User:Epipelagic/sandbox/current2

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  Looking down from above a man o' war, showing its sail. Sails can be left-handed or right-handed.
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  A typical square-rigged caravel (Livro das Armadas)]]

References

Zooplankton vertical migration

• Bandara, Kanchana; Varpe, Øystein; Wijewardene, Lishani; Tverberg, Vigdis; Eiane, Ketil (2021-05-04). "Two hundred years of zooplankton vertical migration research". Biological Reviews. 96 (4). Wiley: 1547–1589.
  ISSN 1464-7931. Material was copied from this source, which is available under a Creative Commons Attribution 4.0 International License
  .

Symbioses of cyanobacteria

in marine environments

Cyanobacteria are a diversified phylum of nitrogen-fixing, photo-oxygenic bacteria able to colonize a wide array of environments. In addition to their fundamental role as diazotrophs, they produce a plethora of bioactive molecules, often as secondary metabolites, exhibiting various biological and ecological functions to be further investigated. Among all the identified species, cyanobacteria are capable to embrace symbiotic relationships in marine environments with organisms such as protozoans, macroalgae, seagrasses, and sponges, up to ascidians and other invertebrates. These symbioses have been demonstrated to dramatically change the cyanobacteria physiology, inducing the production of usually unexpressed bioactive molecules. Indeed, metabolic changes in cyanobacteria engaged in a symbiotic relationship are triggered by an exchange of infochemicals and activate silenced pathways. Drug discovery studies demonstrated that those molecules have interesting biotechnological perspectives. In this review, we explore the cyanobacterial symbioses in marine environments, considering them not only as diazotrophs but taking into consideration exchanges of infochemicals as well and emphasizing both the chemical ecology of relationship and the candidate biotechnological value for pharmaceutical and nutraceutical applications.^[2]

Cyanobacteria are a wide and diversified phylum of bacteria capable of photosynthesis. They are found in symbiosis with a remarkable variety of hosts, in a wide range of environments (Figure 1). Symbiotic relationships concern advantages and disadvantages for the organisms involved. Symbiosis, indeed, can be advantageous for only one of the involved organisms (commensalism, parasitism), or for both (mutualism) [1]. Symbiotic interactions are widespread and involve organisms among life domains, in both Eukaryota and Prokaryota (Archaea and Bacteria). Among prokaryotes, various species have been demonstrated to be associated with invertebrates such as sponges [2,3], corals [4,5,6,7], sea urchins [8], ascidians [9,10], and mollusks [11,12,13]. In addition, symbiotic relationships between bacteria and various microorganisms such as Retaria [14,15], Myzozoa [16], Ciliophora, and Bacillariophyceae [17] were investigated in the frame of the peculiar N2 fixing process performed by various associated prokaryotes. In fact, cyanobacteria are able to perform nitrogen fixation and, among all the symbiotic interactions they are able to establish, the nitrogenase products represent the major contribution to the partnership [18]. Nitrogen-fixing organisms are often called diazotrophs and their diazotroph-derived nitrogen (DDN) gives their hosts the advantage to populate nitrogen-limited environments [19,20]. Cyanobacterial symbionts (also named cyanobionts) are active producers of secondary metabolites and toxins [21], able to synthesize a large array of bioactive molecules, such as photoprotective and anti-grazing compounds [4,22]. In addition, cyanobionts have the advantage to be protected from environmental extreme conditions and from predation/grazing. In parallel, hosting organisms grant enough space to cyanobionts for growing at low competition levels. Several investigations demonstrated an influence of host organisms on the production of cyanobiont secondary metabolites, as in the case of the symbiotic interaction of Nostoc cyanobacteria with the terrestrial plant of Gunnera and Blasia genera [23]. Indeed, changes in the expression of secondary metabolites, as in the cases of the cyanobacterial nostopeptolide synthetase gene and the altered secretion of various nostopeptolide variants, were recorded in Nostoc punctiforme according to the presence of the host [24]. Changes in the metabolic profiles have probably a clear role in the formation of cyanobacterial motile filaments (hormogonia) and, most probably, they affect the infection process and the symbiotic relationship itself [24]. This suggests that cyanobacterial secondary metabolites may play a key role in host–cyanobacterium communications.^[2]

There are lines of evidence that cyanobionts produce novel compounds of interest to pharmaceutical research [25,26], exhibiting cytotoxic and antibacterial activities. Some of these molecules are produced by cyanobacteria only in a symbiotic relationship, as in the case of polyketide nosperin (Figure 2) [27].^[2]

Cyanobacteria are capable of establishing various types of symbiosis, with variable degrees of integration with the host, and probably symbiosis emerged independently with peculiar characteristics [28,29,30]. Symbionts are transferred to their hosts by a combination of vertical and horizontal transmission, with some strains passed down from ancestral lineage, while others are acquired by the surrounding environment [31]. However, cyanobacteria are less dependent on the host than other diazotrophs, such as rhizobia, due to the presence of specialized cells (i.e., heterocysts) and a cellular mechanism to reduce the oxygen concentration in the cytosol [32]. Nostoc species are heterocystic nitrogen-fixing cyanobacteria, producing motile filaments called hormogonia, and are considered the most common cyanobacteria in symbiotic associations [33,34]. The ability of diazotrophs cyanobacteria to fix nitrogen through various oxygen-sensitive enzymes, such as molybdenum nitrogenase (nifH), vanadium nitrogenase (vnfH), and iron-only nitrogenase (anfH), is a key point to fully understand the relationships between cyanobionts and their hosts [28].^[2]

Multicellular organisms coevolved with a plethora of symbiotic microorganisms. These associations have a crucial effect on the physiology of both [35] and, in some cases, the host-associated microbiota can be considered as a meta-organism forming an intimate functional entity [36]. This means that there are coevolutive factors that led to the evolution of signals, receptors, and infochemicals among the organisms involved in symbiosis. Host–symbionts communication, based on this complex set of dose-dependent [37] and evolutionarily evolved [38] infochemicals, influences many physiological aspects of symbiosis; some examples are the microbiota composition, defensive mechanisms, development, morphology, and behavior (Figure 3) [39]. The main interactions occurring between cyanobacteria and host organisms are summarized in Table 1.^[2]

Protists

Photosynthetic eukaryotes are the product of an endosymbiotic event in the Proterozoic oceans, more than 1.5 billion years ago [86,87]. For this reason, all eukaryotic phytoplankton can be considered an evolutive product of symbiotic interactions [87] and the chloroplast, as the remnant of an early symbiosis with cyanobacteria [86]. Nowadays, the associations among these unicellular microorganisms range from simple interactions among cells in close physical proximity, often termed “phycosphere” [88], to real ecto- and endosymbiosis. The study of these associations is often neglected, partially because symbiotic microalgae and their partners show an enigmatic life cycle. In most of these partnerships, it is unclear whether the relationships among partners are obligate or facultative [89]. The symbiotic associations between cyanobacteria and planktonic unicellular eukaryotes, both unicellular and filamentous, are widespread, in particular in low-nutrient basins [89]. It is assumed that cyanobacteria provide organic carbon through photosynthesis, taking advantage of the special environmental conditions offered by the host. In contrast, some single-celled algae are in symbiotic association with diazotrophic cyanobacteria, providing nitrogen-derived metabolites through N2 fixation [90]. This exchange is important for nitrogen acquisition in those environments where it represents a limiting factor, both in terrestrial and in aquatic systems, as well as in open oceans [91]. In fact, in marine environments, cyanobacteria are associated with single-celled organisms such as diatoms, dinoflagellates, radiolarians, and tintinnids [52,92]. The exchange of nitrogen between microalgae and cyanobacterial symbionts, although important, is probably flaked by other benefits such as the production of metabolites, vitamins, and trace elements [49,93]. In fact, available genomic sequences indicate bacteria, archaea, and marine cyanobacteria as potential producers of vitamins [94], molecules fundamental in many symbiotic relationships. Moreover, about half of the investigated microalgae have to face a lack of cobalamin, and other species require thiamine, B12, and/or biotin [95,96]; these needs may be satisfied, in many cases, by the presence of cyanobionts [97].^[2]

The first case described of marine planktonic symbiosis was represented by the diatom diazotrophic associations (DDAs) among diatoms and filamentous cyanobacteria provided of heterocysts [98]. Although this kind of interaction is the most studied, little is known about the functional relationships of the symbiosis. Recent studies are mainly focused on the symbiotic relationships between the diazotroph cyanobacteria Richelia intracellularis and Calothrix rhizosoleniae with several diatom partners, especially belonging to the genera Rhizosolenia, Hemiaulus, Guinardia, and Chaetoceros [18,40]. The location of the symbionts varies from externally attached to partially or fully integrated into the host [41]. Indeed, it has been demonstrated through molecular approaches that morphology, cellular location, and abundances of symbiotic cyanobacteria differ depending on the host and that the symbiotic dependency and the location of the cyanobionts R. intracellularis and C. rhizosoleniae seems to be linked to their genomic evolution [99]. In this regard, it was demonstrated a clear relationship between the symbiosis of diatom–cyanobacteria symbiosis and the variation of season and latitude suggesting that diatoms belonging to the genus Rhizosolenia and Hemiaulus need a symbiont for high growth rates [40]. The reliance of the host seems closely related to the physical integration of symbionts: endosymbiotic relationships are mainly obligatory, while ecto-symbiosis associations tend to be more facultative and/or temporary [89]. Another interesting cyanobacteria–diatoms symbiosis involves the chain-forming diatom Climacodium frauenfeldianum, common in oligotrophic tropical and subtropical waters [100]. In this case, diatoms establish symbiotic relationships with a coccoid unicellular diazotroph cyanobacterial partner that is similar to Crocosphaera watsonii in morphology, pigmentation, and nucleotide sequence (16S rRNA and nifH gene) [41]. In addition, it has been demonstrated that nitrogen, fixed by cyanobionts is transferred to diatom cells [90]. Occasionally, C. watsonii has been reported as symbiotic diazotroph in other marine chain-forming planktonic diatoms, such as those belonging to the genera Streptotheca and Neostrepthotheca [42]. One of the most peculiar symbiosis is represented by the three-part partnership between the unicellular cyanobacterium Synechococcus sp., Leptocylindrus mediterraneus, a chain-forming centric diatom, and Solenicola setigera, an aplastidic colonial protozoa [43,44]. This peculiar association is cosmopolitan and occurs primarily in the open ocean and the eastern Arabian Sea; nevertheless, it remained poorly studied and exclusively investigated by means of microscopy techniques. Electron microscopy observations (SEM) reveal that in presence of S. setigera, the diatom can be apochlorotic (it lacks chloroplasts), thus offering refuge to the aplastidic protozoan, benefiting, and nourishing from the exudates it produces. It is assumed that the cyanobacterial partner, Synechoccus sp., supports the protozoan by supplying reduced nitrogen. It is also speculated that the absence of the cellular content of L. mediterraneus can be due to parasitism by S. setigera [44]. Recent studies reported a novel symbiotic relationship between an uncultivated N2-fixing cyanobacterium and a haptophyte host [45,46,47,48,49]. The host is represented by at least three distinctly different strains in the Braarudosphaera bigelowii group, a calcareous haptophyte belonging to the class of Prymnesiophyceae [101,102,103]. The cyanobiont, first identified in the subtropical Pacific Ocean through the analysis of nifH gene sequence, is UCYN-A or “Candidatus Atelocyanobacterium Thalassa,” formerly known as Group A. For many years, the lifestyle and ecology of this cyanobiont remained unknown, because cannot be visualized through fluorescence microscopy. Furthermore, the daytime maximum nifH gene expression of UCYN-A opposite with respect to unicellular diazotroph organisms [104,105]. The entire genome of the UCYN-A cells was sequenced, leading to the discovery of the symbiosis: the genome is unusually small (1.44 Mbp) and revealed unusual gene deletions, suggesting a symbiotic life history. Indeed, the genome completely lacks some metabolic pathways, oxygen-evolving photosystem II (PSII), RuBisCo for CO2 fixation, and tricarboxylic acid (TCA), revealing that the cyanobiont could be a host-dependent symbiont [47,48].^[2]

Symbiotic relationships include interactions between cyanobacteria and nonphototrophic protists. Heterotrophic protists include nonphotosynthetic, photosynthetic and mixotrophic dinoflagellates, radiolarians, tintinnidis, silicoflagellates, and thecate amoebae [51,52,92,106,107]. In dinoflagellates, cyanobionts were observed using transmission electron microscopy with evidence of no visible cell degradation, the presence of storage bodies and cyanophycin granules, nitrogenase, and phycoerythrin (confirmed by antisera localization), confirming that these cyanobionts are living and active and not simple grazed prey [52,108,109]. In addition, these cyanobionts are often observed with coexisting bacteria, suggesting a potential tripartite symbiotic interaction [52,109]. A cyanobiont surrounding the outer sheath was observed in rare cases, suggesting an adaptation to avoid cell degradation in symbiosis [52]. Despite the presence of N2 fixing cyanobacteria, molecular analyses demonstrated the presence of a vast majority of phototrophic cyanobionts with high similarity to Synechococcus spp. and Prochlorococcus spp. [50,51]. The complex assemblage of cyanobacteria and N2 fixing proteobacteria suggests a puzzling chemical and physiological relationship among the components of symbiosis in dinoflagellates, with an exchange of biochemical substrates and infochemicals, and the consequent coevolution of mechanisms of recognition and intracellular management of the symbionts. In tintinnid, ciliates able to perform kleptoplastidy, epifluorescent observations of Codonella species demonstrated the presence of cyanobionts, with high similarities with Synechococcus, in the oral grove of the lorica and, in addition, the presence of two bacterial morphotypes [52]. In radiolarians (Spongodiscidae Dictyocoryne truncatum), the presence of cyanobionts has been demonstrated, initially identified as bacteria or brown algae [110,111]. In addition, several non-N2-fixing cyanobionts have been identified using autofluorescence, 16s rRna sequence, and cell morphology, resembling Synecococcus species [51,52]. In agreement with associations observed in dinoflagellates, mixed populations of cyanobacteria and bacteria are common in radiolarian species, although their inter-relationship is still unknown.^[2]

Macroalgae and seagrasses

Mutual symbioses between plants and cyanobacteria have been demonstrated in macroalgae and seagrasses, as is the case of Acaryochloris marina and Lynbya sp., in which cyanobacteria contribute to the epiphytic microbiome of the red macroalgae Ahnfeltiopsis flabelliformis [53] and Acanthophora spicifera [54], respectively. Epiphytic relationships have been demonstrated as well with green and brown algae [112]. In Codium decorticatum, endosymbionts cyanobacteria belonging to genera Calothrix, Anabaena, and Phormidium, have been shown to fix nitrogen for their hosts [55,56]. Cyanobacteria are also common as seagrass epiphytes, for example, on Thalassia testudinum, where organic carbon is produced by cyanobacteria and other epiphyte symbiotic organisms rather than the plant itself [57,58]. In many cases, the presence of phosphates stimulates the cyanobionts growth on seagrasses and other epiphytes [113,114]. In oligotrophic environments, nitrogen-fixing cyanobacteria are advantaged against other seagrass algal epiphytes [115], and these cyanobacteria may contribute to the productivity of seagrass beds [116]. In addition, a certain level of host specificity can be determined in many plant–cyanobacteria symbioses [59], for example, among heterocystous cyanobacteria such as Calothrix and Anabaena, and the seagrass Cymodocea rotundata. A few cyanolichens live in marine littoral waters [92], and they play a role in the trophism of Antarctic environments, where nitrogen inputs from atmospheric deposition are low [117,118,119].^[2]

Sponges

Marine sponges are among the oldest sessile metazoans, known to host dense microbial communities that can account for up to 40–50% of the total body weight [31]. These microbial communities are highly species-specific, and characterized by the presence of several bacterial phyla; cyanobacteria constitute one of the most important groups [120,121,122]. Sponges with cyanobionts symbionts can be classified as phototrophs when they are strictly depending on symbionts for nutrition or mixotrophs when they feed also by filter feeding [92]. These “cyanosponges” are morphologically divided into two categories—the phototrophs present a flattened shape, while the mixotrophs have a smaller surface area to volume ratio [29]. Cyanobacteria are located in three main compartments in sponges: free in the mesohyl, singly or as pairs in closed-cell vacuoles, or aggregated in large specialized “cyanocytes” [123]. Their abundance decreases away from the ectosome, while it is null in the endosome of the sponge host [124]. Cyanobacteria belonging to the genera Aphanocapsa, Synechocystis, Oscillatoria, and Phormidium are usually found in association with sponges and most species are located extracellularly, while others have been found as intracellular symbionts benefiting sponges through fixation of atmospheric nitrogen [92]. Indeed, some cyanobacteria located intracellularly within sponges showed to own nitrogenase activity [124]. Most of the sponges containing cyanobionts, however, are considered to be net primary producers [125]. Cyanobacteria in sponges can be transmitted vertically (directly to the progeny) or horizontally (acquired from the surrounding environment), depending on the sponge species [29]. For instance, the sponge Chondrilla australiensis has been discovered to host cyanobacteria in its developing eggs [126]. Caroppo et al., instead, isolated the cyanobacterium Halomicronema metazoicum from the Mediterranean sponge Petrosia ficiformis, which has been later found as a free organism and isolated from leaves of the seagrass Posidonia oceanica [119,127], highlighting that horizontal transmission of photosymbionts can occur in other sponge species [128]. Cyanobacteria associated with sponges are polyphyletic and mostly belonging to Synechoccoccus and Prochlorococcus genera [129]. Synechococcus spongiarum is one of the most abundant symbionts found in association with sponges worldwide [130,131]. In some cases, however, the relationship between symbionts and host sponges can be controversial. Some Synechococcus strains seem to be mostly “commensals”, whereas symbionts from the genus Oscillatoria are involved in mutualistic associations with sponges [3,132]. In the past, many researchers performed manipulative experiments to demonstrate the importance of cyanobacteria associations for the metabolism of the host [3,128,133]. A case study from Arillo et al. performed on Mediterranean sponges revealed that Chondrilla nucula, after six months in the absence of light, displayed metabolic collapse and thiol depletion [63]. This highlights that symbionts are involved in controlling the redox potential of the host cells transferring fixed carbon in the form of glycerol 3-phosphate and other organic phosphates. Instead, Petrosia ficiformis, which is known to live in association with the cyanobacterium Aphanocapsa feldmannii [62], showed the capability to perform heterotrophic metabolism when transplanted in dark conditions [63]. In some tropical environments, the carbon produced by cyanobionts can supply more than 50% of the energy requirements of the sponge holobiont [122]. Cyanobacteria, moreover, can contribute to the sponge pigmentation and production of secondary metabolites (e.g., defensive substances) [134], as in the case of the marine sponge Dysidea herbacea [64]. Thus, symbiotic associations could result in the production of useful compounds with biotechnological potential [134,135]. Meta-analysis studies on sponge–cyanobacterial associations revealed that several sponge classes could host cyanobacteria, although most of the knowledge in this field remains still unknown, and mostly hidden in metagenomics studies [136]. Sponge-associated cyanobacteria hide a reservoir of compounds with biological activity, highlighting an extraordinary metabolic potential to produce bioactive molecules for further biotechnological purposes [137].^[2]

Cnidarians

It is widely accepted that reef environments rely on both internal cycling and nutrient conservation to face the lack of nutrients in tropical oligotrophic water [138]. A positive ratio in the nitrogen export/input between coral reefs and surrounding oceans has been observed [139,140]. Tropical Scleractinia are able to obtain nitrogen due to various mechanisms that include the endosymbiont Symbiodinium [141], the uptake of urea and ammonium from the surrounding environment [142], predation and ingestion of nitrogen-rich particles [143,144,145,146], or diazotrophs itself through heterotrophic feeding [147] and nitrogen fixation by symbiotic diazotrophic communities [4,7,68,69,73,148]. In addition to nitrogen fixation, coral-associated microbiota performs various metabolic functions in carbon, phosphorus, sulfur, and nitrogen cycles [74,149,150,151]; moreover, it plays a protective role for the holobiont [152,153,154], possessing inhibitory activities toward known coral pathogens [155]. These complex microbial communities that populate coral surface mucopolysaccharide layers show a vertical stratification of population resembling the structure of microbial mats, with a not-dissimilar flux of organic and inorganic nutrients [156]. It is reasonable to believe that microbiota from all the compartments, such as tissues and mucus, can contribute to the host fitness and interact with coral in different ways, ranging from the direct transfer of fixed nitrogen in excess to the ingestion and digestion of prokaryotes [20]. Diazotrophs, and in particular cyanobionts, are capable of nitrogen fixation and they can use glycerol, produced by zooxanthellae, for their metabolic needs [4,73]. The relationship between corals and cyanobacteria is yet to be fully explored and understood but some lines of evidence regarding Acropora millepora [69,70] suggest coevolution between corals and associate diazotrophs (cyanobionts). This relationship appears to be highly species-specific. In hermatypic corals, a three-species symbiosis can be observed, with diazotrophs in direct relation with Symbionidium symbiont. In Acropora hyacinthus and Acropora cytherea, cyanobacteria-like cells, characterized by irregular layered thylakoid membranes and with a remarkable similarity to the ones described by previous authors [4], were identified in strict association with Symbiodinium, within a single host cell, especially in gastrodermal tissues [67]. The high density of these cells closely associated with Symbiodinium suggests that the latter is the main user of the nitrogen compounds produced by the cyanobacterium-like cells. The presence of these cyanobacterium-like cells is more widespread than assumed in the past and this symbiosis was found in many geographic areas, for example, in the Caribbean region and the Great Barrier Reef [67].^[2]

Microbial communities inhabiting the coral surface can greatly vary due to environmental conditions [147,157,158]. Diazotroph-derived nitrogen assimilation by corals varies on the basis of the autotrophic/heterotrophic status of the coral holobiont and with phosphate availability in seawater. Consequently, microbial communities increase when corals rely more on heterotrophy or when they live in phosphate-rich waters [147]. This suggests that diazotrophs can be acquired and their population managed according to the needs of corals [159]. This view was confirmed by the identification of a first group of organisms that form a species–specific, temporarily, and spatially stable core microbiota and a second group of prokaryotes that changes according to environmental conditions and in accordance with the host species and physiology state [160]. Experimental lines of evidence, using N2-labelled bacteria, demonstrated that diazotrophs are transferred horizontally and very early in the life cycle, and it is possible to identify nifH sequences, in larvae and in one-week-old juveniles [70], and in adult individuals [69] of the stony coral Acropora millepora. About coral tissues, the distribution of microbiota, and cyanobacteria as well, is not the same in all the tissue districts. Species that live in the mucus resemble the species variety and abundance that can be found in the surrounding water. On the contrary, the microbiota of internal tissues including also calcium carbonate skeletons is made, at least partially, of species that cannot be easily found free in the environment [68,69]. This plasticity might as well characterize cyanobacteria hosted in cnidarians, although such multiple relationships are still scarcely investigated.^[2]

Synechococcus and Prochlorococcus cyanobacteria have been identified in association with Montastraea cavernosa [4], through molecular approaches and genes belonging to filamentous cyanobacteria [6]. Filamentous and unicellular diazotrophic cyanobacteria belonging to the orders Chroococcales, Nostocales, Oscillatoriales, and Proclorales were found, using pyrosequencing approach, as associated organisms to the shallow water coral Porites astreoides [6] and Isopora palifera [71]. On the contrary, in Montipora flabellate, Montipora capitate [7], Acropora millepora [69,70], Acropora muricate, and Pocillopora damicornis [69], cyanobacteria are present in various tissues and in the skeleton, but their contribution in terms of nitrogen fixation is minimal [5]. In Montastraea cavernosa, Montastraea franksi, and in species of the genus Diploria and Porites, cyanobacterial sequences belonging to various genera (e.g., Anabaena, Synechoccus, Spirulina, Trichodesmium, Lyngbya, and Phormidium) have been found in coral tissues by PCR amplification [4,73,74,75,161]. In Montastraea cavernosa, the orange fluorescence protein, peaking at 580 nm, was attributed to phycoerythrin, a cyanobacterial photopigment produced by a cyanobacterium living in the host epithelial cells [4]. The different colors, especially of fluorescent proteins in corals, suggest specific biological functions for these compounds. Moreover, it is not clear if they act as photoprotective compounds, antenna pigments, or if they photoconvert part of the light spectrum to help zooxanthellae photosynthesis. These results are contested by some authors who excluded the role of phycoerythrin as a pigment compound in corals [5]. In order to determine the presence and the activity of cyanobacteria in corals, the following aspect should be considered: nonquantitative approaches cannot assure accurate values of abundance; moreover, the presence of nifH gene is not necessarily linked to the fixation and the transfer of nitrogen performed by diazotrophs. H [20]. Endolithic cyanobacteria have been found in Porites cylindrica and Montipora monasteriata, but their role in the relationship with host corals is unknown [162]. In contrast, in other cnidarians, it has been demonstrated that endolithic cyanobacteria establish symbiotic relationships with coral hosts: this is the case of Plectonema terebrans, a cyanobacterium belonging to the order Oscillatoriales [72]. Cold-water corals are ecosystem engineers providing a habitat for thousands of different species. Their trophism is related to the low energy, partially degraded, organic matter that derives from the photic zone of oceans [163]. To face the lack of nutrients, cold-water corals evolved, on one hand, from an opportunistic feeding strategy [164,165], and on the other hand, from a symbiosis with various diazotrophs, including cyanobacteria [166,167,168]. Plectonema terebrans filaments, visible as pinkish to violet staining, are able to colonize the entire skeleton of the cold-water corals Desmophyllum dianthus and Caryophyllia huinayensis; however, their density is higher at the skeleton portion covered with polyp tissue [72]. The close contact between coral tissues and cyanobacteria obliges the endoliths to exchange nutrients with the surrounding water through the polyp itself. This close relationship is advantageous for the cyanobacterium because the coral nematocysts protect it from the grazers [169], and it is mutualistic because such a close relationship inevitably includes exchanges of metabolites between organisms [170]. These metabolites produce benefits for the host and play a trophic and/or protective role in the symbiotic mutualistic relationship. Middelburg et al. suggested that in cold-water corals, a complete nitrogen cycle occurs similar to that inferred for tropical reefs, ranging from ammonium production and assimilation to nitrification, nitrogen fixation, and denitrification [166].^[2]

The effects of environmental changes on the nitrogen fixation rates are still poorly explored, especially if specifically related to the symbiotic diazotrophs and to cyanobacteria. Ocean acidification enhances nitrogen fixation in planktonic cyanobacteria, as in the case of Crocosphaera watsoni, due to enhancement of photosynthetic carbon fixation [171]. It is interesting to underline that in the planktonic diazotroph cyanobacterium Trichodesmium sp., which forms symbiotic association with diatoms [172], the nitrogen fixation is enhanced under elevated CO2 conditions [173], but it is strongly reduced if there is an iron limitation [174]. On the contrary, Seriatopora hystrix diazotrophs are sensible to ocean acidification, with a decline of the nitrogen fixation rate at high CO2 concentration, leading to consequences on coral calcification and potential starvation for both the coral and the Symbiodinium spp. [175]. In addition, environmental changes can increase in coral symbionts, the abundance of microbial genes involved in virulence, stress resistance, sulfur and nitrogen metabolisms, and production of secondary metabolites. These changes that affect the physiology of symbionts can also affect the composition of the coral-associated microbiota [74], with the substitution of a healthy-associated coral community (e.g., cyanobacteria, Proteobacteria), playing a key role in mediating holobiont health and survival upon disturbance [176], with a community related to coral diseases (e.g., Bacteriodetes, Fusobacteria, and Fungi).^[2]

Ascidians and other tunicates

Tunicates are considered rich in biologically active secondary metabolites [177,178,179,180], but it is unclear if these bioactive compounds were produced by tunicates themselves or by associated microorganisms [181,182], although strong direct and indirect lines of evidence show that defensive compounds and other secondary metabolites are produced by various symbiotic prokaryotes and not by the tunicates themselves. Among tunicate symbionts, cyanobacteria have been found in symbiotic relationships with various tunicates, ranging from tropical to temperate environments. In fact, obligate associations with cyanobacteria of Prochloron and Synechocystis genus have been found in some species of ascidians belonging to the genera Didemnum, Lissoclinum, Diplosoma, and Trididemnum [77], with cyanobacterial cells distributed in the cavities and/or tunic [78]. These cyanobionts have been demonstrated to be part of the core microbiome, in which species and populations do not reserve the water–column ones and microbiome–host relationship is species specific and not correlated to the geographical location [9]. In colonial ascidians, such as Botryllus schlosseri and Botrylloides leachii, an abundant population of Synechococcus-related cyanobacteria have been identified [79], while in the Mediterranean ascidian Didemnum fulgens, a coral-associated cyanobacterium has been observed in its tissues [183]. In some cases, the cyanobiont completely or partially lacks the nitrogen-fixation pathway. This is the case of Prochloron didemni, in symbiosis with the tunicate Lissoclinum patella, which is probably involved in carbon fixation and in the ammonia incorporation and not in the nitrogen fixation [80,81]. In fact, in contrast with the presence of genes for the nitrate reduction pathway and all primary metabolic genes required for free-living, Prochloron seems to lack the capability to fix nitrogen and to live outside the host [80]. Prochloron sp. also protects the host versus active forms of oxygen, which can be formed during photosynthesis processes. The cyanobacterium produces a cyanide-sensitive superoxide dismutase, a Cu-Zn metalloprotein, that has been demonstrated to prevent the toxicity of superoxide radicals, hydrogen peroxide, and hydroxyl radicals in the host ascidians [82]. In Lissoclinum patella, other cyanobacteria were abundant in various tissues and one of these is Acaryochloris marina, a chlorophyll d-rich cyanobacterium, able to sustain oxygenic photosynthesis under near-infrared radiation that propagates through Prochloron cells and ascidian tissue [83]. The Caribbean tunicate Trididemnum solidum produces a peculiar biologically active molecule, the acyl-tunichlorine (Figure 2) [84,85], that contains both nickels accumulated by the tunicate and pheophytin, which is produced by organisms with photosynthetic machinery and suggests a dual origin of this compound. In fact, this tunicate hosts the cyanobacterium Synechocystis trididemni, which contributes to the production of acyl-tunichlorine synthesizing the pheophytin through an intermediate molecule, the pyropheophorbide [84,85]. In addition, behavioral tests demonstrated the presence of deterring compounds in ascidian larvae able to distaste predatory fishes. These compounds have been identified to be didemnin B (Figure 2) and nordidemnin [65]. Didemnin B was found in various tunicates, and it is similar to a bioactive molecule produced by other cyanobacteria, enforcing the idea that the predation-deterring compounds can be produced by cyanobionts [184], although the possibility of a horizontal gene transfer cannot be totally rejected [185,186]. The tunicate–cyanobacteria symbiosis is evidenced by the presence, in the host tunicate, of a cellulose synthase gene, similar to the one found in cyanobacteria, which probably derives from horizontal transfer between the two organisms [187,188] and that may have a role in the tunicates evolutive radiation and in the development of adult and larvae body plans [188,189,190]. The presence of a rich and bio-diversified microbiome makes tunicates promising models for various purposes and important for drug discovery [10,191].^[2]

References

Sea surface microlayer processes

As with other marine ecosystems, life in the SML is dominated by microorganisms, which are collectively referred to as the “neuston,” whereas “plankton” describes the microscopic organisms that inhabit the underlying water column. So far, most recent studies have focused on the diversity and abundance of bacterioneuston assemblages, principally through the application of molecular ecology tools (for a review see Cunliffe et al., 2011). The consensus view is that bacterioneuston assemblages form by recruitment from the underlying bacterioplankton; however, the species composition and activity of bacterioneuston assemblages can be very different compared to those in the underlying water column (Agogue et al., 2005; Franklin et al., 2005; Obernosterer et al., 2008; Stolle et al., 2011; Taylor et al., 2014). What exactly controls bacterioneuston species diversity is at present not fully understood, but evidence from both field observations (Cunliffe et al., 2009a) and large-scale mesocosm experiments (Cunliffe et al., 2009c) suggests that the development of bacterioneuston assemblages is ecologically regulated and not random (Cunliffe et al., 2011). Possible controls of bacterioneuston diversity and activity include the prevailing meteorological conditions, incident UV light, organic matter availability and aerosol deposition (Carlucci et al., 1985; Agogue et al., 2005; Stolle et al., 2010, 2011; Nakajima et al., 2013; Astrahan et al., 2016). It is, however, not clear whether solar and UV irradiance promote or inhibit neuston activity (Bailey et al., 1983; Carlucci et al., 1985; Santos et al., 2012).[1]

The bacterioneuston has been shown to directly influence air-sea gas exchange by consuming and producing trace gases (e.g., CO, H2, CH4, N2O) (Conrad and Seiler, 1988; Frost, 1999; Upstill-Goddard et al., 2003; Nakajima et al., 2013). The addition of methanotrophs in a laboratory gas exchange tank led to an enhancement of the apparent transfer velocity of CH4 by 12 ± 10% (Upstill-Goddard et al., 2003). Later work reported bacterial growth efficiency in the SML and underlying water to be similar, but also showed higher respiration in the SML that pointed to some potential control of the air-sea exchange of CO2 and O2 by the bacterioneuston (Reinthaler et al., 2008). Through the application of functional gene probes to a range of SML environments, the metabolic potential for trace gas cycling in the SML has since been established as being widespread (Cunliffe et al., 2008, 2011). It can therefore be concluded that for some trace gases such as CH4 and CO2, the bacterioneuston is intimately involved in their cycling and that it may potentially act as either a small gas source or sink in response to the prevailing microbial and biogeochemical conditions. The bacterioneuston may also modulate the enrichment of surfactants in the SML and thus indirectly influence air-sea gas exchange, as indicated by a recent study relating SML surfactant enrichment and the occurrence of specific bacterial taxa (Kurata et al., 2016).^[1]

The second major biological component of SML ecosystems is phytoneuston, historically studied using microscopes (Hardy and Valett, 1981; Hardy, 1982), and more recently through high-throughput sequencing (Taylor and Cunliffe, 2014). High-throughput sequencing of phytoneuston assemblages in the coastal SML of the English Channel showed that biodiversity at the interface was very different to that in the underlying water (Taylor and Cunliffe, 2014). Two major algal groups, the chrysophytes and the diatoms, were significantly enriched in the neuston, with diatom diversity in particular being distinctly different to that of planktonic diatoms. While the functional impacts of the differences between phytoneuston and phytoplankton abundance and diversity are not yet fully understood, it is believed that specific processes such as the maintenance of air-sea CO2 disequilibria could be controlled, at least in part, by the phytoneuston (Calleja et al., 2005).^[1]

Other components of SML ecosystems such as heterotrophic protists and zooneuston have also been studied, but to a lesser extent. Looking forward, the application of contemporary tools, such as high-throughput sequencing, hold great promise in revealing new insights into the biology of the SML. The recent English Channel study discussed above also showed that the diversity of neustonic fungi (myconeuston) is also distinctly different from mycoplankton diversity (Taylor and Cunliffe, 2014). Relative to other marine microbial groups, the marine fungi remain poorly understood. A recent study of the coastal mycoplankton implicated marine fungi in a range of ecosystem functions, including nutrient recycling and parasitism (Taylor and Cunliffe, 2016). Exactly what functional roles myconeuston may play at the ocean-atmosphere interface still remain to be revealed.^[1]

Marine aerosols that are formed from the SML via bubble bursting are a vector for the transfer of microbial life from the lower water column and SML into the atmosphere. Measurements of bacteria and viruses in sea spray aerosols (SSA) show large enrichments relative to both the SML (~5) and subsurface waters (~10) (Aller et al., 2005). A key factor in this process appears to be the embedding of microorganisms within the organic gel matrix that forms in the SML (discussed further below) (Aller et al., 2005). The global-scale implications for ocean-atmosphere exchange of microorganisms are profound because this process may have a major impact on the long-distance dispersal of marine microbial life. For example, phytoplankton viruses that are transported into the atmosphere from the SML can remain highly infective under prevailing meteorological conditions and can be spread across hundreds of kilometers to subsequently infect distal phytoplankton (Sharoni et al., 2015).^[1]

Organisms in the SML interact with the surface accumulation of organic matter produced by biological processes in the underlying water column (Galgani et al., 2014). Some dissolved compounds have surface active (surfactant) properties or adsorb onto floating compounds and selectively become enriched in the SML (Hunter and Liss, 1977). The accumulation of surfactants in the SML was found to be related to the occurrence of certain bacterioneuston taxa, indicating microbial contribution to surfactant production and decomposition (Kurata et al., 2016). In addition to soluble surfactants, the SML also accumulates a variety of colloidal and particulate organic matter that may serve as substrates for the bacterioneuston. Sieburth (1983) proposed the SML to be a complex hydrated “gel” of macromolecules and colloidal material.^[1]

The “gel” material has now been identified and studied in greater detail. Included in the gelatinous matrix are transparent exopolymer particles (TEP), i.e., polysaccharide-rich microgels that are produced primarily by the abiotic coagulation of phytoplankton exudates (Passow, 2002), and Coomassie stainable particles (CSP) containing proteinaceous polymers released during cell lysis and decomposition (Long and Azam, 1996). The aggregation of TEP precursors in the SML is enhanced due to the constant compression and dilation of the air-sea interface (Wurl et al., 2011), which may explain, at least in part, the gelatinous nature of the SML. Gel particles may serve as grazing protection for the phytoneuston and bacterioneuston, as has been shown for their planktonic counterparts (Passow and Alldredge, 1999; Dutz et al., 2005). In addition, TEP have been suggested to protect neuston from high irradiance (Ortega-Retuerta et al., 2009), possibly helping the neuston withstand periods of high irradiation of the SML.^[1]

Surface microlayer

The surface microlayer (SML) is an ecotone, which constitutes an interface between the hydrosphere and the atmosphere. It is a thin layer of maximum 1000 µm in thickness [1]. The SML is unique due to its physical, chemical and biological properties, which differ from those of subsurface water (SUB) [1,2]. The SML is capable of accumulating microorganisms and chemical substances at a rate as high as 100-fold greater than that observed in the SUB [3]. The SML is the zone of matter and energy exchange between the hydrosphere and the atmosphere, and, as such, it is affected by global climate changes [4].^[3]

The accumulation of chemical substances in the SML may vary, as it is dependent on many factors such as the chemical composition of water, salinity, the nutrient status and the presence of neustonic organisms (organisms that live upon the upper surface of waters or beneath its surface film, the SML) [5,6]. The SML may also accumulate toxic substances at greater amounts than observed in the pelagic zone [7]. At the same time, it is the zone that accumulates nutrients [5,8]. In turn, such substances as calcium, magnesium, potassium or sodium, being macroelements found at high concentrations in the pelagic zone, are usually not accumulated in the SML [9]. The SML is inhabited by neustonic autotrophic and heterotrophic microorganisms, which exhibit both enzymatic and high respiratory activity [5,10,11,12]. The SML structure may be disturbed as a result of physical non-equilibrium processes such as temperature fluxes, irradiance, wind and wave action that influence its biogeochemical properties. However, it is well fit for a rapid self-reconstruction of its original structure [3]. Physical forces such as adhesion, cohesion, surface tension, vortex motion and the Langmuir circulations, associated with its unique chemical and biological structure, form the specific properties of SML, including its stability [2].^[3]

Chemical contaminants are accumulated in the SML because of physical, chemical and biological processes. These processes include, e.g., hydrosphere–atmosphere exchange, convection, upwelling of underlying waters, transport by bubbles, atmospheric deposition, simple diffusion, buoyant particles, turbulent mixing, scavenging and chelation of inorganic components by organic matter [3]. Important sources of contaminants in urban objects are connected with urban pollutants supplied with wet and dry atmospheric deposition [13,14].^[3]

The taxonomic composition of phytoplankton is dependent on the chemical composition of the aquatic environment, which it colonizes [5,15]. Consequently, communities of the phytoneuston and phytoplankton vary in the SML and the SUB in terms of their taxonomic composition and population size [16].^[3]

^[4]

Ocean surface ecosystem

Above and below

Air-sea interface

Food webs

Organisms that live at the surface are a nexus for food webs both above and below (Fig 3). From the air, seabirds prey on neuston, including fulmars, storm petrels, and sooty shearwaters (see review in [32]). For the Pacific ocean Laysan albatross, nearly 30% of their diet is neuston, including Velella, Janthina, Halobates, and the eggs and larvae of flying fish [10]. Even ducks [33] and sea-going bats [34] prey on floating neuston when they drift close to shore. Below the surface, diverse sea turtles eat neuston (see review in [32]), including olive ridley (Lepidochelys olivacea), which prey upon Janthina [9,35], green turtles (Chelonia mydas), which prey upon Porpita [36], and loggerhead turtles (Caretta caretta), which prey upon Velella and Janthina [9]. Neuston are among the most important prey for central North Pacific loggerhead sea turtles [9]. Fish like coho salmon Oncorhynchus kisutch and spiny dogfish Squalus acanthias prey on Velella (see review in [32]), and animals of the deep-scattering layer also prey upon neuston [37]. Diverse larval fish from a wide variety of ecologically and economically important species live as or prey on neuston [11] (Table 1).^[5]

Neuston themselves reach into the waters below, capturing non-neustonic prey and further linking deeper waters to this thin surface layer. Velella feed on a variety of foods, including fish eggs and larvae [69], while Porpita and

calanoid copepods [66,70]. Unlike Velella and Porpita, which each have tentacles extending only a few centimetres, Physalia can extend tentacles many meters below the surface, and prey primarily on fish [71].^[5]

Many species of the neuston also prey on one another, creating an interconnected food web stretching into the broader world around it. Janthina and Glaucus prey on Physalia, Velella, and Porpita. Janthina have also been observed trying to eat each other, suggesting they have the capacity to be cannibalistic [65]. The only true open ocean insect, the neustonic Halobates, preys upon other neuston by sucking nutrients from organisms with piercing mouthparts [72].[5]

Ecoregions

The ocean’s surface possesses diverse floating ecosystems within different regions. The only well-known neustonic ecoregion, the Sargasso Sea, covers an area in the western North Atlantic where the neustonic Sargassum concentrates. Multiple endemic species live in the Sargasso Sea, many of them adapted to shelter among the neustonic seaweed [14,15]. The Sargasso Sea contributes to a variety of ecosystem goods and services, and its valuation ranges from over US$200 million for fisheries services to US$2.7 billion for all services [16]. The distribution of surface life in the Sargasso Sea changes by season [17,18] and may be subject to annual and decadal trends [19,20]. These trends can impact both the ecology and economy of the region, as well as stakeholders further afield that rely on species that shelter in the Sargassum.^[5]

Beyond the Sargasso Sea, the most detailed survey of Pacific neuston occurred in the 1950s. Scientists in the USSR crisscrossed the Pacific, collecting nearly 500 samples from 50°N to over 40°S [12]. In this massive survey, 7 distinct ecoregions were discovered, with different species of neuston showing different ranges that likely reflect their ability to move with wind, thermal optima, seasonality, and life cycles [12].^[5]

A linear survey from Fiji to the Bay of Biscay also found considerable geographic variation. The tropical seas of the Indian Ocean were dominated by neustonic species including Halobates, Physalia, Velella, and Porpita [21]. In contrast, the eastern North Atlantic was dominated by small quick-moving crustaceans that made up over 90% of neustonic organisms [21,22]. This suggests these regions have distinct neustonic communities.^[5]

Ecological variation across regions also includes how the surface changes through time. On short time scales, the surface habitat is part of the diel vertical migration of marine life from the deep sea: the largest migration on Earth, which happens twice each day [23]. Because of this migration, significant differences in surface life occur between day and night at basin-wide scales. Ostracods, mysids, isopods, heteropods, various crustacean and bryozoan larvae, are all more abundant at the surface at night [21,22]. In contrast, some surface-associated species, such as Sapphirina copepods, which use complex visual cues for mating, migrate to the surface only during the day [24]. These migratory species add to the diversity at the ocean’s surface. On larger time scales, neustonic Sargassum abundance changes seasonally [19,25,26], and some neuston, such as Velella, strand more often in certain seasons than others [27], possibly due to seasonal variation in distribution.^[5]

Differences in neuston across space and time may be due to real population and species boundaries. For example, while some species, such as the nudibranch Glaucus atlanticus, are globally distributed, closely relatives Glaucus bennettae and Glaucus mcfarlanei have thus far been identified only in the North Pacific subtropical gyre system [28], and represent cryptic species. The sea skater Halboates shows remarkable population- and species-level isolation both across oceans and ocean basins [29,30], while neustonic Sargassum represent a genetic and morphotype species complex with diverse and distinct distribution patterns [31]. It is clear that neuston are not uniformly distributed, and there is evidence for both species and population isolation as well as sympatric speciation. However, for the majority of neustonic species, no genetic or population data exist. Are individuals of the “same species” half a planet away part of an interconnected global population, or isolated and distinct enough to be considered different species with unique adaptations to the conditions in their region of the world?^[5]

Poorly studied neuston ecoregions should be considered in the context of the Sargasso Sea: We know this comparatively well-studied region is critical for both the ecology and economy of the North Atlantic, its services valued in the billions. What ecological and economic services are neuston ecosystems providing in other ocean regions?^[5]

Threats

The ocean surface is a concentrating front for floating pollutants from plastic to petroleum. Metals and toxicants concentrate on the ocean’s surface, particularly

organotin compounds, polycyclic aromatic hydrocarbons (PAH), and heavy metals at the surface can reach concentrations up to 500 times higher than those in the water column.^[7] Many of these compounds are concentrated in the sea surface microlayer (0 to 1,000 μm depth). In general, pollutants are at lower concentrations in the open ocean than in areas closer to shore,^[7] and while this may bode well for open-ocean neustonic species, it presents challenges to coastal or benthic species with neustonic eggs or larvae.^[5]

One large threat to both coastal and open-ocean surface organisms comes from oil. An estimated 741 kilotonnes of oil is released into the ocean each year from both natural and human sources,[8] with unknown effects on surface ecosystems. Because hydrophobic molecules concentrate at the ocean’s surface,^[9][10] neustonic species will face orders of magnitude higher oil exposure than animals even a meter below the surface. Additionally, neuston species may also be vulnerable to dispersants used in breaking down oil spills, as is the case with jellyfish,^[11] which die at significantly higher rates in the presence of dispersants, and Sargassum, which sinks in the presence of dispersants.^[12] However, as of 2021 studies on diverse neustonic species in the presence of oil or dispersants have not been conducted.^[5]

Floating plastic is another widespread petroleum product on the ocean’s surface.^[13][14] There are an estimated 14.9 to 51.2 trillion pieces of plastic on the ocean’s surface,^[15] representing upwards of 250,000 tons, largely concentrated in oceanic subtropical gyres (known colloquially as "Garbage Patches", which includes the Sargasso Sea).^[16] These plastics are consumed by surface-hunting species like the Laysan albatrosses of Midway Atoll, which feed nearly 5 tons of ocean plastic to their chicks each year.^[17][18] Such high plastic consumption makes sense only in light of these birds’ predation on neuston [10]. Larval neustonic fish and rafting barnacles have been found with plastic in their gut [11,97], though the impact of this plastic on these organisms, or the animals that feed on them, is not known. Some neustonic species, such as Halobates, may benefit from plastic, which provides a hard surface for laying eggs [98]. Larval fish may also shelter around plastic debris [73].^[5]

With this complexity in mind, we must proceed cautiously when attempting to restore or conserve the ocean’s surface. For example, multiple organizations pledge to remove plastic from the ocean using unmanned collection devices inspired by pool skimmers or technology used to catch algae and jellyfish [99]. It should be no surprise then that one organization trapped hundreds of neustonic animals in their prototype, visible in their press release photo [100].^[5]

Of all the human impacts on the ocean’s surface, climate change will have the farthest reach, and it is unclear what impact it will have on neuston. The ocean’s surface is directly exposed to the atmosphere, and changes in temperature will be felt first at the surface. This region is also uniquely exposed to atmospheric carbon dioxide and the ravages of storms, which are predicted to increase in intensity and frequency under climate change [101].^[5]

Overall, threats to the neuston are poorly understood, and for every likely threat listed here, there are no doubt many that are largely unknown (e.g., ballast water, localized pollution, deep-sea mining impacts on pelagic life-cycle stages, geoengineering, etc.).^[5]

Gallery

paper nautilus Aurgonaut sp. viewed from the side and reflecting off the water’s surface, (k) a shrimp in the family Hippolytidae, clinging to a discarded Janthina bubble raft, (l) seaweed Sargassum sp. with a small sargassum crab Portunus sayi

pleuston

not a jellyfish but a colony of smaller organisms