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Authors thank NERC, BBSRC, AXA research fund, Royal Society (AB & AG) and ERC 370 (AG) for funding. Heterogeneity in resources is a ubiquitous feature of natural landscapes affecting many aspects of biology. However, the effect of environmental heterogeneity on the evolution of cooperation has been less well studied. Here, using a mixture of theory and experiments measuring siderophore production by the bacterium Pseudomonas aeruginosa as a model for public goods based cooperation, we explore the effect of heterogeneity in resource availability. We show that cooperation in metapopulations that were spatially heterogeneous in terms of resources can be maintained at a higher level than in homogeneous metapopulations of the same average resource value. The results can be explained by a positive covariance between fitness of cooperators, population size, and local resource availability, which allowed cooperators to have a disproportionate advantage within the heterogeneous metapopulations. These results suggest that natural environmental variation may help to maintain cooperation. Publisher PDF

Quantifying variation in ecosystem metabolism is critical to predicting the impacts of environmental change on the carbon cycle. We used a metabolic scaling framework to investigate how body size and temperature influence phytoplankton community metabolism. We tested this framework using phytoplankton sampled from an outdoor mesocosm experiment, where communities had been either experimentally warmed (+ 4 °C) for 10 years or left at ambient temperature. Warmed and ambient phytoplankton communities differed substantially in their taxonomic composition and size structure. Despite this, the response of primary production and community respiration to long‐ and short‐term warming could be estimated using a model that accounted for the size‐ and temperature dependence of individual metabolism, and the community abundance‐body size distribution. This work demonstrates that the key metabolic fluxes that determine the carbon balance of planktonic ecosystems can be approximated using metabolic scaling theory, with knowledge of the individual size distribution and environmental temperature.

26 pages, 2 figures, 3 tables.-- This is the pre-peer reviewed version of the following article: Brown, A.R., Lilley, M., Shutler, J., Lowe, C., Artioli, Y., Torres, R., Berdalet, E. and Tyler, C.R. (2020), Assessing risks and mitigating impacts of harmful algal blooms on mariculture and marine fisheries. Rev Aquacult, 12: 1663-1688. doi:10.1111/raq.12403, which has been published in final form at https://doi.org/10.1111/raq.12403. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions, Aquaculture is the fastest growing food sector globally and protein provisioning from aquaculture now exceeds that from wild capture fisheries. There is clear potential for the further expansion of marine aquaculture (mariculture), but there are associated risks. Some naturally occurring algae can proliferate under certain environmental conditions, causing deoxygenation of seawater, or releasing toxic compounds (phycotoxins), which can harm wild and cultured finfish and shellfish, and also human consumers. The impacts of these so-called harmful algal blooms (HABs) amount to approximately 8 $billion/yr globally, due to mass mortalities in finfish, harvesting bans preventing the sale of shellfish that have accumulated unsafe levels of HAB phycotoxins and unavoided human health costs. Here, we provide a critical review and analysis of HAB impacts on mariculture (and wild capture fisheries) and recommend research to identify ways to minimise their impacts to the industry. We examine causal factors for HAB development in inshore versus offshore locations and consider how mariculture itself, in its various forms, may exacerbate or mitigate HAB risk. From a management perspective, there is considerable scope for strategic siting of offshore mariculture and holistic Environmental Approaches for Aquaculture, such as offsetting nutrient outputs from finfish farming, via the co-location of extractive shellfish and macroalgae. Such pre-emptive, ecosystem-based approaches are preferable to reactive physical, chemical or microbiological control measures aiming to remove or neutralise HABs and their phycotxins. To facilitate mariculture expansion and long-term sustainability, it is also essential to evaluate HAB risk in conjunction with climate change, This review was funded by the European Martime and Fisheries Fund (EMFF) grant ENG2360: Assessing and mitigating the future risks of harmful algal blooms (HABs) to wild fisheries and aquaculture. […], With the funding support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), of the Spanish Research Agency (AEI)

Evolutionary theory suggests that the conditions required for the establishment of mutualistic symbioses through mutualism alone are highly restrictive, often requiring the evolution of complex stabilising mechanisms. Exploitation, whereby initially the host benefits at the expense of its symbiotic partner and mutual benefits evolve subsequently through trade-offs, offers an arguably simpler route to the establishment of mutualistic symbiosis. In this review, we discuss the theoretical and experimental evidence supporting a role for host exploitation in the establishment and evolution of mutualistic microbial symbioses, including data from both extant and experimentally evolved symbioses. We conclude that exploitation rather than mutualism may often explain the origin of mutualistic microbial symbioses., The authors review the theoretical and experimental evidence supporting exploitation as an alternative route to the evolution of mutualistic symbioses.

Trophic interactions are important determinants of the structure and functioning of ecosystems. Because the metabolism and consumption rates of ectotherms increase sharply with temperature, there are major concerns that global warming will increase the strength of trophic interactions, destabilizing food webs, and altering ecosystem structure and function. We used geothermally warmed streams that span an 11°C temperature gradient to investigate the interplay between temperature-driven selection on traits related to metabolism and resource acquisition, and the interaction strength between the keystone gastropod grazer, Radix balthica, and a common algal resource. Populations from a warm stream (~28°C) had higher maximal metabolic rates and optimal temperatures than their counterparts from a cold stream (~17°C). We found that metabolic rates of the population originating from the warmer stream were higher across all measurement temperatures. A reciprocal transplant experiment demonstrated that the interaction strengths between the grazer and its algal resource were highest for both populations when transplanted into the warm stream. In line with the thermal dependence of respiration, interaction strengths involving grazers from the warm stream were always higher than those with grazers from the cold stream. These results imply that increases in metabolism and resource consumption mediated by the direct, thermodynamic effects of higher temperatures on physiological rates are not mitigated by metabolic compensation in the long term, and suggest that warming could increase the strength of algal-grazer interactions with likely knock-on effects for the biodiversity and productivity of aquatic ecosystems.

Phytoplankton photosynthesis is a critical flux in the carbon cycle, accounting for approximately 40% of the carbon dioxide fixed globally on an annual basis and fuelling the productivity of aquatic food webs. However, rapid evolutionary responses of phytoplankton to warming remain largely unexplored, particularly outside the laboratory, where multiple selection pressures can modify adaptation to environmental change. Here, we use a decade-long experiment in outdoor mesocosms to investigate mechanisms of adaptation to warming (+4 °C above ambient temperature) in the green alga Chlamydomonas reinhardtii, in naturally assembled communities. Isolates from warmed mesocosms had higher optimal growth temperatures than their counterparts from ambient treatments. Consequently, warm-adapted isolates were stronger competitors at elevated temperature and experienced a decline in competitive fitness in ambient conditions, indicating adaptation to local thermal regimes. Higher competitive fitness in the warmed isolates was linked to greater photosynthetic capacity and reduced susceptibility to photoinhibition. These findings suggest that adaptive responses to warming in phytoplankton could help to mitigate projected declines in aquatic net primary production by increasing rates of cellular net photosynthesis. Isolates of the green alga Chlamydomonas reinhardtii adapted to a decade of chronic warming in outdoor mesocosms have higher competitive fitness than ambient controls, due to greater photosynthetic capacity and reduced susceptibility to photoinhibition

Gross primary production (GPP) is the largest flux in the carbon cycle, yet its response to global warming is highly uncertain. The temperature sensitivity of GPP is directly linked to photosynthetic physiology, but the response of GPP to warming over longer timescales could also be shaped by ecological and evolutionary processes that drive variation community structure and functional trait distributions. Here, we show that selection on photosynthetic traits within and across taxa dampen the effects of temperature on GPP across a catchment of geothermally heated streams. Autotrophs from cold streams had higher photosynthetic rates and after accounting for differences in biomass among sites, rates of ecosystem-level GPP were independent of temperature, despite a 20 ºC thermal gradient. Our results suggest that thermal adaptation constrains the long-term temperature dependence of GPP, and highlights the importance of considering physiological, ecological and evolutionary mechanisms when predicting how ecosystem-level processes respond to warming.

Through the merger of previously independent lineages, symbiosis promotes the acquisition of new traits and exploitation of inaccessible ecological niches [1, 2], driving evolutionary innovation and important ecosystem functions [3–6]. The transient nature of establishment makes study of symbiotic origins difficult, but experimental comparison of independent origins could reveal the degree of convergence in the underpinning mechanisms [7, 8]. We compared the metabolic mechanisms of two independent origins of Paramecium bursaria-Chlorella photosymbiosis [9–11] using a reciprocal metabolomic pulse-chase method. This showed convergent patterns of nutrient exchange and utilization for host-derived nitrogen in the Chlorella genotypes [12, 13] and symbiont-derived carbon in the P. bursaria genotypes [14, 15]. Consistent with a convergent primary nutrient exchange, partner-switched host-symbiont pairings were functional. Direct competition of hosts containing native or recombined symbionts against isogenic symbiont-free hosts showed that the fitness benefits of symbiosis for hosts increased with irradiance but varied by genotype. Global metabolism varied more between the Chlorella than the P. bursaria genotypes and suggested divergent mechanisms of light management. Specifically, the algal symbiont genotypes either produced photo-protective carotenoid pigments at high irradiance or more chlorophyll, resulting in corresponding differences in photosynthetic efficiency and non-photochemical quenching among host-symbiont pairings. These data suggest that the multiple origins of P. bursaria-Chlorella symbiosis use a convergent nutrient exchange, whereas other photosynthetic traits linked to functioning of photosymbiosis have diverged. Although convergence enables partner switching among diverse strains, phenotypic mismatches resulting from divergence of secondary symbiotic traits could mediate host-symbiont specificity in nature. Sørensen et al. compare multiple independent evolutionary origins of Paramecium-Chlorella symbiosis to reveal the underpinning metabolic mechanisms. Although the independent origins use a convergent nutrient exchange, they have diverged in traits linked to photosynthesis, which could mediate host-symbiont specificity in nature.

We use strains recently collected from the field to establish cultures; then, through laboratory studies we investigate how among strain variation in protozoan ingestion and growth rates influences population dynamics and intraspecific competition. We focused on the impact of changing temperature because of its well-established effects on protozoan rates and its ecological relevance, from daily fluctuations to climate change. We show, first, that there is considerable inter-strain variability in thermal sensitivity of maximum growth rate, revealing distinct differences among multiple strains of our model species Oxyrrhis marina. We then intensively examined two representative strains that exhibit distinctly different thermal responses and parameterised the influence of temperature on their functional and numerical responses. Finally, we assessed how these responses alter predator–prey population dynamics. We do this first considering a standard approach, which assumes that functional and numerical responses are directly coupled, and then compare these results with a novel framework that incorporates both functional and numerical responses in a fully parameterised model. We conclude that: (i) including functional diversity of protozoa at the sub-species level will alter model predictions and (ii) including directly measured, independent functional and numerical responses in a model can provide a more realistic account of predator–prey dynamics.

Understanding, and therefore measuring, factors that determine fitness is a central problem in evolutionary biology. We studied a natural population of Coenagrion puella (Odonata: Zygoptera) over two entire breeding seasons, with over a thousand individuals uniquely marked and genotyped, and all mating events at the rendezvous site recorded. Using a parentage analysis, fitness of individuals in the first generation was quantified as the numbers of offspring that survived to maturity. Although mating behaviour can be predicted by environmental and demographical variables, the numbers of mature offspring produced (fitness) cannot, and crucially, are poorly correlated with behavioural observations of mating. While fitness of both sexes was positively related to mating behaviour and to female's ectoparasite burden, these behavioural observations explained little more variance in offspring production than environmental and demographical variables. Thus, we demonstrate that behavioural measures of reproductive success are not necessarily reliable estimates of fitness in natural populations.

International audience; The heterotrophic marine flagellate Oxyrrhis marina, is a popular and tractable model organism, being common in the intertidal environment and relatively straight forward to maintain in the laboratory. In this report, based on our experience of collecting and culturing ~400 isolates, we provide a quantification of how 'easy' O. marina is to locate and isolate from the environment and subsequently maintain in culture. In addition, we provide a brief review of the literature pertaining to O. marina culturing, stressing in particular the broad physiological tolerance and wide diet range of this organism. Finally, in light of the increasing interest in the genetics and genomics of O. marina, and heterotrophic protists more generally, we highlight existing strategies for culturing heterotrophs for '-omic' applications, and summarise our specific culturing approaches, for genomic and proteomic studies of O. marina. In this context, we outline a simplified version of Droop's medium for axenic culturing and offer details on the use of heat-killed Escherichia coli as a simple and robust food source for culturing O. marina in the absence of other eukaryotes and live prokaryotes.

Many "model" protists are maintained in culture and used, experimentally, to answer questions associated with planktonic processes. Given the current interest and rapidly increasing amount of literature on the heterotrophic dinoflagellate Oxyrrhis marina, we present in this special issue a series of focused, interlinked research articles. Being written by experts in their respective fields, the authors have included unpublished data and in all cases have offered a synthesis of data and ideas. Furthermore, we have encouraged cross-paper discourse, emphasizing the interdisciplinary nature of our work and the utility of O. marina to this end; we also offer guidance, both practical and intellectual, on how future research related to O. marina might progress. In this introduction, however, we raise the wider issue of which criteria are required to consider a taxon as a "model species". We then assess the extent to which O. marina can fill this role. In general, we recognize O. marina as a model in three distinct disciplines: ecology, evolution/genomics and biogeography. Of possibly greater importance, we recognize that if O. marina continues to be studied at an escalating rate, there will be a concomitant increase in realized and potential synergies across these fields. © The Author 2010. Published by Oxford University Press. All rights reserved.

1. Age-dependent increases in mortality have been documented in a variety of species of insect under laboratory conditions. However, while strong statistical evidence has been presented for senescence in vertebrate populations in the wild, we know little about the rate and shape of senescence in wild populations of insects. 2. Odonates (damselflies and dragonflies) provide excellent candidate species for evaluating demographic senescence as they are large enough to be marked individually and they are easily re-sighted without recapture. The prevailing opinion - based entirely on qualitative examination of the declines in log numbers alive with time since marking - is that odonates exhibit age-independent daily survivorship. 3. Here, we examine mark-recapture data on the Azure Damselfly Coenagrion puella over two consecutive seasons. For the first time, we evaluate and compare the fit of quantitative models that not only account for weather-dependent daily variation in daily re-sighting rates, but also age-dependent variation in daily survivorship. 4. Models with age-dependent declines in daily survivorship provide a more parsimonious explanation for the data than similar models without these age-dependent effects. In general, models in which mortality increases in an exponential (Gompertz) fashion explain the mark-recapture sequences more efficiently than a range of alternative models, including those in which mortality increases as a power function (Weibull) or reaches a plateau (logistic). These results are indicative of a general senescent decline in physiological functioning, which is particularly marked after 15 days as a mature adult. 5. Weather (temperature, sun and precipitation) and initial mite load influenced the probability of daily re-sighting. Weather and mite load also influenced daily survivorship, but their effects differed between seasons. 6. Overall, fitting models with age as an explicit covariate demonstrates that odonates do indeed senesce. This contradicts previously held assumptions that Odonata do not exhibit age-dependent survivorship in the wild.

Purpose: del(17p), del(11q), and associated p53 dysfunction predict for short survival and chemoresistance in B-cell chronic lymphocytic leukemia (CLL). DNA-dependent protein kinase (DNA-PK) is activated by DNA damage and mediates DNA double-strand break repair. We hypothesized that inhibiting DNA-PK would sensitize CLL cells to drug-induced DNA damage and that this approach could increase the therapeutic index of agents used to treat CLL. Experimental Design: Fifty-four CLL cases were characterized for poor prognosis markers [del(17p), del(11q), CD38, and ZAP-70]. In selected cases, DNA-PK catalytic subunit (DNA-PKcs) expression and activity and p53 function were also measured. Ex vivo viability assays established sensitivity to fludarabine and chlorambucil and also tested the ability of a novel DNA-PK inhibitor (NU7441) to sensitize CLL cells to these drugs. The effects of NU7441 on fludarabine-induced DNA damage repair were also assessed (Comet assays and detection of γH2AX). Results: DNA-PKcs levels correlated with DNA-PK activity and varied 50-fold between cases but were consistently higher in del(17p) (P = 0.01) and del(11q) cases. NU7441 sensitized CLL cells to chlorambucil and fludarabine, including cases with del(17p), del(11q), p53 dysfunction, or high levels of DNA-PKcs. NU7441 increased fludarabine-induced double-strand breaks and abrogated drug-induced autophosphorylation of DNA-PKcs at Ser2056. High DNA-PK levels predicted for reduced treatment-free interval. Conclusions: These data validate the concept of targeting DNA-PKcs in poor risk CLL, and demonstrate a mechanistic rationale for use of a DNA-PK inhibitor. The novel observation that DNA-PKcs is overexpressed in del(17p) and del(11q) cases indicates that DNA-PK may contribute to disease progression in CLL.

Coenagrionid damselflies are in general decline in the British Isles. Numerous factors have been implicated in the loss of these species including recent speculation that hybridisation between congeners may result in species decline. Here we use a panel of 12 microsatellite loci to examine levels of genetic divergence and the likely occurrence of hybridisation in five populations of Coenagrion puella and C. pulchellum using samples from four sites in south-east England. Coenagrion puella and C. pulchellum were highly genetically divergent, and there was no evidence of hybridisation between any of the populations examined, even where C. puella and C. pulchellum were sympatric. There was some suggestion that C. pulchellum was less genetically diverse than C. puella, though this may have been a result of ascertainment bias associated with cross-species application of microsatellite markers. We conclude that there is no evidence that hybridisation between C. puella/pulchellum could be responsible for the on-going demographic decline in C. pulchellum. Nevertheless, further genetic studies such as this one are likely to provide estimates of diversity, population structure and dispersal capacity that will be invaluable in future conservation management strategies for coenagrionid damselflies.

Summary 1. High levels of intraspecific variation are commonly observed in natural microbial populations, yet the consequences of this variation for ecological and evolutionary processes remain poorly understood. Protists are excellent experimental models for investigating fundamental and applied questions in ecology and evolution, but studying intraspecific variation remains a challenge due to a lack of molecular resources to aid in quantifying and distinguishing strains during experiments. 2. Here we present a molecular method, quantitative microsatellite genotyping, to accurately quantify strain-specific frequencies from microcosm experiments of the marine flagellate Oxyrrhis marina, both between many pairs of strains and between strains in a multistrain mixture. 3. We find that for pairs of strains, the method is effective for relative frequencies as low as 0� 02 and with around 99% accuracy. The method is able to quantify four strains reasonably well, though less accurate than for pairs (range 92–97% accuracy). 4. This makes accessible a cheap and easy-to-implement method for quantifying strain (or allele) frequencies and is suitable for use in a broad range of single-celled eukaryotes (protists) where copy number should correlate well with number of individuals (i.e. cells). This opens up the possibility of examining the role of intraspecific variation using experimental protist microcosms.

Environmental salinity is important in defining Brachionus plicatilis sibling species distributions. However, while salinity influences distributions, sibling species often co-exist. Three different mechanisms potentially account for the partial co-occurrence of sibling species: (1) siblings have differing salinity tolerances that partially overlap; (2) siblings physiological tolerances may be commonly broad, but relatively small differences in tolerances differentiate distributions via interactions e.g. competition; or (3) siblings distributions may be influenced by physical factors other than salinity. Here, we assess the extent of salinity tolerance in three B. plicatilis sibling species (B. plicatilis 6TUR, B. plicatilis IOM and B. rotundiformis 6TOS) by measuring population growth rate (μ, day−1) and egg development time in response to salinity (5–60‰) and salinity fluctuations (≤ Δ40‰). Sibling species were identified by analysis of the mitochondrial COI gene, and salinity responses were compared by regression analysis. Responses differed significantly between siblings, although the broad trends were similar. Positive growth occurred at all salinities, and highest growth rates ranged between 0.93 and 1.08 day−1 at 16–18‰. Rapid changes in salinity reduced growth rates, but net mortality occurred only in one treatment (100% mortality on transfer from 10 to 40‰). Egg development time was largely invariant with salinity except for B. plicatilis IOM and where rotifers were transferred from 30 to 60‰. We indicate that several siblings are similarly euryhaline and tolerate salinity fluctuations. Undoubtedly, wide tolerances in B. plicatilis are adaptations to ephemeral and seasonally variable habitats. Given common broad salinity tolerances, it is unlikely that the differential distributions of sibling species are a direct result of physiological constraints. Instead, we illustrate using a simple model that subtle differences in physiological tolerances may have important impacts on interactions between sibling species, which may in turn influence distributions.

Oxyrrhis marina, a widely distributed marine protist, is used to model heterotrophic flagellate responses in microbial food webs. Although clonal variability occurs in protists, assessments of intraspecific diversity are rare; such assessments are critical, particularly where species are used as models in ecological studies. To address the extent of intraspecific variation within O. marina, we assessed diversity among 11 strains using 5.8S rDNA and ITS sequences. The 5.8S rDNA and ITS regions revealed high divergence between strains: 63.1% between the most diverse. To compare O. marina diversity relative to other alveolates, 18S rDNA sequences for five strains were analysed with sequences from representatives of the major alveolate groups. 18S rDNA also revealed high divergence in O. marina. Additionally, consistent with phylogenies based on protein coding genes, maximum likelihood analysis indicated that O. marina was monophyletic and ancestral to the dinoflagellates. To assess ecophysiological differences, growth rates of seven O. marina strains were measured at 10 salinities (10-55 per thousand). Two salinity responses occurred: one group achieved highest growth rates at high salinities; the other grew best at low salinities. There was no clear correlation between molecular, ecophysiological, or geographical differences. However, salinity tolerance was associated with habitat type: intertidal strains grew best at high salinities; open-water strains grew best at low salinities. These data indicate the need to examine many strains of a species in both phylogenetic and ecological studies, especially where key-species are used to model ecological processes.

Free-living microbes are arguably ubiquitously dispersed. Molecular phylogeographies have been used to assess microbial distributions but can be difficult to interpret. Combinations of molecular and ecophysiological data have, however, proven useful in assessing the influence of dis- persal, local adaptation and historical contingency on distributions. A logical extension of this approach is to develop molecular markers for ecophysiological traits, which would allow the assess- ment of adaptations in large numbers of environmental isolates without requiring extensive cultur- ing. To assess this approach, we compared enzyme activity and gene expression of Na + /K + ATPase in response to salinity, and compared Na + /K + ATPase mRNA sequences, in 3 Brachionus plicatilis (Rotifera) sibling species. B. plicatilis siblings display different salinity tolerances, which in turn influence their distributions. Na + /K + ATPase is an important component of salinity tolerance and a potentially useful marker for ecophysiological variation. In all cases, Na + /K + ATPase enzyme activity and gene expression increased in response to salinity (5 to 50‰) and paralleled growth rate differ- ences: highest enzyme activity/gene expression occurred in the sibling species that displayed high- est growth. However, sequence variation in Na + /K + ATPase mRNAs was minor (~4%) and did not match gene expression patterns; thus, differences between siblings in Na + /K + ATPase occurred as a result of differential expression of highly similar gene transcripts. While we were not able to develop functional molecular markers for salinity tolerance in B. plicatilis, we highlight that the application of phylogenetic and functional markers will be a powerful tool for assessing the distributions of free- living microorganisms.

The rotifer Brachionus plicatilis is euryhaline (growing between 2 and 97 ppt) and has previously been considered an osmoconformer. We suggest that B. plicatilis is an osmoregulator, exhibiting a pattern of Na+/K+ ATPase activity in response to salinity consistent with that of other osmoregulating euryhaline invertebrates. To examine salinity tolerance, growth rates between 5 and 60 ppt were determined. The activity of Na+/K+ ATPase was examined, over the same range of salinities, by measuring ATPase activity in rotifer homogenates in the presence and absence of a Na+/K+ ATPase inhibitor. Maximum specific growth rate (0.95 day−1) occurred at 16 ppt, highest mean amictic eggs per female (1.41) occurred at 20 ppt, and both parameters decreased rapidly as salinity increased. Egg development time was constant with salinity at 0.92 days. The activity of Na+/K+ ATPase per milligram protein increased from 3.9 µmol h−1 at 5 ppt to 6.8 µmol h−1 at 50 ppt and accounted for 15 and 30% of total ATPase activity, respectively. We suggest that these observations are consistent with increasing stress at high salinities and the occurrence of a hypo-osmoregulatory response. Given the high ATP consumption of Na+/K+ ATPase at high salinities, it is possible that a proportion of the corresponding decreases in growth rate and egg production are a direct cost of regulation.

This study developed a field-based system to examine the influence of cyclical distur- bances on populations and metapopulations. We have examined the population dynamics of a tide- pool ciliate that possesses several cycles, endogenously and exogenously induced, ranging in scale from hours to years. Strombidium oculatum Gruber exhibits an endogenous circatidal behaviour: for ~6 h, at low tide, S. oculatum is free-swimming in pools, and ~20 to 60 min before flushing of the pools it encysts on a substrate. Encystment lasts for ~19 h: 2 high tides and 1 intervening low tide. Excyst- ment then occurs the next day ~30 to 40 min after the pools are isolated. This behaviour allows S. ocu- latum to remain in pools and creates isolated populations; a matrix of such pools on the shore then comprises a metapopulation. Over a 5 yr period, we conducted a study to examine (1) the timing of division and the cell cycle; (2) the endogenous, 25 h encystment-excystment cycle; (3) the tidally induced fortnightly behaviour; (4) the seasonally induced long-term variation in population abun- dance; and (5) the seasonal change in distribution on the shore. We found that ciliates on the Isle of Man exhibit an excystment-encystment pattern similar to that previously described for populations of S. oculatum in France: cells divide almost immediately after excysting, allowing the ciliate population to rapidly exploit potential food resources; 2 populations exist in each pool, each isolated to a single low tide; the ciliate exhibits seasonal trends in population dynamics, appearing in the spring and dis- appearing in the autumn; and ciliates appear to survive seasonal changes by remaining at low den- sities in upper-shore pools during the winter. We have used these data and other measurements of the population dynamics to discern how populations are maintained on the shore, and we discuss these mechanisms in the context of population-metapopulation survival.

We isolated and characterized 10 polymorphic microsatellite loci from the azure damselfly Coenagrion puella (Zygoptera; Coenagrionidae) as part of a study assessing reproductive success and genetic structure in an isolated population of this species. Levels of genetic diversity were assessed in 50 individuals collected from Queen Elizabeth Country Park, Hampshire, UK. The number of alleles per microsatellite loci ranged from three to 22 and the observed and expected heterozygosities varied between 0.26 and 0.84 and between 0.23 and 0.91, respectively. Two loci showed significant ( P < 0.05) heterozygote deficits, likely because of null (non-amplifying) alleles; one pair of loci was in linkage disequilibrium.

It is timely to evaluate the role of protozoa as model organisms given their diversity, abundance and versatility as well as the economic and ethical pressures placed on animal-based experimentation. We first define the term model organism and then examine through examples why protozoa make good models. Our examples reflect major issues including evolution, ecology, population and community biology, disease, the role of organelles, ageing, space travel, toxicity and teaching. We conclude by recognising that although protozoa may in some cases not completely mimic tissue- or whole-animal-level processes, they are extremely flexible and their use should be embraced. Finally, we offer advice on obtaining emergent model protozoa.

International audience; It is often argued that microscopic organisms typically have cosmopolitan distributions. This argument has been criticised as it fails to acknowledge the many protists that have limited geographic distributions. Oxyrrhis marina, a common heterotrophic dinoflagellate in many intertidal and coastal habitats, is regarded as globally-distributed, yet despite its prominent role as an experimental planktonic organism there are few explicit studies of this species' geographic range. An analysis of 846 water samples (from across the World) confirmed the wide geographic distribution of O. marina and extends this morphospecies' range into the southern hemisphere. We did not find O. marina beyond 63º N latitude, implying that it is rare, or even absent, in Polar seas. Despite being rare in open water, O. marina inhabits the coastal waters of remote islands, such as Hawaii and the Azores. Beneath the apparent broad spatial range are some emerging patterns on the distribution of distinct genetic clades that form two divergent lineages that may represent different species. Two of the clades have discrete, non-overlapping distributions (i in the European Atlantic and western Mediterranean, and ii in the eastern Mediterranean Sea): one possesses a broad distribution (both coasts of North America and also in the eastern Mediterranean Sea), and one is limited to culture collections from the Baltic Sea and Red Sea. Thus, the Mediterranean Basin appears to be a diversity hotspot for Oxyrrhis, and there is a mixture of wide distributions and endemicity. These issues are discussed with respect to protist biogeography.

International audience; Protists are major consumers of phytoplankton and bacterioplankton in the marine environment, playing a key role in carbon cycling and nutrient regeneration. Consequently, the feeding activities of planktonic protists have been intensively studied. In particular, the dinoflagellate, Oxyrrhis marina, has been used extensively as a model predator in laboratory-based feeding studies. This is predominately because it is easy to culture and manipulate but also because its plasticity in feeding behaviour allows it to represent a broad range of free-living heterotrophic protists. Prey preferences of this versatile, but selective, predator have been well documented. However, our ability to determine the factors driving prey selectivity is limited by a poor understanding of the detailed mechanisms underlying O. marina feeding. Within this study, we explore known aspects of O. marina feeding behaviour and highlight areas that require further research to exploit this species as an effective model protist predator.

We examine the literature on Oxyrrhis marina cell and life cycles, population growthand production. We then provide an overview of what is known regarding aspectsof O. marina growth, indicate where information is needed and suggest ways inwhich this species can and cannot be used as a general model, in this respect.Little is known about the O. marina life cycle; it is even unknown if cells arehaploid, diploid or polyploid, although there is one report that sex occurs byhomothallic isogamy. There is considerable information on the cell cycle, whichwe briefly review and provide a guide to the literature for details. We brieflydiscuss and provide guidance to information on: (i) population cell size distri-butions; (ii) cannibalism; (iii) our first report of “mini-cells” in cultures ( 8 mm)and their possible role in the life cycle; (iv) cysts, and their possible role in the lifeand cell cycles; (v) biotic influences on growth, such as food type and abundance,assimilation efficiency, prey stiochiometry, strain differences, population growthand starvation and nutritional shifts; (vi) abiotic factors that affect growth, such astemperature, salinity, pH, light and turbulence. We then reflect on the conse-quences of interactions between the above factors and review data on populationgrowth of O. marina in the field and laboratory. Finally, we evaluate the use ofO. marina as a model organism to examine cell and life cycles and ecological pro-cesses. Throughout the paper, we suggest areas that need evaluation.KEYWORDS: model organism; dinoflagellate; protozoa; life history; Oxyrrhis

Oxyrrhis marina is an extensively studied morphospecies and a common protist model used to examine a range of ecological processes. Further, as a result of a number of unusual cytological and genetic features, Oxyrrhis is increasingly a target for the study of evolutionary patterns and genome organization within the Alveolata. However, a small number of early morphological studies and recent phylogenetic data suggest that 0. marina represents more than one species. As different research groups employ different 0. marina isolates (which are potentially highly divergent strains or different species), the context in which comparisons between isolates can be made is difficult to assess. In this paper, we explore the literature that has contributed to the definition of 0. marina, highlighting the unusual characteristics possessed by 0. marina that have motivated much of the study on this organism and informed its key phylogenetic position. In addition, we assess historical and contemporary evidence for multiple Oxyrrhis species. Based on this assessment, in particular recent molecular genetic data, we assert that 0. marina represents two species: 0. marina and 0. maritima. Based on historical observations, we also indicate that a third species (0. tenticulifera) may occur, although there are no contemporary data to support or refute this designation. Extensive cryptic diversity has important implications for researchers studying Oxyrrhis: caution must be exercised in characterizing Oxyrrhis isolates for experimental study (i.e. it is inappropriate to report assessments concerning poorly characterized isolates), and comparative studies of multiple isolates are required to assess individual, population and species level variation in the genus. Finally, in a broader context, the ecological and evolutionary processes driving diversity in free-living protists remains poorly understood. Model protists such as 0. marina and 0. maritima for which we are beginning to recognize and characterize an extensive pool of variation present ideal opportunities to unravel these fundamental processes.

1. The extent to which individuals are parasitised is a function of exposure to parasites and the immune response, which in ectotherms may be associated with temperature. 2. We test the hypothesis that seasonal variation in ectoparasite burden is driven by temperature using an extensive mark-release-recapture study of adult Coenagrion puella (L.) (Zygoptera) as a model system. Mite counts were taken both at capture and on a subset of subsequent recaptures over two entire, consecutive breeding seasons. 3. Emergence date was the most significant factor in determining individual differences in mite burden, and mean counts for individuals emerging on the same days showed strong unimodal relationships with time of season. Subsequent recounting of mites on a subset of individuals showed that patterns of loss of mites were similar between seasons. 4. While temperature did not significantly affect mite burdens within seasons and ectoparasite prevalence was very similar across the two seasons, intensity of infection and rate of mite gain in unparasitised individuals were significantly higher in the cooler season. 5. We demonstrate that, while temperature may modulate the invertebrate immune response, this modulation does not manifest in variations in mite burdens in natural populations.

By a combination of detailed behavioral observations and molecular genetic approaches we have assessed development time, timing of first maturity, and the extent of genetic structure through the flying season in a wild population of the damselfly Coenagrion puella in England. This work provides the first estimate of development time (egg to mature adult) in the field based on individual damselflies. Development time was significantly longer for females than males. In contrast to reported laboratory studies, there was no difference in development times between different female color morphs. Development time ranged between 347 and 396 days and was negatively correlated with egg-laying date. As a result eggs laid early in one season reach adult maturity relatively late in the next; concurrently individuals developing from eggs laid late mature relatively early. We speculate that this pattern of development is a direct physiological response to seasonal environmental variation and results in reproductive synchrony within a population. Size, specifically hind wing length, declined with development time in males, but not in females. In one of the two years of the study there was evidence for weak clustering of related individuals during the reproductive season. This appeared to be the result of developmental synchronization within families: variance in timing of maturation was smaller in full-sib families than in half-sib families or randomly assigned unrelated groups.

Oxyrrhis marina is an important model in ecological studies of free-living protists. Despite this, O. marina has rarely been studied in the environment and no explicit distributional studies exist. Further, phylogenetic data for a small number of isolates indicate that O. marina constitutes two divergent lineages. Here, we quantify phylogenetic variation between 58 globally distributed O. marina isolates using 5.8S – internal transcribed spacer 1 and 2 rDNA (5.8S ITS) and cytochrome c oxidase I (COI) partial sequences. 5.8S ITS and COI phylogenies both partitioned O. marina into four clades, which formed two lineages; mean sequence identity for 5.8S ITS and COI respectively was ∼40 and 90% between these two lineages. Sequence identities for 5.8S ITS/ COI between clades within lineages were 66.3/99.4% (lineage 1: clade 1 vs 2) and 42.3/99.1% (lineage 2: clade 3 vs 4). rDNA mutation rates in O. marina appear to be abnormally high and were not interpreted in a species delineation context. Based on variation in COI sequence and comparisons with other protists, we suggest that O. marina lineages may constitute two species. In a geographic context, evidence of spatial restriction but also extensive overlap between O. marina clades occurred. Further, clade abundances varied considerably: clades 1 and 2 (belonging to one lineage) were abundant and widespread; in contrast, clades 3 and 4 (belonging to the second lineage) were rare and spatially restricted (occurring only in the Mediterranean or in culture collection). There is need for further phylogenetic and taxonomic studies to assess species delineation in O. marina , and for the application of high resolution genetic markers to resolve processes driving genetic diversity in this important model organism.

SUMMARYA panel of microsatellites mapped to the Leishmania genome might make it possible to find associations between specific loci and phenotypic traits. To identify such loci, a Perl programme was written that scans the sequence of a genome and writes all loci containing microsatellites to a MySQL database. The programme was applied to the sequences of the L. braziliensis, L. infantum and L. major genomes. The database is publicly available over the internet: http://www.genomics.liv.ac.uk/tryps/resources.html ‘Microsatellite Locus Extractor’, and allows the selection of mapped microsatellites that meet user-defined criteria from a specified region of the selected genome. The website also incorporates a primer design pipeline that will design primers to amplify the selected loci. Using this pipeline 12 out of 17 primer sets designed against the L. infantum genome generated polymorphic PCR products. A tailed primer protocol was used to label all microsatellite primers with a single set of labelled primers. To avoid the culture of parasites prior to genotyping, sets of nested PCR primers were developed to amplify parasite DNA eluted from microscope slides. The limit of detection was approximately 1·6 parasite equivalents. However, only 6/56 DNA from slides stored at ambient temperature for over 6 months gave positive PCR results.

Oxyrrhis marina, a widely distributed marine protist, is used to model heterotrophic flagellate responses in microbial food webs. Although clonal variability occurs in protists, assessments of intraspecific diversity are rare; such assessments are critical, particularly where species are used as models in ecological studies. To address the extent of intraspecific variation within O. marina, we assessed diversity among 11 strains using 5.8S rDNA and ITS sequences. The 5.8S rDNA and ITS regions revealed high divergence between strains: 63.1% between the most diverse. To compare O. marina diversity relative to other alveolates, 18S rDNA sequences for five strains were analysed with sequences from representatives of the major alveolate groups. 18S rDNA also revealed high divergence in O. marina. Additionally, consistent with phylogenies based on protein coding genes, maximum likelihood analysis indicated that O. marina was monophyletic and ancestral to the dinoflagellates. To assess ecophysiological differences, growth rates of seven O. marina strains were measured at 10 salinities (10-55 per thousand). Two salinity responses occurred: one group achieved highest growth rates at high salinities; the other grew best at low salinities. There was no clear correlation between molecular, ecophysiological, or geographical differences. However, salinity tolerance was associated with habitat type: intertidal strains grew best at high salinities; open-water strains grew best at low salinities. These data indicate the need to examine many strains of a species in both phylogenetic and ecological studies, especially where key-species are used to model ecological processes.

International audience; Aquatic microbes, like all organisms, have biogeographies, but this subject has attracted relatively little attention. In this review, recent results exploiting techniques of molecular biology are summarized to place in perspective the studies of this Theme Section. The studies considered concern large-scale patterns of spatial distribution among heterotrophic planktonic prokaryotic and eukaryotic microbes. For freshwater bacterioplankton communities, reported patterns are inconsistent. Taxonomic richness may increase with system size, and composition may be related among neighboring bodies of water. However, inconsistencies in patterns may be due to differences in the temporal and spatial scales considered. Among planktonic marine prokaryotes, biogeographic patterns are known only in terms of high level groups, e.g. Archea are perhaps dominant in deep oceanic waters. However, studies of large-scale patterns have just begun and they suggest that some ribotypes or species may be restricted to certain oceanic areas. Eukaryotic microbes appear to characterised by high capacities for both dispersal and gene flow. Recent studies appear to conclude that we can form morphological, genetic and physiological groupings but their inter-relationships are obscure at this point in time.

The marine, tide pool-dwelling ciliate Stombidium oculatum was redescribed using live, stained, SEM, and TEM material prepared from samples collected from pools on the Isle of Man (Irish Sea) and Brittany (France). Also, we reviewed the older German and French works that reported on ciliates collected in the Mediterranean and Brittany, respectively. The Brittany and Isle of Man populations of the ciliate were considered identical. Some morphological and behavioural differences exist between the Brittany-Isle of Man populations and the Mediterranean populations, but they were insufficient to distinguish different taxa. Thus, taxa from all three locations were considered to be conspecific. Key features used to describe the ciliate were: morphology and ultrastructure of the free-swimming ciliate; cyst morphology; presence of mixotrophic-chloroplasts; presence of an eye spot composed of stigma obtained from chlorophyte prey; division, morphogenesis, and nuclear structure; live observations and behaviour, including the encystment-excystment cycle. Based on morphological and behavioural characteristics the taxon was distinguished from other similar species, and a neotype has been designated as no type material exists.

The mechanisms that underpin the varied spatial genetic structures exhibited by free-living marine microorganisms remain controversial, with most studies emphasizing a high dispersal capability that should redistribute genetic diversity in contrast to most macroorganisms whose populations often retain a genetic signature of demographic response to historic climate fluctuations. We quantified the European phylogeographic structure of the marine flagellate Oxyrrhis marina and found a marked difference in spatial genetic structure, population demography, and genetic diversity between the northwest Atlantic and Mediterranean Sea that reflects the persistent separation of these regions as well as context-dependent population responses to contrasting environments. We found similar geographic variation in the level of genetic diversity in the sister species Oxyrrhis maritima . Because the capacity for wide dispersal is not always realized, historic genetic footprints of range expansion and contraction persist in contemporary populations of marine microbes, as they do in larger species. Indeed, the well-described genetic effects of climatic variation on macroorganisms provide clear, testable hypotheses about the processes that drive genetic divergence in marine microbes and thus about the response to future environmental change.

Abstract 433 Defects in ataxia telangiectasia mutated kinase (ATM) confer poor survival in B-cell chronic lymphocytic leukaemia (CLL). ATM gene deletion (11q23) occurs in approximately 20% of cases, and of these, 38% harbor an ATM mutation on the second allele, resulting in complete loss of ATM function. This prevents signaling of DNA double strand break (DSB) damage and compromises homologous recombinational (HR) repair. Importantly, del(11q) cases without corresponding ATM mutation display wild type ATM activity, with an intact DNA damage response and better outcome than ATM mutated cases1. Thus, in terms of chemotherapeutic resistance, it has been established that not all del(11q) cases should be placed in one group, since those with wild type ATM function have a distinct response. DNA-dependent protein kinase (DNA-PK) is also important for DSB repair and mediates the alternative non homologous end-joining (NHEJ) pathway. We previously showed that increased DNA-PK activity and expression is frequent in poor prognosis CLL and is associated with shorter survival2. We hypothesized that targeting the NHEJ pathway (via DNA-PK inhibition), in ATM deficient CLL cells that already have a defective DNA damage response, would completely compromise the ability of these cells to perform DSB repair, resulting in selective killing of ATM mutant CLL cells. The ATM status of CLL cases was determined using denaturing HPLC followed by confirmatory direct sequencing. Only those cases with pathogenic mutations (e.g. predicted to cause truncations or change in protein structure, or previously reported in A-T patients) were classed as ‘ATM mutant'. The resulting defects in ATM activity in these cases was confirmed by quantifying phosphorylation of downstream ATM targets (e.g autophosphorylation of ATM (ser1981) or phosphorylation of SMC-1) in lysates of ionizing-radiation-treated cells. Ex vivo cytotoxicity using freshly isolated CLL cells was possible in 77 cases of known ATM status. Cells were exposed to increasing concentrations of Fludarabine in combination with the DNA-PK inhibitor, NU7441 (1μ M), for 72 hr, and viability was assessed using an XTT kit. NU7441 potentiated the cytotoxicity of Fludarabine in 31 cases, including 8 cases with a known ATM mutation. As expected, ATM mutant CLL cells were more drug-resistant than those with wild type ATM, but even highly resistant (>20μ M) ATM mutant cases were resensitized up to 20-fold by NU7441. Pilot data using a similar approach showed ATM mutant cases to be 5-fold more resistant to mitoxantrone, but NU7441 potentiated the cytotoxicity of mitoxantrone in ATM mutant cases (n = 3) and ATM wild type cases (n = 5) by up to 6-fold or 3-fold respectively. Preliminary data (n =18) using an ex vivo assay dot blot assay to measure DNA-PK activity correlated DNA-PK activity to expression of the DNA-PK catalytic subunit as determined by Western blotting. Furthermore, DNA-PK activity was significantly higher in ATM mutant, compared to wild type CLL cells (p = 0.008). Current studies are exploring the formation and longevity of γH2AX foci in ATM mutant CLL cells treated with DNA damaging agents, and the role of DNA-PK in repair of DSB in these cells. Also, the link between DNA-PK activity and ATM mutation will be further examined as it provides a possible mechanism and proof of concept of increased sensitization by DNA-PK inhibitors. These results suggest that DNA-PK inhibition can sensitize poor prognosis, ATM mutant CLL cells to chemotherapeutics. Our data are consistent with the concept of synthetic lethality, where tumor cells harboring a DNA repair defect can be killed by targeting the compensatory DNA repair pathway, and suggest a group of patients that may benefit from this combination. 1 Austen et al., Journal of Clin. Oncol. 2008 2 Willmore et al., Clin. Can. Res. 2008 Disclosures: No relevant conflicts of interest to declare.

Background The heterotrophic dinoflagellate Oxyrrhis marina is increasingly studied in experimental, ecological and evolutionary contexts. Its basal phylogenetic position within the dinoflagellates make O. marina useful for understanding the origin of numerous unusual features of the dinoflagellate lineage; its broad distribution has lent O. marina to the study of protist biogeography; and nutritive flexibility and eurytopy have made it a common lab rat for the investigation of physiological responses of marine heterotrophic flagellates. Nevertheless, genome-scale resources for O. marina are scarce. Here we present a 454-based transcriptome survey for this organism. In addition, we assess sequence read abundance, as a proxy for gene expression, in response to salinity, an environmental factor potentially important in determining O. marina spatial distributions. Results Sequencing generated ~57 Mbp of data which assembled into 7, 398 contigs. Approximately 24% of contigs were nominally identified by BLAST. A further clustering of contigs (at ≥ 90% identity) revealed 164 transcript variant clusters, the largest of which (Phosphoribosylaminoimidazole-succinocarboxamide synthase) was composed of 28 variants displaying predominately synonymous variation. In a genomic context, a sample of 5 different genes were demonstrated to occur as tandem repeats, separated by short (~200-340 bp) inter-genic regions. For HSP90 several intergenic variants were detected suggesting a potentially complex genomic arrangement. In response to salinity, analysis of 454 read abundance highlighted 9 and 20 genes over or under expressed at 50 PSU, respectively. However, 454 read abundance and subsequent qPCR validation did not correlate well - suggesting that measures of gene expression via ad hoc analysis of sequence read abundance require careful interpretation. Conclusion Here we indicate that tandem gene arrangements and the occurrence of multiple transcribed gene variants are common and indicate potentially complex genomic arrangements in O. marina. Comparison of the reported data set with existing O. marina and other dinoflagellates ESTs indicates little sequence overlap likely as a result of the relatively limited extent of genome scale sequence data currently available for the dinoflagellates. This is one of the first 454-based transcriptome surveys of an ancestral dinoflagellate taxon and will undoubtedly prove useful for future comparative studies aimed at reconstructing the origin of novel features of the dinoflagellates.