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.     

Complex predator-prey interactions and predator intimidation among crayfish,piscivorous fish,and small benthic fish

Summary Predator-prey interactions were studied among a small prey fish (the johnny darter Etheostoma nigrum) and two predators (crayfish Orconectes rusticus and smallmouth bass Micropterus dolomieui) with complementary foraging behaviors. When only smallmouth bass were present, darters reduced activity to 6% of control rates and spent most of the time hiding under tile shelters. When only crayfish were present, darter activity and shelter-use were similar to controls. When both crayfish and bass were present, an interaction occurred. Darters, normally inactive in the presence of bass, were often forced to move by approaching crayfish and thus activity increased to 19% of control rates. Also, darters were often evicted from shelters by intruding crayfish. Thus, crayfish increased the vulnerability of small fish to bass by evicting them from shelters and causing increased activity. Conversely, bass increased the vulnerability of small fish to crayfish by forcing these fish to seek cover under shelters occupied by crayfish. Intimidation effects of bass on darters last for some time. After a 30-min exposure to bass, darters showed reduced activity and increased shelter use lasting at least 24 h after the bass was removed. Thus predators, throught intimidation, can influence prey behavior even though the predators are no longer present.The Unit is sponsored jointly by the United States Fish and Wildlife Service, the Ohio Department of Natural Resources, and The Ohio State University     

Austropotamobius pallipes reduction vs. Procambarus clarkii spreading: Management implications

One of the main threats to native European crayfish is the spread of alien crayfish species that are immune carriers of the detrimental crayfish plague Aphanomyces astaci. In this study we analyzed both at a regional and at a local scale factors determining native and alien crayfish distribution in an area where the species Procambarus clarkii is currently spreading. We aim to understand which management strategies could prevent the extinction of native populations. We sampled 81 waterbodies in Lombardy (NW Italy) to assess the Austropotamobius pallipes complex and alien crayfish occurrence; we also monitored the only first order stream in our study area that we found to be currently inhabited by A. pallipes upstream and by P. clarkii downstream. A. pallipes generally occurred in small brooks, near the spring, and was linked to low periphyton abundance, while P. clarkii occurred in sites at lower altitudes, far from the spring area, mainly in lentic waterbodies with large widths; this allochthonous species was positively related to periphyton abundance. Our monitoring showed that physical barriers such as dams may prevent P. clarkii dispersal upstream. Our results confirm that A. pallipes and P. clarkii have different ecological requirements and show that, at least during the first years of invasion, P. clarkii spreading involves lowland or downstream altered sites. We suggest the need for management strategies maximizing connectivity among native populations, to avoid isolation, and promoting the separation of native populations from those of P. clarkii.     

Interactions among juveniles of two freshwater crayfish species and a predatory fish

Two freshwater crayfish species, Astacus astacus L. and Pacifastacus leniusculus Dana, co-occur in some Swedish lakes. Observational studies indicate that the introduced, North American species P. leniusculus may gradually replace the native A. astacus, but the mechanism behind the replacement is not known. This study examined the direct effects of interspecific competition between the crayfish, and indirect effects of competitive interactions and fish (European perch, Perca fluviatilis L.) predation. Three different experiments with young-of-the-year (YOY) crayfish were performed. P. leniusculus was strongly dominant over similar-sized A. astacus in interference competition for shelter in a laboratory experiment. However, in a 35-day experiment in outdoor pools, A. astacus growth and survival were about equally affected by interactions with conspecifics and P. leniusculus. In contrast, P. leniusculus was significantly more affected by intraspecific competition than by competition with A. astacus, suggesting asymmetric competition between the two species. The presence of perch in outdoor ponds with mixed-species groups of the two crayfish species resulted in considerably higher predation rates on A. astacus than on P. leniusculus. Both species showed strong antipredator responses to perch by increasing refuge use. I suggest that higher perch predation rates on A. astacus originate from P. leniusculus being the superior species in interspecific competition for shelter. Because of displacement from refuges, A. astacus individuals become more exposed to the predator. This indirect effect of interactions among the two cray-fish species and the predator may be important in the observed in situ replacement of A. astacus by P. leniusculus.     

A highly virulent pathogen, Aeromonas hydrophila, from the freshwater crayfish Pacifastacus leniusculus

Aeromonas spp. are characteristic bacteria of freshwaters and many of them can be components of the bacterial flora of aquatic animals and may become pathogens on animals including humans. In this study Aeromonas hydrophila was isolated from the freshwater crayfish, Pacifastacus leniusculus, and was found to be a highly pathogenic bacterium among many isolated bacteria. Mortality reached 100% within 6 h when 200 μl of 1.24 × 10⁷ CFU/ml was applied by injection. Histopathological studies of moribund crayfish showed that extensive necrotic nuclei and clump-infiltrated hemocytes were found in observed tissues including gill, heart, hepatopancreas and the circulatory system. To verify how crayfish are susceptible to this bacterium, crude extracellular products (ECPs) obtained from culture supernatant of A. hydrophila was studied either in vivo or in vitro. ECPs (200 μl) were able to kill crayfish by injection. In an in vitro study, ECPs induced cytotoxicity of hemocytes as well as hematopoietic cells in a dose- and time-dependent manner after 30 min post inoculation. Two genes coding for endotoxins were also found in this isolate of A. hydrophila. This indicates that the bacterial endotoxins are the causative agents of crayfish mortality. Moreover, the effect of temperature on the infectivity of A. hydrophila to crayfish was also studied. At 4 °C, all crayfish survived, whereas at 20 °C the animals died rapidly after bacterial challenge. At this low temperature A. hydrophila did not replicate or replicated at a very low degree and hence crayfish could probably mount effective cellular reactions towards A. hydrophila.     

Haemolymph constituents and osmolality as functions of moult stage,body weight,and feeding status in marron,Cherax cainii () and yabbies,Cherax destructor (Clark, 1936)

The study investigates the change in osmolality and haemolymph constituents in marron Cherax cainii and yabbies Cherax destructor associated with moult stages, body weights and their feeding status. A total of 582 haemolymph samples from 5 moult stages (postmoult-AB, intermoult-C, and premoult stages – D₀, D₁, D₂), two body weight classes (2–15 g and 61–75 g) and nutritional status were used for analysis of osmolality, protein, glucose, and ionic concentrations of potassium and chloride following the standard biochemical procedures. The haemolymph protein, glucose, potassium and chloride levels were highest at intermoult and early premoult stages, and lowest at postmoult in both crayfish species. Except protein, no significant differences were seen in analyzed parameters between various weight classes and two species. Haemolymph osmolality, protein and glucose were significantly higher in fed crayfish, whereas no variations in haemolymph potassium and chloride concentrations were observed between the fed and unfed crayfish. Maximum osmolality was recorded at 7–8 h after feeding in both crayfish species. The results showed that the biochemical changes in the haemolymph of marron and yabbies are related to moult stages, body weight and feeding and thus can be used as tools for determining suitable diets.     

Spatio-temporal pattern of Pentastiridius leporinus migration in an ephemeral cropping system

• 1 Cixiid planthoppers (Hemiptera: Fulgoromorpha: Cixiidae) are considered to be important economic pests because of their ability to transmit phloem‐restricted prokaryotes causing emerging plant diseases worldwide. However, little information is available on the biology and ecology of such species. This is the case for Pentastiridius leporinus (Linnaeus), a cixiid planthopper reported to live on common reed across Countries of Central and Northern Europe. However, in the east of France, the same planthopper species appears to complete its life cycle in the sugar beet‐wheat cropping system and has been repeatedly shown to transmit prokaryotic plant pathogens that are associated with an emerging disease of sugar beet called syndrome ‘basses richesses'.
• 2 To gather evidence on the biology of the planthopper in the cropping rotation, we analysed the flight activity of adults. We used transparent sticky traps for sampling migrating adults and quantified nymphs as well as emerging adults on the roots of wheat plants.
• 3 Results showed a significant correlation between disappearance of nymphs and emerging adults from wheat roots and the occurrence of migrant adults in nearby sugar beet fields. Planthoppers migrated more abundantly and colonized sugar beet for longer periods than any other crop available. Flight activity was very pronounced during the migratory phase that was extended from the middle of June to the middle of July. A geographic information system and geostatical analysis revealed that planthoppers flew and colonized the centre of the sugar beet field rather than the borders.
• 4 Overall, results obtained in the present study suggest that the ecology and biology of the planthopper vector in the cropping rotation is a primary factor that leads to the emergence of the syndrome ‘basses richesses' disease of sugar beet.

     

Phylogenetic relationships among plant and animal parasites,and saprotrophs in Aphanomyces (Oomycetes)

Molecular phylogenetic relationships among 12 species of Aphanomyces de Bary (Oomycetes) were analyzed based on 108 ITS sequences of nuclear rDNA. Sequences used in the analyses belonged to the major species currently available in pure culture and GenBank. Bayesian, maximum likelihood, and maximum parsimony analyses support that Aphanomyces constitutes a monophyletic group. Three independent lineages were found: (i) plant parasitic, (ii) animal parasitic, and (iii) saprotrophic or opportunistic parasitic. Sexual reproduction appeared to be critical in plant parasites for survival in soil environments while asexual reproduction seemed to be advantageous for exploiting specialization in animal parasitism. Repeated zoospore emergence seems to be an advantageous property for both plant and animal parasitic modes of life. Growth in unspecific media was generally faster in saprotrophs compared with parasitic species. A number of strains and GenBank sequences were found to be misidentified. It was confirmed molecularly that Aphanomyces piscicida and Aphanomyces invadans appear to be conspecific, and found that Aphanomyces iridis and Aphanomyces euteiches are closely related, if not the same, species. This study has shown a clear evolutionary separation between Aphanomyces species that are plant parasites and those that parasitize animals. Saprotrophic or opportunistic species formed a separate evolutionary lineage except Aphanomyces stellatus whose evolutionary position has not yet been resolved.     

Aphanomyces astaci mtDNA: insights into the pathogen’s differentiation and its genetic diversity from other closely related oomycetes

The causative agent of crayfish plague, Aphanomyces astaci (Saprolegniales, Oomycota), is one of the 100 world’s worst invasive alien species and represents a major threat to freshwater crayfish species worldwide. A better understanding of the biology and epidemiology of A. astaci relies on the application of efficient tools to detect the pathogen and assess its genetic diversity. In this study, we validated the specificity of two recently developed PCR-based approaches used to detect A. astaci groups. The first relies on the analysis of mitochondrial ribosomal rnnS (small) and rnnL (large) subunit sequences and the second, of sequences obtained by using genotype-specific primers designed from A. astaci whole genome sequencing. For this purpose, we tested the specificity against 76 selected isolates, including other oomycete species and the recently described species Aphanomyces fennicus, which, when used in nrITS-based specific tests for A. astaci, is known to result in a false positive. Under both approaches, we were able to efficiently and accurately identify A. astaci and its genetic groups in both pure cultures and clinical samples. We report that sequence analysis of the rnnS region alone is sufficient for the identification of A. astaci and a partial characterization of haplogroups. In contrast, the rnnL region alone is not sufficiently informative for A. astaci identification as other oomycete species present sequences identical to those of A. astaci.     

Response of chlorophyll fluorescence parameters to drought stress in sugar beet seedlings

We compared the parameters of chlorophyll fluorescence between two sugar beet (Beta vulgaris L.) species differing in drought tolerance. Our results indicated that there were different responses to the drought stress of these sugar beet species. In drought-tolerant sugar beet, the F ₀ increased slightly, while qN increased substantially, indicating that these plants can protect PSII reaction centers from the damage. F _v/F _m and qP decreased slightly during the initial period of drought stress; this suggests that there is a slight impact of drought stress on the openness of PSII reaction centers, and thus the plants did not suffer seriously. This was further shown by the decreased Yield and electron transfer rate. The parameters of chlorophyll fluorescence were stable and can be used as an important indicator for sugar beet seedlings in the early drought tolerance.     

Quantitative trait locus responsible for resistance to Aphanomyces root rot (black root) caused by Aphanomyces cochlioides Drechs. in sugar beet

Aphanomyces root rot, caused by Aphanomyces cochlioides Drechs., is one of the most serious diseases of sugar beet (Beta vulgaris L.). Identification and characterization of resistance genes is a major task in sugar beet breeding. To ensure the effectiveness of marker-assisted screening for Aphanomyces root rot resistance, genetic analysis of mature plants’ phenotypic and molecular markers’ segregation was carried out. At a highly infested field site, some 187 F₂ and 66 F₃ individuals, derived from a cross between lines ‘NK-310mm-O’ (highly resistant) and ‘NK-184mm-O’ (susceptible), were tested, over two seasons, for their level of resistance to Aphanomyces root rot. This resistance was classified into six categories according to the extent and intensity of whole plant symptoms. Simultaneously, two selected RAPD and 159 ‘NK-310mm-O’-coupled AFLP were used in the construction of a linkage map of 695.7 cM. Each of nine resultant linkage groups was successfully anchored to one of nine sugar beet chromosomes by incorporating 16 STS markers. Combining data for phenotype and molecular marker segregation, a single QTL was identified on chromosome III. This QTL explained 20% of the variance in F₂ population (in the year 2002) and 65% in F₃ lines (2003), indicating that this QTL plays a major role in the Aphanomyces root rot resistance. This is the first report of the genetic mapping of resistance to Aphanomyces-caused diseases in sugar beet.     

Diseases of crayfish: a review

A systematic review of parasites, pathogens and commensals of freshwater crayfish has been conducted. All major groups of disease causing agents have been covered including viruses, bacteria, fungi, protistans and metazoans. Most agents tend to cause limited problems for crayfish. Exceptions to this include fungi, bacteria and viruses. However, in many cases, these tend to be isolated reports in either a specific geographical location or in individual animals. The apparent absence of pathology associated with these agents in crayfish should not be taken to suggest that movements of crayfish to new geographical areas is necessarily acceptable. Several examples are given where seemingly healthy animals have been moved to new areas leading to mortality of other crayfish within the same area as a direct result of transmission of pathogens to naïve hosts. Some future research needs are proposed, including the need for pathogen characterisation and production of disease-free crayfish for aquaculture.     

The behavioural response of the white-clawed crayfish (Austropotamobius pallipes) to five standard marking techniques over a 14-day period

ABSTRACT

The white-clawed crayfish (Austropotamobius pallipes), is classified as endangered in the 2010 IUCN Red List and is protected under Irish and European legislation due to a drastic decline in abundance and range over the past century. Mark-recapture methods play a vital role in the process of estimating population size, and in the monitoring and conservation of mobile species. A variety of marking techniques are routinely used in a range of crayfish species for ecological research. The majority of information on the effects of these marking techniques on the physiology of crayfish species focuses on survivability and growth, whereas information is scarce on the effects on crayfish behaviour. This study investigates the behavioural changes of crayfish in reaction to five commonly used marking techniques in laboratory conditions. The marking techniques used were internal and external passive integrated transponder (PIT) tags, visible implant elastomer (VIE) tags, cauterisation and ablation. Effects of marking technique on behaviour were evaluated immediately after marking, and over a 14-day period, on 78 crayfish. Results indicated that the internal passive integrated transponder (PIT) tags negatively affected crayfish behaviour by increasing resting levels. Ablation and cauterisation also increased grooming levels, while marking reduced activity levels in general. These effects on behaviour occurred immediately after marking, but quickly dissipated. These results suggest that the use of internal PIT tags for marking white-clawed crayfish may need to be avoided as increased resting levels could lead to increased exposure out of refuge and predation. This may have a detrimental impact on the welfare and conservation of white-clawed crayfish and also lead to biased population estimates during mark-recapture projects. The remaining marking techniques (external PIT tag, VIE tag, ablation, cauterisation) may be used, as changes in behaviour will not have a detrimental impact and therefore should not lead to biased population estimates.     

Development of real-time PCR method for the detection and the quantification of a new endogenous reference gene in sugar beet “Beta vulgaris L.”: GMO application

Key message

Here, we describe a new developed quantitative real-time PCR method for the detection and quantification of a new specific endogenous reference gene used in GMO analysis.

Abstract

The key requirement of this study was the identification of a new reference gene used for the differentiation of the four genomic sections of the sugar beet (Beta vulgaris L.) (Beta, Corrollinae, Nanae and Procumbentes) suitable for quantification of genetically modified sugar beet. A specific qualitative polymerase chain reaction (PCR) assay was designed to detect the sugar beet amplifying a region of the adenylate transporter (ant) gene only from the species of the genomic section I of the genus Beta (cultivated and wild relatives) and showing negative PCR results for 7 species of the 3 other sections, 8 related species and 20 non-sugar beet plants. The sensitivity of the assay was 15 haploid genome copies (HGC). A quantitative real-time polymerase chain reaction (QRT-PCR) assay was also performed, having high linearity (R ² > 0.994) over sugar beet standard concentrations ranging from 20,000 to 10 HGC of the sugar beet DNA per PCR. The QRT-PCR assay described in this study was specific and more sensitive for sugar beet quantification compared to the validated test previously reported in the European Reference Laboratory. This assay is suitable for GMO quantification in routine analysis from a wide variety of matrices.     

Saprolegnia parasitica, an oomycete pathogen with a fishy appetite: new challenges for an old problem

Water moulds (oomycetes) of the order Saprolegniales, such as Saprolegnia and Aphanomyces species, are responsible for devastating infections on fish in aquaculture, fish farms and hobby fish tanks. Members of the genus Saprolegnia cause Saprolegniosis, a disease that is characterised by visible white or grey patches of filamentous mycelium on the body or fins of freshwater fish. Up till 2002, Saprolegnia infections in aquaculture were kept under control with malachite green, an organic dye that is very efficient at killing the pathogen. However, the use of malachite green has been banned worldwide due to its carcinogenic and toxicological effects and this has resulted in a dramatic re-emergence of Saprolegnia infections in aquaculture. As a consequence Saprolegnia parasitica is now, economically, a very important fish pathogen, especially on catfish, salmon and trout species, and warrants further investigation to develop new alternative control strategies.     

Monosomic addition lines of Beta corolliflora Zoss in sugar beet: cytological and molecular-marker analysis

Beta corolliflora is a wild relative of sugar beet (Beta vulgaris) with 2n=4x=36 chromosomes. Monosomic addition lines (2n=19) of B. corolliflora in B. vulgaris were identified from backcross progenies between triploid hybrids (genome constitution VVC) and sugar beet. They were characterized by DNA-fingerprinting using nine different B. corolliflora-specific repetitive sequences as probes and by fluorescence in situ hybridization (FISH) using two B. corollifora specific sequences and two rDNA probes. Unique banding patterns obtained after genomic Southern hybridization enabled the classification of monosomic addition lines into 11 clusters, three of which proved to have a wild beet chromosome fragment in addition to the sugar beet chromosomes as revealed by FISH. Repetitive sequences pBC216 and pBC1416 were found to be present only on wild beet chromosomes IV and V. Chromosomes I and IV were found to carry genes for 18S and 5S rRNA, respectively. An idiogram of B. corolliflora was established in the triploid VVC hybrid on the basis of chromosome size and FISH. Eight B. corolliflora addition lines could be unequivocally identified by Southern hybridization and FISH, one addition line carrying the missing wild beet chromosome is probably not viable under greenhouse conditions. The monosomic addition lines will serve as a bridge for transferring genes from wild species to sugar beet and will help to uncover genetic relationships between species of the genus Beta.     

Impact of Field Dodder (<Emphasis Type="Italic">Cuscuta campestris</Emphasis> Yunk.) on Chlorophyll Fluorescence and Chlorophyll Content of Alfalfa and Sugar Beet Plants

The impact that the parasitic plant field dodder (Cuscuta campestris Yunk.) has on chlorophyll fluorescence and chlorophyll content of infested alfalfa (Medicago sativa L.) and sugar beet (Beta vulgaris L.) was examined under controlled conditions. Several parameters of chlorophyll fluorescence were measured in infested and non-infested alfalfa and sugar beet plants over a period of twenty days, beginning with the day of infestation. Chlorophyll contents (total, relative and ratio of chlorophyll a to b) were determined 1, 7, 14 and 20 days after infestation (DAI). Field dodder was found to affect both the total and relative chlorophyll contents in infested alfalfa and sugar beet, causing significant reduction in chlorophyll content in both host plants. This parasitic plant also affects a number of parameters of chlorophyll fluorescence (F_o, F_v/F_m, Φ_PSII, F_v and IF), showing that these parameters may be considered sensitive indicators of the impact that field dodder has on its host plants.     

The identification of allergen proteins in sugar beet (Beta vulgaris) pollen causing occupational allergy in greenhouses

Background

During production of sugar beet (Beta vulgaris) seeds in greenhouses, workers frequently develop allergic symptoms. The aim of this study was to identify and characterize possible allergens in sugar beet pollen.

Methods

Sera from individuals at a local sugar beet seed producing company, having positive SPT and specific IgE to sugar beet pollen extract, were used for immunoblotting. Proteins in sugar beet pollen extracts were separated by 1- and 2-dimensional electrophoresis, and IgE-reactive proteins analyzed by liquid chromatography tandem mass spectrometry.

Results

A 14 kDa protein was identified as an allergen, since IgE-binding was inhibited by the well-characterized allergen Che a 2, profilin, from the related species Chenopodium album. The presence of 17 kDa and 14 kDa protein homologues to both the allergens Che a 1 and Che a 2 were detected in an extract from sugar beet pollen, and partial amino acid sequences were determined, using inclusion lists for tandem mass spectrometry based on homologous sequences.

Conclusion

Two occupational allergens were identified in sugar beet pollen showing sequence similarity with Chenopodium allergens. Sequence data were obtained by mass spectrometry (70 and 25%, respectively for Beta v 1 and Beta v 2), and can be used for cloning and recombinant expression of the allergens. As for treatment of Chenopodium pollinosis, immunotherapy with sugar beet pollen extracts may be feasible.