Molecular characterization and functional analysis of a beta-1,3-glucan recognition protein from oriental fruit moth Grapholita molesta (Lepidoptera: Tortricidae).

The beta-1,3-glucan recognition protein (BGRP) is an important pattern recognition protein (PRP), which plays an important role in immune recognition and signaling pathway of insect innate immunity. Herein, a BGRP gene was obtained from the transcriptome of Grapholita molesta and its expression was verified by PCR. The full cDNA of the GmBGRP gene was 1691 bp encoding 486 amino acid residues. The calculated molecular mass of the mature protein was 54.83 kDa with an estimated pI of 6.14. The amino acid sequence of GmBGRP was highly homologous to BGRPs of other lepidopterans including Leguminivora glycinivorella BGRP-3. Expression profile of GmBGRP at different developmental stages and different tissues of 5th instar larvae showed that the expression level of this gene tends to slightly increase and then decrease at the adult stage, with the highest at the pupa stage; and mainly expressed in the epidermis, fat body and hemocytes compared with other tissues. In addition, we investigated the transcription levels of other immune-related genes, such as Serine-1, Serine-2, Serine-3, Serpin, SRCB (scavenger receptor gene), Toll, PPO (prophenoloxidase) upon GmBGRP gene silencing, indicating that GmBGRP expression is associated with immune responses of G. molesta. This was further supported by the upregulation of the mRNA level of GmBGRP following fungal infection. Taken together, these results provide experimental evidence for the role of GmBGRP gene in immune defense in G. molesta larvae.

Molecular characterization and functional analysis of the ecdysone receptor isoform (EcR) from the oriental fruit moth Grapholita molesta (Lepidoptera: Tortricidae).

20-Hydroxyecdysone (20E) plays a vital role in a series of biological processes, via the nuclear receptors, EcR/USP by activating the ecdysone regulatory cascade. To clarify the role of EcR during the development of Grapholita molesta, the complementary DNA of ecdysone receptor isoform B1 (GmEcR-B1) was obtained from the transcriptome of G. molesta and verified by PCR. Alignment analysis revealed that the deduced protein sequence of GmEcR-B1 was highly homologous to EcR proteins identified in other lepidopteran species, especially the EcR-B1 isoform in Spodoptera litura. Quantitative real-time PCR showed that GmEcRs was expressed at all test developmental stages, and the expression level of GmEcRs was relatively higher during the period of the 3rd day of fifth instar larvae to 2nd of pupa than those in other stages. Moreover, the messenger RNA of GmEcRs was much more strongly expressed in the Malpighian tubule and epidermis than those in other tissues, which suggests that this gene may function in a tissue-specific manner during larval development. Silencing of GmEcRs could significantly downregulate the transcriptional level of ecdysone-inducible genes and result in increased mortality during metamorphosis and prolonged prepupal duration. Taken together, the present results indicate that GmEcRs may directly or indirectly affect the development of G. molesta.

Mitochondrial genomes of Nemourinae species (Plecoptera: Nemouridae) and the phylogenetic implications.

Currently, the classification system of 2 subfamilies within Nemouridae has been widely accepted. However, monophyly of 2 subfamilies has not been well supported by molecular evidence. To date, only mitogenomes from genus Nemoura of the subfamily Nemourinae were used in previous phylogenetic studies and produced conflicting results with morphological studies. Herein, we analyzed mitogenomes of 3 Nemourinae species to reveal their mitogenomic characteristics and to examine genus-level classification among Nemouridae. In this study, the genome organization of 3 mitogenomes is highly conserved in gene order, nucleotide composition, codon usage, and amino acid composition. In 3 Nemourinae species, there is a high variation in nucleotide diversity among the 13 protein-coding genes (PCGs). The Ka/Ks values for all PCGs were far lower than 1, indicating that these genes were evolving under purifying selection. The phylogenetic analyses highly support Nemurella as the sister group to Ostrocerca. Meanwhile, Nemoura is recovered as the sister group of Malenka; they are grouped with other Amphinemurinae and emerged from a paraphyletic Nemourinae. More molecular data from different taxonomic groups are needed to understand stoneflies phylogeny and evolution.

Two Complete Mitochondrial Genomes From Leuctridae (Plecoptera: Nemouroidea): Implications for the Phylogenetic Relationships Among Stoneflies.

The family-level relationships within Plecoptera have been a focused area of research for a long time. Its higher classification remains unstable, and the phylogenetic relationships within Plecoptera should be re-examined. Here, we sequenced and analyzed two complete mitochondrial genomes (mitogenomes) of Paraleuctra cercia and Perlomyia isobeae of the family Leuctridae. We reconstructed the phylogeny of Plecoptera based on 13 protein-coding genes (PCGs) from published stoneflies. Our results showed that the Bayesian inference and maximum-likelihood tree had similar topological structures except for the positions of two families, Peltoperlidae and Scopuridae. The Plecoptera is divided into two clades, the suborder Antarctoperlaria and the suborder Arctoperlaria. The two suborders subsequently formed two groups, Eusthenioidea and Gripopterygoidea, and Euholognatha and Systellognatha, which is consistent with the results of morphological studies. In addition, the Leuctridae is the earliest branch within the superfamily Nemouroidea. But the monophyly of Perloidea and Pteronarcyoidea are still not well supported.

Complete Mitochondrial Genome of Suwallia teleckojensis (Plecoptera: Chloroperlidae) and Implications for the Higher Phylogeny of Stoneflies.

Stoneflies comprise an ancient group of insects, but the phylogenetic position of Plecoptera and phylogenetic relations within Plecoptera have long been controversial, and more molecular data is required to reconstruct precise phylogeny. Herein, we present the complete mitogenome of a stonefly, Suwallia teleckojensis, which is 16146 bp in length and consists of 13 protein-coding genes (PCGs), 2 ribosomal RNAs (rRNAs), 22 transfer RNAs (tRNAs) and a control region (CR). Most PCGs initiate with the standard start codon ATN. However, ND5 and ND1 started with GTG and TTG. Typical termination codons TAA and TAG were found in eleven PCGs, and the remaining two PCGs (COII and ND5) have incomplete termination codons. All transfer RNA genes (tRNAs) have the classic cloverleaf secondary structures, with the exception of tRNASer(AGN), which lacks the dihydrouridine (DHU) arm. Secondary structures of the two ribosomal RNAs were shown referring to previous models. A large tandem repeat region, two potential stem-loop (SL) structures, Poly N structure (2 poly-A, 1 poly-T and 1 poly-C), and four conserved sequence blocks (CSBs) were detected in the control region. Finally, both maximum likelihood (ML) and Bayesian inference (BI) analyses suggested that the Capniidae was monophyletic, and the other five stonefly families form a monophyletic group. In this study, S. teleckojensis was closely related to Sweltsa longistyla, and Chloroperlidae and Perlidae were herein supported to be a sister group.

Identification and Evaluation of Suitable Reference Genes for Normalization of MicroRNA Expression in Helicoverpa armigera (Lepidoptera: Noctuidae) Using Quantitative Real-Time PCR.

More and more studies have focused on microRNAs (miRNAs) expression in the pest Helicoverpa armigera (Lepidoptera: Noctuidae) recently. Quantitative real-time PCR (qRT-PCR) is being widely used in miRNA expression studies. Suitable reference genes are necessary for the correct analysis of results. In this study, 10 candidate genes of H. armigera were selected and analyzed for their expression stability under different biotic and abiotic conditions with 3 statistical methods, including geNorm, NormFinder, and Bestkeeper. Combination the best number of reference genes was calculated by geNorm. One target gene, let-7, was used to validate the selection of reference genes. The suitable candidate reference genes were shown as follows: miR-9 and U6 snRNA for developmental stages, miR-100 and U6 snRNA for larval tissues, miR-100 and miR-305 for adult tissues, miR-9 and miR-279 for parasitic treatment, miR-998 and U6 snRNA for nuclear polyhedrosis virus infection, miR-9 and U6 snRNA for insecticide treatment, miR-92a, miR-100, and miR-279 for temperature treatment, miR-92a, miR-305, and miR-998 for starvation treatment, miR-9 and miR-279 for light treatment, miR-305 and miR-998 for hormone treatment, and there was not one reference gene suitable for all samples. This study could promote future research on miRNAs expression in H. armigera with optimal reference genes under different experimental conditions.

Identification of the Population Structure of Myzus persicae (Hemiptera: Aphididae) on Peach Trees in China Using Microsatellites.

In this study, we characterized the genetic structure of Myzus persicae (Sulzer) (Hemiptera: Aphididae) populations in China using microsatellites. We expected that these data will reveal the genetic relationships among various populations of M. persicae and will be of value in the development of better methods for pest control. Four hundred sixty individuals from 23 areas over 13 provinces were collected in the early spring of 2010, all from their primary host, Prunus persicae. The markers analyzed were highly polymorphic, as demonstrated by the expected heterozygosity value (He = 0.861) and the Polymorphism Information Content (PIC = 0.847), which indicated that M. persicae maintains a high level of genetic diversity. Analysis of molecular variance revealed an intermediate level of population differentiation among M. persicae populations (F(ST) = 0.1215). Geographic isolation existed among these populations, and, consequently, the genetic structure of the populations was split into a southern group and a northern group divided by the Yangtse River.

The mitochondrial genome of Neoperla bimaculata (Li et al. 2021) (plecoptera: perlidae) from Tibet of southwest China and its phylogenetic analysis.

The complete mitochondrial genome (mitogenome) of Neoperla bimaculata was sequenced and annotated in this study. We found that the mitogenome of N. bimaculata is 15,774 bp in length with an A + T content of 64.3%. It exhibits the classic structure of a mitogenome. Most protein-coding genes (PCGs) of the mitogenome initiate with the standard start codon ATN. Ten PCGs use the standard stop codon TAA/TAG, while the COI, COII, and ND5 genes terminate with a single T nucleotide. Phylogenetic analyses suggested that N. bimaculata, along with two unpublished Neoperla species, formed a cluster within the phylogenetic tree. Our results indicated that the genus Neoperla and Neoperlops were sister groups. Meanwhile, the monophyly of Perlinae and Acroneuriinae was supported in the mitochondrial phylogeny.

Metabolomics analysis reveals metabolite diversity of the rare cliff plant Oresitrophe rupifraga unge.

Oresitrophe is monotypic, with the only species, Oresitrophe rupifraga Bunge, which is exclusive to China, having special growth and developmental traits due to its habitat. Furthermore, it has bright flowers and medicinal benefits. This study investigated the metabolites present in various tissues of Oresitrophe rupifraga Bunge. Using a widely targeted metabolomics approach, 1965 different metabolites were identified in Oresitrophe rupifraga Bunge. Based on principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA), the aboveground and underground metabolites of Oresitrophe rupifraga differed significantly. The comparison between bulblets and leaves revealed the differential expression of 461 metabolites, whereas the comparison between rhizomes and leaves showed the differential expression of 423 metabolites, and the comparison between bulblets and rhizomes showed the differential expression of 249 metabolites. The bulblets exhibited 49 metabolites that were higher and 412 metabolites that were lower than those of the leaves, whereas the rhizomes showed 123 upregulated and 300 downregulated metabolites. Bulblets showed an increase in 18 metabolites and a decrease in 231 metabolites compared to the rhizomes. Leaves contain more phenolic acids than the rhizomes and bulblets, whereas the rhizomes and bulblets contain more terpenoids than the leaves. KEGG pathway analysis showed an association between metabolites and metabolic pathways, as well as their effect on the progression and maturation of Oresitrophe rupifraga Bunge. The research findings can provide some insight into the growth and developmental traits of Oresitrophe rupifraga Bunge, thus providing a theoretical foundation for cultivating and utilising this plant.

Mitochondrial genomes provide insights into the Euholognatha (Insecta: Plecoptera).

BACKGROUND: Euholognatha is a monophyletic group within stoneflies comprised by a superfamily Nemouroidea and a family Scopuridae. Based on morphological data, the family-level phylogenetic relationships within Euholognatha are widely accepted, but there is still controversy among different molecular studies. To better understand the phylogeny of all six extant euholognathan families, we sequenced and analyzed seven euholognathan mitogenomes. RESULTS: The sequence heterogeneity analysis observed a low degree of compositional heterogeneity in euholognathan mitogenomes. Meanwhile, leuctrid mitogenomes were more heterogeneous than other euholognathan families, which may affect the phylogenetic reconstruction. Phylogenetic analyses with various datasets generated three topologies. The Leuctridae was recovered as the earliest branching lineage, and the sister relationship of Capniidae and Taeniopterygidae was supported by most tree topologies and FcLM analyses. When separately excluding sparsely sampled Scopuridae or high heterogeneity leuctrid taxa, phylogenetic analyses under the same methods generated more stable and consistent tree topologies. Finally, based on the results of this study, we reconstructed the relationships within Euholognatha as: Leuctridae + (Scopuridae + ((Taeniopterygidae + Capniidae) + (Nemouridae + Notonemouridae))). CONCLUSION: Our research shows the potential of data optimizing strategies in reconstructing phylogeny within Euholognatha and provides new insight into the phylogeny of this group.

Expression analysis of plvap in mouse heart development, homeostasis and injury.

Plasmalemma vesicle associated protein (PLVAP) is commonly considered to be specifically expressed in endothelial cells in which it localized to diaphragms of caveolae, fenestrae, and transendothelial channels. PLVAP is reported to be an important regulator of heart development and a novel target to promote cardiac repair in the ischemic heart. However, the dynamics of plvap expression in heart development, homeostasis and pathology have not been comprehensively described. In this study, we analyzed the temporal and spatial expression of plvap in mouse heart under different conditions. We found that, during embryonic and neonatal stages, PLVAP was detected in endocardial endothelial cells, epicardial mesothelial cells, and a small amount of coronary vascular endothelial cells. In adult heart, PLVAP was also identified in endocardial cells and a few coronary vascular endothelial cells. However, epicardial expression of PLVAP was lost during postnatal heart development and cannot be detected in mouse heart by immunostaining since 3-week-old. We also analyzed the expression of plvap in a model of cardiac hypertrophy and failure induced by transverse aortic constriction surgery, and identified expression of PLVAP in endocardial cells and coronary vascular endothelial cells in the injured heart. This study provides new evidence to better understand the role of plvap in mouse heart development and injury.

Population structure of Aphis spiraecola (Hemiptera: Aphididae) on pear trees in China identified using microsatellites.

The spiraea aphid (Aphis spiraecola Patch) is a primary pest of fruit trees, particularly pear trees in China. Despite the economic importance of this pest, little is known about its genetic structure or its patterns of dispersal at local and regional scales; however, knowledge of these characteristics is important for establishing effective control strategies for this pest. The genetic variability of 431 individuals from 21 populations on pear trees in China was investigated using eight polymorphic microsatellite loci. The high polymorphism of these markers was evident from the expected heterozygosity value (He = 0.824) and the Polymorphism Information Content (PIC = 0.805), indicating that the spiraea aphid maintains a high level of genetic diversity. The analysis of molecular variance revealed a middle level of population differentiation (F(ST) = 0.1478) among A. spiraecola populations. This result is consistent with the results of the STRUCTURE analysis (K = 3), the unweighted pair-group method with arithmetic average tree and the Mantel test (r = 0.6392; P < 0.05). Our results indicate high levels of genetic exchange in the spiraea aphid, possibly facilitated by geography and climate. Our findings emphasize the importance of considering regional differences in studies of population structure, even when strong isolation-by-distance influences the genetic population structure of species.

Complete Mitochondrial Genome of Malenka flexura (Plecoptera: Nemouridae) and Phylogenetic Analysis.

The genus-level relationships within the subfamily Amphinemurinae have been controversial, although attempts have been made based on morphology and limited molecular data. With the establishment of two new genera, the phylogenetic relationships within Amphinemurinae should be re-examined. In this study, the complete mitochondrial genome (mitogenome) of Malenka flexura of the genus Malenka was firstly sequenced and analyzed. The phylogeny of Amphinemurinae was also reconstructed using 13 proteincoding genes (PCGs) from previously published stoneflies. This mitogenome was 15,744 bp long and encoded the typical 37 genes, as well as a putative control region. The gene arrangement of M. flexura mitogenome is identical with the putative ancestral mitogenome in Drosophila yakuba. Most PCGs used standard ATN as start codons and TAA/TAG as termination codons. All tRNA genes exhibited the typical cloverleaf secondary structure, except for tRNASer(AGN), whose dihydrouridine (DHU) arm was lacking. Some structural elements in the control region were founded, such as tandem repeat regions, stemloop structures, polyN stretch and microsatellite structure, etc. Phylogenetic analyses of sequenced Amphinemurinae mitogenomes unsupported the sister relationship of Amphinemura and Malenka. Finally, the phylogenetic analyses inferred a relationship within Amphinemurinae: Amphinemura + (Malenka + (Protonemura + (Indonemoura + (Sphaeronemoura + Mesonemoura)))).

Contributions to the winter stoneflies (Plecoptera: Taeniopterygidae amp; Capniidae) of China.

A complementary re-description of Kyphopteryx dorsalis Kimmins, 1947 is presented on the basis of a fresh male and additional females from southeastern Tibet, southwestern China. The hitherto unknown inner structures of the female terminalia are described, as well as further details of the male genitalia. In this study, we also present the first records of Capnia s.l. yunnana Li Yang, 2011 from Tibet and a formal description of an unassociated Capnia s.l. female collected together with the above species. An updated checklist of the named species of Capniidae and Taeniopterygidae from China is also given.

Two new species of the genus Flavoperla (Plecoptera: Perlidae) from Guangxi, China.

Two new species, Flavoperla retusata Mo, Li Wang, sp. nov. and F. yangi Mo, Li Muryi, sp. nov. are proposed from the Guangxi Zhuang Autonomous Region of southern China. Distinctness of the new species is based on morphological characters and DNA sequence comparisons with their closest known relative, F. galerispina Mo, Wang Li, 2020. The taxonomic relationships of the two new species and related congeners are discussed.

The complete mitochondrial genome of Amphinemura bulla Shimizu, 1997 (Plecoptera: Nemouridae) from Japan.

The genus Amphinemura belongs to the family Nemouridae (Plecoptera) and has 205 species in the Holarctic and Oriental Regions. We sequenced the fourth complete mitochondrial genome of A. bulla Shimizu, 1997. The mitogenome is 15,827 bp long with 37 genes plus a control region with an A + T content of 68.9%. There are 10 intergenic spacers (75 bp total) and 13 gene overlaps (43 bp total). All protein-coding genes (PCGs) use normal initiation codons, except ND1 and ND5 which begin with TTG and GTG. Two PCGs (COII and ND5) use a single T as a partial termination codon. Phylogenetic analyses showed that Nemoura and Amphinemura were sister group resulting in a paraphyletic Amphinemurinae different from the morphological classification.

The mitochondrial genome analysis of Flavoperla hatakeyamae (Plecoptera: Perlidae).

In this study, we analyzed the entire mitochondrial genome of Flavoperla hatakeyamae, which has 15,730 bp in length. This complete mitochondrial genome possessed 13 protein coding genes (PCGs), 2 ribosomal RNA genes, 22 transfer RNA genes, and 1 control region. The nucleotide composition of the complete sequence of mitochondrial genome was accounting for 35.0%, T for 31.3%, C for 21.2%, G for 12.5%, and A + T for 66.3%. Among the 13 protein-coding genes, the starting codon of 12 protein-coding genes was ATN except the starting codon of COII was ACC. In addition, 11 of the protein-coding genes used conservative termination codon TAA/TAG, except for COII and ND5 which terminated by a single T. Based on Bayesian (BI) and maximum-likelihood (ML) methods, we found that the genus Flavoperla and Niponiella were gathered into one branch and the species of Perlinae were clustered in one clade.

Characterization of the complete mitochondrial genome of Nemoura meniscata Li & yang (Plecoptera: Nemouridae) from China.

We sequenced the third complete mitochondrial genome of Nemoura meniscataby using the high-throughput sequencing method. The mitochondrial genome harbored 37 typical code genes and a control region with 15,895 bp in length was a double-stranded and circular genome. The nucleotide composition is partial to A and T. Seventy-eight nucleotides were dispersed in 10 intergenic spacers and gene overlaps were also found at 13 gene junctions with 48 nucleotides. In phylogenetic trees, the 13 Nemouridae species form a clade diverged from the outgroup clade. The genus Nemoura and Amphinemura were sister groups which is consistent with the previous study.

The mitochondrial genome analysis of Sphaeronemoura elephas (Plecoptera: Nemouridae) from Jiangxi Province of southeastern China.

We sequenced and annotated the species of Sphaeronemoura elephas which represents the first record for continental China from Jiangxi Province in this study to provide mitochondrial genome data for future studies. The entire mitochondrial genome of S. elephas harbored 37 typical code genes and one control region with 15,846 bp in length. The A + T account of total nucleotide, PCGs, tRNAs, rRNAs and control region were 67.1, 64.5, 70.5, 71.0, 82.4%, respectively and the A + T content was the highest in control region. The start codon of all PCGs used ATN except ND5 and ND1 started with GTG and TTG. Eleven PCGs used typical terminal codon TAA or TAG while the COII and ND5 stopped with the single T. Based on 13 PCGs by using Bayesian (BI) and maximum-likelihood (ML) methods, we found that the genus Sphaeronemoura and Mesonemoura were sister groups and the species of Amphinemurinae was monophyletic group.

The mitochondrial genome of the stonefly Togoperla limbata Pictet (Plecoptera: Perlidae).

We sequenced the complete mitochondrial genomes of Togoperla limbata which is the unique species distributed in Japan to provide supplementary data for future study in phylogenetic studies of Perlidae. This complete mitogenome of T. limbata is 15,915 bp long, including 37 typical genes and a control region as other stoneflies. Through the data analysis, the A + T content of the whole mitogenome, PCGs, tRNAs, rRNAs, and control region was 63.7%, 61.3%, 68.7%, 66.8%, and 74.8%. Most PCGs used typical start or stop codon except COI, ND5, and ND1 used the exceptional start codon and the COII and ND5 used the single T as the stop codon. Phylogenetic tree was constructed by using Bayesian inference (BI) and maximum-likelihood (ML) analysis based on the sequences of the 13 PCGs and two rRNAs and the result showed that subfamily Perlinae was a monophyletic group and the clade Togoperla and Paragnetina had a closer relationship.