Identification and immune analysis of antimicrobial peptides from the cigarette beetle (Lasioderma serricorne).

Antimicrobial peptides (AMPs) in insects are endogenous peptides that are effector components of the innate defense system of the insect. AMPs may serve as antimicrobial agents because of their small molecular weight and broad-spectrum antimicrobial activity. In this study, we performed transcriptome analysis of cigarette beetle (Lasioderma serricorne) larvae, parasitized by the ectoparasitic wasp, Anisopteromalus calandrae. Several AMP genes were significantly upregulated following A. calandrae parasitism, postulating the hypothesis that the parasitization enhanced the host's resistance against pathogenic microorganisms through the regulation of host AMP genes. Specifically, 3 AMP genes (LsDef1, LsDef2, and LsCole) were significantly upregulated and we studied their immune function in L. serricorne. Immune challenge and functional analysis showed that LsCole was responsible for the immune response against Gram-negative and Gram-positive bacteria, while LsDef1 and LsDef2 were involved in insect defense against Gram-positive bacteria. Purified recombinant LsCole exhibited antimicrobial activities against the Gram-negative bacterium Escherichia coli and the Gram-positive bacterium Staphylococcus aureus. LsDef2 showed an antibacterial effect against S. aureus. LsCole and LsDef2 exhibited antibiofilm activity against S. aureus. The 2 AMPs disrupted cell membranes and caused leakage of S. aureus cell contents. The results indicated that the 3 AMPs in L. serricorne are involved in the innate immunity of this pest insect. These AMPs may have potential as antimicrobial agents for bacterial infection chemotherapy. Hence, data are discussed in relation to new control strategies with greater biosafety against pest insects with use of microbial biocontrol agents in combination with RNA interference against the insect's defensive AMP genes.

Immune function analysis of LsSd, a transcription factor of the Hippo signaling pathway, in the cigarette beetle Lasioderma serricorne.

The Scalloped (Sd) is a transcription factor that regulates organ size control in the Hippo-signaling pathway. Recent studies have showed that Hippo signaling also functions in the innate immune response. Although the Sd gene has been reported in many insects, their immune functions remain unexplored. In this study, the LsSd gene of Lasioderma serricorne, with a complete open reading frame that encodes a protein composed of 402 amino acids was identified. LsSd was predominantly expressed in early pupae. Tissue-specific analyses revealed that the highest concentrations of LsSd were detected in the midgut and brain. At 1-24 h after Escherichia coli infection, LsSd expression increased substantially. However, LsSd expression was downregulated 3-12 h after Staphylococcus aureus infection. RNA interference-mediated silencing of the LsSd transcript resulted in deformed, considerably smaller, and degenerated wings. Meanwhile, LsCycE, LsDiap1, and LsVg, which are involved in cell proliferation and wing development, were drastically reduced when LsSd was depleted. In a survival assay, the LsSd knockdown considerably decreased the susceptibility to S. aureus, a gram-positive bacterium. In addition, knockdown of LsSd remarkably downregulated the transcription of LsCact in response to S. aureus stimulation, while upregulating the expression of five immune-related genes. Our results provide conclusive proof for the important roles of LsSd in the immune response of L. serricorne.

Mitogenomic phylogeny of Typhlocybinae (Hemiptera: Cicadellidae) reveals homoplasy in tribal diagnostic morphological traits.

The subfamily Typhlocybinae is a ubiquitous, highly diverse group of mostly tiny, delicate leafhoppers. The tribal classification has long been controversial and phylogenetic methods have only recently begun to test the phylogenetic status and relationships of tribes. To shed light on the evolution of Typhlocybinae, we performed phylogenetic analyses based on 28 newly sequenced and 19 previously sequenced mitochondrial genomes representing all currently recognized tribes. The results support the monophyly of the subfamily and its sister-group relationship to Mileewinae. The tribe Zyginellini is polyphyletic with some included genera derived independently within Typhlocybini. Ancestral character state reconstruction suggests that some morphological characters traditionally considered important for diagnosing tribes (presence/absence of ocelli, development of hind wing submarginal vein) are homoplastic. Divergence time estimates indicate that the subfamily arose during the Middle Cretaceous and that the extant tribes arose during the Late Cretaceous. Phylogenetic results support establishment of a new genus, Subtilissimia Yan & Yang gen. nov., with two new species, Subtilissimia fulva Yan & Yang sp. nov. and Subtilissimia pellicula Yan & Yang sp. nov.; but indicate that two previously recognized species of Farynala distinguished only by the direction of curvature of the processes of the aedeagus are synonyms, that is, Farynala dextra Yan & Yang, 2017 equals Farynala sinistra Yan & Yang, 2017 syn. nov. A key to tribes of Typhlocybinae is provided.

Comparison of mitogenomes of three Petalocephala species (Hemiptera: Cicadellidae: Ledrinae) and their phylogenetic analysis.

Ledrinae is a unique group of leafhoppers with a distinct appearance. Petalocephala is the largest Ledrinae genus that is difficult to identify except by dissecting the male genitals. To date, research on Ledrinae is relatively less compared with other leafhoppers. Therefore, to better understand this group, we sequenced and analyzed three complete Petalocephala mitochondrial genomes. We comparatively analyzed these general Petalocephala genomic features (including size, AT content, AT/GC skew, 13 protein-coding gene nucleotide compositions, etc.), and predicted 22 transfer RNA secondary structures. We obtained highly consistent phylogenetic results within Cicadellidae based on mitogenomic data using the maximum likelihood and Bayesian methods. Our results showed that all subfamilies were monophyletic and had a high node support rate, and there was a sister group relationship between Ledrinae and all other leafhopper groups. Furthermore, treehoppers were found to originate from leafhoppers and showed sister group relationships with Megophthalminae. Within Ledrinae, all phylogenetic trees supporting phylogenetic relationships were as follows: ([P. dicondylica + P. gongshanensis] + [Tituria pyramidata + [Ledra auditura + P. gongshanensis]]) Based on the complete mitogenome phylogenetic analysis and the comparison of morphological characteristics, we propose that Petalocephala is not monophyletic.

The Phylogenetic Implications of the Mitochondrial Genomes of Macropsis notata and Oncopsis nigrofasciata.

Macropsinae are forest pests that feed on woody plants. They can damage the growth of trees and crops, and some species can also spread plant pathogens. Due to their widespread effects, these leafhoppers are of great economic significance, which is why there is a need to study their genomes. To fill the gap in the mitochondrial genomic data of the subfamily Macropsinae, we sequenced the complete mitochondrial genomes of Macropsis notata and Oncopsis nigrofasciata (which were 16,323 and 15,927 bp long, respectively). These two species are representative species of the leafhoppers group (Cicadellidae); the mitochondrial genomes of these species range from a length of 15,131 bp (Trocnadella arisana) to 16,811 bp (Parocerus laurifoliae). Both mitogenomes contained 37 typical insect mitochondrial genes and a control region; there were no long non-coding sequences. The genes within the mitogenome were very compact. The mitogenomes from both species contained two kinds of parallel repeat units in the control region. The whole mitogenomes of Macropsinae showed a heavy AT nucleotide bias (M. notata 76.8% and O. nigrofasciata 79.0%), a positive AT Skew (0.15 and 0.12), and a negative GC Skew (-0.14 and -0.08). Upon comparative ML and BI analysis, some clade relationships were consistent among the six trees. Most subfamilies were reconstructed into monophyletic groups with strong support in all analyses, with the exception of Evacanthinae and Cicadellinae. Unlike the results of previous research, it was shown that although all Deltocephalinae species are grouped into one clade, they were not the sister group to all other leafhoppers. Further, Cicadellinae and Evacanthinae were occasionally reconstructed as a polyphyletic and a paraphyletic group, respectively, possibly due to the limited numbers of samples and sequences. This mitogenome information for M. notata and O. nigrofasciata could facilitate future studies on the mitogenomic diversity and evolution of the related Membracoidea, and eventually help to control their effects on plants for the betterment of society at large.