The Complete Mitochondrial Genome of Chilo infuscatellus (Lepidoptera: Pyralidae), and Related Phylogenetic Analysis.

The Chilo infuscatellus (Lepidoptera: Pyralidae) is a significant pest of sugarcane in China. The genome-level characteristics of this pest are important genetic resources for identification, phylogenetic analysis, and even management. In the present study, the complete mitogenome of C. infuscatellus was sequenced and characterized. The assembled mitochondrial genome is 15,252 bp in length and includes 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (rRNAs), and an A + T-rich region. Except for the CGA codon for the cox1 gene, the PCGs are initiated with ATN codons (ATG, ATT, and ATA). These PCGs are terminated with TAA or an incomplete termination codon of a single T. Except for the loss of the "DHU" arm for trnS1, the tRNA genes were folded into the typical cloverleaf structure. The A + T-rich region has a high AT content of 96.19% and contains the motifs "ATAGA" and "ATTTA", as well as a 19 bp poly-T stretch and microsatellite regions. The C. infuscatellus mitogenome exhibits a conserved gene order among lepidopteran insects, with a rearrangement of the trnM gene compared to the ancestral insect gene order. Phylogenetic analysis based on the 13 PCGs using Bayesian inference (BI) and maximum likelihood (ML) methods confirmed the monophyly of Pyralidae and Crambidae within Pyraloidea. The relationships between subfamilies in Pyralidae can be described as (Galleriinae + (Phycitinae + (Pyralinae + Epipaschiinae))). The "PS clade" and "non-PS clade" were formed within the family Crambidae. These findings provide valuable genetic resources for the identification, phylogenetic analysis, and management of sugarcane borers, contributing significantly to our understanding of the phylogeny of Pyraloidea insects and their evolution.

Deletion of IL-27p28 induces CD8 T cell immunity against colorectal tumorigenesis.

Colorectal cancer (CRC) is a leading cause of cancer-related deaths worldwide, characterized by molecular and clinical heterogeneity. Interleukin (IL)-27, a heterodimeric cytokine composed of p28 and EBI3 subunits, has been reported to exert potent antitumor activity in several cancer models. However, the precise role of IL-27 in the pathogenesis of CRC remains unclear. Here, we show that during the azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced CRC development, IL-27p28 levels are dramatically increased in peripheral blood and tumor tissues, and the cytokine is mainly produced by tumor-infiltrating myeloid cells. IL-27p28 deficient mice display tumor resistances in both inflammation-associated CRC model and syngeneic MC38 colon cancer model. Administration with IL-27p28 neutralizing antibody also reduces the tumor formation in AOM/DSS-treated mice. Mechanically, CD8+ T cells in IL-27p28-/- mice exhibit enhanced tumor infiltration and cytotoxicity, which can be largely attributed to activation of the Akt/mTOR signaling pathway. Furthermore, selective depletion of CD8+ T cells in IL-27p28-/- mice markedly accelerate tumor growth and almost abrogate the protective effects of IL-27p28 deficiency. Most interestingly, the expression of IL-27p28 is also upregulated in tumor tissues of CRC patients and those with high expression of IL-27p28 tend to have a poorer overall survival. Our results suggest that loss of IL-27p28 suppresses colorectal tumorigenesis by augmenting CD8+ T cell-mediated anti-tumor immunity. Targeting IL-27p28 could be developed as a novel strategy for the treatment of colorectal cancers.

Induced expression modes of genes related to Toll, Imd, and JAK/STAT signaling pathway-mediated immune response in Spodoptera frugiperda infected with Beauveria bassiana.

Spodoptera frugiperda is one of the most harmful pests that attack maize and other major food crops and causes huge economic loss every year in China and other countries and regions. Beauveria bassiana, a kind of entomological fungus that is highly pathogenic to pests, is harmless to the environment and human beings. However, at present, S. frugiperda has gradually developed resistance to many pesticides and microbial insecticides. In this study, transcriptome sequencing was conducted to analyze the differences in gene expression between B. bassiana-infected and -uninfected S. frugiperda. More than 160 Gb of clean data were obtained as 150-bp paired-end reads using the Illumina HiSeq™ 4000 platform, and 2,767 and 2,892 DEGs were identified in LH36vsCK36 and LH144vsCK144, respectively. In order to explore the roles of JAK/STAT, Toll, and Imd signaling pathways in antifungal immune response in S. frugiperda against B. bassiana infection, the expression patterns of those signaling pathway-related genes in B. bassiana-infected S. frugiperda were analyzed by quantitative real-time PCR. In addition, antifungal activity experiments revealed that the suppression of JAK/STAT, Toll, and Imd signaling pathways by inhibitors could inhibit the antifungal activity to a large extent and lead to increased sensitivity of S. frugiperda to B. bassiana infection, indicating that JAK/STAT, Toll, and Imd signaling pathways and their associated genes might be involved in the synthesis and secretion of antifungal substances. This study implied that JAK/STAT, Toll, and Imd signaling pathways played crucial roles in the antifungal immune response of the S. frugiperda larvae, in which the related genes of these signaling pathways could play special regulatory roles in signal transduction. This study would improve our understanding of the molecular mechanisms underlying innate immunity and provide the basis for a wide spectrum of strategies against antifungal resistance of S. frugiperda.

Novel gene rearrangement in the mitochondrial genome of Anastatus fulloi (Hymenoptera Chalcidoidea) and phylogenetic implications for Chalcidoidea.

The genus Anastatus comprises a large group of parasitoids, including several biological control agents in agricultural and forest systems. The taxonomy and phylogeny of these species remain controversial. In this study, the mitogenome of A. fulloi Sheng and Wang was sequenced and characterized. The nearly full-length mitogenome of A. fulloi was 15,692 bp, compromising 13 protein-coding genes (PCGs), 2 rRNA genes, 22 tRNA genes and a control region (CR). The total A + T contents were 83.83%, 82.18%, 87.58%, 87.27%, and 82.13% in the whole mitogenome, 13 PCGs, 22 tRNA genes, 2 rRNA genes, and CR, respectively. The mitogenome presented negative AT skews and positive GC skews, except for the CR. Most PCGs were encoded on the heavy strand, started with ATN codons, and ended with TAA codons. Among the 3736 amino acid-encoding codons, TTA (Leu1), CGA (Arg), TCA (Ser2), and TCT (Ser2) were predominant. Most tRNAs had cloverleaf secondary structures, except trnS1, with the absence of a dihydrouridine (DHU) arm. Compared with mitogenomes of the ancestral insect and another parasitoid within Eupelmidae, large-scale rearrangements were found in the mitogenome of A. fulloi, especially inversions and inverse transpositions of tRNA genes. The gene arrangements of parasitoid mitogenomes within Chalcidoidea were variable. A novel gene arrangement was presented in the mitogenome of A. fulloi. Phylogenetic analyses based on the 13 protein-coding genes of 20 parasitoids indicated that the phylogenetic relationship of 6 superfamilies could be presented as Mymaridae + (Eupelmidae + (Encyrtidae + (Trichogrammatidae + (Pteromalidae + Eulophidae)))). This study presents the first mitogenome of the Anastatus genus and offers insights into the identification, taxonomy, and phylogeny of these parasitoids.

Transcriptome responses to heat and cold stress in prepupae of Trichogramma chilonis.

Trichogramma is a useful species that is widely applied in biocontrol. Temperature profoundly affects the commercial application of T. chilonis. Different developmental transcriptomes of prepupae and pupae of T. chilonis under 10, 25, and 40°C were obtained from our previous study. In this study, transcriptomic analysis was further conducted to gain a clear understanding of the molecular changes in the prepupae of T. chilonis under different thermal conditions. A total of 37,295 unigenes were identified from 3 libraries of prepupae of T. chilonis, 17,293 of which were annotated. Differential expression analysis showed that 408 and 108 differentially expressed genes (DEGs) were identified after heat and cold treatment, respectively. Under heat stress, the pathway of protein processing in endoplasmic reticulum was found to be active. Most of the genes involved in this pathway were annotated as lethal (2) essential for life [l(2)efl] and heat shock protein genes (hsps), which were both highly upregulated. Nevertheless, most of the genes involved in another significantly enriched pathway of starch and sucrose metabolism were downregulated, including 1 alpha-glucosidase gene and 2 beta-glucuronidase genes. Under cold stress, no significantly enriched pathway was found, and the significantly enriched GO terms were related to the interaction with host and immune defenses. Together, these results provide us with a comprehensive view of the molecular mechanisms of T. chilonis in response to temperature stresses and will provide new insight into the mass rearing and utilization of T. chilonis.

Genetic Diversity and Population Structure of Gynaephora qinghaiensis in Yushu Prefecture, Qinghai Province Based on the Mitochondrial COI Gene.

Gynaephora qinghaiensis (Lepidoptera: Lymantriidae: Gynaephora), a serious economic pest in alpine meadows, is mainly distributed in Yushu prefecture, Qinghai province, China. In this study, we aimed to investigate the genetic diversity and population structure of G. qinghaiensis through analyzing the sequence of 194 mitochondrial cytochrome oxidase subunit (COI) genes (658 bp in length) identified from 10 geographic populations located in three different countries, including Zhiduo, Zaduo, and Chengduo, of Yushu prefecture. Eleven haplotypes were identified from all populations of G. qinghaiensis with high levels of haplotype diversity (0.78500) and low levels of nucleotide diversity (0.00511). High levels of genetic differentiation and low levels of gene flow were also detected among the populations of G. qinghaiensis. Analysis of molecular variance (AMOVA) showed that 90.13% of the variation was attributed to distribution among groups (Chengduo, Zhiduo, and Zaduo), and 5.22% and 4.65% were, respectively, attributed to distribution among populations, within group, and within populations. The result of mantel test showed a highly significant positive correlation (P < 0.01) between FST and geographical distance. A maximum likelihood tree showed that most haplotypes were grouped into three clusters corresponding to the three counties, suggesting a significant phylogeographic structure in the populations of G. qinghaiensis. The haplotype networks revealed that H2 may be the most primitive haplotype and the most adaptable in nature. Populations 7# and 8# had haplotype H2 and higher haplotype diversity; therefore, we speculated that the G. qinghaiensis in both populations were more adaptable to the environment and had greater outbreak potential and, therefore, should be focused on in terms of prevention and control. Our findings provide valuable information for further study of the population structure and phylogeny of G. qinghaiensis and provide a theoretical basis for the control of G. qinghaiensis.

Transcriptome Characterization and Expression Analysis of Chemosensory Genes in Chilo sacchariphagus (Lepidoptera Crambidae), a Key Pest of Sugarcane.

Insect chemoreception involves many families of genes, including odourant/pheromone binding proteins (OBP/PBPs), chemosensory proteins (CSPs), odourant receptors (ORs), ionotropic receptors (IRs), and sensory neuron membrane proteins (SNMPs), which play irreplaceable roles in mediating insect behaviors such as host location, foraging, mating, oviposition, and avoidance of danger. However, little is known about the molecular mechanism of olfactory reception in Chilo sacchariphagus, which is a major pest of sugarcane. A set of 72 candidate chemosensory genes, including 31 OBPs/PBPs, 15 CSPs, 11 ORs, 13 IRs, and two SNMPs, were identified in four transcriptomes from different tissues and genders of C. sacchariphagus. Phylogenetic analysis was conducted on gene families and paralogs from other model insect species. Quantitative real-time PCR (qRT-PCR) showed that most of these chemosensory genes exhibited antennae-biased expression, but some had high expression in bodies. Most of the identified chemosensory genes were likely involved in chemoreception. This study provides a molecular foundation for the function of chemosensory proteins, and an opportunity for understanding how C. sacchariphagus behaviors are mediated via chemical cues. This research might facilitate the discovery of novel strategies for pest management in agricultural ecosystems.

Prepupae and pupae transcriptomic characterization of Trichogramma chilonis.

The egg parasitoid, Trichogramma chilonis, has significant control effects on agriculture and forestry pests and is widely employed in southern China for the biological control of lepidopteran pests. In this study, transcriptomic analysis was used to gain a clear understanding of the molecular changes in prepupae and pupae of T. chilonis. A total of 16.88 Gb of clean data were obtained and finally assembled into 43,136 unigenes, 18,880 of which were annotated. After FPKM standardization, 117 and 838 specific expression genes were found in prepupae and pupae, respectively. There were 3129 differentially expressed genes between prepupae and pupae. Compared to pupae, 806 genes were up-regulated and 2323 were down-regulated in prepupae. Background on the T. chilonis transcriptome, the enriched GO function and KEGG pathway analysis of DEGs were considered. As indicated by GO classification, up-regulated genes were mainly involved in chitin metabolism, cell adhesion and endocytic, while most down-regulated genes were involved in synthesis of cell components, ion transport and biological regulation. KEGG enrichment analysis showed that 458 DEGs were enriched in 94 metabolic pathways. DEGs involved in nucleotide replication and transcription, substance metabolism, insect hormone biosynthesis, cell growth and death, reproductive metabolism, circadian rhythms and signal transduction pathways were up-regulated or down-regulated to different degrees, indicating that these genes played important roles during the process of metamorphosis in T. chilonis. This study provides a rich data source for the future study of T. chilonis molecular and biological mechanisms. A large number of genes related to metamorphosis were found based on comparison analysis between prepupae and pupae transcriptomes. This study lays a good foundation for in-depth study of gene transcription and regulation mechanisms during T. chilonis metamorphosis.

Identification and functional characterization of D-fructose receptor in an egg parasitoid, Trichogramma chilonis.

In insects, the gustatory system has a critical function not only in selecting food and feeding behaviours but also in growth and metabolism. Gustatory receptors play an irreplaceable role in insect gustatory signalling. Trichogramma chilonis is an effective biocontrol agent against agricultural insect pests. However, the molecular mechanism of gustation in T. chilonis remains elusive. In this study, we found that T. chilonis adults had a preference for D-fructose and that D-fructose contributed to prolong longevity and improve fecundity. Then, We also isolated the full-length cDNA encoding candidate gustatory receptor (TchiGR43a) based on the transcriptome data of T. chilonis, and observed that the candidate gustatory receptor gene was expressed from the larval to adult stages. The expression levels of TchiGR43a were similar between female and male. A Xenopus oocyte expression system and two-electrode voltage-clamp recording further verified the function analysis of TchiGR43a. Electrophysiological results showed that TchiGR43a was exclusively tuned to D-fructose. By the studies of behaviour, molecular biology and electrophysiology in T. chilonis, our results lay a basic fundation of further study on the molecular mechanisms of gustatory reception and provide theoretical basis for the nutritional requirement of T. chilonis in biocontrol.

Molecular characterization and expression of six heat shock protein genes in relation to development and temperature in Trichogramma chilonis.

Trichogramma is a kind of egg parasitoid wasp that is widely used to control lepidopterous pests. Temperature is one of the main factors that determines the various life activities of this species, including development, reproduction and parasitism efficiency. Heat shock proteins (HSPs) are highly conserved and ubiquitous proteins that are best known for their responsiveness to temperature and other stresses. To explore the potential role of HSPs in Trichogramma species, we obtained the full-length cDNAs of six HSP genes (Tchsp10, Tchsp21.6, Tchsp60, Tchsp70, Tchsc70-3, and Tchsp90) from T. chilonis and analyzed their expression patterns during development and exposure to temperature stress. The deduced amino acid sequences of these HSP genes contained the typical signatures of their corresponding protein family and showed high homology to their counterparts in other species. The expression levels of Tchsp10, Tchsp21.6 and Tchsp60 decreased during development. However, the expression of Tchsc70-3 increased from the pupal stage to the adult stage. Tchsp70 and Tchsp90 exhibited the highest expression levels in the adult stage. The expression of six Tchsps was dramatically upregulated after 1 h of exposure to 32 and 40°C but did not significantly change after 1 h of exposure to 10 and 17°C. This result indicated that heat stress, rather than cold stress, induced the expression of HSP genes. Furthermore, the expression of these genes was time dependent, and the expression of each gene reached its peak after 1 h of heat exposure (40°C). Tchsp10 and Tchsp70 exhibited a low-intensity cold response after 4 and 8 h of exposure to 10°C, respectively, but the other genes did not respond to cold at any time points. These results suggested that HSPs may play different roles in the development of this organism and in its response to temperature stress.

Transcriptome characterization and gene expression analysis related to chemoreception in Trichogramma chilonis, an egg parasitoid.

Chemoreception is critical for the survival of insects. Insects have a variety of behavioral responses, such as mating, host searching and ovipositing, in response to different odor signals detected in their living environment. Trichogramma chilonis, an egg parasitoid, acts as an efficient and effective biocontrol reagent for many agricultural and forestry insect pests in many parts of China. However, little is known about the molecular mechanism of the olfaction-evoked behavior in T. chilonis. In the present study, we conducted transcriptome profiling analysis of T. chilonis based on the Illumina high-throughput sequencing platform in order to explore differences of chemoreception between male and female T. chilonis. In this study, a total of 85 chemosensory genes were identified from transcriptomic data, including 45 odorant receptors (ORs), 22 odorant binding proteins (OBPs), 14 ionotropic receptors (IRs), 2 sensory neuron membrane proteins (SNMPs) and 2 chemosensory proteins (CSPs). From the analysis of the transcriptome, most of the candidate olfactory genes had similar expression levels in males and females, including a few OR and OBP genes (TchiOR38, TchiOR39, TchiOR40, TchiOR41, TchiOR42, TchiOR43, TchiOR44, TchiOR45, TchiOBP1, TchiOBP4, TchiOBP10, TchiOBP12, TchiOBP18 and TchiOBP19) which showed male-biased expression. Some annotated unigenes were chosen randomly to have qRT-PCR, which verified the correctness of analysis of transcriptome in T. chilonis. This is the first study to obtain and identify candidate genes related to chemoreception in T. chilonis. Our work lays a solid foundation for related future research on the chemosensory system of T. chilonis at the molecular level and helps advance the use of T. chilonis as biological control agents.

Molecular Characterization and Expression of Vitellogenin and Vitellogenin Receptor of Thitarodes pui (Lepidoptera: Hepialidae), an Insect on the Tibetan Plateau.

Vitellogenin (Vg) and vitellogenin receptor (VgR) play important roles in the vitellogenesis of insects. In this study, we cloned and characterized the two corresponding genes (TpVg and TpVgR) in an economically important insect, Thitarodes pui (Lepidoptera: Hepialidae), from the Tibetan plateau. The full length of TpVg is 5566 bp with a 5373 bp open reading frame (ORF) encoding 1,790 amino acids. Sequence alignment revealed that TpVg has three conserved domains: a Vitellogenin_N domain, a DUF1943 domain, and a von Willebrand factor type D domain (VWD). The full length of TpVgR is 5732 bp, with a 5397 bp ORF encoding 1798 amino acids. BLASTP showed that TpVgR belongs to the low-density lipoprotein receptor (LDLR) gene superfamily. Structural analysis revealed that TpVgR has a group of four structural domains: a ligand-binding domain (LBD), an epidermal growth factor (EGF)-precursor homology domain, a transmembrane (TM) domain, and a cytoplasmic domain. In addition, TpVgR has four cysteine-rich LDL repeats in the first ligand-binding site and seven in the second. Quantitative real-time polymerase chain reaction analysis revealed that the expression levels of TpVg and TpVgR are much higher in later pupa than in either the larval or adult stage, implying that the synthesis and uptake of Vg in T. pui occurs in the later pupal stage. These results will help us to understand the molecular mechanism of the reproductive capacity and will provide new insight into the mass rearing and utilization of T. pui.

Transcriptome characterization and gene expression analysis related to sexual dimorphism in the ghost moth, Thitarodes pui, a host of Ophiocordyceps sinensis.

Thitarodes pui is one of the host species of the Chinese caterpillar fungus Ophiocordyceps sinensis as a traditional Chinese medicine with economic and medical importance. The pupal and adult stages of T. pui are sexually dimorphic. In order to elucidate the molecular mechanisms involved in the sexually dimorphic development of T. pui, we compared the transcriptomes of female and male pupae and adults. We obtained 15,881,734, 16,962,086, 17,514,743, and 17,770,904 clean reads from female pupae, male pupae, female adults, and male adults, respectively. The reads obtained from the four samples were pooled and assembled into 65,165 unigenes, 23,597 of which were annotated. Candidate genes involved in sexual development were identified and analysed. Gene expression analysis revealed that 1406 genes were differentially expressed in male and female pupae, 448 of which were up-regulated in males and 958 were up-regulated in females. A total of 2025 genes were differentially expressed in male and females adults, 1304 of which were up-regulated in males and 721 were up-regulated in females. The functional enrichment of the differentially expressed genes indicated that reproduction and cuticle synthesis were regulated differently between the sexes. The transcriptome data obtained provide significant information regarding the genes involved in sexually dimorphic development, which will improve our understanding of the molecular mechanisms related to sexual dimorphism and helpful for the moth mass rearing which would provide enough host insects for the sustainable utilization of O. sinensis.

Complete mitochondrial genome of Aleuroglyphus ovatus (Acari: Acaridae).

In this study, the complete mitochondrial genome (mitogenome) sequence of Aleuroglyphus ovatus (Acarinae: Aleuroglyphus) was determined by long PCR and primer walking methods. The complete mitochondrial genome is 14,305 bp in length and contains 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes as well as a displacement loop (D-loop). The overall base composition of the genome is A (23.61%), T (44.60%), C (13.03%) and G (18.76%) respectively. Stop codon was missed for the CYTB gene and the gene overlaps were suggested between ten pairs of the contiguous genes in A. ovatus. The mitogenome would contribute to resolving phylogenetic position and interrelationships of Aleuroglyphus.

Complete mitochondrial genome sequence of Napialus hunanensis (Lepidoptera: Hepialidae), the host insect of Cordyceps hawkesii.

Although Napialus hunanensis is important as the host of Cordyceps hawkesii (a valuable medicine in China), it remains poorly known especially on molecular level. To get a better understanding of it, the complete mitogenome was sequenced and annotated. It was 15,301 bp in length, contained 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and an AT rich region. The base composition was 40.68% for A, 41.17% for T, 7.56% for G and 10.59% for C on the heavy strand. Except the COI with CGA and ND1 with TTG, the remaining protein-coding genes started with typical ATN. These coding genes ended up with TAA as well as an incomplete stop codon T (COII and ND3). According to other Hepialidae species, the rearrangement of tRNA(Met) in N. hunanensis is the ancestral type. The mitochondrial genome of N. hunanensis is an important source for species identification and phylogenetic analyses.

Complete mitochondrial genome of Thitarodes pui (Lepidoptera: Hepialidae).

To know the genetic structure and phylogeny status, the complete mitochondrial genome of Thitarodes pui was the first time to be sequenced. The genome was 15,064 bp in length and contained 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, as well as an AT rich region. The base composition of the genome was A (40.93%), T (39.80%), C (11.72%) and G (7.55%), with an AT content of 80.73%. It is interesting to that the COI would start with CGA. The tRNA order between ND2 and AT rich region was tRNA(Ile)-tRNA(Gln)-tRNA(Met), which confirms the thesis that Thitarodes has the ancestral gene arrangement. It is surprised that AT-rich region was 287 bp in length and became the shortest in contrast to other insects of Hepialidae.

SEASONAL CHANGES OF FATTY ACID COMPOSITION IN Thitarodes pui Larvae, A HOST OF Ophiocordyceps sinensis.

BACKGROUND: Thitarodes larvae are the host of Ophiocordyceps sinensis and exist in the permafrost region of the Tibetan Plateau. OBJECTIVE: To understand the adaptation mechanism of Thitarodes larvae to seasonal fluctuations of ambient temperatures in the Tibetan Plateau by studying seasonal changes of the fatty acids composition in the larvae of T. pui. MATERIALS AND METHODS: The profile of fatty acids in the 6th instar T. pui larvae collected at the mid-month in a whole year were examined by GC-MS. RESULTS: There was a negative correlation between the total lipid and ambient (soil) temperature. Further study indicated that oleic, palmitic, linoliec, palmitoleic, stearic were the major fatty acids. The ratio of unsaturated fatty acids to saturated fatty acids (U/S) and the unsaturated index (UI) in triacylglycerols remain stable during the whole year, while the U/S and UI in phospholipids vary dramatically in response to soil temperature. CONCLUSION: The fluctuations in phospholipids were attributed to seasonal changes of oleic and linoleic. The changes of the fatty acid composition may result from their adaptation to the variation of temperature in different seasons.