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Yong-Gang Hu, Yi-Hong Shen, and Ze Zhang

State Key Laboratory of Silkworm Genome Biology, The Institute of

Sericulture and Systems Biology, Southwest University, Chongqing, China

Gui-Qin Shi

School of Life Sciences, Chongqing University, Chongqing, China The phenomenon that epidermal cells under the white stripes rather than black stripes contain many uric acid granules was found in larvae of several Lepidopteran species. However, the biological mechanism of this phenomenon is still unknown. In the present study, we take advantage of several silkworm (Bombyx mori) body color mutant strains to investigate the deposition patterns and biological mechanism of urate and melanin in the integuments of these mutant larvae. By imaging with transmission electron microscope, we found that there were some melanin granules in the larval cuticle in black body color mutant plain Black (pB ), but not in background strain plain (p) with white larval body color. In contrast, the larval epidermal cell of background strain had much more urate granules than that of black one. Furthermore, the uric acid content under the black stripes was significantly lower than that under the white stripes in a single individual of mottled stripe (pS ) with black and white stripes in each segment. Ultraviolet A (UVA) exposure experiments showed that the distinct oily (od) mutant individuals with translucent larval integument were more sensitive to the UVA damage than black body color mutant and background strain without any pigmentation in the larval cuticle. This is likely due to the absence of melanin granules and few urate granules in the integument of od mutant. Thus, both the deposited melanin granules in Contract grant sponsor: Program of Introducing Talents of Discipline; contract grant number: B07045.

Correspondence to: Dr. Yi-Hong Shen, State Key Laboratory of Silkworm Genome Biology, The Institute of Sericulture and Systems Biology, Southwest University, Chongqing 400715, China.

E-mail: yhshen1964@126.com ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY, Vol. 83, No. 1, 41–55 (2013) Published online in Wiley Online Library (wileyonlinelibrary.com).

C 2013 Wiley Periodicals, Inc. DOI: 10.1002/arch.21096 r 42 Archives of Insect Biochemistry and Physiology, May 2013 the cuticle and the abundant urate granules in the epidermis cells constitute effective barriers for the silkworm to resist UVA-induced damage.

C 2013 Wiley Periodicals, Inc.

Keywords: silkworm; pigment pattern; melanin; urate; UV; protection


Insect pigmentation is a highly variable character, and varies between species, between populations of the same species, between individuals within a population, between life stages of a single individual, and even between body parts of the same individual life stage (Wittkopp and Beldade, 2009). Pigmentation is not only variously used for purpose of mimicry (Rettenmeyer, 1970; Futahashi and Fujiwara, 2008), sexual selection (Wiernasz, 1989), and thermoregulation (True, 2003; Talloen et al., 2004) but also plays a major role in wound healing (Ashida and Brey, 1995) and cuticle hardening (Sugumaran, 1998, 2009).

The larval body color in the silkworm (Bombyx mori) is mainly determined by concentrations of melanin in the cuticle and ommochromes and sepiapterins in the epidermis (Mazda et al., 1980; Ohashi et al., 1983). Black body color is mainly due to the deposition of melanin in the cuticle. Melanin is produced by epidermal cells through the melanin synthesis pathway (True, 2003; Wittkopp et al., 2003). Tyrosine hydroxylase (TH) and dopa decarboxylase (DDC) are two important genes for the formation of dopamine (the main precursor of melanin) and their expression patterns coincide with the black stripes (Futahashi and Fujiwara 2005; Futahashi et al., 2010; Liu et al., 2010; Shirataki et al., 2010; Yu et al., 2011). Besides these pigments, urate granules in the epidermis are also important for larval body coloration in Lepidoptera because they make the larval integument white or opaque (Tamura and Sakate, 1983). In larvae of black swallowtail (Papilio polyxenes), it was found that uric acid content was the highest in the integument in white saddle of the third instar, followed by brown-colored integument, green stripes, and yellow spots, the accumulation of uric acid in the black stripes was the lowest (Timmermann and Berenbaum, 1999). A similar pattern was also observed in the larvae of the armyworm Pseudaletia separate (Ninomiya et al., 2006; Ninomiya and Hayakawa, 2007). The electron microscopic observation showed that the epidermal cells under gap cuticle region (white stripe) between black stripes contained many urate granules while there were no urate granules in the epidermal cells under the black stripes (Ninomiya et al., 2006; Ninomiya and Hayakawa, 2007). However, the reason why epidermal cells under black stripes have no or few urate granules is still unclear.

The accumulation of pigments could protect individuals from harmful effects of ultraviolet A (UVA) irradiation. Previous studies showed that photon absorption produces reactive oxygen species (ROS) which in turn cause protein, lipid, and DNA oxidation, leading to the protein denaturation, DNA lesions, and cell damage (Afaq and Mukhtar, 2001; Trautinger, 2001; Nishigori, 2006; Svobodova et al., 2006). Melanin in the skin was suggested to play an important role for protecting against UV-induced skin damage (Kvam and Dahle, 2003). Photoprotection for the skin by melanin is due to its role as a shield against damaging UV light and its likely scavenging or quenching activity against ROS such O2.− and 1 O2 (Tada et al., 2010). Furthermore, uric acid is the main end product of nitrogen metabolism in insects (Bursell, 1967). Although uric acid is excreted in Lepidopteran faeces, a considerable amount is transported to the larval epidermis, where

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it accumulates as white urate granules. Urate is well known as an antioxidant (Frei et al., 1988). In Drosophila, urate-null mutants are more sensitive to oxidative stress or natural sunlight, pointing to an antioxidative role of urate in insects (Hilliker et al., 1992; Arakawa et al., 2006). In the blood-sucking insect Rhodnius prolixus, urate is the most important low molecular weight antioxidant present in the hemolymph. The maintenance of high urate titer in its extracellular fluids was suggested to be an important protective biochemical adaptation to oxidative damage caused by the intake of large amounts of hemin in a blood meal (Souza et al., 1997; Graca-Souza et al., 1999). However, whether the protective effect of melanin and urate against UV damage exists in the silkworm remains to be investigated.

The silkworm, Bombyx mori, is a highly domesticated Lepidopteran insect. It has been found that several silkworm mutant strains exhibit different body color phenotypes.

The larval body of the plain Black (pB ) mutant shows strong overall black pigmentation (Fig. 1A) while the background strain, plain (p), shows white larval body color pattern (Fig. 1B). However, the body colors of pupa and adult moth of pB are the same as those of p. The mottled striped (pS ) mutant has black stripe on each larval segment (Xiang et al., 2005). In addition, there is another mutant silkworm, the distinct oily (od) mutant strain;

its larval integument is translucent because of the inability to construct urate granules in the epidermal cell (Fig. 1C). The responsible gene (BmBLOS2) for this mutant has been recently identified (Fujii et al., 2010). In the present study, we take advantage of different body color mutants of the silkworm to investigate the melanin and uric acid deposition in the integument and to see whether the accumulation of melanin and urate granules in the integument is related to the adaptation to the UVA damage. Our results suggest that both melanin and urate granules act as UVA shield in the integument of the silkworm.

A possible mechanism of few urate granules under the black stripes is discussed.


Chemicals The uric acid and dopamine hydrochloride were purchased from Sigma (Shanghai, China). All other chemicals were reagent grade (Sangon Biotech (Shanghai) Co., Ltd., China).

Silkworm Strains The pB (pB /p), pS (pS /pS ), p (p/p), and od (od/od) strains were obtained from the silkworm resource pool at the Institute of Sericulture and Systems Biology, Southwest University. The pB strain, a dominant mutant, is homozygous lethal and arrests during late embryogenesis.

So it can only be kept in heterzygote. All silkworm larvae were reared on fresh mulberry leaves under a 16 h light: 8 h dark photoperiod at 25◦ C. The staging of molting period was based on the time when head capsule slippage occurred, as well as spiracle index, which represented the characteristic sequence of new spiracle formation (Kiguchi and Agui, 1981).

Transmission Electron Microscopy For electron microscopic observation, pieces of dorsal integument of larvae on day 3 of the fifth-instar were fixed with 4% glutaraldehyde in 0.1 M phosphate buffered saline buffer (pH 7.2) for 4 h and postfixed with OsO4 for 2 h at room temperature. Then Archives of Insect Biochemistry and Physiology r 44 Archives of Insect Biochemistry and Physiology, May 2013 Figure 1. The phenotypes of p, pB, and od strains and the TEM micrographs of larval integuments among these three strains. (A, B, C) Larvae of pB (A), p (B), and od (C) on day 3 of 5th instar. (D, E, F) TEM images of dorsal cuticle of 5th instar pB (D), p (E), and od (F) showing some melanin granules existing in the distal part of exocuticle of pB, not in p and od. (G, H, I) TEM images of epidermal cells under the larval cuticle of pB (G), p (H), and od (I). Note that only epidermis cells under the cuticle of p contain numerous uric acid granules.

Bar = 1 cm (A, B, C) and 2 μm (D, E, F, G, H, I).

the fixed pieces were dehydrated in graded concentrations of acetone and embedded in Epon618 resin. Ultra-thin sections were made by cutting the resin with a diamond knife (Reichert-Jung, Germany), stained with both lead citrate and uranyl acetate, then observed under the H7500 election microscope (Hitachi, Japan) and photographed.

Quantitative Analysis of Uric Acid by High Performance Liquid Chromatography

To prepare uric acid stock standard solution (1 g/L), we dissolved 500 mg of uric acid (Sigma) and 375 mg of lithium carbonate in 500 ml warm distilled water. After complete dissolution, this solution was cooled at room temperature and then stored at –20◦ C (Zhiri et al., 1985). It was further diluted with 0.7 g/L lithium carbonate into the following concentrations: 200, 100, 50, 25, 12.5, and 6.25 mg/l. The standards were mixed with an

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equal volume of acetonitrile before injection into the chromatograph, and then 5 μl of each standard was used for analysis (Ingebretsen et al., 1982). For sample preparation, the faeces and the integuments of several developmental stages were dried at 150◦ C and then ground. Hundred milligrams of the fine fraction was suspended in 10 ml 0.7 g/l lithium carbonate for 10 min, and subsequently centrifuged at 13,000 × g for 10 min at room temperature. Supernatant was also mixed with an equal volume of acetonitrile.

Again, 5 μl of each preparation was injected into the chromatograph for analysis. A 1260 liquid chromatograph (Agilent) with a SymmetryShieldTM RP18 (5 μm, 4.6 × 250 mm) column was used to detect at 292 nm. The solvent was sodium acetate, 35 mmol/L, pH 5.0, and acetonitrile (9/1 by volume). The flow rate was 1 ml/min. Before each high performance liquid chromatography (HPLC) measurement, the retention time of uric acid standard was confirmed as 26.4 min. Uric acid was quantified by comparing the peak height in the chromatograms with the value from the standard curve.

Reverse Transcription Polymerase Chain Reaction Analysis of BmTH and BmDDC

A whole integument was dissected from a larva. After fat body and muscle attached to epidermis were carefully removed, total epidermal RNA was isolated using TRIzol reagent (Invitrogen, China). The RNA samples were treated with Dnase I to remove any contamination of genomic DNA, and then were reversely transcribed with oligo (dT) and an M-MLV reverse Transcriptase (Promega, China) according to the manufacture’s instructions. Expression profiles for BmTH and BmDDC in two different stages were analyzed by reverse transcription polymerase chain reaction (RT-PCR). The primers were designed based on the complete coding DNA sequence of BmTH (AB439286) and BmDDC (AF372836). Bombyx mori actin3 gene (BmActin3) was used as an internal control for normalization of equal sample loading (Meng

et al., 2009; Dai et al., 2010). The PCRs were taken with the following primers:

5 -GCTTCCGTCTATCGCATACACT-3 and 5 -CAGTCCGAACTCAACCGTAAAC-3 for BmTH, 5 -GCTAAAATCACTACAGCCAGACG-3 and 5 -AATCGCAAAACGAACCACAACfor BmDDC, and 5 -AACACCCCGTCCTGCTCACTG-3 and 5 -GGGCGAGACGTGTG ATTTCCT-3 for BmActin3. The PCR condition was 94◦ C for 4 min followed by 26 cycles of 94◦ C for 30 sec, 55–57.5◦ C for 30 sec, 72◦ C for 1 min.

Quantitative Analysis of Dopamine by HPLC

Dopamine was extracted following former methods (Koch et al., 2000; Arakane et al.,

2009) and was measured according to Qiao’s Method (Qiao et al., 2012). HPLC was performed on a SymmetryShieldTM RP18 (5 μm, 4.6 × 250 mm) column (Waters, Ireland) using the 1260 liquid chromatograph (Agilent) at 208 nm. The flow rate of mobile phase was 0.8 ml/min. Ten microliters of each standard and sample was injected into the chromatograph for analysis. Dopamine was identified based on retention time of dopamine standard (6.7 min) and was quantified by comparing the peak area with the value from a standard curve.

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