Optical and Paramagnetic Properties of Size-Controlled Ink Particles Isolated from Sepia officinalis

The optical and paramagnetic properties of size-controlled ink particles isolated from ink sacs of Sepia officinalis were investigated. Topographic images of atomic force microscopy (AFM) revealed that the average heights of the large and small ink particles were 156 nm and 5.3 nm respectively. The ultraviolet-visible (UV-VIS) spectral features of aqueous solutions of ink particles were dependent on particle size. Electron spin resonance (ESR) spectra suggested that the ink particles are highly pure for paramagnetic species and are of reliable quality. These size-controlled ink particles are suitable for a basic study of melanin-related materials.     

饥饿和再投喂对虎斑乌贼幼体存活、生长和消化酶活力的影响

本文探究了饥饿胁迫与饥饿后再投喂对虎斑乌贼幼体存活率、生长、行为、肝体比、摄食率以及消化酶活力的影响.在室内控制条件下开展了幼体(初始体质量为4.95±0.48 g)的饥饿(0、1、2、3、4、5、6 d)和再投喂(15 d)试验.结果表明: 不同饥饿时间对虎斑乌贼的幼体存活率、体质量降低率、肝体比和消化酶活力影响显著.随着饥饿胁迫时间的增加,其存活率、肝体比呈下降趋势,其中饥饿3 d后,存活率开始明显下降,体质量降低率明显增大,幼体出现喷墨、互相残杀等异常行为;4种消化酶活力呈先下降后上升的趋势,淀粉酶活力以饥饿4 d组最低 (0.07±0.02 U·mg^-1·prot^-1);脂肪酶活力以饥饿2 d组最低(18.47±2.07 U·g^-1·prot^-1),饥饿6 d组最高(57.60±3.98 U·g^-1·prot^-1),胃蛋白酶活力和胰蛋白酶活力以饥饿5 d组(1.98±0.59 U·mg^-1·prot^-1)和饥饿4 d(186.68±20.72 U·mg^-1·prot^-1) 最低.饥饿处理结束后,经15 d再投喂,各试验组存活率、特定生长率、肝体比和摄食率差异显著,幼体的存活率、特定生长率、肝体比和摄食率均与饥饿处理时间呈负相关;饥饿1和2 d组与对照组的存活率、特定生长率和肝肝体比无显著差异,而饥饿3～6 d组显著低于对照组;饥饿1～2 d组的摄食率明显高于对照组,而饥饿6 d组的摄食率明显小于对照组;各组淀粉酶与脂肪酶活力无显著差异,胃蛋白酶与胰蛋白酶活力差异显著,均以对照组最高(胃蛋白酶活力7.06±0.64 U·mg^-1·prot^-1,胰蛋白酶活力914.67±26.54 U·mg^-1·prot^-1),饥饿6 d组最低(胃蛋白酶活力3.21±0.57 U·mg^-1·prot^-1,胰蛋白酶活力660.04±37.92 U·mg^-1·prot^-1).说明虎斑乌贼的幼体饥饿不可逆点(PNR)为第6天,且不能补偿生长.     

Elektronenmikroskopische und histochemische Untersuchungen zur Funktion des Kiemenherzanhanges (Pericardialdrüse) von Sepia officinalis

Zusammenfassung Die elektronenmikroskopischen Untersuchungen am Kiemenherzanhang von Sepia officinalis zeigen, daß diesem Organ wie den Venenanhängen exkretorische Funktionen zukommen. Die Pericardialdrüse erweist sich als ein Derivat des Kiemenherzens. Ihre Hauptmasse besteht aus einem polar organisierten Faltenepithel, dessen Ultrastrukturmerkmale (basaler Faltenapparat, Gehalt an Mitochondrien und Lysosomen, sekretorisch aktiver Mikrovillisaum) anderen Exkretionsorganen entsprechend auf aktive Ionentransportvorgänge hindeuten. Es ist dem äußeren Kiemenherzepithel homolog und kommt in ähnlicher Ausbildung auch in anderen Organen (Zentralherz, Kieme) vor. Die polygonalen Zellen der Randzone sind dem zentralen Gewebe des Kiemenherzens zu homologisieren; ihr Reusensystem in Nachbarschaft der Blutlakunen ist ähnlich dem der Podocyten in der Vertebratenniere und macht eine Druckfiltration wahrscheinlich. Als Filtermembranen funktionieren neben dem Plasmalemm auch noch die Basalmembran und Zwischenmembran, die porenartige, für Myofer durchlässige Lücken von 30 Å Weite zeigen. Daneben werden an der Grenze zwischen Epithel und Lakune wie auch in der Intima größerer Gefäße besondere Dichtezellen dargestellt, die ähnlich wie Macrophagen größere Partikel (z.B. Ferritin und Myofer) aufnehmen und intraplasmatisch als dense bodies ablagern.Die enzymhistochemischen Befunde über die Verteilung von G-6-PD, SDH, IDH, MDH und MAO und der sauren und alkalischen Phosphatase in Faltenepithel und Lakunen stützen diese Befunde und sprechen ebenfalls für eine exkretorische Funktion.

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Electron microscopic and histochemical studies on the function of the branchial heart appendage (pericardial gland) in Sepia officinalis (l.)

Summary Our electron microscopic studies of the appendages of the branchial heart and veins of Sepia officinalis suggest an excretory function for these organs. The pericardial gland appears to be a derivate of the branchial heart; it mostly consists of a folded polarized epithelium. Ultrastructural features of the epithelium such as the basal folding system, contents of mitochondria and lysosomes, and secretory microvilli suggest active ion transport in these cells of the pericardial gland. The pericardial gland epithelium is homologous with the external epithelium of the branchial heart; it is also present in a similar form in the central heart and branchia. Polygonal cells present in the peripheral zone of the pericardial gland are homologous with the central branchial heart tissue. The system of basal cell processes of the polygonal cells near blood lacunae is similar to the podocyte-capillary relationship in the vertebrate kidney and suggests the possibility of pressure filtration. The plasmalemma, as well as the basal and intermediate membranes, appear to be filtration membranes; they contain pore-like structures about 30 Å in diameter which are permeable to Myofer. Furthermore, special dense cells are present at the epithelium-lacunae border and in the intima of larger vessels; these cells, similar to macrophages, take up large particles, such as ferritin and Myofer, and deposit them in the cytoplasm as dense bodies.Histochemical findings on the distribution of enzymes G-6-PD, SDH, IDH, MDH, and MAO, and the distribution in the folded epithelium and lacunae of alkaline and acid phosphatases confirm the ultrastructural results and suggest an excretory function of the pericardial gland.

Mit Unterstützung durch die Deutsche Forschungsgemeinschaft. — Frau Beyerle-von Wehren sei für wertvolle technische Unterstützung gedankt.     

Melanin Standard Method: Titrimetric Analysis

Melanin isolated from the ink sac of Sepia officinalis (Sepia melanin) has been proposed as a standard for natural eumelanin, and a standard mild isolation and purification protocol for Sepia melanin has been developed (Zeise, doctoral dissertation, Johns Hopkins University, 1991). The goal of the present work, developed using Sepia melanin, was to quantify the bioavailable carboxylic acid groups present in melanin particles. Bioavailability is governed by the accessibility of carboxy groups to the surrounding biological milieu, and is expressed as microequivalents of carboxy group per gram of melanin. The present work was carried out using an heterogeneous slurry of melanin in a nonaqueous system. A standard acidic titrant, and an automatic titrator operating in an equilibrium titration mode were used to characterize and quantify the carboxy group content of Sepia melanins and several other commonly used melanins purified by a standard method (Zeise et al., Pigment Cell Res. [Suppl] 2:48–53, 1992).     

Functional response of early stages of the cuttlefish Sepia officinalis preying on the mysid Mesopodopsis slabberi

The functional response of Sepia officinalis early stages, preying on mysids of the species Mesopodopsis slabberi was investigated. The effects of five prey densities (12.5, 25, 37.5, 50 and 125 mysids l^-1) and two hatchling ages (1-day-old and 7-day-old) on consumption rate and the frequency of non-feeding animals were tested. Older animals were approximately 50% heavier than newly hatched ones. Hatchlings were individually assayed under 0.25 W m^-2 natural light, 37.8 psu in salinity and 19°C. The effect of prey density on consumption rate was highly significant and no effect of age was detected within the age range tested. Maximal values recorded for consumption rate were about 0.45 mysids h^-1. The frequency of non-feeding individuals was strongly reduced at saturating prey densities. The functional response curve showed an interval of prey densities for which density-dependent prey mortality is probable.     

Molecular analysis of a novel FMRFamide-related peptide gene (SOFaRP(2)) and its expression pattern in the brain of the European cuttlefish Sepia officinalis

FMRFamide-related peptides (FaRPs) are among several neurotransmitters known to regulate the chromatophore function in the European cuttlefish Sepia officinalis. Here we report the cloning and sequencing of a novel S. officinalis FaRP gene (SOFaRP(2)). The complete 835-base pair cDNA sequence of the SOFaRP(2) gene contains an open reading frame of 567 base pairs encoding 188 amino acids and four putative FaRPs, NSLFRFamide, GNLFRFamide, TIFRFamide and PHTPFRFamide. All except TIFRFamide cause chromatophore expansion when assayed in an in vitro chromatophore bioassay. To investigate the expression pattern of SOFaRP(2) gene in the cuttlefish brain, in situ hybridization was performed using a full length RNA probe. The SOFaRP(2) gene was expressed primarily in the posterior chromatophore, anterior chromatophore, lateral basal and optic lobes among other brain locations. The SOFaRP(2) gene appears to be expressed in all brain regions involved in chromatophore regulation. These data suggests that some or all of the four FaRPs encoded by SOFaRP(2) might be involved in controlling chromatophore activity in cuttlefish.     

拟目乌贼幼体日摄食量及其对体重影响

在人工养殖条件下,对拟目乌贼幼体的日摄食量和日增重率进行了分析。结果显示,在水温、盐度、pH等条件相对稳定的环境下,10～15d的拟目乌贼幼体平均日摄食虾苗(体长0.5mm)的条数分别是8、11、12、13、78、83条,在这种摄食量下,拟目乌贼幼体的日增重率分别为6.8%、5.68%、6.44%、8.81%、12.5%。其胴长、体重的生长符合S型曲线生长规律。     

Egg capsule secretion in invertebrates: a new ovarian regulatory peptide identified by mass spectrometry comparative screening in Sepia officinalis

Mass spectrometry comparative screening was used to identify ovarian regulatory peptides involved in the successive steps of egg-laying in the cuttlefish Sepia officinalis. The peptide content of full-grown oocytes (FGO) was compared with that of oocyte-conditioned medium, which resulted in the detection of peptides that were present in both samples. These peptides, which are suspected of being released by the oocyte in the genital tract, were submitted to a structural analysis. This strategy led to the characterization of a new ovarian regulatory peptide (EISLDKD) able to inhibit the contractions of the whole female genital tract and of the main nidamental glands (MNG). As EISLDKD appeared to be the first regulatory peptide directly involved, at physiological concentrations, in the secretion of the egg capsule by the main nidamental glands, it was named SepCRP for Sepia Capsule Releasing Peptide. Mass spectrometry analysis clearly demonstrated that SepCRP was expressed during vitellogenesis by the ovarian follicles and released by the FGO in the lumen of the female genital tract. In association with the ovarian 5-HT, SepCRP would be responsible for the storage of FGO to avoid the spawning of unfertilized oocytes before mating. Released by the distal oviduct in the mantle cavity, SepCRP probably in association with a cocktail of ovarian regulatory factors targets the MNG to regulate the egg capsule secretion. Thus, the ovary appeared to be one of the main sources of regulatory peptides involved in the successive steps of egg-laying in the cephalopod mollusk S. officinalis.     

Toxicity of melanin-free ink of Sepia officinalis to transformed cell lines: identification of the active factor as tyrosinase

The melanin-free ink of the cephalopod Sepia officinalis is shown to contain a heat labile proteinaceous component toxic to a variety of cell lines, including PC12 cells. Gel filtration chromatography indicated that the toxic component was concentrated in those fractions eluted at a molecular weight higher than 100 kDa and exhibiting the highest tyrosinase activity. SDS-PAGE analysis of the active fractions displayed a single major band migrating at an approximate molecular weight of 100 kDa, identical with that of the single tyrosinase band in the melanin-free ink. These data unambiguously demonstrated the identity of the toxic component with tyrosinase. Treatment of purified Sepia as well as of mushroom tyrosinase with an immobilized version of proteinase K resulted in a parallel loss of tyrosinase activity and cytotoxicity. Sepia apotyrosinase was ineffective in inducing cytotoxicity in PC12 cells. Purified Sepia tyrosinase was found to induce a significant increase in caspase 3 activity in PC12 cells, leading eventually to an irreversible apoptotic process. Overall, these results disclose a hitherto unrecognized property of tyrosinase that may lead to a reappraisal of its biological significance beyond that of a mere pigment producing enzyme.     

Fertilization in Sepia officinalis: the first mollusk sperm-attracting peptide

Egg mass extract was used to characterize regulatory peptides, involved in the successive steps of egg-laying of the cuttlefish Sepia officinalis. Among these peptides, a C-terminally amidated hexapeptide revealed a sperm-attracting activity. MALDI-TOF MS (matrix-assisted laser desorption ionization-time of flight mass spectrometry) and Edman degradation led to a peptide of m/z 596.6 and the following primary sequence: Pro-Ile-Asp-Pro-Gly-Val-CO(NH2). From concentrations as low as 10(-17)M, the PIDPGVamide was able to attract freshly dissected spermatozoa. Nano-ESI-Q-TOF MS (nano-electrospray ionization-quadrupole-time-of-flight mass spectrometry) analysis established the quantitative occurrence of this peptide in different egg structures. The PIDPGVamide appears to be synthesized in oocytes during vitellogenesis and released by the embedded oocytes in the external media during egg-laying to facilitate fertilization by increasing chances of gamete collision. This novel peptide called SepSAP for Sepia sperm attracting peptide is the first sperm-attracting peptide, identified in mollusks or even in protostomians.