Melanin And Urate Act As Potent Ultraviolet-A Preventer In The Integument Of The Silkworm,Bombyx Mori

Insect pigmentation is a highly variable character, and it varies between species, between populations of the same species, between individuals within a population, even between life stages of single individuals and between body parts of individual life stages. Diversity exists in terms of which colors are used as well as how these colors are arranged into patterns. Insect melanization is very common, the process of which is variously used for purposes of mimicry, sexual selection, and thermoregulation, but also play a major role in wound healing and cuticular hardening. The phenomenon that epidermal cells under the white stripes rather than black stripes contain many urate granules was found in larvae of several Lepidopteran species. However, the biological mechanism of this phenomenon is still unknown.The silkworm, Bombyx mori, is a highly domesticated Lepidopteran insect. There are about 700 mutant systems kept in our silkworm resource pool, part of them are color mutants. In this study, we take advantage of several silkworm body color mutant strains to investigate deposition patterns and biological mechanism of urate and melanin in the integuments of these mutant larvae. The main results are as follows:There is significant difference of body color at the larval stage between plain Black (pB) and background strain (p). The larval body of pB shows strong overall black pigmentation while the larval body of p shows white. However, during the pupa and adult moth stage, the body color pattern of pB is consistent with p. By imaging with transmission electron microscope (TEM), we found that there were some melanin granules in the larval cuticle in pB with black body color, not in p with white larval body color. In contrast, the larval epidermal cell of p has much more urate granules than that of black one. The analysis of integument ultrastructure elucidated the material basis of different larval body color between pB and p, black body color of pB is because of a lot of black melanin granules in exocuticle and little urate granules in the epidermis white body color of p is due to a great quantity of white uric acid granules in epidermis and no melanin granules in exocuticle.Then, we compare the expression patterns of melanin synthesis related genes between pB and p. The results showed that the peaks of expression of melanin synthesis related genes existed in the molting period, but the pattern varies among different genes. The expression peaks of yellow, yellow-f and laccase2 were in the middle of molting period. However, the expression peaks of TH, DDC, tan, ebony, dat and black were latter than that of yellow, yellow-f and laccase2, in the latter half of the molting period. When compared the expression profiles, we found that the expression profiles of TH, DDC, yellow, yellow-f and laccase2 in pB were higher than in p, which promoted melanization. While the expression profile of ebony, which inhibited melanization, in pB was less than in p. And the larval integument of pB contained significantly higher dopamine than integument of p. Therefore, the black larval body color of pB is probably due to the large amount of dopamine-melanin oxidized from dopamine.In order to confirm that white granules we observed were made up of urate, we used HPLC to measure the uric acid content in the integuments of several developmental stages between pB and p. The results showed that the the white integument of p contained over seven times the amount of uric acid in the black integument of pB on the day 3 of the 5th instar larvae. As developmental stage proceeds, the accumulation of uric acid in the integument of pB increased and then stabilized at approximately the same level as the counterpart in p in pupa and adult moth. This implied that the uric acid deposition in the integument was related to the body color, we speculated that a large amount of dopamine might inhibit the urate deposition in the epidermis of pB because we observed that the epidermis of pB does have significantly higher content of dopamine than that of p. We supposed that it should be ubiqutious that the deposition of uric acid under white integument was higher than that under black integument in some insects which viewed uric acid as the excretory product.Ultraviolet-A (UVA) exposure experiments reflected that od mutant larvae were more sensitive to the UVA irradiation than p and pB. To test the oxidative damage caused by UVA, we examined the content of MDA in blood of these three strains, which could reflect the content of free radical indirectly. Overall, the content, of MDA increased gradually with exposure time increase. More MDA was detected in od rather than in p and pB. Thus, the deposition of urate or melanin in the integument plays an important role in protecting the silkworm from the oxidative damage caused by UVA irradiation.In the present study, we found the opposite deposition patterns of melanin and urate granules in the larval integuments of pB and p. Furthermore, the uric acid content under the black stripes was significantly lower than that under the white stripes in a single individual of mutant (ps). It appears that an antagonism exists between melanin and uric acid. UVA exposure experiments indicated that deposition of either melanin and urate granules in integuments of silkworm larvae reduced damage of UVA to silkworm. Thus, we propose that melanin and urate act as potent UVA preventer in the integument of the silkworm.