Transcriptome analysis of the eggs of the silkworm pale red egg (rep-1) mutant at 36 hours after oviposition
Abstract
The egg stage is one of the most critical periods in the life history of silkworms, during which physiological processes such as sex determination, tissue organ formation and differentiation, diapause and pigmentation occur. In addition, egg color gradually emerges around 36h after oviposition. The red egg mutant rep-1, which was recently discovered in the C1(H) wild-type, C1(H) exhibits a brown egg color. In this study, the transcriptome of the eggs was analyzed 36h after oviposition. Between the rep-1 mutant and the C1(H) wild-type, 800 differentially expressed genes (DEGs) were identified, including 325 up-regulated genes and 475 down-regulated genes. These DEGs were mainly involved in biological processes (metabolic process, cellular process, biological regulation and regulation of biological process and localization), cellular components (membrane, membrane part, cell, cell part and organelle) and molecular functions (binding, catalytic activity, transporter activity, structural molecule activity and molecular transducer activity). The pathway enrichment of these DEGs was performed based on the KEGG database, and the results indicated that these DEGs were mainly involved in pathways in the following categories: metabolic pathways, longevity-regulating pathway-multiple species, protein processing in endoplasmic reticulum, peroxisome, carbon metabolism and purine metabolism. Further analysis showed that a large number of silkworm growth- and development-related genes and ommochrome synthesis- and metabolism-related genes were differentially expressed, most of which were up-regulated in the mutant. Our research findings provide new experimental evidence for research on ommochrome pigmentation and lay the foundation for further research on the mechanism of the rep-1 mutant.
1. Introduction
The silkworm (Bombyx mori) is an economically important insect and a fully metamorphic lepidopteran model insect [1]. The entire life cycle involves 4 stages, including eggs, larvae, pupae and moths. Diapause occurs in the silkworm egg stage. Therefore, the egg stage is one of the most important stages in the life cycle of the silkworm. Initially, the eggs appear light yellow, whereas diapause eggs gradually become gray-green, gray-purple or light brown. Other uncommon egg colors, such as red, white, brown, orange, and purple, are caused by mutations. Silkworm eggs are mainly composed of the egg shell and complex contents, including the yolk membrane, serosa, egg yolk, and embryo in addition to other components. Egg color arises from a number of expression events in the egg shell, serosa pigment, embryo, and yolk. However, ommochrome in the serosa is the determining factor [2]. Ommochrome is a tryptophan metabolite in insects that prevents tryptophan poisoning [3]. Ommochrome forms different colors by participating in redox reactions and is one of the main pigment substances in insects [4]. Tryptophan is converted into formylkynurenine via the opening of the pyrrole ring by a tryptophan pyrrolase (TRPO), after which kynurenine formamidase (KFase) causes a change in kynurenic, which is further transformed into 3-hydroxy-canine with the aid of kynurenine 3-hydroxylase (K3H) ureine. Eventually, two molecules of 3-hydroxy-kynurenine are synthesized by phenoxazinone synthetase (PHS) to produce xanthommatin [5–8]. Ommochrome is mainly found in compound eyes and eggs. Additionally, it exists in the epidermis and central nervous system of silkworm larvae [9]. 3-Hydroxykynurenine in the blood in the hemolymph of the pupal stage enters the ovaries and compound eyes to affect egg color and adult compound eye color, respectively [10].
Egg color mutants are good model systems for investigating pigment metabolism. A large number of egg color mutants are maintained in the silkworm mutant library, and many new mutants are continually being discovered. The initial silkworm white egg (w-1) phenotype identified cannot produce ommochrome because the lack of kynurenine-3-hydroxylase leads to an accumulation of kynurenine, which cannot be converted into 3-hydroxy kynurenine [11]. In contrast, another mutant silkworm white egg (w-2) phenotype is yellow-white in the early stage and then develops a reddish color. Researchers have shown that the mutant form results from a mutation in a ATP-binding transporter. Dimers formed by ATP-binding cassette transporters and products encoded by the white gene are responsible for ommochrome transport [12]. Multiple alleles contribute to the third type of mutant silkworm egg phenotype (w-3), including the white egg worm (w-3oe) mutant, which is caused by a single-base deletion in exon 2 of the white3 gene [13]. The serosa cells of the red egg mutant (re) are red and contribute to the eggs turning dark red. Map-based cloning has proven that a new member of the transmembrane protein family causes the mutation [14].
The pale red egg mutant (pale red egg, rep-1) is a new egg color mutant found in the silkworm breed C1(H). Its color gradually changes to pale red over approximately 36h to a color similar to that of the red egg mutant (re) albeit a different intensity (Fig 1). By hybridizing the rep-1 mutant with the wild type p50 silkworm variety, the genetic model indicated that the pale red egg mutation was controlled by an autosomal recessive gene [15]. When the rep-1 (female) mutant was crossed with the re mutation, or the re (female) mutant was crossed with the rep-1 mutation, all of the F1 eggs were the same color as the female parent, whereas the F2 eggs were all red. The F3 egg color, however, segregated, with different degrees of red that were difficult to distinguish from red eggs and pale red eggs. Gene mapping showed that the key gene controlling the rep-1 mutant is very close to the MFS (major facilitator superfamily) gene that controlled the re mutation (data not shown).
(A) Normal type C1(H). (B) Pale red egg (rep-1) mutant. (C) Red egg (re) mutant. (D) Normal type C1(H) after 36h of oviposition. (E) Pale red egg (rep-1) mutant after 36h of oviposition. Finally, the eggs of normal type C1(H) was black colors, the eggs of rep-1 mutant were pale red colors, the eggs of re mutant was red colors.
Because the egg color change starts 36h after oviposition, to understand the gene expression profile of the mutant at this time, eggs of the rep-1 silkworm mutant laid within 36h were used as the experimental materials, and C1(H) as a control group. Fifty eggs from each silkworm strain were pooled for transcriptome analysis. Three experiments were repeated for each group, and the transcriptome data to NCBI with the login number of PRJNA622872. Differentially expressed genes (DEGs) were identified according to the sequencing results and further analyzed. At the same time, the expression levels of genes related to ommochrome synthesis and metabolism, egg development and structure and major genes associated with egg color mutants were analyzed for possible roles of these genes in the rep-1 pale red egg mutant. The study provides a foundation for understanding the formation mechanism of rep-1 mutants and as well as a theoretical basis for the study of the molecular mechanism controlling egg color formation.
2. Materials and methods
2.1. The silkworm strains
The C1(H) and rep-1 silkworm strains were provided by the Chinese Academy of Agricultural Sciences (Zhenjiang. China). Larvae were reared on fresh mulberry leaves in an environment with a 12-h light/12-h dark cycle at 25 ± 1°C and 70–85% relative humidity. After 36h of oviposition, 50 eggs of each silkworm strain were pooled for RNA-Seq and quantitative analysis. Each assay was repeated three times. Eggs at 12h, 24h and 36h after oviposition were used to examine the expression of genes of interest. This experiment was approved by the Jiangsu University of Science and Technology Animal Care and Use Committee.
2.2. RNA extraction, library construction and RNA-Seq
RNA was extracted using RNAiso Plus (TaKaRa, China) and dissolved in RNase-free water. RNA purity was assessed by using a NanoDrop 1000 microspectro- photometer (Thermo, USA). Then total mRNA treated with RNase was enriched using oligo (dT) magnetic beads. The next step was to shear mRNA into short fragments by adding hybridization interruption reagents. Double-stranded cDNA was synthesized using random six-base primers and the interrupted mRNA as the template. The purified double-stranded cDNA was end repaired, and a tail was added and connected with a sequencing connector. PCR amplification was carried out to generate cDNA library. The library constructed in the previous step was loaded onto the sequencing chip of an Illumina HiSeq TM 2000 sequencer for sequencing (NanJing Decode Genomics). Finally, 150-bp paired end reads data of the processing group and the control group library were obtained.
2.3. Sequence assembly
After filtering out low quality reads and sequence reads containing connectors or poly-A / T tails, clean reads were assembled by using the short reads assembler Trinity-v2.8.4. All experiments were performed with Trinity using parameters: minimum contig length of 100 bases and average fragment length of 300 bases [16]. The unigenes from two sample groups were gathered together and clustered using the TGI clustering tool [17]. For functional annotation, BLASTX (E-value cut-off = 10−5) was used to retrieve single gene sequences from various protein databases including Nr, Swiss-Prot, COG and KEGG [18], and BLASTN was used to search the nucleotide database (E-value<10−5). The coding sequence (CDS) was extracted from unigenes by searching BLAST results and translated into peptide sequence. When unigene failed to generate a corresponding BLAST hit, the sequence direction was determined through ESTScan software 2.1 [19]. In addition, a Gene Ontology (GO) Association was performed using the Nr database according to BLASTX, and each gene was annotated with Blast2GO [20, 21].
2.4. Quality control of RNA-Seq data
Quality control analysis was performed on the raw data, which were filtered to obtain clean reads. The filtering rules were as follows: removal of reads with adapter sequences at first; then removal of reads with N bases (N means that the base information cannot be determined); finally removal of reads with low quality (in which the number of reads with a quality value of Qphred ≤ 5 is more than 50% of the total reads). At the same time, the distribution of base quality values, the base composition and the average GC content of the original sequence data were analyzed through ESTScan software 2.1 to ensure that the sequencing data met the requirements for subsequent analysis.
The clean reads were compared with the reference genome (NCBI, https://www.ncbi.nlm.nih.gov/)), the statistical reads in the distribution of different segments of the genome and the results of the second quality analysis, including read coverage uniformity, insert sequence fragment length and sequencing saturation analyses, to ensure that the reads met the requirements for subsequent gene expression data and DEG analyses.
2.5. Analysis of differentially expressed genes
The number of reads obtained from the genome was counted, and the RPKM (reads per kilobase per million reads) method using RSEM program of Trinity software-v2.8.4 was used to standardize the expression level of each gene [22]. The boxplot and density distribution of the RPKM values and the correlation analysis of each sample were performed to compare the differences in gene expression levels among different samples according to the overall level of gene expression. Then, an FDR≤0.05 (FDR: false discovery rate) was used as the threshold for the screening of DEGs [23]. GO and KEGG pathway enrichment analyses were performed for the differentially expressed genes encoding proteins. The expression levels of genes related to ommochrome synthesis and metabolism, genes related to egg development and structure and major genes of the egg color mutants were analyzed simultaneously to elucidate the possible roles of these genes in rep-1 pale red egg mutants.
2.6. Quantitative Reverse Transcription PCR (qRT-PCR)
Eggs were produced by C1(H) and rep-1 mutant silkworms for 36h. For each sample, 50 eggs were pooled. The RNAiso plus method was used to extract the total RNA, followed by treatment with DNase, and cDNA was obtained using an M-MLV Reverse Transcriptase (RNase H-) kit. The cDNA was diluted to 100 ng/μl as a template for qRT-PCR. All qRT-PCR reactions were performed in a 20 μl volume containing 1μl specific primers (10 μM), 1μl cDNA, 10 μl NovoStart®SYBR qPCR SuperMix (Novoprotein, Nanjing, China) and H2O in a LightCycler® 96 quantitative PCR instrument. The reaction conditions were as follows: predenaturation at 95°C for 10 min, 40 three-step cycles including denaturation at 95°C for 10 s, annealing at 58°C for 10 s, and extension at 72°C for 10 s (58°C to 95°C in an increment of 0.5°C every 5 s). Finally, the melting curve was generated. The relative expression of each gene was calculated in relation to the average values for two housekeeping genes, RPL-3 (BGIBMGA013567) and GAPDH (BGIBMGA007490). The 2-ΔΔCt method [24] was used to examine differential expression of the genes of interest. qRT-PCR expression in the wild-type was compared with rep-1 to verify the accuracy of the DEG data.
3. Results
3.1. General information for RNA-Seq
Eggs that hatched after 36h were used for RNA-seq. raw reads were obtained from the wild-type C1(H) group (55.72 ± 6.22), and Mega clean reads (55.54 ± 6.19) were obtained following quality control. Similarly, raw reads (64.08 ± 7.50) and Mega clean reads (63.88 ± 7.50) were obtained from the rep-1 mutant. The ratios of clean reads and raw reads were 99.67% and 99.68%, respectively. The GC content of the reads for both the wild-type C1(H) group and the rep-1 mutant was approximately 43%, and that of adapter reads was approximately 3% (Table 1).
Table 1
| Category | Parameter | Wildtype | rep-1 mutant |
|---|---|---|---|
| Raw data | Total Reads (Megabases) | 55.72 ± 6.22 | 64.08 ± 7.50 |
| Total bases (Gigabases) | 8.35 ± 0.92 | 9.61 ± 1.13 | |
| Q20 (%) | 97.56 ± 0.05 | 97.41 ± 0.06 | |
| Q30 (%) | 93.38 ± 0.11 | 93.08 ± 0.13 | |
| Adapter reads (%) | 3.13 ± 0.09 | 3.03 ± 0.09 | |
| GC (%) | 43.33 ± 0.09 | 43.32 ± 0.20 | |
| Clean data | Total Reads (Mega) | 55.54 ± 6.19 | 63.88 ± 7.50 |
| Total bases (Gigabit) | 8.31 ± 0.92 | 9.56 ± 1.12 | |
| Q20 (%) | 97.76 ± 0.05 | 97.62 ± 0.05 | |
| Q30 (%) | 93.61 ± 0.10 | 93.31 ± 0.12 | |
| GC (%) | 43.27 ± 0.09 | 43.27 ± 0.20 | |
| Clean data/Raw data (%) | 99.67 | 99.68 | |
| Quality Control | Clean Read Q20(%) ≥ 95 (Y or N) | Y | Y |
| Clean Read Q30(%) ≥ 90 (Y or N) | Y | Y | |
| Clean Reads ≥ 10M (Y or N) | Y | Y | |
| Gene Unique Mapping Ratio(%) ≥ 80 (Y or N) | Y | Y | |
| Genome-Mapping | Total Mapped Reads (%) | 87.09 ± 0.01 | 87.46 ± 0.01 |
| Unique Match (%) | 83.44 ± 0.01 | 83.90 ± 0.01 | |
| Multi-position Match (%) | 2.72 ± 0.01 | 2.69 ± 0.01 | |
| Total Unmapped Reads (%) | 12.91 ± 0.01 | 12.54 ± 0.01 | |
| Gene expression | Total Genes | 15884 | 15884 |
| Expressed genes (%) | 12403 (78.08%) | 12488 (78.62%) | |
| Both expressed genes (%) | 12007 (75.59%) | ||
| Not expressed genes (%) | 3481 (21.91%) | 3396 (21.38%) | |
| Neither expressed genes (%) | 3000 (18.89%) | ||
| Expressed only in C1(H) (%) | 396 (2.49%) | — | |
| Expressed only in rep-1 (%) | — | 481 (3.03%) | |
The analysis of distribution of the base mass values of the sequencing data (S1A Fig, S1D Fig), the distribution of the composition of each base (S1B Fig, S1E Fig) and the distribution of the average GC content (S1C Fig, S1F Fig) showed that the base mass was good; the base composition was uniform; and the GC content was stable, thus satisfying the requirements for subsequent analysis. In addition, the Q20 values of the raw reads and clean reads of C1(H) and rep-1 were greater than 95%. The Q30 values of the raw reads and clean reads of C1(H) and rep-1 were greater than 90% (Table 1), thus meeting the requirements for subsequent analysis.
3.2. Read mapping analysis
The clean reads obtained from RNA-seq were mapped with the NCBI reference genome seventy-six. and the accession number is ASM15162v1. In C1(H) and rep-1, 87.09% and 87.46% of the reads were mapped to the reference genome. A total of 83.44% and 83.90% of the reads, respectively, were mapped to the single-copy reference sequence, and 2.72% and 2.69% of the reads were mapped to the multicopy reference sequence. The number of clean reads exceeded 55 megabases, reaching the minimum requirement of 10 megabases, and a unique gene mapping ratio reached 80%.
The distribution statistics of the reads in different regions of the genome showed that more than 87% of the reads matched to a CDS, and some of the reads matched intron and gene intervals, possibly due to the presence of unknown genes and alternative splicing (S2A Fig, S2E Fig) The coverage uniformity analysis of the reads showed that the coverage of 10 windows at the 5' end was limited. The coverage of 10 to 60 windows was best. The coverage of the 3' end gradually decreased (S2B Fig, S2F Fig). The analysis of insert length showed that the average distances between two reads in C1(H) and rep-1 were 11 BP and 14 BP, respectively, which showed that the selected length could basically cover two reads and achieved the maximum utilization of the sequence (S2C Fig, S2G Fig). The saturation analysis indicated that saturation was reached when the sequencing percentage was approximately 40%, and all of the sequencing requirements were met (S2D Fig) Therefore, mapping results met the requirements for subsequent analysis.
3.3. General gene expression information
In total, we analyzed the expression levels of 15884 gene (S2 Table). Among these genes, 12403 were expressed in wild-type C1(H), accounting for 78.08% of the total, while 12488 (78.62%) were expressed in the rep-1 mutant (3 replicates could be detected). A total of 12007 (75.60%) genes were expressed in both C1(H) and rep-1 (6 replicates could be detected); 396 (2.50%) genes were expressed in C1(H) but not in rep-1; and 481 genes were expressed in rep-1 but not in C1(H), accounting for 3.03% of the total. Finally, 3000 genes were not expressed in C1(H) and rep-1 (could not be detected in at least one of the six samples), accounting for 18.89% of the total (Table 1).
The correlation analysis results for sample expression showed that the correlations between the six samples were very close; the correlation coefficient was close to 1 (S3A Fig); the box plot analysis results showed that the repeatability of the gene expression results of the six samples was good. The maximum value, the upper quartile, the median value, the lower quartile and the minimum value showed good uniformity among the six samples (S3B Fig). A horizontal density distribution diagram of gene expression was generated. Genes showing an expression value of approximately 10 were the most abundant (the first peak). Genes with an expression value of approximately 0.1 were also highly expressed (the second peak) (S3C Fig).
3.4. Analysis of differentially expressed genes
An FDR ≤ 0.05 was used as a threshold for screening differentially expressed genes indicated in S3 Table. We identified 800 differentially expressed genes (S3 Table). Compared with the C1(H) wild-type, 475 genes in the rep-1 mutant were down-regulated, and 325 genes were up-regulated (S3D Fig–S3F Fig). GO cluster analysis was carried out on the differentially expressed genes. Among these genes, the identified biological processes mainly included the following terms: metabolic process (103 up-regulated genes and 115 down-regulated genes), cellular process (85 up and 115 down), biological regulation (27 up and 66 down), regulation of biological process (24 up and 59 down) and localization (22 up and 46 down). The identified cellular component terms mainly included the following: membrane (67 up and 110 down), membrane part (60 up and 96 down), cell (62 up and 80 down), cell part (61 up and 80 down) and organelle (41 up and 35 down). The molecular function terms were mainly related to binding (100 up and 142 down), catalytic activity (104 up and 133 down), transporter activity (8 up and 28 down), structural molecule activity (6 up and 7 down) and molecular transducer activity (3 up and 12 down) (Fig 2, S4 Table). Based on a difference multiple greater than 2, that was |log2(FOLD change) | > 1, 114 genes were up-regulated and 120 were down-regulated, and the 234 differentially expressed genes were mainly related to binding proteins (56) and catalytic activity (64).
These DEGs were mainly involved in biological process, cellular component and molecular function.
These differentially expressed genes were analyzed by using the KEGG database to evaluate pathways, and 218 genes were clustered into 105 pathways. The top six clustered genes belonged to the following categories: metabolic pathways (89), longevity regulating pathway—multiple species (16), protein processing in the endoplasmic reticulum (16), peroxisome (14), carbon metabolism (14) and purine metabolism (14) (Fig 3, S5 Table). Among the metabolic pathways, a large number of amino acid metabolic pathways showed changes, including those of serine, cysteine, methionine, leucine, lysine, arginine, proline, tyrosine, polyamines, p-aminobutyric acid and glutathione.
3.5. Verifying the accuracy of the RNA-Seq data by qRT-PCR
To verify the accuracy of the RNA-seq data, we analyzed the expression of three housekeeping genes, and selected certain genes for qRT-PCR analysis, including representative differentially expressed genes, pigment synthesis genes, metabolism-related genes, egg shell protein genes, yolk membrane protein genes, and major genes affected by egg color mutants (Table 2, S1 Table).
Table 2
Three housekeeping genes, bmactin3 (fold-change = 1.07). Glyceraldehyde-3-phosphate dehydrogenase (fold-change = 0.81) and ribosomal protein L3 (fold-change = 0.97), showed fold-changes in all databases that were approximately equal to 1, providing initial verification that the RNA-seq data were accurate. Twelve up-regulated genes and twelve down-regulated genes with fold-change differences greater than 2 were randomly selected for qRT-PCR validation. The results for 20 out of 24 DEGs were identical to the qRT-PCR results, whereas the qRT-PCR results for 2 out of 4 genes were inconsistent with the DEG results. However, the fold-change difference was not significant (0.5 < fold-change < 2). Therefore, the results for 22 of the DEGs genes were consistent with the qRT-PCR results. The accuracy was 91.67%, which indicated that the DEG results were reliable. The expression levels of select genes are shown in Fig 4.
4. Discussion
4.1. Differentially expressed genes based on RNA-Seq data
Serotonin is a widespread distribution in the insect nervous system [25]. It affects sensory processing, information coding and behavior [26]. The glutamate-gat chloride channel is only found in invertebrates, mediating fast inhibitory neurotransmission [27]. The genes encoding these two proteins involved in the nervous system were down-regulated (fold-change = 0.49) and significantly up-regulated (fold-change = 11.69), respectively, suggesting that the development of the nervous system might be affected in the mutant. Troponin plays a role in muscle contraction [28]. Dynein plays a role in the movement of flagella and cilia [29]. The genes encoding these two proteins are related to motility and were up-regulated in the mutant (fold-change = 3.71 for troponin, while dynein was expressed only in the mutant). These results indicate that material transport and metabolism in the mutant are relatively similar. Other up-regulated genes among the genes involved in the metabolism of these substances included those encoding a key enzyme in the urea cycle, arginine succinate lyase, was also up-regulated (argininosuccinate lyase, fold-change = 16.83) [30], the Krebs cycle (tricarboxylic acid cycle) component isocitrate dehydrogenase (isocitrate dehydrogenase, fold-change = 3.28) [31], a protein with a role in cell adhesion (cytoadherence) (integrin, fold-change [32] = 7.48), the signal transduction-related enzyme serine-threonine protein kinase (serine/threonine—protein kinase, fold-change = 4.89) [33, 34] and Phosphoglyceryl acyltransferase (old-change = 26.66) involved in glycerolipid synthesis [35], further confirming the similar cellular activity and material metabolism of the mutant.
4.2. Differentially expressed genes involved in amino acid metabolism
KEGG pathway clustering analysis of the differentially expressed genes revealed 89 DEGs clustering to metabolic pathways. The analysis revealed many amino acid metabolic pathways with gene expression differences (S5 Table), including the serine, betaine, cysteine, arginine, proline, polyamine and glutathione biosynthesis pathways, methionine, leucine, lysine and tyrosine degradation pathways and aminobutyric acid metabolic pathways. Amino acids are not only the basic substances necessary for biological activities but are also the components of basic molecules such as proteins, which are involved in almost all physiological processes. Abnormal metabolism will affect the growth and development of the silkworm.
4.3. Differentially expressed genes related to ommochrome synthesis
Ommochrome is the key substance responsible for the formation of egg color. 3-Hydroxykynurenine, which is synthesized from tryptophan through a series of catalytic reactions, is transported to pigment particles by the ABC transporter heterodimer. Then, it is transformed via oxidative condensation to form purple ommin pigment and red-brown xanthommatin pigment [10] (Fig 5). In silkworms the scarlet protein encoded by the Bmw-2 gene and the white protein encoded by the Bmwh3 gene form a white/scarlet dimer, which transports 3-hydroxykynurenine, an ommochrome precursor, to pigment granules [12, 13]. The Bmcardinal gene encodes phenoxazinone synthase, which catalyzes the synthesis of xanthommatin. The other 3-hydroxykynurenine is used to synthesize ommin, which plays a key role in red egg mutants. The red egg mutant gene MFS (major facilitator superfamily) is related to the formation of ommin [14]. Kynurenine 3-hydroxykynurenine can simultaneously be hydrolyzed to produce anthranilic acid and 3-hydroxy-2-aminobenzoic acid via the action of the kynureninase gene bmkynu [36] (Fig 5).
Ommochrome is synthesized through a series of enzymatic reactions employing tryptophan as the substrate. Most genes encoding these enzymes were up-regulated in the rep-1 mutant especially at 24h after oviposition. The vertical axis is defined as the ratio of expression of rep-1 mutant / expression of wildtype. If the ratio> 2 or <0.5, it indicates that there are differences in expression quantity.
The quantitative analysis of gene expression in the ommochrome synthesis and metabolism pathways at 12h, 24h and 36h after oviposition showed that the expression levels of most genes were up-regulated in the rep-1 mutant. Compared to the C1(H) wild-type, there was a trend of increasing expression levels in eggs in the 12h postpartum period. Expression was significantly increased at 24h and tended to return to a level similar to that in the wild-type at 36h (Fig 5). According to the analysis of gene expression, the main effector gene of the rep-1 mutant caused changes in gene expression in the ommochrome synthesis pathway. A series of genes were up-regulated in the mutant, causing more ommochrome to be produced. In relation to ovary coloration and the phenotypic changes in mutant pale red eggs, the changes in gene expression at different developmental stages were analyzed. The genes related to egg pigment synthesis began to be expressed at high levels at 12h after oviposition, reaching their highest expression at 24h, after which their expression began to decrease. At 36h, ommochrome synthesis tended to be complete. Egg color began to appear, and gene expression was reduced to the lowest level. In particular, the expression levels of the two ABC transporters responsible for transporting 3-hydroxykynurenine to pigment granules (white and scarlet) reached a maximum at 24h. The results showed that 24h after oviposition may be the key period of ommochrome synthesis in silkworm eggs.
4.4. Expression analysis of key genes affected in several egg color mutants
Silkworm eggs represent not only the end of life but also the beginning of a new cycle. Egg diapause is useful for surviving in harsh environments, and the egg stage is therefore a very important developmental period. Egg color is a critical physiological characteristic of silkworm eggs. Normal silkworm eggs are gray-green, gray-purple or light brown. Many different egg color mutants have been identified through silkworm breeding. These egg color mutants are essential for studying silkworm egg pigment deposition. The color mutations and mutant genes identified thus far are shown in Table 2.
The mutant egg color genes that were identified were all related to the ommochrome synthesis pathway. Quantitative analysis showed that these genes were up-regulated in the pale red egg mutant. Most of the genes were up-regulated at 24h after oviposition (Fig 5). The results indicated that the egg color mutants largely result from mutations in the ommochrome synthesis pathway. The white egg mutant is unable to complete ommochrome synthesis because of gene mutations during the early stage of the synthesis pathway. However, compared to the white egg mutant, egg color mutations are caused by later gene mutations that result in different egg color phenotypes according to different ratios of the mixture of ommin (purple) and xanthommatin (red-brown).
4.5. Differentially expressed genes encoding structural proteins of eggs
Insect eggs are mainly composed of the shell and its contents, including the yolk membrane, protoplasm, vitellus, cell nucleus and other structures. The membrane protoplasm as noncellular structure is located close to the egg shell. The vitellus is very important because of its complex components, which provide nutrition for the development of the egg. The nucleus is responsible for the storage of genetic information. The egg shell is a complex protective structure on the surface of the egg with the physiological function of protecting embryos, gas exchange in the eggs [37]. The egg shell protein is the main component of the egg shell. A total of 96% of the dry weight of the silkworm is contributed by the egg shell protein. More than 100 egg shell proteins have been identified in silkworms to date [38, 39]. According to RNA-Seq data analysis, eight chorion-related genes were expressed. However, there was no significant difference in the expression of these genes (|log2(fold-change) | < 0.5). The results are consistent with egg shell construction being completed 36h after oviposition. Only a few egg shell proteins were expressed. The mutant genes of the pale red egg mutant did not affect the regulation and expression of egg shell proteins (S6 Table). The yolk protein precursor is mainly derived from the female-specific hemolymph protein vitellogenin (Vg) of the adipose body. After synthesis by the adipose body, vitellogenin is transferred to the ovary through the hemolymph, which is absorbed by oocytes and deposits vitellogenin in the egg to provide nutrients and functional substances for embryonic development [40–42]. By analyzing the expression of the vitellogenin gene, we found that the expression of all vitellogenin genes was up-regulated in the light red egg mutants except for one gene (101746088).
The skin an essential organ for the avoidance of harm, growth reproduction and adaptation to complex environments in insects [43, 44]. In the insect genome, cuticular protein genes (CPG) are characterized by a number of structural features. There are more than 200 genes encoding CPGs in the silkworm genome [43, 45]. Approximately 30 CPGs were expressed in the 36h silkworm eggs. However, there was no significant difference between the wild-type and the mutants. In conclusion, the expression of genes encoding silkworm egg structural proteins was not significantly affected apart from some genes encoding vitellogenin.
5. Conclusion
In this study, the silkworm eggs of the light red pale egg mutant rep-1 at 36h after oviposition were used as the experimental group, and those of the C1(H) wild-type were used as the control group for RNA-seq data analysis. The qRT-PCR results verified the accuracy of the RNA-seq data. A total of 800 DEGs were identified by RNA-seq. Compared with C1(H), 475 genes were down-regulated and 325 genes were up-regulated in rep-1. It was found that the expression of genes related to the growth and development of silkworms and ommochrome synthesis and metabolism changed significantly. The overall trend was that the expression level was up-regulated in the mutants. At the same time, numerous DEGs related to amino acid metabolism pathway were found.
Supporting information
S1 Fig
Quality control of sequencing base.(A) Base quality value distribution in wildtype C1(H). (B) Base composition distribution in wildtype C1(H). (C) Average GC content distribution in wildtype C1(H). (D) Base quality value distribution in mutant rep-1. (E) Base composition distribution in mutant rep-1. (F) Average GC content distribution in mutant rep-1.
(TIF)
S2 Fig
Quality control of genome alignment.(A) Distribution of reads in different regions of genome the wild type genome. (B) Homogeneous analysis of read coverage in wild type. (C) Length analysis of insert fragment in wildtype. (D) Saturation analysis in wild type. (E) Read distribution in different regions of the mutant. (F) Homogeneous analysis of read coverage in the mutant. (G) Length analysis of insert fragments in the mutant. (H) Saturation analysis in the mutant.
(TIF)
S3 Fig
Analysis of general expression and differentially expressed genes.(A) Expression correlation analysis between different samples. (B) Box plots of gene expression level. (C) Density graphic of gene expression level. (D) Cluster analysis of differentially expressed genes. (E) MA plot of differentially expressed genes. (F) Volcano Plot of differentially expressed genes.
(TIF)
S1 Table
Primers.Expression levels and descriptions of differentially expressed genes.
(XLSX)
S2 Table
Read number and RPKM values of all genes.(XLSX)
S4 Table
Gene ontology analysis of differentially expressed genes.(XLSX)
S6 Table
Genes encoding chorion. Cpgs (Cuticular protein gene). Vitellogenin and ommochrome synthesis related enzymes.(XLSX)
Funding Statement
This work was supported by grants from the National Natural Science Foundation of China (No. 31972616) (Qiaoling Zhao)and the Innovation Plan of Jiangsu Province (KYCX19_ 1656) (Meina Wu). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Data Availability
The transcriptome data has been deposited at NCBI under accession number PRJNA622872.
References
Decision Letter 0
25 Mar 2020
PONE-D-20-03586
Transcriptome analysis of the eggs of the silkworm pale red egg (rep-1) mutant at 36 hours after oviposition
PLOS ONE
Dear professor Zhao,
Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Reviewer 1, in particular, had questions regarding the nomenclature used regarding the term rep-1, which would suggest that the mutant examined is a derivative of the re mutant. If so, this was not fully demonstrated. Reviewer 1 also had a number of suggestions regarding the inclusion of data/images describing the mutant phenotype relative to WT and/or other mutants that would greatly strengthen the manuscript.
In addition, I had a number of comments/suggestions for you to consider.
1) As currently written, the Methods section lacks sufficient details (and references) to allow for replication. I recommend looking at the relevant sections of the following papers for examples on the details needed for sequencing/quality control/quantification of the Illumina data.
Thoma et al Front Ecol Evol 2019 “Transcriptome Surveys in Silverfish Suggest a Multistep Origin of the Insect Odorant Receptor Gene Family” https://doi.org/10.3389/fevo.2019.00281
Dou et al PLOS ONE 2019 “Pheromone gland transcriptome of the pink bollworm moth, Pectinophora gossypiella: Comparison between a laboratory and field population” https://doi.org/10.1371/journal.pone.0220187
Yang et al PLOS ONE 2018 “Transcriptome analysis in different developmental stages of Batocera horsfieldi (Coleoptera: Cerambycidae) and comparison of candidate olfactory genes” https://doi.org/10.1371/journal.pone.0192730
Kozma et al PLOS ONE 2020 “Comparison of transcriptomes from two chemosensory organs in four decapod crustaceans reveals hundreds of candidate chemoreceptor proteins” https://doi.org/10.1371/journal.pone.0230266
2) Similarly, qPCR methods and data should include amplification efficiencies (see Bustin et al Clin Chem 2009 The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments).
3) Related to Reviewer 1 comments re the nomenclature used. If rep-1 is a derivative of the re mutant, why was the re mutant not included in the analyses?
4) It is not clear if qualitative differences were examined. Relatedly, since this is referred to as rep-1, is the same/similar mutation in the MFS transporter reported in the original Osanai-Futahashi manuscript present? Line 357 states that the mutant is “unable to complete ommochrome synthesis because of gene mutations during the early stage..”. What are these mutations? As written, it suggests that the mutations are qualitative. If not, this should be re-phrased so that is more clear that the phenotype is driven by quantitative differences in transcript expression, which could indicate potential changes to transcription factors. If so, were these examined?
5) Indicate in Figure 7 the genes that differ from WT, perhaps by addition of colors or some other type of marker. It is not clear in the current version where the differences occur.
We would appreciate receiving your revised manuscript by May 09 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.
If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.
To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols
Please include the following items when submitting your revised manuscript:
- A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
- A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
- An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.
Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.
We look forward to receiving your revised manuscript.
Kind regards,
J Joe Hull, Ph.D.
Academic Editor
PLOS ONE
Journal Requirements:
When submitting your revision, we need you to address these additional requirements:
1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at http://www.plosone.org/attachments/PLOSOne_formatting_sample_main_body.pdf and http://www.plosone.org/attachments/PLOSOne_formatting_sample_title_authors_affiliations.pdf
2. We note that you are reporting an analysis of a microarray, next-generation sequencing, or deep sequencing data set. PLOS requires that authors comply with field-specific standards for preparation, recording, and deposition of data in repositories appropriate to their field. Please upload these data to a stable, public repository (such as ArrayExpress, Gene Expression Omnibus (GEO), DNA Data Bank of Japan (DDBJ), NCBI GenBank, NCBI Sequence Read Archive, or EMBL Nucleotide Sequence Database (ENA)). In your revised cover letter, please provide the relevant accession numbers that may be used to access these data. For a full list of recommended repositories, see http://journals.plos.org/plosone/s/data-availability#loc-omics or http://journals.plos.org/plosone/s/data-availability#loc-sequencing.
[Note: HTML markup is below. Please do not edit.]
Reviewers' comments:
Reviewer's Responses to Questions
Comments to the Author
1. Is the manuscript technically sound, and do the data support the conclusions?
The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.
Reviewer #1: No
Reviewer #2: Yes
**********
2. Has the statistical analysis been performed appropriately and rigorously?
Reviewer #1: I Don't Know
Reviewer #2: Yes
**********
3. Have the authors made all data underlying the findings in their manuscript fully available?
The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.
Reviewer #1: No
Reviewer #2: Yes
**********
4. Is the manuscript presented in an intelligible fashion and written in standard English?
PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.
Reviewer #1: Yes
Reviewer #2: Yes
**********
5. Review Comments to the Author
Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)
Reviewer #1: In this paper, the authors conducted RNAseq for C1 (H) and rep-1 silkworm strains 36hours after oviposition, and conducted qRT-PCR for with the same strains at the same stage.
I have two major concerns for this study.
One is that the authors are using the name rep-1 for the newly found egg color mutant without offering basis that it is an allele of the re mutant. They refer to a paper that this mutant is controlled by a recessive gene on the autosome, but the paper could not be easily accessed at least by google scholar search. Nor the information on the chromosome number the gene is located is provided.
To use the name rep-1, confirmation by complementation test with re is necessary to confirm that it is an allele of re (cross re with rep-1 and confirm that WT egg pigmentation does not occurr in the next generation. If the F1 eggs have WT color, rep-1 is not an allele of re. In this case, the authors should name this mutant with another name which is not used for already existing mutants.).
Another concern is that this paper needs more experiments and data before publishing in PLOS One.
I suggest the authors at add the following data.
Egg photos of C1 (H) and rep-1 strains at 36h and at least 1 time point each before and after 36h post oviposition.
photos of adult eyes of C1 (H) and rep-1 strains
complementation test with re mutant
If it is not an allele of re, linkage analysis for rep-1(RAD-seq using different WT strain may be effective)
conduct qRT-PCR for fig 6 or RNAseq for other stages (at the least, one point before 36h)
I have some other comments to improve the manuscript.
The first sentence of the abstract should be deleted, because it has little relevance with the main subject of this paper.
Figures 1, 2, 3 should be moved to Supplementary data.
line 66-68
"Eventually, two molecules of 3-hydroxy-kynurenine are synthesized by phenoxazinone synthetase (PHS) to produce lutein."
This is incorrect. The authors seem to mistaken xanthommatin for lutein. Lutein is carotenoid, not ommochrome.
line 64: "pyrrolasc" may be typo (unfamiliar term to me)
figure 6 (seems to be the most important figure at present): Presenting only NCBI gene ID is not common. The name of the gene or should be stated. If it is not a previously studied gene, at least kind of the gene, such as non-coding RNA, and the chromosome number the gene is located should be stated.
figure 7: please state clearly in the text whether the expression data for 12 and 24 hours post oviposition were conducted by the authors for this manuscript (If so, the results should in the results section and stated clearly in the materials and methods section) or refers to data from a previous study. Also, the label for vertical axis should be clearly stated (is this expression data of rep-1 or WT?)
Reviewer #2: I have 3 concerns in the Manuscript
1. In Materials and methods, line no 127 onwards, the numbering can be brought down, instead of running text as it confuses in continuous reading
2. Flow chart can be made for the various processes involved in Quality check of the data and its analysis
3. In references, the complete author names can be mentioned instead of et al like in 2nd, 8th, 11,12, 14, 15,20, 21, 32, 35 and 36.
**********
6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.
If you choose “no”, your identity will remain anonymous but your review may still be made public.
Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.
Reviewer #1: No
Reviewer #2: Yes: A.H Manjunatha Reddy
[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]
While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at gro.solp@serugif. Please note that Supporting Information files do not need this step.
Author response to Decision Letter 0
22 Apr 2020
Dear Editor and Reviewers:
For our detailed response to this manuscript, please refer to the Response to Reviewer document for details. (As some Figures involved in the response which cannot be uploaded.) Thank you!
Attachment
Submitted filename: Response to reviewers.doc
Decision Letter 1
21 May 2020
PONE-D-20-03586R1
Transcriptome analysis of the eggs of the silkworm pale red egg (rep-1) mutant at 36 hours after oviposition
PLOS ONE
Dear Dr. Zhao,
Thank you for submitting your revised manuscript to PLOS ONE. Although the revisions addressed many of mine and the Reviewer's comments, there remain a number of relatively minor points that could be more fully considered.
While I appreciate the author's direct replies, it would perhaps be more useful to the manuscript audience if the replies were incorporated directly into the manuscript. In addition, some confusion remains regarding the allele and its functionality. From my reading, I agree with the Reviewer that the color change would most likely be associated with a loss of pigmentation upstream. It would be most helpful if this was further clarified.
While the back crosses suggested by the Reviewer would undoubtedly provide further insights (and are greatly encouraged if possible), I think that by addressing the concerns discussed above regarding rep-1 should be sufficient for this study. Lastly, I agree with the Reviewer that the manuscript is better served by having photos of the egg phenotypes within the paper rather than as a supplementary figure.
Please submit your revised manuscript by Jul 05 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at gro.solp@enosolp. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.
Please include the following items when submitting your revised manuscript:
- A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
- A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
- An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.
If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.
If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols
We look forward to receiving your revised manuscript.
Kind regards,
J Joe Hull, Ph.D.
Academic Editor
PLOS ONE
[Note: HTML markup is below. Please do not edit.]
Reviewers' comments:
Reviewer's Responses to Questions
Comments to the Author
1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.
Reviewer #1: (No Response)
**********
2. Is the manuscript technically sound, and do the data support the conclusions?
The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.
Reviewer #1: Yes
**********
3. Has the statistical analysis been performed appropriately and rigorously?
Reviewer #1: I Don't Know
**********
4. Have the authors made all data underlying the findings in their manuscript fully available?
The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.
Reviewer #1: Yes
**********
5. Is the manuscript presented in an intelligible fashion and written in standard English?
PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.
Reviewer #1: Yes
**********
6. Review Comments to the Author
Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)
Reviewer #1: 1)
From the authors information on the result of complementation test, I assume there is a possibility of rep-1 is an individual allele of re mutant with independent origin (though the coloration of F2 and F3 eggs may not be explained only by Bmre gene). However, this information is not provided in the revised text but only in the answers to reviewer and editor.
These are very important information and the original paper is not easily accessible to non-chinese researchers.
The genetic relationship between rep-1 and re (i.e. the results of complementation test), and also the mutation in Bmre gene in rep-1 mutant should be described and explained in the last paragraph of introduction section so that the general readers are given enough background information to evaluate the results of this study.
2)
Bm-re is not a transcription factor or a signal transducer, but a transporter. Since the authors stand on the basis that rep-1 is an allele of re and there is enough possility, they should discuss the link between the mutation in Bm-re gene in rep-1 mutant and the differential expression of genes between rep-1 and C1H, if they aim to say the differential expression of these genes are caused by Bm-re mutation.
However, there may be a possibility that differential gene expression detected in this study (i.e. Fig. 3) is caused by a locus other than Bm-re. Expression analysis between individuals obtained by several rounds of backcross may answer this.
3)
In the last sentence of introduction, the authors state that “The study aims to build a foundation for understanding the formation mechanism of rep-1 mutants and provides a theoretical basis for the study of the molecular mechanism of egg color formation.”
If we stand on the basis that rep-1 is an allele of re, the most simple explanation for color change in rep-1 is the lack of a pigment precursor due to the disruption of Bmre gene.
In the current version of the manuscript, the results of this study do not seem to add information on the cause of color change in the rep-1 mutant nor egg color formation.
4)
In the revised Figure 3, it is preferred that the name of the gene in addition to accession number is provided in the figure panel or in the figure legend.
5)
Photos of eggs are better included in the main figure than supplementary figure.
**********
7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.
If you choose “no”, your identity will remain anonymous but your review may still be made public.
Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.
Reviewer #1: No
[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]
While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at gro.solp@serugif. Please note that Supporting Information files do not need this step.
Author response to Decision Letter 1
3 Jun 2020
For more information, please check the "Response to Reviewers".
Attachment
Submitted filename: Response to Reviewers.docx
Decision Letter 2
10 Jun 2020
PONE-D-20-03586R2
Transcriptome analysis of the eggs of the silkworm pale red egg (rep-1) mutant at 36 hours after oviposition
PLOS ONE
Dear Dr. Zhao,
Thank you for considering the Reviewer comments during the revision process. Although the comments/suggestions have been sufficiently addressed, because PLOS ONE does not utilize a copy editor, I would ask that you consider the edits (see attached) I have made to the manuscript that address a number of copyediting issues and/or unclear word usage. As such, we feel that although the manuscript has merit it does not yet fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses my suggested edits and questions.
Please submit your revised manuscript by Jul 25 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at gro.solp@enosolp. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.
Please include the following items when submitting your revised manuscript:
- A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
- A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
- An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.
If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.
If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols
We look forward to receiving your revised manuscript.
Kind regards,
J Joe Hull, Ph.D.
Academic Editor
PLOS ONE
[Note: HTML markup is below. Please do not edit.]
[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]
While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at gro.solp@serugif. Please note that Supporting Information files do not need this step.
Attachment
Submitted filename: PONE-D-20-03586_R2-jh.pdf
Author response to Decision Letter 2
22 Jul 2020
Dear Editor and Reviewers:
Thank you for your valuable suggestions, we have answered the questions responses to each ponit raised by the academic editor and reviewers and made changes in the uploaded file ('Manuscript' and 'Revised Manuscript with Track Changes'). Please refer to the uploaded file for more details.
Attachment
Submitted filename: Response to Reviwers.doc
Decision Letter 3
23 Jul 2020
Transcriptome analysis of the eggs of the silkworm pale red egg (rep-1) mutant at 36 hours after oviposition
PONE-D-20-03586R3
Dear Dr. Zhao,
We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.
Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.
An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at gro.solp@gnillibrohtua.
If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact gro.solp@sserpeno.
Kind regards,
J Joe Hull, Ph.D.
Academic Editor
PLOS ONE
Additional Editor Comments (optional):
Reviewers' comments:
Acceptance letter
28 Jul 2020
PONE-D-20-03586R3
Transcriptome analysis of the eggs of the silkworm pale red egg (rep-1) mutant at 36 hours after oviposition
Dear Dr. Zhao:
I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.
If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact gro.solp@sserpeno.
If we can help with anything else, please email us at gro.solp@enosolp.
Thank you for submitting your work to PLOS ONE and supporting open access.
Kind regards,
PLOS ONE Editorial Office Staff
on behalf of
Dr. J Joe Hull
Academic Editor
PLOS ONE