NO signaling is involved in many physiological processes in invertebrates. In crustaceans, it plays a role in the regulation of the nervous system and muscle contraction. Nested reverse transcription-polymerase chain reaction(RT-PCR) and 5′ and 3′ rapid amplification of cDNA ends (RACE) PCR generated a full-length cDNA sequence (3982 bp) of land crab NO synthase(Gl-NOS) from molting gland (Y-organ) and thoracic ganglion mRNA. The open reading frame encoded a protein of 1199 amino acids with an estimated mass of 135 624 Da. Gl-NOS had the highest sequence identity with insect NOS. The amino acid sequences for binding heme and tetrahydrobiopterin in the oxygenase domain, binding calmodulin and binding FMN, FAD and NADPH in the reductase domain were highly conserved. Gl-NOS had single amino acid differences in all three highly conserved FAD-binding sequences, which distinguished it from other NOS sequences. RT-PCR showed that the Gl-NOS mRNA was present in testis,ovary, gill, eyestalk neural ganglia, thoracic ganglion and Y-organ. NOS mRNA varied between preparations of Y-organ, thoracic ganglion and gill, while NOS mRNA was at consistently high levels in the ovary, testis and eyestalk ganglia. Immunohistochemistry confirmed that the Gl-NOS protein was expressed in Y-organ, ovary and gill. These results suggest that NOS has functions in addition to neuromodulation in adults, such as regulating or modulating ecdysteroid synthesis in the Y-organ.

Nitric oxide (NO) appears to have evolved as a signaling molecule before the radiation of the metazoans (Feelisch and Martin, 1995; Torreilles,2001). NO is generated by nitric oxide synthase (NOS) from l-arginine, O2 and NADPH and diffuses freely across the cell membrane to induce responses in neighboring cells(Colasanti and Venturini,2000). The best-known NO signaling pathway is one in which NO activates a soluble class I guanylyl cyclase (GC-I; Baranano and Snyder, 2001). Activated GC-I produces cyclic 3′,5′-guanosine monophosphate(cGMP), which in turn activates cGMP-dependent protein kinase. In mammals,NO/cGMP signaling is involved in vasodilation, neurotransmission and the immune response (Ahern et al.,2002; Baranano and Snyder,2001; Bredt and Snyder,1994). In insects, NO signaling is involved in many physiological processes (Davies, 2000). NO regulates nervous system development and integration(Bicker, 2001; Haase and Bicker, 2003; Schachtner et al., 1999; Seidel and Bicker, 2002; Truman et al., 1996). The hematophagous insect Rhodnius prolixus produces NO, which dilates blood vessels and inhibits platelet aggregation in the host(Ribeiro and Nussenzveig,1993). Recent studies show that the NO/cGMP pathway is involved in the insect immune response (Imamura et al., 2002; Luckhart et al.,1998; Weiske and Wiesner,1999) and activation of NO/cGMP signaling inhibits steroid synthesis in the ovary of blow fly (Phormia regina; Maniere et al., 2003).

In mammals, there are three NOS genes: neuronal NOS(nNOS), endothelial NOS (eNOS) and inducible NOS(iNOS) (Bogdan, 2001; Mungrue et al., 2003; Nathan and Xie, 1994). Although their expression and biological roles vary, they share a common structural organization (Ghosh and Salerno, 2003; Kone et al.,2003; Torreilles,2001). The native enzyme is a homodimer of 130–160-kDa subunits (Torreilles, 2001). The N-terminal oxygenase domain contains the binding motif for a P450-like cysteine thiolate-ligate heme and tetrahydrobiopterin (H4). The C-terminal reductase domain contains the binding motifs for FAD, FMN and NADPH. These two domains are linked by a calmodulin (CaM) binding motif. nNOS and eNOS are constitutively expressed and their enzymatic activities are regulated by the intracellular Ca2+ concentration through binding of Ca2+to CaM (Roman et al., 2002). They contain a 40–50 amino acid sequence linked to the FMN binding motif that acts as an autoinhibitory loop, blocking electron transfer from FMN to the heme in the absence of Ca2+/CaM(Craig et al., 2002; Ghosh and Salerno, 2003; Nishida and de Montellano,2001; Salerno et al.,1997). By contrast, iNOS lacks the autoinhibitory loop and binds CaM with high affinity at low Ca2+ levels; its activity is regulated predominantly at the transcriptional level(Chen and Wu, 2003; Nathan and Xie, 1994).

Insect NOSs have the highest sequence identity with mammalian nNOS and share the same organization in the oxygenase, CaM-binding and reductase domains (Davies, 2000; Torreilles, 2001). Insect NOS requires NADPH, Ca2+ and CaM for enzymatic activity. It is expressed in a variety of adult and embryonic tissues, including abdominal nerve cord, optic lobes, fat body, antenna, hemocytes, midgut and Malpighian tubule (Broderick et al.,2003; Gibbs and Truman,1998; Imamura et al.,2002; Luckhart et al.,1998; Nighorn et al.,1998). Isoforms of NOS are generated by alternative splicing. The Drosophila NOS gene contains at least four alternative promoters(Stasiv et al., 2001). Some truncated alternative splicing variants of the Drosophila NOS lacking the reductase domain may act as dominant negative regulators, as heterodimers would lack enzyme activity (Stasiv et al.,2001).

In crustaceans, NO/cGMP signaling plays a role in neuronal development and neuron, skeletal muscle and cardiac muscle regulation(Aonuma et al., 2000; Aonuma and Newland, 2001, 2002; Erxleben and Hermann, 2001; Hermann and Erxleben, 2001; Johansson and Mellon, 1998; Mahadevan et al., 2004;Scholz, 1999, 2001; Scholz et al., 1998, 2001). NOS is expressed in neurons of the cerebral, stomatogastric, eyestalk, abdominal terminal and cardiac ganglia (Christie et al.,2003; Johansson and Carlberg,1994; Johansson and Mellon,1998; Lee et al.,2000; Scholz et al.,2002; Schuppe et al., 2001a,b, 2002; Talavera et al., 1995; Zou et al., 2002). The NO/cGMP signaling pathway is required for the dynamic assembly of the neuronal circuit that drives rhythmic movement in crabs(Scholz, 2001; Scholz et al., 2002), alters ion channel properties of skeletal muscle(Erxleben and Hermann, 2001; Hermann and Erxleben, 2001)and decreases heartbeat amplitude and frequency(Mahadevan et al., 2004). The biochemical properties of crustacean NOS are similar to those of mammalian nNOS and insect NOS, as it also requires NADPH, Ca2+ and CaM for activity (Johansson and Carlberg,1994; Lee et al.,2000; Scholz et al.,2002; Zou et al.,2002).

The NO/cGMP signaling pathway may be involved in regulating molting in crustaceans. Molt inhibiting hormone (MIH), a neuropeptide synthesized in a neurosecretory center (X-organ/sinus gland complex) in the eyestalk acts as a negative regulator of ecdysteroidogenesis in the Y-organ(Lachaise et al., 1993; Skinner, 1985). The signal transduction pathway is poorly understood. Cyclic nucleotides mediate MIH inhibition of Y-organ ecdysteroidogenesis (Spaziani et al., 1999, 2001). There are species differences in the relative importance of cAMP and cGMP, although both cyclic nucleotides probably play a role (Lachaise et al., 1993; Sedlmeier and Fenrich, 1993; Spaziani et al., 1999). MIH induces an increase in cAMP and cGMP, with subsequent activation of protein kinases in Y-organs in vitro(Baghdassarian et al., 1996; Böcking and Sedlmeier,1994; Saïdi et al.,1994; Sedlmeier and Fenrich,1993; Von Gliscynski and Sedlmeier, 1993).

Given the wide expression of NOS in insect and mammalian tissues, we hypothesized that crustacean NOS is expressed in non-neuronal tissues and functions in regulating a variety of physiological processes. A cDNA encoding a full-length crustacean NOS (Gl-NOS) was cloned from land crab(Gecarcinus lateralis) Y-organ and thoracic ganglion mRNA using RT-PCR and 3′ and 5′ RACE PCR. The tissue expression of NOS mRNA was determined with RT-PCR. Immunohistochemistry was used to localize NOS protein in Y-organ, gill and ovary. The results show that NOS is more widely distributed than was previously supposed and suggest that NO is involved in regulating diverse functions, including MIH signaling in the Y-organ.

Animals

Adult land crabs (Gecarcinus lateralis Fréminville) were collected from San Miguel Reserve near Fajardo, Puerto Rico. They were kept in covered plastic cages with aspen bedding moistened with tap water at 27°C and 30–40% humidity and were fed cat chow, carrots and lettuce twice a week. A 12 h:12 h dark:light cycle was used.

Cloning of Gl-NOS cDNA

A partial NOS cDNA was initially obtained by nested RT-PCR using degenerate primers directed to highly conserved sequences in a wide variety of NOS genes in the GenBank database(http://www.ncbi.nlm.nih.gov),including those from six insect species and three human forms (nNOS, eNOS,iNOS) and aligned using the ClustalW program(http://www.ebi.ac.uk/clustalw/index.html). Two sets of degenerate primers were designed to anneal to DNA sequences encoding F(S/N)GWYM, VF(H/F)QEM or TFGNG(E/D)PP(Fig. 1, broken lines with solid arrowheads): NOS F1, 5′-TT(C/T) (A/T)(G/C/A)(A/G/T/C) GG(A/G/T/C)TGG TA(C/T) ATG-3′; NOS F2, 5′-GT(A/G/T/C) TT(C/T) (C/T)(A/T)(C/T)CA(G/A) GA(G/A) ATG-3′; NOS R1, 5′-GG(A/G/T/C) GG(A/G/T/C)TC(A/G/T/C) CC(G/A) TT(A/G/T/C) CC-3′; R2, 5′-G(A/G/T/C)TC(A/G/T/C) CC(G/A) TT(A/G/T/C) CC(G/A) AA(A/G/T/C) G-3′. All the primers were synthesized and purified by Integrated DNA Technologies, Inc.(Des Moines, IA, USA).

Total RNA was isolated from thoracic ganglia and Y-organ using RNeasy Protect mini kit (Qiagen, Inc., Valencia, CA, USA). About 20 mg of tissue and 600 μl of RTL reagent were used for each spin column unit. Total RNA (100μg) was used for isolating mRNA with an Oligotex mRNA isolation kit(Qiagen). cDNA was synthesized according to the manufacturer's protocol using the Superscript II RNase H-reverse transcriptase first-strand synthesis system(Invitrogen, Inc., Carlsbad, CA, USA). Briefly, 12 μl of a mixture containing 1 μl oligo (dT)12–18 (500 μg ml–1),100 ng RNA and 1 μl of 10 mmol l–1 dNTPs was heated to 65°C for 5 min and chilled on ice for 1 min. 4 μl of 5×First-Strand Buffer, 2 μl of 0.1 mol l–1 dithiothreitol(DTT) and 1 μl RNaseOUT, recombinant ribonuclease inhibitor (40 unitsμl–1) were added. The mixture was incubated at 42°C for 2 min. The reaction was initiated by the addition of 1 μl (200 units)SuperScript II at 42°C for 50 min. The reaction was inactivated by heating at 70°C for 15 min. PCRs were performed using an ABI 9600 thermal cycler(Perkin-Elmer, Inc., Wellesley, MA, USA). The first-round PCR contained 3μl of cDNA, 3 μl of 10× Takara EX Taq buffer, 2 μl of 250μmol l–1 dNTPs, 1 μl of forward degenerate primer (NOS F1), 1 μl of reverse degenerate primer (NOS R1), 0.2 μl of Takara EX Taq DNA polymerase (5 units μl–1) and 18.8 μl of PCR-grade deionized water. Initial denaturation (95°C for 5 min) was followed by 35 amplifying cycles (95°C for 30 s, 53°C for 30 s and 72°C for 1 min) and final extension at 72°C for 7 min. For the second PCR, 0.1 μl of the first PCR reaction and the nested degenerate NOS F2 and NOS R2 primers were used. Other reaction components and PCR conditions were the same as those in the first reaction.

PCR products were separated by 2% agarose gel electrophoresis and stained with ethidium bromide. The PCR products were purified from gel slices using QIAquick Gel Extraction mini kit (Qiagen), ligated into PCR2.1 vector with the TOPO TA Cloning kit (Invitrogen) and transformed into One Shot TOP 10 E. coli strain (Invitrogen). Plasmids were purified using Qiagen spin mini prep kit and sequenced using T7 and M13 reverse vector primers (Davis Sequencing, Davis, CA, USA).

A semi-nested RT-PCR strategy was used to obtain more of the ORF 3′to the initial nested RT-PCR product. The reactions used two sequence-specific forward primers (cNOS F5, 5′-CAAGTCAGAGATGTACGCCAAGAAG-3′, and cNOS F6, 5′-TCTTCGGTCACACCTTCAATGCTC-3′) and a degenerate reverse primer (NOS R3, 5′-RAADATRTCYTCRTGRTANC-3′) to a highly conserved sequence in the reductase domain (Fig. 1, broken lines with open arrowheads). First-round PCR used cNOS F5 and NOS R3 primers and the original cDNA synthesized from thoracic ganglia and Y-organ mRNA (see above). Second-round PCR used cNOS F6 and NOS R3 primers and 0.1 μl of the first-round PCR. The PCR conditions were the same as described above, except that the extension time for the amplification cycles was 2 min instead of 1 min. PCR products were separated on 1% agarose gels,purified, cloned and sequenced as described above.

RACE (rapid amplification of cDNA ends) of mRNA was used to obtain full-length sequences. Poly(A+) RNA was isolated from total RNA using Oligotex mRNA kit (Qiagen). For the 3′ sequence, the RACE System(Invitrogen) was used. Briefly, first-strand cDNA synthesis reactions contained 200 ng poly(A+) RNA and adaptor primer(5′-GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT-3′). First-round PCR on the cDNA (20 ng) included a universal amplification primer(5′-CUACUACUACUAGGCCACGCGTCGACTAGTAC-3′) and gene-specific forward primer, cNOS F1 (5′-CACTATGGCTGAGTGTGTCTACCAGAAG-3′), under the following conditions: denaturation at 96°C for 5 min, 35 amplification cycles (96°C for 30 s, 60°C for 30 s and 72°C for 2 min) and final extension at 72°C for 10 min. Nested PCR (30 μl total volume) was conducted with a gene-specific primer, cNOS F2(5′-AGCTGAGGTCCATTGTGCAGGAGCATG-3′), and an abridged universal amplification primer (5′-GGCCACGCGTCGACTAGTAC-3′) under the same conditions as the first-round PCR. PCR products were separated by agarose gel electrophoresis and stained with ethidium bromide.

The SMART™ RACE cDNA amplification kit (BD Biosciences, Inc., San Jose, CA, USA) was used to obtain the 5′ sequence. The first-strand cDNA synthesis reaction contained 3 μl poly(A+) RNA (100 ng), 1 μl 5′CDS primer [10 mmol l–1, 5′-(T)25N-1N-3′] and 1μl SMART II A oligo (10 mmol l–1,5′-AAGCAGTGGTATCAACGCAGAGTACGCGGG-3′) and was incubated at 68°C for 2 min. After cooling the reaction on ice for 2 min, 2 μl of 5× First-Strand buffer [250 mmol l–1 Tris-HCl (pH 8.3),375 mmol l–1 KCl and 30 mmol l–1MgCl2], 1 μl DTT (20 mmol l–1), 1 μl dNTPs(10 mmol l–1) and 1 μl PowerScript Reverse Transcriptase were added. The reaction was covered with 20 μl paraffin oil and incubated at 42°C for 1.5 h in an ABI 9600 DNA thermal cycler (Perkin-Elmer). The reaction mixture was diluted 10-fold with autoclaved distilled water and was used for first-round PCR with 10× universal primer A mix (0.4 mmol l–15′-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3′ and 2 mmol l–1 5′-CTAATACGACTCACTATAGGGC-3′) and gene-specific reverse primer, cNOS R1(5′-CGAAGTCCTCCCCATTCTCAGGAG-3′), under the following conditions:denaturation at 96°C for 5 min, 35 amplification cycles (96°C for 30 s, 66°C for 15 s and 72°C for 3 min) and final extension at 72°C for 10 min. Second-round PCR was conducted using nested gene-specific primer cNOS R2 (5′-AGCTTACTTGTGAACTTGACGGCTCTG-3′) and nested universal primer A (10 mmol l–1,5′-AAGCAGTGGTATCAACGCAGAGT-3′). The PCR conditions were the same as those used for first-round PCR. PCR products were separated by agarose gel electrophoresis and stained with ethidium bromide. Purified products were sequenced to confirm identities.

Gl-NOS expression by RT-PCR

Integument, thoracic ganglia, testis, ovary, heart, digestive gland, gill,claw muscle, eyestalk neural ganglia and Y-organ were dissected from 3–5 crabs and immediately placed in RNAlater RNA stabilization reagent (Qiagen). Tissues were stored at –20°C until RNA extractions could be performed. Total RNA was isolated from pooled tissues using either the RNeasy mini or midi kit according to the manufacturer's instructions (Qiagen). RNA concentration was determined by UV absorbance at 260 nm and stored at–80°C. About 1 μg of total RNA was used for the reverse transcription reaction. RNA was first treated with DNase I to degrade any contaminating genomic DNA. First-strand cDNA was synthesized in a 20 μl reaction volume containing 50 mmol l–1 Tris-HCl, 75 mmol l–1 KCl, 3 mmol l–1 MgCl2, 10 mmol l–1 DTT, 0.5 mmol l–1 of each dNTP, 40 units of RNaseOUT ribonuclease inhibitor, 1 ng oligo dT primer and 200 units of Moloney murine leukemia virus reverse transcriptase (Invitrogen). The reaction was incubated for 50 min at 37°C, heat-inactivated and stored at–20°C.

The quality of the cDNA was first verified by performing PCR with land crab elongation factor 2 (EF2; GenBank accession #AY552550) primers (cEF F1,5′-TTCTATGCCTTTGGCCGTGTCTTCTC-3′; cEF R1,5′-TGATGGTGCCCGTCTTAACCAGATAC-3′). The PCR conditions were an initial denaturation at 95°C for 2 min, 35 amplification cycles(denaturation at 94°C for 30 s, annealing at 61°C for 30 s and extension at 72°C for 30 s) and 2 min at 72°C as a final extension.

NOS PCR was then performed in a 20-μl reaction mixture as described above using 2 μl of the first-strand cDNA and an NOS gene-specific primer pair (cNOS EXF, 5′-CAACTTGAGAAGGAATAAAAGGGGAGGATG-3′; cNOS R31, 5′-CTGCTGAAGCTGCTGCCTCTGTCTTGAG-3′), each at a final concentration of 0.2 μmol l–1. The PCR conditions were an initial denaturation at 95°C for 2 min, 35 amplification cycles(denaturation at 96°C for 20 s, annealing at 62°C for 20 s and extension at 72°C for 90 s) and 4 min at 72°C as a final extension. This primary PCR reaction was then used as template with a nested NOS primer pair (cNOS F1, 5′-GTACAAGCAGGAGGACGGGAG-3′; cNOS R5,5′-AGCTTACTTGTGAACTTGACGGCTCTG-3′), each at a final concentration of 0.2 μmol l–1 as described above. The primary reaction was diluted 1:10 000 in water, and 2 μl was used in the reaction. The PCR conditions were an initial denaturation at 95°C for 2 min, 35 amplification cycles (denaturation at 96°C for 20 s, annealing at 62°C for 20 s and extension at 72°C for 50 s) and 4 min at 72°C as a final extension. All PCR reactions were analyzed by separating some or all of the 20μl reaction volume on 1–2% agarose gels with a 100 bp PCR Molecular Ruler DNA size ladder (Bio-Rad, Inc., Hercules, CA, USA).

Immunohistochemistry

Tissues used for immunohistochemistry were dissected and fixed for 24–48 h in Bouin's fixative or Histochoice MB fixative (Amresco, Inc.,Solon, OH, USA) containing 0.15 mol l–1 NaCl. The tissue was then dehydrated through a graded ethanol series, cleared and embedded in paraffin. Sections (∼9 μm) were placed on clean glass slides and allowed to dry at 40°C for 24 h. Sections were then deparaffinized in xylene and rehydrated through an ethanol series into a phosphate-buffered saline (137 mmol l–1 NaCl, 2.7 mmol l–1 KCl,4.3 mmol l–1 Na2HPO4, 1.4 mmol l–1 KH2PO4; pH 7) with 15 mmol l–1 glycine (PBSG). Sections were incubated at room temperature in 1% bovine serum albumin (BSA) and normal goat serum in PBSG for 45 min and then with primary antibody (rabbit anti-NOS antibody; Affinity BioReagents, Inc., Golden, CO, USA; 1:1000–2000 dilution in PBSG) at 4°C overnight. The NOS antibody was a universal antibody (generated against a portion of murine nNOS/iNOS) that crossreacts with crustacean NOS(Christie et al., 2003; Scholz et al., 1998, 2002). After four rinses (5 min each) in PBSG, sections were incubated with either a goat anti-rabbit or mouse anti-rabbit biotin-conjugated antibody (Pierce, Inc., Rockford, IL, USA;diluted 1:1000–1500) at room temperature for 2 h, rinsed four times in PBSG and incubated with an avidin/alkaline phosphatase reagent (Vector Laboratories, Inc., Burlingame, CA, USA). Sections were rinsed four times in PBSG and incubated with BCIP/NBT (Gibco/Invitrogen) or Vector Red (Vector Laboratories, Inc.) substrate until sufficient color development was attained. Control incubations were also done on adjacent tissue sections and included either omitting the primary antibody or substituting a non-immune rabbit serum for the primary antibody.

Cloning and tissue expression of Gl-NOS cDNA

Nested PCR using NOS F2 and R2 primers amplified an initial product of∼400 bp. The deduced amino acid sequence had 72% identity with the CaM-binding and flavodoxin-like regions of Drosophila NOS (accession number AAC46882). A cDNA (1913 bp) was amplified by semi-nested PCR to obtain more sequence 3′ to the initial 400-bp product. The deduced amino acid sequence of the 1913-bp product was 50% identical to that of NOS from the insect Rhodnius prolixus (accession number Q26240). A 1.5-kb product containing the 5′ UTR and the remainder of the 5′ ORF was obtained from 5′ RACE PCR using a sequence-specific reverse primer (cNOS R2; Fig. 1). A 500-bp product containing the 3′ UTR and the remainder of the 3′ ORF was obtained from 3′ RACE PCR using a sequence-specific forward primer (cNOS F2; Fig. 1). The combined PCR products contained a full-length 3982-bp sequence of land crab NOS cDNA(Gl-NOS; GenBank accession #AY552549).

The Gl-NOS cDNA encoded a protein containing 1199 amino acids with an estimated mass of 135 624 Da (Fig. 1). The Gl-NOS amino sequence was aligned with NOS sequences from five insects, a mollusk and three human types(Fig. 2). The N-terminal region varied among different NOS genes, but the oxygenase domain in Gl-NOS was 70% identical to the Drosophila NOS, 68%identical to Aplysia NOS and 66% identical to human nNOS. In the oxygenase domain, the heme-binding motif was well conserved, including the cysteine residue that acts as an axial ligand. The motifs for binding tetrahydrobiopterin (H4) cofactor and CaM were also well-conserved. The reductase domain contained all conserved binding motifs for FMN, FAD and NADPH. Interestingly, the Gl-NOS had single amino acid substitutions in all three motifs for binding FAD that differed from other NOS cDNAs(Fig. 2). The domain organization of Drosophila NOS and Gl-NOS is compared in Fig. 3 to show the high amount of similarity between the two sequences.

The phylogenetic relationships of various NOS sequences were determined using sequence alignments of the oxygenase domains(Fig. 4; residues #54–455 in Gl-NOS). Insect NOS sequences clustered according to major taxonomic groups: Lepidoptera (M. sexta and B. mori), Diptera (A. stephensi and D. melanogaster) and Hemiptera (R. prolixus). Molluskan NOS (A. californica) and vertebrate NOS formed distinct groups. Within the vertebrates, the inducible NOS (iNOS) and noninducible NOS (nNOS, eNOS) were divided. Since few NOS genes have been obtained from lower invertebrates (e.g. nematode), Gl-NOS could not be grouped with any other NOS, although overall sequence comparison showed that Gl-NOS was most closely related to Drosophila NOS.

Gl-NOS was expressed in both neuronal and non-neuronal tissues. Initial RT-PCR using cNOS EXF and R31 primers generated a 2110-bp product amplified from RNA isolated from testis, gill, ovary, eyestalk neural ganglia and Y-organ (Fig. 5A, lanes c,f–i). RT-PCR with an elongation factor 2 (EF2) primer pair served as a positive control (Fig. 5C). Nested PCR on the first-round PCR product using cNOS F1 and R5 primers generated a product of the expected size (795 bp), which confirmed the identity of the initial product as the NOS sequence(Fig. 5B). Gl-NOS mRNA varied in Y-organ, thoracic ganglion and gill; in some preparations, no PCR product was detected in these tissues. Although no PCR product was obtained from the thoracic ganglion mRNA preparation in Fig. 5 (lane b), other thoracic ganglion mRNA preparations yielded an NOS PCR product (data not shown). By contrast, the NOS mRNA was present at consistently high levels in all RNA preparations from eyestalk ganglia, ovary and testis.

Immunohistochemistry

Immunohistochemistry was used to confirm that the NOS protein is present in Y-organs and other non-neuronal tissues. A pair of Y-organs is located adjacent to the branchial chamber on one side and a hemolymph sinus and connective tissue on the other. A thin cuticle separates the Y-organ from the branchial chamber. A universal anti-NOS antibody reacted with the nuclei and cytoplasm of all Y-organ cells (Figs 6A, 7A). Some nuclei in the connective tissue (Fig. 6A,C,D)were stained, as well as the tendinous cells, which anchor connective tissue to the cuticle lining the branchial chamber(Fig. 6D).

In gill, the NOS protein was localized in the epithelium(Fig. 7B). Staining was more intense in the epithelium lining the central axis between the gill lamellae(Fig. 7B, arrows) and in the pillar cells (Fig. 7B,arrowheads). In the ovary, the NOS protein was confined to the perinuclear cytoplasm of oocytes (Fig. 7D). Control sections without primary antibody showed a low amount of staining in oocytes, indicating some non-specific binding of the biotinylated secondary antibody and/or the avidin/alkaline phosphatase reagent(Fig. 7C).

NOS produces NO by catalyzing the conversion of l-arginine to l-citrulline and oxidation of NADPH. Three types of NOS are found in vertebrates (Bogdan, 2001; Kone, 2001). Both nNOS and eNOS are Ca2+/CaM-dependent, whereas iNOS is Ca2+-independent. All are homodimers consisting of a 130–160-kDa subunit. Invertebrates appear to have only a Ca2+/CaM-dependent form(Davies, 2000; Korneev et al., 1998; Luckhart and Rosenberg, 1999; Nighorn et al., 1998; Regulski and Tully, 1995; Ribeiro and Nussenzveig, 1993; Stasiv et al., 2001). We have cloned a cDNA encoding a full-length sequence of land crab NOS (Gl-NOS) from Y-organ and thoracic ganglion mRNA (Fig. 1). The ORF codes for a 1199 amino acid protein with an estimated mass of ∼136 kDa, which is similar to the mass of a 138-kDa NOS-immunoreactive protein in crayfish eyestalk neural ganglia extract(Lee et al., 2000). The domain organization is highly conserved, with a CaM-binding domain located between the oxygenase and reductase domains (Figs 2, 3). Interestingly, Gl-NOS has single amino acid changes in all three FAD binding motifs(Fig. 2). In the FAD(ADP) site,the Gl-NOS has an alanine (A), whereas all the other sequences have a glycine(G) in that location. This is a conservative substitution, as both alanine and glycine are nonpolar residues. The differences in the other two sites are greater, as a nonpolar residue in Gl-NOS replaces a polar residue in the other NOS sequences. An alanine replaces a serine (S) or threonine (T) in the FAD(flavin) site, and glycine (G) replaces a lysine (K) or glutamine (Q) in the FAD(ribose) site. Since the FAD binding motif participates in electron transfer within the reductase domain, these changes may affect NO production by altering the binding affinity for FAD.

NOS genes are regulated at the transcriptional, translational and post-translational levels (Hall et al.,1994; Kone, 2001; Wang et al., 1999). Numerous isoforms of human nNOS are produced by alternative promoters and alternative splicing and differ in tissue expression patterns(Kone, 2001; Wang et al., 1999). Multiple isoforms of Drosophila NOS (dNOS) result from alternative mRNA splicing; the truncated isoforms lack NOS activity and those that have sequences required for dimerization in the vicinity of the CaM-binding domain act as dominant negative regulators(Regulski and Tully, 1995; Stasiv et al., 2001). The dNOS isoforms are differentially expressed during Drosophila development(Stasiv et al., 2001). In the mosquito Anopheles stephensi, 18–22 NOS alternative transcripts are expressed (Luckhart and Li,2001). The number of alternative transcripts expressed in land crab has not been determined. Our preliminary results indicate that a truncated isoform was expressed in the Y-organ. In this cDNA, alternative splicing introduced a stop codon in the reductase domain, resulting in a polypeptide of 720 amino acids in length. This isoform may act as a dominant negative regulator, as it retains the dimerization sequence but lacks a complete reductase domain. It was not characterized further, because it was expressed at low levels in the Y-organ and thoracic ganglion.

In insects, the NO/cGMP signaling pathway is involved in such diverse functions as phototransduction, olfaction, neuronal development, ecdysis, food search behavior and epithelial fluid transport(Davies, 2000; Morton and Hudson, 2002). Until recently, NOS had only been reported in the nervous system of crustaceans, in which it is involved in regulating neuronal activity(Aonuma et al., 2000; Aonuma and Newland, 2002; Johansson and Carlberg, 1994; Johansson and Mellon, 1998; Lee et al., 2000; Scholz et al., 1998, 2001, 2002; Schuppe et al., 2001a,b, 2002; Talavera et al., 1995; Zou et al., 2002). In lobster,NO produced by the cardiac muscle reduces bursting activity of the cardiac gangion, which decreases heartbeat amplitude and frequency(Mahadevan et al., 2004). In addition to nervous tissue, we have shown that the land crab Y-organ, gill,testis and ovary express NOS by RT-PCR(Fig. 5) and immunohistochemistry (Figs 6, 7). However, we did not detect NOS mRNA in land crab heart, even after a second round of PCR with nested primers (Fig. 5B). Although this is the first report of NOS in these tissues from a crustacean,non-neuronal NOS has been reported in insect Malpighian tubules, imaginal discs and salivary glands (Davies et al.,1997; Kuzin et al., 1996, 2000; Ribeiro and Nussenzveig, 1993; Stasiv et al., 2001).

The presence of NOS in crustacean non-neuronal tissues suggests that NO signaling is involved in physiological processes in addition to neuromodulation. NOS was not detected in skeletal muscle(Fig. 5), although NO and cGMP increase inward Ca2+ current and both early and delayed outward K+ currents in skeletal muscle of a marine isopod(Erxleben and Hermann, 2001; Hermann and Erxleben, 2001). The functions of NOS in Y-organ, ovary, testis and gill are not known. In gonadal tissue, NOS may regulate gametogenesis and/or steroidogenesis. In blowfly, for example, steroid synthesis is inhibited by NO and cGMP. However,crustacean ovary accumulates ecdysteroid, but there is little evidence that it is a significant site for ecdysteroid synthesis(Gunamalai et al., 2003; Spaziani et al., 1997; Subramoniam, 2000; Suzuki et al., 1996; Warrier et al., 2001). The localization of NOS in connective tissue and epithelia in Y-organ and gill suggests that it functions in an immune response to pathogens. In insects,hemocytes express an NOS that is activated upon bacterial infection(Weiske and Wiesner, 1999). Plasmodium infection in mosquitoes leads to a rapid induction of NOS activity (Luckhart et al.,1998). The pillar cells, which extend across each lamella and anchor in the cuticle, restrict distension caused by hemolymph pressure(Copeland, 1968; Taylor and Taylor, 1992). The localization of NOS in tendinous and pillar cells suggests that NOS is involved in the regulation of blood pressure, as it is in vertebrates.

The presence of NOS in the Y-organ suggests that NO regulates ecdysteroidogenesis via the activation of a soluble NO-sensitive GC-I. A hypothetical pathway is presented in Fig. 8, which is consistent with the available data. Increased cAMP, cGMP and Ca2+ levels inhibit ecdysteroid synthesis in crustacean Y-organ (Spaziani et al., 1999, 2001). We propose that MIH binds to a G protein-coupled receptor, leading to activation of NOS by the combined effects of dephosphorylation by calcineurin and binding of CaM. The regulation of NOS by phosphorylation/dephosphorylation is complex. Several protein kinases phosphorylate NOS in different regions(Bredt et al., 1992), which may have different effects on enzyme activity(Kone, 2001; Nakane et al., 1991). For example, CaM kinase II phosphorylates nNOS at Ser847, which inhibits enzyme activity by reducing its binding affinity for CaM(Hayashi et al., 1999; Komeima et al., 2000). The NO activates a GC-I, resulting in an increase in cGMP. This is similar to the signaling mechanism that stimulates fluid secretion in DrosophilaMalpighian tubules by the decapeptide cardioacceleratory peptide 2b(Davies, 2000; Davies et al., 1995, 1997; Dow et al., 1994; Kean et al., 2002; MacPherson et al., 2001; Rosay et al., 1997) and inhibits steroidogenesis in blowfly ovary(Maniere et al., 2003). We have recently cloned a cDNA encoding a GC-I that is expressed in land crab Y-organ (H.-W.K. and D.L.M., data not shown). Studies are now in progress to determine the role of NOS and GC-I in the MIH signaling pathway.

This research was supported by a grant from the National Science Foundation(IBN-9904528). L.A.B. was supported in part by a Research Experience for Undergraduates supplement from NSF. We thank Hector C. Horta (Puerto Rico Department of Natural Resources) and Hector J. Horta for collecting land crabs; Marian Allen for technical assistance; Laura Baker, Emma James,Stephanie Spenny and Kristin Van Ort for animal care; and Dr Scott Medler for critical reading of the manuscript.

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