Bombyx mori can synthesize ascorbic acid through the l-gulose pathway to varying degrees depending on developmental stage.

Vitamin C (VC) is an essential nutrient for many animals. However, whether insects, including Bombyx mori, can synthesize VC remains unclear. In this article, the optimized HPLC method was used to determine the content of l-ascorbic acid (AsA) in silkworm eggs, larvae and pupae, and the activity of l-gulono-1,4-lactone oxidase (GULO), a key enzyme in VC synthesis. The RNA interference method was used to determine the effect of the BmGulo-like gene on embryonic development and GULO activity in the pupal fat body. The AsA content increased significantly during E144 h-E168 h in the late embryonic stage and P48 h-P144 h in the middle-late pupal stage, in which exogenous VC was not ingested. Furthermore, the body AsA content in larvae fed VC-free feed also increased with larval stage. The GULO enzymatic activity was present in eggs and the fat bodies of larvae and pupae, even when the larvae were reared with fresh mulberry leaves. Moreover, the activity was higher in the later embryonic stages (E144 h-E168 h) and the early pupal stage (before P24 h). The GULO activity in the pupal fat body dramatically decreased when the screened BmGulo-like gene (BGIBMGA005735) was knocked down with small interfering RNA; in addition, the survival rate and hatching rate of eggs significantly decreased 21% and 44%, respectively, and embryonic development was delayed. Thus, Bombyx mori can synthesize AsA through the l-gulose pathway, albeit with low activity, and this synthesis ability varies with developmental stages.

Differences in gut microbiota between silkworms (Bombyx mori) reared on fresh mulberry (Morus alba var. multicaulis) leaves or an artificial diet.

Artificial diets for silkworms have many potential applications and they are important in sericulture. However, the challenges of weak larvae and low silk protein synthesis efficiency in silkworms reared on artificial diets have not been resolved. Here, we used high-throughput sequencing to analyse the differences between the gut microbiota of 5th-instar larvae reared on mulberry leaves and larvae reared on an artificial diet. The results showed that at the phylum level, Cyanobacteria, Firmicutes, Proteobacteria, Bacteroidetes and Actinobacteria are the dominant bacteria in the intestines of silkworm larvae of all the strains. But the abundance of dominant bacteria in the gut microbiota differed between the silkworm strains that were reared on mulberry leaves, as well as between the silkworm strains that were reared on the artificial diet, while the gut microbiota diversity was lower in the silkworm strains that were reared on the artificial diet. Prediction of the functions of the gut microbiota in the hosts indicated that there was no significant difference between the silkworm strains that were reared on mulberry leaves, while there were significant differences between silkworm strains reared on the artificial diet. When the silkworm diet changed from mulberry leaves to the artificial diet, changes in gut microbiota in the silkworms affected host nutrient metabolism and immune resistance. These changes may be related to the adaptation of silkworms to their long evolutionary history of eating mulberry leaves.

Metabolomics differences between silkworms (Bombyx mori) reared on fresh mulberry (Morus) leaves or artificial diets.

Silkworms (Bombyx mori) reared on artificial diets have great potential applications in sericulture. However, the mechanisms underlying the enhancement of metabolic utilization by altering silkworm nutrition are unclear. The aim of this study was to investigate the mechanisms responsible for the poor development and low silk protein synthesis efficiency of silkworms fed artificial diets. After multi-generational selection of the ingestive behavior of silkworms to artificial diets, we obtained two strains, one of which developed well and another in which almost all its larvae starved to death on the artificial diets. Subsequently, we analyzed the metabolomics of larval hemolymph by gas chromatography/liquid chromatography-mass spectrometry, and the results showed that vitamins were in critically short supply, whereas the nitrogen metabolic end product of urea and uric acid were enriched substantially, in the hemolymph of the silkworms reared on the artificial diets. Meanwhile, amino acid metabolic disorders, as well as downregulation of carbohydrate metabolism, energy metabolism, and lipid metabolism, co-occurred. Furthermore, 10 male-dominant metabolites and 27 diet-related metabolites that differed between male and female silkworms were identified. These findings provide important insights into the regulation of silkworm metabolism and silk protein synthesis when silkworms adapt to an artificial diet.

Transcription factor SGF1 is critical for the neurodevelopment in the silkworm, Bombyx mori.

FoxA transcription factors play vital roles in regulating the expression of organ-specific genes. BmSGF1, the sole FoxA family member in Bombyx mori, is required for development of the silk gland. However, the function of BmSGF1 in development of the nervous system in the silkworm remains unknown. Here, we show that the amino acids sequence of BmSGF1 is evolutionarily conserved in its middle region from Trichoplax adhaerens to human and diverged from the homologues in most other species in its N-terminal region. BmSGF1 expresses in the nervous system at the embryonic stage. Knockdown of Bmsgf1 by RNA interference (RNAi) results in abnormal development of axons. Therefore, our results demonstrate that BmSGF1 is an indispensable regulator for neurodevelopment.

BmRobo2/3 is required for axon guidance in the silkworm Bombyx mori.

Axon guidance is critical for proper wiring of the nervous system. During the neural development, the axon guidance molecules play a key role and direct axons to choose the correct way to reach the target. Robo, as the receptor of axon guidance molecule Slit, is evolutionarily conserved from planarians to humans. However, the function of Robo in the silkworm, Bombyx mori, remained unknown. In this study, we cloned robo2/3 from B. mori (Bmrobo2/3), a homologue of robo2/3 in Tribolium castaneum. Moreover, BmRobo2/3 was localized in the neuropil, and RNAi-mediated knockdown of Bmrobo2/3 resulted in the longitudinal connectives forming closer to the midline. These data demonstrate that BmRobo2/3 is required for axon guidance in the silkworm.

BmRobo1a and BmRobo1b control axon repulsion in the silkworm Bombyx mori.

The development of the nervous system is based on the growth and connection of axons, and axon guidance molecules are the dominant regulators during this course. Robo, as the receptor of axon guidance molecule Slit, plays a key role as a conserved repellent cue for axon guidance during the development of the central nervous system. However, the function of Robo in the silkworm Bombyx mori is unknown. In this study, we cloned two novel robo genes in B. mori (Bmrobo1a and Bmrobo1b). BmRobo1a and BmRobo1b lack an Ig and a FNIII domain in the extracellular region and the CC0 and CC2 motifs in the intracellular region. BmRobo1a and BmRobo1b were colocalized with BmSlit in the neuropil. Knock-down of Bmrobo1a and Bmrobo1b by RNA interference (RNAi) resulted in abnormal development of axons. Our results suggest that BmRobo1a and BmRobo1b have repulsive function in axon guidance, even though their structures are different from Robo1 of other species.

Evolutionarily conserved repulsive guidance role of slit in the silkworm Bombyx mori.

Axon guidance molecule Slit is critical for the axon repulsion in neural tissues, which is evolutionarily conserved from planarians to humans. However, the function of Slit in the silkworm Bombyx mori was unknown. Here we showed that the structure of Bombyx mori Slit (BmSlit) was different from that in most other species in its C-terminal sequence. BmSlit was localized in the midline glial cell, the neuropil, the tendon cell, the muscle and the silk gland and colocalized with BmRobo1 in the neuropil, the muscle and the silk gland. Knock-down of Bmslit by RNA interference (RNAi) resulted in abnormal development of axons and muscles. Our results suggest that BmSlit has a repulsive role in axon guidance and muscle migration. Moreover, the localization of BmSlit in the silk gland argues for its important function in the development of the silk gland.

Evidence for the coevolution of axon guidance molecule Netrin and its receptor Frazzled.

Coevolution of a ligand and its receptor is critical for maintaining their function in different species, but how ligand and its receptor coevolve is poorly understood. The axon guidance molecule Netrin and its receptor Frazzled (Fra) are useful to study the mechanisms of ligand-receptor coevolution. Here, we have applied codon substitution models to identify positive selection of the netrin and fra genes. The sites under positive selection in netrin and fra were detected in same lineage, such as nematode, dipteran, hymenopteran, hemichordate, and teleost. Several amino acid residues that are under positive selection were identified in the interaction domains. Here we provide evidence that positive selection is essential for the coevolution of Netrin and Fra during central nervous system evolution.

[Progress in the role of oxidative stress in the pathogenesis of type 2 diabetes].

Diabetes mellitus (DM) is characterized by hyperglycemia and disturbances of carbohydrate and fat metabolism resulted from an absolute or relative deficiency of insulin and insulin resistance. Recent studies indicate that oxidative stress may have a central role in the pathogenesis of type 2 diabetes. Currently, the diagnosis of body oxidative stress level mainly depends on the detection of oxidative stress markers including reactive oxygen species (ROS), reactive nitrogen species (RNS) and lipid peroxide in clinical and experimental studies with methods combining physical and chemical means. The mechanism underlying oxidative stress-induced diabetes mainly may be through two ways. Firstly oxidative stress damages the normal function of islet ß cells, through the destruction of mitochondrial structure and inducing apoptosis, activation of nuclear transcription factor kappa B (NF-κB) signaling pathway, causing cell inflammatory response, and reducing insulin synthesis and secretion by inhibiting pancreatic and duodenal homeobox 1 (PDX-1) nuclear cytoplasm translocation as well as inhibiting energy metabolism; Secondly, oxidative stress induces insulin resistance by interfering physiological activities related to insulin signaling including phosphorylation of insulin receptor (InsR) and insulin receptor substrate (IRS), the activation of phosphatidylinositol 3-kinase (PI3K) and the translocation of glucose transporter 4 (GLUT4), as well as injuring the cytoskeleton. Studying the role of oxidative stress in the pathogenesis of diabetes not only helps to reveal the pathogenesis of diabetes, but also provides a theoretical basis for the prevention and treatment of diabetes.

Alternative splicing of the antitrypsin gene in the silkworm, Bombyx mori.

Alternative splicing plays an important role in expanding protein diversity. In the present study, different splice variants of the antitrypsin gene (sw-AT) in the silkworm were identified by bioinformatics analyses using expressed sequence tags and genomic information. Four splice variants were obtained by RT-PCR with suitably designed primers, confirmed by sequencing, and designated as sw-AT-1, sw-AT-2, sw-AT-3, and sw-AT-4. The sw-AT gene contains 10 exons and nine introns. The splice variants differ in exon 9, with sw-AT-1, sw-AT-2, and sw-AT-3 using different versions of the exon, namely exon 9a, 9b, and 9c, respectively. In sw-AT-4, exon 9 consists of the combination of exons 9b and 9c. The expression patterns of the four isoforms in different tissues, at different developmental stages, and under different stress conditions (temperature, starvation, and mycotic infection) were characterized and quantified. The sw-AT isoforms showed tissue-specific expression patterns, with sw-AT-1 present in almost all tissues and sw-AT-4 found in only a few tissues. The four isoforms were predominantly expressed in the fat body, body wall, and testes of larvae, and exhibited similar expression profiles during development of the fat body. Among the stress treatments, low temperature had the greatest effect on isoform expression, and expression was also upregulated with mycotic infection.

Accumulation of 1-deoxynojirimycin in silkworm, Bombyx mori L.

1-deoxynojirimycin (1-DNJ) contents in the silkworm, Bombyx mori, at different developmental stages and tissues were investigated by using reverse-phase high-performance liquid chromatography. The 1-DNJ contents of silkworm larvae change significantly with their developmental stages. The male larvae showed higher accumulation efficiency of 1-DNJ than the females and also a significant variation was observed among the silkworm strains. The present results show that tissue distribution of 1-DNJ was significantly higher in blood, digestive juice, and alimentary canal, but no 1-DNJ was observed in the silkgland. Moreover, 1-DNJ was not found in silkworms fed with artificial diet that does not contain mulberry leaf powder. This proves that silkworms obtain 1-DNJ from mulberry leaves; they could not synthesize 1-DNJ by themselves. The accumulation and excretion of 1-DNJ change periodically during the larval stage. There was no 1-DNJ in the newly-hatched larvae and 1-DNJ was mainly accumulated during the early and middle stages of every instar, while excreted at later stages of larval development. Further, it is possible to extract 1-DNJ from the larval feces and it is optimal to develop the 1-DNJ related products for diabetic auxiliary therapy.