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 (SrBLOS2KO) mutants.
(A) Schematic presentation of the spacer and binding sequences of TALEN targeting SrBLOS2. (B) Larval phenotype of SrBLOS2KO 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 SrBLOS2KO 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.
Table 1.
Efficiency of transcription activator-like effector nuclease-mediated knockout of Samia ricini biogenesis of lysosome-related organelles complex 1, subunit 2.
Fig 6.
Transmission electron microscopy of the epidermis of wild-type and Samia ricini biogenesis of lysosome-related organelles complex 1, subunit 2 knockout (SrBLOS2KO) 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 SrBLOS2KO 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.