The dual roles of three MMPs and TIMP in innate immunity and metamorphosis in the silkworm, Bombyx mori.

The matrix metalloproteinases (MMPs) and their endogenous inhibitory factors, tissue inhibitors of metalloproteinases (TIMPs), are implicated in many diseases. However, the mammalian MMPs (> 20) and TIMPs (> 3) are larger in number, and so little is known about their individual roles in organisms. Hence, we have systematically studied the roles of all three MMPs and one TIMP in silkworm innate immunity and metamorphosis. We observed that MMPs and TIMP are highly expressed during the pupation stage of the silkworms, and TIMP could interact with each MMPs. High-activity MMPs and low-activity TIMP may enhance the infection of B. mori nucleopolyhedrovirus in both in vitro and in vivo. MMPs' knockout and TIMP overexpression delayed silkworm development and even caused death. Interestingly, different MMPs' knockout led to different tubular tissue dysplasia. These findings provide insights into the conserved functions of MMPs and TIMP in human organogenesis and immunoregulation.

Accumulation and translocation of food chain in soil-mulberry (Morus alba L.)-silkworm (Bombyx mori) under single and combined stress of lead and cadmium.

In recent years, heavy metal pollution has caused immeasurable harm to the environment. As an emerging technology, phytoremediation technology has gained a place in the treatment of heavy metal pollution with its unique advantages. This study analyzes the toxic effects of mulberry (Morus alba) seeds, seedling growth and silkworm under heavy metal stress of lead (Pb) and cadmium (Cd), and explore the accumulation and migration of Pb and Cd in the soil-mulberry tree-silkworm system. The main results were as follows: (1) Seed germination and potted seedling experiments were conducted under heavy metal Pb and Cd stresses, and it was found that Pb and Cd had inhibitory effects on mulberry seed germination, growth and photosynthesis of mulberry seedlings, and as the concentration of heavy metals increased, the stronger the inhibitory effect. Moreover, Pb and Cd have a synergistic effect under compound stress. (2) The accumulation and transfer rules of Pb and Cd ions in mulberry were different. The content of Pb in mulberry was root > leaf > stem and the content of Cd was root > stem > leaf. The combined stress promoted the transfer of Pb and Cd from the underground part to the aerial portion of mulberry. (3) The silkworm feeds on mulberry leaves contaminated with heavy metals in this experiment and found that: with the increase of silkworm feeding, the heavy metal content in the silkworm body increased significantly, but the content remained in the silkworm body was less, most of it was excreted with silkworm excrement. Combined stress has no significant effect on the detoxification mechanism of silkworm. It is indispensable to think of the synergistic effect of heavy metals on plants germination when seeds are used for phytoremediation.

Insect-machine hybrid robot.

Recently, insect-machine hybrid robots have been developed that incorporate insects into robots or incorporate machines into insects. Most previous studies were motivated to use the function of insects for robots, but this technology can also prove to be useful as an experimental tool for neuroethology. We reviewed hybrid robots in terms of the closed-loop between an insect, a robot, and the real environment. The incorporated biological components provided the robot sensory signals that were received by the insects and the adaptive functions of the brain. The incorporated artificial components permitted us to understand the biological system by controlling insect behavior. Hybrid robots thus extend the roles of mobile robot experiments in neuroethology for both model evaluation and brain function analysis.

A re-evaluation of silk measurement by the cecropia caterpillar (Hyalophora cecropia) during cocoon construction reveals use of a silk odometer that is temporally regulated.

The late 5th instar caterpillar of the cecropia silk moth (Hyalophora cecropia) spins a silken cocoon with a distinct, multilayered architecture. The cocoon construction program, first described by the seminal work of Van der Kloot and Williams, consists of a highly ordered sequence of events. We perform behavioral experiments to re-evaluate the original cecropia work, which hypothesized that the length of silk that passes through the spinneret controls the orderly execution of each of the discrete events of cocoon spinning. We confirm and extend by three-dimensional scanning and quantitative measurements of silk weights that if cocoon construction is interrupted, upon re-spinning, the caterpillar continues the cocoon program from where it left off. We also confirm and extend by quantitative measurements of silk weights that cecropia caterpillars will not bypass any of the sections of the cocoon during the construction process, even if presented with a pre-spun section of a cocoon spun by another caterpillar. Blocking silk output inhibits caterpillars from performing normal spinning behaviors used for cocoon construction. Surprisingly, unblocking silk output 24-hr later did not restart the cocoon construction program, suggesting the involvement of a temporally-defined interval timer. We confirm with surgical reductions of the silk glands that it is the length of silk itself that matters, rather than the total amount of silk extracted by individuals. We used scanning electron microscopy to directly show that either mono- or dual-filament silk (i.e., equal silk lengths but which vary in their total amount of silk extracted) can be used to construct equivalent cocoons of normal size and that contain the relevant layers. We propose that our findings, taken together with the results of prior studies, strongly support the hypothesis that the caterpillar uses a silk "odometer" to measure the length of silk extracted during cocoon construction but does so in a temporally regulated manner. We further postulate that our examination of the anatomy of the silk spinning apparatus and ablating spinneret sensory output provides evidence that silk length measurement occurs upstream of output from the spinneret.

Steroid hormone 20-hydroxyecdysone induces the transcription and complex assembly of V-ATPases to facilitate autophagy in Bombyx mori.

Vacuolar-type H + -adenosine triphosphatases (V-ATPases) are indispensable for lysosome acidification and participate in autophagic processes. The steroid hormone 20-hydroxyecdysone (20E) predominantly induces autophagy and regulates insect larval molting and metamorphosis; however, the specific mechanism of lysosome acidification regulation by 20E remains unclear. Here, we showed that the developmental profiles of Bombyx V-ATPases were in accordance with autophagy occurrence and lysosome acidification in the fat body during larval-pupal metamorphosis. BmV-ATPase-A and BmV-ATPase-B were required for lysosome acidification and autophagic flux. Both 20E treatment and starvation were able to induce lysosome acidification. Furthermore, BmV-ATPase transcription was induced by 20E treatment and reduced by RNAi targeting the 20E receptor BmUsp. On the one hand, 20E upregulated the transcription of BmV-ATPases through inducing Bombyx transcription factor EB (TFEB) and its nuclear translocation; on the other hand, 20E inhibited mTOR signaling to induce the transcription and assembly of BmV-ATPase subunits. Overall, 20E induces lysosome acidification by upregulating the transcription and assembly of V-ATPase subunits via activating BmTFEB and cooperating with nutrient signaling. These findings improve our understanding of the regulatory mechanisms underlying lysosome acidification and autophagic flux in Bombyx mori.

Survival of silk worm, Bombyx mori in azaserine induced oxidative stress.

Cells under stress generate reactive oxygen species (ROS) in excess, which causes mitochondrial dysfunction and stimulates the apoptotic cascade. However, mild stress or pre-conditioning lead to the evasion of apoptosis by activating mitogenic signaling, including the signaling of inhibitors of apoptosis proteins (IAPs), or by inactivating certain apoptotic molecules. The silkworm (Bombyx mori) is an important economic insect which serves as a model organism in biological research. Bombyx mori apoptotic protease inducing factor (BmApaf1), a death-related ced-3/Nedd2-like protein (BmDredd), and BmSurvivin-2 (BmSvv2) are known to play significant roles in metamorphosis. Azaserine is an analogue of glutamine and irreversibly inhibits glutamine-utilizing enzymes and cysteine-glutamate transporter genes EAAT2. In the present study, we experimentally demonstrated stress induced by azaserine along with the capacity of antioxidants to modulate apoptotic/anti-apoptotic gene expression in determining the fate of the larvae. We observed higher larval survival with higher azaserine dosages and attributed this to the quantum of ROS generated and AOEs response, which favoured the BmSvv2 expression. Meanwhile higher levels of ROS with concomitant changes in AOEs were found to be responsible for BmApaf1 and BmDredd expression, which reflected a higher mortality rate.

An insulin-like growth factor-like peptide promotes ovarian development in the silkmoth Bombyx mori.

Insulin family peptides are known to be key regulators of growth and metabolism in insects and vertebrates. Insects have two types of insulin family peptides: insulin-like peptides and insulin-like growth factor (IGF)-like peptides (IGFLPs). We recently demonstrated that an IGFLP in the silkmoth, Bombyx mori (BIGFLP) promotes the growth of the genital imaginal disc ex vivo. However, the role of BIGFLP in the regulation of insect growth remains unclear because no in vivo study has been performed. Therefore, we analysed the functions of BIGFLP in vivo by constructing BIGFLP knock-out (KO) B. mori using the clustered regularly interspaced palindromic repeats (CRISPR) and CRISPR-associated protein 9 (CRISPR-Cas9) system. The KO moths exhibited decreased body weights and size of the appendages compared wild-type (wt) moths. Interestingly, KO females also had drastically lower ovary weights and number of eggs than wt females. However, mutant ovaries that were transplanted into wt host pupae reached a similar weight to wt ovaries that were transplanted into the wt hosts, suggesting that IGFLP in the haemolymph promotes ovarian development. These findings show that BIGFLP regulates the growth and development of adult organs, particularly the ovaries, in B. mori.

Aggregation State of Residual α-Helices and Their Influence on Physical Properties of S. c. ricini Native Fiber.

Formation of the α-helical conformation in the poly-l-alanine (PA) sequence regions, subsequent structural transition to ß-sheet during natural spinning, and presence of residual α-helices in Samia cynthia ricini (S. c. ricini) native silk fiber have been experimentally proven. However, the aggregation state of the residual α-helices, and their influence on the mechanical deformation behavior in native fiber remain unclear. Here we show that the α-helices form an ordered aggregation state with a hexagonal packing in the aqueous solution, some of which remain during natural spinning. X-ray scattering and differential scanning calorimetry (DSC) analyses revealed occurrence of a structural transition of the residual α-helices to the ß-sheet structure, accompanied by disappearance of the plateau region in the force-strain curve, due to heat-treatment at ~220 °C. On the basis of X-ray scattering before and after tensile stretching of S. c. ricini native silk, a direct connection between the plateau region and the α-helix to ß-sheet structural transition was confirmed. Our findings demonstrate the importance of the PA sequence regions in fiber structure formation and their influence on the tensile deformation behavior of S. c. ricini silk, features believed to be essentially similar in other saturniid silks. We strongly believe the residual ordered α-helices to be strategically and systematically designed by S. c. ricini silkworms to impart flexibility in native silk fiber. We anticipate that these knowledge forms a basis for fruitful strategies in the design and development of amino acid sequences for artificial silks with desired mechanical properties.

HN1L is essential for cell growth and survival during nucleopolyhedrovirus infection in silkworm, Bombyx mori.

Hematological and neurological expressed 1-like (HN1L) protein is an evolutionarily conserved protein that plays an important role in embryonic development. It has been reported that HN1L is involved in the process of cell growth and cancer formation and that cell cycle arrest occurs during suppression of HN1L expression. Previous studies have demonstrated that the expression levels of the Bombyx mori HN1L protein were significantly downregulated in Bombyx mori Nucleopolyhedrovirus (BmNPV) infected silkworm cells. Transient transfections were performed with plasmids for pIEX-1-HN1L expression in Bombyx mori ovarian cells (BmN) in order to explore the effect of the HN1L protein on the growth of silkworm cells and its regulatory role in the process of viral infection. Cellular localization analysis revealed that HN1L was localized in the cytoplasm and that its upregulation could significantly enhance cellular activity. Furthermore, HN1L could promote G1/S phase conversion, thereby contributing to cell proliferation. Upon infection of BmN cells with BmNPV, the induction of apoptosis increased, although HN1L overexpression could inhibit DNA fragmentation, suggesting that the HN1L protein could inhibit cell apoptosis induced by viral invasion. In addition, Western blotting indicated that the HN1L protein inhibited the activation of caspase-9 zymogen and the expression of Bax protein, although it promoted Bcl-2 expression. Flow cytometry analysis further confirmed that overexpression of HN1L significantly inhibited apoptosis induced by BmNPV infection. Consequently, we demonstrated that BmN HN1L is a protein with multiple functions, which enhanced cell activity, regulated the cell cycle and induced an anti-apoptotic response by BmNPV infection.

Bombyxin/Akt signaling in relation to the embryonic diapause process of the silkworm, Bombyx mori.

Our previous study showed that phosphorylation of glycogen synthase kinase (GSK)-3ß is related to the embryonic diapause process in Bombyx. However, the upstream signaling pathway was not clearly understood. In the present study, we examined bombyxin/Akt signaling in relation to the embryonic diapause process of B. mori. Results showed that GSK-3ß phosphorylation stimulated by dechorionation was blocked by LY294002, a specific phosphatidylinositol 3-kinase (PI3K) inhibitor, indicating involvement of PI3K in GSK-3ß phosphorylation in dechorionated eggs. Direct determination of Akt phosphorylation showed that dechorionation stimulated Akt phosphorylation. The Akt phosphorylation was blocked by LY294002. Temporal changes in Akt phosphorylation showed that different changing patterns exist between diapause and developing eggs. Relatively higher phosphorylation levels of Akt were detected between days 3 and 5 after oviposition in non-diapause eggs compared to those at the same stages in diapause eggs. Upon treatment with HCl, which prevents diapause initiation, Akt phosphorylation levels exhibited a later and much broader peak compared to diapause eggs. Examination of expression levels of the bombyxin-Z1 gene showed that in diapause eggs, a major peak occurred 1 day after oviposition, and its level then sharply decreased on day 2. However, in both non-diapause and HCl-treated eggs, a major broad peak was detected between days 1 and 4 after oviposition. These temporal changes in bombyxin-Z1 gene expression levels during embryonic stages coincided with changes in Akt phosphorylation, indicating that bombyxin-Z1 is likely an upstream signaling component for Akt phosphorylation. Taken together, our results indicated that PI3K/Akt is an upstream signaling pathway for GSK-3ß phosphorylation and is associated with the diapause process of B. mori eggs. To our knowledge, this is the first study to demonstrate the potential correlation between bombyxin/Akt signaling and the embryonic diapause process.

Expression profile of several genes on ecdysteroidogenic pathway related to diapause in pupal stage of Bombyx mori bivoltine strain.

Ecdysone is involved in regulation of embryonic diapause in the silkworm, Bombyx mori. However, its mechanism still remains unclear. To explore the role of ecdysteroidogenic pathway (EP) genes in diapause process of bivoltine B. mori, the eggs of "Qiufeng", a bivoltine strain, were used as the study materials and arranged into diapause eggs producers (DEPs) and non-diapause eggs producers (NDEPs), respectively. The differential expression of EP genes between two groups was analysed during the early pupal stage. The expression of Shadow was significantly increased in the NDEPs in day-3 pupae and reached the peak simultaneously, indicating that Shadow was in coincidence with diapause process. To validate this hypothesis, a repression of Shadow by RNA interference was performed in day-2 pupae of NDEPs. The expression of Shadow was downregulated by RNAi, and ßFtz-F1, a downstream gene of EP, was also decreased. Furthermore, the genes encoding the kynurenine-synthetase were upregulated in the ovary, and Brown, AdenoK which link Shadow to the kynurenine-synthase gene were also upregulated in the fat body. The progeny eggs appeared a light purple colour at 48 h after oviposition, revealing a certain tendency to diapause. We speculate that inhibition of Shadow upregulates 3-hydroxy-kynurenine synthesis by increasing the expression of Brown and AdenoK. In addition, Shadow was cloned, and expressed in E. coli for further functional study of Shadow protein. Our study provided insight into the role of EP genes in the process of diapause of B. mori.

Negative impact of Novaluron on the nontarget insect Bombyx mori (Lepidoptera: Bombycidae).

Due to increased use of agrochemicals and growing concerns about ecotoxicology, the development of new insecticides, moving away from those with neurotoxic and broad spectrum effects towards insecticides that are safer for the environment and nontarget beneficial species, has been a research priority. Novaluron stands out among these newer insecticides, is an insect growth regulator that is used for the control of insect pests in crops grown close to mulberry plantations. Mulberry serves as food for the silkworm Bombyx mori, which is a nontarget insect of great economic importance to silk production. We investigated the lethal and sublethal effects of Novaluron on the development of B. mori. Larvae were segregated into experimental groups: the control groups (CGs) and the treatment groups (TGs), which were treated with the Novaluron concentration of 0.15 mL/L. Following exposure, we analyzed: larval mortality, changes in the insect life cicle and cytotoxic effects on the midgut cells. This is the first report about the Novaluron's effects on B.mori. We detected rupture in the integument, complete cessation of feeding, late development, incomplete ecdysis and production of defective cocoons. After 240 h of exposure, there was 100% mortality in TG larvae exposed in the 3rd instar and 20% mortality from larvae exposed in the 5th instar. Cytotoxic effects was observed, such as dilation of cells, emission of cytoplasmic protrusions, extreme rarefaction of the cytoplasm and nuclei, dilation of the endoplasmic reticulum in addition to changes in mitochondria, the presence of large digestive vacuoles and intercellular spaces and the presence of active caspase. Novaluron exposure impairs the midgut and may affect the physiological functions of this organ. Novaluron additionally compromises several phases of insect development, indicating the importance of toxicology studies that utilize different life stages of nontarget species to evaluate the safe use of insecticides.

Resistant silkworm strain block viral infection independent of melanization.

Melanization mediated by the prophenoloxidase-activating system (proPO) is an important immune response in invertebrates. However, the role of melanization on viral infection has not been wildly revealed in Bombyx mori (B. mori), silkworm. Here, we investigated the extent of melanization of susceptible (871) and resistant (near-isogenic line 871C) B. mori strains. The result showed that the extent of melanization was significantly higher in the susceptible strain than in the resistant strain. A majority of Serpins were up-regulated in the resistant strain through iTRAQ-based quantitative proteomics, comparing with susceptible strain. Our data further identified that Serpin-5, Serpin-9 and Serpin-19 reduced PO activity, indicating that the menlanization pathway was inhibited in the resistant strain. Moreover, our results indicated that the hemolymph of 871 reduced viral infection in the presence of PTU, indicating that melanization of 871 strain hemolymph blocked viral infection. However, viral infection was significantly suppressed in the hemolymph of 871C strain regardless of the presence of PTU or not, which implied that the resistant strain inhibited viral infection independent of the melanization pathway. Taken together, our findings indicated that the melanization pathway was inhibited in resistant strain. These results expend the analysis of melanization pathway in insects and provide insights into understanding the antiviral mechanism.

Metformin prolongs lifespan through remodeling the energy distribution strategy in silkworm, Bombyx mori.

Metformin is a hypoglycemic agent used clinically in the treatment of type 2 diabetics. In addition, metformin is being investigated as a potential geroprotector. Here, we investigated the effects of metformin silkworm lifespan and the underlying molecular pathways involved. We found that metformin prolonged the lifespan of the male silkworm without reducing body weight, which suggests metformin can increase lifespan through remodeling of the animal's energy distribution strategy. Consistent with that idea, metformin reduced silk production and thus the energy devoted to that process. Metformin also increased fasting tolerance and levels of the antioxidant glutathione, and also activated an adenosine monophosphate-activated protein kinase-p53-forkhead box class O signaling pathway in silkworm. These results suggest that activity in this pathway may contribute to metformin-induced lifespan extension in silkworm by increasing stress resistance and antioxidative capacity while reducing energy output for silk product. The results also show that the silkworm is a potential useful animal model for evaluating the effects of small molecules with potential clinical utility.

Silkworm serpin32 functions as a negative-regulator in prophenoloxidase activation.

The extracellular serine protease cascade is an essential component of insect humoral immunity. Serine protease inhibitors (serpins) play an important regulatory role in the process of insect immunity by regulating the serine protease cascade pathway. We aimed to clarify the function of Bmserpin32 in this study. First, we performed homologous sequence alignment and phylogenetic analysis of Bmserpin32. Bmserpin32 was found to share 64% amino acid sequence identity with Manduca sexta serpin7, an immunomodulatory protein. Bmserpin32 cDNA was cloned, and the recombinant Bmserpin32 protein was expressed in Escherichia coli and purified by nickel-nitrilotriacetic acid affinity and gel filtration chromatography. The activity assay showed that Bmserpin32 had significant inhibitory activity against trypsin. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry and site-directed mutagenesis combined with activity assays indicated that the cleavage site of Bmserpin32 is between Arg359 and Ile360. After infection with E. coli or Micrococcus luteus, the expression level of Bmserpin32 in immune-related tissues was significantly upregulated. In addition, Bmserpin32 could delay or inhibit the melanization of hemolymph by inhibiting the activation of prophenoloxidase in larval hemolymph. Furthermore, a physiological target of Bmserpin32 was identified as the clip protease, BmPAP3, an apparent ortholog of M. sexta propenoloxidase-activating protease-3. Our observations enable a better understanding of the physiological role of Bmserpin32 in regulating melanization in silkworm.

Effects of sodium fluoride on the reproductive development of Bombyx mori.

Bombyx mori was used as a model to evaluate the reproductive toxicity of NaF in insects. Significant reduction in cocoon quality, survival rate, fecundity, and hatchability were observed upon NaF treatment groups. Fluoride determination indicated that F- has a cumulative effect on the gonad of silkworm. High-performance liquid chromatography revealed that the testosterone content of males was decreased in NaF-treated groups, and enzyme-linked immunosorbent assay showed that the estradiol content was decreased in NaF-treated females. Ultrastructural observation of testicles of silkworm larvae revealed mitochondrial turgescence, endoplasmic reticulum destruction, the appearance of vacuoles and lysosomes, and apoptosis and necrosis of cells in NaF-treated groups. Altered tail length and tail DNA content in Comet assays further confirmed DNA damage in NaF-treated larvae. We demonstrated reproductive toxicity of fluoride toward silkworm at physiological and biochemical levels, and the results provide a theoretical basis for revealing the reproductive toxicity of fluoride in terrestrial insects.

Bombyx mori homolog of tumor suppressor p53 is involved in apoptosis-mediated antiviral immunity of B. mori cells infected with nucleopolyhedrovirus.

Apoptosis is important in antiviral immunity and affects viral multiplication and pathogenesis. Here, we showed that Bombyx mori cells transiently expressing B. mori homolog of the tumor suppressor p53 (Bm-p53) protein underwent apoptosis accompanied by elevated caspase-3-like protease activity and processing of B. mori Dronc (Bm-Dronc). RNAi-mediated silencing of bm-p53 expression, which significantly diminished accumulation of bm-p53 transcript and Bm-p53 protein, prevented apoptosis of B. mori cells infected with a recombinant B. mori nucleopolyhedrovirus defective in the anti-apoptotic p35 gene (vBmΔp35) and abolished the activation of caspase-3-like protease and processing of Bm-Dronc. Apoptosis in vBmΔp35-infected B. mori cells is associated with viral DNA replication, suggesting involvement of the DNA damage response. The Bm-p53 pro-apoptotic function is also found in Spodoptera frugiperda and Lymantria dispar cells. These results indicate that apoptosis induction in vBmΔp35-infected B. mori cells is a Bm-p53-mediated process promoted by the commencement of viral DNA replication.

Refined Crystal Structure of Samia cynthia ricini Silk Fibroin Revealed by Solid-State NMR Investigations.

Samia cynthia ricini is one of the wild silkworms and its silk fibroin (SF) consists of alternatively repeating poly-l-alanine (PLA) sequences as crystalline domain and glycine-rich sequences as noncrystalline domain; the structure is similar to those of spider silk and other wild silkworm silks. In this paper, we proposed a new staggered model for the packing arrangement of the PLA sequence through the use of the Cambridge Serial Total Energy Package program and a comparison of the observed and calculated chemical shifts of the PLA sequence with the Gauge Including Projector Augmented Wave method. The new model was supported by the interatomic distance information from the cross peaks of Ala Cß dipolar-assisted rotational resonance (DARR) spectrum of the PLA sequences in S. c. ricini SF fiber. In addition, three 13C NMR peaks observed in the ß-sheet region were assigned to the carbons with different environments in the same model, but not assigned to different ß-sheet structures.

Physiological correlates of chill susceptibility in Lepidoptera.

The majority of insects enter a state of reversible coma if temperature is lowered sufficiently. If the cold treatment is not too severe these insects recover gradually when returned to benign temperatures in a time-dependent manner that often depends on the duration and intensity of the cold exposure. Previous studies have associated these phenotypes to changes in membrane potential (Vm) and ion balance, and especially hemolymph [K+] is known to be of importance for the recovery time. In the present study we examined this link in three species of Lepidoptera as insects from this order are known to possess resting hemolymph [K+] that would severely compromise Vm in other insects. Specifically, we exposed larval and adult Manduca sexta, larval Bombyx mori, and adult Heliconius cydno to stressful cold (0°C) for extended periods of time. Subsequently we measured chill coma recovery time (CCRT), ion- and water balance, and muscle Vm. As expected we find that resting hemolymph [K+] is high and that resting hemolymph [Na+] is low compared to most other insect species. Muscle Vm depolarised considerably during acute cold exposure, but did so in a manner that was not associated with changes in ion balance. However, prolonged cold exposure coincided with an increase of hemolymph [K+] and further depolarisation of Vm which correlated well with prolongation of CCRT. Combined this demonstrates how insects with different ionic compositions generally suffer from similar consequences of cold stress as other species, such that cold tolerance of chill-susceptible insects within Lepidoptera is also intimately linked to maintenance of ion balance and membrane polarisation.

Antheraea pernyi silk sericin mediating biomimetic nucleation and growth of hydroxylapatite crystals promoting bone matrix formation.

Bone biomineralization is well-regulated processes mediated by extracellular matrix proteins. The materials that can direct nucleation of hydroxylapatite (HAp) crystals and assembly of well-structured material-minerals complex are the key to mimicking the natural mineralization. This study used sericin from Antheraea pernyi (A.pernyi), non-mulberry silkworm cocoon as template to mediate nucleation of HAp crystals. Here we find out that AS (Antheraea pernyi sericin) can nucleate the formation HAp crystals in simulated body fluid verified by XRD and FTIR observations. The HAp crystals are organized into nano-rods oriented with c-axis preferentially parallel to the long axis of AS due to hydrogen bonds and electrostatic interaction and finally aggregated into HAp globule. The cell culture of human bone marrow-derived mesenchymal stem cells (BMSCs) showed that the HAp crystals mediated by AS not only stimulate cell adhesion and proliferation but also promote 0f osteogenic differentiation, suggesting that the resultant mineralized AS biomaterial has potential in promoting bone formation. Thus our work will provide significant implication on biomineralization of A. pernyi silk sericin as a potential scaffold for tissue engineering.