Synthesis of 2-benzyl N-substituted anilines via imine condensation-isoaromatization of (E)-2-arylidene-3-cyclohexenones and primary amines.

A catalyst- and additive-free synthesis of 2-benzyl N-substituted anilines from (E)-2-arylidene-3-cyclohexenones and primary amines has been reported. The reaction proceeds smoothly through a sequential imine condensation-isoaromatization pathway, affording a series of synthetically useful aniline derivatives in acceptable to high yields. Mild reaction conditions, no requirement of metal catalysts, operational simplicity and the potential for scale-up production are some of the highlighted advantages of this transformation.

Transcriptomic data recover a new superfamily-level phylogeny of Cucujiformia (Coleoptera, Polyphaga).

Cucujiformia, the largest taxon in the order Coleoptera, exhibits extraordinary morphological, ecological, and behavioral diversity. This infraorder is currently divided into seven superfamilies, but considerably incongruent relationships among superfamilies have been reported by recent phylogenomic studies. Here, we combined the 21 newly sequenced transcriptomes representing six superfamilies with nine previously published cucujiform genomes/transcriptomes to elucidate the phylogeny and evolution of Cucujiformia. The monophyly of each of five superfamilies were consistently supported by all phylogenetic analyses based on the twelve datasets (matrix occupancy, amino acid and nucleotide data) and the two analytical methods (maximum likelihood method and Bayesian inference). Both the amino acid datasets and the RY recoded nucleotide datasets recovered the monophyly of Cucujoidea. Topology test results statistically supported the following robust superfamily-level phylogeny in Cucujiformia: (Coccinelloidea, (Cleroidea, (Tenebrionoidea, (Cucujoidea, (Chrysomeloidea, Curculionoidea))))). Our divergence time analyses recovered a Permian origin of Cucujiformia and a Jurassic-Cretaceous origin of most superfamilies. The diversification of phytophagous beetles that occurred in the Cretaceous can be attributed to its co-evolution with angiosperms, supporting the hypothesis of a Cretaceous Terrestrial Revolution.

Confusoside from Anneslea fragrans Alleviates Acetaminophen-Induced Liver Injury in HepG2 via PI3K-CASP3 Signaling Pathway.

Confusoside (CF), a major chemical compound in the leaves of Anneslea fragrans Wall., is a dihydrochalcone glycoside with excellent antioxidant and anti-inflammatory effects. However, the hepatoprotective effect of CF has not been described. This study aimed to explore the hepatoprotective effect of CF against acetaminophen (APAP)-induced hepatic injury in HepG2 cells. First, the potential hepatoprotective effect mechanisms of CF were predicted by network pharmacology and were thought to involve reducing inflammation and inhibiting apoptosis. Target proteins (phosphatidylinositol3-kinase (PI3K) and caspase-3 (CASP3)) were found via molecular docking analysis. To verify the predicted results, an analysis of biological indicators was performed using commercial kits and Western blotting. The results showed that CF significantly decreased the levels of liver injury biomarkers (ALT, AST, and LDH), strongly inhibited the production of inflammatory cytokines (IL-1ß, IL-6, and TNF-α) and the NO level via inhibiting the activation of the NF-κB signaling pathway, and markedly regulated the expression levels of Bcl2, Bax, and cleaved-CASP3/9 proteins by activating the PI3K-CASP3 apoptosis pathway. The results demonstrated that CF has a therapeutic effect on APAP-induced liver injury by inhibiting intracellular inflammation and cell apoptosis, indicating that CF may be used as a potential reagent for the prevention and treatment of APAP-induced liver injury.

Genome-wide gene expression profiles of the pea aphid (Acyrthosiphon pisum) under cold temperatures provide insights into body color variation.

Cold temperatures are one of the factors influencing color polymorphisms in Acyrthosiphon pisum, resulting in a change from a red to greenish color. Here we characterized gene expression profiles of A. pisum under different low temperatures (1°C, 4°C, 8°C, and 14°C) and durations (3, 6, 12, and 24 h). The number of differentially expressed genes (DEGs) increased as temperatures decreased and time increased, but only a small number of significant DEGs were identified. Genes involved in pigment metabolism were downregulated. An interaction network analysis for 506 common DEGs in comparisons among aphids exposed to 1°C for four durations indicated that a cytochrome P450 gene (CYP, LOC112935894) significantly downregulated may interact with a carotenoid metabolism gene (LOC100574964), similar to other genes encoding CYP, lycopene dehydrogenase and fatty acid synthase. We proposed that the body color shift in A. pisum responding to low temperatures may be regulated by CYPs.

Mitochondrial genomes of three Bostrichiformia species and phylogenetic analysis of Polyphaga (Insecta, Coleoptera).

Here we determined mitogenomes of three Bostrichiformia species. These data were combined with 51 previously sequenced Polyphaga mitogenomes to explore the higher-level relationships within Polyphaga by using four different mitogenomic datasets and three tree inference approaches. Among Polyphaga mitogenomes we observed heterogeneity in nucleotide composition and evolutionary rates, which may have affected phylogenetic inferences across the different mitogenomic datasets. Elateriformia, Cucujiformia, and Scarabaeiformia were each inferred to be monophyletic by all analyses, as was Bostrichiformia by most analyses based on two datasets with low heterogeneity. The large series Staphyliniformia was never recovered as monophyletic in our analyses. The Bayesian tree using a degenerated nucleotide dataset (P123_Degen) and a site-heterogeneous mixture model in PhyloBayes was supported as the best Polyphaga phylogeny: (Scirtiformia, (Elateriformia, ((Bostrichiformia, Cucujiformia), (Scarabaeiformia + Staphyliniformia)))). For Cucujiformia, the largest series, we inferred a superfamily-level phylogeny: ((Cleroidea, Coccinelloidea), (Tenebrionoidea, (Cucujoidea + Curculionoidea + Chrysomeloidea))).

Comparative transcriptomic analysis of fireflies (Coleoptera: Lampyridae) to explore the molecular adaptations to fresh water.

Aquatic insects are well adapted to freshwater environments, but the molecular basis of these adaptations remains largely unknown. Most firefly species (Coleoptera: Lampyridae) are terrestrial, but the larvae of several species are aquatic. Here, larval and adult transcriptomes from Aquatica leii (freshwater) and Lychnuris praetexta (terrestrial) were generated to test whether the genes associated with metabolic efficiency and morphology have undergone adaptive evolution to fresh water. The aquatic fireflies had a significantly lower ratio of nonsynonymous to synonymous substitutions than the terrestrial insects, indicating a genomewide evolutionary constraint in the aquatic fireflies. We identified 341 fast-evolving genes and 116 positively selected genes in the aquatic fireflies. Of these, 76 genes exhibiting both fast evolution and positive selection were primarily involved in ATP production, energy metabolism and the hypoxia response. We identified 7,271 differentially expressed genes (DEGs) in A. leii (adults versus larvae) and 8,309 DEGs in L. praetexta (adults versus larvae). DEGs specific to the aquatic firefly (n = 1,445) were screened via interspecific comparisons (A. leii versus L. praetexta) and were significantly enriched for genes involved in metabolic efficiency (e.g., ATP production, hypoxia, and immune responses) and certain aspects of morphology (e.g., cuticle chitin, tracheal and compound eye morphology). These results indicate that sequence and expression-level changes in genes associated with both metabolic efficiency and morphological attributes related to the freshwater lifestyle contributed to freshwater adaptation in fireflies. This study provides new insights into the molecular mechanisms of aquatic adaptation in insects.

Mitochondrial phylogeny, divergence history and high-altitude adaptation of grassland caterpillars (Lepidoptera: Lymantriinae: Gynaephora) inhabiting the Tibetan Plateau.

Grassland caterpillars (Lepidoptera: Lymantriinae: Gynaephora) are the most important pests in alpine meadows of the Tibetan Plateau (TP) and have well adapted to high-altitude environments. To further understand the evolutionary history and their adaptation to the TP, we newly determined seven complete TP Gynaephora mitogenomes. Compared to single genes, whole mitogenomes provided the best phylogenetic signals and obtained robust results, supporting the monophyly of the TP Gynaephora species and a phylogeny of Arctiinae + (Aganainae + Lymantriinae). Incongruent phylogenetic signals were found among single mitochondrial genes, none of which recovered the same phylogeny as the whole mitogenome. We identified six best-performing single genes using Shimodaira-Hasegawa tests and found that the combinations of rrnS and either cox1 or cox3 generated the same phylogeny as the whole mitogenome, indicating the phylogenetic potential of these three genes for future evolutionary studies of Gynaephora. The TP Gynaephora species were estimated to radiate on the TP during the Pliocene and Quaternary, supporting an association of the diversification and speciation of the TP Gynaephora species with the TP uplifts and associated climate changes during this time. Selection analyses revealed accelerated evolutionary rates of the mitochondrial protein-coding genes in the TP Gynaephora species, suggesting that they accumulated more nonsynonymous substitutions that may benefit their adaptation to high altitudes. Furthermore, signals of positive selection were detected in nad5 of two Gynaephora species with the highest altitude-distributions, indicating that this gene may contribute to Gynaephora's adaptation to divergent altitudes. This study adds to the understanding of the TP Gynaephora evolutionary relationships and suggests a link between mitogenome evolution and ecological adaptation to high-altitude environments in grassland caterpillars.

[Progress in innate immunity-related genes in insects].

Insects have evolved a strong innate immune system to defense pathogens and adverse conditions. Insect innate immune system comprises of humoral immunity and cellular immunity. Humoral immunity mainly includes three signaling pathways, i.e., Toll, IMD and JAK/STAT, by which signal transduction and immune pathways regulate expression of immune-related genes and activate antimicrobial peptides and other effectors. Cellular immunity contains phagocytosis, encapsulation and nodulation of pathogens, which is mediated by hemolymph cells. In recent years, with the rapid development of insect genomics, a large number of immune-related genes have been characterized from insect genome data using bioinformatic methods. Studies on these genes have significantly deepened the understanding of molecular mechanisms of insect innate immune system. Insect immune-related genes can be divided into seven categories by gene functions: recognition, signaling transduction, modulator, effectors, melanization, RNA interference and other genes. The humoral immunity and cellular immunity are regulated by the interactions among these immune-related genes. In this review, we summarize the classification, function and evolution of insect immune-related genes, and propose future research directions of insect innate immunity, which will be helpful for understanding molecular mechanisms of insect innate immune system and developing new strategies for controlling pest insects.

High-level phylogeny of the Coleoptera inferred with mitochondrial genome sequences.

The Coleoptera (beetles) exhibits tremendous morphological, ecological, and behavioral diversity. To better understand the phylogenetics and evolution of beetles, we sequenced three complete mitogenomes from two families (Cleridae and Meloidae), which share conserved mitogenomic features with other completely sequenced beetles. We assessed the influence of six datasets and three inference methods on topology and nodal support within the Coleoptera. We found that both Bayesian inference and maximum likelihood with homogeneous-site models were greatly affected by nucleotide compositional heterogeneity, while the heterogeneous-site mixture model in PhyloBayes could provide better phylogenetic signals for the Coleoptera. The amino acid dataset generated more reliable tree topology at the higher taxonomic levels (i.e. suborders and series), where the inclusion of rRNA genes and the third positions of protein-coding genes improved phylogenetic inference at the superfamily level, especially under a heterogeneous-site model. We recovered the suborder relationships as (Archostemata+Adephaga)+(Myxophaga+Polyphaga). The series relationships within Polyphaga were recovered as (Scirtiformia+(Elateriformia+((Bostrichiformia+Scarabaeiformia+Staphyliniformia)+Cucujiformia))). All superfamilies within Cucujiformia were recovered as monophyletic. We obtained a cucujiform phylogeny of (Cleroidea+(Coccinelloidea+((Lymexyloidea+Tenebrionoidea)+(Cucujoidea+(Chrysomeloidea+Curculionoidea))))). This study showed that although tree topologies were sensitive to data types and inference methods, mitogenomic data could provide useful information for resolving the Coleoptera phylogeny at various taxonomic levels by using suitable datasets and heterogeneous-site models.

Comparative mitogenomic analysis of the superfamily Pentatomoidea (Insecta: Hemiptera: Heteroptera) and phylogenetic implications.

BACKGROUND: Insect mitochondrial genomes (mitogenomes) are the most extensively used genetic marker for evolutionary and population genetics studies of insects. The Pentatomoidea superfamily is economically important and the largest superfamily within Pentatomomorpha with over 7,000 species. To better understand the diversity and evolution of pentatomoid species, we sequenced and annotated the mitogenomes of Eurydema gebleri and Rubiconia intermedia, and present the first comparative analysis of the 11 pentatomoid mitogenomes that have been sequenced to date. RESULTS: We obtained the complete mitogenome of Eurydema gebleri (16,005 bp) and a nearly complete mitogenome of Rubiconia intermedia (14,967 bp). Our results show that gene content, gene arrangement, base composition, codon usage, and mitochondrial transcription termination factor sequences are highly conserved in pentatomoid species, especially for species in the same family. Evolutionary rate analyses of protein-coding genes reveal that the highest and lowest rates are found in atp8 and cox1 and distinctive evolutionary patterns are significantly correlated with the G + C content of genes. We inferred the secondary structures for two rRNA genes for eleven pentatomoid species, and identify some conserved motifs of RNA structures in Pentatomidea. All tRNA genes in pentatomoid mitogenomes have a canonical cloverleaf secondary structure, except for two tRNAs (trnS1 and trnV) which appear to lack the dihydrouridine arm. Regions that are A + T-rich have several distinct characteristics (e.g. size variation and abundant tandem repeats), and have potential as species or population level molecular markers. Phylogenetic analyses based on mitogenomic data strongly support the monophyly of Pentatomoidea, and the estimated phylogenetic relationships are: (Urostylididae + (Plataspidae + (Pentatomidae + (Cydnidae + (Dinidoridae + Tessaratomidae))))). CONCLUSIONS: This comparative mitogenomic analysis sheds light on the architecture and evolution of mitogenomes in the superfamily Pentatomoidea. Mitogenomes can be effectively used to resolve phylogenetic relationships of pentatomomorphan insects at various taxonomic levels. Sequencing more mitogenomes at various taxonomic levels, particularly from closely related species, will improve the annotation accuracy of mitochondrial genes, as well as greatly enhance our understanding of mitogenomic evolution and phylogenetic relationships in pentatomoids.

A facile and mild synthesis of trisubstituted allylic sulfones from Morita-Baylis-Hillman carbonates.

An efficient and catalyst-free synthesis of trisubstituted allylic sulfones through an allylic sulfonylation reaction of Morita-Baylis-Hillman (MBH) carbonates with sodium sulfinates has been developed. Under the optimized reaction conditions, a series of trisubstituted allylic sulfones were rapidly prepared in good to excellent yields (71%-99%) with good to high selectivity (Z/E from 79:21 to >99:1). Compared with known synthetic methods, the current protocol features mild reaction temperature, high efficiency and easily available reagents.

Physio-mechanical properties of an active chitosan film incorporated with montmorillonite and natural antioxidants extracted from pomegranate rind.

An active film was prepared from chitosan incorporated with montmorillonite (MMT) and pomegranate rind powder extract (PRP). The effect of MMT (1 %, 3 %, and 5 % w/w chitosan) and PRP (1 %, 1.5 %, and 2 % w/v chitosan) on the physical, mechanical and antioxidant properties of the chitosan-based films was studied. Fourier transform infrared (FTIR) spectra revealed that good interactions occurred between functional groups of chitosan with MMT or with PRP. The results showed that the water vapor barrier property of the films was significantly improved by incorporation of MMT and PRP (p < 0.05). When compared to pure chitosan film, the WVP of M3P2 film (Chitosan/3 % MMT/2 % PRP) decreased by 25.2 %. Tensile strength of the films was affected by the addition of MMT and PRP. However, percent elongation at break was not significantly changed by addition of PRP. The film incorporated with 3 % MMT and 2 % PRP that contained the highest amount of total phenolic (15.2 mg GAE/g DW), was found to be the most active radical scavenger. These results suggest that chitosan films containing MMT and PRP can be used for development of active food packaging materials.

Comparative mitogenomic analysis provides evolutionary insights into Formica (Hymenoptera: Formicidae).

Formica is a large genus in the family Formicidae with high diversity in its distribution, morphology, and physiology. To better understand evolutionary characteristics of Formica, the complete mitochondrial genomes (mitogenomes) of two Formica species were determined and a comparative mitogenomic analysis for this genus was performed. The two newly sequenced Formica mitogenomes each included 37 typical mitochondrial genes and a large non-coding region (putative control region), as observed in other Formica mitogenomes. Base composition, gene order, codon usage, and tRNA secondary structure were well conserved among Formica species, whereas diversity in sequence size and structural characteristics was observed in control regions. We also observed several conserved motifs in the intergenic spacer regions. These conserved genomic features may be related to mitochondrial function and their highly conserved physiological constraints, while the diversity of the control regions may be associated with adaptive evolution among heterogenous habitats. A negative AT-skew value on the majority chain was presented in each of Formica mitogenomes, indicating a reversal of strand asymmetry in base composition. Strong codon usage bias was observed in Formica mitogenomes, which was predominantly determined by nucleotide composition. All 13 mitochondrial protein-coding genes of Formica species exhibited molecular signatures of purifying selection, as indicated by the ratio of non-synonymous substitutions to synonymous substitutions being less than 1 for each protein-coding gene. Phylogenetic analyses based on mitogenomic data obtained fairly consistent phylogenetic relationships, except for two Formica species that had unstable phylogenetic positions, indicating mitogenomic data are useful for constructing phylogenies of ants. Beyond characterizing two additional Formica mitogenomes, this study also provided some key evolutionary insights into Formica.

Mitochondrial phylogeography of grassland caterpillars (Lepidoptera: Lymantriinae: Gynaephora) endemic to the Qinghai-Tibetan plateau.

Grassland caterpillars (Lepidoptera: Lymantriinae: Gynaephora) are the most damaging pests to alpine meadows in the Qinghai-Tibetan Plateau (QTP). Here, we conducted extensive sampling from 39 geographic populations covering almost the entire distribution of the eight QTP Gynaephora (Hübner) species to investigate phylogeographic patterns and speciation based on two mitochondrial genes (COI and ND5). A total of 40 haplotypes were detected in the 39 populations, with >70% of all haplotypes not shared between populations. The monophyletic QTP Gynaephora migrated from non-QTP regions during the Pliocene, corresponding to the uplift of the QTP, suggesting a mode of transport into the QTP. Among the eight QTP Gynaephora species described by morphological characteristics, two species (G. alpherakii and G. menyuanensis) were recovered as monophyletic groups (Clades B and C), while the remaining six formed two monophyletic clades: Clade A (G. qinghaiensis, G. jiuzhiensis, and G. qumalaiensis) and Clade D (G. aureata, G. ruoergensis, and G. minora). These results suggested that the number of the QTP Gynaephora species may be overestimated and further studies based on both morphological and nuclear gene data are needed. Genetic differentiation and speciation of the QTP Gynaephora were likely driven by the QTP uplifts and associated climate fluctuations during the Pleistocene, indicated by divergence time estimation, suggesting that isolation and subsequent divergence was the dominant mode of speciation. The Sanjiangyuan region (i.e., Clade A, characterized by high genetic diversity) may have been a glacial refugium of the QTP Gynaephora, as supported by analyses of gene flow and biogeography. High levels of genetic diversity were found in QTP Gynaephora, without population expansion, which may explain the high-altitude adaptation and outbreaks of grassland caterpillars in alpine meadows of the QTP. This study provides the largest phylogeographic analysis of QTP Gynaephora and improves our understanding of the diversity and speciation of QTP insects.

Comparative mitogenomic and evolutionary analysis of Lycaenidae (Insecta: Lepidoptera): Potential association with high-altitude adaptation.

Harsh environments (e.g., hypoxia and cold temperatures) of the Qinghai-Tibetan Plateau have a substantial influence on adaptive evolution in various species. Some species in Lycaenidae, a large and widely distributed family of butterflies, are adapted to the Qinghai-Tibetan Plateau. Here, we sequenced four mitogenomes of two lycaenid species in the Qinghai-Tibetan Plateau and performed a detailed comparative mitogenomic analysis including nine other lycaenid mitogenomes (nine species) to explore the molecular basis of high-altitude adaptation. Based on mitogenomic data, Bayesian inference, and maximum likelihood methods, we recovered a lycaenid phylogeny of [Curetinae + (Aphnaeinae + (Lycaeninae + (Theclinae + Polyommatinae)))]. The gene content, gene arrangement, base composition, codon usage, and transfer RNA genes (sequence and structure) were highly conserved within Lycaenidae. TrnS1 not only lacked the dihydrouridine arm but also showed anticodon and copy number diversity. The ratios of non-synonymous substitutions to synonymous substitutions of 13 protein-coding genes (PCGs) were less than 1.0, indicating that all PCGs evolved under purifying selection. However, signals of positive selection were detected in cox1 in the two Qinghai-Tibetan Plateau lycaenid species, indicating that this gene may be associated with high-altitude adaptation. Three large non-coding regions, i.e., rrnS-trnM (control region), trnQ-nad2, and trnS2-nad1, were found in the mitogenomes of all lycaenid species. Conserved motifs in three non-coding regions (trnE-trnF, trnS1-trnE, and trnP-nad6) and long sequences in two non-coding regions (nad6-cob and cob-trnS2) were detected in the Qinghai-Tibetan Plateau lycaenid species, suggesting that these non-coding regions were involved in high-altitude adaptation. In addition to the characterization of Lycaenidae mitogenomes, this study highlights the importance of both PCGs and non-coding regions in high-altitude adaptation.

Comparative Mitogenomic Analyses of Darkling Beetles (Coleoptera: Tenebrionidae) Provide Evolutionary Insights into tRNA-like Sequences.

Tenebrionidae is widely recognized owing to its species diversity and economic importance. Here, we determined the mitochondrial genomes (mitogenomes) of three Tenebrionidae species (Melanesthes exilidentata, Anatolica potanini, and Myladina unguiculina) and performed a comparative mitogenomic analysis to characterize the evolutionary characteristics of the family. The tenebrionid mitogenomes were highly conserved with respect to genome size, gene arrangement, base composition, and codon usage. All protein-coding genes evolved under purifying selection. The largest non-coding region (i.e., control region) showed several unusual features, including several conserved repetitive fragments (e.g., A+T-rich regions, G+C-rich regions, Poly-T tracts, TATA repeat units, and longer repetitive fragments) and tRNA-like structures. These tRNA-like structures can bind to the appropriate anticodon to form a cloverleaf structure, although base-pairing is not complete. We summarized the quantity, types, and conservation of tRNA-like sequences and performed functional and evolutionary analyses of tRNA-like sequences with various anticodons. Phylogenetic analyses based on three mitogenomic datasets and two tree inference methods largely supported the monophyly of each of the three subfamilies (Stenochiinae, Pimeliinae, and Lagriinae), whereas both Tenebrioninae and Diaperinae were consistently recovered as polyphyletic. We obtained a tenebrionid mitogenomic phylogeny: (Lagriinae, (Pimeliinae, ((Tenebrioninae + Diaperinae), Stenochiinae))). Our results provide insights into the evolution and function of tRNA-like sequences in tenebrionid mitogenomes and contribute to our general understanding of the evolution of Tenebrionidae.

Differential gene expression patterns between the head and thorax of Gynaephora aureata are associated with high-altitude adaptation.

Grassland caterpillars (Lepidoptera: Erebidae: Gynaephora) are important pests in alpine meadows of the Qinghai-Tibetan Plateau (QTP). These pests have morphological, behavioral, and genetic adaptations for survival in high-altitude environments. However, mechanisms underlying high-altitude adaptation in QTP Gynaephora species remain largely unknown. Here, we performed a comparative analysis of the head and thorax transcriptomes of G. aureata to explore the genetic basis of high-altitude adaptation. We detected 8,736 significantly differentially expressed genes (sDEGs) between the head and thorax, including genes related to carbohydrate metabolism, lipid metabolism, epidermal proteins, and detoxification. These sDEGs were significantly enriched in 312 Gene Ontology terms and 16 KEGG pathways. We identified 73 pigment-associated genes, including 8 rhodopsin-associated genes, 19 ommochrome-associated genes, 1 pteridine-associated gene, 37 melanin-associated genes, and 12 heme-associated genes. These pigment-associated genes were related to the formation of the red head and black thorax of G. aureata. A key gene, yellow-h, in the melanin pathway was significantly upregulated in the thorax, suggesting that it is related to the formation of the black body and contributed to the adaptation of G. aureata to low temperatures and high ultraviolet radiation in the QTP. Another key gene, cardinal, in the ommochrome pathway was significantly upregulated in the head and may be related to red warning color formation. We also identified 107 olfactory-related genes in G. aureata, including genes encoding 29 odorant-binding proteins, 16 chemosensory proteins, 22 odorant receptor proteins, 14 ionotropic receptors, 12 gustatory receptors, 12 odorant degrading enzymes, and 2 sensory neuron membrane proteins. Diversification of olfactory-related genes may be associated with the feeding habits of G. aureata, including larvae dispersal and searching for plant resources available in the QTP. These results provide new insights into high-altitude adaptation of Gynaephora in the QTP and may contribute to the development of new control strategies for these pests.

Comparative mitogenomic analyses provide evolutionary insights into the retrolateral tibial apophysis clade (Araneae: Entelegynae).

The retrolateral tibial apophysis (RTA) clade is the largest spider lineage within Araneae. To better understand the diversity and evolution, we newly determined mitogenomes of ten RTA species from six families and performed a comparative mitogenomics analysis by combining them with 40 sequenced RTA mitogenomes available on GenBank. The ten mitogenomes encoded 37 typical mitochondrial genes and included a large non-coding region (putative control region). Nucleotide composition and codon usage were well conserved within the RTA clade, whereas diversity in sequence length and structural features was observed in control region. A reversal of strand asymmetry in nucleotide composition, i.e., negative AT-skews and positive GC-skews, was observed in each RTA species, likely resulting from mitochondrial gene rearrangements. All protein-coding genes were evolving under purifying selection, except for atp8 whose Ka/Ks was larger than 1, possibly due to positive selection or selection relaxation. Both mutation pressure and natural selection might contribute to codon usage bias of 13 protein-coding genes in the RTA lineage. Phylogenetic analyses based on mitogenomic data recovered a family-level phylogeny within the RTA; {[(Oval calamistrum clade, Dionycha), Marronoid clade], Sparassidae}. This study characterized RTA mitogenomes and provided some new insights into the phylogeny and evolution of the RTA clade.

Mitochondrial phylogenomics provides insights into the phylogeny and evolution of spiders (Arthropoda: Araneae).

Spiders are among the most varied terrestrial predators, with highly diverse morphology, ecology, and behavior. Morphological and molecular data have greatly contributed to advances in the phylogeny and evolutionary dynamics of spiders. Here, we performed comprehensive mitochondrial phylogenomics analysis on 78 mitochondrial genomes (mitogenomes) representing 29 families; of these, 23 species from eight families were newly generated. Mesothelae retained the same gene arrangement as the arthropod ancestor ( Limulus polyphemus), while Opisthothelae showed extensive rearrangement, with 12 rearrangement types in transfer RNAs (tRNAs) and control region. Most spider tRNAs were extremely truncated and lacked typical dihydrouridine or TΨC arms, showing high tRNA structural diversity; in particular, trnS1 exhibited anticodon diversity across the phylogeny. The evolutionary rates of mitochondrial genes were potentially associated with gene rearrangement or truncated tRNAs. Both mitogenomic sequences and rearrangements possessed phylogenetic characteristics, providing a robust backbone for spider phylogeny, as previously reported. The monophyly of suborder, infraorder, retrolateral tibial apophysis clade, and families (except for Pisauridae) was separately supported, and high-level relationships were resolved as (Mesothelae, (Mygalomorphae, (Entelegynae, (Synspermiata, Hypochilidae)))). The phylogenetic positions of several families were also resolved (e.g., Eresidae, Oecobiidae and Titanoecidae). Two reconstructions of ancestral web type obtained almost identical results, indicating that the common ancestor of spiders likely foraged using a silk-lined burrow. This study, the largest mitochondrial phylogenomics analysis of spiders to date, highlights the usefulness of mitogenomic data not only for providing efficient phylogenetic signals for spider phylogeny, but also for characterizing trait diversification in spider evolution.

A method for isolating highly purified and active mitochondria from insects.

So far, methods that yield the high purity and activity of the isolated mitochondria from insects have not been reported and determined. Here, we develop methods that combine differential centrifugation and discontinuous Nycodenz density gradient centrifugation to isolate highly purified mitochondria from the thorax muscle of insects, and the methods were widely validated across three orders (Coleoptera, Hymenoptera, and Blattaria) covering four insect species using Western blot and transmission electron microscopy (TEM) analysis. The results showed the removal of the residual contamination with nonmitochondrial components such as nucleus, sarcolemma, cytosol, and endoplasmic reticulum. Furthermore, TEM, mitochondria staining, fluorescence detection, and flow cytometry analyses were employed to assess membrane integrity and activity of the isolated mitochondria. The results showed no loss of mitochondria activity/integrity after isolation. In addition, temporal dynamics in activity of the isolated mitochondria under commonly used laboratory temperature (-20 °C, 4 °C, and 25 °C) were respectively detected using a fluorescence microplate reader. The results showed that it should be avoided to store the isolated mitochondria at room temperature, and the mitochondria can meet the requirements of the most downstream experiments when they were stored at -20 °C. Overall, the study presented a method for isolating highly purified and active mitochondria from insects. This study firstly described a high-speed discontinuous density gradient centrifugation-based method that could be widely applied for mitochondria isolation in insects. The present study also provided an example to assess purity and integrity/activity of the isolated mitochondria.