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In this study we successfully constructed a full-length cDNA library from Chinese oak silkworm, Antheraea pernyi, the most well-known wild silkworm used for silk production and insect food. Total RNA was extracted from a single fresh female pupa at the diapause stage. The titer of the library was 5 x 10(5) cfu/ml and the proportion of recombinant clones was approximately 95%. Expressed sequence tag (EST) analysis was used to characterize the library. A total of 175 clustered ESTs consisting of 24 contigs and 151 singlets were generated from 250 effective sequences. Of the 175 unigenes, 97 (55.4%) were known genes but only five from A. pernyi, 37 (21.2%) were known ESTs without function annotation, and 41 (23.4%) were novel ESTs. By EST sequencing, a gene coding KK-42-binding protein in A. pernyi (named as ApKK42-BP; GenBank accession no. FJ744151) was identified and characterized. Protein sequence analysis showed that ApKK42-BP was not a membrane protein but an extracellular protein with a signal peptide at position 1-18, and contained two putative conserved domains, abhydro_lipase and abhydrolase_1, suggesting it may be a member of lipase superfamily. Expression analysis based on number of ESTs showed that ApKK42-BP was an abundant gene in the period of diapause stage, suggesting it may also be involved in pupa-diapause termination.
Hypoxia not only plays a critical role in multiple disease conditions; it also influences the growth and development of cells, tissues and organs. To identify novel hypoxia-related mechanisms involved in cell and tissue growth, studying a precise hypoxia-sensitive time window can be an effective approach. Drosophila melanogaster has been a useful model organism for studying a variety of conditions, and we focused in this study on the life cycle stages of Drosophila to investigate their hypoxia sensitivity. When normoxia-grown flies were treated with 4% O2 at the pupa stage for 3, 2 and 1 day/s, the eclosion rates were 6.1%, 66.7% and 96.4%, respectively, and, when 4% O2 was kept for the whole pupa stage, this regimen was lethal. Surprisingly, when our hypoxia-adapted flies who normally live in 4% O2 were treated with 4% O2 at the pupa stage, no fly eclosed. Within the pupa stage, the pupae at 2 and 3 days after pupae formation (APF), when treated for 2 days, demonstrated 12.5 ± 8.5% and 23.6 ± 1.6% eclosion, respectively, but this was completely lethal when treated for 3 days. We conclude that pupae, at 2 days APF and for a duration of a minimum of 2 days, were the most sensitive to hypoxia. Our data from our hypoxia-adapted flies clearly indicate that epigenetic factors play a critical role in pupa-stage hypoxia sensitivity.
The proliferation inhibition effects of the hydrolysates from silkworm pupa proteins on MGC-803 gastric cancer cells were investigated in this study. The specific morphological changes (cell membrane, cell nucleus and cytoskeleton) of cells were measured. In vitro, the proliferation of MGC-803 cells was inhibited by silkworm pupa protein hydrolysates (SPPHs) in a dose-dependent manner. The flow cytometry analysis showed that the blocking effect of SPPHs on the MGC-803 cells was mainly in the G0/G1-phase. The morphological changes, disintegration of the cytoskeleton and retardant cell cycles were probably related to the activation of apoptosis. Thus, SPPHs could be promising as a chemopreventive agent due to their ability to promote apoptosis of tumor cells.
This protocol describes the step-by-step generation of tumors with specific genotypes on the dorsal thorax epithelium of the fly. This in vivo system allows the imaging of tumor cell morphology and behavior in high spatial and temporal resolution. Phenotypes such as cell invasion, cell division, and tumor size can be quantified and compared to specific controls or to the neighboring wild-type tissue. Thus, this model allows the study of conserved genes that enhance or suppress epithelial tumor progression. For complete details on the use and execution of this protocol, please refer to Canales Coutiño et al. (2020).
The pigmentation pattern of Lepidoptera varies greatly in different development stages. To date, the effects of key genes in the melanin metabolism pathway on larval and adult body color are distinct, yet the effects on pupal pigmentation remains unclear. In the silkworm, Bombyx mori, the black pupa (bp) mutant is only specifically melanized at the pupal stage. Using positional cloning, we found that a mutation in the Aspartate decarboxylase gene (BmADC) is causative in the bp mutant. In the bp mutant, a SINE-like transposon with a length of 493 bp was detected ~2.2 kb upstream of the transcriptional start site of BmADC. This insertion causes a sharp reduction in BmADC transcript levels in bp mutants, leading to deficiency of β-alanine and N-β-alanyl dopamine (NBAD), but accumulation of dopamine. Following injection of β-alanine into bp mutants, the color pattern was reverted that of the wild-type silkworms. Additionally, melanic pupae resulting from knock-down of BmADC in the wild-type strain were obtained. These findings show that BmADC plays a crucial role in melanin metabolism and in the pigmentation pattern of the silkworm pupal stage. Finally, this study contributes to a better understanding of pupa pigmentation patterns in Lepidoptera.
Erectile dysfunction (ED) is a highly prevalent disorder that affects millions of men worldwide. ED is now considered an early manifestation of atherosclerosis, and consequently, a precursor of systemic vascular disease. This study was designed to investigate the effects of male silkworm pupa powder (SWP) on the levels of nitric oxide synthase (NOS) expression, nitrite, and glutathione (GSH); lipid peroxidation; libido; and erectile response of the corpus cavernosum of the rat penis. We induced ED in the study animals by oral administration of 20% ethanol over 8 weeks. The SWP-treated male rats were divided into 3 groups that were orally administered 200, 400, and 800 mg/kg. The libido of the SWP-administered male rats was higher than that of the ethanol control group. In addition, the erectile response of the corpus cavernosum was restored in males on SWP administration, to a level similar to that of the normal group without ED. The testosterone concentration did not increase significantly. The lipid peroxidation in the corpus cavernosum of the male rats administered SWP decreased significantly. In contrast, compared to the ethanol group, SWP-administered male rats showed increased GSH levels in the corpus cavernosum. The level of nitrite and NOS expression in the corpus cavernosum of SWP-administered male rats increased significantly. These results indicated that SWP effectively restored ethanol-induced ED in male rats.
Insect ecdysis triggering hormone (ETH) receptors (ETHRs) are rhodopsin-like G protein-coupled receptors. Upon binding its ligand ETH, ETHR initiates a precisely programed ecdysis behavior series and physiological events. In Drosophila melanogaster, the ethr gene produces two functionally distinct splicing isoforms, ethra and ethrb. ETH/ETHRA activates eclosion hormone (EH), kinin, crustacean cardioactive peptide (CCAP), and bursicon (burs and pburs) neurons, among others, in a rigid order, to elicit the behavioral sequences and physiological actions for ecdysis at all developmental stages, whereas ETH/ETHRB is required at both pupal and adult ecdysis. However, the role of ETHRB in regulation of molting has not been clarified in any non-drosophila insects. In the present paper, we found that 20-hydroxyecdysone (20E) signaling triggers the expression of both ethra and ethrb in a Coleopteran insect pest, the Colorado potato beetle Leptinotarsa decemlineata. RNA interference (RNAi) was performed using double-stranded RNAs (dsRNAs) targeting the common (dsethr) or isoform-specific (dsethra, dsethrb) regions of ethr. RNAi of dsethr, dsethra, or dsethrb by the final-instar larvae arrested larva development. The arrest was not rescued by feeding 20E. All the ethra depleted larvae stopped development at prepupae stage; the body cavity was expanded by a large amount of liquid. Comparably, more than 80% of the ethrb RNAi larvae developmentally halted at the prepupae stage. The remaining Ldethrb hypomorphs became pupae, with blackened wings and highly-expressed burs, pburs and four melanin biosynthesis genes. Therefore, ETHRA and ETHRB play isoform-specific roles in regulation of ecdysis during larva-pupa transition in L. decemlineata.
Holorusia Loew, 1863 (Diptera: Tipulidae) is a relatively large crane fly genus with a wide distribution in the Afrotropic, Australasian-Oceanian, Eastern Palearctic, Oriental and Nearctic Regions. Although the genus is well known to include the largest crane fly species, the immature stages are, thus far, only described for the larva and pupa of the North American Holorusia hesperea Arnaud & Byers, 1990.
Acetaminophen (APAP) overdose causes severe hepatotoxicity and acute liver failure. The current study aims to investigate the protection effects of silkworm pupa oil (SPO) against acute hepatic injury in APAP-exposed Kunming mice. Our results showed that the liver index and the levels of serum alanine transaminase (ALT) and aspartate transaminase (AST) in mice subjected to APAP treatment were decreased by SPO. Supplement of SPO also restored hepatic histopathological alterations induced by APAP. The APAP-induced increase in proinflammatory cytokines, including TNF-α, IL-6, and IL-12, was reversed by SPO, which was mediated by the reduction of nuclear factor (NF)-κB p65 expression and the increase in the expression of IκB-α in liver tissue. Moreover, SPO inhibited APAP-triggered oxidative stress by decreasing MDA level and increasing the activities of SOD and GSH-Px. Collectively, SPO attenuated hepatic injury induced by APAP, which attributed to the suppression of oxidative stress-mediated NF-κB signaling. Our findings suggest that SPO supplementation may be potential strategy against acute hepatic injury.
The larvae, pupae, and adults of Laenahaigouica Schawaller, 2001 were collected during recent fieldwork in the Sichuan Province (China). Since the pupal morphology of Laena has never been investigated this created an opportunity to provide the first description. Moreover, prior to this study larval characters of only one species, Laenastarcki Reitter, 1887, were known. Therefore, description of the larva of L.haigouica enabled the first verification of the intrageneric stability of larval characters revealed for other Laena species. Association of the studied immature stages with the adults was confirmed by analysing COI sequences. Additionally, three other loci (16S, Cytb, 28S) were sequenced for L.haigouica during this study.
A new species of Pseudopyrochroa Pic, 1906, P. reni Pan & Young, n. sp., is described from the western region of Mt. Qinling, China. Larvae, pupae, and adults were associated using molecular phylogenetic analyses based on mtDNA COI barcode sequences. All three stages are described and illustrated. Additionally, preliminary phylogenetic relationships among five genera and 14 species of Pyrochroidae, including Pseudopyrochroa, are hypothesized based on COI sequence data. The fauna of Pyrochroidae from the Mt. Qinling biodiversity conservation area is discussed.
In this study, peptides were prepared from defatted Antheraea pernyi (Lepidoptera: Saturniidae) pupa protein via hydrolysis with combined neutral proteases. Single-factor tests and response surface methodology (RSM) were used to determine the optimal hydrolysis condition suitable for industrial application. Optimal hydrolysis of the defatted pupa protein was found to occur at an enzyme concentration of 4.85 g/liter, a substrate concentration of 41 g/liter, a hydrolysis temperature of 55°C, and a hydrolysis time of 10 h and 40 min. Under these conditions, the predicted and actual rates of hydrolysis were 45.82% and 45.75%, respectively. Peptides with a molecular weight of less than 2,000 Da accounted for 90.5% of the total peptides generated. Some of the peptides were antioxidant peptides as revealed by sequencing and functional analysis. The antioxidant activity of the mixed peptides was subsequently confirmed by an antioxidant activity assay. The results showed that peptides with high antioxidant activity could be obtained from the hydrolysis of A. pernyi pupa protein.
It is estimated that animals pollinate 87.5% of flowering plants worldwide and that managed honey bees (Apis mellifera) account for 30-50% of this ecosystem service to agriculture. In addition to their important role as pollinators, honey bees are well-established insect models for studying learning and memory, behavior, caste differentiation, epigenetic mechanisms, olfactory biology, sex determination, and eusociality. Despite their importance to agriculture, knowledge of honey bee biology lags behind many other livestock species. In this study, we have used scRNA-Seq to map cell types to different developmental stages of the worker honey bee (prepupa at day 11 and pupa at day 15) and sought to determine their gene expression signatures. To identify cell-type populations, we examined the cell-to-cell network based on the similarity of the single-cells transcriptomic profiles. Grouping similar cells together we identified 63 different cell clusters of which 17 clusters were identifiable at both stages. To determine genes associated with specific cell populations or with a particular biological process involved in honey bee development, we used gene coexpression analysis. We combined this analysis with literature mining, the honey bee protein atlas, and gene ontology analysis to determine cell cluster identity. Of the cell clusters identified, 17 were related to the nervous system and sensory organs, 7 to the fat body, 19 to the cuticle, 5 to muscle, 4 to compound eye, 2 to midgut, 2 to hemocytes, and 1 to malpighian tubule/pericardial nephrocyte. To our knowledge, this is the first whole single-cell atlas of honey bees at any stage of development and demonstrates the potential for further work to investigate their biology at the cellular level.
The adult, pupa and larva of a new species, Gnaptorina (Gnaptorina) lhorongica Li, sp. nov., from northeastern Xizang, China are described and illustrated. The species was identified using molecular phylogenetic analyses based on three mitochondrial fragments and one nuclear gene fragment (COI, Cytb, 16S, and 28S-D2). The taxonomic status of the new species is confirmed using a combination of molecular and morphological datasets. This study provides valuable molecular and morphological data for phylogenetic studies of the tribe Blaptini.
A new species, Pleurodesmospora lepidopterorum, isolated from a pupa, is introduced. Morphological comparisons and phylogenetic analyses based on multigene datasets (ITS+RPB1+RPB2+TEF) support the establishment of the new species. Pleurodesmospora lepidopterorum is distinguished from P. coccorum by its longer conidiogenous pegs located in the terminal or lateral conidiophores, and smaller subglobose or ellipsoidal conidia. A combined dataset of RPB1, RPB2, and TEF confirmed the taxonomic placement of Pleurodesmospora in Cordycipitaceae for the first time.
Human growth hormone (hGH) is a peptide hormone secreted by eosinophils of the human anterior pituitary, and a regulatory factor for a variety of metabolic pathways. A 30-kD protein from the pupa stage of silkworm was detected by Western blotting and confirmed by immunoprecipitation based on its ability to bind to anti-hGH antibody. This protein, named BmhGH-like protein, was purified from fresh silkworm pupas through low-temperature homogenization, filtration, and centrifugation to remove large impurity particles. The supernatants were precipitated, resuspended, and passed through a molecular sieve. Further purification by affinity chromatography and two-dimensional electrophoresis resulted in pure protein for analysis by MS MALDI-TOF-MS analysis. An alignment with predicted proteins indicated that BmhGH-like protein consisted of two lipoproteins, which we named hGH-L1 and hGH-L2. These proteins belong to the β-trefoil superfamily, with β domains similar to the spatial structure of hGH. Assays with K562 cells demonstrated that these proteins could promote cell division in vitro. To further validate the growth-promoting effects, hGH-L2 was cloned from pupa cDNA to create recombinant silkworm baculovirus vBmNPV-hGH-L2, which was used to infect silkworm BmN cells at low titer. Flow cytometric analysis demonstrated that the protein shortened the G0/G1 phase of the cells, and enabled the cells to rapidly traverse the G1/S phase transition point to enter S phase and promote cell division. Discovery of hGH-like protein in silkworm will once again arouse people's interest in the potential medicinal value of silkworm and establish the basis for the development of new hormone drugs.
The silkworm maggot, Exorista sorbillans, is a well-known larval endoparasitoid of the silkworm Bombyx mori that causes considerable damage to the silkworm cocoon crop. To gain insights into the response mechanism of the silkworm at the protein level, we applied a comparative proteomic approach to investigate proteomic differences in the hemolymph of the female silkworm pupae parasitized by E. sorbillans. In total, 50 differentially expressed proteins (DEPs) were successfully identified, of which 36 proteins were upregulated and 14 proteins were downregulated in response to parasitoid infection. These proteins are mainly involved in disease, energy metabolism, signaling pathways, and amino acid metabolism. Eight innate immune proteins were distinctly upregulated to resist maggot parasitism. Apoptosis-related proteins of cathepsin B and 14-3-3 zeta were significantly downregulated in E. sorbillans-parasitized silkworm pupae; their downregulation induces apoptosis. Quantitative PCR was used to further verify gene transcription of five DEPs, and the results are consistent at the transcriptional and proteomic levels. This was the first report on identification of possible proteins from the E. bombycis-parasitized silkworms at the late stage of parasitism, which contributes to furthering our understanding of the response mechanism of silkworms to parasitism and dipteran parasitoid biology.
Some parasites have an ability to fabricate the behavior of their host and impel the host to guard parasites' offspring, which is popularly called as bodyguard manipulation. Psalis pennatula larva parasitized by a braconid parasitoid wasp Microplitis pennatula exhibits some behavioral changes including the guarding of the parasitoid pupa from its natural enemies. We hypothesized that these behavioral change exhibited by the parasitized host larva are induced by the parasitoid and can be considered as an example of bodyguard manipulation. Even though hyperparasitoids are the more specialized natural enemy of parasitoids than predators, very few studies tested the success of guarding parasitoid pupa against hyperparasitoids. This study analyzed the success of guarding behavior of the parasitized host against hyperparasitoids. The onsets of parasite-induced phenotypic alterations (PIPAs) in the parasitized host were inspected to analyze whether these behavioral changes in the host larva manifests gradually or abruptly. The study concludes that parasitized host larva defends the parasitoid pupa from hyperparasitoids and the PIPAs in the parasitized host develops abruptly only after the egression of parasitoid prepupa.
Insects undergo metamorphosis, involving an abrupt change in body structure through cell growth and differentiation. Rice stem stripped borer (SSB), Chilo suppressalis, is one of the most destructive rice pests. However, little is known about the regulation mechanism of metamorphosis development in this notorious insect pest. Here, we studied the expression of 22,197 SSB genes at seven time points during pupa development with a customized microarray, identifying 622 differentially expressed genes (DEG) during pupa development. Gene ontology (GO) analysis of these DEGs indicated that the genes related to substance metabolism were highly expressed in the early pupa, which participate in the physiological processes of larval tissue disintegration at these stages. In comparison, highly expressed genes in the late pupal stages were mainly associated with substance biosynthesis, consistent with adult organ formation at these stages. There were 27 solute carrier (SLC) genes that were highly expressed during pupa development. We knocked down SLC22A3 at the prepupal stage, demonstrating that silencing SLC22A3 induced a deficiency in pupa stiffness and pigmentation. The RNAi-treated individuals had white and soft pupa, suggesting that this gene has an essential role in pupal development.
Silkworm pupae (Bombyx mori) are a high-protein nutrition source consumed in China since more than 2 thousand years ago. Recent studies revealed that silkworm pupae have therapeutic benefits to treat many diseases. However, the ability of the compounds of silkworm pupae to inhibit tumourigenesis remains to be elucidated. Here, we separated the protein of silkworm pupae and performed alcalase hydrolysis. Silkworm pupa protein hydrolysate (SPPH) can specifically inhibit the proliferation and provoke abnormal morphologic features of human gastric cancer cells SGC-7901 in a dose- and time-dependent manner. Moreover, flow cytometry indicated that SPPH can induce apoptosis and arrest the cell-cycle in S phase. Furthermore, SPPH was shown to provoke accumulation of reactive oxygen species (ROS) and depolarization of mitochondrial membrane potential. Western blotting analysis indicated that SPPH inhibited Bcl-2 expression and promoted Bax expression, and subsequently induced apoptosis-inducing factor and cytochrome C release, which led to the activation of initiator caspase-9 and executioner caspase-3, cleavage of poly (ADP-ribose) polymerase (PARP), eventually caused cell apoptosis. Moreover, SPPH-induced S-phase arrest was mediated by up-regulating the expression of E2F1 and down-regulating those of cyclin E, CDK2 and cyclin A2. Transcriptome sequencing and gene set enrichment analysis (GSEA) also revealed that SPPH treatment could affect gene expression and pathway regulation related to tumourigenesis, apoptosis and cell cycle. In summary, our results suggest that SPPH could specifically suppress cell growth of SGC-7901 through an intrinsic apoptotic pathway, ROS accumulation and cell cycle arrest, and silkworm pupae have a potential to become a source of anticancer agents in the future.
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