Traços funcionais do zooplâncton (copépodos) determinam duração da facilitação na dominância de cianobactérias filamentosas

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(1)UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE DEPARTAMENTO DE ECOLOGIA PROGRAMA DE PÓS-GRADUAÇÃO EM ECOLOGIA. EWALDO LEITÃO DE OLIVEIRA JÚNIOR. COPEPODS FUNCTIONAL TRAITS DETERMINE DURATION OF FACILITATION ON FILAMENTOUS CYANOBACTERIA TRAÇOS FUNCIONAIS DE ZOOPLÂNCTON (COPÉPODOS) DETERMINAM DURAÇÃO DA FACILITAÇÃO NA DOMINÂNCIA DE CIANOBACTÉRIAS FILAMENTOSAS. Natal - RN Fevereiro / 2018.

(2) Universidade Federal do Rio Grande do Norte - UFRN Sistema de Bibliotecas SISBI Catalogação de Publicação na Fonte. UFRN - Biblioteca Setorial Prof. Leopoldo Nelson - Centro de Biociências - CB. Oliveira Júnior, Ewaldo Leitão de. Traços funcionais do zooplâncton (copépodos) determinam duração da facilitação na dominância de cianobactérias filamentosas / Ewaldo Leitão de Oliveira Júnior. - Natal, 2018. 34 f.: il. Dissertação (Mestrado) - Universidade Federal do Rio Grande do Norte. Centro de Biociências. Programa de Pós-Graduação em Ecologia. Orientadora: Profa. Dra. Renata de Fátima Panosso. Coorientador: Prof. Dr. Kemal Ali Ger. 1. Dinâmica predador-presa - Dissertação. 2. Zooplankton tropical - Dissertação. 3. Notodiaptomus iheringi - Dissertação. 4. Thermocyclops decipiens - Dissertação. 5. Cylindrospermopsis raciborskii - Dissertação. 6. Teia trófica artificial - Dissertação. I. Panosso, Renata de Fátima. II. Ger, Kemal Ali. III. Universidade Federal do Rio Grande do Norte. IV. Título. RN/UF/BSE-CB. CDU 591.5.

(3) EWALDO LEITÃO DE OLIVEIRA JÚNIOR. COPEPODS FUNCTIONAL TRAITS DETERMINE DURATION OF FACILITATION ON FILAMENTOUS CYANOBACTERIA TRAÇOS FUNCIONAIS DE ZOOPLÂNCTON (COPÉPODOS) DETERMINAM DURAÇÃO DA FACILITAÇÃO NA DOMINÂNCIA DE CIANOBACTÉRIAS FILAMENTOSAS. Dissertação de Mestrado em Ecologia Programa de Pós Graduação em Ecologia (PPGEco) Universidade Federal do Rio Grande do Norte (UFRN) Orientador: Renata Panosso Co-Orientador: Kemal Ali Ger. Natal - RN Fevereiro / 2018.

(4) Agradecimentos Gostaria de deixar meus profundos agradecimentos por todos aqueles que sempre me incentivaram a seguir essa carreira acreditando em meu potencial e com palavras que me ajudaram muito. Desde a graduação, a UFRN e o LAMAQ tem sido de fundamental importância para meu crescimento como mente científica, e cada conversa cruzada foi um bloco de concreto para a fundação de meu conhecimento crítico. Primeiramente ao grupo de limnólogos tão incríveis que tive o prazer de trocar experiências e amizades. Obrigado Iagê, Radmila, Gabizão, Bruno, Fabíola (Fafá), Regina, Fabiana (Bibinha), Léo Teixeira, Anízio, Letícia, Camila, Marcolina, Maricota, Léo (Rafael) e Pedro. Vocês ajudaram imensamente a construir os pilares de meu conhecimento. Gostaria também de agradecer a Walter, Aninha, Gabi, Layana, Rayane e, principalmente, Natália, companheiros de laboratório. Natália, sua ajuda foi fundamental para que eu conseguisse rodar esse laboratório. Sem você eu certamente teria enlouquecido e não teria terminado meus afazeres no prazo. Tenho certeza que estou deixando meus "bixinhos" em boas mãos. :) Obrigado pessoal da pós-graduação da Ecologia. Obrigado pelo café, por tentar responder minhas perguntas sobre seus projetos, e por escutar pacientemente quando eu tinha uma ideia maluca ou algum pitaco, mesmo que esse fosse muitas vezes sem sentido pelo meu desconhecimento metodológico de suas respectivas áreas. O fato de haver pessoas dispostas a falar de ciência sempre foi muito motivador e apenas engrandeceu meu conhecimento. Aos meus amigos de graduação que a Ecologia me proporcionou Anízio, João Lucas, Leo Hobbit e, em especial, Maiara, por eu ter acompanhado mais de perto seu crescimento. Maiara, saiba que ver você superar todas as suas dificuldades e continuar seguindo me deu uma força que você sequer faz ideia. Observar seu crescimento sempre que você apresenta seu trabalho me traz um sorriso besta ao rosto por ver a brilhante pesquisadora que você vem se tornando, o quanto você aprendeu, e o quanto ama o que faz apesar das dificuldades. Você é uma inspiração. Um grande abraço aos meus amigos de longa data, Raphael, Leonardo, Felipe e Clênio, pelas perspectivas antropológicas, corporativistas e até mesmo religiosas. Sem nossas discussões socio-filosóficas e companheirismo, esses dois anos teriam sido mais difíceis. Assim como Maressa, que sempre me apoiou, independente do que eu fizesse, sempre me motivando (e inflando meu ego). Agradeço também aos meus pais, Ewaldo Leitão e Edmar Oliveira, que apesar de não entenderem muito o que eu faço da minha vida, nunca hesitaram em acreditar em mim e sempre me deram muito suporte. Obrigado aos professores que tive em meus anos de estudo até agora, em especial Marcio Zikan, Hugo Sarmento, André Megali e Vanessa Bécker. Ter professores que tratam seus alunos em tom de igualdade é uma qualidade de um respeito profundo..

(5) Obrigado ao meu companheiro, Eno, por estar sempre presente e ter me ajudado tanto emocionalmente e intelectualmente a terminar essa fase e abrir novas portas para meu futuro. Obrigado por acreditar em mim e sempre me falar para eu parar de reclamar e voltar para meus estudos. Isso foi muito importante. Ευχαριστω πάρα πολύ. Agradeço também a banca de defesa tanto da qualificação Guilherme Ortigara (com sua mente afiadíssima sempre contextualizando trabalhos com teorias ecológicas amplas) e Adriano Caliman (por dissecar meu texto e me dar dicas preciosas de organização das minhas ideias). E pelos Professores Rosemberg Menezes e Juliana Dio, por terem aceitado fazer parte dessa banca e contribuir para meu trabalho. Obrigado aos meus orientadores Renata Panosso e Ali Ger. Obrigado por sempre acreditarem muito em mim e pelo, sempre necessário de escutar, "vai dar certo". Renata, muito obrigado pela paciência (enorme) por sempre dar um jeito para que as coisas dessem certo, mesmo quando eu aparecia com problemas em cima da hora. Agradeço em especial ao Ali, por ter tido papéis múltiplos de co-orientador, psicólogo, amigo e biblioterapeuta. Você foi o que alguém chamaria de mentor. Não tenho palavras pra expressar a gratidão por todo o conhecimento instruído nesses quase seis anos de parceria. O mérito de minhas conquistas jamais poderia ser designado somente a mim. Obrigado a todos vocês..

(6) “[...]He then took me into his laboratory and explained to me the uses of his various machines, instructing me as to what I ought to procure and promising me the use of his own when I should have advanced far enough in the science not to derange their mechanism. He also gave me the list of books which I had requested, and I took my leave. Thus ended a day memorable to me; it decided my future destiny.” Frankenstein, by Mary Shelley.

(7) Title: Copepods feeding mode and body size determine duration of facilitation on filamentous cyanobacteria Ewaldo Leitão1,2, Kemal Ali Ger1,2, Renata de Fátima Panosso1,2. 1. Graduate Program of Ecology, Federal University of Rio Grande do Norte, Natal, RN, Brazil. 2. Department of Microbiology, Federal University of Rio Grande do Norte, Natal, RN, Brazil. Abstract Top-down regulations in trophic chain depend on herbivore grazing traits and producers edibility. By actively selecting nutritious eukaryotic phytoplankton and rejecting toxic cyanobacteria, grazing copepods may contribute to cyanobacteria blooms and dynamics. In tropical environments, for instance, copepods commonly cooccur with cyanobacteria blooms, raising the question whether they can facilitate cyanobacteria dominance. We experimentally tested the effects of two groups of copepods with different feeding modes – calanoid Notodiaptomus iheringi (active filter feeding) and cyclopoid Thermocyclops decipiens (ambush feeder) – on the competition of an eukaryotic phytoplankton Cryptomonas and the filamentous cyanobacteria Cylindrospermopsis raciborskii. We assessed grazing in 1L batch cultures for seven days, starting with 10-fold initial dominance of Cryptomonas. Copepods demonstrated initial rejection of Cylindrospermopsis filaments, but while cyclopoids slightly increased grazing on cyanobacteria in extended experimental periods, calanoids reversed to clear more particles of cyanobacteria. Despite differences on grazing, both zooplankton decreased cyanobacteria filaments size similarly, reducing it in ~70%. We also performed experiments testing competition between phytoplankton that showed no interference on each other growth rates, assuring that results from grazing experiment are addressed to zooplankton feeding. Here, we demonstrated that copepod selectively avoidance of filamentous cyanobacteria Cylindrospermopsis may facilitate its dominance, depending on time and alternative food concentration. Cyclopoid, rather than calanoid, grazing may be an appropriate mechanism to explain filamentous cyanobacteria dominance, as they had lower impact on cyanobacteria abundance. This mechanistic approach understanding trophic dynamics using traits of different groups (i.e. copepods and cyanobacteria) is especially relevant in light of the more intense warmer and eutrophic world that will promote cyanobacteria bloom, and increase complexity of such interactions. Keywords: predator-prey dynamics; tropical zooplankton, Notodiaptomus iheringi, Thermocyclops decipiens, Cylindrospermopsis raciborskii, artificial food-webs.

(8) Introduction Ecological interactions of herbivores and producers are fundamental link to transfer energy through food web, however producer defenses may dampen top-down regulations. That heterogeneity in edibility within a given trophic level, producers, is affected by different herbivores traits (Leibold, 1989). For example, producer fraction of inedible or defended species can be avoided if herbivores have the ability to discriminate prey quality. Herbivores then direct grazing to edible fraction of producers, decreasing competition, leading to dominance of inedible fraction (Ford et al., 2014). In planktonic communities, despite the importance of bottom-up regulations on producers, top-down zooplankton grazing can be an important feature shaping phytoplankton communities (Urrutia-Cordero et al., 2014). A group of phytoplankton that is commonly assumed to be inedible is cyanobacteria. Zooplankton selective grazing can indirect influence on competing cyanobacteria and edible phytoplankton (Haney, 1987; Boon et al., 1994), and cyanobacteria component may be positively affected, characterizing as a facilitation process (Bruno et al., 2003). Yet little is studied about cyanobacteria facilitation mediated by zooplankton selective grazing. Environmental conditions such as climate change and nutrient enrichment are increasing the duration and frequency of harmful cyanobacteria bloom (O’Neil et al., 2012), but biotic interactions may also play a role in their dominance. In tropical warm lakes, cyanobacteria is largely associated long bloom duration, dominating up to 95% of the phytoplankton biomass (Soares et al., 2013). Cyanobacteria dominance is often attributed to traits such as regulation on water column by buoyance and Nfixing, overcoming light and nutrient deficiencies, granting competitive advantages over other phytoplankton taxa (Padisák et al., 2009). Alternatively, cyanobacteria have grazing resistant traits, forming of colonies or filaments, potential toxicity and nutrient deficiency, decreasing loss by predation (Ger et al., 2016a). Despite the prevailing idea that grazing on cyanobacteria is little and inefficient, results are contrasting. Herbivores have been shown to do little or no grazing in cyanobacteria (Engstrom-Ost et al., 2000). Whereas they have also been shown to graze on blooms (Chislock et al, 2013) or promote cyanobacteria dominance (Hong et al., 2015; Leitão et al., 2018.). Therefore it is necessary to avoid generalizations and assess underlying factors, looking for specific traits of both zooplankton (e.g. size, feeding behavior and feeding mode) and cyanobacteria (e.g. colonial, filamentous, toxicity), to understand their interactions mechanistically. An approach to understand the functional complexities in trophic interactions is to study different traits of zooplankton that co-exist with cyanobacteria. Zooplankton traits, such as body size, can affect how herbivores regulate producers, by which an optimal predator prey ratio can indicate preferred prey (Hansen, 1997). The plethora of studies assessing top-down regulations of zooplankton on cyanobacteria are experimental and field studies performed with large generalist grazers, cladoceran Daphnia (Ger et al., 2014). Large zooplankton can graze on large algae, considered to be a trait indicative of grazing resistance. Thus, theoretically, large Daphnia is assumed to control blooms of morphological resistant algae (Leibold, 1989). However, this taxon may misrepresent the zooplankton community that often co-exists with cyanobacteria in eutrophic environments. In eutrophic environments, Daphnia is substituted by small cladocerans and copepods as.

(9) eutrophication increases (Jiang et al., 2017; Jeppesen et al., 2011; Hansson et al., 2007). Small zooplankton can interact with cyanobacteria in a different fashion that are largely unexplored. While large generalist Daphnia is regarded to potentially graze on larger phytoplankton volume, they are more susceptible than smaller zooplankton to deleterious effects of cyanobacteria morphology (Gliwicz, 1990) and toxicity (Hansson et al., 2007). On the other hand, copepods and small cladocerans have been shown to either avoid predation on cyanobacteria or successfully graze on it (Haney, 1987; DeMott and Moxter, 1991; Kâ et al., 2012; Urrutia-Cordero et al., 2014). Those results mount evidence that large generalist zooplankters are unlikely to maintain viable population in cyanobacteria bloom conditions, meriting further studies to understand dynamics of zooplankton that co-exist with cyanobacteria. Despite the recognized importance of zooplankton body size as a master trait (Litchman et al., 2013; Hébért et al., 2016), we need also to acknowledge feeding traits. Herbivore feeding modes can shape producers community composition as a result of specific herbivores targets (Schmitz, 2008). Taxonomic groups within copepods, for example, despite sharing selective feeding behavior, can present distinguishable feeding traits. While calanoids have active current feeding, cyclopoids have strict ambush/raptorial feeding modes. Filter-feeding mode creates currents that actively bring large volume of particles to mouthparts (Kiørboe, 2011). Ambush cyclopoids perceive motile prey via mechanoreceptors remotely, that in some instances can be the primary mechanism of perception rather than chemical cues (DeMott and Watson, 1991; Svensen and Kiørboe, 2000). These different feeding modes largely influence perception and capture of different prey types (Kiørboe et al., 1996). Zooplankton feeding mode trait can help understanding grazer effects on ecosystem processes, such as carbon flow and nutrient recycling (Hebert et al., 2016). But how each grazing trait affect producers, especially cyanobacteria, needs to be explored to understand in more details the coupling copepods–cyanobacteria. Copepods have a remarkable positive correlation with toxic cyanobacteria blooms in field monitoring, often occurring in high abundances (Rangel et al., 2016a; Bouvy et al., 2001; Eskinazi-Sant'Anna et al., 2013). Selective avoidance of noxious particles is one of the accepted explanations by which copepods can co-exist with blooms (Ger et al., 2016b). Experiments tested mechanistically selectivity of copepods against cyanobacteria, especially in toxic strains (Ger et al., 2016b; Kurmayer and Juettner, 1999). Tolerance to toxic particles has also been observed by copepods (Ger et al., 2016b), and can be an indication of local adaptations (Colin and Dam, 2003). By selectively grazing on edible phytoplankton fraction, copepods can have indirect impacts on cyanobacteria. Modeling studies suggested that selective grazing on cyanobacteria competitors and nutrient remineralization via excretion, can act together to facilitate cyanobacteria bloom (Mitra and Flynn, 2006; Irigoien et al., 2005). Also, experiments using field zooplankton assemblages have demonstrated an increase of filamentous cyanobacteria Cylindrospermopsis raciborskii biomass when copepods are more abundant (Hong et al., 2015). Calanoid copepods also showed consistent selective grazing on edible phytoplankton and shift on algal dominance to cyanobacteria (Leitão et al., in prep), however this study was performed with the single-celled cyanobacteria Microcystis. Studies that address effects of selective grazing on cyanobacteria often do not address different defense traits of cyanobacteria (i.e. single-cell vs. filamentous morphology) and feeding traits of zooplankton that may add nuances to such interaction. As far as the authors are concerned, there is only.

(10) one study so far that attempts to use traits on both trophic levels to analyze plankton interactions (Colina et al., 2015). Cyanobacteria morphological traits (i.e. colony and filament formation) can act as grazing deterrent (Fulton and Paerl, 1988; Gliwicz, 1990). While colonial cyanobacteria hardly form aggregation in laboratory conditions, filamentous cyanobacteria can express similar morphology in both nature and laboratory. That can be a proxy to test filamentous morphology as grazing defense. However, filamentous cyanobacteria have contrasting effects on zooplankton grazing. Filaments usually clog large Daphnia apparatus, preventing from feeding on filaments and other available algae, and smaller species are advantaged (Gliwicz, 1990). Nonetheless, this effect seems to arise only in high filamentous abundance (Panosso and Lürling, 2010), or when filaments are long (>50µm) (Bednarska et al., 2014), and negative effects may even be species specific, depending on toxicity of cyanobacteria strain and species of zooplankton (Costa et al., 2013). A meta-analysis showed that despite overall idea of negative effects of filaments, they are less deleterious to cladocerans growth rate than unicellular cyanobacteria (Wilson et al., 2006). On the other hand, filamentous morphology may not be so effective as a defense trait against copepods grazing (DeMott and Moxter, 1991; Fulton, 1988), as they can handle and ingest it (Vanderploeg et al., 1990; Panosso et al., 2003). Copepods were also observed modifying cyanobacteria population structure, decreasing filament length (Burns and Xu, 1990; Bouvy et al., 2001; Kâ et al., 2012), despite no observed impact on filamentous cyanobacteria abundance (Chan et al., 2004). Despite that, toxicity may be decisive to grazing avoidance (Kurmayer and Juettner, 1999; Engström-Ost et al., 2000; Kirk and Gilbert, 1992; DeMott and Moxter, 1991), being even more important than morphology itself (Rangel et al., 2016b), thus facilitation is expected if the filamentous cyanobacteria species is toxic. The majority of the results of zooplankton grazing on cyanobacteria are evaluated in short-term (i.e. few hours) experimental studies (DeMott and Moxter, 1991; Hong et al., 2015; Rangel et al., 2016b; Ger et al., 2011), which are seldom extended to observe prey dynamics. Increases in duration of grazing experiments permits studying dynamics of prey in the system, and how herbivores react to changes in prey relative abundance. Grazing on low quality food can be dependent on relative abundance of alternative phytoplankton (DeMott, 1989), thus herbivores can swap to cyanobacteria if alternative food is scarce (i.e. prey-switching). However, a six-day experiment showed no prey-switching, and selective grazers were consistently avoiding cyanobacteria (Leitão et al., 2018). This study was done with cyanobacteria Microcystis, which did not show morphological defenses. Filamentous cyanobacteria can be affected in other fashion in longer grazing periods. For example, filament shortening can be accentuated in more than a few hours of exposure to grazers (Burns and Xu, 1990), and that may account for overall grazing. Yet, to observe the zooplankton-phytoplankton dynamics in longer period, we have to account for potential interactions (i.e. allelopathy) between phytoplankton growing in batch cultures, as they may chemically interact inhibiting or stimulating growth on each other (Gráneli et al., 2008; Bar-Yosef et al., 2010; Mello et al., 2012; B-Béres et al., 2012), which can be a potential background effect in longer term grazing experiments..

(11) The aim of our study was to assess how different feeding traits of selective grazers affects outcome of two competing algae with different quality status. We used two species of copepods, Notodiaptomus iheringi (calanoid) and Thermocyclops decipiens (cyclopoid), that were used here as proxy for active filter feeding and raptorial feeding modes, respectively. These copepods also differ in body size, being the former bigger than the later. We tested grazing of those two copepods on filamentous cyanobacteria dominance over motile eukaryotic phytoplankton, Cryptomonas obovata. To rule out potential competitive effects between phytoplankton during experiment, competition was assessed by co-culturing Cylindrospermopsis with Cryptomonas in batch cultures in different initial proportions and single cultures. We then tested the effect of the two grazers, separately, and observed the dynamics of the two phytoplankton in the presence or absence of Notodiaptomus and Thermocyclops for seven days. We hypothesized that selective feeding behavior of copepods would facilitate Cylindrospermopsis dominance, decreasing growth of Cryptomonas, but not affecting growth of cyanobacteria. We also hypothesize that, despite rejection, copepods may decrease size structure of filaments (Chan et al., 2004). We also hypothesized that, because of different feeding modes and body size, Thermocyclops and Notodiaptomus grazing will impact differently the dynamics of the two phytoplankton. Material and Methods Phytoplankton cultures The initial culture of Cryptomonas obovata (CCMA- UFSCar 148, WDCM835) was conceded by Federal University of São Carlos (Brazil). Cylindrospermopsis raciborskii strain (ITEP-A3) was isolated from Riacho do Pau (Federal University of Pernambuco, Recife, Brazil) (Gugger et al. 2005). ITEP-A3 genotype has been shown to produce saxitoxin (STX) and neo-saxitoxin (neoSTX) (Soto-Liebe et al. 2013). Phytoplankton were maintained at batch cultures in Wright's Cryptophyte medium (WC) at 23±1 °C under 50 µmol quanta m-2 s-1 12:12 hour light:dark cycle. Cultures were maintained in exponential growth phase, and only cells in those conditions were used in the experiment. Cultures were gently swirled once a day to avoid cell clumping and self-shading. Cryptomonas grew as motile spheroid single cells (mean 12.5 × 6.5 µm). Cylindrospermopsis in cultures typically grows in straight, long filaments, but some aggregated can be found forming clumps. Under culture conditions filaments (n = 126) were on average 549µm (±101µm confidence interval [CI] at 95%) length and 2.5µm diameter. Minimal length was 60µm while maximum was 3045 µm. Only 10% were shorter than 100µm and 75% of the population did not exceed 712.5 µm. Carbon content of cultures was estimated by counting cell and filament density (via hemocytometer) and multiplying by their biovolume, using the formulae pgC. cell-1 = 0.1204 × (µm3)1.051 (Rocha and Duncan, 1985). For carbon estimate of Cylindrospermopsis, 30 filaments length were measured to calculate mean biomass before each experiment to account for slight variations. Zooplankton cultures.

(12) Copepods were collected in surface and sub-surface waters of Armando Ribeiro Gonçalves reservoir (ARG). ARG reservoir is located in semi-arid region of northeastern Brazil (5°47'27'' S, 36°52'43'' W) in the Rio Grande do Norte state. Long-term duration blooms were recorded in ARG reservoir blooms includes various cyanobacteria dominance, including Cylindrospermopsis (Costa et al., 2006; Medeiros et al., 2015). Zooplankton net vault with opening of 68µm was used to concentrate zooplankton in 20L plastic bottles and immediately brought to laboratory. In the laboratory, we concentrated live mesozooplankton using a 200µm mesh, and transferred to a petri dish using distilled water. Healthy (i.e. parasite free and active) gravid females of Notodiaptomus iheringi and Thermocyclops decipiens were isolated in drops and rinsed with distilled water several times to assert cleaning from particles. Those zooplankton genera have been reported to commonly co-exist with cyanobacteria bloom (Eskinazi-Sant'Anna et al., 2013; Rangel et al., 2016b; Silva and Matsumura-Tundisi, 2005; vonRuckert and Giani, 2008). After cleaning, copepods were transferred to a 2L glass beaker with modified WC medium (lacking NO3 and PO4) to acclimate for experimental conditions. Individuals were fed with 0.5 µgC. L-1. day-1 of Cryptomonas. Cultures were maintained in 23±1 °C, 12:12h light:dark cycle, and aeration with bubbling (~5 bubbles sec–1). A new brood of copepods were grown in these conditions prior to experiments. When reached C5 copepodite or adult stage, copepods were used in experiments. Competition experiment Competition was assessed comparing growth rates of Cryptomonas and Cylindrospermopsis in single species (i.e. control) and co-cultures in different proportions (i.e. treatment) over a nine days period under identical maintenance conditions of phytoplankton cultures described above. Controls and treatments were prepared by diluting stock cultures in 500mL glass flasks containing 250mL of WC medium in a total phytoplankton concentration of 0.5 (±0.04 CI) mgC.L–1. Initial composition of controls and treatments are shown in Table 1. Treatment concentrations were chosen to cover a range of ratios likely to occur during grazing experiment (described below), from initial dominance of Cryptomonas (9Crypto:1Cyl) to slight dominance of Cylindrospermopsis (1Crypto:3Cyl), thus ascertaining that competition will not be a factor at any phytoplankton abundance conditions in grazing experiment. Flasks were shaken twice a day to avoid clumps and self-shading, and flasks were randomly re-ordered once a day to account for light. Phytoplankton samples of 10-20mL were collected in days 0, 3, 6 and 9. Experimental time and sampling intervals were chosen to allow time to observe interference between phytoplankton and possible intermediary interference effects. Samples were fixed with gluteraldehyde (1% final concentration) and stored in –4 °C fridge until counting (that did not exceed two weeks after sampling). Samples were filtered in 0.6µm white polycarbonate filters (Millipore IsoporeTM Membrane Filter). Counting was performed in epi-fluorescence microscopy at 100x (Olympus/EX41). Since whole pieces of filament were too variable in length, to assess carbon content of Cylindrospermopsis we counted only the pieces of filament in the field of vision. Pieces of filament that were inside the ocular grid view were measured (total grid area 70µm2). Only filament pieces that were over 18.75µm (ca. 20% of the grid width) were accounted in the sum. Once reached 100 pieces of filament count was stopped..

(13) All pieces of filament length were added to account for actual counted biomass in the field of view. We counted up to 100 cells (Cryptomonas) and 100 filament pieces (Cylindrospermopsis) in either transect or field method according to their density (Lund, 1958). To account for changes in Cylindrospermopsis filament length, we counted 30 whole filament lengths for each sampled day and each replicate. This way we could analyze morphological changes in filament length. Presence of specialized cells (i.e. akinete and heterocyst) was not accounted. Table 1: Composition of controls and treatments of competition experiment showing % of each phytoplankton in initial conditions. Ratios of co-culture (i.e. treatments) are shown in Name, n is the number of replicates and initial total concentration given in mgC.L-1.. %Cryptomonas %Cylindrospermopsis Initial total concentration Control 100 0 0.5 0 100 0.5 Treatment 90 10 0.5 75 25 0.5 50 50 0.5 25 75 0.5. n. Name. 3 3 3 3 3 3. Cry Cyl 9Cry:1Cyl 3Cry:1Cyl 1Cry:1Cyl 1Cry:3Cyl. Facilitation experiment Facilitation experiments were performed using a bi-trophic containing either Notodiaptomus or Thermocyclops and the two phytoplankton species. To assess the effect of different grazers in competing phytoplankton we measured biomass density, growth rates, and relative abundance of Cryptomonas to Cylindrospermopsis over the period of seven days. The no grazer controls (Control), Thermocyclops (Thermo) and Notodiaptomus (Noto) were prepared diluting the two phytoplankton from exponentially growing cultures in 1L glass flask containing 1L of freshly prepared sterilized WC medium, kept at 23(±1) °C, at 50-µmol quanta m-2 s-1 12:12h light:dark cycle. Flasks were placed randomly at an incubator shelf in relation to light source and their position was changed randomly daily to avoid differences in light conditions. Initial ratio had a dominance of Cryptomonas to Cylindrospermopsis of ~9Cry:1Cyl, in both controls and treatments (Thermo and Noto), with an initial total phytoplankton biomass with a mean of 0.44 (±0.02 CI) mgC.L-1. We have started with a dominance of Cryptomonas in order to assess facilitation, a shift in dominance to Cylindrospermopsis caused by selective grazers. Zooplankton was separated into droplets with distilled water and gently poured in treatment flasks. The same extra volume added into the treatments was added into control flasks (~10mL). We adjusted the total zooplankton biomass for each species using allometric equations (Dumont et al., 1975). We measured total body size (prosome + urosome) of 30 individuals of each zooplankton species and calculated the average body mass per individual (Table 2). We used commercial gasified water to sedate organisms and measured their length using a 10x magnification microscopy (Olympus/CX41). We used the individual body mass and calculated the amount organisms for each species that would be equivalent in total dry biomass. Notodiaptomus treatments received 25 individuals (adult or C5 copepodites) each replicate. While cyclopoid Thermocyclops.

(14) treatments received 75 individuals (adult or C5 copepodites) in each replicate. The number of individuals was equivalent in biomass according to Dumont et al. (1975) (Table 2). These abundances were decided based on pilot experiments that showed that a higher number of individuals depleted Cylindrospermopsis by day four. At the end of experiments, we measured all experimental individuals length and zooplankton adults total weight per replicate. Zooplankton total weight per replicate was measured for calculations of grazing measurements per total zooplankton biomass. We used gasified water to lower zooplankton movement, being able to measure their size without killing them. After measuring, we transferred them to a glass fiber filter (0.7µm) (GFF, Whatman), that was previously rinsed, pre-dried in 60 °C for 24h, and weighted, and added the zooplankton of each replicate in those. After adding zooplankton we dried again in 60 °C for 24h and weighted in balance (Shimadzu) of 5 decimals precision. Table 2: Thermocyclops and Notodiaptomus body size measurement of individuals to define zooplankton density of treatment flasks. Body size (prosome+urosome) was measured µm, and weight ind-1 (µg) and total weight (µg) were calculated using Dumont (1975) equation for adult cyclopoid and for adult calanoid, Thermocyclops and Notodiaptomus rows, respectively. Weight Ind Total Zooplankton Body size n Dumont's equation ind-1 treatment-1 weight Thermocyclops 810±39 30 W = 4.9×10-8×L2.75 3.76 75 285 Notodiaptomus 1183±113 30 W = 7.9×10-7×L2.33 10.35 25 285. Experiments ran for 7 days, with samples of 10-20mL taken at days 0, 2, 4 and 7. We decided to run experiments for seven days as experimental controlled conditions can worsen (e.g. organismal bacterial infection, zooplankton nauplii production) decreasing experimental results accuracy as length increases. Phytoplankton samples were fixed with gluteraldehyde (1% final concentration) and further counted in epi-fluorescence microscopy, as described above. Flasks were gently shaken twice a day, manually rotating circularly around 100 times to avoid cell clumping and accumulation at the bottom and walls. Flasks were sealed with 0.2 µm Millex FG vent filter (Millipore, USA), and bubbled around 3-4 bubbles per second. Copepods were investigated every day, and when suspected dead (i.e. checking for motility and heart beating under dissecting scope) were replaced for new adults or C5 copepodites. The relative abundance of Cylindrospermopsis over Cryptomonas (Cyl:Crypto) in biomass was also estimated throughout the experiment to assess for facilitation process. We measured variations in treatments and compared to controls where the ratio is expected to be constant. We made this comparison every sampled day to account for variability in each time point compared to controls. To quantify grazing specific on each phytoplankton we calculated clearance rates (CR) and ingestion rates (IR) of each zooplankton species on each phytoplankton based on relative change from initial to final concentrations among controls and treatments according to Frost (1972): CR = ln( ( [Cf/C0]/[Tf/T0] )/t ) x (V/N). IR = CR x C C = (Tf - T0) / ln(Tf/T0).

(15) Where Cf and C0 are phytoplankton concentrations at the end and beginning of experiment in controls, respectively. Tf and T0 are phytoplankton concentration at end and beginning of experiment in treatments, respectively. t equals grazing period (in hours), V is incubation volume (in mL), and N is total number of grazers in flask. C is the average concentration of phytoplankton biomass in treatment with grazers. We calculated CR and IR for three different days (i.e. 0-2, 0-4 and 0-7) for each grazer species, in order to compare how grazing is affected by changes in prey concentration. The effect of grazing on carbon biomass was also assessed at final experimental day for each phytoplankton prey and each grazer, and further compared to controls. Data analysis Growth rates (GR = ln(Ct2/Ct1)/days) were calculated for both phytoplankton species, where Ct1 and Ct2 are initial and final biomass concentration, respectively. Growth rates were used as proxy to compare effects of competition and grazing experiment on each treatment at each experimental time of sample taken. In the competition experiment, to test effect of initial ratios on interference between phytoplankton, we compared growth rates of controls (single culture) and treatments (co-culture proportions) for each time period (i.e. days 0-3; 3-6; 6-9; and 0-9) using ANOVA one-way, and post-hoc Tukey's HSD (honest significant difference) test if differences were met. For grazing experiment differences in responses among control and treatments (i.e. phytoplankton growth rate, filament size, change in Cyl:Cry biomass and clearance rates) were evaluated by one-way ANOVA or Kruskal-Wallis test, when homoscedasticity was not met after log transformation, and subsequent post-hoc tests Tukey's HSD or Dunn's test, respectively. Growth rates were calculated in different stages (i.e. days 0-2, 2-4, 4-7) to account for subtle changes throughout the experiment length. Differences between control and treatment are addressed to grazing, and growth rates can be proxy of predation in phytoplankton population increase, reduction or maintaining standing stock. We also performed generalized linear models (GLM) to assess slopes at the relative abundances of Cryptomonas and Cylindrospermopsis as continuous variables in control and treatments (Thermo and Noto). Clearance and ingestion rates were compared among three experimental periods (i.e. 0-2, 0-4 and 0-7 days) for each zooplankton to check for potential changes feeding rates as abundances change using ANOVA one-way. We also tested differences between feeding rates on each phytoplankton using Welch's t-test. For all data set quantile-quantile and residual plots were visually inspected, Shapiro-Wilk normality test and Bartlett's test for homogeneity of variances were performed to assess for linearity, normality and homoscedasticity of data. Non-normally distributed data were log transformed. All statistical tests were performed using R software (R Development Team, 2016).. Results Competition experiment No competitive interference was observed in any of the treatments, and both competitors grew equally exponentially at all co-cultures proportions and controls (Fig. 1). There was no statistical difference among single and co-cultures (ANOVA, F4,10 = 0.50, p = 0.73, Fig. 2), and Cryptomonas had a mean growth rate from, day 0 to 9, of 0.43 d-1 (± 0.02, n = 3). Growth rates of Cryptomonas decreased slightly initial experimental period (0.42 ± 0.04 d-1) compared to final period (0.37 ± 0.03 d-1,.

(16) n = 3) likely because stationary phase was beginning to start (Table 3). Cylindrospermopsis showed a slightly lower growth rate when calculated from day 0 to 9, with a mean of 0.35 d-1 (± 0.02 CI, n = 3), but with no difference between control and treatments (ANOVA, F4,10 = 0.14, p = 0.96, Fig. 2). However, at initial experimental period (i.e. days 0-3) Cylindrospermopsis had much higher growth rates (0.51±0.03 d-1, n = 3) than at final experimental days (0.24±0.05 d-1 from days 6-9, n = 3), likely because it reached stationary phase earlier in the experimental time (Table 3). There were no significant differences among control and treatments in none of the experimental periods either overall experimental time (Table 3).. (A) Single 9Crypto:1Cyl 3Crypto:1Cyl 1Crypto:1Cyl 1Crypto:3Cyl. 10.0. Biomass µgC.mL−1. (B) 10.0. 1.0 1.0. 0.1 0.1 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Days Figure 1: Temporal trends of Cryptomonas (A) and Cylindrospermopsis (B) of biomass (µgC.ml-1) for competition experiment (log scale). Single culture controls and co-cultures are showed in proportions. Symbols and bars represent means and confidence interval at 95%, respectively.. 0.6. Cryptomonas Cylindrospermopsis. Growth rate. 0.5 0.4 0.3 0.2 0.1 0 1Crypto:1Cyl 1Crypto:3Cyl 3Crypto:1Cyl 9Crypto:1Cyl. Single.

(17) Figure 2: Growth rate (day-1) of Cryptomonas (black bars) and Cylidrospermopsis (grey bars) in competition experiment. Lines indicate confidence interval 95%.. Table 3: Numerical report of competition experiment. Growth rates (day-1) and confidence intervals (n=3) of Cryptomonas and Cylindrospermosis for each experimental time period (i.e. 0-3, 3-6, 6-9 and 0-9) are shown, and ANOVA one-way results comparing control (single-cultures) and treatments (coculture in different ratios, see Table 1 for details).. Period Control Cryptomonas 0-3 0.51±0.07 3-6 0.39±0.05 6-9 0.34±0.03 0-9 0.41±0.01 Cylindrospermosis 0-3 0.57±0.06 3-6 0.28±0.02 6-9 0.28±0.02 0-9 0.35±0.04. 9Cry:1Cyl. 3Cry:1Cyl. 1Cry:1Cyl. 1Cry:3Cyl. df. F. p. 0.37±0.16 0.57±0.28 0.33±0.04 0.42±0.05. 0.42±0.05 0.46±0.08 0.42±0.06 0.44±0.05. 0.37±0.11 0.53±0.18 0.38±0.08 0.43±0.04. 0.43±0.07 0.55±0.02 0.37±0.10 0.45±0.03. 4,10 4,10 4,10 4,10. 1.20 1.02 1.02 0.50. 0.36 0.43 0.44 0.73. 0.51±0.05 0.32±0.25 0.25±0.08 0.36±0.08. 0.47±0.07 0.34±0.05 0.26±0.09 0.36±0.03. 0.50±0.06 0.37±0.08 0.25±0.12 0.36±0.06. 0.49±0.04 0.31±0.16 0.20±0.19 0.33±0.06. 4,10 4,10 4,10 4,10. 1.32 0.23 0.16 0.15. 0.32 0.91 0.95 0.96. Grazing experiment While controls grew exponentially in our lab cultures, the presence of grazers affected the dynamics of the two phytoplankton in different ways for the two grazers (Fig 3). Cryptomonas was readily suppressed by Thermocyclops and, more expressively, by Notodiaptomus at the first two days of experiment, resulting in negative growth rates compared to control (ANOVA, F2,10 = 13.11, p = 0.001; Fig 4A, Table 4). Cylindrospermopsis biomass, however, was not affected by Thermocyclops, and Notodiaptomus resulted in a small, but non-significant, decrease at Cylindrospermopsis growth rate, from 0.29 d-1 (±0.15 CI, n = 5) in Notodiaptomus compared to 0.44 d-1 (±0.10 CI, n = 4) in controls (Fig.4A). From day two to four Thermocyclops lowered grazing pressure on Cryptomonas, resulting in phytoplankton positive growth rates (0.36 d-1 ± 0.32). Meanwhile, Notodiaptomus decreased but continued suppression on Cryptomonas abundance resulting in equilibrium on standing stock of Cryptomonas, but large variations (mean growth rate of -0.16 d-1 ± 0.39 CI, n = 5) resulted in no statistical difference (ANOVA, F2,10 = 3.40, p = 0.079; Fig. 4B). During this experimental period (day 2-4), Thermocyclops slightly decreased Cylindrospermopsis growth rate (0.33 d-1 ± 0.18 CI, n = 4) compared to control (0.42 -1 ± 0.03 CI, n = 4). Notodiaptomus, on the other hand, decreased significantly the growth rate of Cylindrospermopsis (-0.002 d-1 ± 0.13 CI, n = 5; ANOVA, F2,10 = 9.18, p = 0.007; Table 4; Fig. 4B). The more drastic shift was from day four to seven. Controls kept growing exponentially: Cryptomonas at 0.51 d-1 (±0.10 CI, n = 4) and Cylindrospermopsis at 0.34 d-1 (±0.07, n = 4). While Thermocyclops kept moderately grazing on Cryptomonas, resulting in a positive, yet smaller than control, growth rates (0.30 d-1± 0.12). Notodiaptomus treatment showed an increase in Cryptomonas biomass, resulting in a growth rate from day four to seven significantly higher (0.80 d-1 ± 0.25.

(18) CI, n = 5; ANOVA, F2,10 = 9.48, p = 0.005; Fig. 4C) compared to control. Surprisingly, predation on Cylindrospermopsis occurred by the two copepods. Thermocyclops decreased Cylindrospermopsis growth rate to near zero (0.05 d-1 ± 0.17 CI, n = 4), but not significantly compared to control (Tukey HSD, p = 0.174). While Notodiaptomus grazing pressure resulted in a significant negative growth rate of Cylindrospermopsis, significantly different compared to controls (–0.32 d-1 ± 0.26 CI, n = 5; Tukey HSD, p = 0.013; Table 4; Fig. 4C).. Biomass µgC.mL−1. 10.0. A. 10.0. B. 1.0 1.0 0.1. 0.1. 0. 2. 4. 6. 0. Days. 2. 4. 6. Figure 3: Temporal trends of Cryptomonas (A) and Cylindrospermopsis (B) (µgC.ml-1) for grazing experiment. Circles in black, blue and green represent no-grazers control, Thermocyclops and Notodiaptomus treatments, respectively. Controls are the same for both graphs. Biomass axis is shown in log scale. Lines are smoothed polynomial ('loess') models. Grey bands are confidence intervals at 95%..

(19) Figure 4: Growth rates of Cryptomonas and Cylindrospermopsis at experimental periods of days 0 to 2 (A); 2 to 4 (B); and 4 to 7 (C). Bars in black, blue and green represent no-grazers control, Thermocyclops and Notodiaptomus treatments, respectively. Lines represent confidence interval. Different letters mean statistical difference (Tukey's HSD test results at Table 4).. Table 4: The summary of one-way analysis of variance for growth rate in periods 0-2, 2-4 and 4-7 days to Cryptomonas and Cylindrospermopsis in controls, Thermocyclops (Thermo) and Notodiaptomus (Noto) treatments. Growth rates (GrowthR, day-1) means ± confidence intervals are displayed. Tukey HSD Period. Treatment. Cryptomonas 0-2 Control Thermo Noto 2-4 Control Thermo Noto 4-7 Control Thermo Noto Cylindrospermopsis. GrowthR. n. df. F. p. 0.21±0.14 -0.19±0.12 -0.47±0.23 0.41±0.22 0.36±0.32 -0.16±0.39 0.51±0.10 0.30±0.12 0.77±0.19. 4 4 5 4 4 5 4 4 5. 2,10. 13.108. 0.001*. 2,10. 3.61. 0.066. 2,10. 9.48. 0.005*. Control. Thermo. 0.040* <0.001* ns. 0.130. 0.187 0.098. 0.003*.

(20) 0-2. 2-4. 4-7. Control Thermo Noto Control Thermo Noto Control Thermo Noto. 0.44±0.10 0.40±0.11 0.29±0.15 0.42±0.03 0.33±0.18 -0.002±0.13 0.34±0.07 0.05±0.17 -0.32±0.26. 4 4 5 4 4 5 4 4 5. 2,10. 1.57. 0.25. 2,10. 11.43. 0.002*. 2,10. 9.48. ns. 0.622 0.010*. 0.085. 0.174 0.002*. 0.075. 0.005*. Cylindrospermopsis to Cryptomonas ratio (Cyl:Cry) were constant throughout controls and both algae grew similarly (Fig. 5). Grazing by Thermocyclops increased Cyl:Crypto ratio from initial to day two and kept constant further experimental time (Fig. 5A), but maintaining low total biomass (Fig. 6), indicating that both phytoplankton were being similarly eaten after day two. Yet there was an increase of the contribution of Cylindrospermopsis to total biomass from 8% at initial condition to 21% (±6%) at day 4, but at the end of the experiment contribution of Cylindrospermopsis decreased again to 11% (± 4%), almost reaching initial conditions (Fig. 6). Cyl:Cry ratio increase at Notodiaptomus treatment at days 2 and 4 indicating a shift of dominance towards Cylindrospermopsis (Fig. 5B). But the last day there was a major decrease of Cyl:Cry ratio (reaching lower values than Control), and Cryptomonas again dominated the phytoplankton (Fig. 5). Indeed, the contribution of Cylindrospermopsis grew from 8% (± 6%) to 36%(± 27%) at day 4, but then decreased at last day of experiment to 11% (± 21%) and overall biomass also increase (Fig. 6).. Figure 5: Temporal trends of Cylindrospermopsis to Cryptomonas ratio (Cyl:Crypto) for grazing experiment with Thermocyclops (A) and Notodiaptomus (B). Circles and lines in black, blue and green represent no-grazers control, Thermocyclops and Notodiaptomus treatments, respectively. Controls are the same for both graphs. Ratio axis is shown in log scale for Notodiaptomus (B). Lines and grey bands are determined by smoothing polynomial ('loess') model and confidence interval at 95%, respectively..

(21) Figure 6: Mean percentage of contribution of each phytoplankton, Cryptomonas (grey bars) and Cylindrospermopsis (white bars), at each experimental time, for Controls, Thermocyclops and Notodiaptomus. Black dots and lines indicate the total biomass of both phytoplankton in µgC.ml-1 (right 'y' axis) at each experimental treatment and control. Black vertical lines indicate confidence interval at 95% of total phytoplankton concentration.. Observing the relationship of Cryptomonas and Cylindrospermopsis abundance, GLM results provided that control, as expected, showed a positive relationship between Cryptomonas and Cylindrospermopsis abundance (GLM, slope = 0.698, p < 0.001; Table 5). In treatments with Thermocyclops it was observed a positive relationship between algae abundance (GLM, slope = 0.978, p = 0.005; Table 5). On the other hand, Notodiaptomus grazers resulted in a negative relationship between Cryptomonas and Cylindrospermopsis (GLM, slope = –5.55, p < 0.001; Table 5). Table 5: The summary results of generalized linear models (GLM) for relationship between Cryptomonas and Cylindrospermopsis abundance (µgC.L-1) for all experimental points at control, Thermocyclops and Notodiaptomus treatments. Control Intercept Slope Thermocyclops Intercept Slope Notodiaptomus Intercept Slope. Estimate. CI. df. F. r2. p. 2.564 0.978. 0.559 0.218. 14. 77.1. 0.83. <0.001. 1.026 0.698. 1.118 0.445. 14. 11.32. 0.41. 0.005. -5.515 -1.316. 1.477 0.441. 14. 39.38. 0.67. <0.001. Filament size decreased in both treatments after two days of experiment, and maintained consistently smaller sizes compared to controls (Table 3). Filament sizes in Thermocyclops and Notodiaptomus decreased 59% and 55% (177 µm±27.63 and 196 µm±31.12, mean ±CI, respectively) at day two compared to controls (429 µm±70.60 CI) (Table 5, Fig 7). This shortening pressure decreased, but was continuously throughout the experimental time, and at day seven filaments have.

(22) Filament size (ESD). decreased 67% and 71% in Thermocyclops and Notodiaptomus, respectively. Both grazer treatments showed similar decreased on filament size without difference between them. We also calculated the equivalent spherical diameter (ESD) of filaments for all treatments in time (Table 5), and observed that ESD of Cylindrospermopsis filaments in controls were similar to those of Cryptomonas (15.32 µm; reference line in Fig. 7).. 20.00. 15.32. 10.00. 0. 2. Days. 4. 7. Figure 7: Cylindrospermopsis filament size in equivalent spherical diameter (ESD) in µm for controls and treatments (Thermocyclops in blue and Notodiaptomus in green) at experimental times. Boxplots show median and quartiles. Dashed reference line indicates ESD of Cryptomonas.. Table 5: Summary of one-way analysis of variance results for filament size at each day in controls, Thermocyclops (Thermo) and Notodiaptomus (Noto). Mean values of filament size (µm) ± confidence intervals (CI) are provided, as its equivalent spherical diameter (ESD, µm).. Tukey HSD Day 0. 2. 4. Treatment. Fil. size±CI. ESD. n. df. F. p. Control. Control Thermo Noto Control Thermo Noto Control Thermo Noto. 305±56.13 347±76.50 404±72.07 429±70.60 177±27.63 196±31.12 427±71.21 159±29.65 126±19.34. 13.12±0.66 13.24±0.81 14.01±0.76 14.82±0.71 11.08±0.52 11.35±0.51 14.65±0.77 10.51±0.55 9.74±0.46. 120 120 150 120 120 150 120 120 150. 2. 1.42. 0.24. ns. 2. 46.12. <0.001*. 2. 73.41. Thermo. <0.001* <0.001*. 0.789. <0.001* <0.001*. 0.065. <0.001*.

(23) 7. Control Thermo Noto. 356±51.78 119±20.21 104±15.02. 13.97±0.67 9.75±0.43 9.37±0.36. 120 120 150. 2. 97.20. <0.001* <0.001* <0.001*. 0.385. Mean clearance rates were higher on Cryptomonas (CRCry) than on Cylindrospermopsis (CRCyl) for both Thermocyclops and Notodiaptomus. Thermocyclops clearance rate average on Cryptomonas was 0.16 copepod.mL-1.h-1 (±0.04 CI, n = 12), and was similar across periods (i.e. 0-2, 0-4 and 0-7) (CRCry: F2,9 = 3.61, p = 0.07; Fig 8A). Notodiaptomus had higher average CRCry of 0.97 copepod.mL-1.h-1 (±0.29 CI, n = 15), and was not similar across periods (CRCry: F2,12=5.09, p = 0.02), showing a decrease in final experimental time (Fig. 8B; Table 6). Notodiaptomus significantly the CRCyl from 0-2 to 0-7 in 3-fold and this increase was significant (Table 6). Thermocyclops showed a non-significant 3.5-fold increase in CRCyl (Fig 8A; Table 6). Yet CRCyl values at period 0-7 of CRCyl were 88% smaller than Notodiaptomus CRCyl. Thermocyclops showed significant higher CRCry than CRCyl only at period 0-2 (Fig. 8A). While Notodiaptomus had significantly higher CRCry than CRCyl at periods 0-2 and 0-4 (Fig. 8B). When we compared between grazers (i.e. Notodiaptomus versus Thermocyclops), we observe differences in CRCry at periods 0-2 and 0-4 (t = 4.37; df = 4.19; p = 0.011; t = 4.80; df = 4.15; p = 0.008) and CRCyl at periods 0-4 and 0-7 (t = 5.51, df = 4.13, p = 0.005; t = 5.14, df = 4.21, p = 0.006). Ingestion rates on Cryptomonas (IRCry) were always higher than on Cylindrospermopsis (IRCyl) for both Thermocyclops (Fig. 8C) and Notodiaptomus (Fig. 8D). There were no overall changes in IRCry, only a slight decrease in period 0-4 for Thermocyclops treatment (Fig. 8C; Table 6). IRCyl increased in experimental length for Thermocyclops, but was still 3-fold lower than IRCyl by Notodiaptomus (Fig. 8D, Table 6).. Clearance rate. 0.4 0.3. A. 2.0. *. 1.5. 0.2. *. *. *. 0-2. 0-4. 0.5. 0.0. Ingestion rate. *. 1.0. 0.1. 0.1. B. 0.0. *. *. *. 0.6 0.4. D *. 0.2. 0.0. 0.0 0-2. 0-4. 0-7. 0-7. Fig 8: Clearance (mL.copepod-1.h-1) and Ingestion (µgC.copepod-1.h-1) rates of Thermocyclops (A,C) and Notodiaptomus (B,D) on Cryptomonas (dark grey) and Cylindrospermopsis (light grey). Symbols '*' indicate statistical different of feeding rates between phytoplankton at each period. Table 6: Clearance rate (copepod.mL-1.h-1) values for each zooplankton on each phytoplankton in different experimental lengths (i.e. days 0-2, 0-4 and 0-7). Values show mean ± confidence interval at.

(24) 95%. ANOVA-one way was performed and when p <0.05, post-hoc Tukey HSD test was performed. Different letters mean significantly different groups.. Cryptomonas Notodiaptomus Thermocyclops Cylindrospermopsis Notodiaptomus Thermocyclops. 0-2. Clearance rate 0-4. 0-7. df. ANOVA F p. 1.21±0.43a 0.23±0.07a. 1.27±0.46a 0.13±0.06a. 0.43±0.31b 0.13±0.06a. 2,12 2,9. 5.09 3.60. 0.025* 0.071. 0.28±0.29a 0.03±0.06a. 0.50±0.16ab 0.04±0.02a. 0.77±0.25b 0.09±0.04a. 2,12 2,9. 3.91 2.62. 0.049* 0.126. Ingestion rate Cryptomonas Notodiaptomus Thermocyclops Cylindrospermopsis Notodiaptomus Thermocyclops. 0.289±0.07a 0.073±0.008ab. 0.288±0.11a 0.055±0.019a. 0.219±0.08a 0.089±0.017b. 2,12 2,9. 0.79 4.87. 0.47 0.037*. 0.011±0.010a 0.001±0.003a. 0.021±0.005a 0.003±0.001ab. 0.021±0.005a 0.007±0.003b. 2,12 2,9. 2.26 6.52. 0.146 0.017*. Grazers showed a difference in size, Thermocyclops having an average size of 882.98 µm and Notodiaptomus with an average size of 1159.74 µm (Table 7). Thermocyclops treatments showed a higher adult zooplankton mass in experiments being on average 52% higher than Notodiaptomus treatments. Table 7: Zooplankton body size (prosome+urosome) at the end of experiment for each replicate of Thermocyclops and Notodiaptomus. n is the total of adults measured and used to measure total dry weight. Treatment/ n Size (µm)± CI Size range (µm) Dry weight (µg) Replicate Thermocyclops a 73 881.78±12.19 1000–680 360 b 75 905.20±8.27 1000–840 300 c 75 883.20±8.78 990–800 260 d 75 861.73±8.54 960–780 280 Notodiaptomus a 22 1173.63±32.71 1320–1000 220 b 25 1191.6±33.27 1350–1000 160 c 24 1207.5±38.36 1350–1010 120 d 25 1150.0±35.01 1300–1000 70 e 25 1076.0±26.90 1220–950 150. Discussion Our aim in this study was to investigate the interaction between a common bloom forming filamentous cyanobacteria and selective grazers (i.e. copepods) with different functional traits. We showed that, despite selectivity, zooplankton copepod traits, such as feeding modes, also play a role to understand trophic interactions. While calanoids (Notodiaptomus iheringi) active filter feeding initially rejected cyanobacteria (Cylindrospermopsis), organisms shifted to graze on cyanobacteria from middle to end of experimental length. Meanwhile, smaller raptorial cyclopoids, preferentially fed on motile quality phytoplankton (Cryptomonas) more consistently throughout experimental length, facilitating the dominance of cyanobacteria, slightly.

(25) affecting cyanobacteria biomass only at end of experiment. Although the cyanobacterium species Cylindrospermopsis raciborskii has morphological defenses, i.e. filamentous form, this feature did not interfere in overall handling and consumption by copepods, as length of filaments was readily shortened. Our results showed that copepods facilitated filamentous cyanobacteria only at short experimental length, at greater periods cyanobacteria was manipulated and cleared at equal or higher rates than alternative good quality algae, by cyclopoid and calanoid copepods, respectively. These results highlight the importance of experimental length and evaluation of different zooplankton feeding traits to understand the importance of selective grazers in cyanobacteria harmful algal bloom dynamics. Selective grazing by copepods, especially calanoids, was observed on filamentous cyanobacteria before (Fulton 1988; Burns and Xu, 1990; Panosso et al., 2003). Experiments testing calanoid grazing on filamentous cyanobacteria in short term experiments resulted in similar feeding rates than our initial experimental period, and clearance values here found were within the range of those found in Rangel et al. (2016a) and Hong et al. (2013) (2-3h). However, our results showed increase of clearance on cyanobacteria as incubation period increases. Consumption of cyanobacteria can be attributed to decrease in alternative food, as food becomes scarce, there is less discrimination against low quality food (Burns and Xu, 1990; DeMott, 1989; DeMott and Moxter, 1991), contributing to gradual increase of grazing on cyanobacteria once good food is dampened. As copepods decreased concentration of good food by predominantly grazing on it at beginning of experiment, contribution of cyanobacteria biomass in food suspension increases. Additionally, cyanobacteria filaments are large, and bellow a certain critical food concentration, volume cleared increases as food size particle increase (Frost et al., 1972). Thus, filaments larger onedimensional length became a predominant feature for grazer perception and capture (Vanderploeg et al., 1988). Traits likely determined grazing pressure, as active feeders and larger sized calanoids had higher cyanobacteria consumption. Differences between grazer traits can be attributed to passive rejection leading to smaller grazing rates of cyclopoids on filamentous cyanobacteria, as they have smaller body size and strict raptorial feeding, reducing grazing on large non-motile prey items (Hansen, 1997; Engstrom-Ost et al., 2000). Observed calanoids shifting to graze on cyanobacteria closely followed a reduction of grazing on alternative good food, indicating a prey-switching behavior. Clearance rates on good food and rejection of cyanobacteria in beginning of experiment increased the relative abundance of the later in intermediate experimental periods. This was followed by higher clearance of cyanobacteria and slight decrease of good food consumption in final days – characterizing prey switching. Prey density dependence grazing rates was already observed for some copepods (Kiørboe et al., 1996). In this study, however, shift towards less preferred prey was not expected as the strain used of Cylindrospermopsis was demonstrated to be toxic (Soto-Liebe et al. 2013) and cyanobacteria has lower nutritional value than alternative food (MartinCreuzburg et al., 2008). Also, Cylindrospermopsis, despite increase in concentration in experimental time, did not reach >50% of phytoplankton biomass, and that may indicated low ingestion rates, despite increase in clearance rate. Differences in clearance and ingestion rates may difficult interpretation of possible prey-switching behavior. While ingestion rates use the average concentration of prey biomass in treatment to calculate consumption, clearance rate is calculated using growth rate of.

(26) the phytoplankton (calculated by controls) minus grazing loss. If food concentrations are highly different, as it is in this study, the rates of ingestion will always peak to the highest biomass phytoplankton, because it is very sensitive to average prey biomass. Hence, for comparison of feeding on two prey that have a high discrepancy in biomass, clearance rates may be more representative for changes in grazing behavior by zooplankton. Additionally, it would be unlikely that zooplankton would base its diet and have higher ingestion rates on the prey that has a very small biomass, as they cannot supply nutritional demands in so little carbon. Therefore, we believe that changes in clearance rates are a better indication of prey switching behavior, as previously reported in literature (Kiørboe et al., 1996), and our results indicate that such behavior was observed for calanoids. The increase of cyanobacteria abundance compared to supplementary food did not hinder total feeding rates, however, slightly decreased the cyclopoids average clearance rates on good quality food. Decrease in feeding rates on quality food can be a sub-lethal consequence of cyanobacteria ingestion (Fulton and Paerl, 1987). Cyclopoids might also be consuming cyanobacteria as supplementary food, which would decrease feeding on good food. Cyclopoids slightly increased feeding on filaments only at final experimental period, when cyanobacteria abundance reached ~40% of phytoplankton biomass, reaching higher total clearance rates. Despite grazing on cyanobacteria, cyclopoids showed a more stable facilitation of cyanobacteria dominance. That appears to be a consequence of cyclopoids strict raptorial feeding mode, as the control food used was the motile flagellated Cryptomonas, that likely triggered hydromechanical signal for detection (Saiz et al., 2014), as physical cues seem to be more important for prey detection than chemical cues such as toxicity (Svensen and Kiørboe, 2000). Assessing interactions of cyclopoids and filamentous cyanobacteria changing control algae for a non-motile, such as Scenedesmus, may explain this relationship better. Few studies have been conducted on the feeding behavior of cyclopoids on filamentous cyanobacteria, but examples so far seem to result in a complete rejection of this prey (Kâ et al., 2012; Kurmayer and Juettner, 1999). Body size is an unlikely explanation for nonconsumption of filaments, as bigger sized (1.16 mm; Silva and Roche, 2017) Mesocyclops ogunnus showed no ingestion on filaments of Cylindrospermopsis (Kâ et al., 2012). However, cyclopoids here were able to handle, by cutting filaments, and graze on filamentous cyanobacteria. Initial filaments shortening, but low grazing, was consistent in both copepods and should be considered a similarity of their selective behavior. Both copepods species shortened filaments in similar fashion, and that initial breakage of Cylindrospermopsis filaments can be a result perception bias: copepods use mechanoreceptors to perceive prey and given larger length of filaments they are readily perceived and either pushed through currents or attacked. However once toxicity is sensed in mouthparts prey is rejected (Price and Paffenhoffer, 1985; Vanderploeg et al., 1988), explaining initial rejection despite filament breakage. Recent work has showed that filament length hinders grazing when strain is not toxic (Rangel et al., 2016a). However, that was based on a 2-3h experiment, not allowing time for copepods to handle and modify filament structure, to then consume it. Longer experiments (24h) showed similar 4-fold decrease on filament size by copepods grazing (Burns and Xu, 1990). Initial decrease in filament length followed by negative growth rates of Cylindrospermopsis in subsequent periods may suggest that.

(27) it is necessary for the filaments to be shortened first so that they can be grazed by copepods. Thus, short experiments may not convey all the information about copepod-filamentous cyanobacteria interaction outcome. We initially thought that long filaments of cyanobacteria used here were going deter grazers, as recent work showed that longer and toxic filaments are less consumed by copepods (Rangel et al., 2016a). Long filamentous morphology was shown to hinder some vital rates (i.e. feeding, growth and reproduction) of large generalist cladoceran (Daphnia) (Gliwicz 1990, Panosso and Lurling, 2010). However, smaller cladocerans and copepods seems to be less affected by filamentous forms (Fulton, 1988; Vanderploeg et al., 1988; Engstrom-Ost et al., 2000; Twombly et al., 1990; Kâ et al., 2012; Costa et al., 2013). Rather, some authors claim that elongated filaments may trigger more easily copepods' perceptual bias and be more readily attacked, increasing chances of being captured (Vanderploeg et al, 1988). Recent work showed high clearance rates of calanoid copepods on phytoplankton Morphological Based Functional Group III (Colina et al., 2015), in which filamentous forms, including Cylindrospermopsis, are included. Thus, filamentous morphology only does not seem to play a decisive role for copepods grazing (Fulton, 1988). However, toxicity is still expected to play a role on copepod food selection, as grazing on toxic filaments is usually smaller than on non-toxic (Engstrom-Ost et al., 2000; Rangel et al., 2016a). Nevertheless, presence of toxic filament may not hinder feeding on alternative quality food (Engstrom-Ost et al. 2000), as observed here for both grazers. Moreover, positive effects may be observed when exposed to Cylindrospermopsis, and negative effects of toxicity can be species specific (Costa et al., 2013). More studies on how calanoids perceive and tolerate toxicity of cyanobacteria in filamentous forms are needed to understand this coupling. Filament breakage can lead to impacts on food webs and ecosystem processes. By shortening filament size, copepods can make it fit within the optimal range of prey size for smaller zooplankton (Hansen, 1994) affecting indirectly their mortality rates by predation of other individuals, as smaller sized filaments can be ingested (Bednárska et al., 2014; Panosso and Lurling, 2010). This can be more relevant for tropical environments, where smaller sized zooplankton is more predominant (Fernando, 1994) and they are able to shorten filaments (Kâ et al., 2012). Another consequence of filament shortage is the decrease of photosynthetic rates due to reduction in the number of cells per filament (Chan et al., 2004). That can affect production of differentiated structures, like heterocystis, responsible for nitrogen fixing in diazotrophic cyanobacteria (Chan et al, 2004). Therefore, grazing can impact biogeochemical cycles of nitrogen by decreasing N-fixing capacity of diazotrophic filamentous cyanobacteria. Alternatively, filaments breakage may produce organic debris,, regenerating nutrients to the water column (i.e. sloppy feeding) (Saiz et al., 2014). Short filaments can also be grazed by microzooplankton, like ciliates, that have been observed to feed on and have preference to filamentous cyanobacteria (Sigee et al., 1999). Ciliates, in turn, can be consumed by copepods (Nishibe et al., 2010; Saiz et al., 2014), re-entering the trophic chain via microbial food web. Thus, there is an urge to study fate of cyanobacterial carbon and it is necessary to measure gains and losses of energy when cyanobacteria is directly consumed by zooplankton and indirectly incorporated via microbial food web..

(28) Allometric equations used in this study by Dumont et al. (1975) were suitable for cyclopoids, but were not for calanoids used here (N. iheringi), resulting in overestimation of calanoids weight, and smaller total biomass used in the experiment compared to cyclopoid treatments. The use of an equation for similar genus or region (tropical) would give a better inference for body size-weight relationship (Azevedo et al., 2012). Despite these results, the measured higher biomass in cyclopoid treatment did not result in higher grazing, however, indicating a higher grazing per weight by calanoids. As active feeders, calanoids may increase the overall grazing on particles, as they bring it all to their mouthparts, but in turn may increase metabolic demands and food consumption. Cyclopoids on the other hand are raptorial feeders, thus their particle encounter rate is low (Kiørboe, 2011), but also their metabolic demand is decreased given less activity (Almeda et al., 2011). Additionally, raptorial feeding mode can have a trade-off between predation risk and particles consumed (Kiørboe, 2011). Calanoid higher cyanobacteria ingestion may have resulted in low eggs counted and higher mortality than cyclopoids. Cyclopoids on the other hand had zero mortality, and individuals produced a larger amount of offspring at the end of the experiment (data now shown). Previous studies showed that calanoids are negatively affected by seasonal toxin increase, while raptorial cyclopoids are unaffected (Hansson et al., 2007). More studies are needed to observe possible positive effects of cyanobacteria in diet of cyclopoids to assess whether they may be able to use bioactives from cyanobacteria. Regulation of filamentous cyanobacteria growth rate by copepods can also be a result of acquired resistance due to local eco-evolutionary adaptation. Local adaptation to toxic prey may be an important explanatory variable as organisms used in this experiment were co-existing with long-term blooms of cyanobacteria. Eco evolutionary adaptations can occur in time (Hairston Jr. et al., 1999) and space (Lemaire et al., 2012), and it is an important matter to ecological interactions in freshwater (DeMeester and Pantel, 2014). Higher tolerance of zooplankton to toxic cyanobacteria has been observed when they were previously exposed (non-naïve) in nature and experiments (Hairston Jr et al., 2001; Sarnelle and Wilson, 2005; Jiang et al., 2016; Colin and Dam, 2013). Since organisms here were collected from a reservoir that commonly has cyano-bloom formation (Medeiros et al., 2015) it is likely that clearance rates on cyanobacteria found here are higher than those of naïve organisms. The plethora of studies on the interaction of zooplankton-cyanobacteria is on large cladocerans (Daphnia) and cyanobacteria Microcystis. However, large cladocerans are generalists not being able to discriminate particles quality. On the other hand, selective zooplankton rejects Microcystis in both colonial (Fulton and Paerl, 1988) and single or double-celled forms (DeMott and Moxter 1991; Fulton and Paerl, 1988; Kâ et al., 2012; Ger et al., 2016b). Toxicity is used as chemical cue and non-toxic strains are less discriminated for both Microcystis (Ger et al., 2016b) and filaments (Kurmayer and Juettner, 1999; Rangel et al., 2016a). Exposure to Microcystis shows more deleterious effects on fitness and life-history of cladocerans and on copepods, when compared to filamentous cyanobacteria (Wilson et al., 2006; Twombly, 1998). However, cyanobacteria-zooplankton interactions can be speciesspecific, and specific biocompounds that are not related to toxicity may affect zooplankton fitness differently depending on zooplankton species (Costa et al., 2013)..

(29) Our results focused on the effects of zooplankton upon cyanobacteria, but we also need to assess the ecotoxicological outcomes of zooplankton exposure to high concentrations of cyanobacteria. We started the experiment with low concentrations of cyanobacteria to observe shift in dominance due to hypothesized facilitation. However, copepods often co-exist with toxic cyanobacteria blooms in nature comprising up to 95% of phytoplankton biomass (Soares et al., 2013). Cyanobacteria may increase mortality, reduce somatic growth rates and reproduction on zooplankton (Gliwicz 1990; Bernadska et al., 2014; Ger et al. 2016b), but that was not in the scope of this experiment. Studies conducted with small tropical cladocerans demonstrated species-specific negative and positive effects of toxic and non-toxic Cylindrospermopsis on fitness, suggesting this cyanobacterium regulates zooplankton community composition (Costa et al., 2013). However, little is known about the effects on copepods (Ger et al., 2014). Testing nutritional value of Cylindrospermopsis for copepods, via ecophysiological rates such as respiration, growth, and reproduction, may allow us to know how copepods population dynamics are affected by cyanobacteria. If no negative effects are found then there is no compelling reason for copepods to avoid eating Cylindrospermopsis when they are readily available in food suspension, with potential implications for trophic cascade. Predictions of harmful algal bloom ecology and management efforts have to incorporate predator-prey interactions, especially for the main players in eutrophic systems (i.e. copepods and cyanobacteria). Those may not demonstrate straightforward results, as predatory activity or selective avoidance may respond differently to different communities of predators and prey. Here, we demonstrate that copepod selectively avoidance of filamentous cyanobacteria Cylindrospermopsis is not constant and varies with different zooplankton feeding traits. Filamentous cyanobacteria are facilitated in a higher degree by calanoids (filter feeders), than cyclopoids (raptorial feeders), but while the former has a higher variability in time, the later has a more smooth but constant facilitation on Cylindrospermopsis. Calanoids switched from good food algae to cyanobacteria as its relative abundance increased in food suspension. Cyclopoids, however, were more consistently grazing on good food, and may have higher potential to facilitate cyanobacteria dominance, despite final increase in consumption on cyanobacteria. Copepods were able to shorten filaments of prey that can potentially be subsequently eaten by other zooplankton in nature. Cyclopoid, rather than calanoid, grazing may be an appropriate mechanism to explain filamentous cyanobacteria dominance, as they avoided their consumption consistently. However, filamentous cyanobacteria may still constitute an important source of organic carbon, especially in copepod-dominated systems (Perga et al., 2012), and we should observe traits of cyanobacteria and their grazers before assuming that blooms are dead-ends (Porter, 1972). While environmental correlations due to long term monitoring are important and may reveal interesting trends, a mechanistic approach to understand trophic dynamics of different groups (i.e. copepods and filamentous cyanobacteria) is necessary, and we can use a functional trait approach to understand how zooplankton traits interact with cyanobacterial traits. This way we can adapt management efforts (i.e. biomanipulation) to eutrophic plankton community conditions to achieve better results. This is especially relevant in light of the more intense warmer and eutrophic world that will promote cyanobacteria bloom, and increase complexity of such interactions. References.