Quality status of Bombyx Batryticatus and suggestions for Chinese Pharmacopoeia (2025) / 中国中药杂志

To understand the current quality status and rearing situation of Bombyx Batryticatus, the authors collected 102 batches of Bombyx Batryticatus from different main producing areas and five major Chinese medicine markets from 2016 to 2018, and measured the properties and quality of the silk gland, to clarify the quality status of Bombyx Batryticatus from different producing areas and markets. In addition, 35 batches of Bombyx Batryticatus from 2019 to 2022 were used to verify the silk gland after revision. Moreover, Beauveria Bassiana was inoculated in the silkworm of 4-5 instars, and standardized rearing was carried out until they die. The death rate and the quality of Bombyx Batryticatus were measured to determine the differences in Bombyx Batryticatus of different instars, and explore the rationality of the infection age of Bombyx Batryticatus in Chinese Pharmacopoeia(2020). The results revealed that in the 102 batches of Bombyx Batryticatus, the qualification rate of silk gland was low; the content of total ash far exceeded the standard; the content of beauvericin varied greatly. The qualification rate of the silk gland of the 35 batches of Bombyx Batryticatus was only 47.49%, which could be increased to 73.00% if the number of silk gland was 2 to 4. The death rate of Bombyx Batryticatus at different infection ages was quite different, with uneven quality. Generally, the yield of Bombyx Batryticatus inoculated on the first day of the fifth instar was high with good quality. Therefore, in combination with the quality and actual production of Bombyx Batryticatus, the following suggestions were proposed for revision of Bombyx Batryticatus in Chinese Pharmacopoeia(2025): The number of silk gland should be revised as 2-4 bright brown or bright black silk glands, after which, the quality of Bombyx Batryticatus could be guaranteed, and the "quality identification based on character" could also be reflected scientifically; the content determination index that the content of beauvericin shall not be less than 0.017% should be added to better control the quality of Bombyx Batryticatus; the infection age should be revised as the first day of the fifth instar to narrow the age span, which could better fit the actual production and ensure the quality of Bombyx Batryticatus.

Comparative electrophoretic profile of proteins and esterases in healthy silkworm larvae (Bombyx mori Lineu, 1758) and infected with nucleopolyhedrovirus / Perfil eletroforético comparativo de proteínas e esterases em lagartas de bicho da seda (Bombyx mori Lineu, 1758) saudáveis e infectadas por nucleopoliedrovírus

Total proteins and esterases from silk gland extracts of Bombyx mori silkworm were characterized and compared with electrophoretic profiles of prepared extracts with silkworm glands infected with nucleopolyhedrovirus (BmNPV). SDS-PAGE (7%) gels was used for the total proteins, and 10% native PAGE for esterases. In the silk glands extracts of healthy silkworms, it was observed seven protein zones with molecular weight varying between 10 kDa (P1) and 60 kDa (P7). In the infected silkworms, a new zone named P8 (90 kDa) was also detected. Esterases activity at 5th instar larvae underwent changes after the infection with BmNPV, since there was a reduction (EST-6 and EST-7) and an increase (EST-8) in the intensity of the regions of esterases activity, and specificity of EST-9 to ß-naphthyl acetate. Those alterations observed in the expression of genes after the infection with the nucleoplyhedrovirus can be used as markers to detect infections in B. mori.

Nesse estudo foram identificadas e comparadas as alterações das proteínas totais e esterases em extratos de glândula sericígena de lagartas de Bombyx mori sadias e infectadas por nucleopoliedrovírus (BmNPV), empregando eletroforese SDS-PAGE com géis a 7% para proteínas totais, e PAGE a 10% para esterases. Nos extratos de glândulas sericígenas de lagartas saudáveis, foram observadas sete regiões proteicas com peso molecular que variam entre 10 kDa (P1) a 60 kDa (P7). Naquelas infectadas pelo BmNPV, uma nova região denominada de P8 (90 kDa) foi detectada. A atividade das esterases no 5º instar larval sofreu alterações após a infecção pelo BmNPV porque houve redução (EST-6 e EST-7) e aumento (EST-8) na intensidade das regiões de atividade de esterases; e especificidade da EST-9 com o substrato ß-naftil acetato. Essas alterações na expressão gênica, após a infecção pelo nucleopoliedrovírus, poderão ser utilizadas como marcadores para detecção da infecção em B. mori.

Macromolecular array patterns of silk gland secretion in social Hymenoptera larvae

The cocoon, produced by most holometabolous insects, is built with silk that is usually produced by the larval salivary gland. Although this silk has been widely studied in the Lepidoptera, its composition and macromolecular arrangement remains unknown in the Hymenoptera. The macromolecular array patterns of the silk in the larval salivary gland of some meliponids, wasps, and ants were analyzed with polarized-light microscopy, and they were compared with those of Bombyx mori (Lepidoptera). There is a birefringent secretion in the glandular lumen of all larvae, due to filamentous structural proteins that display anisotropy. The silk in the distal, middle and proximal regions of the secretory portion of Formicidae and Vespidae glands presented a lattice optical pattern. We found a different pattern in the middle secretory portion of the Meliponini, with a zigzag rather than a lattice pattern. This indicates that the biopolymer fibers begin their macromolecular reorganization at this glandular region, different from the Formicidae and the Vespidae, in which the zigzag optical pattern was only found at the lateral duct. Probably, the mechanism of silk production in the Hymenoptera is a characteristic inherited from a common ancestor of Vespoidea and Sphecoidea; the alterations in the pattern observed in the Meliponini could be a derived characteristic in the Hymenoptera. We found no similarity in the macromolecular reorganization patterns of the silk between the Hymenoptera species and the silkworm.

Analysis of nuclear proteins from silk glands of Bombyx mori.

A gentle method for the isolation of nuclei from developing silk glands of Bombyx mori has been standardized. The nuclei, whether isolated or directly visualized in situ within the silk glands, exhibit complex morphology. The nuclei occupy almost the entire volume of the gigantic silk gland cells. Although the isolated nuclei still retain their ramified morphology, being polyploid they are fragile and often become fragmented. The histone and low-salt-extractable proteins from nuclei isolated from the middle and posterior silk glands on different days of the fourth and fifth instars of larval development have been analysed. The histones did not show any stage- or tissue-specific variations whereas the low-salt-extractable proteins showed some developmental stage specific variation. Using the antibody raised against one such protein, its absence in the early stage of development has been confirmed by Western blotting techniques. This developmental stage specific protein may be functionally linked to some activities responsible for boosting up the production of silk or silk-related proteins during the fifth instar of larval development.