Mass spectroscopy reveals compositional differences in copepodamides from limnic and marine copepods.

Marine copepods, the most abundant animals in the global ocean, imprint their surrounding waters with chemical cues, called copepodamides. Copepodamides induce defensive traits such as toxin production, bioluminescence, and colony size plasticity in a variety of marine phytoplankton. The role of copepodamides in freshwater ecosystems is, however, unknown. Here we report the consistent presence of copepodamides in copepods from six Swedish freshwater lakes. Copepodamide concentrations in freshwater copepods are similar to those of marine copepods, around 0.1 ppt of dry mass in millimetre sized individuals. The composition substantially overlaps with marine copepodamides but is also distinctly different. Marine copepods commonly contain both subgroups of copepodamides, the copepodamides (CA) and the dihydro-copepodamides (dhCA), whereas freshwater copepods are dominated by the dhCAs. Taxonomic groups had consistent copepodamide profiles across sampling sites and timepoints, supporting the presence of species-specific copepodamide signatures. We describe 10 new copepodamide structures, four of which were found exclusively in freshwater copepods. The presence of copepodamides in limnic copepods also warrants studies into their potential function as predator alarm cues in freshwater systems.

Roadmap on Deep Learning for Microscopy.

Through digital imaging, microscopy has evolved from primarily being a means for visual observation of life at the micro- and nano-scale, to a quantitative tool with ever-increasing resolution and throughput. Artificial intelligence, deep neural networks, and machine learning are all niche terms describing computational methods that have gained a pivotal role in microscopy-based research over the past decade. This Roadmap is written collectively by prominent researchers and encompasses selected aspects of how machine learning is applied to microscopy image data, with the aim of gaining scientific knowledge by improved image quality, automated detection, segmentation, classification and tracking of objects, and efficient merging of information from multiple imaging modalities. We aim to give the reader an overview of the key developments and an understanding of possibilities and limitations of machine learning for microscopy. It will be of interest to a wide cross-disciplinary audience in the physical sciences and life sciences.

Applying genetic technologies to combat infectious diseases in aquaculture.

Disease and parasitism cause major welfare, environmental and economic concerns for global aquaculture. In this review, we examine the status and potential of technologies that exploit genetic variation in host resistance to tackle this problem. We argue that there is an urgent need to improve understanding of the genetic mechanisms involved, leading to the development of tools that can be applied to boost host resistance and reduce the disease burden. We draw on two pressing global disease problems as case studies-sea lice infestations in salmonids and white spot syndrome in shrimp. We review how the latest genetic technologies can be capitalised upon to determine the mechanisms underlying inter- and intra-species variation in pathogen/parasite resistance, and how the derived knowledge could be applied to boost disease resistance using selective breeding, gene editing and/or with targeted feed treatments and vaccines. Gene editing brings novel opportunities, but also implementation and dissemination challenges, and necessitates new protocols to integrate the technology into aquaculture breeding programmes. There is also an ongoing need to minimise risks of disease agents evolving to overcome genetic improvements to host resistance, and insights from epidemiological and evolutionary models of pathogen infestation in wild and cultured host populations are explored. Ethical issues around the different approaches for achieving genetic resistance are discussed. Application of genetic technologies and approaches has potential to improve fundamental knowledge of mechanisms affecting genetic resistance and provide effective pathways for implementation that could lead to more resistant aquaculture stocks, transforming global aquaculture.

Single-shot self-supervised object detection in microscopy.

Object detection is a fundamental task in digital microscopy, where machine learning has made great strides in overcoming the limitations of classical approaches. The training of state-of-the-art machine-learning methods almost universally relies on vast amounts of labeled experimental data or the ability to numerically simulate realistic datasets. However, experimental data are often challenging to label and cannot be easily reproduced numerically. Here, we propose a deep-learning method, named LodeSTAR (Localization and detection from Symmetries, Translations And Rotations), that learns to detect microscopic objects with sub-pixel accuracy from a single unlabeled experimental image by exploiting the inherent roto-translational symmetries of this task. We demonstrate that LodeSTAR outperforms traditional methods in terms of accuracy, also when analyzing challenging experimental data containing densely packed cells or noisy backgrounds. Furthermore, by exploiting additional symmetries we show that LodeSTAR can measure other properties, e.g., vertical position and polarizability in holographic microscopy.

Microplankton life histories revealed by holographic microscopy and deep learning.

The marine microbial food web plays a central role in the global carbon cycle. However, our mechanistic understanding of the ocean is biased toward its larger constituents, while rates and biomass fluxes in the microbial food web are mainly inferred from indirect measurements and ensemble averages. Yet, resolution at the level of the individual microplankton is required to advance our understanding of the microbial food web. Here, we demonstrate that, by combining holographic microscopy with deep learning, we can follow microplanktons throughout their lifespan, continuously measuring their three-dimensional position and dry mass. The deep-learning algorithms circumvent the computationally intensive processing of holographic data and allow rapid measurements over extended time periods. This permits us to reliably estimate growth rates, both in terms of dry mass increase and cell divisions, as well as to measure trophic interactions between species such as predation events. The individual resolution provides information about selectivity, individual feeding rates, and handling times for individual microplanktons. The method is particularly useful to detail the rates and routes of organic matter transfer in micro-zooplankton, the most important and least known group of primary consumers in the oceans. Studying individual interactions in idealized small systems provides insights that help us understand microbial food webs and ultimately larger-scale processes. We exemplify this by detailed descriptions of micro-zooplankton feeding events, cell divisions, and long-term monitoring of single cells from division to division.

Picture a glass of seawater. It looks clear and empty, but in reality, it contains one hundred million bacteria, about one hundred thousand other single-celled organisms, and a few microscopic animals. In fact, the majority of life in the ocean is microscopic and we know relatively little about it. Nevertheless, these microbes have a major impact on our lives. Microscopic algae known as phytoplankton, for example, produce half of the oxygen we breathe. For animals, birds and other large organisms in the ocean, we have a good understanding of who eats who and where the material ends up. However, for phytoplankton and other microbes, we depend on bulk measurements and averages of large groups. Bachimanchi et al. developed a method to follow individual microbes living in seawater and to observe how they move, grow, consume each other and reproduce. The team combined holographic microscopy with artificial intelligence to follow multiple planktons, diatoms and other microbes throughout their life span and continuously measured their three-dimensional location and mass. This made it possible to estimate how fast the organisms were growing and moving, and to observe what they ate. The experiments revealed new insights into how micro-zooplankton, diatoms and other microbes in the ocean interact with each other. This new method may be useful for researchers who would like to track the movements and whereabouts of microscopic planktons, bacteria or other microbes for extended periods of time. It is also a rapid method for counting, sizing, and weighing cells in suspension. The hardware used in this method is relatively cheap, and Bachimanchi et al. have shared all the computer code with examples and demonstrations in a public database to enable other researchers to use it.

Predatory cues drive colony size reduction in marine diatoms.

Colony formation is a common feature among nonmotile marine phytoplankton. Several theories exist around the potential benefits of larger colonies.Here, we test the hypothesis that predation is one of the drivers behind colony formation and chain length plasticity. We exposed cultures of Thalassiosira rotula, Chaetoceros curvisetus, and Chaetoceros affinis to copepodamides, a chemical alarm cue released by copepods and perceived as an indicator of predation threat by their prey. This was coupled with a grazing experiment, which compared copepod grazing rates on different chain lengths.Our results show that T. rotula and C. curvisetus decreased their chain lengths by 79% and 49%, respectively, in response to copepodamides. Single cells and short chains were grazed at lower rates compared with long chains, and the copepodamide-driven size shift led to 30% and 12% lower grazing in T. rotula and C. curvisetus, respectively. In contrast, C. affinis showed a slight increased chain length in response to copepodamides although nonsignificant.We found that 2 of 3 studied species reduce their chain length in response to the presence of copepod grazers. Altered size structure has implications for the route of carbon in the marine food webs and carbon export to deeper strata.

Predator-induced defence in a dinoflagellate generates benefits without direct costs.

Inducible defences in phytoplankton are often assumed to come at a cost to the organism, but trade-offs have proven hard to establish experimentally. A reason for this may be that some trade-off costs only become evident under resource-limiting conditions. To explore the effect of nutrient limitation on trade-offs in toxin-producing dinoflagellates, we induced toxin production in Alexandrium minutum by chemical cues from copepods under different levels of nitrogen limitation. The effects were both nitrogen- and grazer-concentration dependent. Induced cells had higher cellular toxin content and a larger fraction of the cells was rejected by a copepod, demonstrating the clear benefits of toxin production. Induced cells also had a higher carbon and nitrogen content, despite up to 25% reduction in cell size. Unexpectedly, induced cells seemed to grow faster than controls, likely owing to a higher specific nutrient affinity due to reduced size. We thus found no clear trade-offs, rather the opposite. However, indirect ecological costs that do not manifest under laboratory conditions may be important. Inducing appropriate defence traits in response to threat-specific warning signals may also prevent larger cumulative costs from expressing several defensive traits simultaneously.

Predator Chemical Cue Effects on the Diel Feeding Behaviour of Marine Protists.

We have assessed the effect of copepod chemical cues on the diel feeding rhythms of heterotrophic and mixotrophic marine protists. All phagotrophic protists studied exhibited relatively high diurnal feeding rates. The magnitude of the diel feeding rhythm, expressed as the quotient of day and night ingestion rates, was inversely related to the time that phagotrophic protists were maintained in the laboratory in an environment without predators. In the case of the recently isolated ciliate Strombidium arenicola, the rhythm was lost after a few months. When challenged with chemical alarm signals (copepodamides) from the copepod Calanus finmarchicus at realistic concentrations (0.6-6 pM), S. arenicola partially re-established diurnal feeding. Conversely, the amplitude of the diel feeding rhythm for the ciliate Mesodinium rubrum was not affected by copepodamides, although the 24-h integrated food intake increased by approximately 23%. For the dinoflagellates Gyrodinium dominans and Karlodinium armiger, copepodamides significantly reduced the amplitude of their diel feeding rhythms; significant positive effects on total daily ingestion were only observed in G. dominans. Finally, the dinoflagellate Oxyrrhis marina, isolated >20 years ago, showed inconsistent responses to copepodamides, except for an average 6% increase in its total ingestion over 24 h. Our results demonstrate that the predation risk by copepods affects the diel feeding rhythm of marine protists and suggests a species-specific response to predation threats.

Grazer-induced bioluminescence gives dinoflagellates a competitive edge.

Bioluminescent dinoflagellates grow at one third the rate of their competitors of equivalent size, such as diatoms [1]. Despite this disadvantage, dinoflagellates successfully persist within phytoplankton communities and even form large blooms during favourable conditions. One explanation for this paradox is that bioluminescence acts as a defence that reduces losses to zooplankton grazers, such as copepods [2,3]. Lindström et al.[4] found that the dinoflagellate Lingulodinium polyedra (F.Stein) J.D.Dodge increase their bioluminescence in response to copepodamides [5], polar lipids exuded by copepod grazers, allowing for a brighter flash when bioluminescent capacity is stimulated. Here, we show that copepodamide-induced bioluminescence in L. polyedra causes a marked shift in the grazing preference of the copepod Acartia tonsa Dana. L. polyedra goes from being the preferred prey when non-bioluminescent to near complete rejection when pre-treated with copepodamides to induce a higher bioluminescent capacity. High-speed and low-light-sensitive videos show how L. polyedra cells flash upon contact with the copepod and are subsequently rejected, seemingly unharmed (Videos S1 and S2). Instead, A. tonsa shows compensatory feeding on the alternative prey.

The highly toxic and cryptogenic clinging jellyfish Gonionemus sp. (Hydrozoa, Limnomedusae) on the Swedish west coast.

The clinging jellyfish Gonionemus sp. is a small hydromedusa species known historically from the Swedish west coast but not reported in recent times. This species is thought to be native to the northwest Pacific where it is notorious for causing severe stings in humans and is considered invasive or cryptogenic elsewhere. This year, unlike in the past, severe stings in swimmers making contact with Gonionemus sp. medusae occurred in Swedish waters from a sheltered eelgrass bed in the inner Skagerrak archipelago. To the best of our knowledge, this is only the second sting record of Gonionemus sp. from the Northeast Atlantic-with the first record occurring off the Belgian coast in the 1970s. Stinging Gonionemus sp. medusae have also been recently reported from the northwestern Atlantic coast, where, like on the Swedish coast, stings were not reported in the past. We analyzed sea surface temperature data from the past 30 years and show that 2018 had an exceptionally cold spring followed by an exceptionally hot summer. It is suggested that the 2018 temperature anomalies contributed to the Swedish outbreak. An analysis of mitochondrial COI sequences showed that Swedish medusae belong to the same clade as those from toxic populations in the Sea of Japan and northwest Atlantic. Gonionemus sp. is particularly prone to human-mediated dispersal and we suggest that it is possible that this year's outbreak is the result of anthropogenic factors either through a climate-driven northward range shift or an introduction via shipping activity. We examined medusa growth rates and details of medusa morphology including nematocysts. Two types of penetrating nematocysts: euryteles and b-mastigophores were observed, suggesting that Gonionemus sp. medusae are able to feed on hard-bodied organisms like copepods and cladocerans. Given the now-regular occurrence and regional spread of Gonionemus sp. in the northwest Atlantic, it seems likely that outbreaks in Sweden will continue. More information on its life cycle, dispersal mechanisms, and ecology are thus desirable.

Induction of domoic acid production in diatoms-Types of grazers and diatoms are important.

Grazers can induce toxin (domoic acid, DA) production in diatoms. The toxic response has been observed in two species of Pseudo-nitzschia and was induced by Calanus copepods. In this study, interactions between diatoms and copepods were further explored using different species of diatoms and copepods. All herbivorous copepods induced toxin production, whereas exposure to carnivorous copepods did not. In line with this, increasing the number of herbivorous copepods resulted in even higher toxin production. The induced response is thus only elicited by copepods that pose a real threat to the responding cells, which supports that the induced toxin production in diatoms evolved as an inducible defense. The cellular toxin content in Pseudo-nitzschia was positively correlated to the concentration of a group of specific polar lipids called copepodamides that are excreted by the copepods. This suggests that copepodamides are the chemical cues responsible for triggering the toxin production. Carnivorous copepods were found to produce less or no copepodamides. Among the diatoms exposed to grazing herbivorous copepods, only two of six species of Pseudo-nitzschia and none of the Nitzschia or Fragilariopsis strains responded by producing DA, indicating that not all Pseudo-nitzschia species/strains are able to produce DA, and that different diatom species might have different strategies for coping with grazing pressure. Growth rate was negatively correlated to cellular domoic acid content indicating an allocation cost associated with toxin production. Long-term grazing experiments showed higher mortality rates of grazers fed toxic diatoms, supporting the hypothesis that DA production is an induced defense mechanism.

Grazer-induced transcriptomic and metabolomic response of the chain-forming diatom Skeletonema marinoi.

Diatoms and copepods are main actors in marine food webs. The prey-predator interactions between them affect bloom dynamics, shape marine ecosystems and impact the energy transfer to higher trophic levels. Recently it has been demonstrated that the presence of grazers may affect the diatom prey beyond the direct effect of grazing. Here, we investigated the response of the chain-forming centric diatom Skeletonema marinoi to grazer cues, including changes in morphology, gene expression and metabolic profile. S. marinoi cells were incubated with Calanus finmarchicus or with Centropages typicus and in both cases responded by reducing the chain length, whereas changes in gene expression indicated an activation of stress response, changes in the lipid and nitrogen metabolism, in cell cycle regulation and in frustule formation. Transcripts linked to G protein-coupled receptors and to nitric oxide synthesis were differentially expressed suggesting involvement of these signalling transduction pathways in the response. Downregulation of a lipoxygenase in the transcriptomic data and of its products in the metabolomic data also indicate an involvement of oxylipins. Our data contribute to a better understanding of the gene function in diatoms, providing information on the nature of genes implicated in the interaction with grazers, a crucial process in marine ecosystems.

Effects of predator lipids on dinoflagellate defence mechanisms - increased bioluminescence capacity.

Short flashes of blue light (bioluminescence) from dinoflagellates can reduce copepod grazing of light-emitting cells. Other protective strategies against grazing are toxicity, reduced cell chain length and altered swimming patterns in different phytoplankton. Both toxicity and bioluminescence capacity in dinoflagellates decrease in copepod-free cultures, but toxin production can be restored in response to chemical alarm signals from copepods, copepodamides. Here we show that strains of the dinoflagellates Lingulodinium polyedra and Alexandrium tamarense, kept in culture for 14 and 9 years respectively, are capable of increasing their total bioluminescence capacity in response to copepodamides. The luminescence response to mechanical stimulation with air bubbles also increases significantly in L. polyedra after exposure to copepodamides. Effects on size, swimming speed and rate of change of direction in L. polyedra and A. tamarense were not detected, suggesting that post-encounter mechanisms such as bioluminescence and toxin production may constitute the dominating line of defence in these taxa. To our knowledge, this study provides the first evidence of changes in bioluminescence physiology as a response to chemical cues from natural enemies and emphasizes the importance of bioluminescence as an anti-grazing strategy.

Predator cues reduce intraspecific trait variability in a marine dinoflagellate.

BACKGROUND: Phenotypic plasticity is commonplace and enables an organism to respond to variations in the environment. Plastic responses often modify a suite of traits and can be triggered by both abiotic and biotic changes. Here we analysed the plastic response towards a grazer of two genotypes of the marine dinoflagellate Alexandrium fundyense, evaluated the similarity of this response and discuss potential strain-specific trade-offs. We compared the expression of the known inducible defensive traits paralytic shellfish toxin content, and chain length. The effectiveness of the induced defense was assessed by monitoring grazing rates in both strains. RESULTS: Our results show that the grazer cues diminish phenotypic variability in a population by driving the phenotype towards a common defended morphotype. We further showed that the expression of the sxtA gene that initiates the paralytic shellfish toxin biosynthesis pathway does not correlate with an observed increase in the paralytic shellfish toxin analogue saxitoxin, and that toxin induction differs in its physiological characteristics in both strains. CONCLUSION: Induced defense response in Alexandrium thus can directly affect further species interactions by reducing phenotypic variation and can result in genotype-dependent ecological trade-offs.

Solid phase extraction and metabolic profiling of exudates from living copepods.

Copepods are ubiquitous in aquatic habitats. They exude bioactive compounds that mediate mate finding or induce defensive traits in prey organisms. However, little is known about the chemical nature of the copepod exometabolome that contributes to the chemical landscape in pelagic habitats. Here we describe the development of a closed loop solid phase extraction setup that allows for extraction of exuded metabolites from live copepods. We captured exudates from male and female Temora longicornis and analyzed the content with high resolution LC-MS. Chemometric methods revealed 87 compounds that constitute a specific chemical pattern either qualitatively or quantitatively indicating copepod presence. The majority of the compounds were present in both female and male exudates, but nine compounds were mainly or exclusively present in female exudates and hence potential pheromone candidates. Copepodamide G, known to induce defensive responses in phytoplankton, was among the ten compounds of highest relative abundance in both male and female extracts. The presence of copepodamide G shows that the method can be used to capture and analyze chemical signals from living source organisms. We conclude that solid phase extraction in combination with metabolic profiling of exudates is a useful tool to develop our understanding of the chemical interplay between pelagic organisms.

Predator lipids induce paralytic shellfish toxins in bloom-forming algae.

Interactions among microscopic planktonic organisms underpin the functioning of open ocean ecosystems. With few exceptions, these organisms lack advanced eyes and thus rely largely on chemical sensing to perceive their surroundings. However, few of the signaling molecules involved in interactions among marine plankton have been identified. We report a group of eight small molecules released by copepods, the most abundant zooplankton in the sea, which play a central role in food webs and biogeochemical cycles. The compounds, named copepodamides, are polar lipids connecting taurine via an amide to isoprenoid fatty acid conjugate of varying composition. The bloom-forming dinoflagellate Alexandrium minutum responds to pico- to nanomolar concentrations of copepodamides with up to a 20-fold increase in production of paralytic shellfish toxins. Different copepod species exude distinct copepodamide blends that contribute to the species-specific defensive responses observed in phytoplankton. The signaling system described here has far reaching implications for marine ecosystems by redirecting grazing pressure and facilitating the formation of large scale harmful algal blooms.

A stable-isotope mass spectrometry-based metabolic footprinting approach to analyze exudates from phytoplankton.

Phytoplankton exudates play an important role in pelagic ecology and biogeochemical cycles of elements. Exuded compounds fuel the microbial food web and often encompass bioactive secondary metabolites like sex pheromones, allelochemicals, antibiotics, or feeding attractants that mediate biological interactions. Despite this importance, little is known about the bioactive compounds present in phytoplankton exudates. We report a stable-isotope metabolic footprinting method to characterise exudates from aquatic autotrophs. Exudates from (13)C-enriched alga were concentrated by solid phase extraction and analysed by high-resolution Fourier transform ion cyclotron resonance mass spectrometry. We used the harmful algal bloom forming dinoflagellate Alexandrium tamarense to prove the method. An algorithm was developed to automatically pinpoint just those metabolites with highly (13)C-enriched isotope signatures, allowing us to discover algal exudates from the complex seawater background. The stable-isotope pattern (SIP) of the detected metabolites then allowed for more accurate assignment to an empirical formula, a critical first step in their identification. This automated workflow provides an effective way to explore the chemical nature of the solutes exuded from phytoplankton cells and will facilitate the discovery of novel dissolved bioactive compounds.

Grazer cues induce stealth behavior in marine dinoflagellates.

Chain formation is common among phytoplankton organisms but the underlying reasons and consequences are poorly understood. Here we show that chain formation is strongly impaired by waterborne cues from copepod grazers in the dinoflagellate Alexandrium tamarense. Chains of Alexandrium cells exposed to copepod cues responded by splitting into single cells or shorter chains. Motion analysis revealed significantly lower swimming velocities for single cells compared with chains, with two- to fivefold higher simulated predator encounter rates for two- and four-cell chains, respectively. In addition, the few remaining two-cell chains in grazed treatments were swimming at approximately half the speed of two-cell chains in treatments without grazers, which reduced encounter rates with grazers to values similar to that of single cells. Chain length plasticity and swimming behavior constitute unique mechanisms to reduce encounters with grazers. We argue that dinoflagellates can regulate the balance between motility and predator avoidance by adjusting chain length. The high predator encounter rate for motile chains may have contributed to the low prevalence of chain formation in motile phytoplankton compared with in nonmotile phytoplankton where chain formation is more common.

Induction of toxin production in dinoflagellates: the grazer makes a difference.

The dinoflagellate Alexandrium minutum has previously been shown to produce paralytic shellfish toxins (PST) in response to waterborne cues from the copepod Acartia tonsa. In order to investigate if grazer-induced toxin production is a general or grazer-specific response of A. minutum to calanoid copepods, we exposed two strains of A. minutum to waterborne cues from three other species of calanoid copepods, Acartia clausi, Centropages typicus and Pseudocalanus sp. Both A. minutum strains responded to waterborne cues from Centropages and Acartia with significantly increased cell-specific toxicity. Waterborne cues from Centropages caused the strongest response in the A. minutum cells, with 5 to >20 times higher toxin concentrations compared to controls. In contrast, neither of the A. minutum strains responded with significantly increased toxicity to waterborne cues from Pseudocalanus. The absolute increase in PST content was proportional to the intrinsic toxicity of the different A. minutum strains that were used. The results show that grazer-induced PST production is a grazer-specific response in A. minutum, and its potential ecological importance will thus depend on the composition of the zooplankton community, as well as the intrinsic toxin-producing properties of the A. minutum population.

Copepods induce paralytic shellfish toxin production in marine dinoflagellates.

Among the thousands of unicellular phytoplankton species described in the sea, some frequently occurring and bloom-forming marine dinoflagellates are known to produce the potent neurotoxins causing paralytic shellfish poisoning. The natural function of these toxins is not clear, although they have been hypothesized to act as a chemical defence towards grazers. Here, we show that waterborne cues from the copepod Acartia tonsa induce paralytic shellfish toxin (PST) production in the harmful algal bloom-forming dinoflagellate Alexandrium minutum. Induced A. minutum contained up to 2.5 times more toxins than controls and was more resistant to further copepod grazing. Ingestion of non-toxic alternative prey was not affected by the presence of induced A. minutum. The ability of A. minutum to sense and respond to the presence of grazers by increased PST production and increased resistance to grazing may facilitate the formation of harmful algal blooms in the sea.