Enemy at the gates: introduction potential of non-indigenous freshwater crayfish in Greece via the aquarium trade

Indigenous freshwater crayfish species (ICS) are important biodiversity components and desirable fishery targets. However, ICS populations are increasingly threatened by various anthropogenic stressors. Moreover, established populations of non-indigenous freshwater crayfish species (NICS) and new ‘waves’ of NICS introductions exert significant pressure on ICS populations at a pan-European level. These effects include direct competition for space and resources as well as crayfish ‘plague’ transmission from introduced North American species. Given low public knowledge of this problematic, considerable risk of future introductions exist as a result of conventional and internet-based aquarium trade, which often lead to deliberate and/or accidental releases of NICS into the wild. In 2011, we conducted a survey of freshwater crayfish species in eleven large-size pet shops located in three major cities and in three large internet-based aquarium companies in Greece. Overall, eight species belonging to three genera (Procambarus, Cherax and Cambarellus) were recorded, originating from the USA, Australia, New Guinea and Mexico. The invasion potential of the three most popular species was assessed using the Freshwater Invertebrate Invasiveness Scoring Kit (FI-ISK). Two species were determined to constitute a ‘very high risk’ of invasion. As such, regulatory measures need to be implemented to monitor the ornamental trade of NICS in Greece and a national framework developed for protecting ICS.     

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.     

A synthetic phylogeny of freshwater crayfish: insights for conservation

Phylogenetic systematics is heading for a renaissance where we shift from considering our phylogenetic estimates as a static image in a published paper and taxonomies as a hardcopy checklist to treating both the phylogenetic estimate and dynamic taxonomies as metadata for further analyses. The Open Tree of Life project (opentreeoflife.org) is developing synthesis tools for harnessing the power of phylogenetic inference and robust taxonomy to develop a synthetic tree of life. We capitalize on this approach to estimate a synthesis tree for the freshwater crayfish. The crayfish make an exceptional group to demonstrate the utility of the synthesis approach, as there recently have been a number of phylogenetic studies on the crayfishes along with a robust underlying taxonomic framework. Importantly, the crayfish have also been extensively assessed by an IUCN Red List team and therefore have accurate and up-to-date area and conservation status data available for analysis within a phylogenetic context. Here, we develop a synthesis phylogeny for the world''s freshwater crayfish and examine the phylogenetic distribution of threat. We also estimate a molecular phylogeny based on all available GenBank crayfish sequences and use this tree to estimate divergence times and test for divergence rate variation. Finally, we conduct EDGE and HEDGE analyses and identify a number of species of freshwater crayfish of highest priority in conservation efforts.     

The ornamental fish trade and fish conservation

The global trade in ornamental fish and associated aquarium and pond accessories is in excess of U.S. $7 × 10⁹ each year. Although the majority of freshwater fish involved in the trade are from captive-bred sources, significant numbers are still removed from the wild. In addition, almost all of the marine fish in the trade are wild-caught. Whilst habitat destruction, pollution and overfishing for food are the main threats facing fish populations in the wild, the ornamental fish trade may have adverse effects as a result of the introduction of nonnative organisms, and by the direct depletion of wild stocks. The ornamental fish trade is ideally placed to raise public awareness of, and actively support, fish conservation efforts, and must maintain a close liaison with legislative-forming organizations seeking to impose further trade restrictions.     

Detection of Deefgea chitinilytica in freshwater ornamental fish

Aim: To identify and characterize six chitinolytic bacterial strains isolated from ornamental fish. Methods and Results: Six different isolates of Deefgea chitinilytica were detected in healthy as well as diseased ornamental fish in Germany over a period of 2 years. Bacterial strains were identified using 16S rRNA partial gene sequencing and further characterized using different biochemical microtest systems and additional standard biochemical tests. Conclusion: We show that commercially available biochemical microtest systems are useful for identification of D. chitinilytica, supplemented by 16S rRNA partial gene sequencing. Furthermore, this study provides new information about the occurrence of D. chitinilytica, as this is the first isolation of D. chitinilytica from animals and first described isolation in Europe. Significance and Impact of the Study: Deefgea chitinilytica may be isolated regularly in fish diagnostic laboratories. Therefore, accurate identification of this bacterial species is important. Involvement of D. chitinilytica in opportunistic infections of aquatic organisms cannot be excluded and has to be further investigated.     

Phenotypical characterization of indigenous freshwater crayfish populations

The morphology of an animal is known to reflect both genetic variation and adaptation to the environment. Thus, phenotypic criteria have been used to characterize indigenous crayfish populations. Twenty-one morphometric parameters were measured on crayfish from 25 waterbodies in the Austrian and Italian parts of Tyrol. They were analysed with hierarchical cluster analysis to obtain the population structure based on morphological similarity between and within the freshwater crayfish species Astacus astacus (noble crayfish), Austropotamobius torrentium (stone crayfish) and Austropotamobius pallipes (white-clawed crayfish). Furthermore, a stepwise discriminant analysis was applied to the morphometric data to test their differentiating power between populations. Both analyses resulted in a clear differentiation of species and populations, and reflected geographic separations. Thus, the developed morphometric methods were shown to be applicable to characterize the phenotype in freshwater crayfish and seem appropriate to be used for stock identification and the effective characterization of management units in decapod crustaceans.     

Behavioural determinants of agonistic success in invasive crayfish

Ecosystems today increasingly suffer invasions by multiple invasive species, some of which may share similar advantageous life history traits and ecological niche. In such cases, direct competition can influence invasion success of both species, and provide insights into competition without co-evolution in species equally novel to the environment. We used two widespread crayfish invaders of freshwater ecosystems of Europe, signal crayfish (Pacifastacus leniusculus) and spiny cheek crayfish (Orconectes limosus), to investigate how behavioural decisions in agonistic encounters contribute to competitive advantages in the absence of adaptation to either opponents or an environment. In direct competition against novel but comparable opponents, the key factor for establishing clear dominance of P. leniusculus in interspecific bouts was its greater tendency towards continued engagement in high-intensity fights. With O. limosus individuals consistently retreating from staged bouts as fights became more intense, P. leniusculus individuals did not need to adapt their strategy to be successful, suggesting that their agonistic behaviour intrinsically predisposed them to win. While both species are detrimental to invaded ecosystems, our results indicate that aggressive behaviour of P. leniusculus against unfamiliar opponents could allow it to more easily outcompete other comparable species and consequently present a potentially greater threat for native ecosystems.     

Laboratory investigations of the pathogenicity of Aphanomyces astaci for Irish freshwater crayfish

Crayfish plague, caused by the oomycete Aphanomyces astaci, was first diagnosed in Irish stocks of Austropotamobius pallipes from a midlands limestone lake and crayfish farm in October, 1987. Behavioural activity of infected crayfish was monitored and the position at death noted. Crayfish showed no gross, clinical or behavioural signs although they were somewhat lethargic for about 14 days after infection before a rapid deterioration in their condition. Death followed after about 18 and 21 days at 10 ° and 5 °C respectively, with approximately 85% of the animals dying in the open. Spores remained viable and infective in tanks for between 6 and 9 days after death of an infected crayfish at 10 °C. Based on these experiments, suggestions are given for containing the Irish plague outbreak.     

Zooplankton preference of two species of freshwater ornamental fish larvae

The study aimed at evaluating the ascorbic acid (AA) concentrations in the liver and blood of the Mediterranean sea bream (Sparus aurata L.) after being fed Rovimix Stay C‐25 (RS C‐25) at three different levels (0, 200 and 800 mg kg^?1 of AA) over 45 days. RS C‐25 is a mixture of equal parts of AA monophosphate, diphosphate and polyphosphate, containing a 25% AA equivalent. Increasing RS C‐25 levels in the diet yielded an increasing ascorbic acid content in the plasma and liver, showing a good sea bream utilization of this source of vitamin C. Dietary vitamin C did not affect the growth rate or feed efficiency during the 45‐day experiment. After 75 days, fish fed a vitamin C‐free diet displayed a severe depletion of AA in the liver.     

Iridovirus disease in two ornamental tropical freshwater fishes: African lampeye and dwarf gourami

Many species of ornamental freshwater fishes are imported into Japan from all over the world. We found African lampeye Aplocheilichthys normani and dwarf gourami Colisa lalia suffering from an iridovirus infection just after being imported by tropical fish wholesalers from Singapore. African lampeye were cultured on the Indonesian Island of Sumatra and dwarf gourami were cultured in Malaysia before export. Diseased fishes displayed distinct histopathological signs of iridovirus infection: systemic appearance of inclusion body-bearing cells, and necrosis of splenocytes and hematopoietic cells. Electron microscopy revealed viral particles (African lampeye:180 to 200 nm in edge to edge diameter; dwarf gourami: 140 to 150 nm in diameter) in an inclusion body within the cytoplasm of inclusion body-bearing cells as well as in the cytoplasm of necrotized cells. Experimental infection with an iridovirus isolate from African lampeye (ALIV) revealed pathogenicity of ALIV to African lampeye and pearl gourami Trichogaster leeri. Polymerase chain reaction (PCR) products from ALIV and an iridovirus isolate from dwarf gourami (DGIV) using iridovirus-specific primers were indistinguishable. The nucleotide sequence of PCR products derived from ALIV (696 base pairs) and DGIV (701 base pairs) had 95.3% identity. These results indicate that ALIV and DGIV have a single origin.     

Nitrogen metabolism and excretion in the freshwater crayfish Cherax destructor

• 1.1. The pathway leading to the excretion of ammonia in the crustacean, Cherax destructor, has been investigated.
• 2.2. Injection of ammonium chloride and of serine into the haemolymph always led to a rapid increase in the excretion of ammonia. This was not the case with other amino acids. Isolated gills incubated with serine and threonine but not glutamate and glutamine produced ammonia.
• 3.3. Serine dehydratase was present in the gills, midgut gland and tail muscle and serine (0.1 mM) was present in the haemolymph.
• 4.4. The hypothesis is put forward that serine may be the ultimate source of ammonia and that this deaminating reaction is not restricted to the gill. Such a hypothesis sees the formation and deamination of serine—from 3-phosphoglycerate to pyruvate (lactate) as a modified glycolytic pathway.

     

Genetic and morphological evidence for substantial hidden biodiversity in a freshwater crayfish species complex

North America is a center of biodiversity for freshwater crayfish, with >300 described species, mostly in the family Cambaridae. However, the phylogenetic and phylogeographic relationships among these taxa are poorly understood, despite their ubiquitous occurrence in North American freshwater habitats. Here, we target the geographically widespread Orconectesvirilis and its sister taxa for genetic and morphological investigation. We used sequence data from two mitochondrial genes (16S rRNA and cytochrome oxidase I) and from one nuclear gene (glyceraldehyde-3-phosphate dehydrogenase), as well as morphological data from the male gonopod, to gain insights into phylogenetic relationships among individuals collected in the northeastern United States, where this taxon is considered to be invasive, and from Kansas. Our data reveal evidence for three cryptic species in our collections, in addition to O.virilis sensu stricto; all four clades are separated by both mitochondrial and nuclear sequence data. In addition, two of the newly discovered species, O.sp. nov. A and O.sp. nov. B, are distinguishable from O.virilis by significant differences in gonopod morphology (no data on gonopod morphology were available for O.sp. nov. C). O.sp. nov. A was collected at numerous sites in Massachusetts; additional sampling is required to determine if this species is native to New England. Two other taxa, O.sp. nov. B and O.sp. nov. C, were both collected from sites in eastern Kansas, and historically have probably been considered to be O.nais. In summary, this investigation indicates that this species complex may harbor a great deal of unknown biodiversity over the rest of its described range, and suggests that further attention to the phylogenetics and phylogeography of North American crayfishes may yield interesting insights into the evolution of biodiversity.     

Complex impact of an invasive crayfish on freshwater food webs

Invasive alien species can have complex effects on native ecosystems, and interact with multiple components of food webs, making it difficult a comprehensive quantification of their direct and indirect effects. We evaluated the relationships between the invasive crayfish, Procambarus clarkii, amphibian larvae and predatory insects, to quantify crayfish impacts on multiple levels of food webs, and to evaluate whether crayfish predation of aquatic insects has indirect consequences for their preys. We used pipe sampling to assess the abundance of crayfish, amphibian larvae and their major predators (Ditiscidae, Notonectidae and larvae of Anisoptera) in invaded and uninvaded ponds within a human dominated landscape. We disentangled the multivariate effects of P. clarkii on different components of food web through a series of constrained redundancy analyses. The crayfish had a negative, direct impact on both amphibian communities and their predators. Amphibian abundance was negatively related to both predators. However, the negative, direct effects of crayfish on amphibians were much stronger than predation by native insects. Our results suggest that this crayfish impacts multiple levels of food webs, disrupting natural prey-predator relationships.     

Population regulation and demography in a harvested freshwater crayfish from Madagascar

Despite advances in statistical techniques for investigating population dynamics based on mark–recapture data, the majority of our understanding about demography and regulation comes from relatively few taxa. Most proposed generalisations about the association between demography and variation in population size are based on data from vertebrates, there are few sufficiently detailed invertebrate studies to examine whether these generalisations are widely supported. The population biology of freshwater invertebrates is especially poorly known. We present a large-scale mark–recapture study of an endemic freshwater crayfish from Madagascar ( Astacoides granulimanus ). Variation in density, caused by difference in fishing pressure due to local taboos, allowed us to investigate density-dependent regulation. We found evidence of density dependence in fecundity operating through the proportion of reproductive females by size but no significant evidence of density dependence in growth. Using a prospective analysis based on the elasticities from a size-structured matrix model, we found that both recruitment rates and survival rates of large individuals were strongly associated with deterministic population growth – a result that differs from generalisations drawn from vertebrate studies. A central assumption in mark–recapture studies is that handling does not affect mortality. By treating the number of times an individual was captured as an individual covariate, easily done using the freeware program MARK, we were able to test for, and take account of, handling-induced mortality. Our results show interesting similarities, and important differences, to generalisations based on vertebrate studies and emphasise the importance of population studies on poorly known taxa.     

Repeated matings and sperm depletion in the freshwater crayfish Austropotamobius italicus

1. In sexually reproducing organisms, the energetic costs of spermatogenesis can be considerable, and can limit the reproductive potential of the males. In species where males mate more than once during the reproductive season, the costs of sperm production are generally predicted to result in a decrease of ejaculate size and quality with successive fertilizations. 2. In this study we examined the variation in ejaculate size among successive fertilizations in a long‐lived freshwater crayfish species, Austropotamobius italicus. 3. Sexually active adult males of various sizes were allowed to mate repeatedly with different females on consecutive days. Trials for a given male ended when he copulated but did not release any sperm or refused to mate. 4. Males fertilized between 0 and 4 females, and most (42.5%) fertilized a single female. The overall number of females fertilized by a given male decreased with increasing male body size. Ejaculate size decreased markedly with consecutive fertilizations in a similar fashion among both large and small males, while simultaneously increasing with female body size. The total ejaculate size over successive fertilizations decreased with increasing male size. 5. Our study indicates that either sperm production or release involves non‐trivial costs in freshwater crayfish, and suggests that large/old males may face greater difficulties in gamete release than small/young ones, as shown by the lower number of females fertilized by large compared with small males, which may reflect the ongoing senescence of their reproductive performance.     

The freshwater aquarium trade as a vector for incidental invertebrate fauna

The aquarium trade has a long history of transporting and introducing fish, plants and snails into regions where they are not native. However, other than snails, research on species carried “incidentally” rather than deliberately by this industry is lacking. I sampled invertebrates in the plankton, and from water among bottom stones, of 55 aquaria from 43 New Zealand households. I recorded 55 incidental invertebrate taxa, including copepods, ostracods, cladocerans, molluscs, mites, flatworms and nematodes. Six were known established non-indigenous species, and eight others were not previously recorded from New Zealand. Of the latter, two harpacticoid copepod species, Nitokra pietschmanni and Elaphoidella sewelli, are not native to or known from New Zealand, demonstrating the aquarium trade continues to pose an invasion risk for incidental fauna. The remaining six species were littoral/benthic rotifers with subtropical/tropical affinities; these may or may not be native, as research on this group is limited. A variety of behaviours associated with the set-up and keeping of home aquaria were recorded (e.g., fish and plants in any home were sourced from stores, wild caught, or both, and cleaning methods varied), which made prediction of “high risk” behaviours difficult. However, non-indigenous species had a greater probability of being recorded in aquaria containing aquatic plants and in those that were heated. Methods for disposal of aquarium wastes ranged from depositing washings on the lawn or garden (a low risk for invasion) to disposing of water into outdoor ponds or storm-water drains (a higher risk). It is recommended that aquarium owners be encouraged to pour aquarium wastes onto gardens or lawns—already a common method of disposal—as invasion risk will be minimised using this method.