Study of circadian activity in the crayfish <Emphasis Type="Italic">Pontastacus Leptodactylus</Emphasis> during their multimonth maintenance in the river water flow

By the method of non-invasive on-line recording and processing of photoplethysmograms of testaceous invertebrates, the circadian rhythm of cardioactivity was studied in crayfish Pontastacus leptodactylus by recording for several month of the heart rate (HR) and stress-index (characteristics of variational pulsometry). The crayfish were kept in the natural running water in regime of the natural illumination alternation (the first group) or at constant artificial illumination of low intensity (the second group). The circadian rhythm was more frequent and more pronounced in crayfish of the first group. The criteria were established to determine the appearance and stabilization of the nocturnal, active rhythm phase: an increase of HR by more than 30% as compared with the daytime rest period and duration of such increase for at least 2.5 h. The stress-index has been shown to be a reliable parameter of the beginning of the nocturnal phase of cardioactivity, while preservation of the typical circadian rhythm can be considered as a bioindicator in the biomonitoring systems of the quality of surface waters.     

Total protein in hemolymph of crawfish <Emphasis Type="Italic">Pontastacus leptodactylus</Emphasis> as a parameter of the functional state of animals and a biomarker of quality of habitat

Analysis of total protein in hemolymph was performed by Lowry method on sexually mature crawfish Pontastacus leptodactylus. The total protein content in the crawfish hemolymph was studied for one year. The protein concentration varied widely, amounted to from 12 to 95 mg/ml, and depended on season and the molting cycle phase. There are presented histograms for distribution of animals for the protein level during different seasons and their character is analyzed. In summer the amount of protein is maximal prior to molt and decreases by 40% at once after it. There is studied the diapason of total protein concentrations in hemolymph, in which survival of crawfish at unfavorable changes in habitat is maximal. The adaptive possibilities of crawfish with the low protein content are attenuated. The crawfish with the protein concentration in hemolymph lower than the «critical» one were submitted for different time by action of hydroquinone (1 g/l) used as a model toxicant. A brief action did not affect the protein content in hemolymph. At a long toxic action the protein level in hemolymph fell, on average, by 40%, which preceded death of the animals. Possible mechanisms of positive correlation of the protein concentration in the crawfish hemolymph and of their survival at deterioration of quality of the water medium are discussed.     

Total protein in crawfish hemolymph as a parameter of functional state of animals and biomarker of quality of habitat

Analysis of total protein in hemolymph was performed by Lowry method on sexually mature crawfish Pontastacus leptodactylus. The total protein content in the crawfish hemolymph was studied for one year. The protein concentration varied widely, amounted to from 12 to 95 mg/ml, and depended on season and the moulting cycle phase. There are presented histograms for distribution of animals for the protein level for different seasons and their character is analyzed. In summer the amount of protein is maximal prior to moult and decreases by 40 % at once after it. There is studied the diapason of total protein concentrations in hemolymph, in which survival of crawfish at unfavorable changes in habitat is maximal. The adaptive possibilities of crawfish with the low protein content are reduced. The crawfish with the protein concentration in hemolymph lower than the "critical" one were submitted for different time by action of hydroquinone (1 g/l) used as a model toxicant. A brief action did not affect the protein content in hemolymph. At a long toxic action the protein level in hemolymph fell, on average, by 40%, which preceded the death of the animals. Possible mechanisms of positive correlation of the protein concentration in the crawfish hemolymph and of their survival at deterioration of quality of the water medium are discussed.     

Heart rate within male crayfish: social interactions and effects of 5-HT

Behaviors, such as those that establish dominant and subordinate social status, are thought to be driven by various neuromodulators and hormones. In crustaceans, the level of serotonin (5-HT) in the hemolymph is correlated with degree of aggressiveness. The crustacean heart is neurogenic and is modulated by neural secretion of 5-HT in the hemolymph, which bathes the cardiac tissue. We discuss and present the results of measuring heart rate (HR) of crayfish during interactions, as an indication of their state of excitability. HR is the result of multiple influences: a cocktail of hormones and modulators. HR was monitored during the periods in which crayfish established aggressive and submissive social status, during sham injections, and following injections of various doses of 5-HT. Crayfish, during an interaction to establish social status, can increase HR. Both the aggressive and submissive crayfish can dampen their HR within seconds during a pause in the interaction, while still posturing in an aggressive or submissive state. Injections of 5-HT to obtain systemic levels of approximately 100 nM-10 microM increase HR substantially for hours. This suggests that aggressive interactions and the establishment of a dominant posture may not be related to large increases in the free concentrations of 5-HT within the circulating hemolymph, since a sustained HR is not observed in aggressive animals. Instead, the results may demonstrate that inhibitory cardiac regulation is present in the aggressors during interactions and that a regulator is possibly 5-HT.     

Changes in heart rate and circadian cardiac rhythm as physiological biomarkers for estimation of functional state of crayfish <Emphasis Type="Italic">Pontastacus leptodactylus</Emphasis> Esch. upon acidification of the environment

The changes in heart rate and circadian cardiac rhythm of crayfish Pontastacus leptodactylus Esch. kept in a lightning regime that is close to natural under optimal or low pH values were studied. The heart rate was registered in real time using an original noninvasive fiberoptic method. Upon acidification, disorders in circadian cardiac rhythm and organism reaction (by heart rate) in the suspension test were detected. The characteristics of cardiac activity are considered criteria for estimating the crayfish’s functional state at normal and stress conditions caused by the changes in the quality of the environment.     

Cardiac activity of freshwater crayfish at wakefulness, rest, and “animal hypnosis”

In laboratory experiments, simultaneous continuous recording of the circadian cycle of behavioral reactions and cardiac activity of freshwater crayfish Astacus astacus and Procambarus clarkii was carried out. A non-invasive fiber-optic method of recording of the heart rate (HR) was used. The obtained data were analyzed by the method of variational pulsometry (VP). It was revealed that certain values of HR and characteristics of VP corresponded to the physiological states of active wakefulness and rest. It was found that during long immobilization of crayfish it is possible to identify the states of operative rest and sleep by the animal behavioral reactions and parameters of cardiac activity. Crayfish were studied in the state of artificially evoked immobilization (“animal hypnosis”). During this state, a high HR level, an increase of indexes of tension and autonomic equilibrium, and changes of other VP characteristics were observed. It is suggested that the different level of cardiac activity in different physiological states of crayfish (active wakefulness, operative rest, sleep, and “animal hypnosis”) is regulated by nervous influences analogous to sympathetic and parasympathetic influences in vertebrates. It was concluded that freshwater crayfish as a representative of the highly organized invertebrates can serve an effective model for studying mechanisms of sleep-like states and “animal hypnosis” in animals.     

Circadian rhythm in melatonin release as a mechanism to reinforce the temporal organization of the circadian system in crayfish

Melatonin (MEL) is a conserved molecule with respect to its synthesis pathway and functions. In crayfish, MEL content in eyestalks (Ey) increases at night under the photoperiod, and this indoleamine synchronizes the circadian rhythm of electroretinogram amplitude, which is expressed by retinas and controlled by the cerebroid ganglion (CG). The aim of this study was to determine whether MEL content in eyestalks and CG or circulating MEL in hemolymph (He) follows a circadian rhythm under a free-running condition; in addition, it was tested whether MEL might directly influence the spontaneous electrical activity of the CG. Crayfish were maintained under constant darkness and temperature, a condition suitable for studying the intrinsic properties of circadian systems. MEL was quantified in samples obtained from He, Ey, and CG by means of an enzyme-linked immunosorbent assay, and the effect of exogenous MEL on CG spontaneous activity was evaluated by electrophysiological recording. Variation of MEL content in He, Ey, and CG followed a circadian rhythm that peaked at the same circadian time (CT). In addition, a single dose of MEL injected into the crayfish at different CTs reduced the level of spontaneous electrical activity in the CG. Results suggest that the circadian increase in MEL content directly affects the CG, reducing its spontaneous electrical activity, and that MEL might act as a periodical signal to reinforce the organization of the circadian system in crayfish.     

CIRCADIAN MODULATION OF CRUSTACEAN HYPERGLYCEMIC HORMONE IN CRAYFISH EYESTALK AND RETINA

Previous studies suggested the retina could be a putative locus of daily crustacean hyperglycemic hormone (CHH) secretion, as it possesses its own metabolic machinery and is independent of the well-known CHH eyestalk locus responsible for the circadian secretion of this peptide. However, it has been proposed that hemolymph glucose and lactate concentrations play a dual role in the regulation of CHH in crayfish. To elucidate the temporal relationship between these two different CHH production loci and to examine their relationship with glucose regulation, we investigated the expression of CHH daily and circadian rhythms in the eyestalk and retina of crayfish using biochemical methods and time series analysis. We wanted to determine whether (1) putative retina and eyestalk CHH rhythmic expressions are correlated and if the oscillations of the two metabolic products of lactate and glucose in the blood due to CHH action on the target tissue correlate, and (2) retina CHH (RCHH) and the possible retinal substrate glycogen and its product glucose are temporally correlated. We found a negative correlation between daily and circadian changes of relative CHH abundance in the retina and eyestalk. This correlation and the cross-correlation values found between eyestalk CHH and hemolymph and glucose confirm that CHH produced by the X-organ sinus gland complex is under the previously proposed dual feedback control system over the 24?h time period. However, the presence of both glycogen and glucose in the retina, the cross-correlation values found between these parameters and hemolymph lactate and glucose, as well as RCHH and hemolymph and retina metabolic markers suggest RCHH is not under the same temporal metabolic control as eyestalk CHH. Nonetheless, their expression may be linked to common rhythms-generating processes. (Author correspondence: mlfm@hp.fciencias.unam.mx; mfajul@gmail.com)     

Erg circadian rhythm in the course of ontogeny in crayfish

• 1.1. The objective of the present work was to study the ontogeny of the ERG circadian rhythm in crayfish.
• 2.2. Long-term recordings of ERG and shielding retinal pigments position measured from the instar, the second instar, the third instar and the adult crayfish were obtained.
• 3.3. In the youngest animals (1–8 days old) an ultradian rhythm (15min-4hr periods) in the ERG amplitude was detected.
• 4.4. Older animals showed a progressive increment in the period length before they exhibited a circadian pattern. This last appeared, the first time, in 30-day-old animals and showed noticeable differences in the adult crayfish. At the same time, the crayfish began to show photomotor reflex. Later on (140-day-old crayfish) the circadian rhythm attained its final parameters.
• 5.5. The SD was used as a measure of lability in periods. The 4 hr ultradian rhythm and the 22.4 hr circadian rhythm showed the lowest SD indicating that they are the most precise period values.
• 6.6. Our results support the idea that the ERG circadian rhythm results from the coupling among high frequency (ultradian) oscillators, particularly those of 4 hr periods and that the coupling depends on the action of neurosecretions released from the sinus gland.

     

The Rate- and Species-Dependence of Short-Term Memory in Cardiac Myocytes

Short-term memory is an intrinsic property of paced cardiac myocytes that reflects the influence of pacing history, and not just the immediately preceding diastolic interval (DI), on the action potential duration (APD). Although it is recognized that short-term memory affects the dynamics of cardiac myocytes in general, and the onset of irregular cardiac rhythm in particular, its has never been adequately quantified or measured directly in experiments or numerical simulations, mainly due to the absence of appropriate techniques. As a result, very little is known about the rate- and species dependent behavior of short-term memory. In this study, we introduce a new approach that allows one to estimate how much short-term memory, M _S, is present in the cardiac myocyte at different pacing rates. The new quantification is based on the fact that pacing history affects not only the APD, but the entire dynamics of paced cardiac myocytes, in particular the restitution curve. Using the patch clamp technique and numerical simulations, we measured short-term memory restitution—the dependence of M _S on the cycle length—in isolated rabbit and guinea pig ventricular myocytes. In both species, M _S is rate- and species-dependent, displaying a biphasic behavior as a function of cycle length. Moreover, our results indicate that there is a significant difference in M _S measured between both species at small cycle lengths. Numerical simulations suggest that the kinetics of the rapidly activating delayed rectifier potassium current I _Kr is partially responsible for this difference.     

Role of the olfactory pathway in agonistic behavior of crayfish, <Emphasis Type="Italic">Procambarus clarkii</Emphasis>

Crayfish establish social dominance hierarchies through agonistic interactions, and these hierarchies are maintained through assessment of social status. Chemical signals influence several aspects of fighting behavior, but the specific chemosensory sensilla involved in detecting these signals in crayfish are unknown. The goal of our study was to examine the importance of aesthetasc sensilla—olfactory sensors on the antennules of decapod crustaceans—in regulating changes in fighting behavior in crayfish, Procambarus clarkii, over the course of repeated pairings. We selectively ablated aesthetascs from pairs of crayfish after the first day of trials and compared the behavior of these ablated animals to that of pairs of intact controls. Results show that unablated crayfish significantly decreased the number and duration of fights over repeated pairings, whereas crayfish lacking aesthetascs continued to engage in similar amounts of fighting across all three trial days. This difference shows that aesthetascs regulate fighting behavior in P. clarkii.     

Measuring sub-lethal effects on freshwater crayfish (<Emphasis Type="Italic">Paranephrops planifrons</Emphasis>) behaviour and physiology: laboratory and in situ exposure to modified zeolite

Various behavioural and physiological tests were applied to determine whether an aluminised modified zeolite clay product (Z2G1), used to cap phosphorus release from lake sediments, had sub-lethal effects on adult freshwater crayfish (Paranephrops planifrons). In situ exposures of caged crayfish before and after application of modified zeolite to Lake Okaro, Central North Island, New Zealand, at a nominal application concentration of 350 g m⁻², did not impact the survival of freshwater crayfish or produce any significant sub-lethal effects on crayfish mobility or physiology that we could detect. In laboratory sediment tests, crayfish did not show any consistent effect from short-term (10 days) exposure to modified zeolite (at concentrations of 350, 700 or 2,100 g m⁻²) or to alum. When combined, the field and laboratory data provide strong evidence that applications of 350 g m⁻² modified zeolite will have no short-term effect on adult crayfish, with a ‘margin of safety’ for higher application rates. Chronic exposure effects of capping agents and the indirect impacts of lake treatment (e.g. food web responses) are yet to be determined. Quantitative measures of shade- and shelter-seeking behaviour, righting behaviour, and length of escape response, could be improved by the addition of standardised behavioural observations. Physiological measures of ammoniacal-N production and oxygen consumption are less variable than behavioural tests, but comparisons between treatments are best conducted simultaneously. A suite of behavioural and physiological sub-lethal measures using field and laboratory exposures is recommended to provide an integrated assessment of crayfish health.     

Generation of reactive oxygen species and activity of antioxidants in hemolymph of the moth larvae <Emphasis Type="Italic">Galleria mellonella</Emphasis> (L.) (Lepidoptera: Piralidae) at development of the process of encapsulation

Activities of enzymatic antioxidants—superoxide dismutase, glutathione-S-transferase, and catalase—as well as generation of reactive oxygen species (ROS) in lymph of the honeycomb moth Galleria mellonella L. were studied at development of the process of encapsulation of nylon implants. It has been established that as soon as 15 min after piercing of cuticle with the implant the capsule is formed on its surface. Active melanization of the capsule has been shown to last for 4 h. A statistically significant increase of the ROS generation in lymph and a decrease of the enzymatic antioxidant activities in the insect hemocytes have been revealed after the implant incorporation. The authors suggest that the key role in maintenance of the oxidation-reduction balance in hemolymph at development of the encapsulation process is played by non-oxidative antioxidants.     

Physiological biomarkers of hypoxic stress in red swamp crayfish Procambarus clarkii from field and laboratory experiments

The crayfish industry in Louisiana is the largest in the United States, with crayfish frequently harvested from waters that experience episodic or chronic hypoxia (dissolved oxygen [DO]≤ 2 mg/l). We examined physiological biomarkers (hemolymph lactate, glucose, and protein concentrations) of hypoxic stress in the red swamp crayfish Procambarus clarkii from chronically hypoxic natural habitats and laboratory hypoxia experiments. P. clarkii from normoxic and hypoxic areas in the Atchafalaya River Basin were sampled monthly from April to July 2010. Laboratory experiments subjected P. clarkii to severe hypoxia (1 mg/l DO), moderate hypoxia (2 mg/l DO), or normoxic conditions (control: DO>7.5 mg/l) for 12, 24, and 48 h. P. clarkii from normoxic and hypoxic natural habitats did not display significantly different hemolymph lactate or glucose concentrations; however, mean hemolymph protein concentration was significantly lower in crayfish from hypoxic areas. P. clarkii exposed to severe hypoxia in laboratory experiments had significantly higher hemolymph lactate and glucose concentrations for all three exposure times, whereas large differences in protein concentrations were not observed. These results suggest that elevated hemolymph lactate and glucose concentrations are responses to acute hypoxia in P. clarkii, while differences in protein concentrations are the result of chronic hypoxic exposure.     

Correction factors used for estimating prey biomass in the diet of American minkMustela vison

Feeding experiment on nine feral minkMustela vison Schreber, 1777 was carried out to find differences in digestibility of particular prey types. The values of correction factors (CF), which show the ratio between consumed prey biomass and undigested remains, were calculated for seven food types. The experiment revealed that results obtained by commonly used percentage of occurrence methods differed from the real intake of food. Food types which were intensively digested were underestimated by percentage of occurrence methods and prey which had hardly digestive elements of their body were overestimated. The calculated values of CF are as follows: crayfish — 14.8, fish — 30.8, frog — 61.3, small passerine bird — 17.2, chicken — 41.3, rodent — 17.3, egg — 687.5. The variability in CF values did not depend on mink individuals but could be explained by the mean weight of prey items eaten by mink. The positive correlation between the prey size and its digestibility was recorded for crayfish, fish and chicken. The biomass of frog, passerine and rodent remains was not related to the body mass of eaten prey. The use of evaluated correction factors enables more accurate estimates of the food composition of wild living mustelids.     

Molecular cloning and daily variations of the <Emphasis Type="Italic">Period</Emphasis> gene in a reef fish <Emphasis Type="Italic">Siganus guttatus</Emphasis>

As the first step in understanding the molecular oscillation of the circa rhythms in the golden rabbitfish Siganus guttatus—a reef fish with a definite lunar-related rhythmicity—we cloned and sequenced a Period gene (rfPer). The rfPer gene contained an open reading frame that encodes a protein consisting of 1,452 amino acids; this protein is highly homologous to PER proteins of vertebrates including zebrafish. Phylogenetic analyses indicated that the rfPER protein is related to the zebrafish PER1 and PER4. The expression of rfPer mRNA in the whole brain, retina, and liver under light/dark (LD) conditions increased at 06:00 h and decreased at 18:00 h, suggesting that its robust circadian rhythm occurs in neural and peripheral tissues. When daily variation in the expression in rfPer mRNA in the whole brain and cultured pineal gland were examined under LD conditions, similar expression patterns of the gene were observed with an increase around dawn. Under constant light condition, the increased expression of rfPer mRNA in the whole brain disappeared around dawn. The present results demonstrate that rfPer is related to zPer4 and possibly zPer1. The present study is the first report on the Period gene from a marine fish.     

Dependence of protective effect of α-tocopherol on its concentration and time of action on pc12 cells under conditions of oxidative stress

At the short-term incubation (0.5 and 1.5 h) of cells of the PC12 neuronal line with α-tocopherol, its protective effect against the cytotoxic hydrogen peroxide action was increased with rise of its concentration in samples; the protection was practically absent at action of nanomolar antioxidant concentrations, but was well expressed at its micromolar concentrations. These data agree with the concept that α-tocopherol increases the cell viability by reacting directly with free radicals, which leads to formation of the less reactive compounds deprived of non-paired electron. The evidence is obtained that at the long-term action on PC12 cells, α-tocopherol not only in micro-, but also in nanomolar concentrations increases significantly the cell viability under conditions of oxidative stress. As follows from the obtained data, an important role in realization of the α-tocopherol protective effect at the long-term incubation seems to be played by modulation by this antioxidant of activity of protein kinase activated by extracellular signaling, phosphatidylinosite 3-kinase, and protein kinase C.     

Variations circadiennes des activités estérases de l'hémolymphe de Penaeus japonicus

The esterase activities of Penaeus japonicus hemolymph, revealed by a mixture of α- and β-naphtylacetate exhibit tricircadian variations under an LD:12-12 photoperiod. During the 24 h period, the total esterase activities can vary by 0,12 to 0,64 V/μg of protein. Under the experimental conditions adopted, circadian variations of the hemolymph esterase activities are observed whatever the sex and molt stage; the sex factor seems to be predominant for the esterase activities studied. Concurrently, a circadian rhythm of individual variabilities is observed for both sexes. The variabilities are assessed from the ratio of standard deviations on the esterase activity means, for each hour of experimentation. The minimum variabilities correspond to maximum total or specific esterase activities for the females whereas they precede the maximum esterase activities for males.     

Melatonin modulates the ERG circadian rhythm in crayfish

One of the most important functions modulated by melatonin is the synchronization of circadian rhythms. In crayfish (Procambarus clarkii), we have obtained evidence that the amplitude of the electrical response to light of the retinal photoreceptors the receptor potential, is modified by the action of melatonin and that the magnitude of this action depends on the circadian time of melatonin application. In contrast, the electroretinogram (ERG) circadian rhythm can be synchronized by either single or periodic melatonin application. In this work we hypothesized that, in crayfish, melatonin acts on effectors and on pacemaker of ERG circadian rhythm as a non-photic synchronizer. Melatonin could be a hormone that sends a signal of darkness to the ERG circadian system.     

一种新的螯虾丝氨酸蛋白酶抑制物基因的克隆表达及其活性分析

根据本课题组从克氏原螯虾中新发现的丝氨酸蛋白酶抑制物的基因序列(GenBank登录号CD644775)设计一对引物,应用逆转录-聚合酶链式反应(RT-PCR)技术,从螯虾血淋巴细胞中扩增出丝氨酸蛋白酶抑制物基因PCI188,将其连入原核表达载体pET-32a,转化至大肠杆菌Rosetta株和BL21株中进行蛋白表达,结果该蛋白只在前者表达。表达产物用免疫转印检测,出现50kD的特异性条带,与螯虾PCI188基因编码的蛋白大小相符。将融合蛋白纯化后免疫新西兰兔,用免疫血清与螯虾血淋巴作用后测定酚氧化酶活力,结果显示,酚氧化酶活力有所升高,从而首次证实螯虾PCI188编码的蛋白对丝氨酸蛋白酶有抑制作用。