The Retina of Crayfish Procambarus clarkii During Development Shows Serotonin and Tryptophan Hydroxylase-like Immunoreactivity Daily Changes

The aim of this work was to investigate possible changes in serotonin (5-HT) and tryptophan hydroxylase (TPH)-like immunoreactivity related to time of day in Procambarus clarkii retina during the first developmental stages. Forty-five animals from postembryonic instars (PO1, PO2) to juvenile stage were kept under LD 12:12 cycles. All animals were anesthetized and decapitated at three times of day, 08:00, 15:00 and 20:00 h. Isolated eyestalks were processed by immunohistochemical methods. The 5-HT-like immunoreactive area of retina was measured using computer-based image analysis. Results indicated 5-HT-like immunoreactive differences among the three crayfish instars studied. In PO1 animals, ANOVA revealed no significant differences in 5-HT-like immunoreactivity in the retina at different times of day. PO2 instars as well as juvenile instars, showed statistically significant retinal 5-HT-like immunoreactivity changes related to time of day. Preliminary results indicated that TPH-like immunoreactivity was located only in the tapetal and retinal cells, and it was related to time of day. These changes suggest a diurnal cyclic regulation in the synthesis of 5-HT in the retina.     

Effect of Skeleton Photoperiods upon the Circadian Locomotor Activity Rhythm During Development in Crayfish Procambarus clarkii

This study was carried out to investigate the non-parametric effect of white light on the activity rhythm of the postembryonic stages of crayfish and to elucidate the characteristics and development of its entrainment mechanisms. Forty-five postembryonical instars of crayfish Procambarus clarkii, separated into three groups according to age and moult, were investigated. They were individually monitored with a motor activity recording system in constant photic conditions. For the three groups of animals, three trials with different skeleton photoperiod (SP) imitating LD 8:16, LD 12:12 and LD 20:4 were conducted, each followed by exposure to a period of constant darkness (DD). The locomotor activity rhythm and its entrainment ability were evaluated. All ages showed a circadian rhythm with a capability to synchronize to the different SP increasing with age. The three groups of crayfish analysed were generally synchronized by the dusk pulse, clustering the onset of activity close to the light pulses considered as dusk. The results presented in this study suggest the involvement of extraretinal pathways in the non-parametric entrainment of crayfish.     

Effect of Skeleton Photoperiods upon the Circadian Locomotor Activity Rhythm During Development in Crayfish Procambarus clarkii

This study was carried out to investigate the non-parametric effect of white light on the activity rhythm of the postembryonic stages of crayfish and to elucidate the characteristics and development of its entrainment mechanisms. Forty-five postembryonical instars of crayfish Procambarus clarkii, separated into three groups according to age and moult, were investigated. They were individually monitored with a motor activity recording system in constant photic conditions. For the three groups of animals, three trials with different skeleton photoperiod (SP) imitating LD 8:16, LD 12:12 and LD 20:4 were conducted, each followed by exposure to a period of constant darkness (DD). The locomotor activity rhythm and its entrainment ability were evaluated. All ages showed a circadian rhythm with a capability to synchronize to the different SP increasing with age. The three groups of crayfish analysed were generally synchronized by the dusk pulse, clustering the onset of activity close to the light pulses considered as dusk. The results presented in this study suggest the involvement of extraretinal pathways in the non-parametric entrainment of crayfish.     

Crayfish invading Europe: the case study of Procambarus clarkii

The red swamp crayfish, Procambarus clarkii, native to northeastern Mexico and southcentral USA, is today the dominant macroinvertebrate in several European countries. While the first introduction of this species into Spain is well-documented, little is known about its pathways of invasion and the reason for its rapid spread in several European countries. Study of the biology of the species has revealed a number of properties that makes this crayfish a successful invader. Procambarus clarkii exhibits properties characteristic of an r-selected species, including early maturity at small body size, rapid growth rates, large numbers of offspring at a given parental size, and relatively short life spans. It is also plastic in its life cycle, able to disperse widely in the habitat and to tolerate environmental extremes. It displays generalist and opportunistic feeding habits, consuming macrophytes and preying on amphibians. Procambarus clarkii can also replace indigenous crayfish by a combination of mechanisms, including competitive exclusion and transmission of the fungus-like Aphanomyces astaci, responsible for the crayfish plague. Finally, this species shows a wide behavioral flexibility when coping with new types of predators. The results of these studies, combined with the increasing information available in the scientific literature on this and other crayfish species, will help us understand invasions in this taxon and make predictions about the identity of future crayfish invaders.     

Crayfish invading Europe: the case study of Procambarus clarkii

The red swamp crayfish, Procambarus clarkii, native to northeastern Mexico and southcentral USA, is today the dominant macroinvertebrate in several European countries. While the first introduction of this species into Spain is well-documented, little is known about its pathways of invasion and the reason for its rapid spread in several European countries. Study of the biology of the species has revealed a number of properties that makes this crayfish a successful invader. Procambarus clarkii exhibits properties characteristic of an r-selected species, including early maturity at small body size, rapid growth rates, large numbers of offspring at a given parental size, and relatively short life spans. It is also plastic in its life cycle, able to disperse widely in the habitat and to tolerate environmental extremes. It displays generalist and opportunistic feeding habits, consuming macrophytes and preying on amphibians. Procambarus clarkii can also replace indigenous crayfish by a combination of mechanisms, including competitive exclusion and transmission of the fungus-like Aphanomyces astaci, responsible for the crayfish plague. Finally, this species shows a wide behavioral flexibility when coping with new types of predators. The results of these studies, combined with the increasing information available in the scientific literature on this and other crayfish species, will help us understand invasions in this taxon and make predictions about the identity of future crayfish invaders.     

Ontogeny of Cardiac and Ventilatory Function in the Crayfish Procambarus clarkii

SYNOPSIS. Ontog eny of cardiac and ventilatory function wasinvestigated in the direct developing crayfish Procambarus clarkiito determine basic developmental patterns and to evaluate diffusionaland convective gas exchange. Animals were exposed to water rangingin Po₂ from 150 to less than 10 mmHg. Ontogeny of cardiac functionfollows a pattern unlike that observed in other developing organisms.Heart rate (f_H) decreases from the mid-point of embryonic developmentuntil hatching, and the decrease in f_H is accompanied by a concomitantloss in cardiac and ventilatory sensitivity to hypoxia. Duringlarval development however, f_H increases until a juvenile stageis reached. Heart rate then decreases again as the animal increasesin mass. Cardiac and ventilatory responses to hypoxia are restoredby the third larval instar. Ventilatory function is initiatedwithin hours of hatching. Scaphognathite movement (f_sc), whichis initially uncoordinated, does not result in appreciable movementof water, but functional pumping is achieved within hours ofhatching. Animals do not exhibit an adult-like response to hypoxicexposure until at least the third larval instar. The ontogenyof both cardiac and ventilatory function indicates that thedirect developing crayfish is not physiologically mature untilan early juvenile stage. The drop in embryonic JFH and lossof hypoxic sensitivity late in development may indicate thatoxygen requirements of embryos exceed the capacity of egg membranescapacity (surface area) to supply oxygen by diffusion.     

鳌虾血细胞中一种新的astakine样造血因子基因的克隆及表达

甲壳动物的血细胞参与多种免疫反应,能够激活酚氧化酶原,产生抗菌肽或进行血细胞的封装、吞噬等. 本研究在克氏原螯虾血细胞中发现一种造血激素astakine,是与脊椎动物细胞因子前动力蛋白prokineticin同源的基因,命名为PcAst,该基因的开放阅读框为357 bp,编码118个氨基酸,具有保守的半胱氨酸残基. 半定量PCR结果显示,该基因只在血细胞中表达. 实时荧光定量PCR结果显示,在金黄色葡萄球菌和鳗弧菌刺激后,其mRNA表达量大幅上调. 白斑综合征病毒刺激后,该基因也呈持续上调表达趋势. 为进一步研究其功能,重组表达了PcAst蛋白,并对其蛋白水平表达模式进行了分析. 所有结果表明,该基因可能参与鳌虾血细胞的固有免疫应答反应,在抗细菌和抗病毒中起重要作用.     

Characterization of a Matrix Protein in the Gastroliths of the Crayfish Procambarus clarkii

As a first step in understanding the calcification mechanism, a matrix protein in the gastrolith of the crayfish Procambarus clarkii was purified and sequenced. The protein was insoluble in acid, but after trypsin digestion, it dissolved in 6 m urea. The trypsin-digested protein dissolved in urea solution was purified by reversed-phase HPLC and designated gastrolith matrix protein fragment. The fragment had a molecular weight of 9658 and a blocked amino terminus. It had tandemly repeated units not reported before at the central part of the sequence, with each unit being Gly-Ser-X1-X2-Phe as the most typical sequence. This peptide was found associated with chitin, a main component of the organic matrix.     

切除眼柄对加速克氏原螯虾卵巢发育进程的组织学研究

应用组织切片和透射电镜技术研究眼柄切除对加速克氏原螯虾(Procambarus clarkii)卵巢发育进程的影响。实验分成4组,选取眼柄切除15 d和30 d两个时间点进行。切除眼柄30 d后,雌虾体长增长显著,从(11.50±2.30)cm增长至(18.20±3.40)cm(P0.05),性腺指数(GSI)从4.56%±2.81%增长至8.05%±2.51%(P0.05),体重和螯肢长也有所增长(P0.05)。电镜观察发现卵母细胞从发育Ⅱ期迅速度过Ⅲ期进入到Ⅵ期阶段,卵母细胞体积变大,变圆;液泡压缩变小;卵黄颗粒加速积累,逐渐占满整个胞内空间;滤泡细胞胞质从黏稠变稀薄。这些结果表明,切除眼柄在短期内显著加快卵母细胞内卵黄颗粒的积累,并且加快个体生长。     

克氏原螯虾

从形态、习性、繁殖和幼体发育等7个方面介绍了克氏原螯虾的生物学特性,指出它对人类有益的方面,以及对水利、农业、淡水养殖业的危害及其可能造成食用者食物中毒的不利方面。     

Changes in the Amounts of the Molt-Inhibiting Hormone in Sinus Glands during the Molt Cycle of the American Crayfish, Procambarus clarkii

The purposes of this study are to determine the molt cycle of the American crayfish, Procambarus clarkii, and to quantify the amounts of the molt-inhibiting hormone (Prc-MIH) in the hemolymph and neurohemal sinus glands during the molt cycle of the American crayfish. The molt cycle was classified into six stages based on the changes in volumes of gastroliths in the stomach and ecdysteroid titers in the hemolymph. A sandwich-type enzyme immunoassay using specific antibodies raised against N-terminal and C-terminal segments of Prc-MIH was developed for the Prc-MIH assay. It is sensitive to as little as 0.5 fmol of Prc-MIH (3.3 x10(-12) M). In the hemolymph, no Prc-MIH could be detected at any of the molt stages tested. However, in the sinus gland, it was demonstrated that the amount of Prc-MIH changes in a molt-stage-specific manner during the molt cycle. It was particularly noteworthy that the initiation of a molting sequence (i.e., entering the early premolt stage) corresponded to the increase in Prc-MIH content in the sinus gland, because the finding is consistent with the hypothesis that crustaceans enter the premolt stage when the MIH secretion from the sinus gland is reduced or ceases.     

Purification and Structural Determination of a Phosphorylated Peptide with Anti-calcification and Chitin-binding Activities in the Exoskeleton of the Crayfish,Procambarus clarkii

Organic matrices in calcified hard tissues have been considered to control calcification. A matrix peptide, designated CAP-1, was extracted and purified by anionexchange and reverse-phase high performance liquid chromatographies from the exoskeleton of the crayfish, Procambarus clarkii. The amino acid sequence of CAP-1 was determined by mass spectral and sequence analyses of the intact peptide and its enzymatically digested peptides. CAP-1 consisted of 78 amino acid residues, including a phosphoserine residue, and was rich in acidic amino acid residues. CAP-1 had a RebersRiddiford consensus sequence, which is conserved in cuticle proteins from many arthropods. CAP-1 inhibited precipitation of calcium carbonate in an in vitro anticalcification assay dose-dependently, and completelyinhibited it at 3×10^-7 M. CAP-1 also showed chitinbinding ability, indicating that this molecule was bifunctional and played an important role in formation of the exoskeleton.     

Prohibitin Interacts with Envelope Proteins of White Spot Syndrome Virus and Prevents Infection in the Red Swamp Crayfish,Procambarus clarkii

Prohibitins (PHBs) are ubiquitously expressed conserved proteins in eukaryotes that are associated with apoptosis, cancer formation, aging, stress responses, cell proliferation, and immune regulation. However, the function of PHBs in crustacean immunity remains largely unknown. In the present study, we identified a PHB in Procambarus clarkii red swamp crayfish, which was designated PcPHB1. PcPHB1 was widely distributed in several tissues, and its expression was significantly upregulated by white spot syndrome virus (WSSV) challenge at the mRNA level and the protein level. These observations prompted us to investigate the role of PcPHB1 in the crayfish antiviral response. Recombinant PcPHB1 (rPcPHB1) significantly reduced the amount of WSSV in crayfish and the mortality of WSSV-infected crayfish. The quantity of WSSV in PcPHB1 knockdown crayfish was increased compared with that in the controls. The effects of RNA silencing were rescued by rPcPHB1 reinjection. We further confirmed the interaction of PcPHB1 with the WSSV envelope proteins VP28, VP26, and VP24 using pulldown and far-Western overlay assays. Finally, we observed that the colloidal gold-labeled PcPHB1 was located on the outer surface of the WSSV, which suggests that PcPHB1 specifically binds to the envelope proteins of WSSV. VP28, VP26, and VP24 are structural envelope proteins and are essential for attachment and entry into crayfish cells. Therefore, PcPHB1 exerts its anti-WSSV effect by binding to VP28, VP26, and VP24, preventing viral infection. This study is the first report on the antiviral function of PHB in the innate immune system of crustaceans.     

克氏原螯虾(Procambarus clarkii)血细胞染色方法的研究

甲壳类血细胞的形态和分类是甲壳类免疫学研究的基础.本文选择三种甲壳类血细胞的常用染料:瑞氏染液、姬姆萨染液和瑞氏-姬姆萨混合染液,研究它们对于克氏原螯虾血细胞的染色效果.通过改变染色时间、染色温度、分色方式以及分色时间等染色条件,观察、比较不同染色条件下血细胞内的颗粒、细胞核和细胞质的着色情况,以及细胞整体轮廓清晰程度,确定适用于克氏原螯虾血细胞染色的理想染色方法,并建立相应的操作程序.     

The effects of acute carbon dioxide on behavior and physiology in Procambarus clarkii

Vertebrates and invertebrates show a similar response of rapid anesthesia with high levels of carbon dioxide. In this study, we use crayfish to examine both behavioral and physiological responses to increasing [CO(2)] to explain the rapid unresponsiveness and cessation of an autonomic response. Hypoxic and low pH environments that are induced by CO(2) exposure were also examined, although neither produced the identified CO(2) effects. In insects, low concentrations play a vital role in providing information for task performance such as food location through attraction cues, whereas high concentrations produce avoidance responses. We found behavioral responses in crayfish that demonstrate a strong repellent effect to high [CO(2)] and that the avoidance behavior decreases with lower [CO(2)]. There was not a preference and/or repellent behavioral response with 5% CO(2), hypoxic or low pH environments. Mechanosensory stimulation showed that only at high [CO(2)] there is an unresponsiveness to stimuli within a 30?min time period. Additionally, the autonomic bioindex of heart and ventilatory rates showed a complete cessation with high acute exposure within in the same time period for unresponsiveness to mechanosensory stimulation.     

Postmortem and Regional Changes of Serotonin, 5-Hydroxyindoleacetic Acid, and Tryptophan in Brain

Using a specific and sensitive high pressure liquid chromatographic technique for the measurement of serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), and tryptophan (TRP), we found that there were no changes in 5-HT or 5-HIAA in the rat cortex when left in situ for 6 h at room temperature or 24 h at 4 degrees C. Only a minimal 14% increase in 5-HT was observed after 24 h at 4 degrees C in the striatum of the same animals. Concentrations of TRP, however, were increased significantly in both brain regions by these postmortem delay procedures. A second study revealed that there were significant regional 5-HT and 5-HIAA concentration differences within the cerebral cortex. The frontal cortex was shown to have the highest concentrations of 5-HT and 5-HIAA. Further, within the frontal cortex, 5-HIAA levels varied, showing apparent progressive rostral to caudal increases. 5-HT concentrations, however, remained constant within the frontal cortex. These results are discussed in reference to the conflicting reports of the previous human suicide and postmortem studies.     

Changes in the Daily Rhythm of Lipid Metabolism in the Diabetic Retina

Disruption of circadian regulation was recently shown to cause diabetes and metabolic disease. We have previously demonstrated that retinal lipid metabolism contributed to the development of diabetic retinopathy. The goal of this study was to determine the effect of diabetes on circadian regulation of clock genes and lipid metabolism genes in the retina and retinal endothelial cells (REC). Diabetes had a pronounced inhibitory effect on the negative clock arm with lower amplitude of the period (per) 1 in the retina; lower amplitude and a phase shift of per2 in the liver; and a loss of cryptochrome (cry) 2 rhythmic pattern in suprachiasmatic nucleus (SCN). The positive clock arm was increased by diabetes with higher amplitude of circadian locomotor output cycles kaput (CLOCK) and brain and muscle aryl-hydrocarbon receptor nuclear translocator-like 1 (bmal1) and phase shift in bmal1 rhythmic oscillations in the retina; and higher bmal1 amplitude in the SCN. Peroxisome proliferator-activated receptor (PPAR) α exhibited rhythmic oscillation in retina and liver; PPARγ had lower amplitude in diabetic liver; sterol regulatory element-binding protein (srebp) 1c had higher amplitude in the retina but lower in the liver in STZ- induced diabetic animals. Both of Elongase (Elovl) 2 and Elovl4 had a rhythmic oscillation pattern in the control retina. Diabetic retinas lost Elovl4 rhythmic oscillation and had lower amplitude of Elovl2 oscillations. In line with the in vivo data, circadian expression levels of CLOCK, bmal1 and srebp1c had higher amplitude in rat REC (rREC) isolated from diabetic rats compared with control rats, while PPARγ and Elovl2 had lower amplitude in diabetic rREC. In conclusion, diabetes causes dysregulation of circadian expression of clock genes and the genes controlling lipid metabolism in the retina with potential implications for the development of diabetic retinopathy.