ABHD5 regulates midgut-specific lipid homeostasis in Bombyx mori.

Lipids are an important energy source and are utilized as substrates for various physiological processes in insects. Comparative gene identification 58 (CGI-58), also known as α/ß hydrolase domain-containing 5 (ABHD5), is a highly conserved and multifunctional gene involved in regulating lipid metabolism and cellular energy balance in many organisms. However, the biological functions of ABHD5 in insects are poorly understood. In the current study, we describe the identification and characterization of the ABHD5 gene in the lepidopteran model insect, Bombyx mori. The tissue expression profile investigated using quantitative reverse transcription polymerase chain reaction (RT-qPCR) reveals that BmABHD5 is widely expressed in all tissues, with particularly high levels found in the midgut and testis. A binary transgenic CRISPR/Cas9 system was employed to conduct a functional analysis of BmABHD5, with the mutation of BmABHD5 leading to the dysregulation of lipid metabolism and excessive lipid accumulation in the larval midgut. Histological and physiological analysis further reveals a significant accumulation of lipid droplets in the midgut of mutant larvae. RNA-seq and RT-qPCR analysis showed that genes related to metabolic pathways were significantly affected by the absence of BmABHD5. Altogether, our data prove that BmABHD5 plays an important role in regulating tissue-specific lipid metabolism in the silkworm midgut.

GASZ is indispensable for gametogenesis in the silkworm, Bombyx mori.

The prominent role of the P-element induced wimpy testis (PIWI)-interacting RNA (piRNA) pathway in animals is to silence transposable elements and maintain genome stability, ensuring proper gametogenesis in gonads. GASZ (Germ cell protein with Ankyrin repeats, Sterile alpha motif, and leucine Zipper) is an evolutionarily conserved protein located on the outer mitochondrial membrane of germ cells and plays vital roles in the piRNA pathway and spermatogenesis in mammals. In the model insect Drosophila melanogaster, GASZ is essential for piRNA biogenesis and oogenesis, whereas its biological functions in non-drosophilid insects are still unknown. Here, we describe a comprehensive investigation of GASZ functions in the silkworm, Bombyx mori, a lepidopteran model insect, by using a binary transgenic CRISPR/Cas9 system. The BmGASZ mutation did not affect growth and development, but led to sterility in both males and females. Eupyrene sperm bundles of mutant males exhibited developmental defects, while the apyrene sperm bundles were normal, which were further confirmed through double copulation experiments with sex-lethal mutants, which males possess functional eupyrene sperm and abnormal apyrene sperm. In female mutant moths, ovarioles were severely degenerated and the eggs in ovarioles were deformed compared with that of wild type (WT). Further RNA-seq and RT-qPCR analysis revealed that amounts of piRNAs and transposon expression were dysregulated in gonads of mutants. In summary, this study has demonstrated vital roles of BmGASZ in gametogenesis through regulating the piRNA pathway in B. mori.

Characterization of the Complete Mitochondrial Genome of Eight Diurnal Hawkmoths (Lepidoptera: Sphingidae): New Insights into the Origin and Evolution of Diurnalism in Sphingids.

In this study, the mitochondrial genomes of 22 species from three subfamilies in the Sphingidae were sequenced, assembled, and annotated. Eight diurnal hawkmoths were included, of which six were newly sequenced (Hemaris radians, Macroglossum bombylans, M. fritzei, M. pyrrhosticta, Neogurelca himachala, and Sataspes xylocoparis) and two were previously published (Cephonodes hylas and Macroglossum stellatarum). The mitochondrial genomes of these eight diurnal hawkmoths were comparatively analyzed in terms of sequence length, nucleotide composition, relative synonymous codon usage, non-synonymous/synonymous substitution ratio, gene spacing, and repeat sequences. The mitogenomes of the eight species, ranging in length from 15,201 to 15,461 bp, encode the complete set of 37 genes usually found in animal mitogenomes. The base composition of the mitochondrial genomes showed A+T bias. The most commonly used codons were UUA (Leu), AUU (Ile), UUU (Phe), AUA (Met), and AAU (Asn), whereas GCG (Ala) and CCG (Pro) were rarely used. A phylogenetic tree of Sphingidae was constructed based on both maximum likelihood and Bayesian methods. We verified the monophyly of the four current subfamilies of Sphingidae, all of which had high support. In addition, we performed divergence time estimation and ancestral character reconstruction analyses. Diurnal behavior in hawkmoths originated 29.19 million years ago (Mya). It may have been influenced by the combination of herbaceous flourishing, which occurred 26-28 Mya, the uplift of the Tibetan Plateau, and the large-scale evolution of bats in the Oligocene to Pre-Miocene. Moreover, diurnalism in hawkmoths had multiple independent origins in Sphingidae.

Novel gene rearrangement pattern in mitochondrial genome of Ooencyrtusplautus Huang & Noyes, 1994: new gene order in Encyrtidae (Hymenoptera, Chalcidoidea).

Studies of mitochondrial genomes have a wide range of applications in phylogeny, population genetics, and evolutionary biology. In this study, we sequenced and analyzed the mitochondrial genome of Ooencyrtusplautus Huang & Noyes, 1994 (Hymenoptera, Encyrtidae). The nearly complete mitogenome of O.plautus was 15,730 bp in size, including 13 PCGs (protein-coding genes), 22 tRNAs, 2 rRNAs, and a nearly complete control region. The nucleotide composition was significantly biased toward adenine and thymine, with an A + T content of 84.6%. We used the reference sequence of Chouioiacunea and calculated the Ka/Ks ratio for each set of PCGs. The highest value of the Ka/Ks ratio within 13 PCGs was found in nad2 with 1.1, suggesting that they were subjected to positive selection. This phenomenon was first discovered in Encyrtidae. Compared with other encyrtid mitogenomes, a translocation of trnW was found in O.plautus, which was the first of its kind to be reported in Encyrtidae. Comparing with ancestral arrangement pattern, wasps reflect extensive gene rearrangements. Although these insects have a high frequency of gene rearrangement, species from the same family and genus tend to have similar gene sequences. As the number of sequenced mitochondrial genomes in Chalcidoidea increases, we summarize some of the rules of gene rearrangement in Chalcidoidea, that is four gene clusters with frequent gene rearrangements. Ten mitogenomes were included to reconstruct the phylogenetic trees of Encyrtidae based on both 13 PCGs (nucleotides of protein coding genes) and AA matrix (amino acids of protein coding genes) using the maximum likelihood and Bayesian inference methods. The phylogenetic tree reconstructed by Bayesian inference based on AA data set showed that Aenasiusarizonensis and Metaphycuseriococci formed a clade representing Tetracneminae. The remaining six species formed a monophyletic clade representing Encyrtinae. In Encyrtinae, Encyrtus forms a monophyletic clade as a sister group to the clade formed by O.plautus and Diaphorencyrtusaligarhensis. Encyrtussasakii and Encyrtusrhodooccisiae were most closely related species in this monophyletic clade. In addition, gene rearrangements can provide a valuable information for molecular phylogenetic reconstruction. These results enhance our understanding of phylogenetic relationships among Encyrtidae.

Complete mitochondrial genome of a parasitoid, Trichogramma chilonis (Hymenoptera: Chalcidoidea: Trichogrammatidae) and phylogenetic analysis.

Trichogramma chilonis is a kind of ovoid parasitic wasp, which has important application value in the biological control of pests. In this study, we sequenced and analyzed the complete mitogenome T. chilonis to compare mitogenomic structures and reconstruct phylogenetic relationships. The complete mitogenome sequence of T. chilonis is circular, 16,176 bp in size and encodes 13 protein-coding genes (PCGs), 2 ribosomal RNA genes (rRNA), 22 transfer RNA genes (tRNA), and a control region (CR). Nucleotide composition is highly biased toward A + T nucleotides (85.2%). All 13 protein-coding genes (PCGs) initiate with the standard start codon of ATN and terminate with the typical stop codon TAA/TAG. Phylogenetic analyses were performed using amino acids of 13 PCGs showed that T. chilonis is closely related to Trichogramma ostriniae.