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.

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.

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 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.

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.

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.

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.

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.

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.

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.

The complete mitochondrial genome of Palomena viridissima (Hemiptera: Pentatomidae) and phylogenetic analysis.

Here, we sequenced and annotated the complete mitochondrial genome (mitogenome) of Palomena viridissima (Hemiptera: Pentatomidae). This mitogenome was 15,118 bp long, comprising of 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (rrnL and rrnS) and a large non-coding control region. The P. viridissima mitogenome with an A + T content of 76.0%, presented a positive AT-skew (0.11) and a negative GC-skew (-0.13). Ten PCGs started with a typical ATN codon, two PCGs started with TTG (atp8, nad1), whereas the remaining one used AAC (cox1). All tRNAs had a typical secondary cloverleaf structure, except for trnS1 which lacked the dihydrouridine arm. The Bayesian phylogenetic analysis based on mitogenomic data supported a sister relationship of P. viridissima and Nezara viridula from the same tribe Nezarini and recovered a phylogeny of Pentatominae: (Menidini + (Strachiini + (Pentatomini + ((Cappaeini + Halyini) + (Eysarcorini + (Nezarini + Carpocori)))))).

Mitochondrial genome of Chrysochares punctatus (Coleoptera: Chrysomelidae: Eumolpinae) and phylogenetic analysis.

Here, we determined the nearly complete mitochondrial genome (mitogenome) of Chrysochares punctatus (Coleoptera: Chrysomelidae: Eumolpinae), an important insect pest on Apocynum venetum in Northwestern China. This mitogenome was 14,451 bp long, encoding 13 protein-coding genes (PCGs), 21 transfer RNA genes (tRNAs), and 2 ribosomal RNA genes. The C. punctatus mitogenome presented an A + T content of 75.11%, with a positive AT-skew (0.064) and a negative GC-skew (-0.192). Ten PCGs started with a typical ATN codon, whereas the remaining three PCGs started with AAC (cox1) and TTG (nad1 and nad2). All tRNAs had a typical secondary cloverleaf structure, except for trnS1 which lacked the dihydrouridine arm. Bayesian phylogenetic analysis based on the nucleotide sequences of 13 PCGs recovered a phylogeny within Chrysomelidae: (((Chrysomelinae + Galerucinae), (((Eumolpinae, Lamprosomatinae), Cassidinae), Criocerinae)), Bruchinae).

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 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))).

Transcriptome-Wide Identification of MicroRNAs and Analysis of Their Potential Roles in Development of Indian Meal Moth (Lepidoptera:Pyralidae).

MicroRNAs (miRNAs) have been reported to play indispensable roles in regulating various developmental processes via the posttranscriptional repression of target genes in insect species. In the present paper, we studied the miRNAs in Indian meal moth (Plodia interpunctella (Hübener)), one of the most economically important stored grains pests around the world. In total, 12 small RNA libraries from four developmental stages of P. interpunctella were constructed, and 178 known and 23 novel miRNAs were identified. In addition, the expression profiles of these miRNAs were assessed across different developmental stages and miRNAs that were highly expressed in eggs, larvae, pupae, and adults were identified. Specifically, 100, 61, and 52 miRNAs were differentially expressed between eggs and larvae, larvae and pupae, and pupae and adults, respectively. The KEGG and GO analysis of the predicted target genes suggested the essential roles of miRNAs in the regulation of complex development of P. interpunctella. Importantly, we also found a set of miRNAs might be involved in the larval metamorphic molting process, with their expressions increasing and then decreasing during the larva-pupa-adult stages of P. interpunctella. In conclusion, the current paper has discovered numerous miRNAs, and some key miRNAs that might be responsible for regulating development in P. interpunctella. To our knowledge, this is the first study to document miRNAs and their expression patterns in interpunctella, and those findings would lay an important molecular foundation for future functional analysis of these miRNAs in P. interpunctella.

Mitogenome evolution in ladybirds: Potential association with dietary adaptation.

Dietary shifts can alter the relative availability of different nutrients and are therefore associated with metabolic adaptation in animals. The Coccinellidae (ladybirds) exhibits three major types of feeding habits and provides a useful model to study the effects of dietary changes on the evolution of mitogenomes, which encode proteins directly involved in energy metabolism. Here, mitogenomes of three coccinellid species were newly sequenced. These data were combined with other ten previously sequenced coccinellid mitogenomes to explore the relationship between mitogenome evolution and diets. Our results indicate that mitogenomic data can be effectively used to resolve phylogenetic relationships of Coccinellidae. Strong codon usage bias in coccinellid mitogenomes was predominantly determined by nucleotide composition. The 13 mitochondrial protein-coding genes (PCGs) globally evolved under negative constraints, with some PCGs showing a stronger purifying selection. Six PCGs (nad3, nad4L, and nad5 from Complex I; cox1 and cox3 from Complex IV; and atp6 from Complex V) displayed signs of positive selection. Of these, adaptive changes in cox3 were potentially associated with metabolic differences resulting from dietary shifts in Coccinellidae. Our results provide insights into the adaptive evolution of coccinellid mitogenomes in response to both dietary shifts and other life history traits.

The complete mitochondrial genome of Nysius fuscovittatus (Hemiptera: Lygaeidae).

Here, we sequenced and annotated the complete mitochondrial genome (mitogenome) of Nysius fuscovittatus (Hemiptera: Lygaeidae). This mitogenome was 14575 bp long, including 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), 2 ribosomal RNA unit genes (rrnL and rrnS), and a putative control region. All genes were arranged in the same order as that of most true bugs. Eleven PCGs started with a typical ATN, and the remaining two PCGs started with TTA (nad4L) and TTG (cox1). The N. fuscovittatus mitogenome with an A + T content of 76.42% showed a positive AT-skew (0.15) and a negative GC-skew (-0.15). With the exception of trnS1 that lacked the dihydrouridine arm, all tRNAs had a typical cloverleaf secondary structure. Phylogenetic analysis based on the concatenated nucleotide sequences of the 13 PCGs showed that N. fuscovittatus clustered with other three Lygaeidae species.

The Complete Mitogenome of Pyrrhocoris tibialis (Hemiptera: Pyrrhocoridae) and Phylogenetic Implications.

We determined the complete mitogenome of Pyrrhocoris tibialis (Hemiptera: Heteroptera: Pyrrhocoridae) to better understand the diversity and phylogeny within Pentatomomorpha, which is the second largest infra-order of Heteroptera. Gene content, gene arrangement, nucleotide composition, codon usage, ribosomal RNA (rRNA) structures, and sequences of the mitochondrial transcription termination factor were well conserved in Pyrrhocoroidea. Different protein-coding genes have been subject to different evolutionary rates correlated with the G + C content. The size of control regions (CRs) was highly variable among mitogenomes of three sequenced Pyrrhocoroidea species, with the P. tibialis CR being the largest. All the transfer RNA genes found in Pyrrhocoroidea had the typical clover leaf secondary structure, except for trnS1 (AGN), which lacked the dihydrouridine arm and possessed an unusual anticodon stem (9 bp vs. the normal 5 bp). A total of three different phylogenetic relationships among the five super-families of Pentatomomorpha were obtained using three analytical methods (MrBayes and RAxML under site-homogeneous models and PhyloBayes under a site-heterogeneous CAT + GTR model) and two mitogenomic datasets (nucleotides and amino acids). The tree topology test using seven methods statistically supported a phylogeny of (Aradoidea + (Pentatomoidea + (Lygaeoidea + (Pyrrhocoroidea + Coreoidea)))) as the best topology, as recognized by both RAxML and MrBayes based on the two datasets.

Adaptive evidence of mitochondrial genomes in Dolycoris baccarum (Hemiptera: Pentatomidae) to divergent altitude environments.

Given mitochondrion is the 'energy and oxygen usage factories', adaptive signatures of mitochondrial genes have been extensively investigated in vertebrates from different altitudes, but few studies focus on insects. Here, we sequenced the complete mitochondrial genome (mitogenome) of Dolycoris. baccarum living in the Tibetan Plateau (DBHC, ∼3200 m above sea level (asl)) and conducted a detailed comparative analysis with another D. baccarum mitogenome (DBQY) from relatively low altitude (∼1300 m asl). All the 37 mitochondrial genes were highly conserved and under purifying selection, except for two mitochondrial protein-coding genes (MPCGs) (atp6 and nad5) that showed positively selected signatures. We therefore further examined non-synonymous substitutions in atp6 and nad5, by sequencing more individuals from three populations with different altitudes. We found that these non-synonymous substitutions were polymorphic in these populations, likely due to relaxed selection constraints in different altitudes. Purifying selection in all mitochondrial genes may be due to their functional importance for the precision of ATP production usually. Length difference in mitochondrial control regions between DBHC and DBQY was also conversed at the population level, indicating that sequence size adjustments in control region may be associated with adaptation to divergent altitudes. Quantitatively real-time PCR analysis for 12 MPCGs showed that gene expression patterns had a significant change between the two populations, suggesting that expression levels of MPCGs could be modulated by divergent environmental pressures (e.g. oxygen content and ambient temperature). These results provided an important guide for further uncovering genetic mechanisms of ecological adaptation in insects.