The tetrapeptide FMRFamide and FMRFamide-related peptides (FaRPs) are widely distributed among invertebrates and vertebrates and form a large neuropeptide family with more than 50 members all of which share the RFamide motif (reviews: [121 -127 (link)]). In malacostracan Crustacea, at least twelve FaRPs have been identified and sequenced from crabs, shrimps, lobsters and crayfish [128 (link),129 (link)]. These peptides range from seven to twelve amino acids in length and most of them share the carboxy terminal sequence LRFamide. The antiserum we used was generated in rabbit against synthetic FMRFamide (Phe-Met-Arg-Phe-NH2) conjugated to bovine thyroglobulin (DiaSorin, Cat. No. 20091, Lot No. 923602). According to the manufacturer, staining with this antiserum is completely eliminated by pretreatment of the diluted antibody with 100 μg/ml of FMRFamide. We repeated this experiment and preincubated the antiserum with 100 μg/ml FMRFamide (Sigma; 16 h, 4°C) and this preincubation abolished all staining. Because the crustacean FaRPs know so far all share the carboxy terminal sequence LRFamide we conclude that the DiaSorin antiserum that we used most likely labels any peptide terminating with the sequence RFamide. Therefore, we will refer to the labeled structures in our specimens as "RFamide-like immunoreactive (RFir) neurons" throughout the paper.
The antiserum against serotonin (ImmunoStar Incorporated; Cat. No. 20080, Lot No. 541016) is a polyclonal rabbit antiserum raised against serotonin coupled to bovine serum albumin (BSA) with paraformaldehyde. The antiserum was quality control tested by the manufacturer using standard immunohistochemical methods. According to the manufacturer, staining with the antiserum was completely eliminated by pretreatment of the diluted antibody with 25 μg of serotonin coupled to BSA per ml of the diluted antibody. We repeated this control with the serotonin-BSA conjugate that was used for generation of the antiserum as provided by ImmunoStar (Cat. No. 20081, Lot No. 750256; 50 μg of lyophilized serotonin creatinine sulfate coupled to BSA with paraformaldehyde). Preadsorption of the antibody in working dilution with the serotonin-BSA conjugate at a final conjugate concentration of 10 μg/ml at 4°C for 24 h completely blocked all immunolabelling. We performed an additional control and preadsorbed the diluted antiserum with 10 mg/ml BSA for 4 h at room temperature. This preadsorption did not affect the staining, thus, providing evidence that the antiserum does not recognize the carrier molecule alone. The manufacturer also examined the cross reactivity of the antiserum. According to the data sheet, with 5 μg, 10 μg, and 25 μg amounts, the following substances did not react with the antiserum diluted to 1:20,000 using the horse radish peroxidase (HRP) labeling method: 5-hydroxytryptophan, 5-hydroxyindole-3-acetic acid, and dopamine.
The monoclonal mouse anti-Drosophila synapsin „SYNORF1“ antibody (provided by E. Buchner, Universität Würzburg, Germany) was raised against a Drosophila GST-synapsin fusion protein and recognizes at least four synapsin isoforms (ca. 70, 74, 80, and 143 kDa) in western blots of Drosophila head homogenates [120 (link)]. In western blot analysis of crayfish homogenates, this antibody stains a single band at ca. 75 kDa (see [130 (link)]). We conducted a western blot analysis comparing brain tissue of Drosophila and Coenobita. The antibody provided identical results for both species staining one strong band around 80–90 kDa and a second weaker band slightly above 148 kDa (Fig.20 ). Our analysis strongly suggests that the epitope which SYNORF 1 recognizes is strongly conserved between the fruit fly and the hermit crab. Similar to Drosophila, the antibody consistently labels brain structures in representatives of all major subgroups of the malacostracan crustaceans [42 (link),131 (link)-134 (link)] in a pattern that is consistent with the assumption that this antibody does in fact label synaptic neuropil in Crustacea. In the crustacean first optic neuropil (the lamina), synapsin labeling is weak compared to the other brain neuropils ([131 (link)]; and present report). Similarly, in Drosophila labeling of the lamina is weak because photoreceptors R1–R6 which have their synapses in the lamina contain very little of the presently known synapsin homolog isoforms [120 (link)]. The antibody also labels neuromuscular synapses both in Drosophila and in Crustacea [131 (link)]. These close parallels in the labeling pattern of SYNORF1 between Drosophila and various Crustacea strengthens the claim that it also recognizes crustacean synapsin homologs. This antibody even labels synaptic neuropil in an ancestral clade of protostomes, the Chaetognatha [135 (link)] suggesting that the epitope that this antiserum recognizes is conserved over wide evolutionary distances.
The monoclonal mouse anti-glutamine synthetase antibody (1:100; BD Biosciences Pharmingen, Cat. No. 610517) was generated using sheep glutamine synthetase, an octamer of identical 45 kDa subunits, as the immunogen. According to the manufacturer, this antibody labels a single 45 kDa in Western blot analysis of rat brain homogenates. In Western blots of crayfish (Procambarus clarkii) brain homogenates, the antibody labels a single band at ca. 44 kDa (see [130 (link)]) which is in the same range as the glutamine synthetase in the spiny lobster Panulirus argus (42 kDa; [55 (link)]) suggesting that the antibody that we used also binds to crustacean glutamine synthetase. Because we did not conduct a western blot analysis in C. clypeatus, we will refer to the labeled structures in our specimens as "glutamine synthetase-like immunoreactivity" (GSir) throughout the paper.
In additional control experiments for possible nonspecific binding of the secondary antiserum, we omitted the primary antiserum, replaced it with blocking solution, and followed the labeling protocol as above. In these control experiments, staining was absent.
The antiserum against serotonin (ImmunoStar Incorporated; Cat. No. 20080, Lot No. 541016) is a polyclonal rabbit antiserum raised against serotonin coupled to bovine serum albumin (BSA) with paraformaldehyde. The antiserum was quality control tested by the manufacturer using standard immunohistochemical methods. According to the manufacturer, staining with the antiserum was completely eliminated by pretreatment of the diluted antibody with 25 μg of serotonin coupled to BSA per ml of the diluted antibody. We repeated this control with the serotonin-BSA conjugate that was used for generation of the antiserum as provided by ImmunoStar (Cat. No. 20081, Lot No. 750256; 50 μg of lyophilized serotonin creatinine sulfate coupled to BSA with paraformaldehyde). Preadsorption of the antibody in working dilution with the serotonin-BSA conjugate at a final conjugate concentration of 10 μg/ml at 4°C for 24 h completely blocked all immunolabelling. We performed an additional control and preadsorbed the diluted antiserum with 10 mg/ml BSA for 4 h at room temperature. This preadsorption did not affect the staining, thus, providing evidence that the antiserum does not recognize the carrier molecule alone. The manufacturer also examined the cross reactivity of the antiserum. According to the data sheet, with 5 μg, 10 μg, and 25 μg amounts, the following substances did not react with the antiserum diluted to 1:20,000 using the horse radish peroxidase (HRP) labeling method: 5-hydroxytryptophan, 5-hydroxyindole-3-acetic acid, and dopamine.
The monoclonal mouse anti-Drosophila synapsin „SYNORF1“ antibody (provided by E. Buchner, Universität Würzburg, Germany) was raised against a Drosophila GST-synapsin fusion protein and recognizes at least four synapsin isoforms (ca. 70, 74, 80, and 143 kDa) in western blots of Drosophila head homogenates [120 (link)]. In western blot analysis of crayfish homogenates, this antibody stains a single band at ca. 75 kDa (see [130 (link)]). We conducted a western blot analysis comparing brain tissue of Drosophila and Coenobita. The antibody provided identical results for both species staining one strong band around 80–90 kDa and a second weaker band slightly above 148 kDa (Fig.
The monoclonal mouse anti-glutamine synthetase antibody (1:100; BD Biosciences Pharmingen, Cat. No. 610517) was generated using sheep glutamine synthetase, an octamer of identical 45 kDa subunits, as the immunogen. According to the manufacturer, this antibody labels a single 45 kDa in Western blot analysis of rat brain homogenates. In Western blots of crayfish (Procambarus clarkii) brain homogenates, the antibody labels a single band at ca. 44 kDa (see [130 (link)]) which is in the same range as the glutamine synthetase in the spiny lobster Panulirus argus (42 kDa; [55 (link)]) suggesting that the antibody that we used also binds to crustacean glutamine synthetase. Because we did not conduct a western blot analysis in C. clypeatus, we will refer to the labeled structures in our specimens as "glutamine synthetase-like immunoreactivity" (GSir) throughout the paper.
In additional control experiments for possible nonspecific binding of the secondary antiserum, we omitted the primary antiserum, replaced it with blocking solution, and followed the labeling protocol as above. In these control experiments, staining was absent.
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