Oral vaccination with envelope protein VP28 against white spot syndrome virus in Procambarus clarkii using Bacillus subtilis as delivery vehicles

Aims: To achieve high‐level expression and secretion of active VP28 directed by a processing‐efficient signal peptide in Bacillus subtilis WB600 and exploit the possibility of obtaining an oral vaccine against white spot syndrome virus (WSSV) using vegetative cells or spores as delivery vehicles. Methods and Results: The polymerase chain reaction (PCR)‐amplified vp28 gene was inserted into a shuttle expression vector with a novel signal peptide sequence. After electro‐transformation, time‐courses for recombinant VP28 (rVP28) secretion level in B. subtilis WB600 were analysed. Crayfish were divided into three groups subsequently challenged by 7‐h immersion at different time points after vaccination. Subgroups including 20 inter‐moult crayfish with an average weight of 15 g in triplicate were vaccinated by feeding coated food pellets with vegetative cells or spores for 20 days. Vaccination trials showed that rVP28 by spore delivery induced a higher resistance than using vegetative cells. Challenged at 14 days postvaccination, the relative per cent survival (RPS) values of groups of rVP28‐bv and rVP28‐bs was 51·7% and 78·3%, respectively. Conclusions: The recombinant B. subtilis strain with the ability of high‐level secretion of rVP28 can evoke protection of crayfish against WSSV by oral delivery. Significance and Impact of the Study: Oral vaccination by the B. subtilis vehicle containing VP28 opens a new way for designing practical vaccines to control WSSV.     

Effects of different methods and times of 17β-estradiol treatment on the feminization success in the narrow-clawed crayfish Astacus leptodactylus (Eschscholtz, 1823)

Sex differentiation is a profitable technique in crayfish aquaculture. Most of the studies have focused on sex differentiation in crayfish by ablation or implantation of androgenic glands. There is no research on sex differentiation using hormones in crayfish. In the present study, the effects of 17β-estradiol (E2) injections (10^?7 mol/crayfish) to females and baths (50 μg 17β-estradiol/L) to pleopodal eggs and stage1, 2, and 3 juveniles on the feminization of freshwater crayfish, Astacus leptodactylus were investigated. Results showed that E2 injection to females, bath to pleopodal eggs and bath to stage 1, 2, and 3 juveniles increased feminization rate significantly (p < 0.05) in comparison to the control in A. leptodactylus. Best feminization rate (male to female, 0.25:1) was observed after hormonal injection to the females combined with application of both for their both pleopodal eggs and stage 1, 2, and 3 juveniles. In conclusion, it is possible to obtain 75% females A. leptodactylus by E2 injection to females and bath to pleopodal eggs and stage 1, 2, and 3 juveniles. However, future studies are needed to investigate the mechanism of E2 in crayfish feminization and the sexual development of juveniles obtained from feminization.     

An ultrastructural analysis of mature sperm from the crayfish Pacifastacus leniusculus,Dana

Sperm from the crayfish, Pacifastacus leniusculus, resemble other reptantian sperm in that they are composed of an acrosome, subacrosomal region, nucleus, membrane lamellar complex, and spikes which radiate from the nuclear compartment. The acrosome (PAS positive vesicle) can be subdivided into three regions: the apical cap, crystalline inner acrosomal material, and outer acrosomal material which is homogeneous except for a peripheral electron dense band. The nucleus contains uncondensed chromatin and bundles of microtubules which project into the spikes. The orientation of the microtubule bundles relative to the nuclear envelope near the base of the subacrosomal region suggests that the nuclear envelope may function in the organization of the spike microtubules.     

Absence of the crayfish plague pathogen (Aphanomyces astaci) facilitates coexistence of European and American crayfish in central Europe

1. Most European crayfish species are strongly threatened, mainly as a result of the introduced pathogen, Aphanomyces astaci, transmitted by invasive North American crayfish. Long‐term coexistence of American and European crayfish species is therefore regarded as almost impossible, even though some coexisting populations have been observed. 2. In this study, crayfish were collected from presently coexisting populations of the introduced spiny‐cheek crayfish (Orconectes limosus) and the native noble crayfish (Astacus astacus) from nine standing waters in central Europe. Our aim was to resolve whether the coexistence resulted from reduced virulence in local strains of A. astaci, increased immunity in the native crayfish or an absence of the pathogen in these populations. We used highly sensitive A. astaci‐specific real‐time PCR to evaluate the crayfish latent carrier status, combined with transmission experiments to further validate the molecular results. 3. From the total of 523 crayfish tested (490 spiny‐cheek crayfish, 33 noble crayfish), none positive for A. astaci was detected. Transmission experiments confirmed these results: No abnormal mortality or behavioural changes were seen in noble crayfish kept together with American crayfish from the coexisting populations. If we assume a very low prevalence of A. astaci of 10% in a carrier population, there is a 98% probability of disease being absent in five of the nine coexisting populations tested. Hence, a consistent absence, or an extremely low prevalence, of A. astaci seems to allow the coexistence of European and American crayfish in these central European populations. 4. The results are important for native crayfish conservation and management and demonstrate that disease transmission risk may vary substantially between the different populations of spiny‐cheek crayfish in central Europe.     

Synchronization by light of the circadian rhythm of motor activity in the crayfish

Abstract

We have studied the pattern for resetting the circadian rhythm in the spontaneous motor activity of the crayfish. Spontaneous motor activity was recorded continously at a constant temperature and under free running conditions in complete darkness. The effect of single light pulses applied at different circadian times, on the circadian rhythm of motor activity was measured in both transient stage and steady state. The results led us to construct a phase‐transition curve and phase‐response curve which were analyzed to obtain information about the oscillators which underlie the circadian rhythm of motor activity.     

Phenological response of a key ecosystem function to biological invasion

Although climate warming has been widely demonstrated to induce shifts in the timing of many biological events, the phenological consequences of other prominent global change drivers remain largely unknown. Here, we investigated the effects of biological invasions on the seasonality of leaf litter decomposition, a crucial freshwater ecosystem function. Decomposition rates were quantified in 18 temperate shallow lakes distributed along a gradient of crayfish invasion and a temperature‐based model was constructed to predict yearly patterns of decomposition. We found that, through direct detritus consumption, omnivorous invasive crayfish accelerated decomposition rates up to fivefold in spring, enhancing temperature dependence of the process and shortening the period of major detritus availability in the ecosystem by up to 39 days (95% CI: 15–61). The fact that our estimates are an order of magnitude higher than any previously reported climate‐driven phenological shifts indicates that some powerful drivers of phenological change have been largely overlooked.     

The first record of the non-indigenous signal crayfish <Emphasis Type="Italic">Pasifastacus leniusculus</Emphasis> in Norway

The non-indigenous signal crayfish Pasifastacus leniusculus was registered for the first time in Norway in October 2006. The location represents an isolated pond about 100 km in air line from the nearest known signal crayfish population in the neighbouring country Sweden without any connecting watercourses. The occurrence is therefore undoubtedly caused by human introduction. Molecular analyses confirmed that tested individuals from the signal crayfish population were carriers of the crayfish plague agent Aphanomyces astaci. Disease carrying signal crayfish represents a severe threat to the indigenous and endangered noble crayfish Astacus astacus. Norwegian authorities are currently considering actions for the eradication of the signal crayfish population.     

Experimental study of the combined effects of pH and salinity on the bioaccumulation of inorganic mercury in the crayfish Astacus leptodactylus

ABSTRACT

The combined effects of pH and salinity were investigated on the bioaccumulation of inorganic mercury in the Turkish crayfish Astacus leptodactylus, at the whole organism and organ levels. Effects on the ionic balance were also analysed during the acclimatization phase and after 3 and 15 days of exposure, via the direct route. The experimental protocol, based on a complete factorial design, took into account nine experimental conditions, resulting from the combination of three levels for pH (6, 7.5 and 9) and for salinity (1, 10 and 100 mM Cl-). The results showed an important Hg accumulation in the organs or tissues in direct contact with the surrounding medium, e.g. gills and carapace. The effects of the nine pH and salinity conditions were complex and variable from one organ to another. Correlations were studied between chemical species concentrations in the water (HgCl₂, HgOHCl, HgCl₃-,HgCl₄², Hg(OH)₂) and amounts of metal accumulated in the different organs. A positive correlation was found between the neutral species HgCl₂ and metal accumulated in the gills and in the carapace. Perturbations of the haemolymph ionic concentrations were no significant, except for Na⁺ after 3 days of exposure.     

Immigration rates of signal crayfish (Pacifastacus leniusculus) in response to manual control measures

1. Crayfish are amongst the most frequently introduced non‐native aquatic organisms, with well‐documented negative effects on a large number of freshwater taxa. Many crayfish‐control strategies make use of manual removal by trapping, a method known preferentially to remove the largest individuals, leaving the juvenile population almost entirely untrapped. 2. A predicted outcome of trapping bias in riparian habitats is that removed individuals could be replaced by large crayfish immigrating from surrounding, untrapped, areas. We tested the hypothesis that removal by trapping of American signal crayfish from a U.K. river would result in increased rates of immigration, and increased distances moved, of crayfish from untrapped areas. 3. We studied four stretches of the River Windrush each 1 km in length and divided into three sections; a 250‐m long upstream section, a 500‐m middle section and a 250‐m downstream section. At two sites (removal sites), signal crayfish were trapped and removed from the 500‐m middle sections; at the other two (non‐removal), they were marked and returned. All crayfish captured in the upstream and downstream sections were marked and returned. 4. Probability of capture was higher for larger individuals with both chelae intact, and larger crayfish were more likely to immigrate from the upstream and downstream sections into the middle. The percentage of captured crayfish immigrating into the middle sections was the same (3.7%) in both removal and non‐removal sites. However, the mean distance that crayfish moved when immigrating was significantly greater at removal sites (239 m) than at non‐removal sites (187 m). 5. These results imply that removal of large individuals may have reduced the potential for interference competition by increasing the relative competitiveness of the immigrating individuals and permitting them to make larger movements. Consequently, the impact of manual removal strategies, both on the signal crayfish population and other biota affected by them, is likely to be reduced at the point of removal, but to extend at least 200 m beyond the trapped length of river.     

The effect of manual removal on movement distances in populations of signal crayfish (Pacifastacus leniusculus)

1. Crayfish are amongst the most frequently introduced non‐native aquatic organisms, with well‐documented negative effects on a large number of freshwater taxa. Many crayfish‐control strategies make use of manual removal by trapping, a method known preferentially to remove the largest individuals, leaving the juvenile population almost entirely untrapped. 2. Removal by trapping may be used in an attempt to delay colonisation of new stretches by invasive crayfish. It is, however, unclear what effects trapping may have on movement distances of crayfish in wild populations. We examine the impacts of removal by trapping on the movements of American signal crayfish in two UK rivers. 3. We studied four 100 m stretches of two rivers, the Evenlode and Thame, comprising two removal and two non‐removal stretches. Each river supported both treatments. Half of the crayfish captured from the removal sections were removed and humanely destroyed by freezing, and half were marked with their trap location and released there. All crayfish captured from the non‐removal sections were marked and returned at the point of capture. 4. Mean movement distances were smaller in the removal stretches than the non‐removal stretches, both within capture sessions (10.8 and 16.0 m, respectively) and between sessions (14.5 and 24.6 m, respectively), suggesting that removal trapping resulted in the remaining crayfish making smaller movements. Larger crayfish under both treatments made substantially larger movements than those with smaller carapace lengths, both within capture sessions (range 7.6–19.6 m) and between range capture sessions (range 8.9–32.6 m). 5. The results of this study are consistent with expectations if removal by trapping lowered population densities, which we speculate may have affected movement distances directly or indirectly through increasing the availability of food and shelter. 6. This study suggests that trapping at the margins of a population may be sufficient to delay colonisation of new stretches by: (i) maintaining low densities and therefore reducing movements, and (ii) preferentially reducing the population of large individuals, which make the largest movements. However, it remains unlikely that any trapping programme can entirely prevent emigration /dispersal, and therefore colonisation, by signal crayfish.