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Background: Caligula japonica, commonly known as Japanese giant silkworm (JGS), is a serious defoliating pest of fruit and forest trees in East Asia. To develop eco-friendly and cost-effective control methods for this pest, we evaluated the potential for biological control of JGS using its egg parasitoid Anastatus gansuensis reared on the Chinese oak silkworm (COS) Antheraea pernyi. We compared the reproductive traits and population increase potential of the parasitoid on JGS and COS eggs, as well as its functional response to host egg densities and mutual interference at different parasitoid densities., Results: Anastatus gansuensis was confirmed to be strictly synovigenic, with most eggs maturing post-emergence, and produced <1% male offspring on both host eggs. Although A. gansuensis females reared from COS eggs had longer longevity and oviposition period, and higher fecundity and net reproductive rate compared to those reared from JGS eggs, the parasitoid had a higher intrinsic rate of increase on JGS than COS eggs. The parasitoid exhibited a type II functional response to increasing host densities, with mutual interference among foraging female wasps occurred at higher parasitoid densities., Conclusion: Our results indicate a high potential for biological control of JGS using A. gansuensis. The parasitoid can be efficiently reared on COS eggs and used against JGS. It may be essential to provide food for emerging adult parasitoids allowing time for egg maturation prior to the rearing or augmentative release of the parasitoid. Some mutual interference at high parasitoid rearing densities likely reduces per capita parasitization efficiency of A. gansuensis. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

The egg parasitoid Mesocomys trabalae Yao, Yang, and Zhao is used as a biocontrol agent against the emerging defoliator pest Caligula japonica Moore in East Asia. It has been proven that the eggs of Antheraea pernyi Guérin-Méneville can be used as a factitious host for the mass production of M. trabalae. We examined the parasitic behavior and morphological characteristics of the developmental stages of M. trabalae reared on A. pernyi eggs. The parasitic behavior of M. trabalae encompasses 10 steps, involving searching, antennation, locating the oviposition site, drilling, probing, detecting, oviposition, host feeding, grooming, and resting, with the oviposition step further divided into 3 stages. We determined that the parasitoid released an egg during the second stage of the oviposition step, while her body remained in a relatively static state. Among all the steps in parasitic behavior, probing occupied the longest time, accounting for 26.33% of the entire parasitism process. It was followed by oviposition (15.88%), drilling (15.10%), antennation (13.09%), detecting (10.79%), host feeding (10.02%), and the remaining steps, each occupying less than 5.00% of the total time in steps. The pre-emergence of adult M. trabalae comprised of 4 stages: egg (0-1 day), larva (2-6 days), prepupa (7-11 days), pupa (12-20 days), followed by the development into an adult, and it usually took 20-22 days to develop from an egg into an adult at 25°C. This study advances our understanding of the biology of Mesocomys parasitoids and their mass-rearing for use in augmentation programs., (© The Author(s) 2024. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Detection of sex pheromones of insects relies on the antennae. The female pheromone signal transmission in the male antennae ultimately initiates the courtship and mating behaviors of males. To investigate the proteins and metabolites involved in this neural transduction, integrative proteomics and metabolomics analysis including tandem mass tag (TMT) proteomic quantification and liquid chromatography tandem mass spectrometry (LC/MS)-based metabolomics was adopted for comparing proteomic and metabolic changes between the antennae of male moths following stimulation by females and the non-stimulated males of Antheraea pernyi (Guérin-Méneville, Lepidoptera: Saturniidae) in this study. A total of 92 differentially expressed proteins (DEPs) containing 52 upregulated and 40 downregulated proteins and 545 differentially expressed metabolites (DEMs) including 218 upregulated and 327 downregulated metabolites were identified from the antennae of female-stimulated male moths based on the proteome and metabolome data, respectively. Bioinformatics analysis was performed for the 45 DEPs and 160 DEMs, including Gene Ontology (GO), Clusters of Orthologous Groups (COG), and Kyoto Encylopaedia of Genes and Genomes (KEGG) enrichment analysis and Human Metabolome Database (HMDB) annotation. A number of DEPs and DEMs related to neural transmission of female pheromone signals in the male antennae of A. pernyi were screened, including tyrosine hydroxylase, cryptochrome-1, tachykinin, arylalkylamine N-acetyltransferase, cadherin-23, glutathione S-transferase delta 3, tyramine, tryptamine, n-oleoyl dopamine, n-stearoyl dopamine, and n-stearoyl tyrosine. The altered expression levels of those proteins or metabolites were speculated involved in regulating the neuron activity for enhanced transmission of neural impulses and continuous perception, reception, and transduction of female pheromone signals. Our findings yielded novel insights into the potential mechanisms in the antennae of male A. pernyi responding to female attraction., (© The Author(s) 2023. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

We present a detailed analysis of the brain anatomy of two saturniid species, the cecropia silk moth, Hyalophora cecropia, and the Chinese oak silk moth, Antheraea pernyi, including 3D reconstructions of the major brain neuropils in the larva and in male and female adults. The 3D reconstructions, prepared from high-resolution optical sections, showed that the corresponding neuropils of these saturniid species are virtually identical. Similarities between the two species include a pronounced sexual dimorphism in the adults in the form of a male-specific assembly of markedly enlarged glomeruli forming the so-called macroglomerular complex. From the reports published to date, it can be concluded that the neuropil architecture of saturniids resembles that of other nocturnal moths, including the sibling family Sphingidae. In addition, compared with previous anatomical data on diurnal lepidopteran species, significant differences were observed in the two saturniid species, which include the thickness of the Y-tract of the mushroom body, the size of the main neuropils of the optic lobes, and the sexual dimorphisms of the antennal lobes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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 .

Neuropeptides and neuropeptide receptors are crucial regulators for the behavior, lifecycle, and physiology of insects and are mainly produced and released from the neurosecretory cells of the central nervous system (CNS). In this study, RNA-seq was employed to investigate the transcriptome profile of the CNS which is composed of the brain and ventral nerve cord (VNC) of Antheraea pernyi. From the data sets, a total of 18 and 42 genes were identified, which respectively encode the neuropeptides and neuropeptide receptors involved in regulating multiple behaviors including feeding, reproductive behavior, circadian locomotor, sleep, and stress response and physiological processes such as nutrient absorption, immunity, ecdysis, diapause, and excretion. Comparison of the patterns of expression of those genes between the brain and VNC showed that most had higher levels of expression in the brain than VNC. Besides, 2760 differently expressed genes (DEGs) (1362 up-regulated and 1398 down-regulated ones between the B and VNC group) were also screened and further analyzed via gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) enrichment analyses. The results of this study could provide comprehensive profiles of the neuropeptides and neuropeptide receptors of A. pernyi CNS and lay the foundation for further research into their functions., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interests., (Copyright © 2023 Elsevier B.V. All rights reserved.)

For a half-century, the commercial wild silkworm, Antheraea pernyi , has been protected by coumaphos, which is an internal organophosphorus insecticide used to kill the potential parasitic fly larvae inside. Knowledge about the detoxification genes of A. pernyi as well as the detoxification mechanism for this species remains severely limited. In this study, we identified 281 detoxification genes (32 GSTs, 48 ABCs, 104 CYPs, and 97 COEs) in the genome of this insect, which are unevenly distributed over 46 chromosomes. When compared to the domesticated silkworm, Bombyx mori , a lepidopteran model species, A. pernyi has a similar number of ABCs, but a greater number of GSTs, CYPs, and COEs. By transcriptome-based expression analysis, we found that coumaphos at a safe concentration level significantly changed the pathways related to ATPase complex function and the transporter complex in A. pernyi . KEGG functional enrichment analysis indicated that protein processing in the endoplasmic reticulum was the most affected pathway after coumaphos treatment. Finally, we identified four significantly up-regulated detoxification genes ( ABCB1 , ABCB3 , ABCG11 , and ae43 ) and one significantly down-regulated detoxification gene ( CYP6AE9 ) in response to coumaphos treatment, suggesting that these five genes may contribute to detoxification of coumaphos in A. pernyi . Our study provides the first set of detoxification genes for wild silkworms from Saturniidae and highlights the importance of detoxification gene repertoire in insect pesticide tolerance.

Antheraea pernyi is one of the most famous edible and silk-producing wild silkworms of Saturniidae. Structural cuticular proteins (CPs) are the primary component of insect cuticle. In this paper, the CPs in the genome of A. pernyi were identified and compared with those of the lepidopteran model species Bombyx mori , and expression patterns were analyzed based on the transcriptomic data from the larval epidermis/integument (epidermis in the following) and some non-epidermis tissues/organs of two silkworm species. A total of 217 CPs was identified in the A. pernyi genome, a comparable number to B. mori (236 CPs), with CPLCP and CPG families being the main contribution to the number difference between two silkworm species. We found more RR-2 genes expressed in the larval epidermis of fifth instar of A. pernyi than B. mori , but less RR-2 genes expressed in the prothoracic gland of A. pernyi than B. mori , which suggests that the hardness difference in the larval epidermis and prothoracic gland between the two species may be caused by the number of RR-2 genes expressed. We also revealed that, in B. mori , the number of CP genes expressed in the corpus allatum and prothoracic gland of fifth instar was higher than that in the larval epidermis. Our work provided an overall framework for functional research into the CP genes of Saturniidae.

Background: Chouioia cunea is a generalist pupal endoparasitoid. Native to Eurasia, the parasitoid has been mass-reared on an alternative lepidopteran host (Antheraea pernyi) to successfully control the exotic invasive lepidopteran pest Hyphantria cunea in China. To solicit more accessible hosts, this study evaluated the potential of an easily cultured coleopteran beetle (Tenebrio molitor) as a substitute for rearing C. cunea through comparing the relative performance of the parasitoids reared from both alternative hosts on H. cunea., Results: Compared with those reared from A. pernyi, the parasitoids reared from T. molitor (i.e., T. molitor vs. A. pernyi groups) performed equally well in terms of parasitism rate (94.4 vs. 88.9%), number of offspring produced per parasitized host (278 vs. 286), and female body length (1.334 vs. 1.351 mm), hind-tibia length (0.322 vs. 0.324 mm) and number of mature oocytes in the ovarioles (171 vs. 187), or even better based on offspring pre-emergence time (16.0 vs 16.9 days) and percentages of emerged offspring (99.8 vs. 99.1%) and female offspring (97.1 vs. 91.3%). Flight performance testing indicated that young C. cunea adults emerged from T. molitor had a similar percentage of actively flying wasps (76.9 vs. 72.9%) and a lower percentage of inactive wasps (2.3 vs. 10.6%) when compared to those reared from A. pernyi., Conclusion: Given the remarkable adaptability of C. cunea and the vast availability of T. molitor as a common resource insect worldwide, this indigenous parasitoid could be mass-reared on T. molitor to improve the prospect of biological control of H. cunea in its invaded regions. © 2022 Society of Chemical Industry., (© 2022 Society of Chemical Industry.)

To evaluate the wound-healing effect of Antheraea pernyi epidermal growth factor (ApEGF), we performed the sequence analysis, cloning, and prokaryotic expression of cDNA from the ApEGF gene, examined the transcriptional changes, and investigated the wound-healing effect of this protein in cells and rat epidermis. Primers were designed based on available sequence information related to the ApEGF gene in a public database, and part of the ApEGF sequence was obtained. The full-length cDNA sequence of ApEGF was obtained using inverse PCR. The gene sequence fragment of ApEGF was 666 bp in length, encoding 221 amino acids, with a predicted protein mass of 24.19 kD, an isoelectric point of 5.15, and no signal peptide sequence. Sequence homology analysis revealed 86.1% sequence homology with Bombyx mori , 92.7% with Manducal sexta , 92.6% with Trichoplusia ni , and 91.8% with Helicoverpa armigera . ApEGF was truncated and then subjected to prokaryotic expression, isolation, and purification. Truncated ApEGF was used for wound-healing experiments in vitro and in vivo. The results showed that after 48 h, transforming growth factor (TGF)-β1 had 187.32% cell growth effects, and the ApEGF group had 211.15% cell growth compared to the control group in vitro. In rat epidermis, truncated ApEGF showed a significantly better healing effect than the control. This result indicated that ApEGF, which exerted a direct wound-healing effect, could be used in wound-healing therapy.

Inhibitor of apoptosis protein 1 (IAP1) of Antheraea pernyi multinucleocapsid nucleopolyhedrovirus (AnpeNPV) belongs to the baculovirus IAP1 type. The function of AnpeNPV-IAP1 in viral replication and occlusion body (OB) production remains unknown. In this study, we demonstrated that AnpeNPV-iap1 is a late gene. AnpeNPV-IAP1 mainly localizes to the nuclear ring zone and exhibits dynamic distribution in the cytoplasm and the virogenic stroma during AnpeNPV infection. AnpeNPV-IAP1 impacted the expression of a variety of viral genes at the very late phase of infection in Tn-Hi5 cells. The deletion of AnpeNPV-iap1 caused decreased expression levels of polyhedrin, morphological changes to OBs and reduced OB production in A. pernyi pupae, along with a lengthening of the lethal time of A. pernyi larvae. These results suggest that AnpeNPV-iap1 is involved in regulating viral gene expression, OB production and morphogenesis in A. pernyi., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The M14 family metal carboxypeptidase genes play an important role in digestion and pathogenic infections in the gut of insects. However, the roles of these genes in Antheraea pernyi (Guérin-Méneville, 1855) remain to be analyzed. In the present study, we cloned a highly expressed M14 metal carboxypeptidase gene (ApMCP1) found in the gut and discovered that it contained a 1,194 bp open reading frame encoding a 397-amino acid protein with a predicted molecular weight of 45 kDa. Furthermore, 14 members of the M14 family metal carboxypeptidases (ApMCP1-ApMCP14) were identified in the A. pernyi genome, with typical Zn&#95;pept domains and two Zn-anchoring motifs, and were further classified into M14A, M14B, and M14D subfamilies. Expression analysis indicated that ApMCP1 and ApMCP9 were mainly expressed in the gut. Additionally, we observed that ApMCP1 and ApMCP9 displayed opposite expression patterns after starvation, highlighting their functional divergence during digestion. Following natural infection with baculovirus NPV, their expression was significantly upregulated in the gut of A. pernyi. Our results suggest that the M14 family metal carboxypeptidase genes are conservatively digestive enzymes and evolutionarily involved in exogenous pathogenic infections., (© The Author(s) 2022. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Cocoonase is a serine protease present in sericigenous insects and majorly involved in dissolving of sericin protein allowing moth to escape. Cocoon structure is made up of sericin protein which holds fibroin filaments together. Cocoonase enzyme hydrolyzes sericin protein without harming the fibroin. However, until date, no detailed characterization of cocoonase enzyme and its presence in wild silk moth Antheraea mylitta has been carried out. Therefore, current study aimed for detailed characterization of amplified cocoonase enzyme, secondary and tertiary structure prediction, sequence and structural alignment, phylogenetic analysis, and computational validation. Several computational tools such as ProtParam, Iterative Threading Assembly Refinement (I-TASSER), PROCHECK, SAVES v6.0, TM-align, Molecular Evolutionary Genetics Analysis (MEGA) X, and Figtree were employed for characterization of cocoonase protein., Results: The present study elucidates about the isolation of RNA, cDNA preparation, PCR amplification, and in silico characterization of cocoonase from Antheraea mylitta. Here, total RNA was isolated from head region of A. mylitta, and gene-specific primers were designed using Primer3 followed by PCR-based amplification and sequencing. The newly constructed 377-bp length sequence of cocoonase was subjected to in silico characterization. In silico study of A. mylitta cocoonase showed 26% similarity to A. pernyi strain Qing-6 cocoonase using Blastp and belongs to member of chymotrypsin-like serine protease superfamily. From phylogenetic study, it was found that A. mylitta cocoonase sequence is closely related to A. pernyi cocoonase sequence., Conclusions: The present study revealed about the detailed in silico characterization of cocoonase gene and encoded protein obtained from A. mylitta head region. The results obtained infer the presence of cocoonase enzyme in the wild silkworm A. mylitta and can be used for cocoon degumming which will be a valuable and cost-effective strategy in silk industry., (© 2022. The Author(s).)

In innate immunity, autophagy is an important molecular mechanism that plays a critical role in the animal defense system. Given the importance of anti-microbial autophagy in the innate immune processes, the relationship between anti-microbial autophagy and LPS-induced innate immunity in A. pernyi was investigated. Quantitative RT-PCR analysis revealed that autophagy-related genes (ATG6, ATG5, and ATG12) were induced following LPS injection. LPS treatment in the Relish knockdown larvae reduced the expression of autophagy-related genes, especially ATG5. Furthermore, ATG5 depletion decreased the innate immune effect, while its over-expression with ATG12 was induced after the LPS challenge. The dual-luciferase assay revealed that Relish could regulate ATG5 expression by binding directly to the promoter of the ATG5 gene. Overall, our findings show that Relish regulates the ATG5 transcription to eliminate Gram-negative bacteria by anti-microbial autophagy, implying a strong connection between autophagy and innate immunity in immunologic homeostasis., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The Antheraea Pernyi silk gland protein originates from natural organisms and synthesized by tussah silk glands and has widely potential biomaterial applications due to the superior biocompatibility. This study investigates the Antheraea Pernyi silk gland protein-based drug-loaded bio-hydrogels for bioengineered tissue fabricated by using an eco-friendly method without the harsh extracting process and the usage of toxic chemicals. The drug-loaded bio-hydrogels exhibited a porous structure and interconnected pore walls. The swelling ratio and water absorption of drug-loaded bio-hydrogels were, respectively, above 95% and 1.5 × 10 3 %. The cumulative release of drug loaded hydrogels all reached more than 90% within 4 h, and this indicates the potential of drug-loaded hydrogels as future drug-carrying biomaterials. RSC96 Schwann cells cultured on drug-loaded hydrogels for 72 h under cell culture medium show no toxic effects and more pro-proliferative effects. The results suggest the suitability of drug-loaded bio-hydrogels as natural biopolymer for the potential in vitro RSC96 cell culture platform and other biomaterial applications.

The pupal diapause of univoltine Antheraea pernyi hampers sericultural and biotechnological applications, which requires a high eclosion incidence after artificial diapause termination to ensure production of enough eggs. The effect of pupal diapause termination using 20-hydroxyecdysone (20E) on the eclosion incidence has not been well-documented in A. pernyi . Here, the dosage of injected 20E was optimized to efficiently terminate pupal diapause of A. pernyi , showing that inappropriate dosage of 20E can cause pupal lethality and a low eclosion incidence. The optimal ratio of 20E to 1-month-old pupae was determined as 6 μg/g. Morphological changes showed visible tissue dissociation at 3 days post-injection (dpi) and eye pigmentation at 5 dpi. Comprehensive transcriptome analysis identified 1,355/1,592, 494/203, 584/297, and 1,238/1,404 upregulated and downregulated genes at 1, 3, 6, and 9 dpi, respectively. The 117 genes enriched in the information processing pathways of "signal transduction" and "signaling molecules and interaction" were upregulated at 1 and 3 dpi, including the genes involved in FOXO signaling pathway. One chitinase, three trehalase, and five cathepsin genes related to energy metabolism and tissue dissociation showed high expression levels at the early stage, which were different from the upregulated expression of four other chitinase genes at the later stage. Simultaneously, the expression of several genes involved in molting hormone biosynthesis was also activated between 1 and 3 dpi. qRT-PCR further verified the expression patterns of two ecdysone receptor genes ( EcRB1 and USP ) and four downstream response genes ( E93 , Br-C , βFTZ-F1 , and cathepsin L ) at the pupal and pharate stages, respectively. Taken together, these genes serve as a resource for unraveling the mechanism underlying pupal-adult transition; these findings facilitate rearing of larvae more than once a year and biotechnological development through efficient termination of pupal diapause in A. pernyi in approximately half a month., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Du, Zhao, Wang, Ma, Yao, Zhu, Yang, Zhang, Sun, Liang, Xing and Duan.)

In insects, melanism plays important roles in defense, immunoreactions, and body color. The underlying molecular mechanisms of melanism in different insects are diverse and remain elusive. In contrast to another silkworm, Bombyx mori , the Chinese oak silkworm, Antheraea pernyi , produces melanic pupae under natural environmental conditions. DOPA and dopamine synthesis are crucial for melanin formation. Disruption of these processes reportedly influences body colors in many insects. Most research focuses on newly emerged pupae, and the larval process preceding pupation remains unknown. Due to the large size and long pupation period in A. pernyi , the entire process was studied at least every 12 h. The expression patterns of tyrosine hydroxylase ( TH ) and DOPA decarboxylase ( DDC ), which are involved in DOPA and dopamine synthesis in the epidermis, were evaluated during larval-pupal metamorphosis. We also performed RNA interference (RNAi) and used enzyme inhibitors to examine morphological changes. The amino acid sequences of TH and DDC share 90.91% and 86.64% identity with those of B. mori . TH and DDC expression was upregulated during the 48-72 h period prior to pupal emergence, especially at 60 h. RNAi of TH and DDC induced partial melanism in some pupae. The inhibitors 3-iodo-tyrosine (3-IT) and L-α-methyl-DOPA (L-DOPA) influenced pupal melanization. Different concentrations of inhibitors led to pupal deformity and even mortality. Four different monoamines, only DOPA and Dopamine synthezed from Tyrosine will be influenced by TH and DDC inhibitor. These results indicate that TH and DDC are key genes associated with pupal melanization during larval-pupal transformation in A. pernyi. Overall, our results suggest that TH and DDC expression alterations in a particular stage can affect body color, setting the molecular basis for artificial control of pupal melanization., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wang, Zhong, Wang, Zheng, Bian, Du, Yang, Liu and Qin.)

Japanese giant silkworm (JGS), Caligula japonica Moore, is an emerging defoliator pest of forest and fruit trees in East Asia, causing severe economic losses. To develop a cost-effective biological control program against JGS, we used eggs of the Chinese oak silkworm (COS) Antheraea pernyi Guérin-Méneville as an alternative host to rear the most dominant JGS egg parasitoid Anastatus japonicus Ashmead. We compared the demographic parameters and total parasitism (killing) rates of A. japonicus parasitizing JGS and COS eggs using an age-stage, two-sex life table method. The results showed that A. japonicus performed differently on these two different hosts. Anastatus japonicus reared from COS eggs had a higher fecundity (369.7 eggs per female) and a longer oviposition period (35.9 days) on the COS than JGS eggs (180.9 eggs; 24.0 days). Consequently, A. japonicus parasitizing COS eggs had a higher intrinsic rate of increase ( r = 0.1466 d -1 ), finite rate of increase (λ = 1.1579 d -1 ) and net reproductive rate ( R 0 = 284.9 offspring) than those parasitizing JGS eggs ( r = 0.1419 d -1 , λ = 1.1525 d -1 , R 0 = 150.0 offspring). The total net parasitism rate (the number of parasitized hosts in which the parasitoids successfully developed) of A. japonicus parasitizing COS eggs was 284.9, significantly higher than that of A. japonicus parasitizing JGS eggs (150.0), while the net non-effective parasitism rate (the number of parasitized hosts in which the parasitoids failed to develop) of the former (0.0) was significantly lower than that of the latter (9.6). These results suggest that A. japonicus can be efficiently reared on the alternative (or factitious) COS eggs, and the reared parasitoids have a high biological control potential against the target JGS.

This study reports the first complete mitochondrial genome of Antheraea pernyi Guérin-Méneville 1855 (Lepidoptera: Saturniidae) strain Dingzhou&#95;1, a silkworm resource serving silk production in North Korea. The mitochondrial genome is circular with 15,573 bp in length encoding 37 typical mitochondrial genes (13 protein-coding genes, two ribosomal RNA genes, and 22 transfer RNA genes). The 553-bp A + T-rich region harbors a repeat region composed of 6 ∼ 38 bp tandem repeat units, as found in other known inbred strains from Chinese populations. The phylogenetic analysis clustered Dingzhou&#95;1 from North Korea together with Chinese Liaoning population, suggesting that oak silkworm in North Korea might be introduced from her neighbor China Liaoning., Competing Interests: The authors declare no competing financial interest. The authors alone are responsible for the content and writing of the article., (© 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

Antheraea pernyi (Guérin-Méneville 1855) is an important resource for silk, food, and biohealth products; however, exogenous pathogens largely affect the commercial application potential of this species. Since the gut is a key organ for the digestion and absorption of nutrients as well as for immune defense, we used comparative transcriptome analysis to screen for a gut-specific molecular tool for further functional research in A. pernyi. In total, 3,331 differentially expressed genes (DEGs) were identified in the gut compared with all other pooled tissues of A. pernyi, including 1,463 upregulated genes in the gut. Among these, we further focused on a lipopolysaccharide-induced tumor necrosis factor-α factor (LITAF) gene because of its high gut-specific expression and the presence of a highly conserved SIMPLE-like domain, which is related to the immune response to pathogenic infections in many species. The cDNA sequence of ApLITAF was 447-bp long and contained a 243-bp open reading frame encoding an 80-amino acid protein. Immune challenge assays indicated that ApLITAF expression was significantly upregulated in the gut of A. pernyi naturally infected with nucleopolyhedrovirus (NPV) or fed leaves infected with the gram-negative bacterium Escherichia coli (Migula 1895) and the gram-positive bacterium Bacillus subtilis (Ehrenberg 1835). Cell transfection showed that ApLITAF localized to the lysosome. Collectively, these results suggested that ApLITAF played a role in the immune response of A. pernyi and could facilitate the future research and breeding application in this species., (© The Author(s) 2021. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The present study for the first time describes the complete mitochondrial (mt) genome of Antheraea pernyi Guérin-Méneville 1855 strain Luhong, a genetic lethal mutant exhibiting especially red skin color. The mt genome is 15,563 bp in length that is the smallest among the sequenced A. pernyi inbred strains. This genome displays an identical genomic component and gene order to other six known A. pernyi mt genomes. The mt genome-based phylogenetic analysis clustered Luhong with four strains exhibiting yellow skin color, consistent with the traditional view that all of them belonged to the yellow blood lineage., Competing Interests: The authors declare no competing financial interest. The authors alone are responsible for the content and writing of the paper., (© 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

In insects, trehalose accumulation is associated with the insulin/insulin-like growth factor signalling (IIS) pathway. However, whether insulin-like peptide is involved in the regulation of the trehalose metabolism during diapause termination remains largely unknown. This study assessed whether insulin-like peptide ( ApILP ) enhances the trehalose catabolism in the pupae of Antheraea pernyi during their diapause termination process. Injection of 10 μg of bovine insulin triggered diapause termination and synchronous adult eclosion in diapausing pupae. Moreover, treatment with bovine insulin increased the expression of trehalase 1A ( ApTre-1A ) and trehalase 2 ( ApTre-2 ), as well as the activity of soluble and membrane-bound trehalase, resulting in a decline in trehalose levels in the haemolymph. Silencing ApILP via RNA interference significantly suppressed the expression of ApTre-1A and ApTre-2 , thus leading to an increase in the trehalose concentration during diapause termination. However, neither injection with bovine insulin nor ApILP knockdown directly affected trehalase 1B ( ApTre-1B ) expression. Moreover, overexpression of the transcription factor forkhead box O ( ApFoxO ) induced an increase in trehalose levels during diapause termination; however, depletion of ApFoxO accelerated the breakdown of trehalose in diapausing pupae by increasing the expression of ApTre-1A and ApTre-2 . The results of this study help to understand the contributions of ApILP and ApFoxO to the trehalose metabolism during diapause termination.

The Chinese oak silkworm is commonly used in pupal diapause research. In this study, a long photoperiod was used to trigger pupal diapause termination. Genes encoding three hormones, namely prothoracicotropic hormone (PTTH), ecdysis triggering hormone (ETH), and eclosion hormone (EH), were studied. Additionally, ecdysteroids (mainly 20-hydroxyecdysone, 20E) were quantified by HPLC. Pupal diapause stage was determined by measuring respiratory intensity. The pupae enter a low metabolic rate, which starts approximately 1 month after pupal emergence. ApPTTH expression showed a small increase at 14 days and then a larger increase from 35 days under the long photoperiod treatment. A similar pattern was observed for the titer of 20E in the hemolymph. However, ApETH expression later increased under the long photoperiod treatment (42 days) just before eclosion. Moreover, ApEH expression increased from 21 to 35 days, and then decreased before ecdysis. These results suggest that hormone-related gene expression is closely related to pupal development. Our study lays a foundation for future diapause studies in A. pernyi .

Aprostocetus brevipedicellus , a eulophid gregarious egg parasitoid of lepidopterous pests, is a potential biological control agent for the control of many forest pests. A dominant factitious host, Antheraea pernyi , has been widely used for mass rearing several parasitoids in China. However, whether A. pernyi eggs are suitable for A. brevipedicellus rearing remains unclear. Here we evaluated A. brevipedicellus parasitism and fitness of their offspring on A. pernyi eggs with five different treatments, including manually-extracted, unfertilized and washed eggs (MUW), naturally-laid, unfertilized and washed eggs (NUW), naturally-laid, unfertilized, and unwashed (NUUW) eggs, naturally-laid, fertilized and washed eggs (NFW), and naturally-laid, fertilized and unwashed eggs (NFUW). The results showed that A. brevipedicellus could parasitize host eggs in all treatments but significantly preferred MUW eggs to other treatments. Moreover, A. brevipedicellus preferred unfertilized eggs to fertilized eggs and parasitized more washed eggs than unwashed. The pre-emergence time of parasitoid offspring emerging from fertilized eggs was shorter than that from unfertilized eggs. More parasitoid offspring emerged from unwashed eggs than that from washed eggs. The offspring emergence rate was high (>95%) and also female-biased (>85%) among all egg treatments. The egg load of female parasitoid offspring emerging from MUW and NUW eggs was 30-60% higher than the remaining treatments. Overall, MUW eggs of A. pernyi are the most suitable for the mass production of A. brevipedicellus .

The cell line NISES-AnPe-428 (AnPe), derived from the Chinese oak silkworm Antheraea pernyi , was characterized for its permissiveness and productivity for six different nucleopolyhedrovirus (NPV) species. These NPVs included homologous Antheraea pernyi NPV (AnpeNPV) and heterologous Autographa californica multiple NPV (AcMNPV), Bombyx mori NPV (BmNPV), Hyphantria cunea MNPV (HycuMNPV), Spodoptera exigua MNPV (SeMNPV), and Lymantria dispar MNPV (LdMNPV), representing viruses that had been isolated from insect species belonging to five different families ( Saturniidae , Noctuidae , Bombycidae , Arctiidae , and Lymantriidae ). We found that AnPe cells supported productive replication of AnpeNPV, AcMNPV, BmNPV, HycuMNPV, and SeMNPV to varying degrees. Upon infection with SeMNPV, a subset of AnPe cell population in the culture underwent apoptosis, while remaining cells produced limited amounts of progeny virions and polyhedra. AnPe cells were refractory to LdMNPV infection and failed to support replication of viral DNA, indicating that viral replication was restricted at or prior to the step of viral DNA replication. These results indicated that AnPe cells have the potential to provide excellent systems for studying the molecular mechanisms underlying cellular permissiveness for NPV replication and host-range determination of NPVs., Competing Interests: Conflict of interestThe authors declare that they have no conflict of interest., (© The Author(s), under exclusive licence to Springer Nature B.V. 2021.)

The Chinese oak silkworm, Antheraea pernyi, has not only been semi-domesticated as an important economical insect but also used for genetic research. The female moths of A. pernyi employ a pheromone blend containing (E,Z)-6,11-hexadecadienal (E6,Z11-16:Ald), (E,Z)-6,11-hexadecadienyl acetate (E6,Z11-16:OAc), and (E,Z)-4,9-tetradecadienyl acetate (E4,Z9-14:OAc). While its biosynthesis pathway is largely unknown. By deep sequencing and de novo assembly of sex pheromone gland (PG) transcriptome, we identified 141 candidate genes that are putatively related to pheromone biosynthesis, degradation, and chemoreception in A. pernyi. Gene expression patterns and phylogenetic analysis revealed that two desaturases (AperDES1 and 2), two fatty acid reductase (AperFAR1 and 2), and three acetyltransferase genes (AperACT1, 2 and 3) showed PG-biased or specific expression and were phylogenetically related to genes known to be involved in pheromone synthesis in other species. Furthermore, two carboxylesterases (AperCOE6 and 11) and two chemosensory protein (AperCSP1 and 6) were also expressed specifically or predominantly in the PGs, which might be related to sex pheromone degradation and transportation, respectively. Based on these results, the sex pheromone biosynthesis and metabolic pathway was proposed in A. pernyi. This study provides some crucial candidates for further functional elucidation, and may be used for interfering sexual communication in other Saturniidae pests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Here, we report the complete mitochondrial (mt) genome of Antheraea pernyi Qing&#95;6, an improved strain serving silk production and edible insect resource for 50 years in Northeast China. The circular mt genome spans 15,572 bp in length and contains 37 typical coding genes (13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes). Its A + T-rich region is 552 bp in size exhibiting identical sequence with the first modern improved strain Qinghuang&#95;1. Comparison analysis identified only 12 variable sites (5 substitutions and 7 indels) between Qing&#95;6 and Qinghuang&#95;1. The phylogenetic analysis also clustered Qing&#95;6 and Qinghuang&#95;1 together first, which was in line with the breeding history of the two strains., Competing Interests: The authors declare no competing financial interest. The authors alone are responsible for the content and writing of the paper., (© 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

Antheraea pernyi is a semi-domesticated lepidopteran insect species valuable to the silk industry, human health, and ecological tourism. Owing to its economic influence and developmental properties, it serves as an ideal model for investigating divergence of the Bombycoidea super family. However, studies on the karyotype evolution and functional genomics of A. pernyi are limited by scarce genomic resource. Here, we applied PacBio sequencing and chromosome structure capture technique to assemble the first high-quality A. pernyi genome from a single male individual. The genome is 720.67 Mb long with 49 chromosomes and a 13.77-Mb scaffold N50. Approximately 441.75 Mb, accounting for 60.74% of the genome, was identified as repeats. The genome comprises 21,431 protein-coding genes, 85.22% of which were functionally annotated. Comparative genomics analysis suggested that A. pernyi diverged from its common ancestor with A. yamamai ~30.3 million years ago, and that chromosome fission contributed to the increased chromosome number. The genome assembled in this work will not only facilitate future research on A. pernyi and related species but also help to progress comparative genomics analyses in Lepidoptera., (© 2020 John Wiley & Sons Ltd.)

Here, we assessed the effect of a systematic change in reactive center loop (RCL) length, N-terminal to the reactive center, on the inhibitory activity of the recombinant Apserpin-6. The domain prediction results indicated that the RCL is located between the amino acid numbered 359-379 at the C-terminal of Apserpin-6. The N-terminal variable region for amino acid positions P7-P1 of the RCL of Apserpin-6 was truncated or extended by residue deletion or insertion using site-directed mutagenesis. The recombinant Apserpin-6 with one or two residues insertion in RCL had no effect on prophenoloxidase (proPO) activity, whereas deletion of one or two residues in RCL lowered the efficiency of inhibition of Apserpin-6. The results of this study will facilitate the understanding of inhibition mechanism of RCL on proPO activity., (© 2020 Wiley Periodicals LLC.)

In this study, we established the Antheraea pernyi multicapsid nucleopolyhedrovirus (AnpeNPV) bacmid system for the construction of a Bac-to-Bac expression system and the generation of virus mutants. The CopyRight pSMART BAC cloning vector harboring the chloramphenicol resistance gene was introduced into the AnpeNPV genome to produce the AnpeNPV bacmid that could be propagated in Escherichia coli with stable replication. The enhanced green fluorescent protein (EGFP) was successfully expressed in both Tn-Hi5 cells and A. pernyi pupae using the AnpeNPV Bac-to-Bac expression system. To generate the AnpeNPV mutants, we developed the AnpeNPV bacmid/λ Red recombination system that facilitated the deletion of viral genes from the AnpeNPV genome. The genes cathepsin and chitinase were deleted and a derivative AnpeNPV Bac-to-Bac expression system was constructed. Furthermore, we demonstrated that the novel expression system could be used to express human epidermal growth factor in A. pernyi pupae. Taken together, the AnpeNPV bacmid system provides a powerful tool to create the AnpeNPV Bac-to-Bac expression system for protein expression in A. pernyi pupae. Further, it helps to knock-out genes from the AnpeNPV genome with λ Red recombination system for identification of the role of viral genes involved in regulating gene expression, DNA replication, virion structure, and infectivity during the AnpeNPV infection process., (© The Author(s) 2020. Published by Oxford University Press on behalf of Entomological Society of America.)

Background: Trichogramma dendrolimi has been widely used in augmentative biocontrol of lepidopteran pests in China. In mass production of T. dendrolimi using Antheraea pernyi eggs as substitutive hosts, which are large in size, as clutch size is a parameter of importance to produce high quality parasitoids. Here, we aimed to determine the optimal clutch size for the bisexual Wolbachia-uninfected line (TdB) and Wolbachia-infected thelytokous line (TdT) of T. dendrolimi reared on A. pernyi eggs., Results: A medium clutch size of 42.75 to 62.27 for TdB and 52.93 to 57.14 for TdT was optimal to maximize fitness-correlated traits of parasitoid individual. The optimal clutch sizes with maximized parameters included adult emergence rate, adult body size, adult longevity, fecundity, and sum of fecundity of all females per brood were 58.31 (86.00%), 42.75 (231.11 μm), 50.92 (2.69 days), 62.27 (150.89 eggs), and 83.25 (7926.33 eggs) for TdB and 57.14 (94.54%), 52.93 (236.97 μm), 53.64 (2.62 days), 56.80 (161.01 eggs), and 70.10 (8579.71 eggs) for TdT. The TdT had a shorter adult longevity, longer development time, and higher adult emergence rate than did its non-infected bisexual counterpart., Conclusion: A medium brood size in a A. pernyi egg host was optimal to produce offspring parasitoids with higher fitness parameters for both bisexual Wolbachia-uninfected and thelytokous Wolbachia-infected lines of T. dendrolimi. The determination of optimal clutch size for T. dendrolimi will provide the reference for the quality control of T. dendrolimi production and improvement of the field performance of the wasps. © 2020 Society of Chemical Industry., (© 2020 Society of Chemical Industry.)

A large number of ecdysteroid-regulated 16 kDa proteins (ESR16s) of insects have been isolated and annotated in GenBank; however, knowledge on insect ESR16s remain limited. In the present study, we characterized an ecdysteroid-regulated 16 kDa protein gene isolated in Chinese oak silkworm, Antheraea pernyi Guérin-Méneville ('ApESR16' in the following), an important silk-producing and edible insect. The obtained cDNA sequence of ApESR16 is 1,049 bp, harboring an open reading frame of 441 bp that encodes a polypeptide of 146 amino acids. CD-search revealed that ApESR16 contains the putative cholesterol/lipid binding sites on conserved domain Npc2&#95;like (Niemann-Pick type C-2) belonging to the MD-2-related lipid-recognition superfamily. Sequence comparison revealed that ApESR16 exhibits 51-57% identity to ESR16s of lepidopteran insects, 36-41% identity to ESR16 or NPC2a of nonlepidopteran insects, and 28-32% identity to NPC2a of vertebrates, indicating a high sequence divergence during the evolution of animals. Phylogenetic analysis found that the used sequences were divided into two groups corresponding to vertebrates and invertebrates, and the used insect sequences were also well clustered according to their families. The A. pernyi ESR16 mRNA is expressed during all four developmental stages and in all tested tissues. Injection of 20-hydroxyecdysone (20-E) into A. pernyi diapausing pupae triggering diapause termination induced upregulation of ESR16 mRNA compared to the diapausing pupae, with the highest expression level at day 2 in the ovaries but day 12 in the fat body. Our results suggested that ApESR16 might be a diapause-related gene and plays a vital role in the pupal diapause of A. pernyi., (© The Author(s) 2020. Published by Oxford University Press on behalf of Entomological Society of America.)

Background: Chouioia cunea Yang (Hymenoptera: Eulophidae) is a parasitic wasp and natural enemy of several lepidopteran pests during their pupal stage. The volatiles from pupae of three hosts, Hyphantria cunea (Arctiidae), Antheraea pernyi (Saturniidae) and Lymantria dispar (Erebidae), were analyzed and compared to elucidate the chemical cues used by C. cunea to locate its hosts., Results: The attraction of C. cunea to H. cunea pupae has no obvious association with the types of plant leaves consumed by H. cunea before pupation. C. cunea exhibited the strongest attraction to the pupae of H. cunea, followed by those of A. pernyi and L. dispar based on behavioral experiments. Gas chromatography-mass spectrometry and GC-electroantennography (GC-EAD) analyses showed that these three host pupae consisted of essentially the same active volatile components but at different relative amounts. Active components derived from these pupae by GC-EAD were alkanes from C12 to C27, and C. cunea showed different levels of attraction to different single compounds., Conclusion: Host location by C. cunea primarily depends on common compounds emanating from the pupae of several host species. The relative amount of each component varies across host species, guiding host preferences by C. cunea. Optimal blends of several components were identified. Understanding the chemical cues used by C. cunea to locate its host could increase the possibility of developing attractants for parasitic wasps and subsequently increasing the parasitism rate of C. cunea on various hosts. © 2019 Society of Chemical Industry., (© 2019 Society of Chemical Industry.)

Hexamerin was originally identified as a storage protein but later confirmed to be involved in many physiological processes. In the present study, we cloned and characterized a novel hexamerin complementary DNA sequence from the Chinese oak silkworm, Antheraea pernyi (Ap-hexamerin), which shows high homology with reported insect methionine-rich hexamerins. The tissue distribution and time course of expression demonstrated that Ap-hexamerin was predominantly synthesized in the fat body and the expression level was significantly increased in response to the microbial challenge, suggesting the relevance of Ap-hexamerin to immune responses. In further immune functional studies, Ap-hexamerin was confirmed to take part in the upregulation of prophenoloxidase (PPO) activation in A. pernyi haemolymph triggered by pathogen-associated molecular patterns (PAMPs). Additional molecular interaction analysis revealed that Ap-hexamerin is capable of binding the PAMPs used in the phenoloxidase assay, suggesting hexamerin in A. pernyi may positively regulate haemolymph PPO activation, acting as a pattern recognition protein., (© 2019 Wiley Periodicals, Inc.)

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., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The Chinese oak silkworm, Antheraea pernyi, is an important alternate host for mass production of Trichogramma parasitoids, which play a vital role in the biological control of lepidopterous pests in China. The quality of A. pernyi eggs is particularly important for mass production of these parasitoids. In this study, a pathogenic bacterium, isolated and purified from A. pernyi eggs that had turned gray in color, was identified as Serratia marcescens. We used morphology, biochemistry and 16S rDNA analysis to characterize the strain, which was named "APE strain". Serratia marcescens APE strain was determined to be the causal bacterium associated with the disease in the eggs, verified by a test based on Koch's Postulates. We tested the pathogenicity of S. marcescens APE strain on A. pernyi eggs; the percentage of diseased (gray) eggs reached 57.78% when uninfected eggs were exposed to a concentration of 1 × 10 9 cfu/mL bacterial suspension for 7 h. S. marcescens was transmitted mechanically by Trichogramma parasitoids. The transmission rate was 25.56%. In a horizontal transmission test, the highest percentage of uninfected eggs that developed infections was 51.43% after being treated with contents of diseased eggs for 12 h. In a vertical transmission test, the number of infected eggs per treated adult female was 63.8-92.3 after treatment with different S. marcescens concentrations, significantly lower than the 304.3 eggs per female in the control group. Furthermore, the percentage of infected (gray) eggs produced by adult moths exposed to bacteria in the larval stage was 80.5-85.3%., (Copyright © 2019 Elsevier Inc. All rights reserved.)

In the Antheraea pernyi multicapsid nucleopolyhedrovirus (AnpeNPV)-based expression vector system, the frequency of homologous recombination events between wild-type AnpeNPV DNA and the transfer vector is low, resulting in a small amount of recombinant virus. Previous reports have indicated that linearized baculovirus DNA can increase the proportion of recombinant virus relative to the total progeny. To improve the recombination efficiency, we constructed a linearized derivative of AnpeNPV, referred to as AnpeNPVPhEGFP-AvrII, in which egfp flanked by AvrII restriction sites was located at the polyhedrin locus and driven by the polyhedrin promoter. Linear AnpeNPV DNA was obtained by the treatment of AnpeNPVPhEGFP-AvrII genomic DNA with AvrII endonuclease. The infectivity and recombinogenic activity between the linearized and circular viral DNA were evaluated by quantitative real-time polymerase chain reactions. We demonstrated that the linearized AnpeNPV DNA produced only small numbers of infectious budded viruses, accounting for approximately 4.5% of the budded virus production of wild-type AnpeNPV DNA in A. pernyi pupae. However, the linearized AnpeNPV DNA substantially increased recombinant virus production after cotransfection with an appropriate transfer vector; relative abundance of the recombinant virus was approximately 5.5-fold higher than that of the wild-type AnpeNPV DNA in A. pernyi pupae. The linearization of AnpeNPV DNA will facilitate the purification of recombinant viruses using the AnpeNPV-based expression vector system and the construction of an AnpeNPV-based bacmid system., (© The Author(s) 2020. Published by Oxford University Press on behalf of Entomological Society of America.)

Four species of Anastatus Motschulsky (Hymenoptera, Eupelmidae, Eupelminae) are newly reported as egg parasitoids of the Japanese giant silkworm, Caligula japonica Moore and, as an alternate laboratory host, the Chinese oak silk moth, Antheraea pernyi (Guérin-Méneville) (Lepidoptera, Saturniidae) in China. The four species, A. fulloi Sheng & Wang, 1997, A. gansuensis Chen & Zang, sp. nov. , A. japonicus Ashmead, 1904, and A. meilingensis Sheng, 1998, were reared initially from eggs of C. japonica collected in Gansu, Jilin and Liaoning provinces and subsequently cultured in the laboratory on eggs of A. pernyi . An illustrated key to differentiate females of the four species, and males of some of the species is provided. Key features are illustrated, both sexes of the new species are described, and diagnoses of females of the other species are given.

Insects have evolved efficient innate immune systems to defend themselves against the invasion of pathogens and parasites. We infected the lepidopteran Antheraea pernyi with Antheraea pernyi nucleopolyhedrovirus (ApNPV) and studied the host's transcriptional response to unravel the cellular and humoral immune responses of A. pernyi larval hemolymph to ApNPV. Comparison of gene expression levels revealed that a total of 3191 genes were differentially expressed, including 1107 up-regulated and 2084 down-regulated genes in ApNPV-infected hemolymph. Of these, 1416 and 982 differentially expressed genes (DEGs) were enriched in 3253 Gene Ontology terms and 258 KEGG pathways, respectively. DEGs involved in immunity related pathways were selected for further analysis. We screened 184 and 63 DEGs involved in cellular and humoral immune-related pathways, respectively, via KEGG analysis. Among the cellular immune-related pathways, autophagy, endocytosis, and lysosome were activated based on the expression profiles of several key regulatory genes, such as the up-regulation of Atg3 and LPS-induced TN factor, and the down-regulation of PIK3R4, HSC70 and V-ATPase. We concluded that phagosome, apoptosis, and ubiquitin-mediated proteolysis were suppressed by ApNPV as a result of down-regulation of most related DEGs. The majority of the DEGs enriched in Toll, Jak-STAT, MAPK, NF-κB and Insulin signaling pathways, complement and coagulation cascades, and melanogenesis were down-regulated, demonstrating that ApNPV infection greatly inhibited humoral immunity in the hemolymph of A. pernyi larvae. Our findings may serve as a basis for further research on the antiviral molecular mechanisms of A. pernyi., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Peptidoglycan recognition proteins (PGRPs) are evolutionarily conserved molecules that act in innate immune responses against invading pathogens. Insect PGRPs activate the Toll and/or immune deficiency (IMD) signal transduction pathways. They induce cellular and humoral immune defense reactions that effectively protect insects from invasion of microorganisms. In this study, four cDNA clones encoding PGRPs (ApPGRP-A, ApPGRP-B, ApPGRP-C, ApPGRP-LE) were isolated from the Chinese oak silkworm, Antheraea pernyi. The deduced amino acid sequences of ApPGRP-B and -C share high identity with enzymatically active PGRP proteins and contain the amino acids required for amidase activity. The spatiotemporal expression patterns of ApPGRPs and their response to immune stimulations were determined, and suggest that PGRPs might act as a broad-spectrum pattern recognition protein and participate in the pathway activation system. Here, using an RNA-interference approach, we examined the function of PGRPs in response to immune stimulation. We observed that RNAi-mediated silencing of ApPGRP-A decreased the tested antimicrobial peptides (Attacin, Attacin 2, Ceropin, Gloverin, lysozyme and Lebocin) in response to Enterococcus pernyi challenge. However, reducing the ApPGRP-B, -C mRNA levels led to a strong increase of the AMP genes (except for Lebocin). These results suggest that ApPGRPs are necessary for pathway initiation complex formation and activate AMP generation. Our data also indicated that PGRP-B and -C down-regulate AMPs generation in the immune response., (Copyright © 2019 Elsevier Inc. All rights reserved.)

In the present study, we isolated a spermidine synthase gene from Antheraea pernyi ( ApSpds ) using expressed sequence tag method. The obtained cDNA sequence of 1483 bp contains an open-reading frame of 864 bp encoding a polypeptide of 287 amino acids. Sequence analysis revealed that ApSpds belonged to class I of AdoMet-MTase family, and exhibited 30% identity to those from bacteria, 45-48% identity to fungi, 36-47% identity to plants, 52-54% identity to vertebrates and 53-80% identity to invertebrates. Phylogenetic analysis found that the used Spds protein sequences were well divided into five groups corresponding to bacteria, fungi, plants, invertebrates and vertebrates, respectively. These results further confirmed that Spds is highly conserved through evolution of life organisms. The ApSpds mRNA is expressed during all four developmental stages and is present in all examined tissues with the highest abundance in the muscle, in which the relative mRNA expression level was 1.6 times higher than in the fat body. Although not significant, the mRNA level decreased after high-temperature exposure suggesting that the Spds gene may not be involved in temperature stress tolerance in A. pernyi . Taken together, our results suggested that ApSpds play an important role in development of silkworm., Competing Interests: Conflict of interestAll the authors declare that they have no conflict of interest.

The published genome sequence of Antheraea yamamai (Saturnnidae) was used to construct a library of long terminal repeat (LTR)-retrotransposons that is representative of the wild silkmoth ( Antherea) genus, and that includes 22,666 solo LTRs and 541 full-length LTRs. The LTR retrotransposons of Antheraea yamamai (AyLTRs) could be classified into the three canonical groups of Gypsy , Copia and Belpao . Eleven AyLTRs contained the env gene element, but the relationship with the env element of baculovirus, particularly A . yamamai and pernyi nucleopolyhedrovirus (AyNPV and ApNPV), was distant. A total of 251 "independent" full-length AyLTRs were identified that were located within 100 kb distance (downstream or upstream) of 406 neighboring genes in A . yamamai . Regulation of these genes might occur in cis by the AyLTRs, and the neighboring genes were found to be enriched in GO terms such as "response to stimulus", and KEGG terms such as "mTOR signaling pathway" among others. Furthermore, the library of LTR-retrotransposons and the A . yamamai genome were used to identify and analyze the expression of LTR-retrotransposons and genes in ApNPV-infected and non-infected A . pernyi larval midguts, using raw data of a published transcriptome study. Our analysis demonstrates that 93 full-length LTR-retrotransposons are transcribed in the midgut of A . pernyi of which 12 significantly change their expression after ApNPV infection (differentially expressed LTR-retrotransposons or DELs). In addition, the expression of differentially expressed genes (DEGs) and neighboring DELs on the chromosome following ApNPV infection suggests the possibility of regulation of expression of DEGs by DELs through a cis mechanism, which will require experimental verification. When examined in more detail, it was found that genes involved in Notch signaling and stress granule (SG) formation were significantly up-regulated in ApNPV-infected A . pernyi larval midgut. Moreover, several DEGs in the Notch and SG pathways were found to be located in the neighborhood of particular DELs, indicating the possibility of DEG-DEL cross-regulation in cis for these two pathways.

Baculovirus causes liquefaction of insect cuticle to enhance the dissemination of progeny virions away from the host cadavers for increasing viral transmission rates. Antheraea pernyi nucleopolyhedrovirus (ApNPV) infects A. pernyi larvae with circular pus blotches formed in cuticle in the early stage of liquefaction. To investigate the formation mechanism of those pus blotches, the transcriptome profile changes of the cuticles between ApNPV-infected and non-infected A. pernyi larvae were analyzed using RNA-Seq. The transcriptome was de novo assembled using the Trinity platform. Comparison of gene expression levels revealed that a total of 2990 and 4427 unigenes were up- and down-regulated respectively in ApNPV-infected cuticle, of which 2620 and 1903 differentially expressed genes (DEGs) could be enriched in different GO terms and KEGG pathways. In this study, we focused on chitin metabolism related DEGs, and screened 10 genes involved in chitin synthesis and degradation with down-regulated trends, indicating that the chitin metabolism pathway was inhibited by ApNPV infection, which may promote liquefaction of A. pernyi cuticle. Besides, we also identified a large number of DEGs involved in immune related pathways via KEGG analysis, indicating that intense immune responses occurred in A. pernyi cuticle. Our research findings will serve as a basis for further researching the molecular mechanisms underlying cuticle liquefaction of A. pernyi induced by ApNPV infection., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

At present, Antheraea pernyi silk fibroin (ASF) has attracted research efforts to investigate it as a raw material for fabrication of biomedical devices because of its superior cytocompatibility. Nevertheless, native ASF is not easily processed into a hydrogel without any crosslinking agent, and a single hydrogel shows poor mechanical properties. In this paper, a series of ASF/poly (N-isopropylacrylamide) (PNIPAAm) composite hydrogels with different ASF contents were manufactured by a simple in situ polymerization method without any crosslinking agent. Meanwhile, the structures, morphologies and thermal properties of composite hydrogels were investigated by XRD, FTIR, SEM, DSC and TGA, respectively. The results indicate that the secondary structure of silk in the composite hydrogel can be controlled by changing the ASF content and the thermal stability of composite hydrogels is enhanced with an increase in crystalline structure. The composite hydrogels showed similar lower critical solution temperatures (LCST) at about 32 °C, which matched well with the LCST of PNIPAAm. Finally, the obtained thermosensitive composite hydrogels exhibited enhanced mechanical properties, which can be tuned by varying the content of ASF. This strategy to prepare an ASF-based responsive composite hydrogel with enhanced mechanical properties represents a valuable route for developing the fields of ASF, and, furthermore, their attractive applications can meet the needs of different biomaterial fields.

Small heat shock proteins (sHSPs) are a class of highly conserved proteins that are ubiquitously found in all types of organisms, from prokaryotes to eukaryotes. In the current study, we identified and characterized the full-length cDNA encoding sHSP 19.1 from the oak silkworm, Antheraea pernyi. Ap-sHSP is 510 bp in length, and encodes a protein of 169 amino acid residues. The protein contains conserved domains found in insect sHSPs, and it belongs to the α-crystallin-HSPs&#95;p23-like superfamily. Recombinant Ap-sHSP was expressed in Escherichia coli cells, and a rabbit anti-Ap-sHSP 19.1 antibody was generated to confirm the biological functions of Ap-sHSP 19.1 in A. pernyi. Real-time polymerase chain reaction and western blot analysis revealed that Ap-sHSP 19.1 expression was highest in the fat body, followed by the midgut, and the lowest expression was found in the Malpighian tubule. Ap-sHSP 19.1 transcript expression was significantly induced following challenge with microbial pathogens. In addition, the expression of Ap-sHSP 19.1 was strongly induced after heat shock. These results suggest that Ap-sHSP 19.1 plays a crucial role in immune responses and thermal tolerance in A. pernyi., (© 2018 Wiley Periodicals, Inc.)

Insect pheromone biosynthesis activating neuropeptide (PBAN) controls the synthesis and actuating of sex pheromones of female adult. In the current examination, the full-length cDNA encoding the PBAN receptor was cloned from the pheromone gland (PG) of Antheraea pernyi (AntpePBANR). The AntpePBANR displayed the characteristic seven transmembrane areas of the G protein-coupled receptor (GPCR) and was closely related to the PBANR from Bombyx mori and Manduca sexta in the phylogenetic tree. The AntpePBANR expressed in mammalian cell lines were enacted by AntpePBAN in a concentration-dependent manner. AntpePBANR activation resulted in the calcium mobilization but did not activate the cAMP elevation pathway. Cells expressing AntpePBANR were profoundly responsive to Antpe-γ-SGNP (suboesophageal ganglion neuropeptides) and Antpe-DH (diapause hormone), different individuals from FXPRLamide (X = T, S or V) family in A. pernyi. Deletion of residues in the C-terminal hexapeptide (FSPRLamide) proved that P, R and L played the key parts in initiating the AntpePBANR, the amination to the last C terminal residues which can also likewise impact the activation of AntpePBAN receptor altogether. The mRNA of the AntpePBANR gene demonstrated the most noteworthy transcript levels in pheromone gland followed by fat body., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The growth and development of the Chinese oak silkworm, Antheraea pernyi, are strongly influenced by environmental conditions, including heavy metal pollution. An excess of heavy metals causes cellular damage through the production of free radical reactive oxygen species. In this study, transcriptome analysis was performed to investigate global gene expression when A. pernyi was exposed to zinc infection. With RNA sequencing (RNA-Seq), a total of 25 795 510 and 38 158 855 clean reads were obtained from zinc-treated and control fat body libraries, respectively. We identified 2399 differential expression genes (DEGs) (1845 upregulated and 544 downregulated genes) in the zinc-treated library. In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that these DEGs were related to the peroxisome pathway that was associated with antioxidant defense. Our results suggest that fat bodies of A. pernyi constitute a strong antioxidant defense against heavy metal contamination.

Innate immunity, the evolutionarily conserved defense system, has been extensively analyzed in insect models over recent decades. The significant progress in this area has formed our dominant conceptual framework of the innate immune system, but critical advances in other insects have had a profound impact on our insights into the mystery of innate immunity. In recent years, we focused on the immune responses in Antheraea pernyi, an important commercial silkworm species reared in China. Here, we review the immune responses of A. pernyi based on immune-related gene-encoded proteins that are divided into five categories, namely pattern recognition receptors, hemolymph proteinases and their inhibitors, prophenoloxidase, Toll pathway factors and antimicrobial peptides, and others. Although the summarized information is limited since the research on A. pernyi immunity is in its infancy, we hope to provide evidence for further exploration of innate immune mechanisms., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Antheraea pernyi is not only an important economic insect, it is increasingly employed as a model organism due to a variety of advantages, including ease of rearing and experimental manipulation compared with other Lepidoptera. Peptidoglycan (PGN) is a major component of the bacterial cell wall, and interactions between PGN and A. pernyi cause a series of physiological changes in the insect. In the present study, we constructed cDNA libraries from a A. pernyi PGN-infected group and a control group stimulated with phosphate-buffered saline (PBS). The transcriptome was de novo assembled using the Trinity platform, and 1698 differentially expressed genes (DEGs) were identified, comprising 894 up-regulated and 804 down-regulated genes. To further investigate immune-related DEGs, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment were performed. GO analysis identified major immune-related GO terms and KEGG enrichment indicated gene responses to three pathways related to the insect immune system. Several homologous genes related to the immune response of the A. pernyi fat body post-PGN infection were identified and categorised. Taken together, the results provide insight into the complex molecular mechanisms of the responses to bacterial infection at the transcriptional level., (Copyright © 2018 Elsevier B.V. All rights reserved.)