Toll-interacting protein participates in immunity and development of the lepidopteran insect Antheraea pernyi.

Toll-interacting protein (Tollip) participates in multiple biological processes. However, the biological functions of Tollip proteins in insects remain to be further explored. Here, the genomic sequence of tollip gene from Antheraea pernyi (named Ap-Tollip) was identified with a length of 15,060 bp, including eight exons and seven introns. The predicted Ap-Tollip protein contained conserved C2 and CUE domains and was highly homologous to those tollips from invertebrates. Ap-Tollip was highly expressed in fat body compared with other determined tissues. As far as the developmental stages were concerned, the highest expression level was found at the 14th day in eggs or the 3rd day of the 1st instar. Ap-Tollip was also obviously regulated by lipopolysaccharide, polycytidylic acid or 20E in different tissues. In addition, the interaction between Ap-Tollip and ubiquitin was confirmed by western blotting and pull-down assay. RNAi of Ap-Tollip significantly affected the expression levels of apoptosis and autophagy-related genes. These results indicated that Ap-Tollip was involved in immunity and development of A. pernyi.

Characterization and Phylogenetic Analysis of the Complete Mitochondrial Genome of Saturnia japonica.

The complete mitochondrial genome (mitogenome) of Saturnia japonica (Lepidoptera: Saturniidae) was sequenced and annotated. It is a circular molecule of 15, 376 bp, composed of 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNA), and an adenine (A) + thymine (T)-rich region. All protein-coding genes (PCGs) are initiated by the ATN codon except for cytochrome c oxidase subunit 1 (cox1) gene that is seemingly initiated by the CGA codon. Except for cox2 and nad4, which were terminated by incomplete stop codon T or TA, the rest were terminated by canonical stop codon TAA. The A + T-rich region is high conservative, including 'ATAGA' motif followed by a 19 bp poly-T stretch, a microsatellite-like element (AT)9 and also a poly-A element, with a total length of 332 bp. The Asn codon was the most frequently used codon, followed by Ile, Leu2, Lys, Met, Phe, and Tyr, while Cys was the least frequently used codon. Phylogenetic relationships analysis based on the 13 PCGs by using maximum likelihood (ML) and neighbor Joining (NJ) revealed that S. japonica belongs to the Saturniidae family. In this study, the annotation and characteristics of the mitogenome of S. japonica were resolved for the first time, which laid a foundation for species classification and the molecular evolution of Lepidoptera: Saturniidae.

Characterization of small GTPase Rac1 and its interaction with PAK1 in crayfish Procambarus clarkii.

Ras-related C3 botulinum toxin substrate 1 (Rac1) participates in many biological processes. In this study, a Rac1 gene was identified in the crayfish Procambarus clarkii with an open reading frame of 579 bp that encoded 192 amino acids. This predicted 21.4 kDa protein was highly homologous to those in other invertebrates. Real-time PCR analysis revealed that Pc-Rac1 was expressed in all examined tissues with the highest expression level in hemocytes. The transcriptional expression level of Pc-Rac1 was significantly upregulated in hemocytes and hepatopancreas after lipopolysaccharide (LPS) or polyinosinic: polycytidylic acid (poly I: C) induction. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis suggested that a recombinant Pc-Rac1 protein was successfully expressed in E. coli. Far-western blot analysis demonstrated that Rac1 can interact with the PBD domain of p21-activated kinase 1 (PAK1). RNA interference of Pc-Rac1 affected the mRNA expression levels of immune-related genes lectin, Toll, crustin, TNF, ALF and cactus. These results suggest that Pc-Rac1 is involved in the innate immune responses in P. clarkii.

Identification and function of a lebocin-like gene from the Chinese oak silkworm, Antheraea pernyi.

Antimicrobial peptides (AMPs) play important roles in the insect innate immune response. To investigate the role of a lebocin-like protein in the oak silkworm, Antheraea pernyi, in response to immune challenge, an Ap-lebocin-like gene with an open reading frame of 489 bp was identified. This gene encodes a protein of 162 amino acid residues and belongs to a family of proline-rich antimicrobial peptides. Real-time PCR analysis found that Ap-lebocin-like was expressed in all tested tissues, with the highest expression in the midgut, followed by the epidermis, and the lowest expression in the silk gland. Different transcription patterns of Ap-lebocin-like were observed in the fat body and midgut after injection of Escherichia coli, A. pernyi nucleopolyhedrovirus, Micrococcus luteus, and Beauveria bassiana. An antibacterial activity assay indicated that the Ap-lebocin-like has high antibacterial activity in vitro, with a greater activity toward gram-positive bacteria (Staphylococcus aureus) than toward gram-negative bacteria (E. coli). These results suggested that Ap-lebocin-like participates in the immune response of A. pernyi.

Role of Antheraea pernyi serpin 12 in prophenoloxidase activation and immune responses.

Serine protease inhibitors play a key role in the immune system of invertebrates by controlling proteolytic cascades. Besides its importance, the knowledge on immune functions of serpins in most of insects is fragmentary. In the present study, we identified serpin-12 from Antheraea pernyi encoding a predicted 402 amino acid residue protein (Apserpin-12). We expressed the recombinant protein in Escherichia coli and the purified protein was used for the synthesis of rabbit anti-Apserpin-12 polyclonal antibodies and functional studies. Quantitative real-time ploymerase chain reaction (qRT-PCR) analysis revealed that the knock-down of Apserpin-12 enhanced the prophenoloxidase (PPO) cascade stimulated by Micrococcus luteus in hemolymph, whereas addition of recombinant Apserpin-12 protein along with same elicitor led to down-regulate PPO activation. Following different microbial challenge (E. coli, Beauveria bassiana, M. Luteus, and nuclear polyhedrosis virus), the expression of Apserpin-12 mRNA was induced significantly. Furthermore, the Apserpin-12 double-stranded RNA administration elicited the expression of antimicrobial peptides, while the treatment with recombinant protein suppressed their expression. Tissue profile of Apserpin-12 indicated that it is expressed in all examined tissues, that is, hemolymph, malpighian tubules, midgut, silk gland, integument, and fat body with variation in their transcript levels. We concluded that Apserpin-12 may regulate PPO activation and inhibit the production of antimicrobial peptides in A. pernyi, suggesting important role in its immune system.

Characterization and functional analysis of serpin-28 gene from silkworm, Bombyx mori.

Serine protease inhibitors (Serpins) are a broadly distributed superfamily of proteins with a SERPIN domain and participate in several immune responses. In this study, a serpin-28 gene was identified in B. mori and its role in immune regulation was investigated. This gene has an open reading frame of 1065 bp that encodes a 354-amino acid residue polypeptide containing one SERPIN domain with a predicted molecular weight of 40.3 kDa. Recombinant Bmserpin-28 protein was expressed in Escherichia coli and used to raise rabbit anti-Bmserpin-28 polyclonal antibodies. Quantitative real-time PCR analysis revealed that Bmserpin-28 was expressed in all examined tissues, with maximum expression in the fat body and silk gland. Expression pattern of different developmental stages showed that the highest expression level was in the pupae, while the lowest expression level was recorded at the egg stage. After challenge with four different microorganisms (Escherichia coli, Beauveria bassiana, Micrococcus luteus and B. mori nuclear polyhedrosis virus), the expression pattern of Bmserpin-28 was investigated in fat body and haemocyte samples. A substantial upregulation of Bmserpin-28 expression level was recorded following pathogen challenge in both the tested tissues. Furthermore, RNA interference of Bmserpin-28 resulted in significant upregulation of antimicrobial peptide genes. In summary, our results indicated that Bmserpin-28 may be involved in the innate immunity of B. mori.

Mitochondrial genome of the sweet potato hornworm, Agrius convolvuli (Lepidoptera: Sphingidae), and comparison with other Lepidoptera species.

In the present study, we sequenced the complete mitochondrial genome (mitogenome) of Agrius convolvuli (Lepidoptera: Sphingidae) and compared it with previously sequenced mitogenomes of lepidopteran species. The mitogenome was a circular molecule, 15 349 base pairs (bp) long, containing 37 genes. The order and orientation of genes in the A. convolvuli mitogenome were similar to those in sequenced mitogenomes of other lepidopterans. All 13 protein-coding genes (PCGs) were initiated by ATN codons, except for the cytochrome c oxidase subunit 1 (cox1) gene, which seemed to be initiated by the codon CGA, as observed in other lepidopterans. Three of the 13 PCGs had the incomplete termination codon T, while the remainder terminated with TAA. Additionally, the codon distributions of the 13 PCGs revealed that Asn, Ile, Leu2, Lys, Phe, and Tyr were the most frequently used codon families. All transfer RNAs were folded into the expected cloverleaf structure except for tRNASer(AGN), which lacked a stable dihydrouridine arm. The length of the adenine (A) + thymine (T)-rich region was 331 bp. This region included the motif ATAGA followed by a 19-bp poly-T stretch and a microsatellite-like (TA)8 element next to the motif ATTTA. Phylogenetic analyses (maximum likelihood and Bayesian methods) showed that A. convolvuli belongs to the family Sphingidae.

Characterization and functional study of a Cecropin-like peptide from the Chinese oak silkworm, Antheraea pernyi.

In present study, a Cecropin-like peptide from Antheraea pernyi (ApCec) was cloned and characterized. The full-length ApCec cDNA encoded a protein with 64 amino acids including a putative 22-amino-acid signal peptide, a 4-amino-acid propeptide, and a 38-amino-acid mature peptide. ApCec gene was highly expressed in Malpighian tubules of A. pernyi after induction for 24 h by Escherichia coli in PBS. Pro-ApCec (including propeptide and mature peptide) and M-ApCec (just mature peptide) were synthesized chemically and analyzed by HPLC and mass spectroscopy. The antibacterial activity of M-ApCec is more potent than pro-ApCec against E. coli K12 or B. subtilus in both minimum inhibitory concentration and inhibition zone assays. Hemolytic assay results showed M-ApCec possessed a low cytotoxicity to mammalian cells. The secondary structure of M-ApCec forms α-helical structure, shown by circular dichroism spectroscopy. Transmission electron microscopy analysis suggested that M-ApCec killed bacteria by disrupting bacterial cell membrane integrity. Our results indicate ApCec may play an important role in defending from pathogenic bacteria in A. pernyi, and it may be as a potential candidate for applications in antibacterial drug development and agriculture.

Identification of ecdysteroid receptor-mediated signaling pathways in the hepatopancreas of the red swamp crayfish, Procambarus clarkii.

The hepatopancreas of crustaceans plays an important role in lipid and carbohydrate metabolism, digestion of food, and biogenesis. In this study, the hepatopancreas transcriptome from the red crayfish Procambarus clarkii was characterized for the first time using high-throughput sequencing, producing approximately 41.4 million reads were obtained. After de novo assembly, 57,363 unigenes with an average length of 725bp were identified, Gene Ontology analysis categorized 22,580 as being involved in biological processes, among which metabolic process and cellular process groups were the most highly enriched. A total of 8034 unigenes were assigned to 223 metabolic pathways following mapping against the Kyoto encyclopedia of genes and genomes (KEGG) database. Ecdysteroid receptor (EcR)-mediated signaling pathways were investigated using digital gene expression (DGE) analysis following RNA interference targeting the EcR. A total of 529 differentially expressed genes (DEGs) were identified, including 322 downregulated and 207 upregulated unigenes. Of these, 445 (84.12%) were annotated successfully by alignment with known sequences, many of which were related to catalytic activity and binding functional categories. Using KEGG enrichment analysis, 183 DEGs were clustered into 78 pathways, and six significantly enriched pathways were predicted. The expression patterns of candidate genes identified by real-time PCR were consistent with the DGE results.

Cathepsin O is involved in the innate immune response and metamorphosis of Antheraea pernyi.

Cathepsins are key members of mammalian papain-like cysteine proteases that play an important role in the immune response. In this study, a fragment of cDNA encoding cathepsin O proteinase (ApCathepsin O) was cloned from Antheraea pernyi. It contains an open reading frame of 1170bp and encodes a protein with 390 amino acid residues, including a conserved I29 inhibitor domain and a peptidase C1A (clan CA of cysteine proteases, papain family C1 subfamily) domain. Comparison with other previously reported cathepsin O proteins showed identity ranging from 45% to 79%. Quantitative real-time PCR (qRT-PCR) and Western blot analysis revealed that ApCathepsin O was highly expressed in the fat body; furthermore, the high expression during the pupal stage indicated that it might be involved during metamorphosis. After exposure to four different heat-killed pathogens (Escherichia coli, Beauveria bassiana, Micrococcus luteus, and A. pernyi nucleopolyhedrovirus), the expression levels of ApCathepsin O mRNA significantly increased and showed variable expression patterns. This indicates that ApCathepsin O is potentially involved in the innate immune system of A. pernyi. Interestingly, ApCathepsin O expression was upregulated after 20-hydroxyecdysone (20E) injection, which suggested that it might be regulated by 20E. In conclusion, ApCathepsin O is a protease that may play an important role in the innate immune response and metamorphosis of A. pernyi.

Gene expression patterns in response to pathogen challenge and interaction with hemolin suggest that the Yippee protein of Antheraea pernyi is involved in the innate immune response.

Yippee was first identified as a protein that physically interacts with the Hemolin protein of Hyalophora cecropia. In this study, we identified a gene with a 366bp open reading frame (ORF) that encodes a 121 amino acid protein containing a conserved Yippee domain. We named this gene Ap-Yippee (Yippee gene from Antheraea pernyi), and investigated the role of the protein in the host immune response. A recombinant Ap-Yippee protein was expressed in Escherichia coli cells, and polyclonal antibodies were produced against the recombinant protein. Real-time PCR and a Western blot analysis revealed that Ap-Yippee is expressed in the hemocytes, Malpighian tubules, midgut, silk gland, epidermis, and fat bodies of A. pernyi, with the highest expression level observed in Malpighian tubules. The fifth instar larvae of A. pernyi were challenged by injecting them with nucleopolyhedrovirus (AP-NPV), the Gram-negative bacterium E. coli, the Gram-positive bacterium Micrococcus luteus, or the entomopathogenic fungus, Beauveria bassiana. These challenges with diverse pathogens resulted in differential expression patterns of the protein. A knockdown of the Ap-Yippee gene by small interfering RNA (siRNA) transfection had a significant influence on the expression of the hemolin in the pupae which was confirmed by qRT-PCR and Western blot. Furthermore, a possible protein-protein interaction between Ap-Yippee and Hemolin was explored by Far-Western blotting. Therefore, our data suggest that the Ap-Yippee protein is involved in a pathway that regulates the immune response of insects.

Venom of the parasitoid wasp Pteromalus puparum contains an odorant binding protein.

Odorant binding proteins (OBPs) are crucial for insects to detect food, mates, predators, or other purposes. They are mostly located on antennae and other olfactory sensilla. In this study, we identified an OBP from the venom of Pteromalus puparum, designated as PpOBP. The cDNA of PpOBP is 517 bp in length, encoding 132 amino acids. Phylogenetic analysis revealed that PpOBP was clustered with OBP68 and OBP67 of Nasonia vitripennis. PpOBP was highly expressed in the venom apparatus at the transcriptional and translational levels. PpOBP was located in all parts of venom apparatus including venom gland, venom reservoir, and Dufour's gland. During 0-6 days post adult eclosion, the PpOBP mRNA level peaked at 2 days in the venom apparatus, whereas the protein remained at a high level. In the venom apparatus, the PpOBP mRNA was significantly upregulated following feeding with honey and parasitization. We propose that PpOBP is involved in parasitoid-host interactions.

IDENTIFICATION AND EXPRESSION ANALYSIS OF VITELLOGENIN RECEPTOR FROM THE WILD SILKWORM, Bombyx mandarina.

The vitellogenin receptor (VgR) plays a key role on embryonic development in oviparous animals. Here, we cloned a VgR gene, which was identified from the wild silkworm Bombyx mandarina (BmaVgR) using reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). Sequence analysis revealed that BmaVgR is 5,861 bp long with an open reading frame encoded by 1,811 amino acid residues. The predicted amino acid sequence has 99.7 and 98.2% identity with the VgRs of Actias selene and Bombyx mori, respectively. The class B domain sequence of BmaVgR was cloned and expressed in Escherichia coli, and purified by a Ni-NTA column. Polyclonal antibodies were produced against the purified recombinant protein, and titer of the antibody was about 1:12,800 measured by enzyme-linked immunosorbent assay (ELISA). Western blot and RT-qPCR showed that BmaVgR was expressed in the ovary and fat body of female larvae and the ovary of moth, and the expression level was highest at the third day and then declined from third day to seventh in fat body of pupa. After knockdown of the BmaVgR gene through RNA interference (RNAi), other three BmaVgR-related genes (Vg, egg-specific protein, and low molecular weight lipoprotein LP gene) were all downregulated significantly.

Molecular characterization of an Apolipophorin-III gene from the Chinese oak silkworm, Antheraea pernyi (Lepidoptera: Saturniidae).

Apolipophorin-III (ApoLp-III) acts in lipid transport, lipoprotein metabolism, and innate immunity in insects. In this study, an ApoLp-III gene of Antheraea pernyi pupae (Ap-ApoLp-III) was isolated and characterized. The full-length cDNA of Ap-ApoLp-III is 687 bp, including a 5'-untranslated region (UTR) of 40 bp, 3'-UTR of 86 bp and an open reading frame of 561 bp encoding a polypeptide of 186 amino acids that contains an Apolipophorin-III precursor domain (PF07464). The deduced Ap-apoLp-III protein sequence has 68, 59, and 23% identity with its orthologs of Manduca sexta, Bombyx mori, and Aedes aegypti, respectively. Phylogenetic analysis showed that the Ap-apoLp-III was close to that of Bombycoidea. qPCR analysis revealed that Ap-ApoLp-III expressed during the four developmental stages and in integument, fat body, and ovaries. After six types of microorganism infections, expression levels of the Ap-ApoLp-III gene were upregulated significantly at different time points compared with control. RNA interference (RNAi) of Ap-ApoLp-III showed that the expression of Ap-ApoLp-III was significantly downregulated using qPCR after injection of E. coli. We infer that the Ap-ApoLp-III gene acts in the innate immunity of A. pernyi.

Venom of the ectoparasitoid, Nasonia vitripennis, influences gene expression in Musca domestica hemocytes.

Insect hosts have evolved potent innate immunity against invasion by parasitoid wasps. Host/parasitoids live in co-evolutionary relationships. Nasonia vitripennis females inject venom into their dipteran hosts just prior to laying eggs on the host's outer integument. The parasitoid larvae are ectoparasitoids because they feed on their hosts within the puparium, but do not enter the host body. We investigated the influence of N. vitripennis venom on the gene expression profile of hemocytes of their hosts, pupae of the housefly, Musca domestica. We prepared venom by isolating venom glands and treated experimental host pupae with venom. We used suppression subtractive hybridization (SSH) to determine the influence of venom on hemocyte gene expression. At 1 h post treatment, we recorded decreases in transcript levels of 133 EST clones derived from forward a subtractive library of host hemocytes and upregulation in transcript levels of 111 EST clones from the reverse library. These genes are related to immune and stress response, cytoskeleton, cell cycle and apoptosis, metabolism, transport, and transcription/translation regulation. We verified the reliability of our data with reverse transcription quantitative real-time PCR analysis of randomly selected genes, and with assays of enzyme activities. These analyses showed that the expression level of all selected genes were downregulated after venom treatment. Outcomes of our experiments support the hypothesis that N. vitripennis venom influences the gene expression in host hemocytes. We conclude that the actions of venom on host gene expression influence host biology in ways that benefit the development and emergence of the next generation of parasitoids.

A Protein Asteroid with PIN Domain in Silkworm Bombyx mori Is Involved in Anti-BmNPV Infection.

Nuclease is a type of protein that degrades nucleic acids, which plays an important role in biological processes, including RNA interference efficiency and antiviral immunity. However, no evidence of a link between nuclease and Bombyx mori nucleopolyhedrovirus (BmNPV) infection in silkworm B. mori has been found. In this study, a protein asteroid (BmAst) containing the PIN domain and XPG domain was identified in silkworm B. mori. BmAst gene was highest expressed in hemocytes and fat body of the 5th instar larvae, and high expression in the pupa stage. The transcriptional levels of the BmAst gene in 5th instar larvae were significantly induced by BmNPV or dsRNA. After knocking down BmAst gene expression by specific dsRNA, the proliferation of BmNPV in B. mori was increased significantly, whereas the survival rate of larvae was significantly lower when compared with the control. Our findings indicate that BmAst is involved in silkworm resistance to BmNPV infection.

Complete biodegradation of the oldest organic herbicide 2,4-Dichlorophenoxyacetic acid by engineering Escherichia coli.

After nearly 80 years of extensive application, the oldest organic herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) has caused many problems of environmental pollution and ecological deterioration. Bioremediation is an ideal method for pollutant treatment. However, difficult screening and preparation of efficient degradation bacteria have largely hindered its application in 2,4-D remediation. We have created a novel engineering Escherichia coli with a reconstructed complete degradation pathway of 2,4-D to solve the problem of screening highly efficient degradation bacteria in this study. The results of fluorescence quantitative PCR demonstrated that all nine genes in the degradation pathway were successfully expressed in the engineered strain. The engineered strains can quickly and completely degrade 0.5 mM 2, 4-D within 6 h. Inspiring, the engineered strains grew with 2,4-D as the sole carbon source. By using the isotope tracing method, the metabolites of 2,4-D were found incorporated into the tricarboxylic acid cycle in the engineering strain. Scanning electron microscopy showed that 2,4-D had less damage on the engineered bacteria than the wild-type strain. Engineered strain can also rapidly and completely remedy 2,4-D pollution in natural water and soil. Assembling the metabolic pathways of pollutants through synthetic biology was an effective method to create pollutant-degrading bacteria for bioremediation.

A Kazal-type serine protease inhibitor mediates innate immunity in wild silkworm Actias selene Hübner.

The prophenoloxidase (PPO) activation system is an important innate immune defense mechanism in arthropods. Actias selene is a rare and important wild silk insect that can spin high-quality cocoon silk, but, other than its morphology, its molecular mechanism is rarely reported. Here, we report the purification and characterization of a novel KSPI gene from A. selene (AsKSPI, which can negatively regulate PPO activation. Its open reading frame (ORF) was 291 bp, encoding 96 amino acids. Real-time quantitative PCR (RT-qPCR) showed that AsKSPI mRNA was significantly expressed in the fat body. Immunostimulatory tests showed that the mRNA levels of AsKSPI in the fat body were up-regulated following injection of Micrococcus luteus, Escherichia coli, Beauveria bassiana, and nuclear polyhedrosis virus (NPV). Enzyme activity experiments showed that the purified recombinant AsKSPI could inhibit the activation of PPO in hemolymph of A. selene, but did not affect phenoloxidase (PO) activity after PPO had been activated. So, AsKSPI could regulate the innate immunity of A. selene through the PPO cascade. These findings will contribute to the understanding of the immune mechanism of wild silkworm and provide a basis for better protection and utilization of special economic insect resources.

Immunological Function of the Antibacterial Peptide Attacin-Like in the Chinese Oak Silkworm, Antheraea pernyi.

BACKGROUND: Antibacterial peptides play important roles in the innate immune system of insects and are divided into four categories according to their structures. Although many antibacterial peptides have been reported in lepidopteran insects, the roles of an attacin-like gene in immune response of Antheraea pernyi remain unclear. OBJECTIVE: In this study, the cloning and immunological functions of an attacin-like gene from Antheraea pernyi were investigated. METHODS: The open reading frame of Ap-attacin-like gene was cloned by PCR using the specific primers and then was ligated to the pET-32a vector to construct the recombinant plasmids Ap-attacin- like-pET-32a. The recombinant Ap-attacin-like protein was expressed in E. coli (BL21 DE3) cells and purified by Ni-NTA affinity chromatography. The expression patterns of Ap-attacin-like in different tissues or under microorganism challenges were investigated by real-time PCR and western blotting. Finally, agar well diffusion assay was performed to determine the antimicrobial activity of the recombinant Ap-attacin-like proteins based on the inhibition rate. RESULTS: The expression level of Ap-attacin-like was highest in the fat body compared with the other examined tissues. The expression of Ap-attacin-like in the fat body was significantly elevated after E. coli, Beauveria bassiana, Micrococcus luteus or Nuclear Polyhedrosis Virus challenges. In addition, the recombinant Ap-attacin-like proteins had obvious antibacterial activity against E. coli. CONCLUSION: Ap-attacin-like was highly expressed in immune-related tissues and its expression level was significantly induced by different microorganism challenges, suggesting that Ap-attacin-like participated in the innate immunity of A. pernyi.

The mitochondrial genome of Smerinthus planus (Lepidoptera: Sphingidae) and its comparative analysis with other Lepidoptera species.

In the present study, the complete mitochondrial genome of Smerinthus planus Walker (Lepidoptera: Sphingidae) was sequenced and analyzed to add additional traits for expanding our knowledge on systematics and phylogenetics of world-wide studied Sphingidae moths. The mitochondrial genome is a circular double-stranded DNA molecule, 15368 bp in size. It includes 13 protein-coding genes (PCGs), two ribosomal RNA (rRNA) genes, twenty-two transfer RNA (tRNA) genes, and an adenine (A) + thymine (T) rich region. All the PCGs start with the typical ATN start codons, except for the nad5 gene, which initiates with TTA. The codon usage analysis revealed that Phe, Ile, Lys, Leu, Asn, and Tys were the most common amino acids, while Cys and Trp were least common. Among the 13 PCGs, nine genes harbor the complete termination codon TAA, whereas the remaining four genes (nad1, cob, nad4, and nad3) terminate with TAG. The A+T rich region of S. planus is 318 bp. This region displays the highest A+T rich content, accounting for 91.50%, with both AT skew (-0.09) and GC skew (-0.26) are negative. Like other Lepidopterans, the A+T-rich region of the S. planus also contains some conserved regions, including the motif 'ATAGA' followed by an 18 bp poly-T stretch, a microsatellite-like (AT)8 and a poly-A element. Phylogenetic relationships, based on nucleotide sequences from the genomes of 31 species, confirmed that S. planus belong to the Sphingidae family. This study is aimed to improve the mitochondrial genome database of moths and provide valuable information for studying the genetic evolution and phylogeny of Lepidopteran species.