A neglected fish stressor: mechanical disturbance during transportation impacts susceptibility to disease in a globally important ornamental fish.

The transport of fish in aquaculture and the ornamental trade exposes fish to multiple stressors that can cause mass mortalities and economic loss. Previous research on fish transport has largely focussed on chemical stress related to deterioration in water quality. However, mechanical disturbance during routine fish transport is unpredictable and is a neglected potential stressor when studying fish welfare. Stress-induced immunosuppression caused by mechanical disturbance can increase the chances of contracting infections and can significantly increase infection burden. Here, using a model host-parasite system (guppy Poecilia reticulata and the monogenean ectoparasite Gyrodactylus turnbulli) and a new method of bagging fish (Breathing Bags™), which reduces mechanical disturbance during fish transport, we investigated how parasite infections contracted after simulated transport impact infection trajectories on a globally important ornamental freshwater species. Guppies exposed to mechanical transport disturbance suffered significantly higher parasite burden compared to fish that did not experience transport disturbance. Unfortunately, there was no significant reduction in parasite burden of fish transported in the Breathing Bags™ compared to standard polythene carrier bags. Thus, transport-induced mechanical disturbance, hitherto neglected as a stressor, can be detrimental to disease resistance and highlights the need for specific management procedures to reduce the impact of infectious diseases following routine fish transport.

The prevalence of Aphanomyces astaci in invasive signal crayfish from the UK and implications for native crayfish conservation.

The crayfish plague agent, Aphanomyces astaci, has spread throughout Europe, causing a significant decline in native European crayfish. The introduction and dissemination of this pathogen is attributed to the spread of invasive North American crayfish, which can act as carriers for A. astaci. As native European crayfish often succumb to infection with A. astaci, determining the prevalence of this pathogen in non-native crayfish is vital to prioritize native crayfish populations for managed translocation. In the current study, 23 populations of invasive signal crayfish (Pacifastacus leniusculus) from the UK were tested for A. astaci presence using quantitative PCR. Altogether, 13 out of 23 (56·5%) populations were found to be infected, and pathogen prevalence within infected sites varied from 3 to 80%. Microsatellite pathogen genotyping revealed that at least one UK signal crayfish population was infected with the A. astaci genotype group B, known to include virulent strains. Based on recent crayfish distribution records and the average rate of signal crayfish population dispersal, we identified one native white-clawed crayfish (Austropotamobius pallipes) population predicted to come into contact with infected signal crayfish within 5 years. This population should be considered as a priority for translocation.

Apparent interspecific transmission of Aphanomyces astaci from invasive signal to virile crayfish in a sympatric wild population.

The crayfish plague pathogen (Aphanomyces astaci) causes mass mortalities of European crayfish when transmitted from its original North American crayfish hosts. Little is known, however, about interspecific transmission of the pathogen between different American crayfish species, although evidence from trade of ornamental crayfish suggests this may happen in captivity. We screened signal and virile crayfish for A. astaci at allopatric and sympatric sites in a UK river. Whilst the pathogen was detected in signal crayfish from both sites, infected virile crayfish were only found in sympatry. Genotyping of A. astaci from virile crayfish suggested the presence of a strain related to one infecting British signal crayfish. We conclude that virile crayfish likely contracted A. astaci interspecifically from infected signal crayfish. Interspecific transmission of A. astaci strains differing in virulence between American carrier species may influence the spread of this pathogen in open waters with potential exacerbated effects on native European crayfish.

Pace of life, predators and parasites: predator-induced life-history evolution in Trinidadian guppies predicts decrease in parasite tolerance.

A common evolutionary response to predation pressure is increased investment in reproduction, ultimately resulting in a fast life history. Theory and comparative studies suggest that short-lived organisms invest less in defence against parasites than those that are longer lived (the pace of life hypothesis). Combining these tenets of evolutionary theory leads to the specific, untested prediction that within species, populations experiencing higher predation pressure invest less in defence against parasites. The Trinidadian guppy, Poecilia reticulata, presents an excellent opportunity to test this prediction: guppy populations in lower courses of rivers experience higher predation pressure, and as a consequence have evolved faster life histories, than those in upper courses. Data from a large-scale field survey showed that fish infected with Gyrodactylus parasites were of a lower body condition (quantified using the scaled mass index) than uninfected fish, but only in lower course populations. Although the evidence we present is correlational, it suggests that upper course guppies sustain lower fitness costs of infection, i.e. are more tolerant, than lower course guppies. The data are therefore consistent with the pace of life hypothesis of parasite defence allocation, and suggest that life-history traits mediate the indirect effect of predators on the parasites of their prey.

Comparing the ecological impacts of native and invasive crayfish: could native species' translocation do more harm than good?

Biological invasions are a principal threat to global biodiversity. Omnivores, such as crayfish, are among the most important groups of invaders. Their introduction often results in biodiversity loss, particularly of their native counterparts. Managed relocations of native crayfish from areas under threat from invasive crayfish into isolated 'ark sites' are sometimes suggested as a conservation strategy for native crayfish; however, such relocations may have unintended detrimental consequences for the recipient ecosystem. Despite this, there have been few attempts to quantify the relative impacts of native and invasive crayfish on aquatic ecosystems. To address this deficiency we conducted a meta-analysis on the effects of native and invasive crayfish on nine ecosystem components: decomposition rate, primary productivity, plant biomass, invertebrate density, biomass and diversity, fish biomass and refuge use, and amphibian larval survival. Native and invasive crayfish significantly reduced invertebrate density and biomass, fish biomass and amphibian survival rate and significantly increased decomposition rates. Invasive crayfish also significantly reduced plant biomass and invertebrate diversity and increased primary productivity. These results show that native and invasive crayfish have wide-ranging impacts on aquatic ecosystems that may be exacerbated for invasive species. Subsequent analysis showed that the impacts of invasive crayfish were significantly greater, in comparison to native crayfish, for decomposition and primary productivity but not invertebrate density, biomass and diversity. Overall, our findings reconfirm the ecosystem altering abilities of both native and invasive crayfish, enforcing the need to carefully regulate managed relocations of native species as well as to develop control programs for invasives.

Relationship between European eel Anguilla anguilla infection with non-native parasites and swimming behaviour on encountering accelerating flow.

The effect of Anguillicola crassus, Pseudodactylogyrus bini and Pseudodactylogyrus anguillae infection on the behaviour of downstream migrating adult European eels Anguilla anguilla as they encountered accelerating water velocity, common at engineered structures where flow is constricted (e.g. weirs and bypass systems), was evaluated in an experimental flume. The probability of reacting to, and rejecting, the velocity gradient was positively related to A. crassus larval, adult and total abundance. High abundance of Pseudodactylogyrus spp. reduced this effect, but A. crassus was the strongest parasitic factor associated with fish behaviour, and abundance was positively related to delay in downstream passage. Delayed downstream migration at hydraulic gradients associated with riverine anthropogenic structures could result in additional energetic expenditure for migrating A. anguilla already challenged by A. crassus infection.

Endemic infection reduces transmission potential of an epidemic parasite during co-infection.

Endemic, low-virulence parasitic infections are common in nature. Such infections may deplete host resources, which in turn could affect the reproduction of other parasites during co-infection. We aimed to determine whether the reproduction, and therefore transmission potential, of an epidemic parasite was limited by energy costs imposed on the host by an endemic infection. Total lipids, triacylglycerols (TAG) and polar lipids were measured in cockroaches (Blattella germanica) that were fed ad libitum, starved or infected with an endemic parasite, Gregarina blattarum. Reproductive output of an epidemic parasite, Steinernema carpocapsae, was then assessed by counting the number of infective stages emerging from these three host groups. We found both starvation and gregarine infection reduced cockroach lipids, mainly through depletion of TAG. Further, both starvation and G. blattarum infection resulted in reduced emergence of nematode transmission stages. This is, to our knowledge, the first study to demonstrate directly that host resource depletion caused by endemic infection could affect epidemic disease transmission. In view of the ubiquity of endemic infections in nature, future studies of epidemic transmission should take greater account of endemic co-infections.

Non-invasive investigation of Spironucleus vortens transmission in freshwater angelfish Pterophyllum scalare.

Spironucleus vortens is a protozoan fish parasite of veterinary and economic importance in the ornamental aquaculture industry. Despite this, key aspects of the life cycle of this organism, including its mode of transmission, have not been fully elucidated. We developed a non-invasive method for quantifying S. vortens in freshwater angelfish, which was then used to investigate parasite transmission and aggregation within host populations. As previously observed for S. meleagridis and S. salmonis, motile S. vortens trophozoites were detected in host faeces using light microscopy. Species-level identification of these flagellates was confirmed using 16S rDNA PCR. Faecal trophozoite counts were significantly correlated with trophozoite counts from the posterior intestine, the primary habitat of the parasite. This novel finding allowed effective prediction of intestinal parasite load from faecal counts. Overall, faecal count data revealed that 20% of hosts harbour 83% of parasites, conforming to the Pareto Principle (80/20 rule) of parasite aggregation with implications for parasite transmission. Trophozoites survived for ≥36 d outside the host within faeces and remained motile at low pH (comparable with that of angelfish stomach). No putative S. vortens cysts were observed in cultures or faecal samples. This calls into question the commonly accepted hypothesis that a protective cyst is required in the life cycle of S. vortens to facilitate transmission to a new host.

Interactions between males guppies facilitates the transmission of the monogenean ectoparasite Gyrodactylus turnbulli.

In a previous study we found that female guppies shoaled more than males and that there was greater transmission of the ectoparasite Gyrodactylus turnbulli between females. Here, to test for a possible sex bias in parasite transmission, we conducted a similar experiment on single sex shoals of male and female guppies, observing host behaviour before and after the introduction of an infected shoal mate. The initial parasite burden was considerably lower in the present experiment (30 worms versus >100 worms previously) and we used a different stock of ornamental guppies (Green Cobra variety versus a Tuxedo hybrid previously). Contrary to our previous finding, males aggregated significantly more than females. Males performed 'sigmoid' displays towards each other, a courtship behaviour that is more generally directed towards females. Due to the high rate of male-male interactions, parasite transmission was 10 times higher between males than between females. Furthermore, shoaling intensity was highest for the most parasitised fish indicating that these infected fish were not avoided by non-parasitised conspecifics. These studies show that certain social behaviours including shoaling and courtship displays, appear to facilitate the transmission of gyrodactylid parasites.

First record of Neoxysomatium brevicaudatum through the non-invasive sampling of Anguis fragilis: complementary morphological and molecular detection.

Relatively few studies have examined the parasite fauna of British reptiles, partly due to the cryptic nature and low population density of these hosts. Here we examined 12 populations of the slow worm Anguis fragilis which, unlike other UK lizards, occurs at locally high population densities. Morphological examination of non-invasively collected faecal samples revealed the presence of Neoxysomatium brevicaudatum and a second unidentified nematode species. Although previously unrecorded from slow worms in the UK, N. brevicaudatum was present in 38% of animals (mean intensity 70.9, range 1-686). Morphological identification was confirmed by sequencing the 18S ribosomal gene. The use of the species-specific, cytochrome oxidase I mitochondrial gene primers proved an efficient alternative to conventional, microscope screening for parasites, although the original identification of N. brevicaudatum was dependent upon morphological characters. Sequencing also identified the second, smaller nematode as belonging to the Rhabdiasidae family: this species was even more common at a prevalence of 83% (mean intensity 102.8, range 1-2000). While increasing our knowledge of the UK macroparasite fauna, this work demonstrates the benefits of a combined morphological-molecular approach.

Daily patterns in parasite processes: diel variation in fish louse transcriptomes.

Parasites, similar to all other organisms, time themselves to environmental cues using a molecular clock to generate and maintain rhythms. Chronotherapeutic (timed treatment) techniques based on such rhythms offer great potential for improving control of chronic, problematic parasites. Fish lice are a key disease threat in aquaculture, with current control insufficient. Assessing the rhythmicity of fish lice transcriptomes offers not only insight into the viability of chronotherapy, but the opportunity to identify new drug targets. Here, for the first known time in any crustacean parasite, diel changes in gene transcription are examined, revealing that approximately half of the Argulus foliaceus annotated transcriptome displays significant daily rhythmicity. We identified rhythmically transcribed putative clock genes including core clock/cycle and period/timeless pairs, alongside rhythms in feeding-associated genes and processes involving immune response, as well as fish louse drug targets. A substantial number of gene pathways showed peak transcription in hours immediately preceding onset of light, potentially in anticipation of peak host anti-parasite responses or in preparation for increased feeding activity. Genes related to immune haemocyte activity and chitin development were more highly transcribed 4 h post light onset, although inflammatory gene transcription was highest during dark periods. Our study provides an important resource for application of chronotherapy in fish lice; timed application could increase efficacy and/or reduce dose requirement, improving the current landscape of drug resistance and fish health while reducing the economic cost of infection.

Parasite fauna of introduced pumpkinseed fish Lepomis gibbosus: first British record of Onchocleidus dispar (Monogenea).

A survey of pumpkinseed Lepomis gibbosus from a stillwater fishery in southern England revealed infections by the ancyrocephalid monogenean Onchocleidus dispar at 100% prevalence. Host specificity of O. dispar to North American centrarchid fish suggests that it is a non-native parasite, introduced to Britain with L. gibbosus. Mean intensity of O. dispar was significantly higher in male (25.5 parasites) compared to female (15) and immature (7) hosts, but was not influenced by host standard length or sampling time. This sex-biased parasitism is likely to be related to both ecological and physiological factors, such as differential exposure to infective stages during nest building, or higher susceptibility to infection due to enhanced host stress levels. O. dispar significantly dominated the external surfaces and the outer gill arches. Analysis of the spatial distribution of the parasite between the 9 regions of gill surface determined that as density increased, higher numbers of parasites were found on the anterior and central gill regions compared to posterior (distal) regions. Apparent microhabitat selection is discussed in terms O. dispar life cycle, maximising oxygen availability, avoiding unstable areas and increasing intraspecific contact. The absence of O. dispar in the gills of roach, rudd and gudgeon sampled from the same fishery supports the assumption that this parasite is currently of little threat to native fish populations.

Temperature surpasses the effects of velocity and turbulence on swimming performance of two invasive non-native fish species.

Global climate change continues to impact fish habitat quality and biodiversity, especially in regard to the dynamics of invasive non-native species. Using individual aquaria and an open channel flume, this study evaluated the effects of water temperature, flow velocity and turbulence interactions on swimming performance of two lentic, invasive non-native fish in the UK, pumpkinseed (Lepomis gibbosus) and topmouth gudgeon (Pseudorasbora parva). Burst and sustained swimming tests were conducted at 15, 20 and 25°C. Acoustic Doppler velocimetry was used to measure the flume hydrodynamic flow characteristics. Both L. gibbosus and P. parva occupied the near-bed regions of the flume, conserving energy and seeking refuge in the low mean velocities flow areas despite the relatively elevated turbulent fluctuations, a behaviour which depended on temperature. Burst swimming performance and sustained swimming increased by up to 53% as temperature increased from 15 to 20°C and 71% between 15 and 25°C. Furthermore, fish test area occupancy was dependent on thermal conditions, as well as on time-averaged velocities and turbulent fluctuations. This study suggests that invasive species can benefit from the raised temperatures predicted under climate change forecasts by improving swimming performance in flowing water potentially facilitating their further dispersal and subsequent establishment in lotic environments.

Population genetic analysis of microsatellite variation of guppies (Poecilia reticulata) in Trinidad and Tobago: evidence for a dynamic source-sink metapopulation structure, founder events and population bottlenecks.

Riverine fish populations are traditionally considered to be highly structured and subject to strong genetic drift. Here, we use microsatellites to analyse the population structure of the guppy (Poecilia reticulata), focussing on the headwater floodplain area of the Caroni drainage in Trinidad. We also analyse the population genetics of guppies in the Northern Drainage in Trinidad, a habitat characterized by rivers flowing directly into the sea, and a small isolated population in Tobago. Upland Caroni populations are highly differentiated and display low levels of genetic diversity. However, we found no evidence to suggest that these upland populations experienced recent population crashes and the populations appear to approach mutation-drift equilibrium. Dominant downstream migration over both short- and long-time frames has a strong impact on the population genetics of lowland Caroni populations. This drainage system could be considered a source-sink metapopulation, with the tributary furthest downstream representing a 'super sink', receiving immigrants from rivers upstream in the drainage. Moreover, the effective population size in the lowlands is surprisingly low in comparison with the apparently large census population sizes.

Distribution of Eurasian otter biliary parasites, Pseudamphistomum truncatum and Metorchis albidus (Family Opisthorchiidae), in England and Wales.

Gall bladders from 273 otter carcasses, collected throughout England and Wales, were screened to assess the status of gall bladder parasites in the Eurasian otter, Lutra lutra. The digenean Pseudamphistomum truncatum had previously been found in UK otters collected between 2000 and 2007. The parasite was established in Somerset and Dorset but its distribution elsewhere in the UK was largely unknown. In the current study, P. truncatum was also found to be abundant in south Wales, with occasional cases elsewhere, but appears to be absent from the north of England. Overall, 11.7% of otters were infected with 1-238 P. truncatum. A second digenean, Metorchis albidus, previously unreported in British otters, was found in the biliary system of 6.6% of otters. M. albidus appears well established in Suffolk, Norfolk and north Essex but was recorded elsewhere rarely. Both parasites are associated with pathological damage to the otter gall bladder. The recent discovery of these two non-native parasites provides a unique opportunity to assess their impact on native British fauna.

Riverine effects on mitochondrial structure of Bornean orangutans (Pongo pygmaeus) at two spatial scales.

We examined mitochondrial DNA control region sequences of 73 Kinabatangan orangutans to test the hypothesis that the phylogeographical structure of the Bornean orangutan is influenced by riverine barriers. The Lower Kinabatangan Wildlife Sanctuary contains one of the most northern populations of orangutans (Pongo pygmaeus) on Borneo and is bisected by the Kinabatangan River, the longest river in Sabah. Orang-utan samples on either side of the river were strongly differentiated with a high Phi(ST) value of 0.404 (P < 0.001). Results also suggest an east-west gradient of genetic diversity and evidence for population expansion along the river, possibly reflecting a postglacial colonization of the Kinabatangan floodplain. We compared our data with previously published sequences of Bornean orangutans in the context of river catchment structure on the island and evaluated the general relevance of rivers as barriers to gene flow in this long-lived, solitary arboreal ape.

Gyro-scope: an individual-based computer model to forecast gyrodactylid infections on fish hosts.

Individual-based computer models (IBM) feature prominently in current theoretical ecology but have only been applied in a small number of parasitological studies. Here we designed an IBM to simulate the infection dynamics of gyrodactylid parasites and immune defence of naïve hosts (i.e. fish previously not exposed to these parasites). We compared the results of the model with empirical data from guppies (Poecilia reticulata) infected with Gyrodactylus parasites. The laboratory experiments on guppies showed that larger fish acquired a heavier parasite load at the peak of the infection. The survival probability declined with increased body size and no fish survived a parasite load of 80 or more worms in this experiment (i.e. lethal load). The model was a good predictor of the Gyrodactylus infection dynamics of guppies and the model output was congruent with previously published data on Gyrodactylus salaris infections of salmon (Salmo salar). Computer simulations indicated that the infections persisted longer on larger hosts and that the parasite load increased exponentially with the body size of the host. Simulations furthermore predicted that the parasite load of fish with a standard length in excess of 17mm (i.e. the size of adult guppies) reached a lethal load. This suggests that in the conditions of the experiment, the immune defence of naïve guppies can offer moderate protection against gyrodactylid infections to juveniles, but not to naïve adult guppies. The model is a useful tool to forecast the development of gyrodactylid infections on single hosts and make predictions about optimal life history strategies of parasites.

Long term Gyrodactylus lomi infection on isolated juvenile chub (Leuciscus cephalus).

Previous studies on long term gyrodactylid infections on isolated fish have shown that for the majority of Gyrodactylus-fish interactions, infections peak at a maximum level and then decline as the host mounts an effective immune response. In the current study, juvenile chub collected from the wild still harboured Gyrodactylus lomi infections after 6 - 10 months in the laboratory despite being individually maintained at 12 C.

Isotope tracing of submarine groundwater discharge offshore Ubatuba, Brazil: results of the IAEA-UNESCO SGD project.

Results of groundwater and seawater analyses for radioactive (3H, 222Rn, 223Ra, 224Ra, 226Ra, and 228Ra) and stable (D and 18O) isotopes are presented together with in situ spatial mapping and time series 222Rn measurements in seawater, direct seepage measurements using manual and automated seepage meters, pore water investigations using different tracers and piezometric techniques, and geoelectric surveys probing the coast. This study represents first time that such a new complex arsenal of radioactive and non-radioactive tracer techniques and geophysical methods have been used for simultaneous submarine groundwater discharge (SGD) investigations. Large fluctuations of SGD fluxes were observed at sites situated only a few meters apart (from 0 cm d(-1) to 360 cm d(-1); the unit represents cm3/cm2/day), as well as during a few hours (from 0 cm d(-1) to 110 cm d(-1)), strongly depending on the tidal fluctuations. The average SGD flux estimated from continuous 222Rn measurements is 17+/-10 cm d(-1). Integrated coastal SGD flux estimated for the Ubatuba coast using radium isotopes is about 7x10(3) m3 d(-1) per km of the coast. The isotopic composition (deltaD and delta18O) of submarine waters was characterised by significant variability and heavy isotope enrichment, indicating that the contribution of groundwater in submarine waters varied from a small percentage to 20%. However, this contribution with increasing offshore distance became negligible. Automated seepage meters and time series measurements of 222Rn activity concentration showed a negative correlation between the SGD rates and tidal stage. This is likely caused by sea level changes as tidal effects induce variations of hydraulic gradients. The geoelectric probing and piezometric measurements contributed to better understanding of the spatial distribution of different water masses present along the coast. The radium isotope data showed scattered distributions with offshore distance, which imply that seawater in a complex coast with many small bays and islands was influenced by local currents and groundwater/seawater mixing. This has also been confirmed by a relatively short residence time of 1-2 weeks for water within 25 km offshore, as obtained by short-lived radium isotopes. The irregular distribution of SGD seen at Ubatuba is a characteristic of fractured rock aquifers, fed by coastal groundwater and recirculated seawater with small admixtures of groundwater, which is of potential environmental concern and has implications on the management of freshwater resources in the region.