(<i>ZnMet</i> RNAi); <i>Znev_01548</i>: P = 6.46E-01 (<i>GFP</i>), 6.25E-01 (<i>ZnMet</i> RNAi)) prior to the use of the ANOVAs.</p

Sampling points for RNA-seq analysis and gene expression profiles during each molting process.

<p>(A) Caste differentiation observed in an incipient colony of <i>Zootermopsis nevadensis</i>. The oldest 3rd instar larva (No. 1 larva) molts into a presoldier, whereas the next-oldest 3rd instar larva (No. 2 larva) molts into the next instar (4th instar larva). Gut-purged individuals are always observed 3 days before the molts. Individuals were sampled at the following 5 developmental stages during each molt; pre-gut-purging (pGP), 0 days after gut-purging (GP0), 3 days after gut-purging (GP3), 0 days after the molt (M0) and 3 days after the molt (M3). (B) MDS plots of 14,204 genes detected by the RNA-seq data from the head during 3 molts in <i>Zootermopsis nevadensis</i>. (C) Numbers of caste-specific highly expressed genes among 3 timings in each molt (pGP, GP0, GP3). (D) Clusters of genes with highly expression levels in the head before the presoldier molt. Cluster 1 was composed of 54 genes showing no clear expression patterns with the molting events. Cluster 2 was composed of 26 genes with highly expression levels after the molt (M0) in each molting event. Cluster 3 and 4 were composed of 32 and 19 genes with highly expression levels before the molt (GP0 and GP3, respectively). The solid black line in each panel indicates the average expression pattern. The similarity was calculated based on the Jensen-Shannon distances using the RPKM (Reads Per Kilo base of exon model per Million mapped reads) values.</p

A schematic model on the role of TGF<i>β</i> signaling for soldier differentiation.

<p>When JH titer is increased in larvae, TGF<i>β</i> signaling may be involved in the formation of presoldier-specific traits (e.g. soft cuticle, low aggression) and the regulation of soldier molt via 20E signaling.</p