Studies On The Mechanisms Of The Scaleless Wings Mutant In The Silkworm, Bombyx Mori

The coating of scales on the surface of the wings is a basic characteristic of lepidopteran adults. Wing scales have important physical function to Lepidoptera, and patterns formed by multicolored scales not only have specifically developmental mechanisms, but also are a key innovation during lepidopteran evolution. So lepidopteran wing scales are valuable for the studies of insect physiology, developmental biology, biologic evolution, and even pest control. In this thesis, we studied the silkworm scaleless wings mutant(scaleless, sl) on morphological, cell biological and molecular biological levels, and tried to clarify the mechanism of the mutant and offer references for the studies on the development of lepidopteran wing scales.We carried out our studies beginning with the development of the wing discs/wings, in size and shape, during the 5th instar larval and pupal period. We found that the wing disc grew slowly in the prophase of the larva, while very fast just before metamorphism. We also found that pupa was a period of the wing buds perfecting, with wing scale formation and pigmentation. Hemopoietic organ is located tightly near or attach to the imaginal wing disc in the silkwom, Bombyx mori, and the two kinds of organs are customarily called by a joint name as hemopoietic organ-wing disc complex. We successfully extirpated the complexes of 5th instar larvae, and found that even when all of the complexes were extirpated, majority of the silkworms could still develop into moths normally. Using this technique, we studied the effects of the extirpation on the hemopoiesis of the silkworm, by investigating the change of density and total of hemocytes circulating in the final instar larval hemolymph. We found that mitosis could offer enough hemocytes for the development of the silkworms whose complexes were totally extirpated. And the collapse of the hemopoietic organs during wandering might be a consequence of metamorphosis, but not a prerequisite for pupation. Exogenous hormones could elevate the mitosis of circulating hemocytes in complexes totally extirpated silkworm; and during early spinning stage, complexes extirpation might cause a certain extent hemopoietic compensation of the remainders. The feasibility of the silkworm wing discs growing and developing in exotic site was proved by reciprocal transplantation technique. The results afforded us the background for the following studies.We investigated the morphology of scaleless and found that it had many fewer wing scales than wild type(WT), and that the remaining scales were smaller in shape with fewer furcations. Reciprocal transplantation of wing discs between scaleless and WT revealed that the WT wing disc could develop into a small wing with scales after transplantation into a scaleless larva; however, the scaleless wing disc developed into a small wing without scales in a WT larva. Upon dissection of WT and scaleless wing discs at different stages from the fifth instar larva to adulthood, no obvious differences were found before pupation. However, after pupation, tracheae produced from WT wing veins extended to the lacunae between the veins and formed a network on the second day after pupation, whereas this did not happen in scaleless. At the same time, no marked difference of the adult body tracheal development was found between the mutant and wild type. Furthermore, if the surface of a wing disc was cut and its veins injured, the resulting wing also had fewer scales than that of WT. Also, we found that higher partial pressure of O₂ could rescue the loss of scales in scaleless. These data suggest that the factors affecting the growth of scales were not produced in the hemolymph, but in the wing disc itself. It is also implied that wing scale development is dependent on the correct organization of tracheal system in the wing disc.We gave data to clarify the mechanism of the mutation of scaleless at the cell biological level. The results of AO/EB dying and Caspase-3/7 analysis showed that programmed cell death participates in the wing scale development during early pupal stage. On 2d, well ranged cells could be seen on the pupal wing, surrounded with vast dead ones. There were significant differences between that of sl and the wild type(WT) at each phase. Well-differentiated scale precursor cells did not form in sl when they had formed in WT. The peak of Caspase-3/7 activity in sl occured one day later than, and ten times as much as that in WT. Apoptotic bodies and DNA ladder studies also showed that there was excessive apoptosis in sl early pupal wing.AS-C(achaete-scute Complex) genes are associated with the development of Drosophila notum bristles, and a homolog of AS-C(ASH), B-ASH1, controls the formation of butterfly wing scales. In order to clarify the mechanism of the scaleless wings mutation of sl at the molecular level, we cloned and analyzed the AS-C homolog in Bombyx mori. Four genes, Bm-ASH1, Bm-ASH2, Bm-ASH3 and Bm-ase were cloned, and the proteins coded by the four genes had a typical bHLH motif each, and the former three also each had a 16-17 aa conservative region at the C-terminal. All of these indicated the four genes according with the characteristic of AS-C homolog. Homologous comparation and evolutional analysis showed that Bm-ASH1 formed earliest among the three preneural genes; while Bm-ASH2 and Bm-ASH3 were mostly homologous, and might both occur later than Bm-ASH1. We investigated the expression level in various larval organs and different embryo developmental ages using RT-PCR and realtime quantitative PCR methods. Bm-ASH1 and Bm-ASH2 were expressed in most of the organs and had a high expression level in proper embryo ages. Bm-ASH3 was more differential, so we deduced it formed at the most near time and gained special function. Bm-ase was also more special and the detected expression level was low. But neither homologous comparation nor functional analysis could correspond the three Bombyx preneural genes to those of Drosophila, so we still named the genes after their discoverd order.Both of the results of semi-quantative RT-PCR and in situ hybridyzation showed that Bm-ASH2 gene expressed abnomal in scaleless pupal wing. The gene expressed highly in 0 d-2 d WT pupal wing, but very low in scaleless. Sequencing results showed that there was a continuous absent mutation of 26 bp, 1 027 bp upstream of Bm-ASH2 initial code in scaleless genome. The transcriptional activity of scaleless's, Bm-ASH2 promoter was much lower than that of the WT, just the same as the truncated 979 bp promoter segment of WT without the 26 bp. EMSA result indicated that the 26 bp had a special binding nuclear protein. Genetic experiments also showed that the 26 bp sequence absent was tightly linked with the scaleless wings mutated phenotype. All of the results proved that the mutation in scaleless's Bm-ASH2 promoter caused the failure of Bm-ASH2 gene's transcription and translation. If artificially made the Bm-ASH2 gene expressed in early scaleless pupal wing with immediate expression system, the wing scales on the adult wing were increased significantly. So Bm-ASH2 gene is a key regulator for the formation of silkwom wing scales.