A revised energy partitioning approach to assess the yields of non-photochemical quenching components

Non-photochemical quenching (NPQ) is a complex and still unclear mechanism essential for higher plants. The intensive research on this subject has highlighted three main components of NPQ: energy-dependent process (qE); state transitions to balance the excitation of PSII and PSI (qT); and photoinhibitory processes (qI). Recently, these components have been resolved as quantum yields according to the energy partitioning approach that takes into account the rate constants of every process involved in the quenching mechanisms of excited chlorophylls. In this study a fully extended quantum yield approach and the introduction of novel equations to assess the yields of each NPQ component are presented. Furthermore, a complete analysis of the yield of NPQ in Beta vulgaris exposed to different irradiances has been carried out. In agreement with experimental results here it is shown that the previous approach may amplify the yield of qE component and flatten the quantitative results of fluorescence analysis. Moreover, the significance of taking into account the physiological variability of NPQ for a correct assessment of energy partitioning is demonstrated.     

Potassium Ions and the Inhibitory Process in the Crayfish Stretch Receptor

The effect of the absence of potassium in the bathing solution on the synaptic inhibitory potentials of the crayfish stretch receptor has been studied. The inhibitory potentials were increased in size, i.e. became more hyperpolarizing, in the absence of potassium. Since the resting potential of the cell is increased in the absence of potassium, the alteration of the inhibitory potentials implies that the potassium conductance of the membrane is increased. While other ions, e.g. Cl^-, may also be involved, it seems that the membrane potential during inhibition is mainly dominated by K⁺.     

Evidence for genetic differentiation of Octopus vulgaris (Mollusca, Cephalopoda) fishery populations from the southern coast of Brazil as revealed by microsatellites

Octopus vulgaris is a cephalopod species in several oceans and commonly caught by artisanal and industrial fisheries. In Brazil, O. vulgaris populations are mainly distributed along the southern coast and have been subjected to intensive fishing during recent years. Despite the importance of this marine resource, no genetic study has been carried out to examine genetic differences among populations along the coast of Brazil. In this study, 343 individuals collected by commercial vessels were genotyped at six microsatellite loci to investigate the genetic differences in O. vulgaris populations along the southern coast of Brazil. Genetic structure and levels of differentiation among sampling sites were estimated via a genotype assignment test and F-statistics. Our results indicate that the O. vulgaris stock consists of four genetic populations with an overall significant analogous F_ST (Φ_CT = 0.10710, P < 0.05) value. The genetic diversity was high with an observed heterozygosity of Ho = 0.987. The negative values of F_IS found for most of the loci examined suggested a possible bottleneck process. These findings are important for further steps toward more sustainable octopus fisheries, so that this marine resource can be preserved for long-term utilization.     

Constant morphological patterns in the hemolymph vascular system of crayfish (Crustacea,Decapoda)

We present a study of the hemolymph vascular system of the marbled crayfish, Procambarus fallax f. virginalis, the only crayfish species known to be parthenogenetic. To identify potential evolutionary patterns, we compared data from a total of 48 specimens of P. fallax with 22 specimens of Orconectes limosus. Visualizations (2D and 3D) were carried out using a combination of classical and modern morphological techniques. Our data were compared to the existing literature.Like all Decapoda, both P. fallax and O. limosus have a hemolymph vascular system, consisting of a globular heart with seven off-branching arteries. We were able to visualize in detail the heart of crayfish for the first time, i.e., the myocard with its clusters of muscles running through the lumen of the heart, the valves and flaps of ostia and arteries. Furthermore, the branching patterns of the seven artery systems were analyzed. Anatomical structures identified to be consistent in all specimens of both species were combined as ground pattern of hemolymph vascular system features for Astacida.     

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.     

Prevalence of the Crayfish Plague Pathogen Aphanomyces astaci in Populations of the Signal Crayfish Pacifastacus leniusculus in France: Evaluating the Threat to Native Crayfish

Aphanomyces astaci, the crayfish plague pathogen, first appeared in Europe in the mid-19^th century and is still responsible for mass mortalities of native European crayfish. The spread of this parasite across the continent is especially facilitated by invasive North American crayfish species that serve as its reservoir. In France, multiple cases of native crayfish mortalities have been suggested to be connected with the presence of the signal crayfish Pacifastacus leniusculus, which is highly abundant in the country. It shares similar habitats as the native white-clawed crayfish Austropotamobius pallipes and, when infected, the signal crayfish might therefore easily transmit the pathogen to the native species. We investigated the prevalence of A. astaci in French signal crayfish populations to evaluate the danger they represent to local populations of native crayfish. Over 500 individuals of Pacifastacus leniusculus from 45 French populations were analysed, plus several additional individuals of other non-indigenous crayfish species Orconectes limosus, O. immunis and Procambarus clarkii. Altogether, 20% of analysed signal crayfish tested positive for Aphanomyces astaci, and the pathogen was detected in more than half of the studied populations. Local prevalence varied significantly, ranging from 0% up to 80%, but wide confidence intervals suggest that the number of populations infected by A. astaci may be even higher than our results show. Analysis of several individuals of other introduced species revealed infections among two of these, O. immunis and P. clarkii. Our results confirm that the widespread signal crayfish serves as a key reservoir of Aphanomyces astaci in France and therefore represents a serious danger to native crayfish species, especially the white-clawed crayfish. The prevalence in other non-indigenous crayfish should also be investigated as they likely contribute to pathogen transmission in the country.     

Is the exotic red swamp crayfish (Procambarus clarkii) a current threat for the Mediterranean tree frog (Hyla meridionalis) in the Camargue (Southern France)?

The non-indigenous red swamp crayfish (Procambarus clarkii) has been shown to be a threat for amphibian conservation. Many amphibian species breed in temporary ponds to diminish predation risk as such ecosystems are free of large predators. However P. clarkii, occurring as an invasive species in the Camargue delta, can readily disperse on the ground and thus colonize isolated ponds. We studied the current impact of the exotic crayfish on the reproductive success of the Mediterranean tree frog (Hyla meridionalis). In a mesocosm experiment, we tested the effect of two crayfish densities (1 and 3 crayfish/m²) on tadpole abundance. We also tested in a field experiment, within a temporary pond, the crayfish’s predation on the tree frog’s eggs. Finally, we developed site occupancy models using data from 20 ponds to assess the effect of crayfish abundance on tadpole abundance. Neither the experiments, nor the site occupancy models showed a negative impact of the current crayfish abundance on the tree frog populations breeding in ponds. We found that recorded crayfish densities were lower than in other areas where crayfish has impacted amphibian populations, but we hypothesize that current crayfish abundance in the area may increase in the future, thus impacting tree frog populations.     

Distribution and selective elimination of paralytic shellfish toxins in different tissues of Octopus vulgaris

Octopus (Octopus vulgaris, Cuvier) plays a central role in the marine food web, being an important consumer with high metabolic rates and at the same time an important food item for higher predators. After harmful algal blooms, octopus can accumulate high levels of marine toxins trough trophic interrelationships. The aim of this study is to characterize the distribution of paralytic shellfish toxins (PSTs) in selected tissues of the O. vulgaris, in order to assess the translocation of toxins among organs with different physiological functions. Different retention times and selective elimination of particular toxin analogues were also investigated. Twenty three specimens of O. vulgaris were captured in Peniche (NW coast of Portugal) after PSTs have been detected in molluscan bivalves. Tissue matrices were dissected from organs with digestive function (digestive gland, stomach and salivary glands) and excretory function (kidneys and branchial hearts) and analyzed for toxin determination. Toxin analysis was carried out by high performance liquid chromatography with fluorescence detection (LC-FLD). PSTs were found in all tissues analyzed. The highest toxin concentrations were found in the digestive gland, reaching a maximum of 2980 μg STX equiv. kg⁻¹. The toxin profile was constituted by dcSTX, B1, C1 + 2, dcGTX2 + 3, dcNEO, STX and GTX2 + 3. A lower number of toxins were identified in the remaining organs, with B1 and dcSTX compromising more than 90% in molar fraction. Decarbamoyl saxitoxin was the most abundant toxin detected in digestive gland, stomach and salivary glands, while B1 was dominant in organs with excretory function. A positive correlation of concentrations of B1 and dcSTX were found in the organs analyzed. Results indicate that B1 and dcSTX are assimilated into the digestive gland in a similar proportion. Selective elimination of toxins with higher elimination of B1 and retention of dcSTX is suggested. This study contributes to better understanding of the dynamics of PSTs in O. vulgaris and the fate of PSTs in the food web.     

First records of the non-indigenous signal crayfish (<Emphasis Type="Italic">Pacifastacus leniusculus</Emphasis>) and its threat to noble crayfish (<Emphasis Type="Italic">Astacus astacus</Emphasis>) populations in Estonia

This study gives an overview of status and distribution of signal crayfish (Pacifastacus leniusculus), the first NICS in Estonia and its influence on native noble crayfish (Astacus astacus) populations. The first specimen of signal crayfish was caught during the monitoring of noble crayfish in North Estonia in 2008. The signal crayfish has since been found in three additional sites. Test fishing has indicated that the abundance of signal crayfish has been fluctuating between years and among localities. It has had strong negative impact on abundance of one noble crayfish population. The disconnected distribution of signal crayfish strongly suggests that these populations are the result of human-assisted introductions. Real-time PCR analyses proved that signal crayfish carry the causative agent of the crayfish plague, an oomycete Aphanomyces astaci, thus contributing to its spread. Mortalities in noble crayfish populations had been caused by A. astaci strains from A, B and E genotype group.     

Effectiveness of dam construction to contain the invasive crayfish Procambarus clarkii in a Mediterranean mountain stream

Invasive crayfish are a worldwide problem for aquatic ecosystems. So far, no control method has proven successful. Field data from invaded streams have suggested that physical structures may hamper the spread rate of invasive crayfish into new areas. However, given the difficulties for carrying out dam construction in natural habitats, data about their effectiveness in incipient invading populations are practically nonexistent. This paper evaluates the effectiveness of small dams to contain the advance of an incipient population of the invasive Procambarus clarkii in a mountain stream harboring the European southernmost population of the endangered Austropotamobius pallipes. The management strategy involved the construction of 3 artificial small (<3 m high) dams that were complemented with specific design solutions to increase their theoretical efficacy. Catch Per Unit Effort (CPUE) obtained during monitoring surveys carried out for 4 years were used as an indicator of containment effectiveness. The dams successfully prevented Procambarus clarkii from extending its range into the area occupied by A. pallipes. In addition, regardless of whether the construction of new dams isolated the leading edge of the invading population between two dams, the CPUE declined to below detectable limits. Therefore, dams are suggested to effectively stop, or at least hinder, the advance of invasive crayfish. Long-term monitoring is recommended to verify the on-going success of the dams in preventing upstream invasion.     

Target molecular size of the red beet plasma membrane ATPase

Radiation inactivation of the red beet (Beta vulgaris L.) plasma membrane ATPase was carried out using γ-ray radiation from a ¹³⁷Cs source. Inactivation of vanadate-sensitive ATPase activity by γ-ray radiation followed an exponential decline with increasing total dose, indicating a single target size calculated to have a molecular weight of about 228,000. Since the catalytic subunit of the red beet plasma membrane ATPase has been demonstrated to have a molecular weight of about 100,000 by dodecyl-sulfate gel electrophoresis following ³²P-phosphorylation, it is suggested that the native enzyme may exist, at least, as a dimer of catalytic subunits.     

Threat of non-native crayfish introductions into Turkey: global lessons

Introductions of crayfish species from their home range to new environments have been carried out in many parts of the world. The most important introduced crayfish species are Procambarus clarkii, Pacifastacus leniusculus, Cherax destructor, C. quadricarinatus, Orconectes limosus, O. rusticus and Astacus leptodactylus. The environmental impact of crayfish introductions can be positive, negative or neutral. However, native crayfish populations in Europe have been negatively affected by introductions of non-indigenous crayfish species from America. Negative effects of non-native crayfish introductions included displacement of native crayfish species, transfer of disease (crayfish plague), consumption of fish eggs, reduction of fish stocks, consumption of large amounts of macrophytes, indirect and direct effects on other invertebrates and upsetting production in rice fields. As a result of non-native crayfish introductions, the natural harvest and crayfish industry in Europe have been severely affected. Large quantities of Turkish A. leptodactylus were harvested (approximately 7,000 tonnes annually) and exported to Europe before the crayfish plague was observed in these populations. The total harvest of A. leptodactylus in Turkey reduced dramatically to 320 in 1991 after the plague. Therefore, although Turkey currently has no known non-native crayfish species, there is a threat of non-native crayfish introduction in order to increase crayfish productions and subsequent harvest. The North American spiny-cheek crayfish, O. limosus, has been spreading quickly down the River Danube and could soon reach neighboring countries including Turkey. The North American signal crayfish, P. leniusculus is known from Greece and could be a threat to native stocks if it is introduced into Turkey for aquaculture. Additional threats may come from the release of other North American species, which are widely available through the aquarium trade. We conclude that the spread of non-native crayfish introductions throughout Turkey will increase local problems, because introductions of non-native crayfish in many parts of the world have been known to have caused important reductions in population density and numbers of native crayfish species. Furthermore, freshwater ecosystems may be altered by such introductions and the economic viability of native crayfish species fisheries could be severely reduced in Turkey.     

The effect of endogeneous proteinase inhibitors on the prophenoloxidase activating enzyme,a serine proteinase from crayfish haemocytes

Three proteinase inhibitors have so far been isolated and purified from crayfish haemolymph. One of these, isolated from crayfish plasma, namely a trypsin inhibitor with a molecular mass of 155 kDa was found to inhibit a serine proteinase, ppA, which is involved in the activation of prophenoloxidase, and is localized in the haemocytes. Another high molecular mass proteinase inhibitor, an α₂-macroglobulin from crayfish plasma, which is a dimer of 190 kDa-subunits, was only inhibitory towards ppA to a lesser extent. A 23 kDa subtilisin inhibitor, purified from haemocytes, did not have any effect on the serine proteinase.We suggest that mainly the trypsin inhibitor, but to some extent also the α₂-macroglobulin, are important in the regulation of the prophenoloxidase activating cascade, as they both inhibit ppA, which in its active form has been shown to mediate prophenoloxidase activation.     

Deletion of the rbo Gene Increases the Oxygen Sensitivity of the Sulfate-Reducing Bacterium Desulfovibrio vulgaris Hildenborough

The rbo gene of Desulfovibrio vulgaris Hildenborough encodes rubredoxin oxidoreductase (Rbo), a 14-kDa iron sulfur protein; forms an operon with the gene for rubredoxin; and is preceded by the gene for the oxygen-sensing protein DcrA. We have deleted the rbo gene from D. vulgaris with the sacB mutagenesis procedure developed previously (R. Fu and G. Voordouw, Microbiology 143:1815–1826, 1997). The absence of the rbo-gene in the resulting mutant, D. vulgaris L2, was confirmed by PCR and protein blotting with Rbo-specific polyclonal antibodies. D. vulgaris L2 grows like the wild type under anaerobic conditions. Exposure to air for 24 h caused a 100-fold drop in CFU of L2 relative to the wild type. The lag times of liquid cultures of inocula exposed to air were on average also greater for L2 than for the wild type. These results demonstrate that Rbo, which is not homologous with superoxide dismutase or catalase, acts as an oxygen defense protein in the anaerobic, sulfate-reducing bacterium D. vulgaris Hildenborough and likely also in other sulfate-reducing bacteria and anaerobic archaea in which it has been found.     

AFLP-PCR and RAPD-PCR evidences of the transmission of the pathogen Aphanomyces astaci (Oomycetes) to wild populations of European crayfish from the invasive crayfish species, Procambarus clarkii

Aphanomyces astaci (Oomycetes) is responsible for the crayfish plague disease. This species is endemic of North America and five genotypes have been described using RAPD-PCR. The red swamp crayfish, Procambarus clarkii, is one of the most widely spread North American species and invasive in the world. However, no outbreaks on its specific genotype, i.e., genotype D, have ever been described in nature. We investigated three major series of crayfish plague outbreaks in indigenous crayfish populations of Austropotamobius pallipes, located in the areas of influence of P. clarkii. All samples collected tested positive for A. astaci using a rnDNA ITS-PCR test. We also performed an AFLP-PCR analysis on 19 isolates, and found that all isolates belong to genotype D. These isolates exhibited similar properties, i.e., adaptation to warm temperatures. We demonstrate, for the first time, the transmission of A. astaci genotype D to indigenous European populations of crayfish, and confirm that the properties of adaptation to warm water temperatures seem to be a specific character of genotype D. The results of this work emphasize once more the need of controlling invasive species and its trade, since they can carry harmful pathogens with specific adaptations or increased virulence in new environments.     

Differences Found in the Macroinvertebrate Community Composition in the Presence or Absence of the Invasive Alien Crayfish,Orconectes hylas

Introductions of alien species into aquatic ecosystems have been well documented, including invasions of crayfish species; however, little is known about the effects of these introductions on macroinvertebrate communities. The woodland crayfish (Orconectes hylas (Faxon)) has been introduced into the St. Francis River watershed in southeast Missouri and has displaced populations of native crayfish. The effects of O. hylas on macroinvertebrate community composition were investigated in a fourth-order Ozark stream at two locations, one with the presence of O. hylas and one without. Significant differences between sites and across four sampling periods and two habitats were found in five categories of benthic macroinvertebrate metrics: species richness, percent/composition, dominance/diversity, functional feeding groups, and biotic indices. In most seasons and habitat combinations, the invaded site had significantly higher relative abundance of riffle beetles (Coleoptera: Elmidae), and significantly lower Missouri biotic index values, total taxa richness, and both richness and relative abundance of midges (Diptera: Chironomidae). Overall study results indicate that some macroinvertebrate community differences due to the O. hylas invasion were not consistent between seasons and habitats, suggesting that further research on spatial and temporal habitat use and feeding ecology of Ozark crayfish species is needed to improve our understanding of the effects of these invasions on aquatic communities.     

Differential expression pattern of heat shock protein 70 gene in tissues and heat stress phenotypes in goats during peak heat stress period

It has been established that the synthesis of heat shock protein 70 (Hsp70) is temperature-dependent. The Hsp70 response is considered as a cellular thermometer in response to heat stress and other stimuli. The variation in Hsp70 gene expression has been positively correlated with thermotolerance in Drosophila melanogaster, Caenorhabditis elegans, rodents and human. Goats have a wide range of ecological adaptability due to their anatomical and physiological characteristics; however, the productivity of the individual declines during thermal stress. The present study was carried out to analyze the expression of heat shock proteins in different tissues and to contrast heat stress phenotypes in response to chronic heat stress. The investigation has been carried out in Jamunapari, Barbari, Jakhrana and Sirohi goats. These breeds differ in size, coat colour and production performance. The heat stress assessment in goats was carried out at a temperature humidity index (THI) ranging from 85.36–89.80 over the period. Phenotyping for heat stress susceptibility was carried out by combining respiration rate (RR) and heart rate (HR). Based on the distribution of RR and HR over the breeds in the population, individual animals were recognized as heat stress-susceptible (HSS) and heat stress-tolerant (HST). Based on their physiological responses, the selected animals were slaughtered for tissue collection during peak heat stress periods. The tissue samples from different organs such as liver, spleen, heart, testis, brain and lungs were collected and stored at ?70 °C for future use. Hsp70 concentrations were analyzed from tissue extract with ELISA. mRNA expression levels were evaluated using the SYBR green method. Kidney, liver and heart had 1.5–2.0-fold higher Hsp70 concentrations as compared to other organs in the tissue extracts. Similarly, the gene expression pattern of Hsp70 in different organs indicated that the liver, spleen, brain and kidney exhibited 5.94, 4.96, 5.29 and 2.63-fold higher expression than control. Liver and brain tissues showed the highest gene expression at mRNA levels as compared to kidney, spleen and heart. HST individuals had higher levels of mRNA level expression than HSS individuals in all breeds. The Sirohi breed showed the highest (6.3-fold) mRNA expression levels as compared to the other three breeds, indicating the better heat stress regulation activity in the breed.     

Mutualistic growth of the sulfate-reducer Desulfovibrio vulgaris Hildenborough with different carbohydrates

Desulfovibrio vulgaris Hildenborough genome presents a phosphotransferase system putatively involved in the transport of carbohydrates. However, utilization of sugars by this sulfate-reducing bacterium has never been reported. Herein, we have observed proliferation of D. vulgaris Hildenborough with some carbohydrates, in mutualism with Stenotrophomonas maltophilia, a non-fermentative, gram-negative gammaproteobacterium, or Microbacterium, a gram-positive actinobacterium. These results suggest the importance of feedback interactions between different heterotrophic bacterial species including the alternative for D. vulgaris of exploiting additional organic resources and novel habitats. Thus, D. vulgaris strongly participates in the mineralization of carbohydrates both in complex natural and artificial systems.