title: Localization and expression of molt-inhibiting hormone and nitric oxide synthase in the central nervous system of the green shore crab, Carcinus maenas, and the blackback land crab, Gecarcinus lateralis.
abstract: In decapod crustaceans, molting is controlled by the pulsatile release of molt-inhibiting hormone (MIH) from neurosecretory cells in the X-organ/sinus gland (XO/SG) complex in the eyestalk ganglia (ESG). A drop in MIH release triggers molting by activating the molting gland or Y-organ (YO). Post-transcriptional mechanisms ultimately control MIH levels in the hemolymph. Neurotransmitter-mediated electrical activity controls Ca2+-dependent vesicular release of MIH from the SG axon terminals, which may be modulated by nitric oxide (NO). In green shore crab, Carcinus maenas, nitric oxide synthase (NOS) protein and NO are present in the SG. Moreover, C. maenas are refractory to eyestalk ablation (ESA), suggesting other regions of the nervous system secrete sufficient amounts of MIH to prevent molting. By contrast, ESA induces molting in the blackback land crab, Gecarcinus lateralis. Double-label immunofluorescence microscopy and quantitative polymerase chain reaction were used to localize and quantify MIH and NOS proteins and transcripts, respectively, in the ESG, brain, and thoracic ganglion (TG) of C. maenas and G. lateralis. In ESG, MIH- and NOS-immunopositive cells were closely associated in the SG of both species; confocal microscopy showed that NOS was localized in cells adjacent to MIH-positive axon terminals. In brain, MIH-positive cells were located in a small number of cells in the olfactory lobe; no NOS immunofluorescence was detected. In TG, MIH and NOS were localized in cell clusters between the segmental nerves. In G. lateralis, Gl-MIH and Gl-crustacean hyperglycemic hormone (CHH) mRNA levels were ~105-fold higher in ESG than in brain or TG of intermolt animals, indicating that the ESG is the primary source of these neuropeptides. Gl-NOS and Gl-elongation factor (EF2) mRNA levels were also higher in the ESG. Molt stage had little or no effect on CHH, NOS, NOS-interacting protein (NOS-IP), membrane Guanylyl Cyclase-II (GC-II), and NO-independent GC-III expression in the ESG of both species. By contrast, MIH and NO receptor GC-I beta subunit (GC-Iβ) transcripts were increased during premolt and postmolt stages in G. lateralis, but not in C. maenas. MIH immunopositive cells in the brain and TG may be a secondary source of MIH; the release of MIH from these sources may contribute to the difference between the two species in response to ESA. The MIH-immunopositive cells in the TG may be the source of an MIH-like factor that mediates molt inhibition by limb bud autotomy. The association of MIH- and NOS-labeled cells in the ESG and TG suggests that NO may modulate MIH release. A model is proposed in which NO-dependent activation of GC-I inhibits Ca2+-dependent fusion of MIH vesicles with the nerve terminal membrane; the resulting decrease in MIH activates the YO and the animal enters premolt.
authors.affiliation: Department of Biology, Colorado State University, Fort Collins, CO 80523, USA.
authors.forename: Natalie L
authors.initials: NL
authors.lastname: Pitts
authors.affiliation: Department of Biology, Colorado State University, Fort Collins, CO 80523, USA. Electronic address: Donald.Mykles@ColoState.edu.
authors.forename: Donald L
authors.initials: DL
authors.lastname: Mykles
mesh_terms: D000818:Animals; D017756:Aquaculture; D060829:Arthropod Proteins / Q000235:genetics / Q000378:metabolism*; D001267:Atlantic Ocean; D003386:Brachyura / Q000254:growth & development / Q000502:physiology*; D002140:California; D002490:Central Nervous System / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D004293:Dominican Republic; D005123:Eye; D017952:Ganglia, Invertebrate / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D007447:Invertebrate Hormones / Q000235:genetics / Q000378:metabolism*; D008297:Male; D018978:Molting; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism*; D066194:Olfactory Cortex / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D009928:Organ Specificity; D010140:Pacific Ocean; D013045:Species Specificity; D013909:Thorax
chemical_list: D060829:Arthropod Proteins; D007447:Invertebrate Hormones; D009419:Nerve Tissue Proteins; C012445:molt-inhibiting hormone; D019001:Nitric Oxide Synthase
keywords: Crustacea; Gene expression; Immunofluorescence; Molt-inhibiting hormone; Neuropeptide secretion; Nitric oxide synthase