Accumulation of uric acid in the epidermis forms the white integument of Samia ricini larvae

Abstract

The white color in the larval integument of the silkworm Bombyx mori is considered the result of uric acid accumulation in its epidermal cells. Larvae of the eri silkworm Samia ricini (Lepidoptera; Saturniidae) also have a white and opaque integument, but little is known about its coloration mechanism. In this study, we first performed a feeding assay of S. ricini larvae using allopurinol, an inhibitor of xanthine oxidase, which catalyzes the degradation of xanthine to uric acid. This treatment induced a clear translucent integument phenotype, indicating that the larval color of S. ricini is also determined by uric acid accumulation. Next, to investigate the genetic basis that controls uric acid accumulation in S. ricini larvae, we isolated and characterized the S. ricini homolog of mammalian biogenesis of lysosome-related organelles complex 1, subunit 2 (BLOS2), which is known to play a crucial role in urate granule biosynthesis. We created a transcription activator-like effector nuclease (TALEN)-mediated gene knockout of S. ricini BLOS2 (SrBLOS2) and succeeded in establishing SrBLOS2 knockout strains (SrBLOS2KO). SrBLOS2KO mutants exhibited a translucent larval integument phenotype and lacked uric acid in the epidermis, as also observed in allopurinol-fed larvae. In addition, electron microscopy revealed that urate granules were rarely observed in the epidermis of SrBLOS2KO larvae, whereas abundant granules were found in the epidermis of wild-type larvae. These results clearly demonstrated that larval S. ricini accumulates uric acid as urate granules in the epidermis and that the genetic basis that controls uric acid accumulation is evolutionarily conserved in S. ricini and B. mori.

Fig 1. Graphical view of Samia ricini larva.

(A) Fifth-instar larva of S. ricini. Its integument is white and opaque. (B) Coloration mechanism of the Bombyx mori larval integument (left) and a hypothetical model of S. ricini integument coloration (right).

Fig 2. Phenotype of Samia ricini larvae treated with allopurinol.

(A) Dorsal view of S. ricini larvae treated with (right) or without (left) allopurinol. (B) Ventral view of S. ricini larvae treated with (right) or without (left) allopurinol. To make observation easier, the ventral integument was dissected, and internal organs were removed. (C) Comparison of uric acid concentrations in the integument of S. ricini larvae treated with or without allopurinol. Data are shown as the means + standard error. n = 6. **p < 0.01 by Student t-test.

Fig 3. Genomic structure and phylogenetic analysis of Samia ricini biogenesis of lysosome-related organelles complex 1, subunit 2 (SrBLOS2).

(A) Genomic structure of SrBLOS2. Numbers show the sizes of exons and introns. (B) Phylogenetic tree of insect BLOS2 homologs. The tree was constructed using MEGA7.0 (Kumar et al., 2015). Human BLOS2 sequence was included as an outgroup.

Fig 4. RT-PCR analysis of Samia ricini biogenesis of lysosome-related organelles complex 1, subunit 2 (SrBLOS2) in S. ricini larval tissues.

Total RNA of fifth-instar day 4 larvae of the wild-type individuals was used for RT-PCR. SrRp49 was used as an internal control. EP, epidermis; MG, midgut; AS, anterior silk gland; MS, middle silk gland; PS, posterior silk gland; OV, ovary; TES, testis; ML, Malpighian tubule; TR, trachea; FB, fat body.

Fig 5. Characterization of transcription activator-like effector nuclease (TALEN)-generated Samia ricini biogenesis of lysosome-related organelles complex 1, subunit 2 knockout (SrBLOS2^KO) mutants.

(A) Schematic presentation of the spacer and binding sequences of TALEN targeting SrBLOS2. (B) Larval phenotype of SrBLOS2^KO individuals. The upper panel shows larvae from strain #22. A wild-type larva is shown on the left, and two translucent individuals are presented on the right. The lower panel shows larvae from strain #28. A wild-type larva is presented on the top, and one translucent individual is shown on the bottom. (C) Mutations introduced in three SrBLOS2^KO strains. The spacer sequence is shown in red. Premature stop codons generated by frame shift are highlighted with red squares. The deduced amino acid sequence of wild-type SrBLOS2 is shown on the top. (D) Comparison of uric acid concentrations in the integument of S. ricini wild-type and strain #22 larvae. Data are shown as the mean + standard error. n = 3. ***p < 0.001 by Student’s t-test.

Fig 6. Transmission electron microscopy of the epidermis of wild-type and Samia ricini biogenesis of lysosome-related organelles complex 1, subunit 2 knockout (SrBLOS2^KO) individuals.

Low- (A) and high-magnification (B) micrographs of the epidermis of a fifth-instar day 4 wild-type larva. Low- (C) and high-magnification (D) micrographs of the epidermis of a fifth-instar day 4 SrBLOS2^KO larva. Red squares in A and C indicate the regions enlarged in B and D, respectively. N, nucleus; CU, cuticle; UG, urate granules; UN, unknown vacuole-like organelles.