Hydrobiologia https://doi.org/10.1007/s10750-018-3521-3

CRUSTACEAN GENOMICS

Evaluation of genes involved in Norway () female sexual maturation using transcriptomic analysis

Guiomar Rotllant . Tuan Viet Nguyen . David Hurwood . Valerio Sbragaglia . Tomer Ventura . Joan B. Company . Silvia Joly . Abigail Elizur . Peter B. Mather

Received: 6 October 2017 / Revised: 17 January 2018 / Accepted: 20 January 2018 Ó Springer International Publishing AG, part of Springer Nature 2018

Abstract The Norway lobster Nephrops norvegicus technology applied to multiple tissues. Ovarian mat- is the most important commercial uration-related differential expression patterns were in Europe. Recent decline in wild captures and a observed for 4362 transcripts in ovary, hepatopan- reduction in total abundance and size at first matura- creas, eyestalk, brain, and thoracic ganglia in N. tion indicate that the species is overexploited. Increas- norvegicus. Transcripts detected in the study include ing knowledge of its reproduction, specifically at the vitellogenin, crustacean hyperglycaemic hormone, molecular level will be mandatory to improving retinoid X receptor, heat shock protein 90 and proteins fisheries management. The current study investigated encoding lipid and carbohydrate metabolizing differences between immature and mature N. norvegi- enzymes. From the study, data were collected that cus females using Next Generation Sequencing can prove valuable in developing more comprehensive knowledge of the reproductive system in this lobster species during the ovarian maturation process. Addi- Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10750-018-3521-3) con- tional studies will be required, however, to identify tains supplementary material, which is available to authorized potential novel genes and to develop a molecular users. toolkit for crustacean species that can be applied to improving sustainable future production. Guiomar Rotllant and Tuan Viet Nguyen have contributed equally to this work. Keywords Oocyte Á Fecundity Á Size at maturity Á Guest editors: Guiomar Rotllant, Ferran Palero, Peter Mather, Reproduction Á Fisheries Á Next generation sequencing Heather Bracken-Grissom & Begon˜a Santos / Crustacean Genomics

G. Rotllant (&) Á J. B. Company Á S. Joly D. Hurwood Institut de Cie`ncies del Mar (ICM-CSIC), PasseigMarı´tim Earth, Environmental and Biological Sciences, Science de la Barceloneta 37-49, 08003 Barcelona, Spain and Engineering Faculty, Queensland University of e-mail: [email protected] Technology, 2 George St, Brisbane 4001,

T. V. Nguyen (&) Á T. Ventura Á A. Elizur P. B. Mather Faculty of Science, Health, Education and Engineering, Australian Rivers Institute, Griffith University, Nathan, GeneCology Research Centre, University of the Sunshine QLD 4111, Australia Coast, 4 Locked Bag, Maroochydore DC, QLD 4558, Australia e-mail: [email protected]

123 Hydrobiologia

Introduction once but with immature ovaries) and had a CL of more than 24 mm (Rotllant et al., 2005) indicating a clear The Norway lobster Nephrops norvegicus (Linnaeus, reduction in size at first maturity (the study did not 1758) (Crustacea: : Pleocyemata) has a wide calculate size at first maturity). distribution in European waters, including the conti- In light of N. norvegicus reproductive biology, a nental shelf and slope (4–754 m) in the Northeast number of studies have been conducted on morphol- Atlantic Ocean as far south as the Canary Islands and ogy of the reproductive system (Rotllant et al., Mediterranean Sea (Johnson et al., 2013). This con- 2005, 2012) and biochemical changes in the ovary stitutes one of the most important fisheries in Euro- (Rosa & Nunes, 2002). These insights included pean waters while being the most valuable harvested characterization of vitellogenin activity (Vg; Rotllant crustacean in the region. This species is closely et al., 2017) and gonad inhibiting hormone (GIH; monitored by the International Council for the Explo- Edomi et al., 2002) and other crustacean hyper- ration of the Sea (ICES) under the Working Group on glycemic hormones (CHH; Mettulio et al., 2004). In Nephrops Surveys (http://www.ices.dk/community/ , oocyte development includes a series of groups/Pages/WGNEPS.aspx). Reproduction takes complex cellular events, and different genes are place either biannually (Iceland) or annually involved that control developmental stage and storage (Mediterranean Sea). During this period, females carry of proteins for embryogenesis (Subramoniam, 2011). eggs on their abdomen after fertilization from sperm Recently, genes that have a potent role in reproduction deposited previously by a male during copulation. and development have been identified in a number of Sperm is stored in the female’s thelycum until crustaceans notably in , , , ` spawning takes place (Sarda, 1995). In the Catalan crayfishes, and (Wong et al., 2008; Wu & Chu, region (Western Mediterranean Sea), oocyte matura- 2008; Wu et al., 2009; Zeng et al., 2011; Brady et al., tion commences in spring, a single spawning event 2012, 2013; Jiang et al., 2014; Wang et al., 2014; occurs in summer and females carry eggs on their Rotllant et al., 2015; Saetan et al., 2016). As an abdomen until eggs hatch in the following early spring example, genes that were differentially expressed (Rotllant et al., 2005). Prior to this, ovaries remain between immature and mature females have been immature while females carry eggs. A steady decline identified in the ovary of black tiger shrimp in the N. norvegicus population has been reported in monodon Fabricius, 1798 (Brady et al., 2013) and the Catalan region over 20 years (27% decline) even banana shrimp Fenneropenaeus merguiensis (de Man, though total trawler activity has diminished 28% and 1888) (Saetan et al., 2016), hepatopancreas of the cod-end mesh size has been increased from 36–38 to shrimp Metapenaeus ensis (De Haan, 1844) (Wong ` 40 mm (Sarda, 1998). Additionally, mean size of et al., 2008) and the swimming Norway lobster individuals has decreased from 4 mm trituberculatus (Miers, 1876) (Wang et al., 2014), carapace length (CL) in males to 3.6 mm CL in cephalothorax and eyestalks of P. monodon (Brady females (a difference of approximately 25%). This et al., 2012) and thoracic ganglia in the mud crab corresponds to a reduction of 1–2 years in age of Estampador, 1950 (Zeng et al., captured individuals. Thus, current exploitation is 2011). Notably transcripts that encode vitellogenin directed at younger individuals closer to size at first (the major egg yolk protein), vitellogenin receptor reproduction event and will remove a substantial (VgR), lipid and carbohydrate metabolism related ` proportion of the spawning stock. Sarda (1991) proteins, C-type lectins (CTLs), and hemocyanins determined size at which 50% of individuals reach have also been identified. their first maturity for females at 30–31 mm CL. Ten In the current study, we employed RNA-sequenc- years later however, a study conducted in the same ing technology to investigate genes involved in sexual area resulted in no females being captured in stage II maturation in N. norvegicus. To our knowledge, this is (adult females that have already reproduced at least the first study that compares multi-tissue transcrip- tomes using Next Generation Sequencing (NGS) from V. Sbragaglia immature and mature females in decapods to under- Department of Biology and Ecology of Fishes, Leibniz- Institute of Freshwater Ecology and Inland Fisheries, stand the molecular mechanism controlling ovarian Mu¨ggelseedamm, 310, Berlin, Germany development. Knowledge of mechanisms governing 123 Hydrobiologia ovarian development processes at the molecular level Table 2 De novo assembly statistics of the N. norvegicus will be valuable not only to expand the molecular reference transcriptome (Rotllant et al., 2017) toolkit available for N. norvegicus in the near future, Number of contig 333,225 but can also serve as reference information for other Total size of transcript (nt) 235,992,830 related crustacean species. N50 (bp) 1272 Longest transcripts (bp) 33,925 Mean transcript size (bp) 708 Materials and methods

Reference transcriptome Bioinformatics The current study used a reference transcriptome generated previously (Rotllant et al., 2017). RNA-seq For downstream analysis, quality-trimmed reads from statistics are summarized in Tables 1 and 2. In brief, all immature and mature female tissue samples were female N. norvegicus were collected offshore from mapped back to the previously generated reference Barcelona harbor (Spain). In the laboratory, large transcriptome assembly using Bowtie (Parra et al., females (CL = 34.3 ± 2.1 mm; total wet 2007). Reads counting was generated using RSEM (Li weight = 26.0 ± 5.0 mg) were selected and classi- & Dewey, 2011). Fragments per kilobase of transcript fied following their maturation stage (Rotllant et al., per million mapped reads value (FPKM) were 2005) as immature [stage II; Gonado-somatic index recorded. Following this, EdgeR software (Ye et al., (GSI) = 1.02 ± 0.17] and mature (stage IV; 2006) was used to measure false discovery rate of GSI = 1.91 ± 0.18). No difference in hepato-somatic multiple-hypothesis testing. Designated threshold for indexes (HIS) between immature and mature females any genes to be confirmed as significantly different was identified (HIS = 4.27 ± 0.68). Tissues (ovaries, was |log(FC)| [ 2, P value \ 0.001, FDR hepatopancreas, eyestalks, brain, and thoracic ganglia) value \ 0.001. Volcano and MA plots were generated from these individuals were dissected and conserved using EdgeR. Heatmaps were illustrated using the in RNAlater (Ambion). Total RNA was then extracted analyse_diff_expr.pl script included in Trinity de novo using a TriZOL-based modified protocol (Rotllant assembler (Grabherr et al., 2011).Venn diagram et al., 2017). cDNA libraries were synthetized accord- illustrating similar differentially expressed genes ing to the manufacturer’s recommendation and (DEG) among multiple tissues tested was created sequencing libraries generated using an Illumina using the webserver which is available online at http:// NextSeq 500. A brief summary of the RNA data is bioinformatics.psb.ugent.be/webtools/Venn/. Based reported in Tables 1 and 2. on the highest fold change and previous knowledge on

Table 1 Raw reads and quality control of reads for N. norvegicus female libraries (MF mature female, IF immature female) Tissue Sex Number of raw reads Raw read length (bp) % GC Number of reads after trimming % reads retained

Ovary MF 56,859,898 151 43 53,448,056 94.00 IF 52,854,378 151 48 49,655,513 93.95 Hepatopancreas MF 54,715,141 151 46 50,574,033 92.43 IF 52,217,561 151 46 48,752,848 93.37 Eyestalk MF 45,799,203 151 42 42,121,851 91.97 IF 53,668,850 151 42 49,410,048 92.07 Brain MF 67,530,741 151 42 62,997,606 93.29 IF 61,357,339 151 42 57,343,088 93.46 Thoracic ganglia MF 58,418,219 151 43 54,460,212 93.23 IF 49,303,270 151 42 45,710,029 92.71

123 Hydrobiologia genes related to oocyte maturation in decapods, other TG). In total, 1891 up-regulated transcripts (i.e., crustaceans or up to 36 genes were iden- higher expression in immature females) were observed tified and further analysis was conducted on them as versus 2471 down-regulated transcripts (i.e., higher per below. expression in mature females). Clusters of transcripts Protein domains were identified using InterProScan with similar expression patterns were grouped (Jones et al., 2014) and Smart Domain (Letunic et al., together in the heatmap analysis (illustrated in 2015). For illustration purposes, cDNA sequences Fig. 2). The heatmap showed that there are groups of were converted to amino acids using the Expasy transcripts that are very highly expressed (either up- or translate tool available online (http://web.expasy.org/ down-regulated) in the hepatopancreas and the ovary, translate/), open reading frames (ORFs) were then and nearly a 90% of these transcripts could not be chosen accordingly. Schematic diagrams of protein annotated. Moreover, clustering analysis revealed that structures were illustrated using the Illustrator for immature female TG was more similar to the imma- biological sequences (IBS) suite (Liu et al., 2015). For ture and mature brain. phylogenetic tree construction, data mining was done predominantly on the NCBI TSA (Transcriptome Candidate genes that were differentially expressed Shotgun Assembly) and NR (non-redundant) data- between immature and mature female N. base. A detailed list of all sequences used in the current norvegicus analysis can be found in supplementary file S1. In brief, FASTA files were imported into MEGA 7.0 From the characterized list of all differentially (Kumar et al., 2016) for phylogenetic analysis. Mul- expressed transcripts, we selected 36 transcripts tiple alignment of sequences were conducted using the manually based on previous literature as well as from MUSCLE alignment plugin implemented in MEGA the analyzed GO database expressed in a single or (Edgar, 2004). For all phylogenetic analyses, a several tissues (Table 3). When subjected to the Neighbor-Joining-based approach was conducted with homology search in the GenBank database using 1000 bootstraps. Evolutionary distances were com- BLASTP, the deduced amino acid sequence of these puted using the JTT matrix-based method (Jones et al., transcripts mainly shared high identity with decapods 1992). Phylogenetic trees were exported as Newick or other crustacean species (Table 4). Transcripts format and imported into iTOL webserver for illus- were annotated in 23 genes. Among these genes, five trative purposes (Letunic & Bork, 2016). played roles in oocyte development: vitellogenin (Vg), vitelline membrane outer layer protein 1 (VMOL), sex-determining protein fem-1 (FEM), piwi-like pro- Results tein 2-like (PIWI), and ccr4-not transcription complex (CCR4). Vitellogenin was highly expressed in both the Differential gene expression analysis ovary and brain of immature females when compared with mature females. Molecular phylogenetic tree Volcano plots as well as MA plots were generated and analysis using amino acid sequence (Fig. 3) showed can be found in supplementary material S2. In that Vg in N. norvegicus was most closely related to summary, differential gene expression analysis Homarus americanus H. Milne Edwards, 1837, and resulted in 967(384/583), 939 (328/611), 876 (379/ secondly to quadricarinatus (von Martens, 497), 861 (477/384), and 719 (323/396) transcripts in 1868), confirming the systematic classification of the the thoracic ganglia (TG), hepatopancreas, ovary, species; the three species belong to the infraorder eyestalks, and brain, respectively (illustrated in Astacidea and N. norvegicus and H. americanus Fig. 1). A Venn diagram that illustrates DEG overlap belong to the family Nephropoidea. At a more distant among tissues can be found in supplementary material level, they were related to crab, , and shrimp S3. While a higher number of differentially expressed sequences. Highest expression of PIWI and CCR4 was transcripts were detected between immature and also observed in the ovaries of immature females. The mature females in the TG, the number of transcripts expression of CCR4 was also higher in the hepatopan- exclusively expressed in a single tissue was higher in creas of immature females but lower in their TG. the hepatopancreas (710 in hepatopancreas vs. 617 in Higher expression of VMOL transcripts was also 123 Hydrobiologia

Fig. 1 Number of differentially expressed genes between Nephrops norvegicus tissues. Orange up-regulated, Gray down-regulated, Br brain, EY eyestalk, TG thoracic ganglia, Hep hepatopancreas, Ov ovary

Fig. 2 Heatmap of all differential gene expression based on the current threshold among five tested tissues in Nephrops norvegicus. The color legend shows the log2 FPKM values each represent. Hierarchical clustering of genes and samples was shown in the dendrogram on the top and side of the heatmap. Br brain, EY eyestalk, FI female immature, FM female mature, Hep hepatopancreas, OV ovary, TG thoracic ganglia

detected in mature female ovaries and for FEM including signal peptide (SP), a precursor sequence transcripts and in the hepatopancreas. (CPRP) and putative cleavage sites in the mature We found two transcripts related to regulation of peptide (CHH-2) (Fig. 4). In addition, E75 expression oocyte maturation, CHH and ecdysone inducible was up-regulated in the hepatopancreas. protein 75 (E75). Differential CHH transcript expres- In relation to lipid metabolism, we manually sion was down-regulated in the brain and TG. curated ten transcripts that were differentially Molecular characterization of N. norvegicus CHH expressed and later annotated to four proteins: showed the typical structure of a neuropeptide, apolipoprotein (APO), Niemann-pick c1 or Neverland

123 Hydrobiologia

Table 3 Selected candidate genes that are differentially expressed during oogenesis in Nephrops norvegicus Gene name Abbr. Contig Tissue |Log ± P-value Length GO annotation FC|

Sex-determining protein FEM c205271_g1_i1 Ov 9.47 ? 5.26E-09 3371 F:molecular_function; fem-1 C:cytoplasm; P:biological_process; C:cellular_component Piwi-like protein 2-like PIWI c201464_g1_i3 Ov 9.47 - 1.19E-08 3253 F:nucleic acid binding Vitellogenin Vg c208407_g1_i1 Ov 4.15 - 1.28E-06 7866 P:lipid transport; F:lipid transporter activity; P:oogenesis Br 11.71 - 2.80E-15 Apolipoprotein d-like APO c197835_g2_i2 Br 8.97 ± 8.64E-08 1112 P:lipid transport; F:lipid TG 8.73 2.97E-07 transporter activity; P:cytoskeletal anchoring at plasma membrane; C:focal adhesion; C:actin cytoskeleton; F:structural constituent of cytoskeleton; C:ruffle; P:cell adhesion; F:structural molecule activity; C:cytoskeleton; F:lipid binding; P:Wnt signaling pathway; C:extracellular region; P:transport Vitelline membrane outer VMOL c201770_g1_i1 Hep 8.72 ? 5.43E-07 663 P:vitelline membrane layer protein 1 formation Serine threonine-protein STPK c204932_g1_i4 EY 7.97 ? 2.25E-05 4314 P:protein phosphorylation; kinase 3-like F:ATP binding; P:signal transduction; F:protein serine/threonine kinase activity Ccr4-not transcription CCR4 c160408_g1_i1 Ov 9.71 - 1.59E-09 4000 P:regulation of transcription complex subunit 6-like from RNA polymerase II promoter; P:nuclear- transcribed mRNA poly(A) tail shortening; C:CCR4-NOT complex; F:protein binding; P:oogenesis; C:cytoplasm Ccr4-not transcription c171766_g1_i1 Hep 9.65 - 1.77E-08 6260 F:metal ion binding complex Ccr4-not transcription c171766_g1_i2 TG 11.87 ? 1.00E-15 6380 F:metal ion binding complex Dorsal–ventral patterning SOG c193736_g1_i1 Ov 9.38 ± 1.19E-08 10071 P:transmembrane receptor protein sog-like Hep 10.11 1.29E-10 protein serine/threonine kinase signaling pathway; P:regulation of transcription from RNA polymerase II promoter; P:organ development; P:regionalization; P:axis specification; P:embryo development

123 Hydrobiologia

Table 3 continued Gene name Abbr. Contig Tissue |Log ± P-value Length GO annotation FC|

Histone h3 HMT c207512_g1_i8 Ov 8.34 - 3.54E-06 4883 P:histone lysine methylation; methyltransferase F:metalloendopeptidase activity; F:histone lysine N-methyltransferase activity; C:extracellular region; P:proteolysis; F:zinc ion binding Swisnf-related matrix- SWI/ c154266_g1_i2 Ov 9.22 - 2.87E-08 4429 F:organic cyclic compound associated actin- SNF binding; F:heterocyclic dependent regulator of compound binding chromatin subfamily a containing dead h box 1-like Heat shock protein HSP90 c205460_g1_i3 Ov 10.80 - 1.52E-12 7632 P:protein folding; P:response Hep 10.73 - 2.28E-11 to oxidative stress; F:ATPase activity; C:lipid particle; F:ATP binding; F:unfolded protein binding; C:mitochondrion Heat shock protein 70 kDa HSP70 c205739_g1_i3 Ov 9.55 ? 3.26E-09 2318 F:ATP binding; P:response to stress c205739_g1_i4 Hep 5.84 ? 3.50E-09 2447 F:ATP binding; P:response to stress Tubulin alpha-1 chain TUBA c203790_g2_i8 Ov 9.00 ? 8.64E-08 2779 C:tubulin complex; P:mitotic spindle organization; F:GTP binding; P:GTP catabolic process; C:astral microtubule; P:protein polymerization; C:centrosome; P:antimicrobial humoral response; P:mitotic spindle assembly checkpoint; F:GTPase activity; F:structural constituent of cytoskeleton c300918_g1_i1 TG 8.25 – 4.28E-06 331 P:microtubule cytoskeleton organization; F:protein binding; P:cellular response to interleukin-4; P:cytoskeleton-dependent intracellular transport; F:GTP binding; P:cell division; P:GTP catabolic process; C:cytoplasmic microtubule; P:protein polymerization; F:GTPase activity; P:’de novo’ posttranslational protein folding; F:structural constituent of cytoskeleton

123 Hydrobiologia

Table 3 continued Gene name Abbr. Contig Tissue |Log ± P-value Length GO annotation FC|

Elav-like protein 2-like ELAV c195365_g2_i7 Ov 8.51 – 1.72E-06 3384 F:nucleotide binding; F:RNA binding c195365_g2_i10 EY 8.78 ? 3.45E-07 3433 F:nucleotide binding; F:RNA binding C-type lectin 2 CTL c208513_g3_i4 Hep 10.91 – 7.05E-12 3940 F:metal ion binding; P:neurogenesis C-type lectin domain c207034_g3_i1 EY 10.24 ? 5.56E-11 2070 F:carbohydrate binding family 4 member m-like Neverland NVD c207639_g1_i11 Hep 10.82 ? 1.33E-12 2630 C:integral component of membrane; C:membrane; F:hedgehog receptor activity Retinoid X receptor RXR c208891_g1_i4 Hep 8.63 – 6.35E-07 3084 P:steroid hormone mediated signaling pathway; F:zinc ion binding; P:regulation of transcription, DNA- templated; F:sequence- specific DNA binding; F:sequence-specific DNA binding transcription factor activity; F:steroid hormone receptor activity; C:nucleus c208891_g1_i1 Br 8.98 – 8.64E-08 3102 P:steroid hormone mediated signaling pathway; F:zinc ion binding; P:regulation of transcription, DNA- templated; F:sequence- specific DNA binding; F:sequence-specific DNA binding transcription factor activity; F:steroid hormone receptor activity; C:nucleus Cytochrome p450 cyp44 CYP c194604_g1_i2 Hep 3.76 – 2.87E-06 F:ion binding; F:catalytic activity Probable cytochrome p450 c201287_g1_i2 TG 8.34 - 2.94E-06 F:metal ion binding; 49a1-like Br 8.03 - 1.17E-05 P:oxidation–reduction process; F:oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen; F:heme binding; F:oxidoreductase activity; F:electron carrier activity; F:iron ion binding; F:monooxygenase activity; F:ecdysone 20-monooxygenase activity; F:cholesterol monooxygenase (side-chain- cleaving) activity

123 Hydrobiologia

Table 3 continued Gene name Abbr. Contig Tissue |Log ± P-value Length GO annotation FC|

Ecdysone inducible E75 c191847_g1_i4 Hep 3.53 ? 2.21E-06 3292 P:steroid hormone mediated protein 75 signaling pathway;F:zinc ion binding; P:regulation of transcription, DNA- templated; F:thyroid hormone receptor activity;F:sequence-specific DNA binding; F:steroid hormone receptor activity; C:nucleus Hyperglycemic hormone CHH c203843_g2_i2 Br 5.59 - 7.37E-12 1758 P:glucose metabolic process; peptide 2 precursor TG 13.34 - 4.52E-20 C:extracellular region; P:neuropeptide signaling pathway; F:neuropeptide hormone activity Chitin synthase CS c202979_g1_i3 EY 8.28 ? 5.19E-06 5751 P:terminal branching, open tracheal system; P:trachea morphogenesis; C:integral component of membrane; P:cuticle chitin biosynthetic process; P:Malpighian tubule morphogenesis; P:response to wounding; P:embryonic epithelial tube formation; P:regulation of tube diameter, open tracheal system; P:regulation of tube length, open tracheal system; P:chitin-based embryonic cuticle biosynthetic process; F:chitin synthase activity Chitinase 2 Chi c173465_g1_i1 Ov 8.64 ? 6.35E-07 1392 F:hydrolase activity, acting on glycosyl bonds; P:organic substance metabolic process Chitinase c166295_g1_i1 Br 3.91 – 9.37E-06 2002 F:hydrolase activity, hydrolyzing O-glycosyl compounds; P:chitin metabolic process Probable chitinase 3-like c208603_g1_i2 EY 5.23 ? 4.09E-11 3156 F:hydrolase activity, hydrolyzing O-glycosyl compounds; P:chitin catabolic process Chitinase 2 c42686_g1_i1 TG 4.93 ? 5.86E-10 1223 P:carbohydrate metabolic process; F:chitinase activity; C:extracellular region; F:chitin binding; P:chitin catabolic process Maltase a3-like Mal c202206_g1_i5 Br 9.35 ? 1.07E-08 6995 F:catalytic activity EY 8.32 ? 4.28E-06 TG 11.46 ? 1.62E-14 Br brain, C cellular component, EY eyestalk, F molecular function, Ov ovary, P biological process, TG thoracic ganglia

123 123 Table 4 Summary of BLASTx results for contigs of the selected candidate genes that are differentially expressed during oogenesis in Nephrops norvegicus Gene name Abbr. Contig Species E-value Similarity (%) HSP/Hit (%) Accession number

Sex-determining protein fem-1 FEM c205271_g1_i1 Eriocheir sinensis 0 95.6 100 AKS25866.1 Piwi-like protein 2-like PIWI c201464_g1_i3 0 83.1 97.4 AGV15455.1 Vitellogenin Vg c208407_g1_i1 Homarus americanus 0 97.8 99.3 ABO09863.1 Apolipoprotein d-like APO c197835_g2_i2 3.44E-23 48.7 83.2 ASY04978.1 Vitelline membrane outer layer protein 1 VMOL c201770_g1_i1 Scylla paramamosain 4.7E-46 62.3 82.7 ACK44247.1 Serine threonine-protein kinase 3-like STPK c204932_g1_i4 Hyalella azteca 0 76.5 71.5 XP_018017552.1 Ccr4-not transcription complex subunit 6-like CCR4 c160408_g1_i1 Daphnia pulex 0 78 100 EFX85626.1 Ccr4-not transcription complex c171766_g1_i1 Hyalella azteca 1.8E-48 52.9 10.9 XP_018023566.1 Ccr4-not transcription complex c171766_g1_i2 Hyalella azteca 1.8E-48 52.9 10.9 XP_018023566.1 Dorsal–ventral patterning protein sog-like SOG c193736_g1_i1 Hyalella azteca 0 95.8 95.8 XP_018017544.1 Histone h3 methyltransferase HMT c207512_g1_i8 Daphnia magna 0 57.2 65.4 KZS03200.1 Swisnf-related matrix-associated actin- SWI/SNF c154266_g1_i2 Hyalella azteca 0 75.1 58.9 XP_018017949.1 dependent regulator of chromatin subfamily a containing dead h box 1-like Heat shock protein HSP90 c205460_g1_i3 Hyalella azteca 0 86.2 86.4 XP_018017641.1 Heat shock protein 70 kDa HSP70 c205739_g1_i3 Homarus americanus 0 99.5 100 ANQ44656.1 c205739_g1_i4 Homarus americanus 0 93.2 100 ANQ44656.1 Tubulin alpha-1 chain TUBA c203790_g2_i8 Penaeus vannamei 0 97.3 98.2 ANJ04739.1 c300918_g1_i1 Gecarcinus lateralis 6.04E-76 99.1 99.7 AAC47524.1 Elav-like protein 2-like ELAV c195365_g2_i7 Hyalella azteca 0 95.0 97.3 XP_018012743.1 c195365_g2_i10 Hyalella azteca 0 93.6 97.6 XP_018012741.1 C-type lectin 2 CTL c208513_g3_i4 Penaeus japonicus 7.3E-42 62.7 61.7 AHA83583.1 C-type lectin domain family 4 member m-like c207034_g3_i1 Penaeus mondon 4.11E-35 65.1 69.2 AAZ29608.1 Neverland NVD c207639_g1_i11 Hyalella azteca 0 72.2 75.2 XP_018017486.1 Retinoid X receptor RXR c208891_g1_i4 Homarus americanus 0 99.8 90.9 AGI15961.1 c208891_g1_i1 Homarus americanus 0 99.3 91.6 AGI15961.1 Cytochrome p450 cyp44 CYP c194604_g1_i2 Tigriopus japonicus 5.45E-93 63.8 76.1 AIL94168.1 Probable cytochrome p450 49a1-like c201287_g1_i2 Hyalella azteca 3.67E-96 55.3 77 XP_018011463.1 Ecdysone inducible protein 75 E75 c191847_g1_i4 Portunus trituberculatus 0 92.9 99.4 AJT60335.1

Hyperglycemic hormone peptide 2 precursor CHH c203843_g2_i2 Nephrops norvegicus 4.72E-70 94.9 100 AAQ22392.1 Hydrobiologia Chitin synthase CS c202979_g1_i3 Eriocheir sinensis 0 94.6 96.3 ALO62091.1 Chitinase 2 Chi c173465_g1_i1 Pandalopsis japonica 3.54E-123 67.4 86.7 AFC17977.1 Chitinase c166295_g1_i1 Portunus trituberculatus 0 72.3 98.4 AHZ97887.1 Hydrobiologia

(NVD), retinoid X receptor (RXR), and cytochrome p450 (CYP450). Higher expression of APO was detected in mature females in the brain and TG and NVD in the hepatopancreas. The other two genes showed higher expression in mature females in the hepatopancreas and brain, and for CYP450 also in the TG. Molecular characterization of N. norvegicus RXR showed that it has the typical protein structure, including a zinc finger binding domain and ligand binding domain of nuclear hormone receptors (Fig. 5). The phylogenetic tree showed that N. norvegicus RXR was most closely related to H. americanus, and secondly to two crayfish species, C. quadricarinatus and (Girard, 1852), then to crabs and and showed lowest degree of homology with Daphnia magna (Straus, 1820) and Danio rerio 162 79.0 92.0 KZS09326.1

- (Hamilton, 1822) (Fig. 6). Regarding carbohydrate metabolism, our curated -value Similarity (%) HSP/Hit (%) Accession number 0 67.3 86.9 AQZ26767.1 0 88.3 10.6 XP_018011674.1 2.47E E list included seven transcripts that annotated with chitinase (Chi), a single chitin synthase (CS), and a single maltase (Mal). Higher expression of CS in mature females was observed in eyestalk and Chi in the ovary, eyestalk and TG, while Chi expression was lowest in brain. Maltase was up-regulated in all nervous tissues studied. In relation to stress proteins, two heat shock Tigriopus japonicus Hyalella azteca Daphnia magna proteins (HSP) were detected that were differentially expressed. First, a newly discovered HSP90 was detected and was differentially expressed in immature and mature N. norvegicus females. While this HSP90 did not match with any previously identified crus- tacean HSP90, it still included an HSP90 domain. We attempted to characterize this protein in a few other crustacean species from the TSA database and successfully retrieved some sequences to build a phylogenetic tree for this newly identified (predicted) protein. Phylogenetic analysis showed that the sequence in N. norvegicus was most closely related to other Astacidea, C. quadricarinatus, and P. clarkii, and secondly to crabs and shrimps (Fig. 7). Four isoforms of HSP70 were detected in the ovary and the hepatopancreas that were all highly expressed in mature females. In the ovary, we also detected chromatin modi- fiers—SWI/SNF-related matrix-associated actin-de- pendent regulator of chromatin subfamily (SWI/SNF), continued histone h3 methyltransferase (HMT), and tubulin alpha (TUBA) that were differentially expressed. Probable chitinase 3-like c208603_g1_i2 Chitinase 2 c42686_g1_i1 Maltase a3-like Mal c202206_g1_i5 Gene name Abbr. Contig Species Table 4 Two SWI/SNF and HMT transcripts were highly 123 Hydrobiologia

Fig. 3 Molecular phylogenetic analysis of Nephrops norvegi- value larger than 50. N. norvegicus VG is highlighted with a red cus vitellogenin (Vg) by neighbor-joining method based on the box. Nn, N. norvegicus; Cq, C. quadricarinatus; Ha, H. JTT matrix-based model. Thousand bootstrap replicates were americanus; Es, E. sinensis; Pt, P. trituberculatus; Sp, S. used to produce the phylogenetic tree using amino acids paramamosain; Mr, M. rosenbergii; Mn, M. nipponense; Me, M. sequence of Vg. Red squares represent clades with bootstrap ensis; Pj, P. japonicus; Pm, P. monodon; Pv, P. vannamei

Fig. 4 Molecular characterization of N. norvegicus crustacean (SP), the mature peptide (yellow), and putative cleavage sites. hyperglycemic hormone (CHH). Schematic diagrams show Precursor sequence alignments are shown with site of the mature structure of neuropeptide precursors, including signal peptide peptide highlighted in yellow

Fig. 5 Molecular characterization of N. norvegicus retinoid X receptor (RXR). Schematics diagram shows structure of the protein, including a Zinc finger binding domain (ZnF) and ligand binding domain of hormone receptors (HOLI)

Fig. 6 Molecular phylogenetic analysis of N. norvegicus highlighted with a red box. Cq, C. quadricarinatus; Dm, D. Retinoid X Receptor (RXR) by neighbor-joining method based magna; Dr, D. rerio; Es, E. sinensis; Fc, F. chinensis; Gl, G. on the JTT matrix-based model. To produce the phylogenetic lateralis; Mn, M. nipponense; Nn, N. norvegicus; Pc, P. clarkii; tree using amino acids sequence of RXR 1000 bootstrap Pj, P. japonicus; Pm, P. monodon; Pt, P. trituberculatus; Pv, P. replicates were used. Red squares represent clades with vannamei bootstrap value larger than 50. N. norvegicus RXR is 123 Hydrobiologia

Fig. 7 Molecular phylogenetic analysis of N. norvegicus novel value larger than 50. N. norvegicus HSP90 neuropeptide are Heat Shock Protein (HSP90) by neighbor-joining method based highlighted with red boxes. Cm, C. maenas; Cq, C. quadricar- on the JTT matrix-based model. Thousand bootstrap replicates inatus; Es, E. sinensis; Hya, H. azteca; Nn, N. norvegicus; Pc, P. were used to produce the phylogenetic tree using amino acids clarkii; Pm, P. monodon; Pt, P. trituberculatus; Pv, P. vannamei sequence of HSP90. Red squares represent clades with bootstrap expressed in immature females. TUBA presented two eyestalk, and ovary tissues from wild-caught and isoforms, one up-regulated in ovary and the other captive-reared P. monodon shrimps was used to down-regulated in TG. identify 495 transcripts (213 in cephalothorax, 171 Four additional annotated sequences were found to in eyestalk, and 111 in ovary) that showed maturation- be differentially expressed between immature and related differential expression patterns (Brady et al., mature females: serine/threonine-protein kinase 2012, 2013). Ongoing development of NGS technol- (STPK), dorsal–ventral patterning protein sog 1 ogy has allowed greater resolution analysis to be (SOG), elav-like protein 2 (ELAV), and C-type lectins conducted, resulting in an order of magnitude of more (CTL). Mature promotion factor STPK was highly differentially expressed transcripts being detected in expressed in eyestalk in mature females. Patterning the current study. In comparison, two recent studies protein SOG was detected in ovary of immature that used NGS detected fewer DEGs than the number females and also was highly expressed when com- reported here. In the swimming crab P. trituberculatus pared with mature female. RNA-binding protein 382 DEGs were detected in the hepatopancreas during ELAV presented two transcripts; one down-regulated maturation; this included 165 up-regulated and 217 in ovary and the other up-regulated in eyestalk. Two down-regulated genes (Wang et al., 2014). In F.mer- transcripts identified as CTL transcripts were defense guiensis, analysis of differentially expressed genes in proteins; one was down-regulated in hepatopancreas the ovary identified 1025 DEGs, of which 694 were while the other was up-regulated in eyestalk. up-regulated and 331 down-regulated (Saetan et al., 2016). In the current study, we identified a total of 1891 up- versus 2471 down-regulated transcripts. The Discussion number of DEGs was lower in Saetan and Wang’ studies because they only compared transcriptomes We utilized a multi-tissue transcriptomic library from a single tissue while in the present study five developed previously for N. norvegicus (Rotllant tissues in N. norvegicus were analyzed. Moreover, in et al., 2017) to identify 4362 differentially expressed N. norvegicus, the number of DEGs was highest in TG annotated transcripts between the early and late stages (967), followed by hepatopancreas (939) and then of ovarian maturation. In a pre-omics era study, eight ovary (876). As reviewed in the current volume differentially expressed genes were identified in (Rotllant et al., 2018), TG is a potential site for hepatopancreas of M. ensis during ovarian maturation synthesis of gonad stimulation factor (GSF) justifying via differential display reverse transcription-poly- the high DEGs in TG in immature females. Neverthe- merase chain reaction (Wong et al., 2008). Later, less, the highest number of genes exclusively from a suppression subtractive hybridization cDNA expressed in a single tissue was detected in the library, 124 differentially expressedgenes were iso- hepatopancreas (710 in the hepatopancreas versus 617 lated, cloned, and annotated in the thoracic ganglion of in the TG; Supplementary material 3). S. paramamosain (Zeng et al., 2011). Later, oligonu- Vitellogenesis involves production of vitellogenin, cleotide microarray analysis of cephalothorax, a female-specific high-density lipoprotein and a

123 Hydrobiologia precursor to lipo-glycol-caroteno-protein vitellin (the higher in mature females. While VgR is involved in major egg yolk protein). In crustaceans, Vg synthe- Vg uptake by oocytes and plays a critical role in egg sized in the hepatopancreas is believed to be secreted development as demonstrated in crayfishes (Jiang into the hemolymph, where it is sequestered into et al., 2014) and shrimps (Tiu et al., 2008; Klinbunga developing oocytes by the Vg receptor via receptor- et al., 2015; Rotllant et al., 2015; Saetan et al., 2016), mediated endocytosis (Subramoniam, 2011). in N. norvegicus VgR was not found to be differen- Recently, Vg was identified in N. norvegicus to be tially expressed between immature and mature the most highly DEG between males and females with females in any of the studied tissues. exclusive expression in the female hepatopancreas In crustaceans, VMO1 proteins are synthesized in (Rotllant et al., 2017). The Vg gene has been identified the hepatopancreas and are then transported via the previously in other decapod species and it is expressed hemolymph to developing oocytes. VMO1 plays a in both hepatopancreas and ovaries in H. americanus major role that precedes Vg expression, possibly (Tiu et al., 2009), C. quadricarinatus (Abdu et al., suggesting an important role in oogenesis timing 2002), Penaeus japonicus Spence Bate, 1888 (Tsutsui (Heckmann et al., 2008). VMO1 transcripts have also et al., 2000), Penaeus vannamei Bonne, 1931 (Tseng been found in the cephalothorax of P. monodon (Brady et al., 2002; Parnes et al., 2004; Raviv et al., 2006), et al., 2012) and hepatopancreas of P. trituberculatus Penaeus semisulcatus De Haan, 1844 (Avarre et al., (Wang et al., 2014) and generally showed elevated 2003), M. ensis (Tsang et al. 2003), P. monodon (Tiu expression at late vitellogenic stages. This was also et al., 2006; Brady et al., 2013), F. merguiensis observed in the current study in N. norvegicus (Phiriyangkul et al., 2007), Fenneropenaeus chinensis hepatopancreas. The sex-determining Fem-1 gene (Osbeck, 1765) (Xie et al., 2009), Pandalus hypsinotus family was highly expressed in fertilized eggs, 2–4 Brandt, 1851 (Tsutsui et al., 2004), Pandalopsis cell and blastula stage compared with Eriocheir japonica Blass, 1914 (Jeon et al., 2010), Macro- sinensis (H. Milne Edwards, 1851) larval stages that brachium rosenbergii (De Man, 1879) (Jayasankar suggests they could be maternal genes (Song et al., et al., 2002; Jasmani et al., 2004), P. trituberculatus 2015). These genes were also expressed in an array of (Yang et al., 2005) and Rathbun, other tissues specifically during late gonadal develop- 1896 (Thongda et al., 2015). In the giant freshwater ment stages. In N. norvegicus, Fem-1 expression was prawn Macrobrachium nipponnensis (De Haan, 1849) also higher in ovaries of mature females. however, Vg is synthesized in the hepatopancreas, but The PIWI family is a large family of RNA-binding not in the ovary or ovarian follicles (Wu et al., 2009). proteins involved in gene regulation mediated by In a closely related species, M. rosenbergii in contrast, 21–30-nucleotide-long small non-coding RNAs that Vg is expressed in the ovary and hepatopancreas. are referred to as PIWI-interacting RNAs (piRNAs). Significant accumulation of Vg mRNAs was observed They primarily map as transposons and repeated in female hepatopancreas only, while mRNA expres- sequence elements. The PIWI genes are important in sion was not detected in male hepatopancreas or any gametogenesis, germ cell development, transposon, other female tissues including the ovary (Jasmani and transcriptional or post-transcriptional silencing et al., 2004). In N. norvegicus, we observed that Vg (Malone et al., 2009; Mani et al., 2014). PIWI genes was highly expressed in the ovary and brain of were identified in gonadal transcriptomes in P. immature females but not in the hepatopancreas. vannamei (Peng et al., 2015) and P. trituberculatus Multiple Vg-like genes were detected in the N. (Xiang et al., 2014). The PIWI sequence in P. norvegicus library (Rotllant et al., 2017) in all studied trituberculatus was 58% identical to the one obtained tissues (ovary, hepatopancreas, eyestalk, brain and in the current study for N. norvegicus females, where it thoracic ganglia) as was previously reported for P. was highly expressed in immature females suggesting monodon (Tiu et al., 2006) and M. ensis (Wong et al., that this gene is necessary for activation of ovarian 2008). In S. paramamosain, Vg was differentially maturation. expressed in the TG (Zeng et al., 2011) suggesting that In crustaceans, vitellogenesis is generally under- its synthesis during ovarian maturation is important in stood to be negatively regulated by the gonad inhibit- the nervous system. This result aligns with N. ing hormone (GIH) that is produced and released from norvegicus where Vg expression in the brain was neurosecretory sites in the eyestalk. The X organ also 123 Hydrobiologia secretes a variety of neuropeptides in addition to GIH reported in immature and mature females (Wang et al., including crustacean hyperglycemic hormone (CHH), 2014). molt-inhibiting hormone (MIH), mandibular organ- Vitellogenesis involves accumulation of additional inhibiting hormone (MOIH), and chro- proteins apart from vitellogenin/vitellin, as well as matophorotropins (Nagaraju, 2011). These genes are carbohydrates, lipids, vitamins, and minerals in all grouped into the CHH family of neuropeptides. The oocytes (Subramoniam, 2011). Lipids that accumulate main function of CHH is not only to regulate in crustacean oocytes provide energy required for hemolymph glucose levels, but they also have other biosynthetic processes and vitellogenesis [reviewed functions including reproduction, molting, lipid meta- by Harrison (1990)]. Their content has been reported bolism, stress responses, and hydromineral regulation to decrease in the hepatopancreas and is transferred to (Webster et al., 2012). In N. norvegicus, CHH had the ovary during maturation (reviewed by Glencross, been identified previously in the eyestalk (Mettulio (2009)). In N. norvegicus hepatopancreas, three lipid et al., 2004) and the sequence showed 94% identity compounds were differentially expressed. CYP450 with the sequence found in our transcriptome for and RXR were highly expressed in immature females CHH. CHH in our transcriptome was found to be both while Neverland was highly expressed in mature highly and differentially expressed in the brain and TG females. Neverland is an evolutionary conserved in immature females. Our CHH sequence also showed Rieske-domain protein that is essential for ecdysone 94% of identity with that of the closely related species, synthesis and thus influences insect growth while also H. americanus (de Kleijn et al., 1995). NGS studies affecting cholesterol trafficking (Yoshiyama et al., have identified CHH precursors in a wide range of 2006). RXR is important for ecdysteroid function, and decapod crustacean including H. americanus (Christie forms a dimer with the ecdysteroid receptor (EcR– & Chi, 2015), M. rosenbergii (Ventura et al., 2014), C. RXR) and regulates transcription of target genes via quadricarinatus (Nguyen et al., 2016), P. clarkii ecdysteroid responsive elements in the DNA (Brown (Veenstra, 2015), and S. paramamosain (Bao et al., et al., 2009). Expression of the two genes in P. 2015). In a later study of H. americanus, two CHH monodon showed the highest levels correlated with isoforms were identified that showed differential hatching rate in the hepatopancreas of females (Rotl- expression during oocyte maturation. The highest lant et al., 2015). This supports it being implicated in CHH-A mRNA levels were observed in the pre- gonad maturation. N. norvegicus RXR sequences have vitellogenic stage, while elevated CHH-B mRNA the zinc finger binding and ligand binding domains of levels were evident in the vitellogenic stage (de Kleijn hormone receptors (Fig. 5) and share 99% identity et al., 1995). The CHH in N. norvegicus that was with H. americanus RXR (Tiu et al., 2012) and high differentially expressed in the brain and TG poten- identity ([ 80%) with RXR from other decapod tially could be another isoform of the CHH identified species (Fig. 6; Chung et al., 1998; Durica et al., first by Mettulio et al. (2004) or the GIH identified by 2002; Kim et al., 2005; Asazuma et al., 2007; Nagaraju Edomi et al. (2002). It could also have another et al., 2011; Techa & Chung, 2013; Qian et al., 2014; function related to induction of oocyte maturation. Girish et al., 2015). To identify CYP450 in N. Molting is inhibited by MIH (a member of the CHH norvegicus we clustered the two CYPs of interest family of neuropeptides) that is synthesized in the using a recently published CYPome sequence from eyestalks. MIH suppresses production and secretion of spiny rock lobster (https://www.ncbi.nlm.nih.gov/ ecdysone by the Y organs. Ecdysteroids can also be pubmed/28428023 and Ventura et al., 2014 in this synthesized in other tissues including the gonads and volume). Results showed that N. norvegicus CYPs hepatopancreas (Nagaraju, 2011), where they have cluster between the CYP302A1 group (disembodied) been implicated in female reproduction (Rotllant & and CYP314A1 (shade) with a high level of confi- Takac, 1999). In the current study, anecdysone dence (Supplementary material 1). This suggests that inducible protein 75 (E75) was highly expressed in they may play roles in ecdysteroidogenesis. Moreover, N. norvegicus immature females, suggesting that E75 the differentially expressed CYP450 sequence in N. may have a role in initiation of oocyte maturation. norvegicus shared 54% identity with the CYP450 Regardless, E75 was detected in the hepatopancreatic identified in Carcinus maenas (Linnaeus, 1758) (Re- transcriptomes of P. trituberculatus but no DEGs were witz et al., 2003) and was also highly expressed in 123 Hydrobiologia brain and TG. The presence of CYP450 in the nervous translated into a HSP90 domain containing protein but systems is correlated with oocyte maturation and was did not hit with any previously documented HSP90. first observed in the TG of S. paramamosain (Wong This sequence was BLAST searched against the TSA et al., 2008). An apolipoprotein transcript was also database to confirm similarity with other decapod found to be differentially expressed in the nervous species. Phylogenetic analysis showed that the N. system of N. norvegicus; this transcript was up-regu- norvegicus HSP90 was homologous to the HSP90 in lated in brain and down-regulated in TG tissue. It crayfish species (Fig. 7). Previously, HSP90 had been showed sequence identity with Astacidea (NCBI) and confirmed to be highly differentially expressed other decapods (Tsang et al., 2003; Tsutsui et al., between immature and mature female in M. ensis 2004, 2005; Raviv et al., 2006) were low (23 ± 3%) (Wu & Chu, 2008) and M. nipponense (Zhao et al., where it had been reported in most of them as a Vg 2011). The role and maturation of this HSP90-like gene. Vg in decapod crustaceans was also reported to sequence detected in the current study will need more show a high sequence similarity with APO (Avarre investigation. In the same HSP superfamily, we also et al., 2007). identified a differentially expressed HSP70 encoding In all Pleocyemate decapods, including N. norvegi- transcript that shared high identity with some crayfish cus, apart from their known effect as an energy reserve sequences (Sun et al., 2009; Ali et al., 2015) and other for oocyte maturation, carbohydrates are required for decapod sequences (Leignel et al., 2007; Mestre et al., attaching of the eggs to the female abdomen just after 2015). HPS70 in N. norvegicus was up-regulated in laying the eggs. Pleocyemata decapods are known to ovary and hepatopancreas in immature females, a contain high levels of N-glycosylation sites on pattern previously reported in the ovary and hep- proteoglycans associated with egg brooding (Roth atopancreas of M. ensis and the cephalothorax of P. et al., 2010). In our study, we also found that genes monodon (Brady et al., 2013; Chan et al., 2014). encoding enzymes related to chitin and maltose were At least three processes control assembly and differentially expressed. High expression of chitinase regulation of chromatin: DNA methylation, histone synthase in mature females was observed in the modification, and SWI/SNF. These complexes are eyestalks and chitin in the ovaries, eyestalks, and TG, crucial for appropriate development in all organisms while Chi expression was lower in the brain. CS in which they have been studied (Ho & Crabtree, showed 86% identity with a CS reported in P. japonica 2010). Transcripts identified as SWI/SNF and histone (Uddowla et al., 2015) while Chi showed [ 60% h3 methyltransferase were up-regulated in the ovaries identity with CS in crab species (Fujitani et al., 2014; of N. norvegicus and shared * 50% identity with Li et al., 2015) and shrimp (Watanabe & Kono, 1997; insect sequences. Tubulin is necessary for cytoskele- Huang et al., 2010; Proespraiwong et al., 2010; Rocha ton synthesis in the ovary as reported in vertebrates et al., 2012). The above-mentioned studies suggest (Maurizii et al., 2004). Two transcripts in N. norvegi- that chitinases may play crucial physiological roles in cus matched a-tubulin sequences. A transcript in N. crustaceans, including digestion of chitin-containing norvegicus found in TG showed high homology with food, molting and defense of decapods against viruses. Tubulin sequences in P. clarkii (95%; Jiang et al., Insects chitinases also play a role in morphogenesis 2015) and Gecarcinus lateralis (Gue´rin, 1832) (97%; (Merzendorfer & Zimoch, 2003). Maltase in N. Varadaraj et al., 1997) while a sequence found in norvegicus was up-regulated in the nervous systems ovary showed 92% identity with an insect sequence and showed * 50% sequence identity with Daphnia (Monochamus alternatus), where it had been corre- pulex (Linnaeus, 1758) (NCBI). Whether digestion of lated with microtubule formation (Song et al., 2008). carbohydrates, however, is important for oocyte Expression of TUBA in N. norvegicus was up- maturation will need further investigation. regulated in ovary and down-regulated in TG and Synthesis of Vg in oviparous vertebrates is regu- was found previously to be differentially expressed in lated by the E2-ER-HSP90-Vg pathway, where an the TG of S. paramamosain (Zeng et al., 2011). A estrogen receptor (ER) and HSP90 mediate enhance- short-gastrulation gene was identified in the amphipod ment of vitellogenin transcription by estrogen or crustacean Parhyale hawaiensis (Danna, 1853) that is estrogen-like hormones (Fliss et al., 2000). We a key factor that mediates development of midline detected a HSP90-like transcript in N. norvegicus that cells during embryogenesis (Vargas-Vila et al., 2010). 123 Hydrobiologia

In N. norvegicus, SOG was down-regulated in ovary were highly expressed in immature females. Higher and up-regulated in hepatopancreas, a pattern that may expression of these genes in hepatopancreas during indicate that it is synthesized in the hepatopancreas undeveloped and developing stages in M. ensis (Wong following which, during maturation it may be trans- et al., 2008) and M. nipponense (Xiu et al., 2015) ported to the ovary. The short-gastrulation gene suggests that their synthesis may be necessary for sequence in N. norvegicus shared 46% identity with oocyte maturation to prevent oocytes contamination a sequence in P.hawaiensis (Vargas-Vila et al., 2010) by some bacteria types. and 34% with Artemia franciscana Kellog, 1906 Apart from differentially expressed genes during (NCBI). maturation in N. norvegicus, other genes were found to DEG of three additional genes (serine/threonine- be differentially expressed between early and late protein kinase, ELAV-like protein and C-type lectins) maturation stages in P. monodon and P. tritubercula- between immature and mature females in N. norvegi- tus as crustacean calcium binding protein, insulin-like cus were up-regulated in eyestalk. During ovarian receptor-like, 5-hydroxytryptamine receptor, riboso- maturation in C. quadricarinatus, changes in ovarian mal protein L10a, 1,3-b-D-glucan-binding high-den- protein kinase C isoenzymes take place in parallel to sity lipoprotein, high densitylipoprotein/1,3-beta-D- yolk accumulation (Soroka et al., 2000) and may glucan-binding, protein precursor2/3-oxoacyl-CoA provide maturation factors. RNA-binding proteins thiolase, sterol carrier protein, farnesoic acid O- (RBPs) are involved in regulation of fundamental methyltransferase, PAT-family of lipidstorage dro- steps in RNA metabolism (e.g., pre-mRNA splicing, plet-associated proteins, glycerol-3-phosphate trans- polyadenilation, nuclear-cytoplasmic shuttling, stabil- porter, chymotrypsin, triacylglycerol lipase, zinc ity, and translation) (Bronicki & Jasmin 2013; Dox- proteinase Mpc1, metallothionein, thioredoxinperox- akis 2014). In particular, ELAV binds to adenosine- idise, shrimp ovarian peritrophin, CP14 a calcified uridine-rich elements (AREs) that are present in the 30 cuticle protein, profilin, ferritin, rhodopsin, opsin, untranslated region (UTR) of mRNAs that inhibit cryptocyanine, kazal-typeproteinase inhibitor, purple ARE-mediated RNA decay (Peng et al., 1998). In acid phosphatase, ATP binding cassette transmem- addition, they are also reported to be involved in brane transporter, and manganese-superoxide dismu- regulation of alternative splicing (Koushika et al., tase (Brady et al., 2012, 2013; Wang et al., 2014; 1996), 30UTR extension (Hilgers et al., 2012), and Saetan et al., 2016). The reason why some of these polyadenylation (Zhu et al., 2007; Mansfield & Keene, genes were not differentially expressed in mature and 2012). In Eukaryotes, ELAV-like RBP genes are immature females in N. norvegicus could be because expressed specifically in neurons (Yao et al., 1993; they are species-specific genes; for example shrimp Kim et al., 1996). In N. norvegicus ELAV, in addition ovarian peritrophin, or may result from trimming and to being up-regulated in eyestalks, was down-regu- experimental conditions in our study were too severe. lated in the ovary. An ELAV gene (of three identified Moreover, since decapods are not model species and previously) was found to also be expressed in ovary as only two draft decapod genomes are currently publicly well as the nervous system in Drosophila melanoga- available (Yu et al., 2015; Song et al., 2016), a high ster (Meigen, 1830) (Kim & Baker, 1993) suggesting a percentage of transcripts remain unannotated, as also role in regulation of oocyte maturation. reported recently for N. norvegicus (Rotllant et al., C-type lectins are a family of calcium-dependent 2017), suggesting that numerous potential novel genes carbohydrate-binding proteins that are believed to may be present in published transcriptomes. Hence, play important roles in innate immunity in crustaceans there is still much to investigate about mechanisms (Huang et al., 2014; Xiu et al., 2015). In mammal that control and influence ovarian maturation activa- reproductive physiology, CTLs are involved in molec- tion and regulation in decapod crustaceans. ular mechanisms that underlie successful fertilization of embryos, a process via which sperm lectins recognize specific carbohydrates on egg surface Conclusions glycoproteins. They are involved in pathogen recog- nition and cellular interactions (Rodeheffer & Shur, The current study compared multi-tissue transcrip- 2004). In the hepatopancreas of N. norvegicus, CTLs tomic profiles in Norway lobster between immature 123 Hydrobiologia and mature females using an RNA-Seq approach. In Bao, C., Y. Yang, H. Huang & H. Ye, 2015. Neuropeptides in total, we identified 4362 differentially expressed genes the cerebral ganglia of the mud crab, Scylla paramamosain: transcriptomic analysis and expression profiles during in ten libraries constructed from two wild populations. vitellogenesis. Scientific Reports 5: 17055. Differentially expressed genes identified here Brady, P., A. Elizur, R. Williams, S. F. Cummins & W. Knibb, involved in ovarian maturation were related to oocyte 2012. Gene Expression Profiling of the Cephalothorax and development and regulation, lipid and carbohydrate Eyestalk in during Ovarian Maturation. Interrnational Journal of Biological Sciencces 8(3): metabolism, stress proteins and chromatin modifiers. 328–343. The data collected can assist developing a more Brady, P., A. Elizur, S. F. Cummins, N. H. Ngyuen, R. Williams comprehensive knowledge of the reproductive system & W. Knibb, 2013. Differential expression microarrays during the ovarian maturation process in this impor- reveal candidate genes potentially associated with repro- ductive dysfunction of captive-reared prawn Penaeus tant commercial lobster species. It is important to monodon. 400–401: 14–28. highlight, however, that further studies will be needed Bronicki, L. M. & B. J. Jasmin, 2013. Emerging complexity of to identify as well as to characterize the roles of a the HuD/ELAVl4 gene; implications for neuronal devel- significant number of potentially novel genes in opment, function, and dysfunction. RNA 19(8): 1019–1037. ovarian maturation and female reproductive Brown, M. R., D. H. Sieglaff & H. H. Rees, 2009. Gonadal processes. Ecdysteroidogenesis in Arthropoda: Occurrence and Reg- ulation Annual Review of Entomology. Annual Review of Acknowledgements The current study was supported by a Entomology, vol 54. Annual Reviews, Palo Alto, 105–125. Marie Curie International Research Staff Exchange Chan, S. F., J.-G. He, K. H. Chu & C. B. Sun, 2014. The shrimp Scheme Fellowship within the 7th European Community heat shock cognate 70 functions as a negative regulator in Framework Programme (612296-DeNuGReC) and a USC vitellogenin gene expression. Biology of Reproduction International PhD scholarship to Tuan Viet Nguyen. The 91(1): 1–11. authors are grateful for the support of the crew of the fishing Christie, A. E. & M. Chi, 2015. Prediction of the neuropep- vessel Maireta for field sampling. We would also like to tidomes of members of the Astacidea (Crustacea, Dec- acknowledge QUT HPC for computational support during the apoda) using publicly accessible transcriptome shotgun current study. assembly (TSA) sequence data. General and Comparative Endocrinology 224: 38–60. Chung, A. C. K., D. S. Durica, S. W. Clifton, B. A. Roe & P. M. Hopkins, 1998. Cloning of crustacean ecdysteroid References receptor and retinoid-X receptor gene homologs and ele- vation of retinoid-X receptor mRNA by retinoic acid. Abdu, U., C. Davis, I. Khalaila & A. Sagi, 2002. The vitel- Molecular and Cellular Endocrinology 139(1–2): 209–227. logenin cDNA of encodes a de Kleijn, D. P., M. C. van den Berg, G. J. Martens & F. van lipoprotein with calcium binding ability, and its expression Herp, 1995. Cloning and expression of two mRNAs is induced following the removal of the androgenic gland in encoding structurally different crustacean hyperglycemic a sexually plastic system. General and Comparative hormone precursors in the lobster Homarus americanus. Endocrinology 127(3): 263–272. Biochimica et Biophysica Acta 1260(1): 62–66. Ali, M. Y., A. Pavasovic, S. Amin, P. B. Mather & P. J. Prentis, Doxakis, E., 2014. RNA binding proteins: a common denomi- 2015. Comparative analysis of gill transcriptomes of two nator of neuronal function and dysfunction. Neuroscience freshwater crayfish, Cherax cainii and C-destructor. Mar- Bulletin 30(4): 610–626. ine Genomics 22: 11–13. Durica, D. S., X. Wu, G. Anilkumar, P. M. Hopkins & A. C. K. Asazuma, H., S. Nagata, M. Kono & H. Nagasawa, 2007. Chung, 2002. Characterization of crab EcR and RXR Molecular cloning and expression analysis of ecdysone homologs and expression during limb regeneration and receptor and retinoid X receptor from the kuruma prawn, oocyte maturation. Molecular and Cellular Endocrinology Marsupenaeus japonicus. Comparative Biochemistry and 189(1–2): 59–76. Physiology: Part B: Biochemistry 148(2): 139–150. Edgar, R. C., 2004. MUSCLE: multiple sequence alignment Avarre, J. C., R. Michelis, A. Tietz & E. Lubzens, 2003. Rela- with high accuracy and high throughput. Nucleic Acids tionship between vitellogenin and vitellin in a marine Research 32(5): 1792–1797. shrimp (Penaeus semisulcatus) and molecular characteri- Edomi, P., E. Azzoni, R. Mettulio, N. Pandolfelli, E. A. Ferrero zation of vitellogenin complementary DNAs. Biology of & P. G. Giulianini, 2002. Gonad-inhibiting hormone of the Reproduction 69(1): 355–364. Norway lobster (Nephrops norvegicus): cDNA cloning, Avarre, J. C., E. Lubzens & P. J. Babin, 2007. Apolipocrusta- expression, recombinant protein production, and cein, formerly vitellogenin, is the major egg yolk precursor immunolocalization. Gene 284(1–2): 93–102. protein in decapod crustaceans and is homologous to insect Fliss, A. E., S. Benzeno, J. Rao & A. J. Caplan, 2000. Control of apolipophorin II/I and vertebrate apolipoprotein B. BMC estrogen receptor ligand binding by Hsp90. Journal of Evolutionary Biology. https://doi.org/10.1186/1471-2148- Steroid Biochemistry and Molecular Biology 72(5): 7-3. 223–230. 123 Hydrobiologia

Fujitani, N., H. Hasegawa, H. Kakizaki, M. Ikeda & M. Mat- by endosulfan exposure. Comparative Biochemistry and sumiya, 2014. Molecular cloning of multiple chitinase Physiology: Part B 157(1): 102–112. genes in swimming crab Portunus trituberculatus. Journal Jiang, H. C., Z. J. Xing, W. Lu, Z. J. Qian, H. W. Yu & J. L. Li, of Chitin and Chitosan Science 2(2): 149–156. 2014. Transcriptome Analysis of Red Swamp Crawfish Girish, B. P., C. H. Swetha & P. S. Reddy, 2015. Induction of Procambarus clarkii Reveals Genes Involved in Gonadal ecdysteroidogenesis, methyl farnesoate synthesis and Development. PLoS ONE 9(8): 9. expression of ecdysteroid receptor and retinoid X receptor Jiang, H., Z. Qian, W. Lu, H. Ding, H. Yu, H. Wang & J. Li, in the hepatopancreas and ovary of the giant mud crab, 2015. Identification and Characterization of Reference by melatonin. General and Comparative Genes for Normalizing Expression Data from Red Swamp Endocrinology 217–218: 37–42. Crawfish Procambarus clarkii. International Journal of Glencross, B. D., 2009. Exploring the nutritional demand for Molecular Sciences 16(9): 21591–21605. essential fatty acids by aquaculture species. Reviews in Johnson, M. P., C. Lordan & A. M. Power, 2013. Chapter Two - Aquaculture 1: 71–124. Habitat and Ecology of Nephrops norvegicus. In Magnus, Grabherr, M. G., B. J. Haas, M. Yassour, J. Z. Levin, D. L. J. & P. J. Mark (eds), Advances in Marine Biology. A. Thompson, I. Amit, X. Adiconis, L. Fan, R. Ray- Academic Press, Cambridge: 27–63. chowdhury, Q. D. Zeng, Z. H. Chen, E. Mauceli, N. Jones, D. T., W. R. Taylor & J. M. Thornton, 1992. The rapid Hacohen, A. Gnirke, N. Rhind, F. di Palma, B. W. Birren, generation of mutation data matrices from protein C. Nusbaum, K. Lindblad-Toh, N. Friedman & A. Regev, sequences. Computer applications in the biosciences: 2011. Full-length transcriptome assembly from RNA-Seq CABIOS 8(3): 275–282. data without a reference genome. Nature Biotechnology Jones, P., D. Binns, H.-Y. Chang, M. Fraser, W. Li, C. McA- 29(7): 644. nulla, H. McWilliam, J. Maslen, A. Mitchell, G. Nuka, S. Harrison, K. E., 1990. The role of nutrition in maturation, Pesseat, A. F. Quinn, A. Sangrador-Vegas, M. reproduction and embryonic development of decapod Scheremetjew, S.-Y. Yong, R. Lopez & S. Hunter, 2014. crustaceans: a review. Journal of Shellfish Research 9: InterProScan 5: genome-scale protein function classifica- 1–28. tion. Bioinformatics 30(9): 1236–1240. Heckmann, L.-H., R. Sibly, R. Connon, H. Hooper, T. Kim, Y. J. & B. S. Baker, 1993. The Drosophila gene rbp9 Hutchinson, S. Maund, C. Hill, A. Bouetard & A. Cal- encodes a protein that is a member of a conserved group of laghan, 2008. Systems biology meets stress ecology: putative RNA binding proteins that are nervous system- linking molecular and organismal stress responses in specific in both flies and humans. Journal of Neuroscience Daphnia magna. Genome Biology 9(2): R40. 13: 1045–1056. Hilgers, V., S.B. Lemke & M. Levine, 2012. ELAV mediates Kim, C. H., E. Ueshima, O. Muraoka, H. Tanaka, S. Y. Yeo, T. 30UTR extension in the Drosophila nervous system. Genes L. Huh & N. Miki, 1996. Zebrafish elav/HuC homologue as and Development 26: 2259–2264. a very early neuronal marker. Neuroscience Letters 216: Ho, L. & G. R. Crabtree, 2010. Chromatin remodelling during 109–112. development. Nature 463(7280): 474–484. Kim, H.-W., S. G. Lee & D. L. Mykles, 2005. Ecdysteroid- Huang, Q.-S., J.-H. Yan, J.-Y. Tang, Y.-M. Tao, X.-L. Xie, Y. responsive genes, RXR and E75, in the tropical land crab, Wang, X.-Q. Wei, Q.-H. Yan & Q.-X. Chen, 2010. Cloning Gecarcinus lateralis: Differential tissue expression of and tissue expressions of seven chitinase family genes in multiple RXR isoforms generated at three alternative Litopenaeus vannamei. Fish and Shellfish Immunology splicing sites in the hinge and ligand-binding domains. 29(1): 75–81. Molecular and Cellular Endocrinology 242(1–2): 80–95. Huang, Y., X. Huang, L. Hou, L. An, K.-M. Hui, Q. Ren & W. Klinbunga, S., K. Sittikankaew, N. Jantee, S. Prakopphet, S. Wang, 2014. Molecular cloning and characterization of Janpoom, R. Hiransuchalert, P. Menasveta & B. Kham- three novel Hemocyanins from , Eri- namtong, 2015. Expression levels of vitellogenin receptor ocheir sinensis. Aquaculture 434: 385–396. (Vtgr) during ovarian development and association Jasmani, S., T. Ohira, V. Jayasankar, N. Tsutsui, K. Aida & M. between its single nucleotide polymorphisms (SNPs) and N. Wilder, 2004. Localization of vitellogenin mRNA reproduction-related parameters of the giant tiger shrimp expression and vitellogenin uptake during ovarian matu- Penaeus monodon. Aquaculture 435: 18–27. ration in the giant freshwater prawn Macrobrachium Koushika, S.P., M.J. Lisbin & K. White, 1996. ELAV, a Dro- rosenbergii. Journal of Experimental Zoology Part A sophila neuron-specific protein, mediates the generation of 301A(4): 334–343. an alternatively spliced neural protein isoform. Current Jayasankar, V., N. Tsutsui, S. Jasmani, H. Saido-Sakanaka, W. Biology 6: 1634–1641. J. Yang, A. Okuno, T. T. T. Hien, K. Aida & M. N. Wilder, Kumar, S., G. Stecher & K. Tamura, 2016. MEGA7: Molecular 2002. Dynamics of vitellogenin mRNA expression and Evolutionary Genetics Analysis Version 7.0 for Bigger changes in hemolymph vitellogenin levels during ovarian Datasets. Molecular Biology and Evolution 33(7): maturation in the giant freshwater prawn Macrobrachium 1870–1874. rosenbergii. Journal of Experimental Zoology 293(7): Leignel, V., M. Cibois, B. Moreau & B. Che´nais, 2007. Iden- 675–682. tification of new subgroup of HSP70 in Bythograeidae Jeon, J. M., S. O. Lee, K. S. Kim, H. J. Baek, S. Kim, I. K. Kim, (hydrothermal crabs) and Xanthidae. Gene 396(1): 84–92. D. L. Mykles & H. W. Kim, 2010. Characterization of two Letunic, I. & P. Bork, 2016. Interactive tree of life (iTOL) v3: an vitellogenin cDNAs from a Pandalus shrimp (Pandalopsis online tool for the display and annotation of phylogenetic japonica): Expression in hepatopancreas is down-regulated 123 Hydrobiologia

and other trees. Nucleic Acids Research 44(W1): W242– Parnes, S., E. Mills, C. Segall, S. Raviv, C. Davis & A. Sagi, W245. 2004. Reproductive readiness of the shrimp Litopenaeus Letunic, I., T. Doerks & P. Bork, 2015. SMART: recent updates, vannamei grown in a brackish water system. Aquaculture new developments and status in 2015. Nucleic Acids 236(1–4): 593–606. Research 43(D1): D257–D260. Parra, G., K. Bradnam & I. Korf, 2007. CEGMA: a pipeline to Li, B. & C. N. Dewey, 2011. RSEM: accurate transcript quan- accurately annotate core genes in eukaryotic genomes. tification from RNA-Seq data with or without a reference Bioinformatics 23(9): 1061–1067. genome. BMC Bioinformatics 12: 323. Peng, S.S., C.Y. Chen, N. Xu & A.B. Shyu, 1998. RNA stabi- Li, X., Z. Xu, G. Zhou, H. Lin, J. Zhou, Q. Zeng, Z. Mao & X. lization by the AU-rich element binding protein, HuR, an Gu, 2015. Molecular characterization and expression ELAV protein. The EMBO Journal 17: 3461–3470. analysis of five chitinases associated with molting in the Peng, J., P. Wei, B. Zhang, Y. Zhao, D. Zeng, X. Chen, M. Li & Chinese mitten crab, Eriocheir sinensis. Comparative X. Chen, 2015. Gonadal transcriptomic analysis and dif- Biochemistry and Physiology: Part B 187: 110–120. ferentially expressed genes in the testis and ovary of the https://doi.org/10.1016/j.cbpb.2015.05.007. Pacific white shrimp (Litopenaeus vannamei). BMC Liu, W., Y. Xie, J. Ma, X. Luo, P. Nie, Z. Zuo, U. Lahrmann, Q. Genomics 16(1): 1006. Zhao, Y. Zheng, Y. Zhao, Y. Xue & J. Ren, 2015. IBS: an Phiriyangkul, P., P. Puengyam, I. B. Jakobsen & P. Utarabhand, illustrator for the presentation and visualization of bio- 2007. Dynamics of vitellogenin mRNA expression during logical sequences. Bioinformatics 31(20): 3359–3361. vitellogenesis in the banana shrimp Penaeus (Fennerope- Malone, C. D., J. Brennecke, M. Dus, A. Stark, W. naeus) merguiensis using real-time PCR. Molecular R. McCombie, R. Sachidanandam & G. J. Hannon, 2009. Reproduction and Development 74(9): 1198–1207. Specialized piRNA pathways act in germline and somatic Proespraiwong, P., A. Tassanakajon & V. Rimphanitchayakit, tissues of the Drosophila Ovary. Cell 137(3): 522–535. 2010. Chitinases from the black tiger shrimp Penaeus Mani, S. R., H. Megosh & H. Lin, 2014. PIWI proteins are monodon: Phylogenetics, expression and activities. Com- essential for early Drosophila embryogenesis. Develop- parative Biochemistry and Physiology: Part B 156(2): mental Biology 385(2): 340–349. 86–96. Mansfield, K.D. & J.D. Keene, 2012. Neuron-specific ELAV/ Qian, Z., S. He, T. Liu, Y. Liu, F. Hou, Q. Liu, X. Wang, X. Mi, Hu proteins suppress HuR mRNA during neuronal differ- P. Wang & X. Liu, 2014. Identification of ecdysteroid entiation by alternative polyadenylation. Nucleic Acids signaling late-response genes from different tissues of the Research 40: 2734–2746. Pacific white shrimp, Litopenaeus vannamei. Comparative Maurizii, M. G., L. Alibardi & C. Taddei, 2004. a-Tubulin and Biochemistry and Physiology: Part B 172: 10–30. acetylated a-tubulin during ovarian follicle differentiation Raviv, S., S. Parnes, C. Segall, C. Davis & A. Sagi, 2006. in the lizard Podarcis sicula Raf. Journal of Experimental Complete sequence of Litopenaeus vannamei (Crustacea: Zoology Part A: Comparative Experimental Biology decapoda) vitellogenin cDNA and its expression in 301A(6): 532–541. endocrinologically induced sub-adult females. General and Merzendorfer, H. & L. Zimoch, 2003. Chitin metabolism in Comparative Endocrinology 145(1): 39–50. insects: structure, function and regulation of chitin syn- Rewitz, K., B. Styrishave & O. Andersen, 2003. CYP330A1 and thases and chitinases. Journal of Experimental Biology CYP4C39 enzymes in the shore crab Carcinus maenas: 206(24): 4393–4412. sequence and expression regulation by ecdysteroids and Mestre, N. C., D. Cottin, R. Bettencourt, A. Colac¸o, S. P. C. xenobiotics. Biochemical and Biophysical Research Correia, B. Shillito, S. Thatje & J. Ravaux, 2015. Is the Communications 310(2): 252–260. deep-sea crab Chaceon affinis able to induce a thermal Rocha, J., F. L. Garcia-Carren˜o, A. Muhlia-Almaza´n, A. stress response? Comparative Biochemistry and Physiol- B. Peregrino-Uriarte, G. Ye´piz-Plascencia & J. ogy: Part B 181: 54–61. H. Co´rdova-Murueta, 2012. Cuticular chitin synthase and Mettulio, R., P. G. Giulianini, E. A. Ferrero, S. Lorenzon & P. chitinase mRNA of Litopenaeus vannamei Edomi, 2004. Functional analysis of crustacean Hyper- during the molting cycle. Aquaculture 330–333: 111–115. glycemic Hormone by in vivo assay with wild-type and Rodeheffer, C. & B. D. Shur, 2004. Characterization of a novel mutant recombinant proteins. Regulatory Peptides 119(3): ZP3-independent sperm-binding ligand that facilitates 189–197. sperm adhesion to the egg coat. Development 131(3): Nagaraju, G. P. C., 2011. Reproductive regulators in decapod 503–512. crustaceans: an overview. Journal of Experimental Biology Rosa, R. & M. L. Nunes, 2002. Biochemical changes during the 214(1): 3–16. reproductive cycle of the deeps-ea decapod Nephrops Nagaraju, G. P. C., B. Rajitha & D. W. Borst, 2011. Molecular norvegicus on the south coast of Portugal. Marine Biology cloning and sequence of retinoid X receptor in the green 141: 1001–1009. crab Carcinus maenas: a possible role in female repro- Roth, Z., S. Parnes, S. Wiel, A. Sagi, N. Zmora, J. S. Chung & I. duction. Journal of Endocrinology 210(3): 379–390. Khalaila, 2010. N-glycan moieties of the crustacean egg Nguyen, T. V., S. F. Cummins, A. Elizur & T. Ventura, 2016. yolk protein and their glycosylation sites. Glycoconjugate Transcriptomic characterization and curation of candidate Journal 27(1): 159–169. neuropeptides regulating reproduction in the eyestalk Rotllant, G. & P. Takac, 1999. Ecdysones in the maturational ganglia of the Australian crayfish. Scientific Reports, moult of juvenile females of the spider crab, Libinia Cherax quadricarinatus. https://doi.org/10.1038/ emarginata Leach, 1815 (Decapoda, Majidae). Crus- srep38658. taceana 72(2): 221–231. 123 Hydrobiologia

Rotllant, G., E. Ribes, J. B. Company & M. Durfort, 2005. The Sun, Y., L. Zhang, M. Li, R. Wu, L. Lei & S. Xie, 2009. Cloning ovarian maturation cycle of the Norway lobster Nephrops and expression analysis of an inducible heat shock protein norvegicus (Linnaeus, 1758) (Crustacea, Decapoda) from 70 gene from red swamp crayfish, Procambarus clarkii. the western Mediterranean Sea. Invertebrate Reproduction Acta Hydrobiologica Sinica 33(4): 627–635. & Development 48(1–3): 161–169. Techa, S. & J. S. Chung, 2013. Ecdysone and retinoid-X Rotllant, G., M. Chiva, M. Durfort & E. Ribes, 2012. Internal receptors of the blue crab, Callinectes sapidus: Cloning and anatomy and ultrastructure of the male reproductive system their expression patterns in eyestalks and Y-organs during of the Norway lobster Nephrops norvegicus (Decapoda: the molt cycle. Gene 527(1): 139–153. Astacidea). Journal of Morphology 273(6): 572–585. Thongda, W., J. S. Chung, N. Tsutsui, N. Zmora & A. Katenta, Rotllant, G., N. M. Wade, S. J. Arnold, G. J. Coman, N. 2015. Seasonal variations in reproductive activity of the P. Preston & B. D. Glencross, 2015. Identification of genes blue crab, Callinectes sapidus: Vitellogenin expression and involved in reproduction and lipid pathway metabolism in levels of vitellogenin in the hemolymph during ovarian wild and domesticated shrimps. Marine Genomics 22: development. Comparative Biochemistry and Physiology 55–61. Part A: Molecular & Integrative Physiology 179: 35–43. Rotllant, G., T. V. Nguyen, V. Sbragaglia, L. Rahi, K. J. Dudley, Tiu, S. H. K., J. H. L. Hui, A. S. C. Mak, J.-G. He & S.-M. Chan, D. Hurwood, T. Ventura, J. B. Company, V. Chand, J. 2006. Equal contribution of hepatopancreas and ovary to Aguzzi & P. B. Mather, 2017. Sex and tissue specific gene the production of vitellogenin (PmVg1) transcripts in the expression patterns identified following de novo tran- tiger shrimp, Penaeus monodon. Aquaculture 254(1–4): scriptomic analysis of the Norway lobster, Nephrops 666–674. norvegicus. BMC Genomics 18(1): 622. Tiu, S. H. K., J. Benzie & S.-M. Chan, 2008. From hepatopan- Rotllant, G., T. V. Nguyen, J. Aizen, S. Suwansa-ard & T. creas to ovary: molecular characterization of a shrimp Ventura, 2018, Towards the identification of Female vitellogenin receptor involved in the processing of vitel- Gonad Stimulating Factors in crustaceans. Hydrobiologia logenin. Biology of Reproduction 79(1): 66–74. (in press). Tiu, S. H. K., H. L. Hui, B. Tsukimura, S. S. Tobe, J. G. He & S. Saetan, U., U. Sangket, P. Deachamag & W. Chotigeat, 2016. M. Chan, 2009. Cloning and expression study of the lobster Ovarian transcriptome analysis of vitellogenic and non- (Homarus americanus) vitellogenin: Conservation in gene vitellogenic female banana shrimp (Fenneropenaeus mer- structure among decapods. General and Comparative guiensis). PLoS ONE 11(10): e0164724. Endocrinology 160(1): 36–46. Sarda, F., 1991. Reproduction and molt synchronism in Tiu, S. H.-K., E. F. Hult, K. J. Yagi & S. S. Tobe, 2012. Far- Nephrops norvegicus (L) (Decapoda, Nephropidae) in the nesoic acid and methyl farnesoate production during lob- Western Mediterranean—is spawning annual or biennial. ster reproduction: Possible functional correlation with Crustaceana 60: 186–199. retinoid X receptor expression. General and Comparative Sarda`, F., 1995. A review (1967–1990) o some aspects of the life Endocrinology 175(2): 259–269. history of Nephrops norvegicus. Marine Science Sym- Tsang, W. S., L. S. Quackenbush, B. K. C. Chow, S. H. K. Tiu, J. posia—ICES 199: 78–88. G. He & S. M. Chan, 2003. Organization of the shrimp Sarda`, F., 1998. Symptoms of overexploitation in the stock of vitellogenin gene: evidence of multiple genes and tissue the Norway lobster (Nephrops norvegicus) on the ‘‘Serola specific expression by the ovary and hepatopancreas. Gene Bank’’ (Western Mediterranean Sea off Barcelona). Sci- 303: 99–109. entia Marina 62(3): 295–299. Tseng, D. Y., Y. N. Chen, K. F. Liu, G. H. Kou, C. F. Lo & C. Song, L., X. X. Liu, Y. A. Zhang, Q. W. Zhang & Z. W. Zhao, M. Kuo, 2002. Hepatopancreas and ovary are sites of 2008. The cloning and expression of a-tubulin in Mono- vitellogenin synthesis as determined from partial cDNA chamus alternatus. Insect Molecular Biology 17(5): encoding of vitellogenin in the marine shrimp, Penaeus 495–504. vannamei. Invertebrate Reproduction & Development Song, C., Z. Cui, M. Hui, Y. Liu & Y. Li, 2015. Molecular 42(2–3): 137–143. characterization and expression profile of three Fem-1 Tsutsui, N., I. Kawazoe, T. Ohira, S. Jasmani, W. J. Yang, M. genes in Eriocheir sinensis provide a new insight into crab N. Wilder & K. Aida, 2000. Molecular characterization of a sex-determining mechanism. Comparative Biochemistry cDNA encoding vitellogenin and its expression in the and Physiology: Part B 189: 6–14. hepatopancreas and ovary during vitellogenesis in the Song, L., C. Bian, Y. Luo, L. Wang, X. You, J. Li, Y. Qiu, X. kuruma prawn, Penaeus japonicus. Zoological Science Ma, Z. Zhu, L. Ma, Z. Wang, Y. Lei, J. Qiang, H. Li, J. Yu, 17(5): 651–660. A. Wong, J. Xu, Q. Shi & P. Xu, 2016. Draft genome of the Tsutsui, N., H. Saido-Sakanaka, W. J. Yang, V. Jayasankar, S. Chinese mitten crab, Eriocheir sinensis. GigaScience 5(1): Jasmani, A. Okuno, T. Ohira, T. Okumura, K. Aida & M. 1–3. N. Wilder, 2004. Molecular characterization of a cDNA Soroka, Y., A. Sagi, I. Khalaila, U. Abdu & Y. Milner, 2000. encoding vitellogenin in the coonstiriped shrimp, Pandalus Changes in protein kinase C during vitellogenesis in the hypsinotus and site of vitellogenin mRNA expression. crayfish Cherax qyadricarinatus—Possible activation by Journal of Experimental Zoology Part A 301A(10): methyl farnesoate. General and Comparative Endocrinol- 802–814. ogy 118(2): 200–208. Tsutsui, N., Y. K. Kim, S. Jasmani, T. Ohira, M. N. Wilder & K. Subramoniam, T., 2011. Mechanisms and control of vitelloge- Aida, 2005. The dynamics of vitellogenin gene expression nesis in crustaceans. Fisheries Science 77(1): 1–21. differs between intact and eyestalk ablated kuruma prawn

123 Hydrobiologia

Penaeus (Marsupenaeus) japonicus. Fisheries Science Part D 4(2): 111–120. https://doi.org/10.1016/j.cbd.2008. 71(2): 249–256. 12.004. Uddowla, H., A. R. Kim, W. G. Park & H. W. Kim, 2015. Xiang, D.-F., J.-Q. Zhu, C.-C. Hou & W.-X. Yang, 2014. cDNAs encoding chitin synthase from shrimp (Pandalop- Identification and expression pattern analysis of Piwi genes sis Japonica): molecular characterization and expression during the spermiogenesis of Portunus trituberculatus. analysis. Journal of Aquaculture Research and Develop- Gene 534(2): 240–248. https://doi.org/10.1016/j.gene. ment 6: 298. 2013.10.050. Varadaraj, K., S. S. Kumari & D. M. Skinner, 1997. Molecular Xie, S., L. Sun, F. Liu & B. Dong, 2009. Molecular character- characterization of four members of the a-tubulin gene ization and mRNA transcript profile of vitellogenin in family of the Bermuda land crab Gecarcinus lateralis. Chinese shrimp, Fenneropenaeus chinensis. Molecular Journal of Experimental Zoology 278(2): 63–77. Biology Reports 36(2): 389–397. Vargas-Vila, M. A., R. L. Hannibal, R. J. Parchem, P. Z. Liu & Xiu, Y., L. Hou, X. Liu, Y. Wang, W. Gu, Q. Meng & W. Wang, N. H. Patel, 2010. A prominent requirement for single- 2015. Isolation and characterization of two novel C-type minded and the ventral midline in patterning the lectins from the oriental river prawn, Macrobrachium dorsoventral axis of the crustacean Parhyale hawaiensis. nipponense. Fish and Shellfish Immunology 46(2): Development 137(20): 3469–3476. 603–611. Veenstra, J. A., 2015. The power of next-generation sequencing Yang, F., H. T. Xu, Z. M. Dai & W. J. Yang, 2005. Molecular as illustrated by the neuropeptidome of the crayfish Pro- characterization and expression analysis of vitellogenin in cambarus clarkii. General and Comparative Endocrinol- the marine crab Portunus trituberculatus. Comparative ogy 224: 84–95. Biochemistry and Physiology - Part B: Biochemistry & Ventura, T., S. F. Cummins, Q. Fitzgibbon, S. Battaglene & A. Molecular Biology 142(4): 456–464. Elizur, 2014. Analysis of the central nervous system tran- Yao, K. M., M. L. Samson, R. Reeves & K. White, 1993. Gene scriptome of the eastern rock lobster ver- elav of Drosophila melanogaster: a prototype for neu- reauxi reveals its putative neuropeptidome. PLoS ONE ronalspecific RNA binding protein gene family that is 9(5): 23. conserved in flies and humans. Developmental Neurobi- Wang, W., X. G. Wu, Z. J. Liu, H. J. Zheng & Y. X. Cheng, ology 24: 723–739. 2014. Insights into hepatopancreatic functions for nutrition Ye, J., L. Fang, H. K. Zheng, Y. Zhang, J. Chen, Z. J. Zhang, J. metabolism and ovarian development in the crab Portunus Wang, S. T. Li, R. Q. Li, L. Bolund & J. Wang, 2006. trituberculatus: Gene Discovery in the comparative tran- WEGO: a web tool for plotting GO annotations. Nucleic scriptome of different hepatopancreas stages. PLoS ONE Acids Research 34: W293–W297. 9(1): e0084921. Yoshiyama, T., T. Namiki, K. Mita, H. Kataoka & R. Niwa, Watanabe, T. & M. Kono, 1997. Isolation of a cDNA Encoding 2006. Neverland is an evolutionally conserved Rieske- a Chitinase Family Protein from Cuticular Tissues of the domain protein that is essential for ecdysone synthesis and Kuruma Prawn Penaeus japonicus. Zoological Science insect growth. Development 133(13):2565–2574. 14(1): 65–68. Yu, Y., X. Zhang, J. Yuan, F. Li, X. Chen, Y. Zhao, L. Huang, H. Webster, S. G., R. Keller & H. Dircksen, 2012. The CHH-su- Zheng & J. Xiang, 2015. Genome survey and high-density perfamily of multifunctional peptide hormones controlling genetic map construction provide genomic and genetic crustacean metabolism, osmoregulation, moulting, and resources for the Pacific White Shrimp, Litopenaeus van- reproduction. General and Comparative Endocrinology namei. Scientific Reports 5: 15612. 175(2): 217–233. Zeng, H., J. Huang, W. Li, H. Huang & H. Ye, 2011. Identifi- Wong, Q. W. L., W. Y. Mak & K. H. Chu, 2008. Differential cation of differentially expressed genes in the thoracic gene expression in hepatopancreas of the shrimp Metape- ganglion of the mud crab, Scylla paramamosain during naeus ensis during ovarian maturation. Marine Biotech- ovarian maturation. Marine Biology Research 7(6): nology 10(1): 91–98. 617–622. Wu, L. T. & K. H. Chu, 2008. Characterization of heat shock Zhao, W. H., L. Q. Chen, J. G. Qin, P. Wu, F. Y. Zhang, E. C. Li protein 90 in the shrimp Metapenaeus ensis: evidence for & B. P. Tang, 2011. MnHSP90 cDNA characterization and its role in the regulation of vitellogenin synthesis. Molec- its expression during the ovary development in oriental ular Reproduction and Development 75(5): 952–959. river prawn, Macrobrachium nipponense. Molecular Wu, P., D. Qi, L. Chen, H. Zhang, X. Zhang, J. Guang Qin & S. Biology Reports 38(2): 1399–1406. Hu, 2009. Gene discovery from an ovary cDNA library of Zhu, H., H.L. Zhou, R.A. Hasman & H. Lou, 2007. Hu proteins oriental river prawn Macrobrachium nipponense by ESTs regulate polyadenylation by blocking sites containing annotation. Comparative Biochemistry and Physiology: U-rich sequences. Journal of Biological Chemistry 282: 2203–2210.

123