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Mechanism of fluorescent cocoon sex identification for silkworms Bombyx mori. Sci China Life Sci, ...... 28 Tamura Y, Nakajima K I, Nagayasu K I, et al. Flavonoid.
SCIENCE CHINA Life Sciences • RESEARCH PAPERS •

November 2010 Vol.53 No.11: 1330–1339 doi: 10.1007/s11427-010-4084-3

Mechanism of fluorescent cocoon sex identification for silkworms Bombyx mori ZHANG YuQing1,2*, YU XiaoHua1,2, SHEN WeiDe1,2, MA YongLei1,2, ZHOU LiXia1,2, XU NaiXi1,2 & YI ShuQian1,2 1

2

State Engineering Laboratory of Modern Silk, Soochow University, Suzhou 215123, China; Silk Biotechnology Key Laboratory of Suzhou City, Medical College of Soochow University, Suzhou 215123, China Received June 26, 2009; accepted September 21, 2009

By using silkworms, Bombyx mori, fluorescent cocoon sex identification (FCSI) as an experimental material, direct fluorescence spectrometry of the cocoon surface indicates that the fluorescent color of silkworm cocoons is made up of two peaks of yellow and blue-purple fluorescence emission. The fluorescent difference between male and female cocoons is attributed to the differential absorption of yellow fluorescent substances by the midgut tissue of 5th instar female silkworms. Thin layer chromatography (TLC) and fluorescent spectra indicate that blue-purple fluorescent substances are composed of at least five blue-purple fluorescent pigments, and yellow fluorescent substances are made up of at least three. UV spectra and AlCl3 color reaction show that the three fluorescent yellow pigments are flavonoids or their glycosides. Silkworm FCSI is due to selective absorption or accumulation of the yellow fluorescent pigments by the posterior midgut cells of female 5th instar larvae. The cells of the FCSI silkworm midgut, especially the cylinder intestinal cells of the posterior midgut have a component which is a yellow fluorescent pigment-specific binding protein that may be vigorously expressed in the 5th instar larvae. silkworm, fluorescence cocoon, sex identification, pigment, midgut, accumulation Citation:

Zhang Y Q, Yu X H, Shen W D, et al. Mechanism of fluorescent cocoon sex identification for silkworms Bombyx mori. Sci China Life Sci, 2010, 53: 1330–1339, doi: 10.1007/s11427-010-4084-3

Under ultraviolet radiation the cocoons of silkworms, Bombyx mori, show various fluorescent colors which are divided into yellow-white and blue-purple categories. In the late 20th century, Japanese scholar discovered this phenomenon [1,2]. Genetic analysis has indicated that cocoon fluorescence is closely related to the reelability of silk [3,4]. Yellow fluorescent cocoons have better reelability than the purple ones. The fluorescent characteristics in cocoons are inheritable, but differences in fluorescence do exist among silkworm species. Therefore, yellow fluorescent cocoons are sought to improve cocoon reelability through breeding. There are significant differences in both the quality and mechanical properties of male and female cocoons. Male *Corresponding author (email: [email protected]) © Science China Press and Springer-Verlag Berlin Heidelberg 2010

cocoon silk has a finer size and better economic traits than those of female cocoon silk and its raw silk is 1−2 grades higher. When baked, cooked and reeled, both male and female cocoons are mixed with a composite fiber obtained. It is difficult to mass produce high-grade raw silk of 6A level. Single male silkworms are being bred by breeding sex-limited silkworms, a sex-linked lethal balanced system, and by the use of thermo-sensitive lethal trait techniques or methods. Most of these technologies have not been widely extended and applied in sericulture production [5]. Tang et al. [6] isolated a new silkworm variety for fluorescent cocoon sex identification (East 34) using Chinese system varieties. By the methods of system separation and cross-breeding, Tang et al. [7] also bred a pair of FCSI silkworms whose female and male cocoons respectively life.scichina.com

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displayed white and yellow fluorescent colors under ultraviolet light. By improving available silkworm varieties, Yu et al. [8] bred several species of silkworm FCSI, e.g., YingSu×YingXiao and SuXiong×YingXiao, whose female and male cocoons respectively displayed blue-purple and yellow-white fluorescent colors under ultraviolet light. It is possible to reel male and female cocoons separately to produce high-quality male silk of 6A grade, with the raw silk quality of female cocoons also being improved [9,10]. It has been indicated that the fluorescent substance appears in the 3th, 4th instar silkworm larvae blood and does not appear in their silkglands until day 3 of the 5th instar larvae [11,12]. It is widely felt that the fluorescent pigments in cocoons are derived from silkworm blood, and are consistent with the fluorescence of their blood. It has also been found that silkworm blood was yellow fluorescent while the cocoon was purple [13], which resulted from different fluorescence in the blood of mature larvae. The silkglands did not selectively absorb the fluorescent pigment [14]. There was no evident fluorescence change in blood prior to silkworm maturation and there was also no clear difference between female and male silkworm blood. The evident fluorescent differences appeared in blood with the aging of silkworms. These inconsistent results resulted from several factors indicating the different silkworm species used, different sampling times, as well as different interpretations of fluorescent colors. Like the relationship of fluorescent colors between silkworm blood, silkglands and cocoons, the results from studies about the characteristics and varieties of fluorescent pigment have been affected by the different silkworm varieties or different research methods used in tests. Paper chromatography showed that the shell of bamboo-colored cocoons had seven pigments, while green cocoons “Dazao”, had nine fluorescent pigments in which there were five flavonoid pigments and four flavonoid analogues [15]. The application of thin-layer chromatography (TLC) combined with UV-scanning, high-performance liquid chromatography (HPLC) and paper chromatography showed that silkworm cocoon shells contain several fluorescent pigments that were the same in both male and female cocoons, and the fluorescent differences were caused by their relative levels in cocoon shells [16,17]. The polarity and hydrophilicity of yellow fluorescent pigments were relatively stronger than those of blue-purple fluorescent pigments [18−20], which was perhaps the reason why the former were superior to the latter in cocoon reelability. A few studies have also proposed that the extracts from the cocoon shell, which belonged to flavonoids and their analogues, had similar UV absorption peaks (λmax=270 nm, λmax=240 nm). MS analysis showed that fluorescent pigments were flavonols with a molecular weight of about 340 Da and had very similar molecular structures [21–23]. It is uncertain as to why FCSI silkworm cocoons show different fluorescent colors between males and females and

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exactly how many fluorescent pigments exist. Through the direct analysis of the fluorescent spectrum of silkworm cocoon surfaces, fluorescent microscopic observation of silkworm tissues, as well as through TLC, fluorescence and UV spectroscopy of the midgut, cocoon shell and their extracts, we explored the mechanism of silkworm FCSI. This provides an empirical basis for the development of novel species of FCSI silkworm with improved economic properties.

1 1.1

Materials and methods Experimental materials

A FCSI silkworm variety, YingSu×YingXiao [24], was reared during June, 2006. YingSu is a Chinese system of bivoltine and four-molted silkworms with FCSI. Its male and female cocoons are respectively yellow and purple fluorescent under ultraviolet light and its sex-identified rate was 100%. YingXiao is a Japanese system of bivoltine and four-molted silkworms with the same FCSI as above. Its male and female cocoons are respectively yellow and purple fluorescent under 365 nm UV irradiation and its sex-identified rate is also 99.8% [25]. The male and female cocoons of YingSu×YingXiao display yellow or white-yellow and purple or white-blue fluorescences under UV irradiation 1.2

Extraction of fluorescent substances

The cocoon shell that was cut into small pieces prior to the test, midgut tissue or silica spots containing separated samples from one- or two-dimensional silica gel chromatography (2-D SGC) was immersed in 100% methanol solution at 50°C and extracted shakily for 1−2 d. The supernatant was harvested by using centrifugal or filtering methods and then concentrated in the rotary evaporator and the final extract was maintained at 4°C for fluorescent analysis. 1.3

2-D TLC

One-dimensional developing of SGTLC was carried out by petroleum ether:ethyl acetate:chloroform=8:3:2 (V/V, 1-D developer) using silica gel plates HSGF254 (200×100 mm) made in the Yantai Chemical Industrial Institute. Two-dimensional developing was carried out by n-butanol:acetate acid:H2O=4:1:2 (V/V). The preparation step of the 2-D developer was as follows: after the three solutions were poured into a separating funnel and fully mixed and balanced, the upper liquid was harvested for the following TLC. Approximately 10 μL of the extract sample was placed onto the silica gel plate. After one-dimensional SGTLC was developed in 1-D developing solvents at room temperature, the developed plate was dried and developed in a 2-D developer. The spots of 1-D and 2-D were identified under 365 nm UV light and the Rf value was calculated.

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Fluorescent and UV spectrum analysis

The cocoon shell was cut into pieces of about 1.5×1.5 cm and the lyophilized midgut was also cut into pieces of about 1.0×1.5 cm. The curved surface of the cocoon or midgut was placed at a 45° angle towards the light source and into a sample cuvette in a fluorescence spectrophotometer (Hitachi F-4500). The fluorescence emission spectra (FEmS) of the cocoon shell surface and intestinal tissue were determined. Before the fluorescence excitation spectra (FExS) of extract samples were determined, the extract solution was placed onto the filter paper and then dried in a 50°C oven. The FEmS and FExS of the extract attached to the filter papers were measured. In general, the fluorescence spectra analysis conditions were as follows: excitation wavelength, 360 nm; emission wavelength, 530 nm for yellow fluorescent material or 430 nm for blue-purple fluorescent material; voltage, 950 V; excitation slit, 2.5 nm; emission slit, 1.0 nm. The UV spectra of various sample extracts were determined by a Hitachi U-3000 Spectrometer. The reference solution was a solvent used in sample extraction for measurement. 1.5

Coloring reaction of AlCl3

The 2% AlCl3 solution was prepared with an ethanol aqueous solution. The AlCl3 solution was sprayed onto the developed silica gel plate. The dried plate was placed under UV light to observe the coloring reaction of sample with AlCl3.

2 2.1

Figure 1 The colors or fluorescent colors of female and male silkworm cocoons under daylight (A), ultraviolet light (B) and Nikon TE2000 Inverted Fluorescent Microscopy (C). The observation (40×) is under UV light. Test variety, YingSu×YingXiao. Left, a female cocoon; Right, a male cocoon.

Results Fluorescent cocoon color

FCSI silkworm cocoons are white in color and show no difference from ordinary cocoons in daylight (Figure 1A). Under UV light in a dark environment the female cocoon shows blue fluorescence (Figure 1B, left) while the male displays yellow-white fluorescence (Figure 1B, right). Although available cocoons show different fluorescent colors under ultraviolet light, no FCSI feature exists in these cocoons. When the cocoon shell surface was observed by an Inverted Fluorescence Microscope (Nikon TE2000U), the surface fibers of female cocoons were observed to be dark blue-purple (Figure 1C, left) while the surface of male cocoons were yellow-white but their fibers were all blue-purple owing to the fluorescent emission of sericin protein under UV light (Figure 1C, right). Judging from the fluorescent sex-identification of both male and female cocoons, the deeper the fluorescent colors of newly bred species and the greater the differences in fluorescent colors between male and female cocoons, the easier it is to identify the sex of the cocoons. YingSu×YingXiao used in this experiment is similar to the available species with regard to economic traits and reelability.

2.2 Fluorescent spectra of the cocoon shell surface and its extract In order to investigate fluorescent differences between male and female sex-identified cocoons, the fluorescence spectra of the outer surface of cocoon shells was directly analyzed. Figure 2 shows that the FEmS of male cocoon shells have two emission spectrum bands. A stronger fluorescence emission band is in the range of about 400–500 nm (purple fluorescent bands), and its emission peak (λ1max), which is not sharp, is at about 440 nm. The second is relatively small, is in the range of 500–600 nm (yellow fluorescent bands), and its emission peak (λ2max) which is sharp, is at 542 nm. The intensity ratio of emission peaks (λ2max/λ1max) of the two emission bands is about 0.4 (red line). When the male cocoon shell was immersed in 100% methanol at 50°C for 1–2 d for shaking extraction, the resulting supernatant was harvested by using centrifugation or filtering methods and highly concentrated in the rotary evaporator. 1.0 mL of the final extract solution was dripped onto filter paper and its FEmS was determined after the filter paper was dried. The results are shown in Figure 2 (red dotted line). The two emission bands and the strength of the purple and yellow fluorescent spectra of male cocoons with direct surface

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measurement were similar to those of the male cocoons with their methanol extract. Their strength ratios (λ2max/ λ1max) were about 0.4, which indicate that FEmS from direct measurement of the cocoon surface was similar to that from methanol extract and that the former was much stronger than the latter in FEmS strength. It was apparent that the yellow-white fluorescent color derived from male cocoons is correlated with relative intensity of the λ2max emission band. The FEmS (blue straight line) from the female cocoon surface in Figure 2 is similar to that of the male cocoon shell, but the relative intensities of the purple and yellow bands are different in male cocoons from direct surface measurement and those from methanol extract. The strength ratio (λ2max/λ1max) of the purple and yellow bands from the female cocoon surface is 0.24, which is identical to the FEmS (blue dotted line in Figure 2) from female cocoon methanol extract. The blue-purple fluorescent color under UV light is primarily derived from a weaker yellow band and stronger blue-purple band. Similarly, the yellow-white fluorescence of male cocoons under UV light is primarily caused by the enhanced intensity of the yellow fluorescent band (λ2max). Whether it is direct fluorescence spectrometry of cocoon surfaces, or fluorescence spectrometry of methanol extracts, the fluorescent color differences between male and female cocoons depends on the intensities of the yellow fluorescent bands (λ2max) in FEmS. Direct fluorescence spectrometry of cocoon surfaces fully reflect actual fluorescent information about silkworm cocoon surfaces. Through the ratio of the two peaks (λ2max/λ1max), the fluorescence color and the differences between female and male cocoons are elucidated.

In order to investigate the distribution of fluorescent substances in male and female silkworms, the fluorescence observation of the silkglands and midgut tissues in the 5th instar larvae one day before maturation is carried out. It is found that under UV irradiation at 365 nm the midgut tissue in female silkworms emits a strong yellow fluorescence. The yellow fluorescence appears in the posterior midgut of silkworm larvae and their silkglands display blue-purple fluorescence which is the same as that of the female cocoons (Figure 3A). The midgut tissues of male silkworms do not show the yellow fluorescent color, and their silkglands display a roughly similar yellow-white fluorescence to that of their cocoon surface (Figure 3B), which suggests that no yellow fluorescent substances accumulate in the midgut tissues of male silkworms. As is shown in Figure 3C, nearly all yellow fluorescent substances exist in the posterior midgut of silkworm larvae which accounts for about 2/5 of the entire intestinal tissue. Thus, further fluorescence observation of the midgut tissue was carried out utilizing an inverted fluorescent microscope (Figure 4). In Figure 4A the posterior intestinal wall is an enlarged image (40×) from the fluorescence microscope. The cylindrical cells on both sides of the midgut are filled with yellow-white fluorescent substances. In daylight and UV irradiation (Figures 4B and C) the accumulation of yellow fluorescent materials in the junction of the middle and posterior midgut is apparent. FCSI differences exist between male and female silkworms. The blue-purple and yellow fluorescent materials do

Figure 2 FEmSs of fluorescent sex-identified cocoon shells and their extracts. Red and dashed lines respectively indicate the shell surface and the methanol extract of male cocoons. Blue and dashed lines respectively indicate the shell surface and the methanol extract of female cocoons. Fluorescent excitation wavelength, 360 nm.

Figure 3 Silkworm silkglands, midgut and the cocoons of fluorescent sex-identified species under UV light. A, Female. B, Male. C, The fluorescent images of the posterior midgut of female silkworm.

2.3 Fluorescent microscope observation of silkworm, cocoons, organs and tissues

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Figure 4 The images of yellow fluorescent accumulation of the posterior midgut wall (A) under UV light and the junction of the middle and the posterior midguts under Vis (B) and UV (C) light by using a fluorescent invertation microscope (40×, Nikon TE2000U).

not accumulate in male silkworm midguts but enter the blood and then penetrate into the silkglands so that their cocoons display the mixed fluorescent colors of the two fluorescent substances––a series of yellow-white fluorescent cocoons whose fluorescent shades depend mainly on the relative levels of the two substances. The female midguts especially the posterior midguts accumulate all yellow fluorescent substances, and only blue-purple fluorescent substances secrete and penetrate into the blood and silkglands, so female cocoons show blue-purple fluorescent colors. It is very possible that the yellow fluorescent substances are not entirely accumulated in the posterior midgut, and a small amount of the yellow fluorescent substances accompany the blue-purple substances into the blood and the silkglands so that female cocoons show a mixed fluorescent color––blue-white fluorescent color. Yellow, white and blue fluorescent cocoons of many available silkworm varieties (non-FSCI silkworms) one day before the maturing of 5th instar silkworm are observable under UV light. It was also found that the intestinal tissues of 5th instar silkworms in such varieties as 7532 also contain various yellow fluorescent substances, but the yellow fluorescence is not female-specific in the midgut. Therefore, the abundant accumulation of such a yellow fluorescent substance in the midgut tissues of female silkworms is probably related to the artificial breeding of silkworms. 2.4 Fluorescent color of blood and midgut of 5th instar larvae During the 5th instar, the fluorescence changes of blood and intestinal tissue of the male and female silkworms were

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observed day-to-day. The pleopod of the 5th instar larvae with scissors to harvest silkworm blood in a small plastic tube cooled in ice. 10 μL of blood was dropped onto the filter paper. The dried filter paper was observed for fluorescent color under UV light. The results showed that during day 1−3 of the 5th instar larvae, there was no evident difference in the fluorescence color (purple color) of male and female silkworm blood under 365 nm UV irradiation. However, the darker fluorescent color in the center of the spot on the filter paper indicate the existence of a yellow fluorescent material (Figure 5A(a), left). On the fourth day, a noticeable difference in blood fluorescent colors between male and female silkworms appeared, which remained until the mature larvae spun. Under UV irradiation, a darker fluorescent color appeared in the blood of the female silkworm in the plastic test tube one day before spinning (Figure 5A(b), left) while a bright yellow fluorescent color appeared in that of the male silkworm (Figure 5A(b), right). When the blood was dropped onto a piece of paper and dried in air, the area around the spot showed a blue fluorescence in both male and female silkworm blood. There was a clear difference in the fluorescence in the centre of the spot. The dark purple spot in female blood seemed to be a mixture of both yellow and purple fluorescent substances while a very bright yellow fluorescence existed in the centre of the male blood spot and a weak blue fluorescence appeared in the area around the spot. This result is basically consistent with what has been observed by Yu et al. [18] suggesting that the fluorescent color difference between male and female silkworm blood occurred from day 4 of the 5th instar larvae, during which yellow fluorescent pigments appear in the silkgland [15,16]. When 5 μL of blood was dropped on a silica gel plate and the dried plate was developed in 2-D developer, there was no apparent difference in the fluorescent color between the female and male blood (Figure 5B, left for female blood and right for male blood). The blue fluorescent spots did not move in situ, and the emerged yellow fluorescent bands shifted at the same speed and their Rf values were all 0.42 whether or not the silkworms were male or female, and their fluorescent intensities were not as apparent as those observed on the filter paper or in test tubes under UV light. This indicates that a very low level of purple and yellow fluorescent pigments existed in silkworm blood which were difficult to distinguish on a silica gel plate. When the above lyophilized blood was repeatedly extracted with methanol and the methanol extract was developed on a silica gel plate under the same conditions as shown in Figure 5B, the two bands of yellow fluorescence were quite close to each other and the fluorescent intensity of the second band in female blood was lower than that in the male (Figure 5C). The other yellow fluorescent band was rapidly moving and was difficult to find due to its low level in male blood.

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Figure 5 Fluorescent colors of the blood and the posterior midgut day-to-day during 5th instar larvae under UV light. A, Female and male blood (b) and on filter paper (a). B, TLC of female and male blood on the silica gel plate. C, Extract TLCs of the lyophilized female and male blood in methanol on the silica gel plate. D, The accumulation of yellow fluorescent pigments during 5th instar larvae.

Figure 5D shows the fluorescent images observed in the posterior midgut tissues of days 2, 3, 4, 5 and 6 during the 5th instar female larvae under UV light. No accumulation of the fluorescent pigments was observed in the intestinal tissues of day 2 and day 3 of the 5th larvae while on day 4, a light yellow fluorescence was observed in the posterior intestinal tissue under 365 nm UV irradiation (see partial amplified image in Figure 5D). Subsequently, the yellow fluorescence became increasingly bright day-by-day and rapidly accumulated in the posterior midgut until spinning. Finally, a very strong yellow fluorescence was seen in the female silkworm on day 6 of 5th instar (Figure 5D), which was almost synchronized with the emergence of yellow fluorescence in its silkgland, as described above. No fluorescence was found in the midgut tissues in 5th instar male larvae when 365 nm UV irradiation. There is little difference in the purple and yellow fluorescent pigments in the blood of both male and female silkworms owing to a small intake of mulberry leaves during days 1−3 of the 5th instar larvae. After a large intake of mulberry leaves on day 4, abundant purple and yellow fluorescent pigments enter the blood and then enter the silkglands of male silkworms. In female larvae, all or most yellow fluorescent pigments accumulate in the midgut especially in the posterior midgut. The purple fluorescent pigments enter alone or with a small amount of yellow pigment into the blood and then into the silkglands of female silkworms.

tissues were used for the direct fluorescent spectrum analysis and methanol extract test. The midgut web tissue and male cocoon shell were respectively extracted in methanol. The two extracts were added dropwise onto the filter paper and dried in a 50°C oven. As shown in Figure 6, the FEmS of the fluorescent substance accumulated in the female posterior midgut was essentially identical to those of its extract and the extract of the male cocoon shell. Their maximal emission peaks orderly shifted towards the UV direction for 5, 542, 538 and 533 nm. Their FExS bands were very similar to each other with their maximal FExS peaks at 377 or 378 nm. This result indicated that the yellow fluorescent substances accumulated in the posterior midgut of female silkworms were essentially identical to those in male cocoon shells.

2.5 Fluorescence spectra of the posterior midgut tissue and its extract In order to determine if the yellow fluorescent substances of female and male cocoon shell extracts in methanol are the same as those accumulated in the posterior midgut of female silkworm, the posterior midgut contents, external skin and subcutaneous fat were first removed under anatomic microscopy as shown in Figure 3. The resulting midgut web

Figure 6 FExSs (left, dashed line) and FEmSs (right, solid line) of the wall surface (black line) and its methanol extract (blue line) of the posterior midgut and yellow fluorescent spot (red line) separated after 1-D TLC of the methanol extract of male cocoon shell on the silica gel plate. Excitation wavelength, 360 nm for FEmS; Emission wavelength, 542 nm for FExS.

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TLC of cocoon shell and midgut extracts

In order to identify the composition and kind of fluorescent pigments in male and female cocoons and their differences and to verify the result of the direct fluorescent spectrum analysis of cocoon shell surfaces, the separation of fluorescent substances in male and female cocoons was carried out using 2-D TLC. As is shown in Figure 7, the methanol extracts of male and female cocoon shells were placed on a silica gel plate in 1-D developer solvents for the first-dimensional TLC. Figure 7A shows a purple fluorescent spot after sampling of the female cocoon extract under UV light irradiation. When 1-D TLC was developed, five fluorescent bands were observed on the plate. The four moving bands are a series of different blue-purple fluorescent colors which are respectively called B2, B3, B4 and B5 with Rf values of 0.92, 0.69, 0.36 and 0.21, in which the level of B4 (Rf=0.69) is the lowest and the other three have a higher level. It was determined that the yellow-blue spot (Rf=0) was immobile in 1-D developer, whose center had a significant fluorescent difference from the surrounding area. The unmoved yellow-blue fluorescent spot (B1) may overlap with a small amount of yellow fluorescent substances, which was indicated by the bands of the male cocoon extract when 1-D TLC was developed (Figure 7B). After sampling on the plate, the methanol extract of male cocoons was a yellow-green spot in which the yellow fluorescent pigments were dominant. When 1-D was developed, the shifting of the four fluorescent bands was entirely the same as that of female cocoon extract except for the intensities of these bands. The unmoved spot in situ was brighter in UV light due to the migration of blue-purple substances in 1-D TLC, indicating that the yellow fluorescent pigments are immobile in the 1-D developer and the polarity of the yellow pigments is much stronger than that of the other four blue-purple fluorescent pigments. Therefore, the immobile spot (B1) was developed in water-saturated n-propanol developer for 2-D TLC. The result indicated that the three yellow fluorescent bands were from the unmoved spots in which the rapid moving and highest level band was the third spot (Y3) and its Rf value was 0.82 in UV light. The Y3 band is a very bright yellow fluorescent spot which is a main component in yellow fluorescent pigments. The other two bands, which move more slowly than the Y3 band were Y2 and Y1, their Rf values respectively being 0.47 and 0.41. Both Y2 and Y1 were not the very bright yellow fluorescence which is not the main component. These bands were entirely consistent in speed and fluorescent intensity with those of midgut methanol extract developed in 2-D developer (Figure 8). When the 2% AlCl3 in 95% ethanol solution was sprayed onto the above sample plate, three yellow fluorescent bands on the dried sample plate became three blue bands, indicating that the three bands of yellow fluorescent pigments were the flavonoids in the 3,5-hydroxyl groups.

Figure 7 1-D TLC of the shell extracts of female and male cocoons on a silica gel plate under UV light. A and B, The TLC spots of female and male cocoon extracts before or after 1-D developing. C, The colored reaction of the spots of male cocoon extract after 1-D developing with AlCl3 reaction before or after 1-D developing.

Figure 8 TLC of the methanol extract of the posterior midgut in female silkworms.

2.7

Fluorescent spectra of fluorescent pigments

In order to further analyze fluorescence and the UV spectra of the various components of the fluorescent pigments, the four bands of purple fluorescence in 1-D TLC (B1–B5) and three bands of yellow fluorescence in 2-D TLC (Y1, Y2 and Y3) were removed from the silica gel plates. When these bands were immersed in methanol for extraction, these extracts were used for the measurement of fluorescence and UV spectra. The fluorescence spectra of the four components of B1, B2, B3 and B5 from band extract are shown in Figure 9. B1, apparently belonging to a blue fluorescent pigment, had an emission peak present only in the vicinity of 440.8 nm. The purple FEmS of components B2 and B3 was similar to that of B1, but their largest emission peaks gradually shifted in the direction of UV, and were respectively 420.2 and 392.6 nm. B1, B2 and B3 had similar FExSs and their largest peaks in FExS were all near 330 nm. The FEmS of B5 existed between 400–500 nm and was similar to those of B1, B2 and B3. B5 had two emission peaks whose maximal peaks were near 420 and 440 nm and were similar to those of B1 and B2. The FExS of B5 was distinct

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ments in molecular structure. Therefore, the blue-purple fluorescent substances in the fluorescent sex-identified cocoons may be composed of eight pigments. The yellow fluorescent substances in the fluorescent sex-identified cocoons are composed of three yellow fluorescent pigments. MS analysis also reveals that the Y3 band contains two similar fluorescent pigments in its molecular structure. Thus, the yellow fluorescent substances in fluorescent cocoons contain at least four yellow fluorescent pigments. 2.8

Figure 9 FExSs (A) and FEmSs (B) of the main components of bluepurple fluorescent pigments in the methanol extracts of silkworm cocoons.

from the above three components and its maximal FExS peak was 383 nm, which may be different from B1, B2 and B3 in molecular structure. B5 had its highest Rf value (0.92) in 1-D developing and moved as quickly as in the 1-D developer, which may be a mixture of two similar pigments. Mass spectrometry analysis EMS(−) confirmed that the B5 band contains two components with respective molecular masses of 299.8 and 295.8 (MS data not shown). The B1 band had a small shoulder peak at 420 nm. EMS(−) confirmed that the B1 band also consisted of two similar pigments whose molecular masses were 406.3 and 422.3. The latter may have a hydroxyl group more than the former (MS data not shown). Similarly, the B3 band had a small shoulder peak at 420 nm, which may be the mixture of two pigments. The band B4 is not analyzed in spectroscopy due to low contents isolated from the cocoon methanol extract. Three yellow fluorescent pigments isolated by 2-D developing were analyzed and compared utilizing fluorescent spectrometry. The resulting data showed that the fluorescent spectra of Y1 and Y2 were almost the same as that of Y3 (Figure 6, red line and red dotted lines respectively express FEmS and FExS) and their only difference was that the fluorescent emission peaks of Y1 and Y2 slightly shifted towards the direction of the short-wavelength and were 2−3 nm less than that of Y3 (533 nm), 528 nm and 530 nm. The FExSs of the three were nearly identical and those peaks were all at about 376 nm. EMS(−) analysis showed that Y3 also contained two pigments, whose molecular masses were respectively 465.5 and 494.5. It is estimated that they are very similar in molecular structure (MS data not shown). The above results show that the blue-purple fluorescent substances in the fluorescent sex-identified cocoons are composed of at least five pigments, in which B1, B2, B3 and B5 are the four primary fluorescent pigments. FEmSs of B1, B3 and B5 all appear to have small shoulder peaks. MS analysis reveals that B1 and B5 consist of two similar pig-

UV spectra

From the UV spectrum analysis of the five components (Figure 10A), B1, B2, B4 and B5 all have strong absorption bands in 260–300 nm of the band II, and stronger absorption shoulder peaks in 300–350 nm of the band I. Band I in B2 shifts towards the direction of long wavelengths and has a strong absorption band in 310–380 nm. When 2% AlCl3 solution is sprayed onto these bands of 5 pigments, the colors of the blue-purple fluorescent spots remain unchanged, which indicates that these five pigments may not belong to flavonoids and their derivatives. The Y1, Y2 and Y3 bands have strong absorption bands in the 245–300 nm (Figure 10B), and have strong shoulder peaks between 300–380 nm. As mentioned above, after spraying with a 2% AlCl3 solution, the three yellow components separated from silica gel plate all become blue under ultra-violet light (image not shown). This indicated that the 3,5-O-phenolic hydroxyl group exists in the benzoyl structure. From the analysis of these features, the three pigments may be the flavonol compounds.

3

Discussion

In this experiment, we first adopted direct fluorescence spectrometry for the cocoon shell surfaces and roughly understood the surface fluorescence spectra composition of the fluorescent cocoon shell. According to the intensity of the emission peaks in two bands of 400–500 nm, especially

Figure 10 UV spectrum analysis of the main components of the female and male cocoon methanol extracts after 2-D TLC developing. A, The blue-purple fluorescent pigments. B, The yellow fluorescent pigments.

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according to the λ542max/λ440max ratio, fluorescence shades and grades of the female and male cocoon were identified. This method may be of important reference value for the classification and grading of fluorescent cocoon color. In addition, we also dropped the methanol extract of fluorescent substances onto the filter paper and after drying and solvent evaporation the dried filter paper was used for the determination of FEmS or FExS. The possible extinction of the solvent on the fluorescent material may be entirely eliminated. This method was applied to fluorescent spectrum analysis for other substances. By the fluorescent observation of lyophilized silkworm blood, organs and tissues, we first discovered that the yellow fluorescent material partly or completely accumulated in the midgut especially in the posterior midgut (approximately 1/3–2/5) in the female 5th instar silkworm. The differential absorption and the accumulation of yellow fluorescent substances in the female silkworm midgut resulted in FCSI. It had been previously agreed that the cylinder-shaped cells of the posterior midgut (approximately 1/3–2/5) are the main components of the epithelium, and those cells are more developed and have not only the secretion function but also the absorption function. The nucleus is oval or almost round and is located in the center of the cell. According to electron microscope observation, the mitochondria, rough endoplasmic reticulum and Golgi apparatus in the cytoplasm are all highly developed. In addition, the cells contained a small amount of lipid granules and vacuoles. We found that the cells of the posterior midgut in which plentiful yellow fluorescent pigments accumulated may be these cylindrical cells. The accumulated yellow fluorescent substances in the posterior midgut of the female silkworm are essentially identical to those existing in cocoon shells of male silkworms with regard to their chromatographic behavior and spectral characteristics. This fully indicated that the metabolism of flavonoids from mulberry leaves in silkworm larvae, especially the digestion in the midgut cell of the 5th instar larvae, had no difference between female and male individuals. Utilizing UV observation, we found that the lyophilized mulberry leaves had a small quantity of slightly light yellow-green fluorescent material which may be flavonoids or their derivatives. However, the chromatographic and fluorescent spectral analysis of the mulberry leaf extract obtained by using the same extraction method showed that these fluorescent substances, especially the yellow fluorescent components from the leaves, were completely different from those of the silkworms. These fluorescent colors were extremely weak (data not shown), which demonstrated that the flavonoids existing in those leaves were likely to be flavonoid glycosides. Most flavonoids have stronger fluorescence. When they are conjugated with a monosaccharide or oligosaccharide, the fluorescence of the resulting flavonoid glycosides tend to be greatly reduced in their intensities or even disappear.

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The existence of many flavonoid glycosides in mulberry leaves [26–30] has been previously confirmed. Through larvae chewing and subsequent intestinal digestion, the flavonoid glycosides in mulberry leaves are likely to be hydrolyzed by glycosidases to produce monosaccharides or oligosaccharides and flavonoids which produce strong yellow or blue fluorescence. These flavonoids with yellow or blue fluorescence go through the midgut into the blood and then arrive at the silkglands and are eventually present in the silkworm cocoons which give off mixed fluorescent colors (i.e., yellow or yellow-white fluorescent cocoons). The yellow fluorescent extract absorbed on the skin of our fingers during the extraction process could not be removed by repeated washing with water or even with detergent (data not shown), while the purple fluorescent extracts were water-soluble and did not attach to the skin of our fingers. Therefore, we speculated that the female silkworm midgut, especially the posterior midgut epithelial tissue contained a similar protein to the collagen of our finger skin epidermis. This protein was able to firmly absorb or combine with most or all of the yellow fluorescent pigments. This protein was a yellow fluorescent pigment-specific and could be vigorously expressed in the midgut especially in the posterior midgut of 5th instar larvae of FCSI silkworm. Thus, female silkworms spun blue-purple fluorescent color cocoons because the yellow fluorescent substances were absorbed in a similar collagen in midgut. However, the male silkworms spun cocoons of mixed fluorescent color, i.e., yellow or yellow-white cocoons owing to the lack of any binding protein in midgut. At present, we are proceeding with the experiments of 2-D protein electrophoresis to study a protein difference between the midgut tissues of male and female larvae, especially this special protein combined with yellow fluorescent pigments in the posterior midgut. We are carrying out the gene location and the expression of this special protein in the posterior midgut. This work was supported by the National High Technology Research and Development Program (Grant No. 2006AA10A118) and the Earmarked Fund for Modern Agro-industry Technology Research System, China.

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