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Transcriptome analysis of the molting gland (Y-

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Article in Comparative Biochemistry and Physiology Part D Genomics and Proteomics · December 2015
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 Comparative Biochemistry and Physiology, Part D 17 (2016) 26–40

Contents lists available at ScienceDirect

Comparative Biochemistry and Physiology, Part D

journal homepage: [Link]/locate/cbpd

Transcriptome analysis of the molting gland (Y-organ) from the

blackback land crab, Gecarcinus lateralis
Sunetra Das a, Natalie L. Pitts a, Megan R. Mudron a, David S. Durica b, Donald L. Mykles a,⁎
a
Department of Biology, Colorado State University, Fort Collins, CO 80523, USA
b
Department of Biology, University of Oklahoma, Norman, OK 73019, USA

a r t i c l e i n f o a b s t r a c t

Article history: In decapod crustaceans, arthropod steroid hormones or ecdysteroids regulate molting. These hormones are synthe-
Received 14 October 2015 sized and released from a pair of molting glands called the Y-organs (YO). Cyclic nucleotide, mTOR, and TGFβ/Smad
Received in revised form 19 November 2015 signaling pathways mediate molt cycle-dependent phase transitions in the YO. To further identify the genes in-
Accepted 29 November 2015
volved in the regulation of molting, a YO transcriptome was generated from three biological replicates of intermolt
Available online 2 December 2015
blackback land crab, Gecarcinus lateralis. Illumina sequencing of cDNA libraries generated 227,811,829 100-base
Keywords:
pair (bp) paired-end reads; following trimming, 90% of the reads were used for further analyses. The trimmed
Blast2GO reads were assembled de novo using Trinity software to generate 288,673 contigs with a mean length of 872 bp
Crustacea and a median length of 1842 bp. Redundancy among contig sequences was reduced by CD-HIT-EST, and the output
Decapoda constituted the baseline transcriptome database. Using Bowtie2, 92% to 93% of the reads were mapped back to the
Ecdysteroid transcriptome. Individual contigs were annotated using BLAST, HMMER, TMHMM, SignalP, and Trinotate, resulting
Gecarcinus in assignments of 20% of the contigs. Functional and pathway annotations were carried out via gene ontology (GO)
Molting and KEGG orthology (KO) analyses; 58% and 44% of the contigs with BLASTx hits were assigned to GO and KO terms,
mTOR
respectively. The gene expression profile was similar to a crayfish YO transcriptome database, and the relative abun-
Transforming growth factor beta
dance of each contig was highly correlated among the three G. lateralis replicates. Signal transduction pathway
Transcriptome
Y-organ orthologs were well represented, including those in the mTOR, TGFβ, cyclic nucleotide, MAP kinase, calcium,
Signal transduction VEGF, phosphatidylinositol, ErbB, Wnt, Hedgehog, Jak-STAT, and Notch pathways.
Molt-inhibiting hormone © 2015 Elsevier Inc. All rights reserved.

1. Introduction Medler, 2015), and limb regeneration (Skinner, 1985; Mykles, 2001;
Hopkins and Das, 2015).
Decapod crustaceans possess a rigid exoskeleton that must be shed The YO undergoes transitions in physiological properties at critical
periodically for organismal growth, a process called ecdysis or molting. stages of the molt cycle. During intermolt, the YO is kept in the basal
Molting is regulated by two endocrine organs: the X-organ/sinus gland state by pulsatile releases of MIH to maintain low hemolymph
(XO/SG) complex, located in the eyestalk ganglia, and a pair of molting ecdysteroid titers (Nakatsuji et al., 2009; Chung et al., 2010). The repres-
glands, or Y-organs (YOs), located in the cephalothorax (Skinner, 1985; sion of ecdysteroid synthesis via MIH signaling in the YO is mediated by
Hopkins, 2012). The interaction between molt-inhibiting hormone cyclic nucleotide second messengers (Covi et al., 2009). MIH binding
(MIH), an inhibitory neuropeptide produced by the XO/SG complex, to a putative G protein-coupled membrane receptor is hypothesized
and steroid molting hormones (ecdysteroids), produced by the YO, to initiate a cAMP-dependent triggering phase, which is followed by
drives the progression through the molt cycle (Webster et al., 2012; an NO/cGMP-dependent summation phase for prolonged inhibition of
Webster, 2015). Hemolymph ecdysteroid titers are low during the ecdysteroidogenesis between MIH pulses (Chang and Mykles, 2011;
intermolt stage, increase during the premolt stage, drop precipitously Covi et al., 2012; Webster, 2015). A reduction in MIH during intermolt
near the end of premolt, and remain at low levels during postmolt de-represses the YO. The activated YO hypertrophies to increase
(Chang and Mykles, 2011; Mykles, 2011). The increase in ecdysteroid ecdysteroid synthesis; the hemolymph ecdysteroid titer increases and
titers initiate and coordinate the physiological processes required the animal transitions to the premolt stage (Chang and Mykles, 2011).
for molting, such as degradation and reformation of the exoskeleton The duration of early premolt depends on environmental and physio-
(Skinner, 1985), claw muscle atrophy (Mykles, 1997; Mykles and logical conditions. Environmental stress, acting through the XO/SG com-
plex, can prolong the premolt period, as the YO remains sensitive to MIH
and a stress neuropeptide, crustacean hyperglycemic hormone (CHH)
⁎ Corresponding author. Tel.: +1 970 491 7616. (Chang and Mykles, 2011; Shrivastava and Princy, 2014). Autotomy of
E-mail address: [Link]@[Link] (D.L. Mykles). a limb regenerate suspends premolt for a few weeks, which allows

[Link]
1744-117X/© 2015 Elsevier Inc. All rights reserved.
 S. Das et al. / Comparative Biochemistry and Physiology, Part D 17 (2016) 26–40 27

time for a new regenerate to form and grow, and the animal molts with for the study of the endocrine regulation molting by neuropeptides
a complete set of walking legs (Mykles, 2001; Yu et al., 2002; Chang and and other factors (Chang and Mykles, 2011). A pipeline for the valida-
Mykles, 2011). A critical transition occurs at mid-premolt, when the tion, analysis, and functional assignment of contigs is described. Genes
animal becomes committed to molt. The YO increases ecdysteroid pro- encoding signal transduction pathways were characterized. As expect-
duction further and becomes insensitive to MIH and CHH (Chang and ed, the genes in the cyclic nucleotide, insulin/mTOR, and TGFβ/Smad
Mykles, 2011; Covi et al., 2012). The animal progresses through to ecdy- pathways were well represented in the annotated database. Genes in
sis without delay. If a limb regenerate is autotomized at this stage, there the MAP kinase, calcium, VEGF, phosphatidylinositol, ErbB, Wnt, Hedge-
is no regeneration and the animal molts without a full complement of hog, Jak-STAT, and Notch pathways were also expressed. The diversity
legs (Mykles, 2001; Yu et al., 2002). of signaling pathways raises the possibility that the YO can integrate en-
Two highly conserved signaling pathways mediate the molt cycle- docrine, paracrine, and autocrine signals in order to respond appropri-
dependent transitions of the YO. The activation of the YO in early pre- ately to environmental and physiological conditions that affect molting.
molt requires mechanistic Target of Rapamycin (mTOR)-dependent
protein synthesis. mTOR is a protein kinase that controls global transla- 2. Methods
tion of mRNA into protein in eukaryotic cells (Baretic and Williams,
2014). Its activity is controlled by a variety of signals that regulate ener- 2.1. Animals
gy allocation to protein synthesis, such as nutrients, cellular energy sta-
tus, growth factors, and stress (Shimobayashi and Hall, 2014; Albert and Adult male G. lateralis were collected in the Dominican Republic,
Hall, 2015; Cetrullo et al., 2015). Cycloheximide, an inhibitor of mRNA shipped to Colorado State University, and maintained as described
translation, and rapamycin, an mTOR inhibitor, repress YO ecdysteroid (Covi et al., 2010). YOs were dissected from the branchial chamber
secretion in vitro (Mattson and Spaziani, 1986, 1987; Abuhagr et al., side of the anterior branchiostegite region of the cephalothorax and
2014b). Moreover, increases in mRNA levels of mTOR signaling compo- stored in 300 μL RNAlater (Life Technologies, Grand Island, NY, USA)
nents Gl-mTOR and Gl-Akt as well as Gl-elongation factor 2 (Gl-EF2) in at −20 °C until processing. Hemolymph ecdysteroid titers were quanti-
mid-premolt and late premolt stages coincide with the increase in fied using a competitive enzyme-linked immunosorbent assay (ELISA)
ecdysteroid production in the committed YO (Abuhagr et al., 2014b). (Kingan, 1989; Abuhagr et al., 2014a).
The transition of the YO from the activated to the committed state in-
volves TGFβ/Smad signaling via an Activin-like membrane receptor 2.2. mRNA isolation, library preparation, and sequencing
(Chang and Mykles, 2011). An Activin receptor inhibitor (SB431542)
blocks YO commitment but has no effect on YO activation in eyestalk- Total RNA was isolated using RNeasy™ Mini Kits (Qiagen, Valencia,
ablated Gecarcinus lateralis in vivo (Abuhagr et al., 2012). Both signaling CA, USA) following the manufacturer's protocol. RNA was quantified
pathways are also important in insect molt regulation. mTOR activity using Quant-iT™ RiboGreen® RNA Assay (Life Technologies, Carlsbad,
controls the size and ecdysteroid synthetic capacity of the prothoracic CA, USA). The mRNA purification and cDNA synthesis was carried out
gland (PG) by prothoracicotropic hormone (PTTH), bombyxin, and with a TruSeq™ Stranded mRNA Library Prep Kit (Illumina). Three
insulin-like peptides (ILPs) (Teleman, 2010; Covi et al., 2012; cDNA libraries, designated Im1, Im2, and Im3, were generated; each li-
Yamanaka et al., 2013; Smith et al., 2014; Gu et al., 2015; Hatem et al., brary was derived from mRNA from six YOs pooled from three intermolt
2015). A recent report of an ortholog of Drosophila Ilp7 and eight animals. Paired-end sequencing of the cDNA libraries using an Illumina
insulin-like growth factor binding proteins in the rock lobster, HiSeq™ 2000 instrument was performed at the Oklahoma Medical
Sagmariasus verreauxi (Chandler et al., 2015), lends support for the im- Research Foundation. All samples were run in a single sequencing lane
portance of ILP/mTOR signaling in crustaceans and raises the possibility with three adaptor tags.
that the YO, like the insect PG, is regulated by insulin-like growth factors
(Yamanaka et al., 2013). Moreover, TGFβ signaling, which is mediated 2.3. Transcriptome assembly and annotation
by Smad transcription factors (Macias et al., 2015), is necessary for the
PG to respond to PTTH and insulin. Blocking Activin/Smad signaling in The quality of paired-end raw reads in fastq format was assessed
the Drosophila PG prevents the PTTH-triggered ecdysteroid peak that using the FASTQC program (Babraham Institute, Cambridge, UK).
initiates metamorphosis (Pentek et al., 2009; Gibbens et al., 2011). Quality reads with a minimum phred (nucleotide base call) score of 28
Taken together, the data suggest that mTOR and TGFβ/Smad pathways and length ranging from 36 bp to 100 bp were extracted by trimming
play essential roles in the regulation of crustacean and insect molting of low quality reads and adapter sequences via Trimmomatic software
glands by neuropeptides. (version number: 0.32) (Bolger et al., 2014). The trimmed reads obtained
RNA-Seq technology has quickly become a powerful tool in decapod from three different biological YO replicates were concatenated into two
crustacean physiology, as it provides a deeper and broader range of files containing forward and reverse sequences, respectively. Further,
transcripts than other methods (Wang et al., 2009). As the field lacks a both paired and unpaired reads were used for downstream analyses.
decapod species with a fully mapped and annotated genome, the de The trimmed forward and reverse reads were then assembled via Trinity
novo assembly of RNA-Seq data allows the cataloging of all the genes software with default settings (version number: r20130814) (Haas et al.,
expressed in a tissue, essentially leapfrogging the genome to the direct 2013). The minimum contig length was set at 201 bp. Following assem-
analysis of genes that define a specific function. This methodology can bly, the contigs were clustered based on a 90% sequence similarity
identify gene ontologies and networks associated with a physiological threshold using the CD-HIT-EST program (version number: 4.6.1) (Li
process, as well as quantify levels of mRNA abundance for all genes tran- and Godzik, 2006). The output of CD-HIT-EST was used as the reference
scriptionally activated within that physiological state. Transcriptomic transcriptome to map the reads from individual libraries. We designated
approaches have revealed genes associated with reproduction (Gao the assembled data as the YO baseline transcriptome.
et al., 2014), development (Wei et al., 2014a; Chandler et al., 2015; For annotation, both nucleotide sequences and predicted protein se-
Christiaens et al., 2015; Li et al., 2015), chitin metabolism in integumen- quences were used to run BLAST queries against NCBI non-redundant
tary tissues (Tom et al., 2014; Abehsera et al., 2015), digestion (Wei (NR), Swiss-Prot (SP), TrEMBL (Uniprot), and Uniprot Uniref90 protein
et al., 2014b), neuroendocrine regulation (Christie, 2014; Ventura databases ([Link] Fig. 1) (Altschul et al.,
et al., 2014), and molting and growth (Tom et al., 2013; Lv et al., 1990; Bairoch and Apweiler, 2000). The NR and SP databases
2014). In the present study, Illumina high-throughput sequencing and were downloaded on April 1, 2015, TrEMBL on October 19, 2015,
de novo assembly was used to create a YO transcriptome database of and Uniref90 on June 26, 2015. Stand-alone software was used for run-
the blackback land crab, G. lateralis. The species is an important model ning BLAST (version: 2.2.28) against the above-mentioned databases
28 S. Das et al. / Comparative Biochemistry and Physiology, Part D 17 (2016) 26–40

using the “Generic” GOSlim Set option to create a summary of the GO

Raw reads terms associated with annotated transcripts in the YO baseline tran-
Illumina HiSeq 2000 scriptome (McCarthy et al., 2006). For the purpose of pathway annota-
tion, we used the output from BLASTx against the NR database for
Paired-end sequencing
assignments to Kyoto Encyclopedia for Genes and Genomes (KEGG)
227,811,829 reads pathways and KEGG orthology (KO). MEtagenome Analyzer (MEGAN
V5.6.6) software (Kanehisa and Goto, 2000; Huson and Mitra, 2012)
was used for generating KEGG annotations. We used the auxiliary map-
ping files for KO and pathway assignments rather than the out-of-date
built-in KEGG pathways ([Link]
Trimmed reads megan5/download/[Link]). In order to compare the pathway
Trimmomatic annotations of the land crab baseline transcriptome to the previously
206,526,472 reads published YO transcriptome of crayfish, we downloaded the assembled
Pontastacus leptodactylus transcriptome (TSA: GAFS00000000 from
NCBI database (Tom et al., 2013). The crayfish transcriptome was proc-
essed in the same manner as the land crab data to generate comparable
KEGG pathway and KO annotations.
De novo assembly TransDecoder (version number: v2.0.1; [Link]
io/) was used to predict coding peptide sequences from the baseline tran-
Trinity scriptome contig sequences. These peptide sequences were annotated via
288,673 contigs BLASTp against known databases with a cutoff of 1e−5. In order to identify
conserved protein families among the predicted peptide sequences,
HMMER hmmscan ([Link]
trunk/documentation/man/[Link]) was used to search for se-
quences against a Pfam-A database (downloaded on June 26, 2015)
Sequence clustering ([Link] In addition, transmembrane helical domains
CD-HIT-EST and cleavage sites for signal peptides were identified using TMHMM (ver-
sion number: 2.0c) and SignalP (version number: 4.1) software, respec-
231,579 contigs tively. All the outputs obtained from the above-mentioned resources
(Baseline transcriptome) like BLAST, HMMER, TMHMM, and SignalP were used to achieve a com-
prehensive annotation for each contig. For this purpose we used Trinotate
(version number: r201407708) to generate a flat file report containing all
annotation information for each contig (Haas et al., 2013).

Read alignment to 2.4. Transcript abundance estimation and statistical analyses

baseline
The relative transcript abundance in the three cDNA libraries
Transcriptome was determined via the following steps. The good quality reads from
Bowtie2 all three intermolt replicates were mapped back to the YO baseline
transcriptome using Bowtie2 (version number 2.0.3) to generate a
SAM (Sequence Alignment/Map) file (Langmead and Salzberg, 2012).
SAMtools software (version number: 0.1.18) was used convert the
SAM files to BAM (binary form of SAM file) files (Li et al., 2009). These
Expression profile BAM files were used to quantify abundances of the mapped transcripts
of contigs for individual libraries using eXpress software (version number: 1.5.1)
(Roberts and Pachter, 2013). Expression levels are defined in fragments
eXpress per kilobase per million reads (FPKM). Further data manipulation and
statistical analyses (correlation coefficients) were performed using R
Fig. 1. De novo assembly pipeline for the G. lateralis YO transcriptome. The steps in the as- statistical software (R-Development-Core-Team, 2015).
sembly of the raw reads from the Illumina sequencing and for the estimation of transcript
abundance are illustrated. The software used for each step is indicated by italics; the num-
2.5. Computational resources
bers indicate the output from each step. See Section 2 for the versions of the software used.

The assembly of the baseline transcriptome was performed at

([Link] the Data-Intensive Academic Grid (DIAG) via Secure Shell (SSH)
TYPE=Download). The output of BLASTx against the NR database was ([Link] Additional computation jobs (trimming,
generated as an xml format (outfmt 5) and was parsed with a python mapping, annotation, and abundance estimation) were performed
script developed by the Palumbi laboratory at Stanford University at the Oklahoma State University High Performance Computing Center
([Link] (De Wit et al., 2012). The results using SSH ([Link]
from BLASTx against SP and Uniprot databases were in tabular format
(outfmt 6). The cutoff for e-value was set for 1e− 5 and 10 hits were 3. Results
assigned per contig.
The functional analysis of the transcriptome via gene ontology (GO) 3.1. Assembly and analysis of the baseline transcriptome
was generated via Blast2GO Basic (version number 3.0.8) using the
BLASTx against the NR database as input (Conesa et al., 2005). The GO The YO baseline biological replicates (designated Im1, Im2, and Im3)
numbers obtained from Blast2GO were entered into GOSlimViewer were selected from intermolt (stage C4) animals using standard molt
([Link] staging criteria (Covi et al., 2010). Each replicate consisted of YOs
 S. Das et al. / Comparative Biochemistry and Physiology, Part D 17 (2016) 26–40 29

Table 1 As there is no reference genome for G. lateralis, the fidelity of the as-
Comparison of the three biological replicates of Y-organs from intermolt (Im) G. lateralis sembly was assessed by comparing the sequences of contigs with those
(land crab) after trimming and mapping results back to each library.
of cDNAs obtained by Sanger sequencing. DNA and encoded peptide se-
Library Total raw reads from Quality reads RMBT following mapping quences were obtained from the NCBI nucleotide and protein databases,
Illumina following trimming via Bowtie respectively, for 13 cDNAs that were previously identified in G. lateralis
Im1 72,080,779 62,823,512 92.96% tissues by RT-PCR cloning. Nucleotide identities were 97% to 100%
Im2 81,221,231 74,977,808 91.70% between sequences obtained via Sanger and Illumina sequencing
Im3 74,509,819 68,725,152 91.80%
(Table 3). Over half of the 13 sequences had greater than 99% identity
in protein alignment. The small discrepancy of 1% to 3% between the nu-
cleotide and protein sequences was attributed to differences between
Table 2 the two technologies, the tissue sources used, novel isoforms, and se-
Statistics summary for land crab YO baseline transcriptome. quencing errors (Durica et al., 2014). These comparisons confirmed
Total number of reads 227,811,829 the accuracy of the sequence generation and assembly protocols, vali-
Total number of contigs 288,673
dating the use of this data for further analysis.
Mean length (bp) 871
Median length (N50) (bp) 1,842
Number of clusters (CD-HIT-EST output) 231,579 3.2. Transcriptome annotation
Range of contig lengths (bp) 201 to 28,392
Number of clusters with one contig 203,954
Number of clusters with N1 contigs 27,625 The baseline transcriptome was annotated using multiple methods
Number of predicted peptides (Transdecoder) 81,481 as shown in the annotation pipeline (Fig. 2). All contigs (231,597)
were annotated via a BLASTx search against the NCBI non-redundant
(NR), UniProtKB/Swiss-Prot (SP), and UniProtKB/TrEMBL (http://
from 3 animals. The mean hemolymph ecdysteroid titers were [Link]/downloads) databases. This resulted in 34,605,
0.91 ± 0.02 pg/μl for Im1, 1.32 ± 0.01 pg/μl for Im2, and 1.41 ± 23,504, and 25,723 significant hits, respectively, using 1e− 5 as the
0.02 pg/μl for Im3. Reads from Illumina sequencing were assembled e-value cutoff (Table 4). Each BLAST output generated top ten hits per
to generate the YO baseline transcriptome (Fig. 1). Table 1 compares annotated contig. About 20% of the contigs were identified. The relative-
the outcomes of the sequence analysis for each library. A total of ly low hit rate was attributed to the lack of an annotated decapod crus-
227,811,829 reads were generated. Following trimming, 90% of the tacean genome and the restriction of the analysis to protein-coding
reads were retained for further analysis. The general features of the base- genes, which constitute less than 1% of the total RNA.
line transcriptome library are summarized in Table 2. The good quality One problem associated with BLASTx analysis against the NR database
reads were assembled via the Trinity program (Haas et al., 2013). This was that the output for many sequences was “hypothetical protein” or
generated 288,673 contigs with a mean length of 871 bp and median “predicted protein.” In order to increase the identification of contigs
length (N50) of 1842 bp. The length distribution of the transcriptome in- with known genes, the BLASTx output was analyzed against the SP and
dicated that 40% of the contigs ranged between 201 and 299 bp in length TrEMBL databases (Fig. 2). Open reading frames (ORFs) were generated
and 30% of all contigs were greater than 500 bp (Appendix Fig. A1). By from the YO baseline transcriptome using TransDecoder. This resulted
comparison, 60% of the contigs that were returned with a BLASTx hit in 81,481 predicted peptides, of which 38,151 (47%) were complete (con-
were greater than 500 bp (Appendix Fig. A2). The median length of the taining both start and stop codons), 19,475 (24%) were five-prime partial
protein-coding sequences was greater (740 bp) than the median length (containing a start codon), 6933 (9%) were three-prime partial (ending
of the entire transcriptome (227 bp), suggesting the presence of small with a stop codon), and 16,922 (21%) were internal (lacking both the
non-coding RNAs and/or assembly artifacts. The distribution of contig start and stop codons). These ORFs were annotated via BLASTp against
length from this study is comparable to published de novo transcriptomes the NR, SP, and Uniprot-UniRef90 databases. These BLAST analyses result-
(Ghaffari et al., 2014; Lenz et al., 2014). ed in 24,850, 19,707, and 24,817 predicted peptides/ORFs with significant
In order to reduce the number of redundant sequences, the CD-HIT- hits, respectively (Fig. 2). In addition to the BLAST analyses, the ORF se-
EST program was used to cluster similar sequences at a threshold set at quences were analyzed to identify predicted Pfam domains (HMMER/
90% nucleotide similarity (Li and Godzik, 2006). The output from the Pfam), transmembrane helices (TMHMM), and signal peptide cleavage
CD-HIT-EST program (231,579 contigs) constituted the YO baseline sites (SignalP). All the outputs from BLASTx, BLASTp, HMMER/Pfam,
transcriptome. Using Bowtie, reads from individual libraries were TMHMM, and SignalP were combined into a single annotation file using
mapped back to the baseline transcriptome. Read alignment was similar Trinotate (Fig. 2). The result was 18,772 contigs with predicted Pfam do-
among the three replicates; about 92% to 93% of the reads mapped back mains, 20,706 contigs with predicted transmembrane helices (1 to 16
to the transcriptome (Table 1). transmembrane helices per contig), 5745 contigs with a signal peptide

Table 3
Comparison of alignments between sequences obtained from Sanger and Illumina sequencing.

Gene (GenBank no.) Contig identification Contig length (bp) ORF length (aa) % Protein identity % Protein positives % Nucleotide identity

Akt (HM989974.3) comp105797_c0_seq1 2638 510 100 100 99

EcR (AY642975.1) comp116905_c1_seq4 5899 550 100 100 99
EF2 (AY552550.1) comp101058_c2_seq1 3018 846 100 100 99
Mstn (EU432218.1) comp112193_c3_seq5 1871 497 100 100 99
Ras (HM989971.1) comp33885_c0_seq1 2044 182 99 100 99
Calpain-B (AY639153.1) comp111795_c1_seq91 4664 1015 99 99 99
E75 (DQ058409.2) comp62979_c0_seq1 3759 721 99 99 100
mTOR (HM989973.3) comp118786_c0_seq1 8306 2475 98 98 99
NO-insensitive GC III (DQ355438.1) comp119965_c0_seq2 2511 326 99 98 99
NOS (AY552549.1) comp119039_c0_seq1 4835 1211 98 98 99
β-actin (L76530.1) comp110624_c1_seq1 1186 332 98 98 97
Calpain-T (AY639154.1) comp112562_c0_seq3 2853 642 97 97 99
S6K (HM989975.3) comp95075_c0_seq1 2532 492 97 97 99
30 S. Das et al. / Comparative Biochemistry and Physiology, Part D 17 (2016) 26–40

Y-organ
Baseline transcriptome
231,579 contigs

BLASTx against known Protein coding regions (ORFs)

databases Transdecoder

Against Against Against

Predicted peptides
NR SP TrEMBL
81,481
34,605 23,504 25,723

Pathway annotation Functional

BLASTp against known Functional annotation
MEtaGenome annotation
databases Trinotate
ANalyzer (MEGAN) (Blast2GO)

KEGG pathway GO Against Against Against TMH Pfam SignalP

assignment assignments NR SP Uniref90 prediction domains Prediction
15,090 20,208 24,850 19,707 24,817 20,706 18,772 5,745

Fig. 2. Annotation pipeline for the G. lateralis YO transcriptome. Contigs were analyzed using either nucleotide (BLASTx) or peptide (Transdecoder and BLASTp) sequences to search against
non-redundant (NR), Swiss-Prot (SP), and TrEMBL or Uniref90 databases. The output from BLASTx against the NR database was used to obtain functional and pathway annotations with
Blast2GO for Gene Ontology (GO) and with MEGAN for KEGG pathway assignments, respectively. Trinotate was used to identify and aggregate protein sequences with transmembrane
helix (TMH), SignalP, and Pfam domains into a single comprehensive file. The numbers indicate the total hits/assignments from the analyses. See Section 2 for the versions of the software
used.

cleavage site, and 9566 contigs with GO annotation from Pfam-A hits category, the most represented GO Slims were ion binding
(Table 4). (GO:0043167) and oxidoreductase activity (GO:0016491) (Fig. 4A). The
most represented GO Slims were cell (GO:0005623) and intracellular
3.3. Functional annotation—Blast2GO (GO:0005622) in the CC category (Fig. 4B) and cellular nitrogen com-
pound metabolic process (GO:0034641) and biosynthetic process
Blast2GO was used to perform functional annotation for the YO (GO:0009058) in the BP category (Fig. 4C). Of the 20,208 GO assignments
baseline transcriptome via gene ontology (GO) (Ashburner et al., within the baseline transcriptome, 7272 were assigned to unique GO
2000; Götz et al., 2008). Of the 34,605 BLASTx hits, 20,208 unique terms; the distribution was 2086 (29%) in MF, 814 (11%) in CC, and
contigs were assigned to at least one of the three parent GO categories: 4372 (60%) in BP.
molecular function (MF), cellular component (CC), and biological pro-
cess (BP). A Venn diagram illustrates the distribution of unique contigs
annotated to one or combination of the three GO categories (Fig. 3). 3.4. Pathway annotation—KEGG and comparative analysis of
Many of the contigs were assigned to two or all three categories. For ex- YO transcriptomes
ample, there was considerable overlap of contigs assigned to the CC and
BP categories, with only 674 contigs assigned only to CC and 749 contigs The pathway annotation for the YO baseline transcriptome was con-
assigned only to BP. One-third (6718) of the contigs were assigned to all ducted via KEGG. KEGG orthology (KO) assignment and pathway anno-
three categories. The overall distribution was 17,841 contigs (51%) in tation was performed on the data from BLASTx hits (34,605 contigs)
MF, 9021 contigs (26%) in CC, and 14,266 contigs (41%) in BP. against the NR database; these are the same data used for the GO anal-
A second level GO analysis assigned the contigs to 41, 33, and 71 ysis (Fig. 2). The G. lateralis KO assignments were compared to those
GO Slims terms in the MF, CC, and BP categories, respectively (http:// from a YO transcriptome generated from the crayfish, P. leptodactylus
[Link]/page/go-slim-and-subset-guide). Within the MF (TSA: GAFS00000000) (Tom et al., 2013). The KO assignment analysis
resulted in annotation of 241 and 232 pathways in land crab and cray-
Table 4 fish, respectively. The total number of G. lateralis contig hits that were
Annotation summary for land crab YO baseline transcriptome. assigned to one or more of the five global KEGG pathways (metabolism
Annotation mode or tool Number of contigs with genetic, information processing, organismal systems, cellular processes,
hits or assignments and environmental information processing) was 15,090, of which 6222
BLASTx against NR 34,605
had no specific pathway assignments (Fig. 5A). By comparison, 6805
BLASTx against SP 23,504 crayfish BLASTx hits were assigned to KEGG pathways, of which 3099
BLASTx against Trembl-Uniprot 25,723 were unclassified (Fig. 5B). The unclassified contigs in both land crab
BLASTp against NR 24,850 and crayfish were assigned with a KO number. However, these contigs
BLASTp against SP 19,707
were not incorporated into any particular KEGG pathway. As the
BLASTp against Uniprot-Uniref90 24,817
Pfam domains 18,772 KEGG pathways are manually curated, it is possible that these contigs
TMHMM 20,706 are part of pathways that have not been assigned in the database.
SignalP 5,745 Although there were fewer numbers of assignments for crayfish com-
GO annotation from BLASTx against NR 20,208 pared to land crab, the percentage of BLASTx hits distributed among
KEGG annotation from BLASTx against NR 15,090
KEGG global pathways was similar in both species (Fig. 5).
 S. Das et al. / Comparative Biochemistry and Physiology, Part D 17 (2016) 26–40 31

Fig. 3. Venn diagram of contig numbers assigned to parent gene ontology (GO) terms. The Venn diagram indicates number of contigs annotated by at least one or two or a combination of
all three parent GO terms (molecular function, cellular component, and biological process). BLAST2GO was used for annotation and GOSlimViewer was used to summarize the hierarchical
GO terms associated with each contig. A total of 20,208 contigs were annotated with least one parent GO term. The diagram is drawn to scale.

The KEGG pathways are gene interaction pathways displayed |gb|ADO17754.1| short gastrulation protein [Parhyale hawaiensis];
in a hierarchical manner (Kanehisa et al., 2014). The G. lateralis and e-value = 0; score = 1817) was detected in the land crab tran-
P. leptodactylus data were further assigned to the KEGG second and scriptome database. Seven KEGG identifiers not assigned in the crayfish
third tier in the pathway hierarchy. In land crab, the metabolism global YO transcriptome were detected in the land crab YO transcriptome:
pathway constituted 32% of the KEGG assignments (Fig. 6A). The signal four in the mTOR (K03259, K07209, K04688, K07298) and three in
transduction pathway had the largest number of assignments of the the TGFβ ((K04661, K03347, K03456) signaling pathways (Fig. 7 and
second-tier pathways, followed by translation, carbohydrate metabolism, Tables A1 and B1). Additionally, the main components of the Notch,
and transport and catabolism pathways (Fig. 6A). The assignments of the Hedgehog, Wnt, and MAPK signaling pathways were identified by
crayfish contigs showed similar trends, with signal transduction and KEGG assignments (Table 5).
translation pathways ranked first and second, respectively (Fig. 6A).
Transport and catabolism pathways were ranked third, and folding, 3.5. Correlation of expression levels among replicates
sorting, and degradation pathway were ranked fourth in terms of number
contigs assigned to KO (Fig. 6A). The YOs used for the baseline trancriptomes were from intermolt
Contigs assigned to the signal transduction second-tier hierarchy (stage C4) animals. Although the libraries were from the same molt
were examined further. There are a total of 28 gene interaction path- stage, the validity of the biological replicates was evaluated by compar-
ways in the signal transduction third-tier hierarchy ([Link] ing the digital expression levels of contigs in the three libraries using
[Link]/kegg/[Link]). The 1079 contigs in signal transduc- both count and FPKM values. Count data are used for downstream dif-
tion were assigned to 16 signaling pathways; the 12 pathways with ferential gene expression analysis using Bioconductor packages, such
the largest number of contigs are shown in Fig. 6B for both G. lateralis as DESeq and EdgeR ([Link]
and P. leptodactylus. Pathway rankings based on number of KEGG bioc/) (Anders and Huber, 2010; Robinson et al., 2010). FPKM is a nor-
assignments were similar for both species; PI3K-Akt, calcium, MAPK, malized value of relative transcript abundance that corresponds to the
and Wnt signaling pathways were ranked first to fourth (Fig. 6B). The RNA concentration at the time of tissue collection. Over one-half
components of the mTOR and TGFβ pathways were well represented (122,485 or 53%) of all the contigs had an FPKM value of zero in at
in the transcriptome. Interaction maps for mTOR and TGFβ least one library, suggesting that either (1) there was no expression of
pathways included comparisons of the KO assigned in land crab that particular transcript or (2) the transcript sequence was an artifact
and crayfish (Fig. 7). All but one of the crayfish KO assignments of de novo assembly. Pearson coefficients were calculated in a pairwise
(K04657; chordin or short gastrulation) was present in land crab statistical analysis of count and FPKM values (Table 6). Both count and
(Fig. 7B, Tables A1 and B1). However, although a chordin/sog assign- FPKM values were positively correlated (P = 0.94–0.99), which indi-
ment was not observed in the KEGG analysis, an ortholog for chordin/ cates the three libraries can be used as biological replicates to represent
sog (contig: comp117728_c0_seq1; BLASTx output: gi|308197284 YO gene expression in intermolt animals.
32 S. Das et al. / Comparative Biochemistry and Physiology, Part D 17 (2016) 26–40

Tom et al., 2013, 2014; Christie, 2014; Groh et al., 2014; Ventura et al.,
2014; Abehsera et al., 2015). Using similar strategies, a de novo tran-
scriptome was assembled to catalog genes that are expressed in the
G. lateralis YO. An objective was to generate a comprehensive database
of genes that are well annotated and could serve as a reference tran-
scriptome for decapod crustaceans. The data built from Illumina technol-
ogy ensures fidelity during sequencing, as indicated by the high sequence
identities between the contigs and the same genes sequenced using
Sanger technology (Table 3). Although only 20% of the contigs from the
YO transcriptome were annotated, over 34 K protein-coding sequences
were identified. The number of annotated contigs in the G. lateralis YO
transcriptome is greater than other decapod crustacean transcriptomes
(Lv et al., 2014; Li et al., 2015; Suwansa-Ard et al., 2015). This indicates
that there is a need for using a standard method that incorporates all
available tools for annotating de novo-assembled transcriptomes in spe-
cies without genome sequence information. In addition to BLASTx and
BLASTp, HMMER, TMHMM, and SignalP were used to identify protein
family domains, transmembrane helices, and signal peptide sequences,
respectively, in the G. lateralis YO transcriptome. Trinotate software was
used to combine the analyses to generate a comprehensive annotation
profile for each contig. This combined method provided a detailed analy-
sis of the YO transcriptome that did not rely solely on BLAST.
Comparison of the land crab and crayfish YO KEGG pathway
annotations revealed that both transcriptomes have similar gene expres-
sion profiles. However, certain contigs were not annotated via KEGG, even
with the presence of a BLASTx hit assigned to the particular contig. These
discrepancies occurred in both crayfish and land crab transcriptomes and
can be overcome by pathway annotation comparison, followed by manu-
ally curating of the orthologs identified via BLAST into a specific pathway.
The high similarity of percentage of KO assignments across global
KEGG pathways between land crab and crayfish indicates that the YO
transcriptomes share a common expressed-gene profile. This further val-
idates the application of RNA-Seq technology, as well as the utilization of
proper annotation methods, for the identification of putative orthologs in
a non-model organism. Thus, the G. lateralis YO transcriptome database is
a useful resource to identify and catalog gene networks controlling YO
ecdysteroidogenesis during the molt cycle.
The activation of the molting gland, which is under the control of
neuropeptide hormones, drives the transition from intermolt to pre-
molt. In insects, the PG is activated by PTTH released from the brain.
In decapod crustaceans, the YO is activated by a decrease in MIH release
from the XO/SG complex. The signaling pathways regulating the
molting glands by neuropeptides differ between crustaceans and in-
sects, most likely necessitated by the opposite actions of MIH and
PTTH, respectively, on ecdysteroidogenesis. PTTH binding to Torso, a re-
ceptor tyrosine kinase, triggers an influx of Ca2+, resulting in the activa-
tion of cAMP and/or MAP kinase pathways, depending on species (Covi
et al., 2012; Yamanaka et al., 2013). By contrast, MIH signaling involves
a cAMP/Ca2+-dependent triggering phase and an NO/cGMP-dependent
summation phase linked by calmodulin (CaM) and calcineurin (CaN),
a CaM-dependent protein phosphatase (Chang and Mykles, 2011;
Covi et al., 2012; Webster, 2015). The activation of NO synthase
(NOS) through the combined actions of Ca2+/CaM binding and CaN-
dependent dephosphorylation increases the production of NO from L-
arginine. NO activates an NO-dependent guanylyl cyclase (GC-I), resulting
in a prolonged increase in cGMP that represses YO ecdysteroidogenesis by
Fig. 4. Number of YO transcriptome contigs distributed among second-tier GO terms. The
cGMP-dependent protein kinase (PKG) (Mykles et al., 2010; Chang and
pie charts show the distribution of second-tier GO terms associated with three parent Mykles, 2011; Webster, 2015). All the components of MIH signaling
terms: molecular function (A), cellular component (B), and biological process (C). The are present in the YO baseline transcriptome. Calcium signaling path-
legends rank the terms from the highest to lowest number of contigs in each second-tier way genes were present in the transcriptome (Fig. 6B). In addition to
GO categories.
cDNAs encoding NOS and GC-Iβ obtained by RT-PCR cloning (Kim
et al., 2004; Lee et al., 2007), transcripts encoding adenylyl cyclase, pro-
4. Discussion tein kinase A, CaM, CaN, and PKG were identified (Table 7). The MIH re-
ceptor has yet to be characterized (Webster, 2015). The annotated
High-throughput sequencing technologies has allowed “omics” stud- transcriptome can be used to identify MIH receptor candidates, as
ies to be more cost-effective for non-model organisms (Jung et al., 2011; well as characterize signaling pathway components, such as cyclic
 S. Das et al. / Comparative Biochemistry and Physiology, Part D 17 (2016) 26–40 33

Fig. 5. KEGG pathway analysis of land crab and crayfish YO transcriptomes. The figure depicts the distribution of total assignments for both land crab (A) and crayfish (B) BLASTx assigned
contigs among global KEGG pathways. Land crab had more contigs in each category, but the proportions of the five global KEGG pathways were similar between the two species, as
depicted by the percentage values.

Fig. 6. Comparison of land crab and crayfish contigs associated with selected second- and third-tier KEGG hierarchical pathways. (A) Second-tier KEGG pathways associated with Metab-
olism, Genetic Information Processing, Environmental Information Processing, and cellular processes. The two pathways with the highest number of BLASTx assigned contigs were signal
transduction and translation in the annotated transcriptomes of both species. (B) Third-tier KEGG pathways in the signal transduction category. The PI3K-Akt pathway had the highest
number of assigned contigs in both species, while the TGFβ, Jak-STAT, and Notch pathways had the least representation.
34 S. Das et al. / Comparative Biochemistry and Physiology, Part D 17 (2016) 26–40

Fig. 7. Identification genes assigned to the mTOR and TGFβ signaling pathways in land crab and crayfish transcriptomes. MEGAN was used to identify genes annotated via BLAST and
assigned to a KEGG pathway. The mTOR (A) and TGFβ (B) signaling pathway components/genes were represented in both species.
 S. Das et al. / Comparative Biochemistry and Physiology, Part D 17 (2016) 26–40 35

Table 5
Components of selected KEGG signal transduction pathways in the land crab YO transcriptome.

Signaling Pathway Total assigned contigs Selected genes represented in the KEGG pathway

Notch (KO04330) 53 Dvl (K02353); Notch (K02599); Fringe (K05948); Delta (K06051); RBPSUH (K06053);
γ-Secretase complex (K04505, K06170, K06171, K06172)
Hedgehog (KO04340) 59 Patched (K06225); Smoothened (K06226); Fused (K06228); Su(fu) (K06229); Ci (K16797)
Wnt (KO04310) 155 Wnt (K00182); APC (K02085); β-catenin (K02105); Axin (K02157); Dvl (K02353);
Frizzled (K02354); GSK (K03083); CK II (K03115); TF 7 (K04491)
MAPK (KO04011) 213 EGF (K04357); EGFR (K04361); GRB (K04364); MEK1 (K04368); ERK (K0437); MNK1/2 (K04372);
CREB (K04374); c-Myc (K04377); SRF (K04378); Ras (K07827); FGFR (K05093); SOS (K03099)

nucleotide phosphodiesterases (PDEs), that contribute to the changes mid-premolt (stage D1) (Chang and Mykles, 2011). Contigs encoding
in MIH sensitivity over the molt cycle (Nakatsuji and Sonobe, 2004; Activin/Smad signaling pathway genes are well represented in the
Nakatsuji et al., 2006). land crab and crayfish YO transcriptomes (Fig. 6B), including a
Hypertrophy of the activated molting gland increases ecdysteroid myostatin-like factor related to Activin (Table A2). SB431542, an antag-
synthetic capacity in premolt. Despite the diametrically opposite control onist of the Activin RII receptor, prevents the transition from the activat-
by neuropeptides between insects and crustaceans, both YO and PG ac- ed to the committed state in eyestalk-ablated animals (Abuhagr et al.,
tivation involve mTOR-dependent protein synthesis. Rapamycin in- 2012). Taken together, the data suggest that Activin regulates the sensi-
hibits YO ecdysteroidogenesis in vitro (Abuhagr et al., 2014b). Contigs tivity of the arthropod molting gland to neuropeptides. The differences
encoding mTOR signaling pathway genes are well represented in the between the two arthropod groups are consistent with their opposing
land crab and crayfish YO transcriptomes (Figs. 6B and 7A). mRNA levels actions. PTTH is a tropic factor, while MIH is a static factor. In insects,
of Gl-mTOR and Gl-Akt in the land crab YO are increased during premolt Activin/Smad signaling prevents precocious molting, as it assures that
(Abuhagr et al., 2014b). Genetic manipulation of mTOR signaling affects the PG is not activated by PTTH and ILPs until an animal reaches its crit-
PG ecdysteroidogenesis in Drosophila. The overexpression of PI3 kinase ical weight (Rewitz et al., 2013). In decapod crustaceans, Activin/Smad
stimulates PG growth, whereas the overexpression of Rheb-GTPase ac- signaling appears to be necessary for the transition of the YO to the
tivating protein inhibits PG growth (Colombani et al., 2005; Mirth committed state. The consequent insensitivity to inhibitory neuropep-
et al., 2005; Layalle et al., 2008). In Bombyx mori, inhibitors of PI3 kinase tides (MIH and CHH) and perhaps other factors assures that molting
and mTOR (rapamycin) block PTTH-stimulated PG ecdysteroid secre- processes, such as exoskeleton synthesis and limb regenerate growth,
tion (Gu et al., 2011, 2012), whereas in Manduca sexta, PI3 kinase inhib- proceed without interruption (Chang and Mykles, 2011; Covi et al.,
itors have no effect on PTTH-stimulated ecdysteroid secretion (Smith 2012). In summary, an Activin-like TGFβ factor drives critical transitions
et al., 2014). In Drosophila and both lepidopteran species, insulin and in the molting cycle. In insects, it makes the PG sensitive to tropic pep-
ILPs stimulate mTOR-mediated PG growth and ecdysteroidgenesis tides, while in decapods it makes the YO insensitive to static peptides.
(Walkiewicz and Stern, 2009; Teleman, 2010; Gu et al., 2011, 2015; Other signaling pathways represented in the transcriptome may
Smith et al., 2014; Hatem et al., 2015). These data show that mTOR sig- regulate YO function. The MAP kinase pathway, via ERK phosphoryla-
naling mediates the activation of the arthropod molting gland, in re- tion, may be involved in inhibition of YO ecdysteroid production during
sponse to either the release of tropic peptide hormones in insects postmolt in the crab, Scylla serrata (Imayavaramban et al., 2007). The ac-
(e.g., PTTH and ILP) or a reduction of inhibitory peptide hormones in tivation of protein kinase C, which is a target of the phosphatidylinositol
crustaceans (e.g., MIH and CHH). The large number of contigs assigned pathway, stimulates YO ecdysteroidogenesis in the rock crab, Cancer
to the PI3K-Akt signaling pathway (Fig. 6B) suggests that the YO may re- antennarius, but inhibits ecdysteroidogenesis in the crayfish, Orconectes
spond to growth factors as well. sp. (Mattson and Spaziani, 1987; Spaziani et al., 2001). Calcium signal-
The activation of TGFβ/Smad signaling is necessary for ecdysteroido- ing is complex, as it can interact with other pathways to control
genesis in molting glands, but its role differs between insects and crus- ecdysteroid synthesis and secretion. The manipulation of intracellular
taceans. In insects, TGFβ/Smad signaling renders the PG competent to Ca2+ concentration with pharmacological reagents yields inconsistent
respond to PTTH and ILPs (Rewitz et al., 2013). In Drosophila, loss of results, suggesting that sustained ecdysteroidogenesis requires Ca2 +
Activin signaling by knockdown of Type I and II receptors (Babo and concentrations within a limited range (Spaziani et al., 2001). YO
Punt, respectively), Co-Smad (Medea), or R-Smad (dSmad2) blocks ecdysteroid secretion increases with increasing extracellular Ca2+ con-
the PTTH-induced ecdysteroid peak that triggers metamorphosis centrations (Spaziani et al., 2001). Moreover, intracellular Ca2+ is corre-
(Gibbens et al., 2011). Conversely, the expression of Activin (Actb) or lated with YO ecdysteroidogenesis in the blue crab, Callinectes sapidus
constitutively active Babo causes precocious pupariation (Gibbens (Chen et al., 2012). It is thought that Ca2+ action is mediated by CaM-
et al., 2011). The knockdown of dSmad2 decreases the mRNA levels of dependent PDEs, thus countering the effects of MIH by keeping cyclic
PTTH receptor (Torso), insulin receptor, and Halloween genes, nucleotide levels low, and/or by activating PKC (Nakatsuji et al., 2009).
supporting the role of TGF-β/Smad signaling in PTTH-dependent stimu- Transcriptomics using RNA-Seq technology makes it possible to explore
lation of PG ecdysteroidogenesis (Gibbens et al., 2011). In crustaceans, the interactions between cyclic nucleotide and Ca2+ second messenger
TGFβ/Smad signaling appears to be necessary for YO commitment, pathways that control YO ecdysteroidogenesis.
which occurs at the transition from early premolt (stage D0) to the There are few studies of the Wnt, Hedgehog, and Notch signal trans-
duction pathways in the arthropod molting gland. Contigs encoding
Wnt, Hedgehog, and Notch signaling genes are reported in the land
Table 6
Pearson correlation coefficients between the biological replicates. crab and crayfish YO transcriptomes (Fig. 6B and Table 4), as well as in
the transcriptomes from other decapod tissues (Ventura et al., 2013;
Library Im2 Im3
Hao et al., 2014; Wei et al., 2014a; Song et al., 2015). To our knowledge,
A. Using Count data these pathways have not been studied in the YO. The insect PG
Im1 0.99 0.94 expresses Wnt and Hedgehog signaling genes, and genetic studies in
Im2 - 0.96
Drosophila suggest these pathways have a role in PG function. Hedgehog
B. Using FPKM data signaling inhibits ecdysteroid biosynthesis in the PG (Rodenfels et al.,
Im1 0.99 0.96 2014). Wnt/β-catenin signaling controls gene expression in the PG
Im2 - 0.95
via Wnt-dependent cis-regulatory modules (Archbold et al., 2014).
36 S. Das et al. / Comparative Biochemistry and Physiology, Part D 17 (2016) 26–40

Table 7
BLASTx hits for putative orthologs involved in MIH signaling.

Gene Contig identification Contig BLASTx hit (Subject) Subject length Score e-value
length (bp)

Calmodulin comp72270_c0_seq1 2838 gi|206597719|gb|ACI15835.1| calmodulin [Procambarus clarkii] 149 759 7.59E-91
Adenylyl cyclase comp113291_c0_seq5 9417 gi|398260007|emb|CCF77365.1| Rutabaga adenylyl cyclase [Calliphora vicina] 2087 202 7.38E-12
Adenylyl cyclase comp120618_c0_seq1 2132 gi|646714914|gb|KDR18711.1| Adenylate cyclase type 5, 910 1316 2.82E-165
partial [Zootermopsis nevadensis]
Protein kinase A comp117998_c0_seq3 3311 gi|91093068|ref|XP_968170.1| cAMP-dependent protein kinase catalytic subunit 353 1495 0
[Tribolium castaneum]
Protein kinase A comp120302_c0_seq5 3191 gi|702441760|gb|AIW09155.1| cAMP-activated protein kinase γ subunit, 369 1514 0
partial [Carcinus maenas]
Protein kinase G comp171371_c0_seq1 544 gi|255349294|gb|ACU09499.1| cGMP-dependent protein kinase [Spodoptera exigua] 744 675 2.22E-80
Calcineurin comp113416_c1_seq2 2183 gi|501291888|dbj|BAN20424.1| calcineurin β subunit [Riptortus pedestris] 189 770 1.26E-93
Calcineurin comp105509_c3_seq1 1920 gi|357614525|gb|EHJ69131.1| calcineurin B [Danaus plexippus] 293 772 2.94E-93

In mammals, Hippo, Wnt, and Notch signaling pathways activate DIAG Data-Intensive Academic Grid
mTORC1 (Shimobayashi and Hall, 2014). These data suggest that Dvl dishevelled (segment polarity protein)
mTOR-dependent ecdysteroidogenesis by the arthropod molting gland ELISA enzyme-linked immunosorbent assay
is controlled by multiple signals. EGF epidermal growth factor
In summary, a comprehensive YO transcriptome was assembled and EGFR epidermal growth factor receptor
annotated from deep sequencing with an Illumina platform. Analyses ERK extracellular regulated kinase
indicated high fidelity of de novo-assembled contigs with G. lateralis FGFR fibroblast growth factor receptor 2
cDNA sequences in GenBank. The database is available on iPlant and is FPKM fragments per kilobase of transcript per million fragments
intended to serve as a resource for the identification of gene networks mapped
controlling YO function. A significant finding was the similarity in the GO gene ontology
representation of numerous signal transduction pathways character- GRB growth factor receptor-binding protein 2
ized in the YO transcriptomes of land crab and crayfish (Fig. 6B). Signal GSK glycogen synthase kinase 2 beta
transduction genes constituted the largest second-tier hierarchical GC-Iβ guanylyl cyclase beta
category of KEGG assignments (Fig. 6A). The presence of genes in the HMMER biosequence analysis using profile hidden Markov models
mTOR, TGFβ, MAPK, calcium, and phosphatidylinositol signal transduc- KEGG Kyoto Encyclopedia of Gene and Genomes
tion pathways is consistent with physiological studies (Spaziani et al., KO KEGG orthology
2001; Covi et al., 2009; Nakatsuji et al., 2009; Chang and Mykles, ILP insulin-like peptides
2011; Webster et al., 2012). However, the discovery of genes in the Im1-3 YO baseline biological replicates
Wnt, VEGF, ErbB, Hedgehog, Jak-STAT, and Notch pathways raises the JAK-STAT Janus kinase-signal transducer and activator of transcription
possibility that YO regulation is more complex. As the YO initiates and MAPK mitogen-activated protein kinases
sustains molting processes, it is reasonable to hypothesize that the YO MAP mitogen-activated protein
is able to integrate a variety of extrinsic and intrinsic signals to effect MEGAN MEtaGenome Analyzer
an appropriate response. The decision to initiate molting is controlled MEK1 mitogen-activated protein kinase kinase I
by MIH, but molting can be interrupted or delayed by adverse condi- MF molecular function
tions in early premolt. The decision to complete molting is made MIH molt-inhibiting hormone
when the animal transitions to mid-premolt, at which point molting MNK1/2 MAP kinase interacting serine/threonine kinase
processes continue and the animal molts without delay. The transition mTOR mechanistic target of rapamycin
of the YO from the activated to committed state appears to require NCBI National Center for Biotechnology Information
Activin signaling. Taken together, the YO may be more like the insect NO nitric oxide
PG, which is controlled by a variety of tropic and static factors NOS nitric oxide synthase
(Marchal et al., 2010; Covi et al., 2012; Rewitz et al., 2013; Yamanaka NR non-redundant (protein database)
et al., 2013). mTOR and Activin are potential targets of these factors. ORF open reading frame
One goal is to use transcriptomic tools to identify genes that drive the PCR polymerase chain reaction
transitions in the physiological states of the YO through the molt cycle. PDE phosphodiesterase
Pfam protein family database
List of abbreviations PG prothoracic gland
PKC protein kinase C
APC adenomatous polyposis coli protein PKG protein kinase G
BAM binary form of a SAM file PTTH prothoracic hormone
BLAST Basic Local Alignment Search Tool RBPSUH recombining binding protein suppressor of hairless
BP biological process Rheb Ras homolog enriched in brain
CaM calmodulin RMBT reads mapped back to transcriptome
cAMP cyclic adenosine monophosphate RT-PCR reverse transcriptase polymerase chain reaction
CaN calcineurin SAM sequence alignment map
CC cellular component SHH secure shell
CD-HIT-EST cluster database at high identity with tolerance SOS Son of Sevenless
Ci cubitus interruptus SP SwissPort
CK II casein kinase II SRF serum response factor
cGMP cyclic gunaosine phosphate Su(fu) suppressor of fused
CHH crustacean hyperglycemic hormone TF 7 transcription factor 7
CREB cyclic AMP-dependent transcription factor ATF-4 TGFβ transforming growth factor beta
 S. Das et al. / Comparative Biochemistry and Physiology, Part D 17 (2016) 26–40 37

TMH transmembrane helices search Facility, for Illumina sequencing. We acknowledge the assistance
TMHMM transmembrane helices Markov model of Richard Casey and Joseph Allison, Colorado State University, for gen-
TSA transcriptome shotgun assembly erating scripts to assemble and annotate the RNA-Seq data and Jesse
Wnt wingless Schafer, Oklahoma State University High Performance Computing
XO/SG X-organ/sinus gland Center (National Science Foundation grant OCI–1126330), for technical
VEGF vascular endothelial growth factor support. We also thank undergraduate students Stephanie Oatman and
YO Y-organ Matthew Donovan for analyzing data from Illumina and Sanger se-
quencing and Clayton Hallman, Colorado State University, for his assis-
Acknowledgments tance with statistical analysis of the transcriptome data using R. We
thank the staff of Consejo Dominicano de Pesca y Acuicultura,
We thank Dr. Ernest S. Chang and Sharon A. Chang, UC Davis Bodega Dominican Republic, for expediting approval of permits and identifying
Marine Laboratory, for ELISA on hemolymph samples; Hector C. Horta suitable sites for collecting G. lateralis. This research was supported by
for collecting G. lateralis; and Graham Wiley, Oklahoma Medical Re- the National Science Foundation (IOS-1257732).

Appendix A

Figs. A1 and A2. Length distribution of assembled and annotated contigs. The number of contigs is plotted against the length distribution of the (A1) assembled contigs in the baseline
transcriptome (231,597) and (A2) annotated contigs via BLASTx against NR (34,605). The majority of the contigs (70%) ranged from 201 bp to 499 bp in Fig. A1; however, only 10% of
these contigs were annotated. Of the 30% (70,769) of all assembled contigs with length ≥500 bp, 29% (20,529) were assigned a BLASTx hit.
38 S. Das et al. / Comparative Biochemistry and Physiology, Part D 17 (2016) 26–40

Table A1
The genes and KO associated with mTOR pathway and their corresponding number of contigs in land crab and crayfish transcriptome.

mTOR signaling pathway Land crab Crayfish

K07207—tuberous sclerosis 2 8 1
K07198—5'-AMP-activated protein kinase, catalytic alpha subunit [EC:[Link]] 8 1
K03259—translation initiation factor 4E 6 0
K02991—small subunit ribosomal protein S6e 6 1
K00922—phosphatidylinositol-4,5-bisphosphate 3-kinase [EC:[Link]] 6 2
K16172—insulin receptor substrate 1 4 1
K07209—inhibitor of nuclear factor kappa-B kinase subunit beta [EC:[Link]] 3 0
K03258—translation initiation factor 4B 3 1
K04456—RAC serine/threonine-protein kinase [EC:[Link]] 3 2
K04688—p70 ribosomal S6 kinase [EC:[Link]] 2 0
K02649—phosphoinositide-3-kinase, regulatory subunit 2 1
K06276—3-phosphoinositide dependent protein kinase-1 [EC:[Link]] 2 1
K16185—Ras-related GTP-binding protein A/B 2 1
K16186—Ras-related GTP-binding protein C/D 2 1
K07206—tuberous sclerosis 1 2 2
K08269—unc51-like kinase [EC:[Link]] 2 3
K02677—classical protein kinase C [EC:[Link]] 2 3
K07298—serine/threonine-protein kinase 11 [EC:[Link]] 1 0
K01110—phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase and dual-specificity 1 1
protein phosphatase PTEN [EC:[Link] [Link] [Link]]
K07208—Ras homolog enriched in brain 1 1
K08266—G protein beta subunit-like 1 1
K07203—FKBP12-rapamycin complex-associated protein 1 1
K07204—regulatory associated protein of mTOR 1 1
K08267—rapamycin-insensitive companion of mTOR 1 1
K08268—hypoxia-inducible factor 1 alpha 1 1
K04371—extracellular signal-regulated kinase 1/2 [EC:[Link]] 1 1
K04373—p90 ribosomal S6 kinase [EC:[Link]] 1 1
K08271—protein kinase LYK5 1 1
K07205—eukaryotic translation initiation factor 4E binding protein 1 1 2
K08272—calcium binding protein 39 1 2

Table B1
The genes and KO associated with TGFβ pathway and their corresponding number of contigs in land crab and crayfish transcriptome.

TGF-beta signaling pathway Land crab Crayfish

K04513—Ras homolog gene family, member A 5 1

K03347—cullin 1 4 0
K03456—protein phosphatase 2 (formerly 2A), regulatory subunit A 4 0
K04679—MAD, mothers against decapentaplegic interacting protein 4 1
K04678—E3 ubiquitin ligase SMURF1/2 [EC:[Link]] 4 4
K04676—mothers against decapentaplegic homolog 1/5/8 3 1
K04382—protein phosphatase 2 (formerly 2A), catalytic subunit [EC:[Link]] 3 3
K04688—p70 ribosomal S6 kinase [EC:[Link]] 2 0
K04662—bone morphogenetic protein 2/4 2 1
K04675—activin receptor type-1 [EC:[Link]] 2 1
K04501—mothers against decapentaplegic homolog 4 2 1
K04681—retinoblastoma-like protein 1 2 1
K03094—S-phase kinase-associated protein 1 2 1
K04677—mothers against decapentaplegic homolog 6/7 2 2
K04377—Myc proto-oncogene protein 2 2
K04661—follistatin 1 0
K04659—thrombospondin 1 1
K04667—inhibin, beta 1 1
K04671—bone morphogenetic protein receptor type-2 [EC:[Link]] 1 1
K13578—bone morphogenetic protein receptor type-1B [EC:[Link]] 1 1
K04674—TGF-beta receptor type-1 [EC:[Link]] 1 1
K04500—mothers against decapentaplegic homolog 2/3 1 1
K04682—E2F transcription factor 4/5 1 1
K03868—RING-box protein 1 1 1
K04371—extracellular signal-regulated kinase 1/2 [EC:[Link]] 1 1
K04657—chordin 0 1

Appendix B. Availability of supporting data References

Abehsera, S., Glazer, L., Tynyakov, J., Plaschkes, I., Chalifa-Caspi, V., Khalaila, I., Aflalo, E.D.,
The assembled baseline transcriptome is available for researchers Sagi, A., 2015. Binary gene expression patterning of the molt cycle: the case of chitin
to download from the iPlant Collaborative™ ([Link] metabolism. Plos One 10, e0122602.
[Link]/). The public sharing link is: [Link] Abuhagr, A.M., Chang, E.S., Mykles, D.L., 2012. Role of mTOR and TGF beta in Y-organ
activation during the crustacean molting cycle. Integr. Comp. Biol. 52, E202.
org/dl/d/9B72D2F1-3F85-43FD-9AB1-B80CE84F1108/Gecarcinus_ Abuhagr, A.M., Blindert, J.L., Nimitkul, S., Zander, I.A., LaBere, S.M., Chang, S.A., MacLea,
lateralis_YO_Baseline_Transcriptome.[Link]. K.S., Chang, E.S., Mykles, D.L., 2014a. Molt regulation in green and red color morphs
 S. Das et al. / Comparative Biochemistry and Physiology, Part D 17 (2016) 26–40 39

of the crab Carcinus maenas: gene expression of molt-inhibiting hormone signaling Götz, S., Garcia-Gomez, J.M., Terol, J., Williams, T.D., Nagaraj, S.H., Nueda, M.J., Robles, M.,
components. J. Exp. Biol. 217, 796–808. Talon, M., Dopazo, J., Conesa, A., 2008. High-throughput functional annotation and
Abuhagr, A.M., MacLea, K.S., Chang, E.S., Mykles, D.L., 2014b. Mechanistic target of data mining with the Blast2GO suite. Nucleic Acids Res. 36, 3420–3435.
rapamycin (mTOR) signaling genes in decapod crustaceans: cloning and tissue ex- Groh, K.C., Vogel, H., Stensmyr, M.C., Grosse-Wilde, E., Hansson, B.S., 2014. The hermit
pression of mTOR, Akt, Rheb, and p70 S6 kinase in the green crab, Carcinus maenas, crab's nose-antennal transcriptomics. Front. Neurosci. 7.
and blackback land crab, Gecarcinus lateralis. Comp. Biochem. Physiol. 168A, 25–39. Gu, S.H., Young, S.C., Lin, J.L., Lin, P.L., 2011. Involvement of PI3K/Akt signaling in PTTH-
Albert, V., Hall, M.N., 2015. Reduced C/EBP beta-LIP translation improves metabolic stimulated ecdysteroidogenesis by prothoracic glands of the silkworm, Bombyx
health. EMBO Rep. 16, 881–882. mori. Insect Biochem. Mol. Biol. 41, 197–202.
Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment Gu, S.H., Yeh, W.L., Young, S.C., Lin, P.L., Li, S., 2012. TOR signaling is involved in PTTH-
search tool. J. Mol. Biol. 215, 403–410. stimulated ecdysteroidogenesis by prothoracic glands in the silkworm, Bombyx
Anders, S., Huber, W., 2010. Differential expression analysis for sequence count data. Ge- mori. Insect Biochem. Mol. Biol. 42, 296–303.
nome Biol. 11. Gu, S.-H., Chen, C.-H., Hsieh, Y.-C., Lin, P.-L., Young, S.-C., 2015. Modulatory effects of
Archbold, H.C., Broussard, C., Chang, M.V., Cadigan, K.M., 2014. Bipartite recognition of bombyxin on ecdysteroidogenesis in Bombyx mori prothoracic glands. J. Insect Phys-
DNA by TCF/pangolin is remarkably flexible and contributes to transcriptional re- iol. 72, 61–69.
sponsiveness and tissue specificity of wingless signaling. Plos Genet. 10. Haas, B.J., Papanicolaou, A., Yassour, M., Grabherr, M., Blood, P.D., Bowden, J., Couger, M.B.,
Ashburner, M., Ball, C.A., Blake, J.A., Botstein, D., Butler, H., Cherry, J.M., Davis, A.P., Eccles, D., Li, B., Lieber, M., MacManes, M.D., Ott, M., Orvis, J., Pochet, N., Strozzi, F.,
Dolinski, K., Dwight, S.S., Eppig, J.T., Harris, M.A., Hill, D.P., Issel-Tarver, L., Kasarskis, Weeks, N., Westerman, R., William, T., Dewey, C.N., Henschel, R., Leduc, R.D.,
A., Lewis, S., Matese, J.C., Richardson, J.E., Ringwald, M., Rubin, G.M., Sherlock, G., Friedman, N., Regev, A., 2013. De novo transcript sequence reconstruction from
Gene Ontology, C., 2000. Gene Ontology: tool for the unification of biology. Nat. RNA-seq using the Trinity platform for reference generation and analysis. Nat. Protoc.
Genet. 25, 25–29. 8, 1494–1512.
Bairoch, A., Apweiler, R., 2000. The SWISS-PROT protein sequence database and its sup- Hao, T., Zeng, Z., Wang, B., Zhang, Y., Liu, Y., Geng, X., Sun, J., 2014. The protein-protein in-
plement TrEMBL in 2000. Nucleic Acids Res. 28, 45–48. teraction network of eyestalk, Y-organ, and hepatopancreas in Chinese mitten crab
Baretic, D., Williams, R.L., 2014. The structural basis for mTOR function. Semin. Cell Dev. Eriocheir sinensis. BMC Syst. Biol. 8.
Biol. 36, 91–101. Hatem, N.E., Wang, Z., Nave, K.B., Koyama, T., Suzuki, Y., 2015. The role of juvenile hor-
Bolger, A., Scossa, F., Bolger, M.E., Lanz, C., Maumus, F., Tohge, T., Quesneville, H., Alseekh, mone and insulin/TOR signaling in the growth of Manduca sexta. BMC Biol. 13.
S., Sorensen, I., Lichtenstein, G., Fich, E.A., Conte, M., Keller, H., Schneeberger, K., Hopkins, P.M., 2012. The eyes have it: a brief history of crustacean neuroendocrinology.
Schwacke, R., Ofner, I., Vrebalov, J., Xu, Y., Osorio, S., Aflitos, S.A., Schijlen, E., Gen. Comp. Endocrinol. 175, 357–366.
Jimenez-Gomez, J.M., Ryngajllo, M., Kimura, S., Kumar, R., Koenig, D., Headland, L.R., Hopkins, P.M., Das, S., 2015. Regeneration in crustaceans. In: Chang, E.S., Thiel, M. (Eds.), The
Maloof, J.N., Sinha, N., van Ham, R.C.H.J., Lankhorst, R.K., Mao, L., Vogel, A., Arsova, Biology of Crustacea: Physiology. Oxford University Press, Oxford, U.K., pp. 168–198.
B., Panstruga, R., Fei, Z., Rose, J.K.C., Zamir, D., Carrari, F.F., Giovannoni, J.J., Weigel, Huson, D.H., Mitra, S., 2012. Introduction to the analysis of enviromental sequences:
D., Usadel, B., Fernie, A.R., 2014. The genome of the stress-tolerant wild tomato spe- metagenomics with MEGAN. Methods Mol. Biol. 856, 415–429.
cies Solanum pennellii. Nat. Genet. 46, 1034–1038. Imayavaramban, L., Dhayaparan, D., Devaraj, H., 2007. Molecular mechanism of molt-
Cetrullo, S., D'Adamo, S., Tantini, B., Borzi, R.M., Flamigni, F., 2015. mTOR, AMPK, and Sirt1: inhibiting hormone (MIH) induced suppression of ecdysteroidogenesis in the Y-
key players in metabolic stress management. Crit. Rev. Eukaryot. Gene Expr. 25, 59–75. organ of mud crab: Scylla serrata. FEBS Lett. 581, 5167–5172.
Chandler, J.C., Aizen, J., Elizur, A., Hollander-Cohen, L., Battaglene, S.C., Ventura, T., 2015. Jung, H., Lyons, R.E., Dinh, H., Hurwood, D.A., McWilliam, S., Mather, P.B., 2011. Tran-
Discovery of a novel insulin-like peptide and insulin binding proteins in the Eastern scriptomics of a giant freshwater prawn (macrobrachium rosenbergii): de novo assem-
rock lobster Sagmariasus verreauxi. Gen. Comp. Endocrinol. 215, 76–87. bly, annotation and marker discovery. Plos One 6.
Chang, E.S., Mykles, D.L., 2011. Regulation of crustacean molting: a review and our per- Kanehisa, M., Goto, S., 2000. KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic
spectives. Gen. Comp. Endocrinol. 172, 323–330. Acids Res. 28, 27–30.
Chen, H.-Y., Dillaman, R.M., Roer, R.D., Watson, R.D., 2012. Stage-specific changes in calci- Kanehisa, M., Goto, S., Sato, Y., Kawashima, M., Furumichi, M., Tanabe, M., 2014. Data, in-
um concentration in crustacean (Callinectes sapidus) Y-organs during a natural formation, knowledge and principle: back to metabolism in KEGG. Nucleic Acids Res.
molting cycle, and their relation to the hemolymphatic ecdysteroid titer. Comp. 42, D199–D205.
Biochem. Physiol. 163A, 170–173. Kim, H.W., Batista, L.A., Hoppes, J.L., Lee, K.J., Mykles, D.L., 2004. A crustacean nitric oxide
Christiaens, O., Delbare, D., Van Neste, C., Cappelle, K., Yu, N., De Wilde, R., Van synthase expressed in nerve ganglia, Y-organ, gill and gonad of the tropical land crab,
Nieuwerburgh, F., Deforce, D., Cooreman, K., Smagghe, G., 2015. Differential tran- Gecarcinus lateralis. J. Exp. Biol. 207, 2845–2857.
scriptome analysis of the common shrimp Crangon crangon: special focus on the nu- Kingan, T.G., 1989. A competitive enzyme-linked immunosorbent assay: application in
clear receptors and RNAi-related genes. Gen. Comp. Endocrinol. 212, 163–177. the assays of peptides, steroids, and cyclic nucleotides. Anal. Biochem. 183, 283–289.
Christie, A.E., 2014. In silico characterization of the peptidome of the sea louse Caligus Langmead, B., Salzberg, S.L., 2012. Fast gapped-read alignment with Bowtie 2. Nat.
rogercresseyi (Crustacea, Copepoda). Gen. Comp. Endocrinol. 204, 248–260. Methods 9, 357–359.
Chung, J.S., Zmora, N., Katayama, H., Tsutsui, N., 2010. Crustacean hyperglycemic hormone Layalle, S., Arquier, N., Leopold, P., 2008. The TOR pathway couples nutrition and develop-
(CHH) neuropeptides family: functions, titer, and binding to target tissues. Gen. mental timing in Drosophila. Dev. Cell 15, 568–577.
Comp. Endocrinol. 166, 447–454. Lee, K.J., Kirn, H.-W., Gomez, A.M., Chang, E.S., Covi, J.A., MykleS, D.L., 2007. Molt-
Colombani, J., Bianchini, L., Layalle, S., Pondeville, E., Dauphin-Villemant, C., Antoniewski, inhibiting hormone from the tropical land crab, Gecarcinus lateralis: cloning, tissue
C., Carre, C., Noselli, S., Leopold, P., 2005. Antagonistic actions of ecdysone and insulins expression, and expression of biologically active recombinant peptide in yeast. Gen.
determine final size in Drosophila. Science 310, 667–670. Comp. Endocrinol. 150, 505–513.
Conesa, A., Götz, S., Garcia-Gomez, J.M., Terol, J., Talon, M., Robles, M., 2005. Blast2GO: a Lenz, P.H., Roncalli, V., Hassett, R.P., Wu, L.-S., Cieslak, M.C., Hartline, D.K., Christie, A.E.,
universal tool for annotation, visualization and analysis in functional genomics re- 2014. De novo assembly of a transcriptome for Calanus finmarchicus (Crustacea,
search. Bioinformatics 21, 3674–3676. copepoda): the dominant zooplankter of the north Atlantic ocean. Plos One 9.
Covi, J.A., Chang, E.S., Mykles, D.L., 2009. Conserved role of cyclic nucleotides in the regu- Li, W., Godzik, A., 2006. Cd-hit: a fast program for clustering and comparing large sets of
lation of ecdysteroidogenesis by the crustacean molting gland. Comp. Biochem. Phys- protein or nucleotide sequences. Bioinformatics 22, 1658–1659.
iol. 152A, 470–477. Li, H., Handsaker, B., Wysoker, A., Fennell, T., Ruan, J., Homer, N., Marth, G., Abecasis, G.,
Covi, J.A., Bader, B.D., Chang, E.S., Mykles, D.L., 2010. Molt cycle regulation of protein syn- Durbin, R., Genome Project Data, P., 2009. The Sequence Alignment/Map format
thesis in skeletal muscle of the blackback land crab, Gecarcinus lateralis, and the dif- and SAMtools. Bioinformatics 25, 2078–2079.
ferential expression of a myostatin-like factor during atrophy induced by molting Li, Y., Hui, M., Cui, Z., Liu, Y., Song, C., Shi, G., 2015. Comparative transcriptomic analysis
or unweighting. J. Exp. Biol. 213, 172–183. provides insights into the molecular basis of the metamorphosis and nutrition me-
Covi, J.A., Chang, E.S., Mykles, D.L., 2012. Neuropeptide signaling mechanisms in crusta- tabolism change from zoeae to megalopae in Eriocheir sinensis. Comp. Biochem. Phys-
cean and insect molting glands. Invertebr. Reprod. Dev. 56, 33–49. iol. 13D, 1–9.
De Wit, P., Pespeni, M.H., Ladner, J.T., Barshis, D.J., Seneca, F., Jaris, H., Therkildsen, N.O., Lv, J., Liu, P., Gao, B., Wang, Y., Wang, Z., Chen, P., Li, J., 2014. Transcriptome analysis of the
Morikawa, M., Palumbi, S.R., 2012. The simple fool's guide to population genomics Portunus trituberculatus: de novo assembly, growth-related gene identification and
via RNA-Seq: an introduction to high-throughput sequencing data analysis. Mol. marker discovery. Plos One 9.
Ecol. Resour. 12, 1058–1067. Macias, M.J., Martin-Malpartida, P., Massague, J., 2015. Structural determinants of Smad
Durica, D.S., Das, S., Najar, F., Roe, B., Phillips, B., Kappalli, S., Anilkumar, G., 2014. Alterna- function in TGF-beta signaling. Trends Biochem. Sci. 40, 296–308.
tive splicing in the fiddler crab cognate ecdysteroid receptor: variation in receptor Marchal, E., Vandersmissen, H.P., Badisco, L., Van de Velde, S., Verlinden, H., Iga, M., Van
isoform expression and DNA binding properties in response to hormone. Gen. Wielendaele, P., Huybrechts, R., Simonet, G., Smagghe, G., Vanden Broeck, J., 2010.
Comp. Endocrinol. 206, 80–95. Control of ecdysteroidogenesis in prothoracic glands of insects: a review. Peptides
Gao, J., Wang, X., Zou, Z., Jia, X., Wang, Y., Zhang, Z., 2014. Transcriptome analysis of the 31, 506–519.
differences in gene expression between testis and ovary in green mud crab (Scylla Mattson, M.P., Spaziani, E., 1986. Regulation of crab Y-organ steroidogenesis in vitro:
paramamosain). BMC Genomics 15. evidence that ecdysteroid production increases through activation of cAMP-
Ghaffari, N., Sanchez-Flores, A., Doan, R., Garcia-Orozco, K.D., Chen, P.L., Ochoa-Leyva, A., phosphodiesterase by calcium-calmodulin. Mol. Cell. Endocrinol. 48, 135–151.
Lopez-Zavala, A.A., Salvador Carrasco, J., Hong, C., Brieba, L.G., Rudino-Pinera, E., Blood, Mattson, M.P., Spaziani, E., 1987. Demonstration of protein kinase C activity in crustacean
P.D., Sawyer, J.E., Johnson, C.D., Dindot, S.V., Sotelo-Mundo, R.R., Criscitiello, M.F., 2014. Y-organs, and partial definition of its role in regulation of ecdysteroidogenesis. Mol.
Novel transcriptome assembly and improved annotation of the whiteleg shrimp Cell. Endocrinol. 49, 159–171.
(Litopenaeus vannamei), a dominant crustacean in global seafood mariculture. Sci. Rep. 4. McCarthy, F.M., Wang, N., Magee, G.B., Nanduri, B., Lawrence, M.L., Camon, E.B.,
Gibbens, Y.Y., Warren, J.T., Gilbert, L.I., O'Connor, M.B., 2011. Neuroendocrine regulation of Barrell, D.G., Hill, D.P., Dolan, M.E., Williams, W.P., Luthe, D.S., Bridges, S.M.,
Drosophila metamorphosis requires TGF beta/Activin signaling. Development 138, Burgess, S.C., 2006. AgBase: a functional genomics resource for agriculture. BMC
2693–2703. Genomics 7.
40 S. Das et al. / Comparative Biochemistry and Physiology, Part D 17 (2016) 26–40

Mirth, C., Truman, J.W., Riddiford, L.M., 2005. The role of the prothoracic gland in deter- Song, C., Cui, Z., Hui, M., Liu, Y., Li, Y., Li, X., 2015. Comparative transcriptomic analysis pro-
mining critical weight to metamorphosis in Drosophila melanogaster. Curr. Biol. 15, vides insights into the molecular basis of brachyurization and adaptation to benthic
1796–1807. lifestyle in Eriocheir sinensis. Gene 558, 88–98.
Mykles, D.L., 1997. Crustacean muscle plasticity: molecular mechanisms determining Spaziani, E., Jegla, T.C., Wang, W.L., Booth, J.A., Connolly, S.M., Conrad, C.C., Dewall, M.J.,
mass and contractile properties. Comp. Biochem. Physiol. 117B, 367–378. Sarno, C.M., Stone, D.K., Montgomery, R., 2001. Further studies on signaling pathways
Mykles, D.L., 2001. Interactions between limb regeneration and molting in decapod crus- for ecdysteroidogenesis in crustacean Y-organs. Am. Zool. 41, 418–429.
taceans. Am. Zool. 41, 399–406. Suwansa-Ard, S., Thongbuakaew, T., Wang, T., Zhao, M., Elizur, A., Hanna, P.J., Sretarugsa,
Mykles, D.L., 2011. Ecdysteroid metabolism in crustaceans. J. Steroid Biochem. Mol. Biol. P., Cummins, S.F., Sobhon, P., 2015. In silico neuropeptidome of female Macrobrachium
127, 196–203. rosenbergii based on transcriptome and peptide mining of eyestalk, central nervous
Mykles, D.L., Medler, S., 2015. Skeletal muscle differentiation, growth, and plasticity. In: system, and ovary. Plos One 10.
Chang, E.S., Thiel, M. (Eds.), The Natural History of Crustacea: Physiology. Oxford Uni- Teleman, A.A., 2010. Molecular mechanisms of metabolic regulation by insulin in Dro-
versity Press, Oxford, U.K., pp. 134–167. sophila. Biochem. J. 425, 13–26.
Mykles, D.L., Adams, M.E., Gade, G., Lange, A.B., Marco, H.G., Orchard, I., 2010. Neuropep- Tom, M., Manfrin, C., Giulianini, P.G., Pallavicini, A., 2013. Crustacean oxi-reductases pro-
tide action in insects and crustaceans. Physiol. Biochem. Zool. 83, 836–846. tein sequences derived from a functional genomic project potentially involved in
Nakatsuji, T., Sonobe, H., 2004. Regulation of ecdysteroid secretion from the Y-organ by ecdysteroid hormones metabolism: a starting point for function examination. Gen.
molt-inhibiting hormone in the American crayfish, Procambarus clarkii. Gen. Comp. Comp. Endocrinol. 194, 71–80.
Endocrinol. 135, 358–364. Tom, M., Manfrin, C., Chung, S.J., Sagi, A., Gerdol, M., De Moro, G., Pallavicini, A., Giulianini,
Nakatsuji, T., Sonobe, H., Watson, R.D., 2006. Molt-inhibiting hormone-mediated P.G., 2014. Expression of cytoskeletal and molt-related genes is temporally scheduled
regulation of ecdysteroid synthesis in Y-organs of the crayfish (Procambarus clarkii): in the hypodermis of the crayfish Procambarus clarkii during premolt. J. Exp. Biol. 217,
Involvement of cyclic GMP and cyclic neucleotide phosphodiesterase. Mol. Cell. 4193–4202.
Endocrinol. 253, 76–82. Ventura, T., Manor, R., Aflalo, E.D., Chalifa-Caspi, V., Weil, S., Sharabi, O., Sagi, A., 2013.
Nakatsuji, T., Lee, C.Y., Watson, R.D., 2009. Crustacean molt-inhibiting hormone: structure, Post-embryonic transcriptomes of the prawn Macrobrachium rosenbergii: multigenic
function, and cellular mode of action. Comp. Biochem. Physiol. 152A, 139–148. succession through metamorphosis. Plos One 8.
Pentek, J., Parker, L., Wu, A., Arora, K., 2009. Follistatin preferentially antagonizes activin Ventura, T., Cummins, S.F., Fitzgibbon, Q., Battaglene, S., Elizur, A., 2014. Analysis of the
rather than BMP signaling in Drosophila. Genesis 47, 261–273. central nervous system transcriptome of the eastern rock lobster Sagmariasus
R-Development-Core-Team, 2015. A language and environment for statistical computing. verreauxi reveals its putative neuropeptidome. Plos One 9.
R Foundation for Statistical Computing, Vienna, Austria. Walkiewicz, M.A., Stern, M., 2009. Increased insulin/insulin growth factor signaling
Rewitz, K.F., Yamanaka, N., O'Connor, M.B., 2013. Developmental checkpoints and feed- advances the onset of metamorphosis in Drosophila. Plos One 4.
back circuits time insect maturation. In: Shi, Y.B. (Ed.), Animal, Metamorphosis, Wang, Z., Gerstein, M., Snyder, M., 2009. RNA-Seq: a revolutionary tool for transcripto-
pp. 1–33. mics. Nat. Rev. Genet. 10, 57–63.
Roberts, A., Pachter, L., 2013. Streaming fragment assignment for real-time analysis of Webster, S.G., 2015. Endocrinology of molting. In: Chang, E.S., Thiel, M. (Eds.), The Natural
sequencing experiments. Nat. Methods 10, 71–73. History of Crustacea: Physiology. Oxford University Press, Oxford, U.K., pp. 1–35.
Robinson, M.D., McCarthy, D.J., Smyth, G.K., 2010. edgeR: a bioconductor package for dif- Webster, S.G., Keller, R., Dircksen, H., 2012. The CHH-superfamily of multifunctional
ferential expression analysis of digital gene expression data. Bioinformatics 26, peptide hormones controlling crustacean metabolism, osmoregulation, moulting,
139–140. and reproduction. Gen. Comp. Endocrinol. 175, 217–233.
Rodenfels, J., Lavrynenko, O., Ayciriex, S., Sampaio, J.L., Carvalho, M., Shevchenko, A., Wei, J., Zhang, X., Yu, Y., Huang, H., Li, F., Xiang, J., 2014a. Comparative transcriptomic
Eaton, S., 2014. Production of systemically circulating Hedgehog by the intestine cou- characterization of the early development in pacific white shrimp Litopenaeus
ples nutrition to growth and development. Genes Dev. 28, 2636–2651. vannamei. Plos One 9.
Shimobayashi, M., Hall, M.N., 2014. Making new contacts: the mTOR network in metabo- Wei, J., Zhang, X., Yu, Y., Li, F., Xiang, J., 2014b. RNA-Seq reveals the dynamic and diverse
lism and signalling crosstalk. Nat. Rev. Mol. Cell Biol. 15, 155–162. features of digestive enzymes during early development of Pacific white shrimp
Shrivastava, S., Princy, S.A., 2014. Pharmacophore based approach to design inhibitors Litopenaeus vannamei. Comp. Biochem. Physiol. 11D, 37–44.
in Crustaceans: an insight into the molt inhibition response to the receptor guanylyl Yamanaka, N., Rewitz, K.F., O'Connor, M.B., 2013. Ecdysone control of developmental tran-
cyclase. Indian J. Exp. Biol. 52, 375–382. sitions: lessons from Drosophila research. Annu. Rev. Entomol. 58, 497–516.
Skinner, D.M., 1985. Molting and regeneration. In: Bliss, D.E., Mantel, L.H. (Eds.), The Biol- Yu, X.L., Chang, E.S., Mykles, D.L., 2002. Characterization of limb autotomy factor-
ogy of Crustacea. Academic Press, New York, pp. 44–146. proecdysis (LAFpro), isolated from limb regenerates, that suspends molting in the
Smith, W.A., Lamattina, A., Collins, M., 2014. Insulin signaling pathways in lepidopteran land crab Gecarcinus lateralis. Biol. Bull. 202, 204–212.
ecdysone secretion. Front. Physiol. 5.

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