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

Jung Lee; Takashi Kiuchi; Munetaka Kawamoto; Toru Shimada; Susumu Katsuma

doi:10.1371/journal.pone.0205758

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 (SrBLOS2^KO). SrBLOS2^KO 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 SrBLOS2^KO 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.

Citation: Lee J, Kiuchi T, Kawamoto M, Shimada T, Katsuma S (2018) Accumulation of uric acid in the epidermis forms the white integument of Samia ricini larvae. PLoS ONE 13(10): e0205758. https://doi.org/10.1371/journal.pone.0205758

Editor: J Joe Hull, USDA Agricultural Research Service, UNITED STATES

Received: June 7, 2018; Accepted: October 1, 2018; Published: October 15, 2018

Copyright: © 2018 Lee et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: The nucleotide sequences have been submitted to the DDBJ/EMBL/GenBank data bank under the accession numbers LC378373, LC378374, and LC378375–LC 378377.

Funding: JL is a recipient of a fellowship from the Japan Society for the Promotion of Science (17J06249). https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17J06249/. This work was supported by Grant-in-Aid for Scientific Research (A) (15H02482) to TK, TS, and SK. https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15H02482/. Additional support for TK and SK from Japan Society for the Promotion of Science (17H06431). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Uric acid is the final product of purine metabolism [1]. Most insects have been considered to merely emit uric acid to discard excessive nitrogen. However, recent studies revealed that insects reuse and utilize uric acid in diverse manners [2–3]. The larvae of Bombyx mori (Lepidoptera: Bombycidae) represent one example. B. mori larvae accumulate uric acid as urate granules in their epidermal cells [4–5]. As urate granules diffuse and reflect external light, the larval integument of B. mori appears white and opaque. Therefore, B. mori mutant strains with defects in uric acid synthesis, transport, or accumulation exhibit a translucent integument phenotype [6–8]. Although the biological significance of uric acid accumulation is unclear, it is widely accepted that urate granules in epidermal cells protect biomolecules against photooxidative stress. Uric acid is a physiological antioxidant [9], and uric acid-deficient B. mori larvae are hyper-susceptible to ultraviolet (UV) light and UV-induced reactive oxygen species [10–11]. Considering that the larvae of lepidopteran insects such as Papilio polyxenes and Spodoptera litura, the integuments of which are not entirely white, also accumulate uric acid [12–13], this theory appears highly plausible because wild insects are continuously exposed to photooxidative stress caused by natural UV light.

Lepidopteran insects commonly accumulate uric acid in the epidermis, but it is still unclear that accumulated uric acid contributes the coloration of the larval integument in the lepidopteran species other than B. mori. Samia ricini (Lepidoptera; Saturniidae) is a gigantic silkworm with a white and opaque larval integument, similarly as B. mori (Fig 1A). If uric acid accumulation is responsible for the whiteness of the larval integument (Fig 1B), the deficiency of uric acid would cause a drastic change of its appearance. In this study, to reveal the coloration mechanism of the larval integument of S. ricini, we utilized both physiological and molecular biological approaches. First, we inhibited uric acid synthesis in S. ricini larvae via feeding with allopurinol, an inhibitor of xanthine oxidase, which catalyzes the degradation of xanthine to uric acid. Second, we performed transcription activator-like effector nuclease (TALEN)-mediated knockout (KO) of the homolog of a gene involved in uric acid accumulation in B. mori.

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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).

https://doi.org/10.1371/journal.pone.0205758.g001

We focused on the S. ricini homolog of mammalian biogenesis of lysosome-related organelles complex 1, subunit 2 (BLOS2) because S. ricini BLOS2 (SrBLOS2) is likely a Z-linked gene due to the highly conserved synteny of the Z chromosome among lepidopteran insects [13–14]. By targeting SrBLOS2, we observed a mutant phenotype in generation 1 (G₁) because G₁ females can be hemizygous at the SrBLOS2 locus.

In this study, we revealed that S. ricini larvae accumulate uric acid as urate granules in the epidermis and that a certain part of the genetic basis that controls uric acid accumulation is evolutionally conserved between S. ricini and B. mori. To the best of our knowledge, this is the first report of successful genome editing in S. ricini.

Materials and methods

Insects

S. ricini larvae were provided from National BioResource Project (NBRP; http://shigen.nig.ac.jp/wildmoth/). S. ricini larvae were reared on artificial diet (Insecta LFS, Nosan, Kanagawa, Japan) under a long-day condition (16 h light/8 h dark) at 25°C. TALEN-mediated gene KO larvae and their progenies (G₀ and G₁ individuals) were reared on Ricinus communis leaves under the same condition. Blue strain was one of the chromosome segment substitution lines, derived from backcrossing of F₁ individuals between S. ricini and Samia cynthia pryeri, which is phylogenetically close species to S. ricini and also preserved in NBRP. Blue strain larvae were reared on R. communis under a short-day condition (12 h light/12 h dark) at 25°C.

Inhibition of uric acid synthesis in S. ricini larvae

We used the method reported by Matsuo and Ishikawa (1999) [10]. Four hundred milligrams of allopurinol (Wako, Osaka, Japan) were added to 300 g of Insecta LFS and fed to S. ricini throughout the larval stage. The growth speed of individuals fed Insecta LFS is not uniform, making it impossible to collect individuals at the same developmental stage. In the experiments of Fig 2, we used individuals of the same age (20 days old).

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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.

https://doi.org/10.1371/journal.pone.0205758.g002

Quantification of uric acid content in the integuments of S. ricini larvae

The integuments of 20-day-old larvae or fifth-instar day 4 larvae were dissected and stored at −30°C until use. After homogenization, 0.002 g of the integuments were boiled in 1000 μL of distilled water for 10 min. The uric acid content was measured using a QuantiChrom Uric Acid Assay kit (BioAssay Systems, CA, USA) according to the manufacturer’s protocols. We measured the optical density of each sample at 595 nm using an iMARK Microplate Reader (BIO-RAD, CA, USA) and calculated the uric acid concentration of each sample. For measurements in SrBLOS2^KO mutants, strain #22 individuals were used.

Determination of the partial nucleotide sequences of SrBLOS2 and SrRp49

The partial nucleotide sequences of SrBLOS2 and SrRp49 cDNA were obtained through a tBLASTn search using S. ricini transcriptome data for the larval midgut (SilkBase, http://silkbase.ab.a.u-tokyo.ac.jp) with the amino acid sequences of B. mori BLOS2 (BmBLOS2, GenBank BAI63077.1) and BmRP49 (GenBank NP_001091752.1) as queries, respectively. In addition, to grasp the genomic structure of SrBLOS2, we designed three sets of primers to amplify its introns (S1 Table) according to the genomic structure of BmBLOS2. Genomic PCR was performed using KOD FX Neo (ToYoBo, Osaka, Japan). The PCR program for SrBLOS2 was as follows: 98°C for 2 min; 40 cycles of 10 s at 98°C, 30 s at 57°C, and 3 min at 68°C; and 68°C for 2 min. Amplified fragments were cloned into pGEM-T Easy vectors (Promega, Madison, WI, USA) and sequenced on a 3130xl Genetic Analyzer (Applied Biosystems, Carlsbad, CA, USA). Sequence analyses were conducted using GENETYX-MAC version 16.0.1 (GENETYX Co., Tokyo, Japan) and ATSQ version 5.1.3 (GENETYX Co.).

Phylogenetic analysis

The phylogenetic tree of insect BLOS2 homologs was constructed using MEGA7.0 [15]. The evolutionary history was inferred using the maximum likelihood method based on the JTT matrix-based model [16]. The tree with the highest log likelihood (−1293.7573) was shown. The percentage of trees in which the associated taxa clustered together was calculated via bootstrapping with 1000 replicates. To generate a phylogenetic tree, we used 78 amino acid sequences, including 12 sequences from lepidopteran insects, 3 sequences from hemipteran insects, 27 sequences from dipteran insects, 7 sequences from coleopteran insects, 28 sequences from hymenopteran insects, and 1 sequence from Homo sapiens. The GenBank accession numbers of these amino acid sequences are listed in S2 Table.

Expression analysis of SrBLOS2 mRNA in S. ricini tissues

Total RNA was extracted from S. ricini embryos or tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol and subjected to reverse transcription using SuperScript III reverse transcriptase (Thermo Fisher Scientific, MA, USA) with oligo-dT primers (TaKaRa Bio, Shiga, Japan). RT-PCR was performed using KOD FX Neo (ToYoBo) or Ex-Taq (TaKaRa). The primers used are listed in S1 Table. The PCR program for SrBLOS2 was as follows: 98°C for 2 min; 40 cycles of 10 s at 98°C, 30 s at 60°C, and 15 s at 68°C; and 68°C for 2 min. The PCR program for SrRp49 was as follows: 94°C for 2 min; 25 cycles of 20 s at 94°C, 30 s at 60°C, and 30 s at 72°C; and 72°C for 10 min. The amplified fragments of SrBLOS2 and SrRp49 were cloned into pGEM-T Easy vectors and sequenced on a 3130xl Genetic Analyzer. Sequence analyses were conducted using GENETYX-MAC version 16.0.1 and ATSQ version 5.1.3.

Construction of a TALEN targeting SrBLOS2

A pair of TALENs was designed to target the coding sequence of SrBLOS2 using TALEN Targeter (https://tale-nt.cac.cornell.edu/node/add/talen). We located the target site, the sequence of which was 5′-CCAGCTTTGAAGTACTGGAtccacatgaccctgtTATAAGTAGGTTAGCAACTC-3′, on exon 2. The nucleotides in capital letters indicate the RVD binding sites of the left and right TALENs. The TALE modules were assembled using Golden Gate TALEN and TAL Effector kit 2.0 (Addgene, Cambridge, MA, USA) in accordance with the highly efficient construction methods developed by Cermak et al. (2011) with some modifications by Takasu et al. (2014) [17,18]. pBlue-TAL (GenBank accession no. KF724948), which was developed for genome editing in B. mori [18], was used as a destination plasmid. The TALEN mRNA was synthesized in vitro using a MEGAscript T7 ultra Kit (Ambion, MA, USA) according to the manufacturer’s protocols. Purified mRNA was dissolved in annealing buffer (100 mM potassium acetate, 2 mM magnesium acetate, 30 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-KOH pH 7.4), adjusted to three different concentrations (200, 400, and 600 ng/μL), and stored at −80°C until use. Immediately before embryonic injection, left and right TALEN mRNAs were mixed.

Embryo preparation and embryonic microinjection

To allow S. ricini moths to copulate, pairs of male and female moths were placed in small paper bags. After 18–24 h of copulation, the pairs were decoupled, and males were removed. Female moths started laying eggs on the wall of the paper bag immediately when the dark period began.

Eggs detached from the paper bag were individually affixed onto a microscope slide using instant glue. Injection was performed using a microinjector (IM 300 Microinjector, Narishige, Tokyo, Japan). Approximately 1–5 nL of each TALEN mRNA solution were injected into each embryo between 4–8 h after oviposition. All procedures were performed at 25°C. The injected embryos were incubated at 25°C in a humidified Petri dish, which promoted their hatching in 10 days.

Crossing of G₀ individuals

Adult G₀ moths were crossed with each other. After virgin individuals of the opposite sex were used up, G₀ moths were crossed with wild-type moths. After 36–48 h of copulation, the pairs were decoupled, and males were removed. All of the parents of the three strains described in the Results section were G₀ individuals.

Detection and DNA sequencing of the mutations in G₁ individuals

Ten newly hatched larvae from each brood were collected in one tube, and genomic DNA was extracted using the HotSHOT method [19]. This step was repeated on the other set of 10 newly hatched larvae. Genomic PCR was performed using KOD FX Neo (ToYoBo) with specific primers (S1 Table). PCR products were diluted 2-fold with 1× TE buffer, denatured, and hybridized as follows: 95°C for 10 min; ramping down at −2°C/s to 85°C and at −0.1°C/s to 25°C; and finally holding at 4°C. Microchip electrophoresis of the re-hybridized PCR products was performed using MultiNA (Shimadzu, Kyoto, Japan). The PCR products of the SrBLOS2 locus were also used for DNA sequencing after being cloned into pGEM-T Easy vectors. For each brood, 20 bacterial colonies were selected and analyzed by DNA sequencing. Regarding strain #28, genomic DNA was extracted from three G₂ male larvae using DNeasy Blood & Tissue kit (QIAGEN, Hilden, Germany) and used for DNA sequencing of their SrBLOS2 loci.

Transmission electron microscopy (TEM)

The integuments of fifth-instar day 4 larvae (SrBLOS2^KO and wild-type) were dissected, fixed for 24 h at 4°C in 4% paraformaldehyde in phosphate buffer solution, and washed twice with phosphate buffer solution for 10 min on ice. Secondary fixation was performed for 3 h at 4°C in 2% osmium (VIII) oxide in phosphate buffer solution, and specimens were washed twice with phosphate buffer solution for 10 min on ice. After fixation, the tissues were dehydrated in a graded ethanol series. In the final step of dehydration, the tissues, soaked in absolute ethanol, were placed at 4°C for overnight. Embedding was performed using a Spurr Low Viscosity Embedding Kit (Polysciences, Warrington, PA, USA) according to the manufacturer’s protocols. Polymerization was performed at 70°C for 24 h. Eighty-nanometer-thick sections were excised and stained with 4% uranyl acetate and lead citrate. The sections were examined at 80 kV under a transmission electron microscope (JEM-1400 plus, Nihondenshi, Tokyo, Japan).

Nucleotide sequence deposition

The partial cDNA sequences of SrBLOS2 and SrRp49 and DNA sequences of three introns of SrBLOS2 are available under the accession numbers LC378373, LC378374, and LC378375– LC 378377, respectively.

Results

Allopurinol treatment induced a translucent integument phenotype in S. ricini larvae

Compared with the control group, the integument of the allopurinol-treated group exhibited a slightly translucent phenotype (Fig 2A and 2B). The uric acid concentration was drastically reduced in the integument of larvae in the allopurinol-treated group (Fig 2C). These results indicate that the white color of the S. ricini larval integument is formed by the accumulation of uric acid in the epidermis.

Identification and characterization of SrBLOS2

Through a BLAST search against S. ricini midgut transcriptome data, we identified a contig that includes a putative coding sequence (CDS) of S. ricini BLOS2 (SrBLOS2). The SrBLOS2 CDS is 438 base pairs in length, and an InterProScan search of the deduced amino acid sequence revealed that SrBLOS2 belongs to the BLOS2 family (IPR019269).

We designed three sets of primers to amplify the intronic regions of SrBLOS2 using the genomic structure of BmBLOS2 as a reference. Genomic PCR revealed that the SrBLOS2 CDS consists of four exons (Fig 3A). In addition, phylogenetic analysis revealed that lepidopteran BLOS2 homologs including SrBLOS2 are clustered into a single clade (Fig 3B), indicating that SrBLOS2 is an ortholog of lepidopteran BLOS2.

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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.

https://doi.org/10.1371/journal.pone.0205758.g003

Expression profile of SrBLOS2 in larval tissues

To investigate in which tissue SrBLOS2 functions, SrBLOS2 expression in 10 larval tissues was examined by RT-PCR. In fifth-instar day 4 larvae, SrBLOS2 expression was detected in all examined tissues (Fig 4). This ubiquitous expression pattern was similar to that of BmBLOS2 [7].

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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.

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Microinjection of the TALEN mRNA for SrBLOS2

We designed the targeting site of TALEN on exon 2 of the SrBLOS2 CDS (Fig 5A). The left and right TALEN mRNA mixture at concentrations of 400 (200 + 200), 800 (400 + 400), and 1200 (600 + 600) ng/μL was injected to three batches of eggs (144, 312, and 164 eggs, respectively). The injected eggs displayed hatching ratios of 20.8 (30/144), 40.1 (125/312), and 22.6% (37/164), respectively (Table 1). Although we expected to observe somatic mosaics in the integument of G₀, we failed to identify individuals exhibiting the mottled and translucent phenotype in their integument.

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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.

https://doi.org/10.1371/journal.pone.0205758.g005

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Table 1. Efficiency of transcription activator-like effector nuclease-mediated knockout of Samia ricini biogenesis of lysosome-related organelles complex 1, subunit 2.

https://doi.org/10.1371/journal.pone.0205758.t001

Detection of mutant alleles in G₁ broods

By crossing the sibling G₀ individuals with each other, we obtained 128 G₁ broods (Table 1). Among them, 20, 79, and 29 were obtained from the egg batches injected with 400, 800, and 1200 ng/μL TALEN mRNA, respectively. Through microchip electrophoresis, we found that two G₁ broods (strains #22 and #29) had mutations in the SrBLOS2 locus (S1 Fig). Of these, only strain #22 included individuals exhibiting the translucent integument phenotype (Fig 5B). In addition, strain #28, in which the mutation was not detected by microchip electrophoresis, contained individuals with the translucent integument phenotype (Fig 5B).

Based on the microchip electrophoresis results, the mutations of strains #22 and #29 were transmitted from either of the parents (S1A and S1B Fig). If different mutations were inherited from both parents, there should have been more heteroduplex bands. Concerning strain #28, translucent G₁ individuals were all female, and G₂ progenies obtained by sibling crossing did not exhibit a translucent phenotype, indicating that the G₀ founder was male.

DNA sequencing of the SrBLOS2 loci of the three strains revealed that each strain had a single different mutant allele. A two-nucleotide deletion, a four-nucleotide deletion, and a three-nucleotide deletion and two-nucleotide substitution were detected in strains #22, #28, and #29, respectively. The former two alleles generated a premature stop codon, whereas the latter generated a single amino acid deletion and two amino acid substitutions (Fig 5C).

Germline transmission rates (GTRs) of TALEN-mediated SrBLOS2 mutations

We calculated the GTR as the number of G₁ broods with mutant alleles divided by the number of total G₀ adult moths. The GTR of TALEN-mediated SrBLOS2 mutation was 3.80% (Table 1).

Accumulation of uric acid as urate granules in the epidermal cells of S. ricini larvae

To confirm whether the translucent integument phenotype of TALEN-mediated SrBLOS2^KO mutants was due to the lack of urate granules, we measured the uric acid concentration in the epidermis of SrBLOS2^KO mutants. Compared with wild-type individuals, the mutants almost completely lacked uric acid accumulation in their epidermis (Fig 5D).

Furthermore, TEM revealed that the epidermal cells of wild-type individuals had numerous oval-shaped urate granules (Fig 6A and 6B), whereas SrBLOS2^KO mutants had an extremely reduced number of such granules (Fig 6C and 6D), indicating that SrBLOS2 is indispensable for the formation of urate granules in the epidermal cells of S. ricini larvae.

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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.

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Discussion

Accumulation of uric acid in the integument of S. ricini larvae

Because the integument of the allopurinol-treated individuals and SrBLOS2^KO-mutants were colorless and translucent (Figs 2 and 5B), we concluded that uric acid is the major substance that forms the uniformly white integument of S. ricini larvae. Conversely, the majority of Saturniidae species exhibit various patterns of larval integument coloration. As represented by Antheraea yamamai (Japanese oak silkmoth) or Antheraea pernyi (Chinese oak silkmoth), the basal color of other saturniid larval integuments is green, which is generated by a mixture of bilins and carotenoids [20]. In these species, uric acid does not likely contribute to the green integument color. Some strains of S. ricini have blue integuments (S2A Fig, “blue” strain). However, the blue strains contain a similar amount of uric acid in their epidermis (S2B Fig). Considering the result that Spodoptera litura, the larval integument of which is uniformly black regardless of the body part, also accumulates uric acid [13], it is concluded that uric acid accumulation is not always correlated with the integument color of lepidopteran larvae.

SrBLOS2 functions in urate granule formation

Some B. mori strains display a translucent integument phenotype. Among these mutant strains, od (distinct oily), the responsible gene of which has been elucidated as BmBLOS2, exhibits relatively high translucency [7, 21]. TEM revealed that od mutants accumulate reduced numbers of smaller urate granules compared with those observed in wild-type larvae [5].

BLOS2 is one of eight subunits of the BLOC-1 complex [22], which is required for normal biogenesis and trafficking of lysosome-related organelles [23]. The BLOC-1 complex is considered to function at an early stage of the melanosome, as BLOC-1 deficiency is associated with a bleached coat color in mice and red eye in humans [24]. Given these previous reports, it is extremely likely that BmBLOS2 functions in the formation of urate granules. In the present study, we observed that SrBLOS2^KO mutants also exhibited a translucent integument, deficient uric acid accumulation, and a reduced number of urate granules in the epidermis (Figs 5D and 6), indicating that the function of BLOS2 is conserved between B. mori and S. ricini. In addition, as reported in BmBLOS2 and S. litura BLOS2 [7, 13], SrBLOS2 was found to be located on the Z chromosome. Compared with the autosomal genes, morphological phenotypes of KO mutants can be observed one generation earlier, demonstrating that the BLOS2 homologs represent ideal targets for applying genome-editing approaches to non-model lepidopteran species.

Absence of SrBLOS2^KO G₀ individuals with a somatic mosaic phenotype

We failed to observe somatic mosaics in the integument of SrBLOS2^KO G₀ individuals. When S. ricini larvae enter the third instar, they begin to produce a crystalline white powder from spines on their backs [25]. This powder spreads from the spines and covers the entire larval body. Production of the white powder stops during the molting period, but other than that period, a huge amount of white powder is produced, making it impossible to distinguish SrBLOS2^KO mutants and wild-type individuals visually. For this reason, we did not find G₀ mosaic larvae even though their mosaic phenotype appeared in the integument. Unlike the larval integument, the crystalline powder is not composed of uric acid, as its major components are straight chain-saturated alcohols, namely n-triacontanol (C₃₀H₆₂O, 92.0%) and n-octacosanol (C₂₈H₅₈O, 0.56%) [25]. Through TEM, vacuole-like large organelles other than urate granules were discovered (Fig 6B). Because similar organelles were not observed in the larval integuments of B. mori [5], these vacuole-like organelles were likely S. ricini-specific. These organelles appeared to emerge from the basement membrane of the epidermal cells (Fig 6B) and existed even in the epidermal cells of SrBLOS2^KO mutants (Fig 6D). The large size of these organelles might imply their role in transporting or synthesizing chemical compounds such as n-triacontanol or n-octacosanol.

GTR of TALEN-mediated mutations in S. ricini

Although Takasu et al. [18] reported that the GTR of TALENs that induced somatic mosaics in G₀ individuals reached 100% in B. mori, that of TALENs in S. ricini was extremely low (3.80%, Table 1). Previous studies reported that the GTR was highly dependent on target sequences regardless of the genome-editing tools. For example, Yang et al. [26] designed two TALEN pairs to knock out odorant receptor co-receptor in O. furnacalis. Although one TALEN pair was highly efficient and the GTR reached 62.9%, the other pair did not induce the generation of even somatic mosaics. Thus, the GTR of TALENs in S. ricini might be improved by changing the target sequence. We also suspect that the structure of the TALEN expression vector pBlue-TAL also contributed to the low GTR observed in S. ricini. pBlue-TAL is customized for mRNA translation in B. mori; i.e., codon usage, the Kozak sequence, and the UTR track were optimized on the basis of the genomic information of B. mori [18]. When the genome of S. ricini is sequenced in the future, we will be able to customize pBlue-TAL for S. ricini and improve the GTR of TALEN.

As their large bodies are suitable for experimentation, entomologists have often used S. ricini as a model for physiological and biochemical research [27–29], and knowledge and experimental techniques for this species have accumulated [30–31]. To increase the utility of S. ricini as a model organism, the whole-genome sequencing of S. ricini is strongly expected.

Supporting information

S1 Table. Primer list used in this study.

https://doi.org/10.1371/journal.pone.0205758.s001

(XLSX)

S2 Table. GenBank accession number list of sequences used for phylogenetic analysis.

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(XLSX)

S1 Fig. Representative images of microchip electrophoresis.

(A) Detection of mutations introduced in generation 1 broods. Their parents are from eggs injected with 800 (400 + 400) ng/μL transcription activator-like effector nuclease mRNA. PCR failed in the second lot of strains #29 and #30.

(B) Higher-magnification images of microchip electrophoresis of strains #22 and #29, which include Samia ricini biogenesis of lysosome-related organelles complex 1, subunit 2 (SrBLOS2^KO) mutants. In addition to the major band (indicated by arrows), two bands (indicated by asterisks) were detected, indicating that the PCR products from SrBLOS2 of strains #22 and #29 are heterozygous.

https://doi.org/10.1371/journal.pone.0205758.s003

(TIFF)

S2 Fig. The blue strain of Samia ricini.

(A) Fifth-instar larvae of S. ricini immediately before the spinning stage. The color of its integument is slightly bluish.

(B) Comparison of uric acid concentrations in the integument of wild-type and blue strains of S. ricini. Data are shown as the mean + standard error. N.S., p > 0.05 by Student’s t-test.

https://doi.org/10.1371/journal.pone.0205758.s004

(TIFF)

Acknowledgments

We thank Takaaki Daimon (Kyoto University) for critical advices for TALEN construction and Fumiko Ishizuna (Technology Advancement Center, Graduate School of Agricultural and Life Sciences, The University of Tokyo) for TEM sample preparation and data acquisition.

References

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- View Article
- PubMed/NCBI
- Google Scholar
22. Falcón-Pérez JM, Starcevic M, Gautam R, Dell’Angelica EC. BLOC-1, a novel complex containing the pallidin and muted proteins involved in the biogenesis of melanosomes and platelet-dense granules. J Biol Chem. 2002; 277: 28191–28199. pmid:12019270
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- PubMed/NCBI
- Google Scholar
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- View Article
- PubMed/NCBI
- Google Scholar
24. Gautam R, Novak EK, Tan J, Wakamatsu K, Ito S, Swank RT. Interaction of Hermansky-Pudlak Syndrome genes in the regulation of lysosome-related organelles. Traffic. 2006; 7: 779–792. pmid:16787394
- View Article
- PubMed/NCBI
- Google Scholar
25. Bowers WS, Thompson MJ. Identification of the major constituents of the crystalline powder covering the larval cuticle of Samia cynthia ricini (Jones). J Insect Physiol. 1965; 11: 1003–1011.
- View Article
- Google Scholar
26. Yang B, Fujii T, Ishikawa Y, Matsuo T. Targeted mutagenesis of an odorant receptor co-receptor using TALEN in Ostrinia furnacalis. Insect Biochem Mol Biol. 2016; 70: 53–59. pmid:26689645
- View Article
- PubMed/NCBI
- Google Scholar
27. Gilbert LI, Schneiderman HA. Prothoracic gland stimulation by juvenile hormone extracts of insects. Nature. 1959; 184: 171–173.
- View Article
- Google Scholar
28. White JF, Sastrodihardjo S. Meiosis in pupal ovarian cells of Samia cynthia cultured in vitro. Nature. 1966; 212: 314–315. pmid:5339132
- View Article
- PubMed/NCBI
- Google Scholar
29. Nagasawa H, Kataoka H, Hori Y, Isogai A, Tamura S, Suzuki A, et al. Isolation and some characterization of the prothoracicotropic hormone from Bombyx mori. Gen Comp Endocrinol. 1984; 53: 143–152. pmid:6714648
- View Article
- PubMed/NCBI
- Google Scholar
30. Kobayashi M, Burdette WJ. Heterologous transplantation of corpora allata between lepidoptera diapausing in egg and pupal stages. Nature. 1962; 194: 401–402. pmid:14457447
- View Article
- PubMed/NCBI
- Google Scholar
31. Mizoguchi A, Ishizaki H. Prothoracic glands of the saturniid moth Samia cynthia ricini possess a circadian clock controlling gut purge timing. Proc Natl Acad Sci U S A. 1982; 79: 2726–2730. pmid:16593181
- View Article
- PubMed/NCBI
- Google Scholar

[ref1] 1. Maiuolo J, Oppedisano F, Gratteri S, Muscoli C, Mollace V. Regulation of uric acid metabolism and excretion. Int J Cardiol. 2016; 213: 8–14. pmid:26316329
View Article
PubMed/NCBI
Google Scholar

[2] View Article

[3] PubMed/NCBI

[4] Google Scholar

[ref2] 2. Kashima T, Nakamura T, Tojo S. Uric acid recycling in the shield bug, Parastrachia japonensis (Hemiptera: Parastrachiidae), during diapause. J Insect Physiol. 2006; 52: 816–825. pmid:16797581
View Article
PubMed/NCBI
Google Scholar

[6] View Article

[7] PubMed/NCBI

[8] Google Scholar

[ref3] 3. Tasaki E, Sakurai H, Nitao M, Matsuura K, Iuchi Y. Uric acid, an important antioxidant contributing to survival in termites. PLoS One. 2017; 12: 1–12.
View Article
Google Scholar

[10] View Article

[11] Google Scholar

[ref4] 4. Tsujita M, Sakurai S. Genetical and biochemical studies of the chromogranules in the larval hypodermis of the silkworm, Bombyx mori. J Seric Sci Japan. 1964; 33: 447–459.
View Article
Google Scholar

[13] View Article

[14] Google Scholar

[ref5] 5. Tamura T, Akai H. Comparative ultrastructure of larval hypodermal cell in normal and oily Bombyx mutants. Cytologia (Tokyo). 1990; 55: 519–530.
View Article
Google Scholar

[16] View Article

[17] Google Scholar

[ref6] 6. Kômoto N. A deleted portion of one of the two xanthine dehydrogenase genes causes translucent larval skin in the oq mutant of the silkworm (Bombyx mori). Insect Biochem Mol Biol. 2002; 32: 591–597. pmid:12020833
View Article
PubMed/NCBI
Google Scholar

[19] View Article

[20] PubMed/NCBI

[21] Google Scholar

[ref7] 7. Fujii T, Daimon T, Uchino K, Banno Y, Katsuma S, Sezutsu H, et al. Transgenic analysis of the BmBLOS2 gene that governs the translucency of the larval integument of the silkworm, Bombyx mori. Insect Mol Biol. 2010; 19: 659–667. pmid:20546041
View Article
PubMed/NCBI
Google Scholar

[23] View Article

[24] PubMed/NCBI

[25] Google Scholar

[ref8] 8. Wang L, Kiuchi T, Fujii T, Daimon T, Li M, Banno Y, et al. Mutation of a novel ABC transporter gene is responsible for the failure to incorporate uric acid in the epidermis of ok mutants of the silkworm, Bombyx mori. Insect Biochem Mol Biol. 2013; 43: 562–571. pmid:23567590
View Article
PubMed/NCBI
Google Scholar

[27] View Article

[28] PubMed/NCBI

[29] Google Scholar

[ref9] 9. Ames BN, Cathcart R, Schwiers E, Hochstein P. Uric acid provides an antioxidant defense in humans against oxidant- and radical-caused aging and cancer: a hypothesis. Proc Natl Acad Sci U S A. 1981; 78: 6858–6862. pmid:6947260
View Article
PubMed/NCBI
Google Scholar

[31] View Article

[32] PubMed/NCBI

[33] Google Scholar

[ref10] 10. Matsuo T, Ishikawa Y. Protective role of uric acid against photooxidativestress in the silkworm, Bombyr mori (Lepidoptera: Bombycidae). Appl Entomol Zool. 1999; 34: 481–484.
View Article
Google Scholar

[35] View Article

[36] Google Scholar

[ref11] 11. Hu YG, Shen YH, Zhang Z, Shi GQ. Melanin and urate act to prevent ultraviolet damage in the integument of the silkworm, Bombyx mori. Arch Insect Biochem Physiol. 2013; 83: 41–55. pmid:23575996
View Article
PubMed/NCBI
Google Scholar

[38] View Article

[39] PubMed/NCBI

[40] Google Scholar

[ref12] 12. Timmerman S, Berenbaum M. Uric acid deposition in larval integument of black swallowtails and speculation on its possible functions. J Lepid Soc. 1999; 53: 104–107.
View Article
Google Scholar

[42] View Article

[43] Google Scholar

[ref13] 13. Zhu G, Peng Y, Zheng M, Zhang X, Sun J. CRISPR / Cas9 mediated BLOS2 knockout resulting in disappearance of yellow strips and white spots on the larval integument in Spodoptera litura. J Insect Physiol. 2017; 103: 29–35. pmid:28927827
View Article
PubMed/NCBI
Google Scholar

[45] View Article

[46] PubMed/NCBI

[47] Google Scholar

[ref14] 14. Traut W, Sahara K, Marec F. Sex chromosomes and sex determination in Lepidoptera. Sex Dev. 2007; 1: 332–346. pmid:18391545
View Article
PubMed/NCBI
Google Scholar

[49] View Article

[50] PubMed/NCBI

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[ref15] 15. Kumar S, Stecher G, Tamura K. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for bigger datasets. Mol Biol Evol. 2016; 33: 1870–1874. pmid:27004904
View Article
PubMed/NCBI
Google Scholar

[53] View Article

[54] PubMed/NCBI

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View Article
PubMed/NCBI
Google Scholar

[57] View Article

[58] PubMed/NCBI

[59] Google Scholar

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View Article
Google Scholar

[61] View Article

[62] Google Scholar

[ref18] 18. Takasu Y, Sajwan S, Daimon T, Osanai-Futahashi M, Uchino K, Sezutsu H, et al. Efficient TALEN construction for Bombyx mori gene targeting. PLoS One. 2013; 8: 1–11.
View Article
Google Scholar

[64] View Article

[65] Google Scholar

[ref19] 19. Truett GE, Heeger P, Mynatt RL, Truett AA, Walker JA, Warman ML. Preparation of PCR-quality mouse genomic DNA with hot sodium hydroxide and tris (HotSHOT). Biotechniques. 2000; 29: 52–54. pmid:10907076
View Article
PubMed/NCBI
Google Scholar

[67] View Article

[68] PubMed/NCBI

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View Article
Google Scholar

[71] View Article

[72] Google Scholar

[ref21] 21. Fujii T, Abe H, Katsuma S, Mita K, Shimada T. Mapping of sex-linked genes onto the genome sequence using various aberrations of the Z chromosome in Bombyx mori. Insect Biochem Mol Biol. 2008; 38: 1072–1079. pmid:19216995
View Article
PubMed/NCBI
Google Scholar

[74] View Article

[75] PubMed/NCBI

[76] Google Scholar

[ref22] 22. Falcón-Pérez JM, Starcevic M, Gautam R, Dell’Angelica EC. BLOC-1, a novel complex containing the pallidin and muted proteins involved in the biogenesis of melanosomes and platelet-dense granules. J Biol Chem. 2002; 277: 28191–28199. pmid:12019270
View Article
PubMed/NCBI
Google Scholar

[78] View Article

[79] PubMed/NCBI

[80] Google Scholar

[ref23] 23. Dell’Angelica EC. The building BLOC(k)s of lysosomes and related organelles. Curr Opin Cell Biol. 2004; 16: 458–464. pmid:15261680
View Article
PubMed/NCBI
Google Scholar

[82] View Article

[83] PubMed/NCBI

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View Article
PubMed/NCBI
Google Scholar

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[87] PubMed/NCBI

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View Article
Google Scholar

[90] View Article

[91] Google Scholar

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View Article
PubMed/NCBI
Google Scholar

[93] View Article

[94] PubMed/NCBI

[95] Google Scholar

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View Article
Google Scholar

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[98] Google Scholar

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View Article
PubMed/NCBI
Google Scholar

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[101] PubMed/NCBI

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View Article
PubMed/NCBI
Google Scholar

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View Article
PubMed/NCBI
Google Scholar

[108] View Article

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Figures

Abstract

Introduction

Materials and methods

Insects

Inhibition of uric acid synthesis in S. ricini larvae

Quantification of uric acid content in the integuments of S. ricini larvae

Determination of the partial nucleotide sequences of SrBLOS2 and SrRp49

Phylogenetic analysis

Expression analysis of SrBLOS2 mRNA in S. ricini tissues

Construction of a TALEN targeting SrBLOS2

Embryo preparation and embryonic microinjection

Crossing of G0 individuals

Detection and DNA sequencing of the mutations in G1 individuals

Transmission electron microscopy (TEM)

Nucleotide sequence deposition

Results

Allopurinol treatment induced a translucent integument phenotype in S. ricini larvae

Identification and characterization of SrBLOS2

Expression profile of SrBLOS2 in larval tissues

Microinjection of the TALEN mRNA for SrBLOS2

Detection of mutant alleles in G1 broods

Germline transmission rates (GTRs) of TALEN-mediated SrBLOS2 mutations

Accumulation of uric acid as urate granules in the epidermal cells of S. ricini larvae

Discussion

Accumulation of uric acid in the integument of S. ricini larvae

SrBLOS2 functions in urate granule formation

Absence of SrBLOS2KO G0 individuals with a somatic mosaic phenotype

GTR of TALEN-mediated mutations in S. ricini

Supporting information

S1 Table. Primer list used in this study.

S2 Table. GenBank accession number list of sequences used for phylogenetic analysis.

S1 Fig. Representative images of microchip electrophoresis.

S2 Fig. The blue strain of Samia ricini.

Acknowledgments

References

Crossing of G₀ individuals

Detection and DNA sequencing of the mutations in G₁ individuals

Detection of mutant alleles in G₁ broods

Absence of SrBLOS2^KO G₀ individuals with a somatic mosaic phenotype