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.     

Effects of Illumination on the Circadian Motor Rhythm of the Chelipeds of Crayfish

The effects of different illumination conditions on the main parameters of the circadian motor rhythms of the two chelipeds of the crayfish, Procambarus digueti , were compared. Under either constant darkness (DD) or constant light (LL) the phase relationship between the two circadian rhythms was more stable than under entrained conditions (LD cycles). These results suggest that the oscillators responsible for these rhythms differ in their sensitivity to light. The role of paired organs in the internal temporal order of the crayfish is discussed.     

Effects of Illumination on the Circadian Motor Rhythm of the Chelipeds of Crayfish

The effects of different illumination conditions on the main parameters of the circadian motor rhythms of the two chelipeds of the crayfish, Procambarus digueti, were compared. Under either constant darkness (DD) or constant light (LL) the phase relationship between the two circadian rhythms was more stable than under entrained conditions (LD cycles). These results suggest that the oscillators responsible for these rhythms differ in their sensitivity to light. The role of paired organs in the internal temporal order of the crayfish is discussed.     

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.     

克氏原螯虾的入侵生态学研究进展

克氏原螯虾(Procambarus clarkii)原产于美国南部和墨西哥北部,是一个著名入侵物种,作为一种水产经济资源物种在世界各地扩散。克氏原螯虾抗逆性强,所具有的广泛生境适应性、生长迅速、高生殖率等特点使它们迅速建立野生种群。近10余年的研究认为,克氏原螯虾通过捕食和资源竞争等机制严重威胁引入地的水生植物、无脊椎动物、两栖类等的生存,显著降低引入地的生物多样性。当前,由于克氏原螯虾的经济价值高,它会借助于人力的作用而继续扩散。为认清和减少克氏原螯虾对引入地的生态影响,应加强以下方面的研究:1)在中国开展克氏原螯虾的生态危害的调查和研究;2)克氏原螯虾种群调节和控制对策研究;3)被入侵地的生态恢复工作。     

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.     

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.     

Population genetic structure and post-establishment dispersal patterns of the red swamp crayfish Procambarus clarkii in China

The red swamp crayfish (Procambarus clarkii) was introduced to China in the early 20(th) century. It has been spread to almost all forms of fresh water bodies including lakes, rivers and even paddyfields in most provinces of China. To clarify issues such as the initial entry point(s), dispersal pattern, genetic diversity and genetic structure of Procambarus clarkii in China, the genetic structure and diversity of P. clarkii populations at 37 sampling sites (35 from China, one from the USA and one from Japan) were analyzed using both mitochondrial gene sequences (COI and 16S rRNA) and 12 nuclear microsatellites. Multiple tests including phylogenetic analyses, Bayesian assignment and analysis of isolation by distance showed that (i) the population from Japan and those collected from China, particularly from NanJing (BGt and XG) and its some neighboring sites (CJr, NT and NB), have similar genetic composition, (ii) relatively high genetic diversity was detected in Chinese populations, (iii) the P. clarkii populations in China did not experience significant population expansions. Taken together, Nanjing, Jiangsu province is the presumed initial entry point, and human-mediated dispersal and adaptive variation are likely responsible for the observed genetic pattern of P. clarkii in China.     

Behavioral effects of serotonin and serotonin agonists in two crayfish species, Procambarus clarkii and Orconectes rusticus

Exogenous serotonin elicits several behaviors in Procambarus clarkii, including a flexed, elevated posture, reduced locomotion, and changes in aggressive behavior. We conducted experiments to determine if several serotonin agonists mimicked the behavioral effects of serotonin in two crayfish species, P. clarkii and Orconectes rusticus. Drugs tested were 1-(3-Chlorophenyl)-piperazine dihydrochloride (mCPP), Oxymetazoline, 2,5-dimethoxy-4-iodoamphetamine (DOI), CGS-12066A, and (+/-)-8-hydroxy-2-(di-n-dipropylamino) tetralin (8-OH-DPAT). In P. clarkii, mCPP most closely mimicked the effects of serotonin, significantly increasing the performance of the flexed, elevated posture and reducing locomotion; 8-OH-DPAT significantly reduced locomotion as well. Both of these drugs produced significant increases in elevated posture and decreases in locomotion in O. rusticus, and in this species, the drugs at test concentrations were more effective in eliciting these effects than serotonin. The effects of the drugs on behaviors performed during fighting bouts were variable. In both species, only 8-OH-DPAT significantly reduced several agonistic behaviors, and no agonist or 5-HT itself produced significant increases in agonistic behavior.     

Glutathione content and GSH S-transferase activity in midgut gland of Procambarus clarkii. Sex differences, the effect of fasting, and their implications in cadmium toxicity

1. Glutathione content and GSH S-transferase activity in the midgut gland of Procambarus clarkii (P. c.) of different sex and body weight are presented. 2. Procambarus clarkii females' GSH concentration in the midgut gland decreases to a higher extent upon fasting, compared with males. 3. Procambarus clarkii females, both in control and fasting conditions, have a slightly higher GSH S-transferase activity than males. 4. Cadmium present in water only affects GSH content and GSH S-transferase activity (after 96 hr) in midgut gland, with cadmium chloride concentrations higher than 100 micrograms/l.     

Bioenergetics of juveniles of red swamp crayfish (Procambarus clarkii).

Procambarus clarkii is an endemic North American crayfish species that was introduced into Spain in 1973 for aquacultural and fishing purposes. Although P. clarkii is a well-studied species for commercial production, there is a great gap in the knowledge of the bioenergetics of juveniles. The aims of this study were to quantify the elements of the energy flow for juveniles of P. clarkii when fed in the laboratory on four different diets. The diets used were: (1) trade commercial feed; (2) various dried algal species; (3) dried Gammarus pulex; and (4) an equal mixture of algal species and G. pulex. The best energetic balance was obtained with diet 3 (greatest energy directed to production P=57%; least energetic investment in respiration R=31%, excretion U=9%, but highest energy loss via faeces F=4%). The poorest energetic balance was observed with diet 4 (P=26.3%; R=55.5%; U=14.7%, but lower energy loss in faeces F=3.5%). The mean O:N relationship was 1.37+/-2.15, implying marked protein catabolism. The utility of studying the bioenergetics of juvenile P. clarkii in laboratory conditions results in the formulation of testable hypotheses about ecological facts and the provision of new insights into the management of their populations in natural environments.     

Effect of temperature on the concentration of Ca2+, Mg2+, Na+ and K+ in the hemolymph and urine of Procambarus clarkii, Girard

The concentration variability of Ca, Mg, Na, K ions in the haemolymph and urine has been analyzed in Procambarus clarkii during interecdysis instar, the animals having been kept under several temperature conditions (10, 15, 20, 25, 30 degrees C) during two different periods of time (48 h and 7 days). The environmental temperature did not affect the concentration of sodium and calcium in the haemolymph. Nevertheless the above parameter had an effect on the concentration of potassium and magnesium in the haemolymph as well as on the concentration of the four cations considered in the urine of Procambarus clarkii. No significant differences have been found in relation to the time of exposure.     

Eleven polymorphic microsatellites isolated from red swamp crayfish, Procambarus clarkii

Eleven microsatellites were isolated from a genomic DNA library enriched for CA- and GA-repeats, and were characterized in 48 individuals of red swamp crayfish Procambarus clarkii. All 11 microsatellites were polymorphic with an average of allele number of 6.6 per locus. Genotypic distributions of these 11 markers were found to be in conformance with the expectations of Hardy-Weinberg equilibrium. All 11 markers were unlinked. These markers are being used to study the invasion routine, genetic diversity and population structure of the species P. clarkii.     

Photoperiodic induction on the growth rate and gonads maturation in the crayfish Procambarus clarkii during ontogeny

Five groups of 20 juvenile instars of crayfish each, were submitted to five experimental photoperiodic cycles of different photophase and scotophase characteristics during 13 weeks. The animals' total length and weight were measured weekly. When the size and maturation of the first animals were adequate, all the animals were sacrificed, and the histological study of gonads was performed. The results indicate that animals submitted to a light-dark (LD) cycle 12:12 with light interruptions in the scotophase, showed the greatest gonadal induction. However the greatest growth induction appeared in animals submitted to control condition (LD 12:12). These results could indicate for both functions two different models of photoperiodic induction.     

Effects of an introduced crayfish, Procambarus clarkii, on the distribution of south-western Iberian amphibians in their breeding habitats

The introduction of exotic species in aquatic habitats is one of the causes for the amphibian declines observed worldwide. In the 1970s, the red swamp crayfish Procambarus clarkii was introduced in the southwest Iberian Peninsula, where no native crayfish occur. In this study we assess the effect of P. clarkii presence in the breeding site distribution of each of the 13 southwest Iberian amphibians, while simultaneously accounting for the effects of potentially confounding habitat variables, as well as for the effects of the other large aquatic predators in the study area – predatory fish. Amphibian species richness was lower in places where P. clarkii was present than in places without P. clarkii , regardless of fish presence. After accounting for habitat variables and fish, crayfish presence was a negative predictor of the breeding probability for all urodeles ( Pleurodeles waltl , Salamandra salamandra , Triturus boscai and T. marmoratus ) and for two anurans ( Pelobates cultripes and Bufo bufo ). The majority of the species affected usually breed in temporary ponds without fish, but that may be colonized by the crayfish. The ongoing expansion of P. clarkii may eventually lead to a growing isolation of amphibian populations and ultimately to local extinctions and a permanent alteration of the amphibian communities in southwest Iberian Peninsula.     

Bioenergetics and phenology of reproduction of the introduced red swamp crayfish, Procambarus clarkii, in Do·ana National Park, Spain, and implications for species management

1. The red swamp crayfish, Procambarus clarkii (Girard), is a North American species which was introduced into the Lower Guadalquivir, Spain, in 1974. Procambarus clarkii has become a key species since it has greatly altered the functioning and structure of the ecosystems it has invaded.
2. Since P. clarkii spawn spontaneously throughout the year, it is to be expected that their ecological impact on the ecosystem may vary depending of the timing of habitat flooding (e.g. temporary freshwater marsh, and temporary or permanent pond). The objective of the present study was to understand the reproductive phenology and bioenergetics of P. clarkii in Do·ana National Park, Spain, freshwater ecosystems, and consequently, propose some management plans for their populations.
3. Five stages of ovarian development were detected. Final ovarian maturation occurred a few days before spawning and spawning was synchronized among mature females of all populations. Egg numbers hatched in pleopods ranged from 450 to 550. Timing of juvenile release from pleopods varied greatly (10–35 days).
4. Gravid females support most of their metabolic needs by catabolizing carbohydrates (overall mean of O:N = 30.39), and to a lesser extent, lipids. The aerobic and excretory metabolisms of gravid females were lower than those of immature animals of similar weight.
5. Analysis suggested that several factors influence the spawning of crayfish at the monitored sites. The major factors are water regime and temperature. Red swamp crayfish appear to be a spontaneous spawning species which may adjust their reproductive cycle to the timing of flooding, enhancing the utilization of available resources.
6. Populations of P. clarkii in Do·ana National Park could be controlled by the management of the water regime and by devising a fishing scheme which would maintain an appropriate population structure.     

Primary structure and relative potency of an analog of beta-PDH (pigment-dispersing hormone) from the crayfish Procambarus clarkii.

A pigment-dispersing hormone (PDH) from eyestalks of the crayfish Procambarus clarkii was purified by gel filtration, cation-exchange chromatography, partition chromatography, and reversed-phase HPLC. Based on automated sequencing and by the identical chromatographic behavior of the native PDH and the synthetic amidated form of the deduced sequence, the primary structure of Procambarus PDH has been established as: Asn-Ser-Glu-Leu-Ile-Asn-Ser-Ile-Leu-Gly-Leu-Pro-Lys-Val-Met-Asn-Glu-Ala- NH2. This peptide differs from beta-PDH of the fiddler crab Uca pugilator at a single position, Glu17 in place of Asp17. Because of this substitution, Procambarus PDH was 4 to 7-fold less potent than beta-PDH in causing pigment dispersion in the erythrophores, leucophores, and melanophores of Uca. In contrast, Procambarus PDH was 4-fold more potent than beta-PDH in eliciting pigment dispersion in the erythrophores of Procambarus. These peptides displayed less marked differences in potency in triggering leucophore pigment dispersion and light-adaptational distal eye pigment movement in Procambarus. These findings indicate that the structural requirements for PDH-receptor interactions vary with the species and with the target cell type within a given species.     

Physiological roles of free D- and L-alanine in the crayfish Procambarus clarkii with special reference to osmotic and anoxic stress responses

Under hyper-salinity stress from freshwater to 17 and 25 ppt seawater, red swamp crayfish Procambarus clarkii largely accumulated D- and L-alanine together with glycine, L-glutamine, and L-proline in both muscle and hepatopancreas. The increases of D- and L-alanine in muscle were the highest in all amino acids and reached 6.8- and 5.4-fold, respectively, from freshwater to 25 ppt seawater. These results indicate that both D- and L-alanine are the most potent osmolytes for intracellular isosmotic regulation in crayfish as well as other crustaceans thus far examined. Under anoxia stress below 0.1 mg/l dissolved oxygen for 12 h and subsequent recovery in normoxia for 12 h in freshwater, 17 and 25 ppt seawater, muscle ATP decreased dramatically in all salinity levels and almost depleted in seawater. Along with the decrease of muscle glycogen level, the significant increase of L-lactate was found in muscle, hepatopancreas, and hemolymph for each salinity level, suggesting the transport of L-lactate from muscle into hepatopancreas via hemolymph. Under anoxia, D- and L-alanine also largely increased in both muscle and hepatopancreas for each salinity level. The increase was much higher in seawater than in freshwater. Thus, both D- and L-alanine are possible to be anaerobic end products during prolonged anaerobiosis of this species.     

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.