Hemolymph melanization is a conserved immune response in insects and other arthropods. However, the physiological process of the hemolymph system in the melanization response is hardly studied. Here, alterations of hemocytes in immune melanization were observed by Escherichia coli infection in Bombyx mori. Results first showed that there were cells aggregating into clusters. However, it vanished, and only part of clustered hemocytes were melanized during the period of intense immunity. The hemocyte numbers immediately decreased following an immune challenge, slowly increased to a peak, then reduced and finally returned to normalization. Granulocytes participated in cells aggregation at the early and later immune stage, while plasmatocytes were responsible for hemocytes agglomerate and melanization for the longest time, and more oenocytoids appeared at the peak stage of melanization. Moreover, hemocytes played a crucial role in resisting invasion of pathogens by agglomerate and melanization, and the circulatory system maintained higher hemocyte numbers and stronger antibacterial activity in fifth than fourth instar larvae after infection. In vitro immune melanization was most likely preferentially implemented in an independent process. These were the main characteristics reflecting the physiological process of hemolymph immune melanization, which provided an important foundation for further study of the complete mechanisms in the immunity of silkworm.

Dopamine is widely distributed in metazoans and is implicated in many physiological functions. Dopaminergic signaling is mediated through two classes of dopamine receptors, D1-like and D2-like. Phylogeny analysis reveals that, the dopamine receptors probably appeared ahead of the cnidarian divergence, two distinct classes of dopamine receptors likely formed prior to the separation of deuterostomes and protostomes, and INDRs probably split from its ancestor before the emergence of nematodes. Two D2-like genes are closely linked on the same scaffold, and the chromosome region around D2-like gene loci show colinearity among different species within Lepidoptera. These indicate two D2-like and their adjunction genes are likely Lepidoptera-specific orthologs, and occur by gene duplication event taken place after Lepidoptera ancestor split from the common ancestor of Lepidoptera and Diptera. In silkworm, two D2-like genes were expressed in examined tissues, and encoded BmDop2R2 having all the features of D2-like receptors and BmDop2R1 being a truncated variant without the region of N-terminal to TM II. Only dopamine distinctly lowered cAMP levels in BmDop2R2-expressing cells, whereas all tested amines for BmDop2R1 had not markedly effect in pharmacological test. These suggest there is functional difference between the two genes, which are likely resulted from subfunctionalization of gene duplication.