Characterization of a molt-inhibiting hormone (MIH) of the crayfish, Orconectes limosus, by cDNA cloning and mass spectrometric analysis

The structure of the precursor of a molt-inhibiting hormone (MIH) of the American crayfish, Orconectes limosus was determined by cloning of a cDNA based on RNA from the neurosecretory perikarya of the X-organ in the eyestalk ganglia. The open reading frame includes the complete precursor sequence, consisting of a signal peptide of 29, and the MIH sequence of 77 amino acids. In addition, the mature peptide was isolated by HPLC from the neurohemal sinus gland and analyzed by ESI-MS and MALDI-TOF-MS peptide mapping. This showed that the mature peptide (Mass 8664.29 Da) consists of only 75 amino acids, having Ala75-NH2 as C-terminus. Thus, C-terminal Arg77 of the precursor is removed during processing, and Gly76 serves as an amide donor. Sequence comparison confirms this peptide as a novel member of the large family, which includes crustacean hyperglycaemic hormone (CHH), MIH and gonad (vitellogenesis)-inhibiting hormone (GIH/VIH). The lack of a CPRP (CHH-precursor related peptide) in the hormone precursor, the size and specific sequence characteristics show that Orl MIH belongs to the MIH/GIH(VIH) subgroup of this larger family. Comparison with the MIH of Procambarus clarkii, the only other MIH that has thus far been identified in freshwater crayfish, shows extremely high sequence conservation. Both MIHs differ in only one amino acid residue ( approximately 99% identity), whereas the sequence identity to several other known MIHs is between 40 and 46%.     

Preliminary results on the genetic variability of mitochondrial DNA in the signal crayfish,Pacifastacus leniusculus Dana

This study presents a revised method for the extraction of mitochondrial genome (mtDNA) from crayfish species of aquacultural interest, Pacifastacus leniusculus. The mean size of the mitochondrial genome is approximately 18 000 bp. Restriction fragment length polymorphism (RFLP) analysis performed on the mtDNA has revealed extensive genetic variability within this species. The estimated percentage of nucleotide sequence divergence among the four haplotypes found for P. leniusculus ranges from 1.16 to 3.99%. These data suggest that RFLP analysis of mtDNA may provide greater resolution than protein electrophoresis for population identification among signal crayfish populations. This marker offers new avenues for aquaculture and for understanding the genetic structure and evolutionary history of crayfish populations.     

Photosensitivity spectrum of crayfish rhodopsin measured using fluorescence of metarhodopsin

Discrepancies exist among spectral measurements of sensitivity of crayfish photoreceptors, their absorption in situ, and the number and absorption spectra of crayfish photopigments that are extracted by digitonin solutions. We have determined the photosensitivity spectrum of crayfish rhodopsin in isolated rhabdoms using long wavelength fluorescence emission from crayfish metarhodopsin as an intrinsic probe. There is no measurable metarhodopsin in the dark-adapted receptor, so changes in the emission level are directly proportional to metarhodopsin concentration. We therefore used changes in metarhodopsin fluorescence to construct relaxation and saturation ("photoequilibrium") spectra, from which the photosensitivity spectrum of crayfish rhodopsin was calculated. This spectrum peaks at or approximately 530 nm and closely resembles the previously measured difference spectrum for total bleaches of dark-adapted rhabdoms. Measurements of the kinetics of changes in rhabdom fluorescence and in transmittance at 580 nm were compared with predictions derived from several model systems containing one or two photopigments. The comparison shows that only a single rhodopsin and its metarhodopsin are present in the main rhabdom of crayfish, and that other explanations must be sought for the multiple pigments seen in digitonin solution. The same analysis shows that there is no detectable formation of isorhodopsin in the rhabdom.     

Amino acid sequence of crayfish troponin I

Troponin I is the actomyosin ATPase inhibitory subunit present in the thin filament regulatory complex. The complete amino acid sequence of crayfish tail muscle troponin I has been determined. The protein is composed of 201 amino acid residues and has a molecular weight of 23,547. The N terminus is blocked, likely by an acetyl group. Crayfish troponin I shows a rather low (20-25%) sequence identity with vertebrate troponin Is as compared to the 60-82% identity within the vertebrate phylum. Similar to vertebrate cardiac troponin I, crayfish troponin I contains a 30-residue-long N-terminal extension. In crayfish troponin I, this segment bears significant sequence homology with the heavy or light chains of particular myosins. The actin-binding domain of crayfish troponin I, which displays 57% sequence homology with vertebrate troponin Is, possesses 2 unusual trimethyllysine residues. The consensus sequence of this domain in five troponin Is is as follows: D-L-R-G-K-F-X-R*-P-X-L-R*-R*-V, where R+ stands for Arg/Lys, R* for Arg/trimethyllysine, and X for any amino acid residue. Troponin I possesses two Ca2+-dependent interactive sites for troponin C; one partly overlaps with the actin binding domain and is highly conserved, and the other, corresponding to the 30-residue-long segment following the N-terminal extension in vertebrate cardiac and crayfish troponin I, is poorly conserved in the different troponin Is. Troponin I also interacts with troponin T. The consensus sequence for the interacting site on troponin I is as follows: h-D- -X-D- -R+-Y-D-h-E-h, where h stands for a hydrophobic residue, D- for Asp/Glu, R+ for Arg/Lys, and X for any residue. The five troponin Is further possess one more 15-residue-long segment of high sequence identity near the C terminus. Its evolutionary conservation suggests that this domain is involved in protein-protein interaction.     

Functional characterization of the neurodepressing hormone in the crayfish.

The effect of the neurodepressing hormone (NDH) was studied on different identified motoneurons in the abdominal ganglia of the crayfish Procambarus bouvieri (Ortmann). Although differences in sensitivity were apparent, all the neurons tested responded to NDH with a reduction in spontaneous firing rate, which lasted as long as NDH was present, and, depending on the concentration and time of action of the hormone, for even longer periods. NDH activity was determined in the various parts of the central nervous system of the crayfish, being highest in the eyestalk, gradually diminishing away from the eyestalk, with a cephalo-caudal gradient, being lowest in the abdominal ganglia. High levels of NDH activity were detected in the blood. After eyestalk ablation, NDH concentration steadily diminishes in the blood and central nervous system, until virtually disappearing after 4 days; from day 5 onwards, the activity is recovered up to its original levels. NDH synthesis takes place with a time constant of approximately 3 hr in cultured isolated segments of central nervous system, being highest in the eyestalk.     

Amino acid sequence of crayfish (Astacus fluviatilis) trypsin If

The complete amino acid sequence of trypsin from the crayfish Astacus fluviatilis has been determined. The protein was fragmented with cyanogen bromide after S-carboxymethylation of the reduced disulfide bonds and by trypsin after S-carboxymethylation as well as after succinylation of lysine residues and aminoethylation of the reduced disulfide bonds. Peptides were purified by gel filtration and by reversed-phase high-performance liquid chromatography. Stepwise degradation was performed in a spinning cup sequencer. The enzyme contains 237 amino acid residues and has a molecular weight of 25 030. In contrast to bovine trypsin, it contains three rather than six disulfide bonds which are paired in the same fashion as those in trypsin from Streptomyces griseus. The constituents of the active site of bovine trypsin are present in corresponding positions in the crayfish enzyme. Crayfish trypsin shows 43.6% sequence identity with the bovine enzyme as compared to 40.0% identity with the S. griseus enzyme. The present analysis affords the first detailed view into the evolution of trypsins at the invertebrate level.     

Positive selection of beautiful invaders: long-term persistence and bio-invasion risk of freshwater crayfish in the pet trade

After interest in keeping crayfish (Crustacea, Decapoda, Astacida) in home aquaria peaked in the mid-2000s, the aquarium trade has become a novel introduction pathway for non-native crayfish species in Germany. Here, we provide an update on the ornamental crayfish trade approximately one decade after the ‘crayfish hype’ to explore the long-term implications in terms of bio-invasion risk. Specifically, species’ availability in e-commerce and potential invasiveness were assessed and compared to previous studies. Morphological and ecological traits of the offered species were compiled and related to their long-term availability (covering 2005–2015). In July 2015, a total of 31 online shops offered 28 crayfish species, which represents a decline of 24 % in species diversity compared to the late 2000s. The estimated rate of import of new species has considerably flattened and approaches pre-hype values (<1 species year^?1). However, the risk associated with the offered species, as assessed by a risk screening tool, has not decreased compared to the late 2000s. Long-term availability in the trade was primarily determined by bright coloration, the ability to reproduce under warm aquarium conditions, and a preference for lentic habitats. Species featuring such traits are likely to persist in the aquarium trade and include four high-risk species, most notably invasive and crayfish plague-carrying red swamp crayfish (Procambarus clarkii) and Marmorkrebs (Procambarus fallax f. virginalis). Persistent propagule pressure from aquaria has substantially contributed to the establishment of both species in Germany, stressing the need for more effective pathway management.     

Trypsin from Pacifastacus leniusculus hepatopancreas: purification and cDNA cloning of the synthesized zymogen.

Trypsin was purified from crayfish, Pacifastacus leniusculus, hepatopancreas, and the gene that encoded this enzyme was cloned from a hepatopancreas cDNA library. Crayfish trypsin is synthesized as a zymogen according to the sequence of the putative precursor peptide. The authenticity of the trypsinogen is supported by the deduced amino acid sequence and confirmed by the N-terminal amino acid sequence of the mature protein. The enzyme has features characteristic of a trypsin, such as a specific binding pocket. Sequence comparison shows that crayfish trypsin is similar to those of other species, with the exception that six cysteine residues present in vertebrates are missing. Some structural characteristics, such as the length of the signal peptide and a calcium binding site, are similar to bacterial trypsin.     

Functional characterization of the neurodepressing hormone in the crayfish

The effect of the neurodepressing hormone (NDH) was studied on different identified motoneurons in the abdominal ganglia of the crayfish Procambarus bouvieri (Ortmann). Although differences in sensitivity were apparent, all the neurons tested responded to NDH with a reduction in spontaneous firing rate, which lasted as long as NDH was present, and, depending on the concentration and time od action of the hormone, for even longer periods. NDH activity was determined in the various parts of the central nervous system of the crayfish, being highest in the eyestalk, gradually diminishing away from the eyestalk, with a cephalo-caudal gradient, being lowest in the abdominal ganglia. High levels of NDH activity were detected in the blood. After eyestalk ablation, NDH concentrations steadily diminishes, in the blood and central nervous systems, until virtually disappearing after 4 days; from day 5 onwards, the activity is recovered up to its original levels. NDH synthesis takes place with a time constant of approximately 3 hr in cultured segments of central nervous system, being highest in the eyestalk.     

Detection and quantification of the crayfish plague agent in natural waters: direct monitoring approach for aquatic environments

Aphanomyces astaci, a specialised parasite of North American freshwater crayfish, is the disease agent of crayfish plague that is lethal to European freshwater crayfish. The life cycle of A. astaci has been inferred from experimental laboratory studies, but less is known about its natural sustainability and ecology. To address such questions, tools for monitoring of A. astaci directly in aquatic environments are needed. Here, we present an approach for detecting and quantifying A. astaci directly from water samples using species-specific TaqMan minor groove binder real-time PCR. Samples of a 10-fold dilution series from approximately 10(4) to approximately 1 spore of A. astaci were repeatedly tested, and reliable detection down to 1 spore was demonstrated. Further, to simulate real-life samples from natural water bodies, water samples from lakes of various water qualities were spiked with spores. The results demonstrated that co-extracted humic acids inhibit detection significantly. However, use of bovine serum albumin or the TaqMan Environmental Master Mix largely removes this problem. The practical application of the approach was successfully demonstrated on real-life water samples from crayfish farms in Finland hosting infected North American signal crayfish Pacifastacus leniusculus. Direct monitoring of A. astaci from aquatic environments may find application in the management of wild noble crayfish Astacus astacus stocks, improved aquaculture practices and more targeted conservation actions. The approach will further facilitate studies of A. astaci spore dynamics during plague outbreaks and in carrier crayfish populations, which will broaden our knowledge of the biology of this devastating crayfish pathogen.     

Purification and characterization of a beta-1,3-glucan binding protein from plasma of the crayfish Pacifastacus leniusculus

The plasma of the crayfish Pacifastacus leniusculus contains a protein which is able to bind to laminarin (a soluble beta-1,3-glucan) and which has been isolated by two independent methods, affinity precipitation with a beta-1,3-glucan or immunoaffinity chromatography. The purified beta-1,3-glucan binding protein was homogenous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is a monomeric glycoprotein with a molecular mass of approximately 100,000 Da and an isoelectric point of approximately 5.0. Amino acid analysis showed a very high similarity with the amino acid composition of beta-1,3-glucan binding proteins recently purified from two insects, the cockroach Blaberus craniifer and the silkworm Bombyx mori. The N-terminal amino acid sequence was determined to be: H2N-Asp-Ala-Gly-X-Ala-Ser-Leu-Val-Thr-Asn-Phe-Asn-Ser-Ala-Lys-Leu-X-X-Ly s--- Using monospecific rabbit polyclonal antibodies, the presence of this protein has also been shown within the blood cells. The purified beta-1,3-glucan binding protein did not show any peptidase or phenoloxidase activity but was able to enhance the activation of hemocyte-derived peptidase and prophenoloxidase only in the presence of the beta-1,3-glucan, laminarin, whereas mannan, dextran (alpha-glucan), or cellulose (beta-1,4-glucan) incubated with the beta-1,3-glucan binding protein had no effect on these enzyme activities. The beta-1,3-glucan binding protein could only be affinity-precipitated from crayfish plasma by the beta-1,3-glucans laminarin or curdlan (an insoluble beta-1,3-glucan), while mannan or dextran did not bind to the beta-1,3-glucan binding protein. No hemagglutinating activity of the purified beta-1,3-glucan binding protein could be detected.     

Life stages and reproductive components of the Marmorkrebs (marbled crayfish), the first parthenogenetic decapod crustacean

Recently, we briefly reported on the first case of parthenogenesis in the decapod Crustacea which was found in the Marmorkrebs or marbled crayfish, a cambarid species of unknown geographic origin and species identity. Curiously, this animal is known only from aquarium populations, where it explosively propagates. By means of light and electron microscopic techniques we have now investigated the reproductive components of this crayfish, using more than 100 specimens ranging from hatchling to repeatedly spawned adult. Additionally, we documented its principal life stages. Our results revealed that the external sexual characters and also the gonads of the marbled crayfish are purely female, making this fast-reproducing species a good model for investigating female reproductive features in crayfish. Testicular tissues, ovotestes, or male gonoducts, gonopores, or gonopods were never found, either in small juveniles or large adult specimens, confirming the parthenogenetic nature of this crayfish. Parthenogenesis may have arisen spontaneously or by interspecific hybridization since Wolbachia-like feminizing microorganisms were not found in the ovaries. The external sexual characters of the marbled crayfish are first recognized in Stage 4 juveniles and are structurally complete approximately 2 months after hatching in specimens of approximately 2 cm total length. In the same life stage the ovary is fully differentiated as well, although the oocytes are in previtellogenic and primary vitellogenic stages only. The architecture of the mature ovary and also the synchronous maturation of cohorts of primary vitellogenic oocytes by secondary vitellogenesis are in general agreement with data published on ovaries of bisexual crayfish. New results were obtained with respect to the muscular nature of the ovarian envelope and its extensive proliferation after the first spawning, the distribution of hemal sinuses in the ovarian envelope and in the interstitium around the oogenetic pouches, the high transport activity of the follicle cells, and the colonization of oogenetic pouches by previtellogenic oocytes that originate in the germaria. Investigation of the nuclei of oocytes in the germaria and oogenetic pouches revealed no signs of meiosis, as usually found in females of bisexual decapods, suggesting that parthenogenesis in the marbled crayfish might be an apomictic thelytoky. The detection of new rickettsial and coccidian infections in the ovary and further organs raises fears that the marbled crayfish might endanger native European species by transmission of pathogens once escaped into the wild.     

An atypical iron-responsive element (IRE) within crayfish ferritin mRNA and an iron regulatory protein 1 (IRP1)-like protein from crayfish hepatopancreas

A putative crayfish iron-responsive element (IRE) is present in the 5'-untranslated region of the crayfish ferritin mRNA. The putative crayfish IRE is in a cap-proximal position and shares most of the structural features of the consensus IRE, but the RNA stem-loop structure contains a bulge of a guanine instead of a cytosine at the expected position, so far thought to be a hallmark of IREs. By using an electromobility shift assay this IRE was shown to specifically bind purified recombinant human iron regulatory protein 1 (IRP1) as well as a factor(s) present in a homogenate of crayfish hepatopancreas, likely to be a crayfish IRP1 homologue. With mutations in the crayfish IRE, the affinity of IRP to IRE was drastically decreased. A cDNA encoding an IRP1-like protein was cloned from the hepatopancreas of crayfish. This protein has sequence similarities to IRP, and contains all the active-site residues of aconitase, two putative RNA-binding regions and a putative contact site between RNA and IRP. These results show that a crayfish IRE, lacking the bulged C, can bind IRP1 in vitro and that an IRP1-like protein present in crayfish hepatopancreas may have both aconitase and RNA-binding activities.     

Analysis of variable genomic loci in white spot syndrome virus to predict its origins in Procambarus clarkii crayfish farmed in China

Variable genomic loci were examined in 4 white spot syndrome virus (WSSV) isolates (08HB, 09HB, 08JS and 09JS) from Procambarus clarkii crayfish collected from Jiangsu and Hubei Provinces in China in 2008 and 2009. In ORF75, sequence variation detected in the 4 isolates, as well as in isolates sequenced previously, suggested that WSSV might have segregated into 2 lineages since first emerging as a serious pathogen of farmed shrimp in East Asia in the early-mid 1990s, with one lineage remaining in East Asia and the other separating to South Asia. In ORF23/24, deletions of 9.31, 10.97, or 11.09 kb were evident compared to a reference isolate from Taiwan (WSSV-TW), and, in ORF14/15, deletions of 5.14 or 5.95 kb were evident compared to a reference isolate from Thailand with the largest genome size (TH-96-II). With respect to these genome characteristics, the crayfish isolates 08HB, 09HB and 08JS were similar to WSSV-TW and the isolate 09JS was similar to a reference isolate from China (WSSV-CN). In addition to these loci, sequence variation was evident in ORF94 and ORF125 that might be useful for differentiating isolates and in epidemiological tracing of WSSV spread in crayfish farmed in China. However, as all 4 crayfish isolates possessed a Homologous Region 9 sequence identical to isolate WSSV-TW and another Thailand isolate (WSSV-TH), and as their transposase sequence was identical to isolates WSSV-CN and WSSV-TH, these 2 loci were not useful in predicting their origins.     

Amino acid sequence of a unique protease from the crayfish Astacus fluviatilis

The amino acid sequence of a protease from the crayfish Astacus fluviatilis has been determined from overlapping sets of peptides derived largely by cleavage at Met, Lys, or Arg residues. The protein comprises 200 amino acid residues in a single polypeptide chain, corresponding to a molecular mass of 22,614 daltons. Two disulfide bonds link Cys-42 to Cys-198 and Cys-64 to Cys-84. The sequence of this invertebrate protease appears to be unique since it has no homologous relationship to any of the known protein sequences.     

Rainfall stochasticity controls the distribution of invasive crayfish and its impact on amphibian guilds in Mediterranean temporary waters

Invasive crayfish have severely impacted invaded aquatic ecosystems worldwide. We studied temporal and spatial variation in the range expansion of the red swamp crayfish at one of the first European localities to which it was introduced: Doñana National Park (SW Spain). In contrast to the rapid range expansion witnessed in other areas, this invasive crayfish has not spread across the entire park. Instead, its distribution has expanded during wet periods, but contracted during drought periods. The red swamp crayfish has caused steep amphibian declines in other invaded areas. However, after approximately 35 years of crayfish presence in Doñana National Park, we have yet to detect a reduction in the number or occurrence of amphibian species. Amphibians may thus be protected by the large abundance of temporary ponds in the area, which provides them with an effective refuge network. We show that natural fluctuations in annual rainfall and in the number of ponds filled can temporarily eliminate invasive crayfish from particular areas. This fact should be taken into account when attempting to reduce the impact of crayfish on aquatic communities, intensifying crayfish removal during particularly dry years, when it is most effective.     

Amino acid sequence around the thiolester of alpha 2-macroglobulin from plasma of the crayfish, Pacifastacus leniusculus

Alpha 2-Macroglobulin (alpha 2 M) was isolated from plasma of the freshwater crayfish, Pacifastacus leniusculus, using ultracentrifugation, ion-exchange chromatography and gel filtration techniques. The Pacifastacus alpha 2 M molecule (P alpha 2 M) was radio-actively labeled in the thiol ester structure with iodo [14C]acetic acid in the presence of methylamine. After reduction and carboxymethylation of the protein, it was digested with trypsin. A 14C-labeled tryptic peptide was sequenced and contained an amino acid sequence very similar to other known thiol ester sequences from human alpha 2 M and related proteins. The N-terminal sequence of P alpha 2 M was related to that recently determined for lobster alpha 2 M [(1987) J. Biol. Chem. 262, 14606-14611].     

Predicting the distribution of invasive crayfish (Pacifastacus leniusculus) in a Kusiro Moor marsh (Japan) using classification and regression trees

Summer distributions of the invasive signal crayfish (Pacifastacus leniusculus) were investigated in relation to physicochemistry in a Kusiro Moor marsh and its inflows and outflows in northern Japan. Maximum crayfish abundance and biomass were 1.04 individuals/m² and 3.56 g dry mass (DM)/m² in littoral marsh habitats, and 5.84 individuals/m² and 13.48 g DM/m² in stream habitats. Classification tree analysis was used to predict crayfish occurrence at 102 sites from all habitats (i.e. littoral marsh, pelagic marsh and stream) while regression tree analyses were used to predict crayfish abundance at littoral marsh and stream sites separately. The classification tree showed that crayfish occurrence was primarily determined by undercut bank volume regardless of habitat identity. When undercut bank volume was <0.0054 m³, crayfish were predicted to be absent at marsh sites, but expected to occur at stream sites where pH and water temperature exceeded 6.5 and 14.3°C, respectively. The regression tree using only littoral marsh sites showed that undercut bank volume, followed by dissolved oxygen level, determined the splits of the tree. Crayfish abundance was highest when undercut bank volume was >0.61 m³, and moderately high when dissolved oxygen was >9.09 mg/l and undercut bank volume was <0.61 m³. On the other hand, the regression tree using only stream sites showed that water temperature was the major predictor that determined the splits. We discuss the roles of physicochemical factors as limiting factors of the distribution pattern of the invasive crayfish.