The health and diversity of plant-feeding insects are strictly linked to their host plants and mutualistic symbionts. However, the study of bacterial symbionts within different insects on the same plant lineage is very limited. This study aimed to investigate the bacterial diversity in insect samples that exclusively feed on Bambusa, representing three insect orders, Hemiptera, Lepidoptera, and Blattodea, each exhibiting distinct dietary preferences. The bacterial community was predominantly composed of Proteobacteria, Spirochaetota, Cyanobacteria, Firmicutes, and Bacteroidota. The study found significant variations in symbiotic organisms among three insect orders: hemipterans had Buchnera, lepidopterans had Acinetobacter, and blattodean had Treponema. Furthermore, the dietary preferences of these insects played a pivotal role in shaping the symbiotic relationship of insects. Proteobacteria are prevalent in sap feeders, Spirochaetota dominate in stem feeders, and Cyanobacteria are abundant in leaf feeders. Seasonal influences also affect bacterial symbionts in P. bambucicola, with Serratia present exclusively in winter. We also observed that the bacterial composition varies across all samples, but their core functions appear to be consistent. This highlights the complex relationship between host phylogeny and diet, with phylogeny being the primary driver, shaping adaptations to specialized diets.

The assembly of insect mitochondrial genomes remains challenging in certain taxa due to extreme AT biases and long repetitive regions. The family Diaspididae (Hemiptera: Coccomorpha) exemplifies this difficulty; despite more than 2,700 described species worldwide, no complete mitogenome has been reported prior to this study. Here, the complete mitogenome is presented of Aulacaspis yasumatsui , assembled using a hybrid approach combining second- and third-generation sequencing platforms. The A. yasumatsui mitogenome is the longest identified to date among scale insects, spanning 21,273 bp. It exhibits several extraordinary features, most notably the highest AT content (92%) yet recorded in Insecta, extensive gene rearrangements, the absence of half of the tRNA genes, and the first identified ultra-long repeat region (> 5,000 bp) in Coccomorpha. The leg degeneration and loss observed in Diaspididae evolution may have contributed to the emergence of such an extreme mitogenome structure. Comparative analysis of sequencing strategies revealed substantial coverage bias in Illumina data, which was effectively mitigated by Oxford Nanopore long reads. We propose a standardized pipeline tailored for efficient and accurate assembly of highly AT-rich and structurally complex mitogenomes. This study provides both methodological insights and a valuable genomic resource for future evolutionary and comparative studies on Diaspididae and other taxa with challenging mitogenomes.

The CFEM (Common in Fungal Extracellular Membrane) domain defines a family of cysteine-rich proteins unique to fungi, playing pivotal roles in host–pathogen interactions. However, the repertoire and functions of CFEM proteins in the broad-host-range necrotrophic pathogen Sclerotinia sclerotiorum remain largely unexplored. Through genome-wide bioinformatic analysis, we identified 13 CFEM-containing proteins (SsCFEM1–13) in S. sclerotiorum. Characterization revealed substantial diversity in their physicochemical properties, domain architecture, and predicted subcellular localization. Ten proteins possess a secretion signal, with six predicted to be GPI-anchored and three classified as high-confidence effectors. Members lacking transmembrane domains were predicted to adopt the conserved CFEM “helical-basket” fold. Phylogenetic analysis grouped SsCFEMs into two distinct clades and indicated a complex evolutionary history involving both conserved ancestry and lineage-specific expansion. Transcriptomic profiling showed that most genes were upregulated during early infection of various host plants, with SsCFEM8 exhibiting particularly strong and consistent induction. Crucially, transient expression assays in Nicotiana benthamiana revealed that several SsCFEM proteins, notably SsCFEM4 and SsCFEM9, function as cell death suppressors, validating their predicted effector roles and identifying key virulence candidates. This study provides the first comprehensive catalog and functional prediction of the CFEM protein family in S. sclerotiorum, establishing a foundation for future mechanistic studies on their roles in the pathogenesis of this devastating fungal pathogen.