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.

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.

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.

A small set of differentially expressed genes was associated with two color morphs in natural populations of the pea aphid Acyrthosiphon pisum.

Color polymorphism is an ecologically important trait, which is related to local adaptation and ecological speciation. The pea aphid Acyrthosiphon pisum shows color polymorphism: the red and green color morphs where differences in ecological adaptation have been observed. Here, we measured genome-wide gene expression profiles of two color morphs in natural populations of A. pisum to explore the genetic basis of differentiated ecological adaptation. The results showed that only 32 genes were significantly differentially expressed between the two morphs, of which 18 had functional annotations. Among them, 13 genes were up-regulated [e.g. genes encoding protoheme IX farnesyltransferase (LOC100570971), carotene dehydrogenase (tor) and V-type proton ATPase subunit B (LOC100169462)] and 5 genes were down-regulated in the red morph (e.g. genes encoding transcription factors and heat shock proteins). To assess the functional importance of these differentially expressed genes (DEGs), we selected three highly expressed DEGs (LOC100169462, LOC100570971 and tor) with functional annotations and analyzed their expression levels in the red morph under three low temperatures (1 °C, 4 °C, and 8 °C) for 24 h. These three DEGs showed an interesting expression response to the cold acclimating conditions which resulted in an obvious phenotypic change of the red individuals to be greenish variants. This study suggests a link between gene expressions and body color polymorphisms in the pea aphid and provides important clues for further studying molecular mechanisms of ecological adaptation in aphids.

Comparative transcriptomic analysis of Tibetan Gynaephora to explore the genetic basis of insect adaptation to divergent altitude environments.

Adaptation of insects to different altitudes remain largely unknown, especially those endemic to the Tibetan Plateau (TP). Here, we generated the transcriptomes of Gynaephora menyuanensis and G. alpherakii, inhabiting different high altitudes on the TP, and used these and the previously available transcriptomic and genomic sequences from low-altitude insects to explore potential genetic basis for divergent high-altitude adaptation in Gynaephora. An analysis of 5,869 orthologous genes among Gynaephora and other three low-altitude insects uncovered that fast-evolving genes and positively selected genes (PSGs) in the two Gynaephora species were enriched in energy metabolism and hypoxia response categories (e.g. mitochondrion, oxidation-reduction process, and response to oxidative stress). Particularly, mTOR signaling pathway involving hypoxia was enriched by PSGs, indicating this well-known pathway in mammal hypoxia adaptation may be an important signaling system in Gynaephora. Furthermore, some PSGs were associated with response to hypoxia (e.g. cytochrome proteins), cold (e.g. dehydrogenase) and DNA repair (e.g. DNA repair proteins). Interestingly, several insect-specific genes that were associated with exoskeleton and cuticle development (e.g. chitinase and ecdysteroids) had experienced positive selection, suggesting the specific adaptive mechanisms in insects. This study is favourable for understanding the adaptive evolution of Gynaephora and even TP insects to divergent altitudes.

Selection of reference genes for qRT-PCR and expression analysis of high-altitude-related genes in grassland caterpillars (Lepidoptera: Erebidae: Gynaephora) along an altitude gradient.

Changes in gene expression patterns can reflect the adaptation of organisms to divergent environments. Quantitative real-time PCR (qRT-PCR) is an important tool for ecological adaptation studies at the gene expression level. The quality of the results of qRT-PCR analysis largely depends on the availability of reliable reference genes (RGs). To date, reliable RGs have not been determined for adaptive evolution studies in insects using a standard approach. Here, we evaluated the reliability of 17 candidate RGs for five Gynaephora populations inhabiting various altitudes of the Tibetan Plateau (TP) using four independent (geNorm, NormFinder, BestKeeper, and the deltaCt method) and one comprehensive (RefFinder) algorithms. Our results showed that EF1-α, RPS15, and RPS13 were the top three most suitable RGs, and a combination of these three RGs was the most optimal for normalization. Conversely, RPS2,ACT, and RPL27 were the most unstable RGs. The expression profiles of two target genes (HSP70 and HSP90) were used to confirm the reliability of the chosen RGs. Additionally, the expression patterns of four other genes (GPI,HIF1A,HSP20, and USP) associated with adaptation to extreme environments were assessed to explore the adaptive mechanisms of TP Gynaephora species to divergent environments. Each of these six target genes showed discrepant expression patterns among the five populations, suggesting that the observed expression differences may be associated with the local adaptation of Gynaephora to divergent altitudinal environments. This study is a useful resource for studying the adaptive evolution of TP Gynaephora to divergent environments using qRT-PCR, and it also acts as a guide for selecting suitable RGs for ecological and evolutionary studies in insects.

Comparative mitogenomic analysis of mirid bugs (Hemiptera: Miridae) and evaluation of potential DNA barcoding markers.

The family Miridae is one of the most species-rich families of insects. To better understand the diversity and evolution of mirids, we determined the mitogenome of Lygus pratenszs and re-sequenced the mitogenomes of four mirids (i.e., Apolygus lucorum, Adelphocoris suturalis, Ade. fasciaticollis and Ade. lineolatus). We performed a comparative analysis for 15 mitogenomic sequences representing 11 species of five genera within Miridae and evaluated the potential of these mitochondrial genes as molecular markers. Our results showed that the general mitogenomic features (gene content, gene arrangement, base composition and codon usage) were well conserved among these mirids. Four protein-coding genes (PCGs) (cox1, cox3, nad1 and nad3) had no length variability, where nad5 showed the largest size variation; no intraspecific length variation was found in PCGs. Two PCGs (nad4 and nad5) showed relatively high substitution rates at the nucleotide and amino acid levels, where cox1 had the lowest substitution rate. The Ka/Ks values for all PCGs were far lower than 1 (<0.59), but the Ka/Ks values of cox1-barcode sequences were always larger than 1 (1.34 -15.20), indicating that the 658 bp sequences of cox1 may be not the appropriate marker due to positive selection or selection relaxation. Phylogenetic analyses based on two concatenated mitogenomic datasets consistently supported the relationship of Nesidiocoris + (Trigonotylus + (Adelphocoris + (Apolygus + Lygus))), as revealed by nad4, nad5, rrnL and the combined 22 transfer RNA genes (tRNAs), respectively. Taken sequence length, substitution rate and phylogenetic signal together, the individual genes (nad4, nad5 and rrnL) and the combined 22 tRNAs could been used as potential molecular markers for Miridae at various taxonomic levels. Our results suggest that it is essential to evaluate and select suitable markers for different taxa groups when performing phylogenetic, population genetic and species identification studies.

The first mitochondrial genome for the subfamily Podopinae (Hemiptera: Pentatomidae) and its phylogenetic implications.

Here, we determined the complete mitogenome of Graphosoma rubrolineata, as the first representative for the subfamily Podopinae. This mitogenome is 15,633 bp long and contains 37 typical mitochondrial genes. The genome size, gene arrangement, A + T content, codon usage and secondary structures of 22 tRNAs of the G. rubrolineata mitogenome were similar to that of other sequenced pentatomoids. This mitogenome exhibited a reverse nucleotide strand bias, i.e. positive GC-skew (0.021) and negative AT-skew (-0.086). Phylogenetic analyses based on mitogenomic data strongly supported the monophyly of each of the five superfamilies within Pentatomomorpha and recognized a phylogeny of (Aradoidea + (Pentatomoidea + (Lygaeoidea + (Pyrrhocoroidea + Coreoidea)))). However, G. rubrolineata clustered with three Pentatominae species, suggesting that Pentatominae probably was not monophyletic, or Podopinae may not be a valid taxonomic group. The mitogenome sequence of G. rubrolineata could contribute for better understanding of population genetics and evolution of this insect pest.

Mitochondrial genome of Sitona callosus (Coleoptera: Curculionidae) and phylogenetic analysis within Entiminae.

In this study, we sequenced and annotated the nearly complete mitochondrial genome (mitogenome) of Sitona callosus (Coleoptera: Curculionidae). This mitogenome was 14,333 bp long and encoded 13 protein-coding genes, 19 transfer RNA genes (tRNAs), and two ribosomal RNA unit genes. Gene rearrangements were presented in a tRNA cluster of six tRNAs between nad3 and nad5, i.e. the ancestral order ARNSEF was changed to be RNSAEF. All tRNAs had a typical secondary cloverleaf structure, except for trnS1 which lacked the dihydrouridine arm. The Bayesian phylogenetic tree of 11 Entiminae species based on the concatenated nucleotide sequences of 13 PCGs showed that S. callosus and S. lineatus formed a clade which was at the basal position in the Entiminae phylogeny.

The complete mitochondrial genome of Enallagma cyathigerum (Odonata: Coenagrionidae) and phylogenetic analysis.

To better understand the diversity and evolution of Odonata, we sequenced and annotated the complete mitochondrial genome (mitogenome) of Enallagma cyathigerum. This mitogenome was 16,661 bp in size and encoded the typical 37 genes, i.e. 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs) and two ribosomal RNA genes. The nucleotide composition of the E. cyathigerum mitogenome was significantly biased toward A and T, with an A + T content of 74.2%. Eleven PCGs started with a typical ATN codon, whereas the remaining two PCGs (nad1 and nad3) used TTG as the initial codon. All the 22 tRNAs had a typical secondary cloverleaf structure. The Bayesian phylogenetic analysis based on the concatenated nucleotide sequences of 13 PCGs strongly supported the sister relationship of E. cyathigerum and two Ischnura species from the same family Coenagrionidae. The phylogenetic tree strongly supported the monophyly of each of the two suborders (Zygoptera and Anisoptera) and recovered a phylogeny of Zygoptera + (Anisoptera + Anisozygoptera).

Mitochondrial genome of Taiwania circumdata (Coleoptera: Chrysomelidae: Cassidinae) and phylogenetic analysis.

In this study we sequenced and annotated the nearly complete mitochondrial genome (mitogenome) of Taiwania circumdata (Coleoptera: Chrysomelidae: Cassidinae), an important insect pest on sweetpotato and water spinach in Southern China. This mitogenome was 13,546 bp long and encoded 13 protein-coding genes (PCGs), 19 transfer RNA genes (tRNAs) and 2 ribosomal RNA unit genes. The T. circumdata mitogenome with an A + T content of 77.9% presented a positive AT-skew (0.126) and a negative GC-skew (-0.160). Eleven PCGs started with a typical ATN codon, whereas the remaining two PCGs used TTG (nad1) and AAT (cox1) as the initial codon. All the 19 tRNAs had a typical secondary cloverleaf structure, except for trnS1 (AGN) which lacked the dihydrouridine arm. Phylogenetic analyses using Bayesian inference and maximum likelihood methods based on the concatenated nucleotide sequences of 13 PCGs recovered a phylogeny of Bruchinae+ ((Galerucinae + Chrysomelinae) + (Criocerinae + Cassidinae)). In Cassidinae, T. circumdata and Laccoptera ruginosa formed a clade, which was sister to three Cassida species.