Phylogeographic insights into the invasion history and secondary spread of the signal crayfish in Japan

Successful invasion by nonindigenous species is often attributed to high propagule pressure, yet some foreign species become widespread despite showing reduced genetic variation due to founder effects. The signal crayfish (Pacifastacus leniusculus) is one such example, where rapid spread across Japan in recent decades is believed to be the result of only three founding populations. To infer the history and explore the success of this remarkable crayfish invasion, we combined detailed phylogeographical and morphological analyses conducted in both the introduced and native ranges. We sequenced 16S mitochondrial DNA of signal crayfish from across the introduced range in Japan (537 samples, 20 sites) and the native range in western North America (700 samples, 50 sites). Because chela size is often related to aggressive behavior in crayfish, and hence, their invasion success, we also measured chela size of a subset of specimens in both introduced and native ranges. Genetic diversity of introduced signal crayfish populations was as high as that of the dominant phylogeographic group in the native range, suggesting high propagule pressure during invasion. More recently established crayfish populations in Japan that originated through secondary spread from one of the founding populations exhibit reduced genetic diversity relative to older populations, probably as a result of founder effects. However, these newer populations also show larger chela size, consistent with expectations of rapid adaptations or phenotypic responses during the invasion process. Introduced signal crayfish populations in Japan originate from multiple source populations from a wide geographic range in the native range of western North America. A combination of high genetic diversity, especially for older populations in the invasive range, and rapid adaptation to colonization, manifested as larger chela in recent invasions, likely contribute to invasion success of signal crayfish in Japan.     

一种新的螯虾丝氨酸蛋白酶抑制物基因的克隆表达及其活性分析

根据本课题组从克氏原螯虾中新发现的丝氨酸蛋白酶抑制物的基因序列(GenBank登录号CD644775)设计一对引物,应用逆转录-聚合酶链式反应(RT-PCR)技术,从螯虾血淋巴细胞中扩增出丝氨酸蛋白酶抑制物基因PCI188,将其连入原核表达载体pET-32a,转化至大肠杆菌Rosetta株和BL21株中进行蛋白表达,结果该蛋白只在前者表达。表达产物用免疫转印检测,出现50kD的特异性条带,与螯虾PCI188基因编码的蛋白大小相符。将融合蛋白纯化后免疫新西兰兔,用免疫血清与螯虾血淋巴作用后测定酚氧化酶活力,结果显示,酚氧化酶活力有所升高,从而首次证实螯虾PCI188编码的蛋白对丝氨酸蛋白酶有抑制作用。     

Effects of acclimation temperature,season, and time of day on the critical thermal maxima and minima of the crayfish Orconectes rusticus

• 1.1. The critical thermal minima (CTMin) and maxima (CTMax) were determined for field-acclimatized and laboratory-acclimated crayfish (Orconectes rusticus) throughout 1984.
• 2.2. The CTMin and CTMax of field-acclimatized crayfish were seasonally adjusted by 9.7 C and 14.7 C respectively.
• 3.3. Seasonal variation in both tolerance regimes persisted in crayfish acclimated in the laboratory at 5 and 25°C for one week; however, no diel variation existed in either the CTMin or CTMax of laboratory-acclimated crayfish.
• 4.4. Integration of thermal acclimation of the CTMin and CTMax with seasonal conditioning may influence the functional capacities of this species when considered in relation to the seasonal ranges in stream temperature.

     

Movement and dispersal of the invasive signal crayfish Pacifastacus leniusculus in upland rivers

1. The American signal crayfish Pacifastacus leniusculus, an invasive species widely introduced throughout Europe, is a major threat to native European crayfish species and is causing increasing concern because of its wide impact on aquatic ecosystems. 2. Whilst various control and management methods have been proposed, very little is known about the factors influencing dispersal and movements of signal crayfish. 3. Sixty‐four adult signal crayfish (carapace length 31.9–63.8 mm) were radiotagged in upland rivers in northern England, during four periods. Tracking was carried out at two sites, a low‐density establishing population and a high‐density established population. Tracking was carried out at both sites concurrently during midsummer (June to August 2002), during late summer (August to September 2001) at the low‐density population site and during autumn to winter (October to February 2000/01) at the high‐density population site. 4. Maximum movement occurred during midsummer. Temperature appeared to be a major factor influencing the timing and extent of movements between tracking periods. 5. The frequency distribution of the maximum distance moved upstream and downstream by radiotagged crayfish showed an inverse power relationship. The median maximal upstream and downstream distances moved were 13.5 m (range 0–283 m) and 15 m (range 0–417 m), respectively. There was a significant difference between the distributions of upstream and downstream ranges, with greater distances moved downstream. 6. All downstream movements made by crayfish appeared to be active movements and not the result of passive movement during periods of high discharge. There was no apparent influence of size, sex or density on the amount of movement recorded. 7. The study provides important information on the spatial and temporal behaviour of introduced crayfish in upland lotic systems. In contrast to lowland rivers, our results suggest that flow or gradient may influence the invasive potential of signal crayfish in an upstream direction in upland rivers.     

New Zealand stream crayfish: functional omnivores but trophic predators?

1. The feeding ecology of the crayfish Paranephrops planifrons in streams draining catchments in native forest and pastoral land was investigated using analyses of both stomach contents and stable isotopes of carbon and nitrogen. We aimed to (1) identify the energy sources of crayfish, (2) determine whether these were affected by ontogeny or land use change, and (3) assess the functional and trophic roles of crayfish in New Zealand hill-country streams.
2. In native forest streams, crayfish stomach contents were dominated volumetrically by leaf detritus (>60%), but in pasture streams aquatic invertebrates constituted more than 40% of stomach volumes and leaf detritus <30%. Stable isotope analyses revealed that crayfish from both native forest and pasture streams incorporated energy from aquatic invertebrates into their body tissue but did not appear to utilize detritus for growth. Therefore, deforestation had little impact on crayfish energy sources.
3. In native forest streams, adult crayfish (≥20 mm orbit-carapace length (OCL)) consumed greater amounts of leaf detritus than juvenile crayfish, but a corresponding change in stable isotope signatures was not detected. Ontogenetic shifts in diet were not consistent between land use suggesting that change in local habitat and food resources, as a result of land use change, affect crayfish food choice more than factors related solely to age or size.
4. Crayfish appear to occupy the trophic position of a predator, but by functioning as omnivores, they have dual roles as both predators and processors of organic matter. The use of gut content analysis in conjunction with stable isotope analyses revealed that the functional and trophic roles of P. planifrons differed, with implications for the interpretation of diet studies and understanding of the role of omnivores in food webs.     

Biogeographic differences between native and non-native populations of crayfish alter species coexistence and trophic interactions in mesocosms

Biogeographical comparisons of native and non-native populations allow researchers to understand the degree to which traits contributing to invasion success are intrinsic or change during the invasion process. Here, we investigate whether traits underlying interspecific competition change following invasion and whether these alter the impacts of two crayfish congeners that have invaded into each other’s native ranges. Specifically, we compared native and non-native populations of rusty (Faxonius rusticus) and virile crayfish (F. virilis). We compared native and non-native populations of each species using laboratory assays to examine aggression and large mesocosms with the congeners in sympatry to examine growth and survival as well as impacts on lower trophic levels. We found that non-native virile crayfish were more aggressive in response to a threat than native virile crayfish and exhibited greater growth and survival in sympatry with rusty crayfish. These intraspecific differences were large enough to alter coexistence between species in the mesocosm experiment, which is consistent with patterns of coexistence between these species in the field. We did not observe differences in traits between native and non-native rusty crayfish, but rusty crayfish were consistently competitively dominant over virile crayfish in paired laboratory assays. Non-native populations of both species had greater impacts on lower trophic levels than native populations. Taken together, these findings provide new evidence that trait changes during invasions may enhance ecological impacts of invasive animals and their ability to compete with closely related native species.     

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.     

Non‐lethal predator effects on the performance of a native and an exotic crayfish species

1. I tested the hypothesis that the potential for non‐lethal effects of predators are more important for overall performance of the fast‐growing exotic signal crayfish (Pacifastacus leniusculus Dana) than for the slower growing native noble crayfish (Astacus astacus L.). I further tested if omnivorous crayfish switched to feed on less risky food sources in the presence of predators, a behaviour that could reduce the feeding costs associated with predator avoidance. 2. In a 2 month long outdoor pool experiment, I measured behaviour, survival, cheliped loss, growth, and food consumption in juvenile noble or signal crayfish in pools with either a caged predatory dragonfly larvae (Aeshna sp.), a planktivorous fish that do not feed on crayfish (sunbleak, Leucaspius delineatus Heckel), or predator‐free controls. Crayfish had access to multiple food sources: live zooplankton, detritus and periphyton. Frozen chironomid larvae were also supplied ad libitum outside crayfish refuges, simulating food in a risky habitat. 3. Crayfish were mainly active during hours of darkness, with signal crayfish spending significantly more time outside refuges than noble crayfish. The proportion of crayfish outside refuges varied between crayfish species, time and predator treatment, with signal crayfish spending more time in refuges at night in the presence of fish. 4. Survival in noble crayfish was higher than in signal crayfish, and signal crayfish had a higher frequency of lost chelipeds, indicating a high level of intraspecific interactions. Crayfish survival was not affected by the presence of predators. 5. Gut‐contents analysis and stable isotope values of carbon (δ¹³C) and nitrogen (δ¹⁵N) indicated that the two crayfish species had similar food preferences, and that crayfish received most of their energy from feeding on invertebrates (e.g. chironomid larvae), although detritus was the most frequent food item in their guts. Signal crayfish guts were more full than those of noble crayfish, but signal crayfish in pools with fish contained significantly less food and fewer had consumed chironomids compared with predator‐free controls. Length increase of signal crayfish (35%) was significantly higher than of noble crayfish (20%), but signal crayfish in pools with fish grew less than in control pools. 6. This short‐term study indicates that fish species that do not pose a lethal threat to an organism may indirectly cause reductions in growth by affecting behaviour and feeding. This may occur even though prey are omnivorous and have access to and consume multiple food sources. These non‐lethal effects of predators are expected to be particularly important in exotic crayfish species that show a general response to fish, have high individual growth rates, and when their feeding on the most profitable food source is reduced.     

PHOTOPERIODIC INDUCTION OF OVARIAN MATURATION IN CRAYFISH PROCAMBARUS CLARKII IS MEDIATED BY EXTRARETINAL PHOTORECEPTION

The current study was carried out to investigate whether thephotoperiodic induction of ovarian maturation in crayfish is based on a photosensitiverhythm related to extraretinal photoreceptors. To test this, two batches of61 juvenile crayfish Procambarus clarkiiconsisting of intact organisms and animals lacking retina and lamina were exposed to 24hlight-dark cycles of different photoperiodic schedules based on a night-breakprotocol for 3 months. Both batches of crayfish showed the greatest ovarianmaturation (size, color, degree and size of oocytes) when the light pulseinterrupted the scotophase at 21:00 and 05:00, showing a bimodal photoinduciblerhythm. Results of the current study indicate that crayfish ovarian maturationdepends on a photoinducible rhythm with two possible states that is relatedto the circadian clock of crayfish. This phenomenon is mediated by extraretinalphotoreceptors. Results are interpreted in the light of models of externalcoincidence. (Chronobiology International, 18(3),423–434, 2001)     

Intraguild predation between spawning smallmouth bass (Micropterus dolomieu) and nest‐raiding crayfish (Orconectes rusticus): implications for bass nesting success

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Transmission of crayfish plague

Two possible means of transmission of crayfish plague were investigated: via fish (as vectors), and via crayfish (as hosts or vectors when dead). The crayfish transmission experiments focussed on both the viability of the fungus in dead crayfish when kept in simulated field conditions, and on the treatments which kill viable forms of Aphanomyces astaci within the recently dead host (cadaver). It was found that A. astaci remains viable for 5 d, and possibly longer in crayfish kept in water at 21 degrees C after dying of crayfish plague. Heating (boiling for 1 min) was the quickest way of decontaminating crayfish cadavers. Freezing took considerably longer: after 48 h at -20 degrees C, viable stages were still present. It was also found that A. astaci is unlikely to survive passage of the gastrointestinal tract of either mammals or birds as no viable stages were found after 12 h at 37 degrees C. Two basic modes of transmission of crayfish plague via fish were investigated: (1) after passage of initially viable forms of A. astaci through the digestive tract of fish and (2) via fish skin. If A. astaci was fed to fish as infected abdominal cuticle, it was still viable after passage through the gastrointestinal tract. When pure mycelium or spores were fed to fish there was no indication of viable forms of A. astaci after passage through the gastrointestinal tract. Transmission via fish skin was not observed under the experimental conditions applied. The results on fish as vectors have practical importance for fish transport and stocking because the present study shows that there is a risk of transmission of crayfish plague via fish faeces. The investigation of crayfish as vectors delivers methods that could be used for treatment of crayfish imported for human consumption into crayfish-plague-free areas. This application is particularly important for the importation of American crayfish, which are in general suspected to carry A. astaci in their cuticle.     

A comparative study of the physiological condition of native and invasive crayfish in Croatian rivers

The physiological condition of two invasive crayfish species identified in Croatian rivers (Orconectes limosus and Pacifastacus leniusculus) was compared with that of native crayfish populations (Astacus astacus and A. leptodactylus). Hepatosomatic, gonadosomatic and abdominal muscle indices were examined for all four species and within a specific invadernative pair. The results demonstrate that invasive crayfish had better overall physiological condition when compared to native species, with a higher energy content of the hepatopancreas, abdominal muscle and gonads in invasive than in native species, with the exception of A. leptodactylus females which demonstrated an increased ability to allocate energy to reproduction in comparison with other species. These results also indicate that A. astacus is physiologically more unstable than A. leptodactylus and thus more likely to be outcompeted and replaced by non-indigenous crayfish species.     

Photoperiodic induction of ovarian maturation in crayfish Procambarus clarkii is mediated by extraretinal photoreception

The current study was carried out to investigate whether the photoperiodic induction of ovarian maturation in crayfish is based on a photosensitive rhythm related to extraretinal photoreceptors. To test this, two batches of 61 juvenile crayfish Procambarus clarkii consisting of [1] intact organisms and [2] animals lacking retina and lamina were exposed to 24h light-dark cycles of different photoperiodic schedules based on a night-break protocol for 3 months. Both batches of crayfish showed the greatest ovarian maturation (size, color, degree and size of oocytes) when the light pulse interrupted the scotophase at 21:00 and 05:00, showing a bimodal photoinducible rhythm. Results of the current study indicate that crayfish ovarian maturation depends on a photoinducible rhythm with two possible states that is related to the circadian clock of crayfish. This phenomenon is mediated by extraretinal photoreceptors. Results are interpreted in the light of models of external coincidence. (Chronobiology International, 18(3), 423-434, 2001)     

Predation by the invasive American signal crayfish, Pacifastacus leniusculus Dana, on the invasive zebra mussel, Dreissena polymorpha Pallas: the potential for control and facilitation

Non-indigenous crayfish often have major ecological impacts on invaded water bodies, and have contributed to the decline of native crayfish species throughout Europe. The American signal crayfish, Pacifastacus leniusculus, is the most widespread invasive crayfish in Great Britain, where the zebra mussel, Dreissena polymorpha, is similarly an invasive pest species. The potential for the American signal crayfish to regulate zebra mussel populations was investigated through a series of laboratory experiments. Crayfish were found to be highly size selective, consuming significantly more of the smallest size class of zebra mussels offered (7–12 mm), over medium (16–21 mm) and large (25–30 mm). Crayfish feeding rate on zebra mussels was not altered when mussels were presented clumped together in natural druses compared with mussels in a disassembled druse. Crayfish spent significantly more time foraging when mussels were unattached, and a greater proportion of attacks were on medium and large than on small mussels (83% of attacks were on medium and large mussels when unattached as opposed to 47% when on druses). Individual crayfish feeding rate decreased significantly at densities of > ~5 crayfish m⁻². Signal crayfish are, therefore, unlikely to be able to significantly impact established populations of zebra mussels in the wild, although zebra mussels have the potential to provide crayfish with a substantial food source.     

The influence of evolution and plasticity on the behavior of an invasive crayfish

Invasion success can be enhanced by evolution and behavioral plasticity, but the importance of these processes for most invasions is not well understood. Previous research suggests there is a genetic basis for differences in growth rate between native and invaded range rusty crayfish (Orconectes rusticus). We hypothesized that invaded range O. rusticus achieve faster growth by allocating more time to foraging and less to defense. We conducted a laboratory experiment to test the effects of range (native or invaded) and plasticity (as induced by exposure to predators) on crayfish behavior. We collected O. rusticus adults and eggs from both ranges, hatched eggs in the lab, and reared juveniles in common conditions either with or without predatory fish. We then quantified adult and juvenile crayfish activity in an experiment with and without predatory fish. In support of our hypothesis, invaded range adults displayed reduced antipredator behavior compared to native range adults. Further, invaded range juveniles were more active than native range juveniles without predators, but all juveniles were inactive with predators. In addition, invaded range juveniles had greater plasticity in behavior than native range juveniles. These results suggest that activity level in the absence of predators has diverged in the invaded range. Because active crayfish consume more prey, this change in behavior may be responsible for rapid growth in the invaded range of O. rusticus, a trait that contributes to the strong ecological impacts of this invasive crayfish.     

Invasions and niche width: does niche width of an introduced crayfish differ from a native crayfish?

1. Human activities have promoted the spread of species worldwide. Several crayfish species have been introduced into new areas, posing a threat to native crayfish and other biota. Invader success may depend on the ability to utilise a wide variety of habitats and resources. Successful invaders are generally expected to have broader niches and to be more plastic than non-invasive species.
2. Using stable isotope ratios of carbon and nitrogen we compared the niche widths of native noble crayfish and introduced signal crayfish, a successful invader of Swedish streams. The calculation of niche width took account of between-site differences in basal resource isotope signature ranges. We also assessed whether population density, prey biomass or prey diversity affected niche width.
3. At the species level, signal crayfish had twice the niche width of noble crayfish. However, individual populations of noble crayfish and signal crayfish in Swedish streams had similar niche widths. This suggests that signal crayfish has greater plasticity with respect to habitat utilisation and feeding than noble crayfish. Niche width in both species correlated positively with benthic invertebrate biomass and diversity, indicating that animal food sources are important for crayfish.
4. We find that assessing niche width in relation to invader success can be a useful tool trying to predict the impact of invasions on different scales. The findings in this study suggest that invaders and natives will have a similar impact on the stream scale whereas the invader will have a larger impact on the regional scale due to the ability to utilise a wider range of streams.     

Extended intermolecular interactions in a serine protease-canonical inhibitor complex account for strong and highly specific inhibition

We have previously shown that a trypsin inhibitor from desert locust Schistocerca gregaria (SGTI) is a taxon-specific inhibitor that inhibits arthropod trypsins, such as crayfish trypsin, five orders of magnitude more effectively than mammalian trypsins. Thermal denaturation experiments, presented here, confirm the inhibition kinetics studies; upon addition of SGTI the melting temperatures of crayfish and bovine trypsins increased 27 degrees C and 4.5 degrees C, respectively. To explore the structural features responsible for this taxon specificity we crystallized natural crayfish trypsin in complex with chemically synthesized SGTI. This is the first X-ray structure of an arthropod trypsin and also the highest resolution (1.2A) structure of a trypsin-protein inhibitor complex reported so far. Structural data show that in addition to the primary binding loop, residues P3-P3' of SGTI, the interactions between SGTI and the crayfish enzyme are also extended over the P12-P4 and P4'-P5' regions. This is partly due to a structural change of region P10-P4 in the SGTI structure induced by binding of the inhibitor to crayfish trypsin. The comparison of SGTI-crayfish trypsin and SGTI-bovine trypsin complexes by structure-based calculations revealed a significant interaction energy surplus for the SGTI-crayfish trypsin complex distributed over the entire binding region. The new regions that account for stronger and more specific binding of SGTI to crayfish than to bovine trypsin offer new inhibitor sites to engineer in order to develop efficient and specific protease inhibitors for practical use.     

Ab initio and DFT conformational study on N-nitrosodiethylamine, (C2H5)2N-N=O

Ab initio (MP2) and DFT (B3LYP) calculations, using the cc-pVTZ and aug-cc-pVTZ basis sets, have been performed to characterize some stationary points on the ground state potential energy surface of the title molecules. Several properties as, for instance, relative energies, the barriers for NO rotation around the NN bond, NBO charges on O and amino N atoms, as well as the dipole moments, have been calculated and analyzed in the light of the structures found. Both computational levels here employed yield three minima, in which the C₂NNO frame is ‘planar’ or ‘quasi-planar’. Important correlations between NBO charges and geometric parameters, as well as between some structural features and dipole moments are also discussed. A total of 17 structures have been found for the (C₂H₅)₂N-N=O molecule. Two ranges of values have been obtained for the dipole moment, with the largest values occurring for the structures in which the nitrogen lone pair is parallel to the NO group π system. For instance, these two ranges are from ~4.1 to 4.5 D, and from ~1.6 to 2.1 D, at the MP2/cc-pVTZ level. These ranges are consistent with a larger and a smaller contribution of a dipolar resonance structure, respectively. As the method or basis set changes the values of the dipole moments change by at most ~0.23 D.     

Synaptic plasticity at the crayfish opener neuromuscular preparation

The crayfish opener neuromuscular preparation exhibits most of the plasticities yet described for any synapse, including facilitation, long-term potentiation, presynaptic inhibition, and modulation. Since the presynaptic terminals and postsynaptic muscle fibers can both be intracellularly penetrated, one can now more easily examine the cellular/molecular bases for these plasticities. Data from such studies suggest that facilitation may be influenced by something other than residual free calcium and that presynaptic inhibition is produced by a conductance increase to chloride in the terminals of the excitor axon. Several drugs (ethanol, pentobarbital) have significant effects on these synaptic plasticities over concentration ranges which produce obvious behavioral effects in crayfish and mammals. Hence, this preparation should be a useful model system to determine cellular/molecular bases for various synaptic plasticities and the effects of drugs on these plasticities.     

Preparation and some properties of immobilized trypsin from the crayfish Cambarus affinis Say (author's transl)]

The anionic tryptic enzyme from the crayfish (crayfish trypsin) was adsorbed to DEAE-Sephadex A-50 and covalently coupled to BrCN-activated Sepharose 4B and porous glass loaded with isothiocyanate propyl groups (ITC-glass). The relative activities against p-tosylarginine methyl ester (TosArgOMe) were found to be 30 to 100% for DEAE-Sephadex crayfish trypsin, 20 to 53% for Sepharose crayfish trypsin, and 17 to 38% for ITC-glass crayfish trypsin. The relative activities rise with declining protein content of the enzyme matrix complexes. The highest relative proteinase activities (substrate: 1% casein) were obtained with Sepharose crayfish trypsin (74%), followed by DEAE-Sephadex crayfish trypsin (68%) and ITC-glass crayfish trypsin (45%). Similar results are obtained with protamine and native lactate dehydrogenase as substrates. In accordance with the Sepharose bovine trypsin complex the apparent Michaelis constant (Km(app)) of the Sepharose crayfish trypsin with TosArgOMe was found to be markedly higher than that of the native enzyme. The pH-activity profiles of the crayfish trypsin derivatives using TosArgOMe as substrate were shown to be displaced towards more alkaline pH values by 0.5 (ITC-glass crayfish trypsin) and 1 (Sepharose crayfish trypsin) pH units, respectively, or towards more acidic pH values (by 1.5 pH units) with the polycationic derivative (DEAE-Sephadex crayfish trypsin) as compared to the native enzyme (optimum pH 8.6). Concerning the temperature stability of the derivatives, Sepharose crayfish trypsin was more stabile, ITC-glass crayfish trypsin behaves like the native crayfish trypsin, and DEAE-Sephadex crayfish trypsin was more sensitive at elevated temperatures as compared to the soluble enzyme. The properties of the crayfish trypsin derivatives are compared with the properties of the bovine analogues.