Isabelle Domaizon
email: isabelle.domaizon@thonon.inra.fr

Orlane Anneville
email: orlane.anneville@thonon.inra.fr

Franck Cattane ´o •
email: franck.cattaneo@hesge.ch

Jean Guillard
email: jean.guillard@thonon.inra.fr

B Sotton
email: bsotton@gmail.com

Á O Anneville

Á J Guillard

F Cattane

Nodularin and cylindrospermopsin: a review of their effects on fish Benoı ˆt Sotton •

Keywords: Cyanobacteria, Cyanotoxins, Cylindrospermopsin, Effects, Fish, Nodularins

Nodularin (NOD) and cylindrospermopsin (CYN) are hepatotoxic cyanotoxins that are present in numerous ecosystems where bloom episodes occur. In this review, the different effects of both of these cyanotoxins on the different ontogenic stages of various fish species were summarised to clarify the state-of-the-art scientific knowledge on this topic. It is clear that fish that are exposed to NOD and CYN were negatively impacted in every studied ontogenic stage. Indeed, these cyanotoxins can accumulate in various organs of fish, leading to deleterious effects on the physiology. This review highlights the fact that all of the previously published studies on the topic have focused only on the short-term effects of a given cyanotoxin on fish. However, during cyanobacterial blooms, fish can be exposed chronically to a variety of toxic compounds with which the fish interact, leading to stronger effects than those observed with a single toxin tested over a short timeframe. Thus, it is essential to conduct additional studies to better understand the actual toxic effects of cyanobacterial blooms on fish populations over medium-and long-term time scales.

Introduction

The anthropogenic eutrophication of aquatic ecosystems during the twentieth century promoted toxic cyanobacterial blooms worldwide, leading to modifications in the stability and in the functioning of affected ecosystems and to water use issues [START_REF] Codd | Cyanobacterial toxins: risk management for health protection[END_REF][START_REF] Ibelings | Cyanobacterial toxins: a qualitative meta-analysis of concentrations, dosage and effects in freshwater, estuarine and marine biota[END_REF]. Due to these negative impacts on ecosystems, the development of toxic cyanobacterial blooms has become a key environmental and sanitary concern in recent decades. In addition, ongoing climate changes are predicted to foster the occurrence and intensity of cyanobacterial blooms [START_REF] Paerl | Climate change: links to global expansion of harmful cyanobacteria[END_REF][START_REF] O'neil | The rise of harmful cyanobacteria blooms: the potential roles of eutrophication and climate change[END_REF], suggesting that the resulting environmental issues will likely increase.

Many cyanobacterial genera produce a wide variety of cyanotoxins that can cause severe impairments in aquatic and terrestrial organisms (Chorus and Bartram 1999;[START_REF] Wiegand | Ecotoxicological effects of selected cyanobacterial secondary metabolites a short review[END_REF][START_REF] Ibelings | Cyanobacterial toxins: a qualitative meta-analysis of concentrations, dosage and effects in freshwater, estuarine and marine biota[END_REF]. Cyanotoxins can be of diverse chemical natures and are usually classified into categories based on their biological targets: hepatotoxins, neurotoxins, cytotoxins, dermatotoxins, and others whose toxicological or ecotoxicological profiles are only partially known [START_REF] Codd | Cyanobacterial toxins: risk management for health protection[END_REF]. When blooms occur, hepatotoxins are the most frequently detected toxic molecules and are most often implicated in intoxication cases. Microcystin (MC) and nodularin (NOD), which are both cyclic peptides, as well as cylindrospermopsin (CYN), which is an alkaloid also known as a cytotoxin, are considered hepatotoxins. The presence of these hepatotoxins in the water has already led to human and livestock acute intoxications, ultimately until the death of the exposed individuals [START_REF] Azevedo | Human intoxication by microcystins during renal dialysis treatment in Caruaru-Brazil[END_REF][START_REF] Codd | Cyanobacterial toxins: risk management for health protection[END_REF].

In aquatic ecosystems, hepatotoxins may adversely affect all of the food web components. Phytoplankton, zooplankton, and fish are impacted by the presence of hepatotoxins [START_REF] Leflaive | Algal and cyanobacterial secondary metabolites in freshwaters: a comparison of allelopathic compounds and toxins[END_REF][START_REF] Ibelings | Cyanobacterial toxins: a qualitative meta-analysis of concentrations, dosage and effects in freshwater, estuarine and marine biota[END_REF]. Information about the bioaccumulation and the negative effects of these toxins on fish is abundant in the literature because fish play a key role in the ecosystem functioning and as human food. The consumption by humans of contaminated fish could therefore lead to significant health hazards. Fish are exposed directly to hepatotoxins by feeding on phytoplankton or contaminated organisms and/or passively via their epithelium (gills and skin) when hepatotoxins are dissolved in the water [START_REF] Malbrouck | Effects of microcystins on fish[END_REF]. The disturbances that are induced by hepatotoxins on the behavioural, physiological, and histological parameters of fish have been widely investigated in recent years, but the results are variable and sometimes contradictory [START_REF] Malbrouck | Effects of microcystins on fish[END_REF][START_REF] Ibelings | Cyanobacterial toxins: a qualitative meta-analysis of concentrations, dosage and effects in freshwater, estuarine and marine biota[END_REF][START_REF] Ferra ˜o-Filho | Cyanotoxins: bioaccumulation and effects on aquatic animals[END_REF]. The effects of MC on the physiological parameters of fish have been intensively investigated, and reviews on this topic are available in the literature [START_REF] Zurawell | Hepatotoxic cyanobacteria: a review of the biological importance of microcystins in freshwater environments[END_REF][START_REF] Malbrouck | Effects of microcystins on fish[END_REF][START_REF] Martins | Microcystin dynamics in aquatic organisms[END_REF][START_REF] Amado | Oxidative stress generation by microcystins in aquatic animals: why and how[END_REF][START_REF] Paskerova | Oxidative stress and detoxification biomarker responses in aquatic freshwater vertebrates exposed to microcystins and cyanobacterial biomass[END_REF]. Conversely, information about the effects of NOD and CYN on fish is still scarce and, to our knowledge, has never before been reviewed.

The aim of this review is to synthesise data regarding the interactions between NOD, CYN and fish, including the accumulation processes, and their effects on the physiological parameters of the different ontogenetic stages of fish. To assemble this review, all of the references were identified using the ISI Database from the Web of Science and the keywords ''nodularin'' or ''cylindrospermopsin'' in combination with ''fish''. The number of references that have been published on the topic during the searched timeframe is given in Fig. 1. For each cyanotoxin, the in situ accumulation and biological effects of the toxin are presented separately. For the biological effects, the results are detailed according to the ontogenetic stages that were investigated.

Cylindrospermopsin

General presentation

Cylindrospermopsin is a stable tricyclic alkaloid of 415 Da that possesses a tricyclic guanidine moiety combined with hydroxymethyluracil (Fig. 2). CYN was first detected in Cylindrospermopsis raciborskii from tropical water samples and later in other cyanobacterial genera originating from various countries in different climates [START_REF] Falconer | Cyanobacterial (Blue-Green Algal) toxins in water supplies: cylindrospermopsins[END_REF]. Currently, two structural variants have been identified: 7-epicylindrospermopsin (7-epi-CYN), which has a similar toxicity as that of the mother molecule, and 7-deoxycylindrospermopsin (7-deoxy-CYN), whose toxicity is still debated [START_REF] Looper | Synthesis of the putative structure of 7-deoxycylindrospermopsin: C7 oxygenation is not required for the inhibition of protein synthesis[END_REF][START_REF] Falconer | Cyanobacterial (Blue-Green Algal) toxins in water supplies: cylindrospermopsins[END_REF].

Compared to other cyanotoxins that are generally intracellular components of cyanobacterial cells, CYN is mainly produced as an extracellular toxin (approximately 90 % of the total CYN) and is detected at variable concentrations (from 0 to more than 800 lg l -1 total CYN) in samples from different ecosystems [START_REF] Van Apeldoorn | Toxins of cyanobacteria[END_REF][START_REF] Ru ¨cker | Concentrations of particulate and dissolved cylindrospermopsin in 21 Aphanizomenon-dominated temperate lakes[END_REF]Gutie ´rrez-Praena et al. 2013a). From these observations, Cylindrospermopsis seems to be the main genera producing CYN, but other cyanobacterial genera such as Aphanizomenon, Anabaena, Umezakia and Rhaphidiopsis have also been identified as producers of this cyanotoxin [START_REF] Falconer | Cyanobacterial (Blue-Green Algal) toxins in water supplies: cylindrospermopsins[END_REF].

CYN has been characterised as a hepatotoxin because the liver of exposed mice is the most affected organ [START_REF] Bernard | Toxicological comparison of diverse Cylindrospermopsis raciborskii strains: evidence of liver damage caused by a French C. raciborskii strain[END_REF]. Nevertheless, this toxin has also been classified as a cytotoxin due to negative effects on other organs, such as the kidneys, lungs, thymus, spleen, adrenal glands, intestine, and heart [START_REF] Falconer | Cyanobacterial (Blue-Green Algal) toxins in water supplies: cylindrospermopsins[END_REF]Guzma ´n-Guille ´n et al. 2013b). The toxic mechanism of CYN has not been completely elucidated, but the results that were obtained from primary hepatocytes suggest a bi-modal action that seems to be both time-and concentrationdependent (Gutie ´rrez- Praena et al. 2011b[START_REF] Gutie ´rrez-Praena | Time-dependent histopathological changes induced in tilapia (Oreochromis niloticus) after acute exposure to pure cylindrospermopsin by oral and intraperitoneal route[END_REF]. At low concentrations, the toxicity of CYN seems to be mediated via the rapid irreversible inhibition of protein synthesis, while at higher concentrations, a rapid toxic process implicating cytochrome P-450 (CYP450) might be the main mechanism [START_REF] Froscio | Cylindrospermopsin-induced protein synthesis inhibition and its dissociation from acute toxicity in mouse hepatocytes[END_REF][START_REF] Falconer | Cyanobacterial (Blue-Green Algal) toxins in water supplies: cylindrospermopsins[END_REF]. Other authors have reported that CYN causes a decrease in glutathione (GSH) synthesis [START_REF] Runnegar | The role of glutathione in the toxicity of a novel cyanobacterial alkaloid cylindrospermopsin in cultured rat hepatocytes[END_REF], inducing oxidative stress (Guzma ´n-Guille ´n et al. 2013a, b). However, despite the wide occurrence of CYN in aquatic systems, few studies are available concerning both the accumulation and toxicity processes in fish.

Accumulation of CYN in fish

The accumulation processes of CYN in aquatic invertebrate and vertebrate organisms have been investigated in the past decade [START_REF] Kinnear | Cylindrospermopsin: a decade of progress on bioaccumulation[END_REF]Gutie ´rrez-Praena et al. 2013a). To our knowledge, the first report of CYN accumulation in fish was published by [START_REF] Saker | The accumulation of cylindrospermopsin from the cyanobacterium Cylindrospermopsis raciborskii in tissues of the redclaw crayfish Cherax quadricarinatus[END_REF]. In this paper, five rainbow fish (Melanotaenia eachamensis) were sampled from a crayfish pond during a Cylindrospermopsis bloom, and a concentration of 1.2 lg g -1 dry weight (DW) of CYN was detected in the viscera of fish (Table 1). More recently, [START_REF] Messineo | Seasonal succession of Cylindrospermopsis raciborskii and Aphanizomenon ovalisporum blooms with cylindrospermopsin occurrence in the volcanic Lake Albano, Central Italy[END_REF] measured the CYN accumulation in the viscera (0.6-2.7 ng g -1 FW), muscle (0.1-0.8 ng g -1 FW), and ovaries (0.07 ng g -1 FW; Table 1) of two brown trout (Salmo trutta) specimens that were collected during an Aphanizomenon ovalisporum bloom in Lake Albano (Italy). [START_REF] Berry | Apparent bioaccumulation of cylindrospermopsin and paralytic shellfish toxins by finfish in Lake Catemaco (Veracruz, Mexico)[END_REF] collected various fish species in Lake Catemaco (Mexico) and found for the first time CYN in muscle ranging from 0.09 to 1.26 lg kg -1 FW (Table 1) and bioaccumulation factors ranging from 4 to 59 depending on the species. The highest levels of CYN accumulation were observed in herbivorous filter-feeding species (Dorosoma mexicana and Bramocharax cabelleroi) and herbivorous/omnivorous species (Vieja fenestratus and Heterandria tuxtlaensis), while the lowest levels were unexpectedly measured in the omnivorous introduced tilapia (Oreochromis aureus). These authors suggested that Cylindrospermopsis filaments could either be too small to be ingested or were specifically avoided by the omnivorous tilapia.

These results show that CYN and its variants can bioaccumulate in various fish species. However, the hydrophilic property of these molecules does not support the potential biomagnification process in the aquatic food web. Furthermore, the CYN concentrations in the different fish species were highly variable. Many factors, such as the concentration of cyanobacteria and of CYN present in the system, the absorption/ elimination rates and pathways, and fish ages and analytical processes, could influence the bioaccumulation rates and measurements, thus explaining the differences that are observed between studies. Due to its bioaccumulation, CYN may impact fish at a physiological scale, leading to significant damages to the individual fish and more widely at the population scale.

Biological effects of CYN on fish

In vitro effects of CYN on hepatic fish cells CYN toxicity on fish cells was investigated by in vitro studies to better understand the cellular interactions of CYN leading to disturbances of cell functioning. Interesting results have been observed in the hepatocytes of fish that were exposed to different CYN concentrations ( 2011). A decrease in the multixenobiotic resistance mechanism (MXR) was also observed for all of the CYN treatments [START_REF] Liebel | Cellular responses of Prochilodus lineatus hepatocytes after cylindrospermopsin exposure[END_REF]. These results show that the first-line defence mechanism, which is responsible for the efflux of xenobiotics, toxins, drugs and endobiotic metabolites, was negatively affected by pure CYN, even at low concentrations, suggesting a possible cellular accumulation of other substances at toxic levels. Similarly, a concentration-dependent increase in the reactive oxygen/nitrogen species 2011). A high concentration of RONS may lead to important damages to lipids (partially explaining the concomitant increase in LPO), proteins and even DNA. However, cells possess a robust protective mechanism to counteract the negative effect of CYN, thus maintaining cell viability. Additionally, glutathione S-transferase (GST) and glucose 6-phosphate dehydrogenase (G6PDH) do not seem to play a role in this protective mechanism because no differences in their activities were observed (Table 2; [START_REF] Liebel | Cellular responses of Prochilodus lineatus hepatocytes after cylindrospermopsin exposure[END_REF]. The absence of protein and DNA damage was corroborated by the unaltered glutathione ratio (2GSH/Glutathione disulphide (GSSG)), suggesting that pure CYN did not affect the synthesis or cycling of glutathione (GSH), an important non-enzymatic antioxidant, and cofactors of glutathione-dependent enzymes that are involved in xenobiotic biotransformation and peroxide degradation [START_REF] Liebel | Cellular responses of Prochilodus lineatus hepatocytes after cylindrospermopsin exposure[END_REF]. However, the effects of CYN on GSH content and synthesis are not clear. Indeed, a decrease in the contents of both GSH and c-glutamylcysteine synthetase (GSC), one of the enzymes that are involved in GSH production, was observed following exposure to pure CYN (Table 2; Gutie ´rrez-Praena et al. 2011b). Contrary to [START_REF] Liebel | Cellular responses of Prochilodus lineatus hepatocytes after cylindrospermopsin exposure[END_REF], these results

suggest that an alteration in GSH contents and synthesis can be induced by the presence of CYN, which could lead to an increase in the contribution of oxidative stress to other damages, such as genotoxicity.

Biological effects of CYN on fish embryos

In every investigated ontogenic stage, reports of the biological effects of CYN on fish species are scarce. CYN is particularly present in the extracellular state; therefore, fish eggs and embryos may naturally come into contact with this toxin and thus be impacted by it.

To our knowledge, only one publication has investigated the biological effects of CYN on young-stage fish [START_REF] Berry | Toxicity of cylindrospermopsin, and other apparent metabolites from Cylindrospermopsis raciborskii and Aphanizomenon ovalisporum, to the zebrafish (Danio rerio) embryo[END_REF]. In this study, zebrafish (Danio rerio) embryos were exposed (1) by immersion to pure CYN (50 lg ml -1 for 5 days), (2) to a cyanobacterial extract containing CYN (14.3, 71.5 and 143 lg lyophilised CYN-containing cyanobacteria ml -1 for 1 day), or (3) by microinjection with various concentrations of pure toxin (50, 5, 1, 0.5, 0.2 and 0.1 lg ml -1 for 1 day). No developmental or mortality effects were observed in embryos that were exposed to pure CYN by immersion. Because CYN is highly hydrophilic, the authors suggest that CYN cannot readily permeate cellular membranes in ; protein content at 24 h for doses equal to or greater than 2.5 lg ml -1 and for all the treatments at 48 h ; NR from 5 lg ml -1 CYN for both doses ; MST from 10 lg ml -1 CYN at 24 h and from 2.5 lg ml -1 at 48 h : ROS in a concentration-dependent manner at 24 h : GSH and GCS at 2 lg ml -1 but ; at 8 lg ml -1 at 24 h : = increase, ; = decrease, $ = no change, MRX multixenobiotic resistance mechanism, RONS reactive oxygen/nitrogen species, LPO lipid peroxidation, GST glutathione S-transferase, G6PDH glucose 6-phosphate dehydrogenase, GSH glutathione, GSH/GSSG reduced and oxidised glutathione ratio, NR neutral red uptake, MST tetrazolium salt reduction, ROS reactive oxygen species, GSC cglutamylcysteine synthetase Gavage and injection with 200 lg kg -1 of CYN and observation at 24 h and 5 days

: NADPH activity at 24 h for both exposure mode (liver and kidneys) and at 5 days for fish that were orally exposed (liver)

: LPO at 24 h for both exposure mode (liver and kidneys) and at 5 days for fish that were orally exposed (liver) and for both exposure mode (kidneys)

: in protein oxidation at 5 days for both exposure mode (liver) and duration for fish that were injected (kidneys)

$ DNA oxidation in every exposure mode and duration

; GSH and GSC contents at 24 h for both mode of exposure and $ at 5 days in every exposure mode [START_REF] Puerto | Acute effects of pure cylindrospermopsin on the activity and transcription of antioxidant enzymes in tilapia (Oreochromis niloticus) exposed by gavage[END_REF] Gavage with 200 and 400 lg kg -1 of CYN and observation at 24 h Dose-dependent : of LPO (liver and kidneys)

$ GPx activity (liver) and ; for fish that were exposed to the highest dose (kidneys)

: GPx gene expression for the lowest dose and ; for the highest (liver), : GPx gene expression for both doses (kidneys)

: GST for the lowest dose (liver) and for both doses (kidneys) Dose-dependent ; GST gene expression (liver) and : GST gene expression for both doses (kidneys)

: GST protein abundance for the lowest dose (liver) and for both doses (kidneys)

Puerto et al. ( 2012b
)
Gavage with 200 and 400 lg kg -1 of CYN and observation at 24 h Dose-dependent : in protein oxidation (liver and kidneys)

: NADPH activity for the lowest dose (liver) and for both dose (kidneys)

: and ; GSH content for the lowest and highest dose respectively (liver) and ; GSH content for both doses (kidneys)

Gutie ´rrez-Praena et al. (2013b)
Gavage and injection with 200 lg kg -1 of CYN and observation at 24 h and 5 days $ GPx activity for every exposure duration and mode (liver and kidneys)

: GPx gene expression at 24 h for fish that were orally exposed and for both exposure modes at 5 days (liver)

: GPx gene expression at 24 h for fish that were orally exposed and ; for both exposure modes at 5 days (kidneys)

: GST activity at 24 h both exposure modes, for injected fish at 5 days (liver) and for injected fish at both exposure durations (kidneys)

: GST gene expression at 24 h for injected fish and for both exposure mode at 5 days (liver)

: GST gene expression for both exposure modes at 24 h but only for injected fish at 5 days (kidneys)

: GST protein abundance for both exposure modes at 24 h but only in orally exposed fish at 5 days (liver) and for fish that were injected at 5 days (kidneys)

Guzma ´n-Guille ´n et al. (2013a) Immersion of fish in cyanobacterial cells containing 10 and 100 lg l -1 of CYN and observation at 7 and 14 days : lipid peroxidation for both doses at 14 days (liver) and for the highest dose for every exposure duration (kidneys)

: protein oxidation for the highest dose at 14 days (liver and kidneys)

: DNA oxidation for the highest dose for every exposure duration (liver and kidneys) and for both doses for every exposure duration (kidneys)

: GST activity for the lowest dose at 7 days and for both doses a 14 days (liver), ; GST activity for the highest dose at 14 days (kidneys)

$ GPx activity whatever the dose and exposure duration (liver), : GPx activity for the highest dose at 7 days and for both doses at 14 days (kidneys)

: SOD for the highest dose at 7 days and for both doses at 14 days (liver), : SOD for the highest dose at 7 days but ; SOD for both doses at 14 days (kidneys)

; and : CAT for lowest and highest dose respectively at 7 days and ; CAT for the highest dose at 14 days (liver), ; CAT for every dose and exposure duration (kidneys)

: GCS for the highest dose at 7 days (liver) and for both doses and exposure durations (kidneys)

; GSH/GSSG ratio for the lowest dose at 7 days and dose-dependent ; at 14 days (liver), dose-dependent ; for every exposure duration (kidneys) : = increase, ; = decrease, $ = no change, LPO lipid peroxidation, GST glutathione S-transferase, GPx glutathione peroxidase, NADPH nicotinamide adenine dinucleotide phosphate-oxidase, GSH glutathione, GSC c-glutamylcysteine synthetase, SOD superoxide dismutase, CAT catalase, GSH/GSSG reduced and oxidised glutathione ratio zebrafish embryos. However, developmental toxicity and strong mortality were observed in embryos that were treated with cyanobacterial extracts, suggesting that other metabolites in cyanobacteria could have more toxic effects on fish than CYN [START_REF] Berry | Toxicity of cylindrospermopsin, and other apparent metabolites from Cylindrospermopsis raciborskii and Aphanizomenon ovalisporum, to the zebrafish (Danio rerio) embryo[END_REF]). To test the real toxicity of CYN on embryos and to circumvent possible difficulties in the penetration of CYN across cellular membranes, microinjections were also used. The obtained results showed a dosedependent toxicity of CYN leading to rapid mortality and to deformations in less than 24 h, even at low concentrations. Nevertheless, CYN does not seem to be a negative factor for embryo development and survival, mainly because it does not easily permeate cellular membranes. However, in ecosystems, CYNproducing cyanobacteria can produce other toxic metabolites. Therefore, future studies need to consider the synergistic effects between these various toxic molecules to better comprehend the potential disturbances of both CYN and toxic metabolites to fish embryos.

Effects of CYN on adult fish

Oxidative parameters Oxidative stress is defined as an excess of pro-oxidants compared to antioxidants, leading to potential molecular damage [START_REF] Amado | Oxidative stress generation by microcystins in aquatic animals: why and how[END_REF]. To study the effects of toxic substances on oxidative stress parameters, the transcription and activity of enzymes that have been implicated in reactive oxygen species (ROS) scavenging or consequences of ROS effects are used as indicators. Superoxide dismutase (SOD) and catalase (CAT) provide the first defence against oxygen toxicity [START_REF] Amado | Oxidative stress generation by microcystins in aquatic animals: why and how[END_REF]. Guzma ´n-Guille ´n et al.

(2013a) exposed tilapia sub-chronically (by immersion) to environmentally relevant concentrations of CYN for 7 or 14 days (Table 3). These authors observed an increase in SOD in the liver at the highest dose (100 lg l -1 CYN) for both exposure durations, to counteract ROS formation. In the kidneys after an initial increase in CYN, the SOD activity decreased after 14 days, correlating with an increased susceptibility of the kidneys to CYN (higher LPO, protein and DNA oxidation). The CAT activity increased in the liver after 7 days of exposure to control oxyradicals but decreased after 14 days when the oxidative damage was more pronounced. In the kidneys, the CAT activity decreased under both doses and exposure durations (Guzma ´n-Guille ´n et al. 2013a). Other ROS biomarkers have been investigated in fish that were exposed to cyanotoxins (Table 3). For example, an increase in NADPH oxidase activity, a biomarker of ROS formation, was observed in the liver and kidneys of fish that were exposed to CYN by gavage and injection. Many studies have investigated the GSH-dependent antioxidant system following of the exposure of fish to CYN. In this way, the activity and gene expression of glutathione peroxidase (GPx), an enzyme catalysing the reaction between GSH and hydrogen peroxide (H 2 O 2 ), have been assessed by different authors [START_REF] Puerto | Acute effects of pure cylindrospermopsin on the activity and transcription of antioxidant enzymes in tilapia (Oreochromis niloticus) exposed by gavage[END_REF] 2013a). Changes in the GPx activity following the exposure of fish to CYN are not clear. In the liver, no change in the GPx activity was observed under any exposure mechanism, CYN dose or exposure duration. However, in the kidneys, the GPx activity decreased [START_REF] Puerto | Acute effects of pure cylindrospermopsin on the activity and transcription of antioxidant enzymes in tilapia (Oreochromis niloticus) exposed by gavage[END_REF] or increased in a dose-dependent manner (Guzma ´n-Guille ´n et al. 2013a) in fish that were exposed to CYN by gavage or immersion, respectively (Table 3). These results clearly demonstrate the influence of the exposure pathways in terms of enzyme responses, as well as the potential adaptive response of the kidneys rather than the liver to compensate for greater injuries. Concerning the gene expression of GPx, fish that were orally exposed and sacrificed after 24 h showed an increase in GPx expression in the liver and kidneys [START_REF] Puerto | Acute effects of pure cylindrospermopsin on the activity and transcription of antioxidant enzymes in tilapia (Oreochromis niloticus) exposed by gavage[END_REF]Gutie ´rrez-Praena et al. 2013b). However, at 5 days post-exposure, the GPx expression increased in the liver but decreased in the kidneys, suggesting a greater defensive capacity of the liver and thus a greater sensitivity of the kidneys to CYN. Both the activity and gene expression of GST were assessed in the liver and kidneys of tilapia (Table 3; [START_REF] Puerto | Acute effects of pure cylindrospermopsin on the activity and transcription of antioxidant enzymes in tilapia (Oreochromis niloticus) exposed by gavage[END_REF]Gutie ´rrez-Praena et al. 2013b; Guzma ´n-Guille ´n et al. 2013a). Concerning the GST activity, 24 h post-gavage, an increase was observed in both the liver and kidneys concomitantly with an increase in the GST protein abundance, but after 5 days, an activity similar to that of the control group was detected [START_REF] Puerto | Acute effects of pure cylindrospermopsin on the activity and transcription of antioxidant enzymes in tilapia (Oreochromis niloticus) exposed by gavage[END_REF]Gutie ´rrez-Praena et al. 2013b). These results suggest a potential recovery of the enzyme activity within a few days, traducing a rapid detoxification of negative cell components that is in accordance with the results of a previous study (Gutie ´rrez-Praena et al. 2011a). However, during blooms, fish are chronically exposed to cyanobacteria and to their toxins and not only to a single one as is most often investigated. Guzma ´n-Guille ´n et al. (2013a) investigated the effects of CYN on fish that were exposed subchronically (7 and 14 days) to environmentally relevant concentrations of cyanobacterial extracts containing CYN. The GST activity increased in the liver of fish that were exposed to CYN for different exposure durations, suggesting a detoxification of peroxidised lipids that were detected in the liver. In the kidneys, the GST activity decreased when the fish were exposed to the highest concentration of CYN for 14 days. This difference between organs may be linked to a higher hepatic conjugation activity compared to that of the kidneys, leading to a greater sensitivity of the kidneys to CYN. Furthermore, GST gene expression was generally increased in both of the organs, depending on the time of measurement, and intraperitoneal injection produced greater effects with an increase in GST expression at every time of sacrifice and in every studied organ [START_REF] Puerto | Acute effects of pure cylindrospermopsin on the activity and transcription of antioxidant enzymes in tilapia (Oreochromis niloticus) exposed by gavage[END_REF]Gutie ´rrez-Praena et al. 2013b). In fish that were exposed both acutely and sub-chronically to CYN, the GSH content showed a decrease in both of the organs but with more pronounced effects in the kidneys, suggesting that CYN inhibits GSH synthesis and/or an increases the detoxification of ROS, implying the oxidation of GSH to GSSG (Gutie ´rrez-Praena et al. 2011a; [START_REF] Puerto | Acute effects of pure cylindrospermopsin on the activity and transcription of antioxidant enzymes in tilapia (Oreochromis niloticus) exposed by gavage[END_REF]Guzma ´n-Guille ´n et al. 2013a). In this way, the GSC activity increased, mainly in the kidneys, at every dose and exposure duration, suggesting that GSH synthesis is not affected when the fish are exposed sub-chronically to environmental concentrations of CYN (Guzma ´n-Guille ´n et al. 2013a). At high exposure doses, GSH synthesis could be affected because a decrease in the GSC activity was observed in fish that were acutely exposed to CYN (Gutie ´rrez-Praena et al. 2011a).

The consequences of ROS formation in the organs of fish that were exposed to CYN have also been investigated (Table 3). An increase in LPO and protein oxidation following a time-and dose-dependent manner were observed in both of the organs of the exposed fish (Gutie ´rrez- Praena et al. 2011a;[START_REF] Puerto | Acute effects of pure cylindrospermopsin on the activity and transcription of antioxidant enzymes in tilapia (Oreochromis niloticus) exposed by gavage[END_REF]Guzma ´n-Guille ´n et al. 2013a). In contrast to fish that were acutely exposed to CYN (Gutie ´rrez-Praena et al. 2011a), DNA damages were observed in the liver and more particularly in the kidneys of fish that were exposed sub-chronically to cyanobacterial extracts containing CYN (Guzma ´n-Guille ´n et al. 2013a). These results confirm that CYN can also affect organs other than the liver and that the kidneys appear to be the most sensitive organ to CYN. It has been suggested that the uracil group of CYN can interact with adenine group in RNA and DNA, leading to disturbances in DNA synthesis and thus inducing mutations by acting as a carcinogen [START_REF] Zegura | Genotoxicity and potential carcinogenicity of cyanobacterial toxins-a review[END_REF]). This study is the first to demonstrate the possible involvement of oxidative stress in the genotoxic effects of CYN.

Histological parameters

The studies investigating the histopathological damages that are induced by CYN contamination are presented in Table 4. Gutie ´rrez-Praena et al. (2012) showed that CYN induced histopathological damages in a wide variety of organs, such as the liver and kidneys, which are the main targets, but also in the heart, gills and intestines. The histological damages were more severe after intraperitoneal injection than after oral exposure (except for in the intestines). Furthermore, the exposure duration also affected the results of the histological parameters with more acute damages being observed after 5 days of exposure. Puerto et al. (2012a) observed structural alterations and degenerative processes in the liver, kidneys, heart, gills, and intestines but with more significant alterations at the highest exposure dose. In parallel with the histopathological alterations, authors have also observed changes in some oxidative biomarkers (increase in protein oxidation and in NADPH oxidase activity) in the liver and kidneys of exposed tilapia. The authors suggest that the increase in the observed NADPH oxidase activity could explain in part the vascular alterations that were observed in both the kidney and heart tissues.

To sum up, previous work unequivocally provided evidence for the deleterious effects of CYN at different biological scales and during various fish ontogenic stages. All of the studies report short-term effects. However, the main challenge for future work will be to assess the medium-to long-term effects of CYN at the fish population scale. While it is unlikely that CYN easily permeates the cellular membranes of fish embryos (which is supposed to be the most sensitive ontogenic stage of fish; e.g., Di [START_REF] Giulio | The toxicology of fishes[END_REF], one should keep in mind that in ecosystems, fish are exposed simultaneously to multiple and potentially interacting toxic cyanobacterial metabolites, which could lead to a greater toxicity with species-dependent effects. However, it could be hypothesised that fish species can adapt (at the genetic scale) or accommodate cyanotoxins to counteract their toxic effects and thus to decrease their negative effects on the fish population.

Nodularin

General presentation

NOD was first isolated from Nodularia spumigena, a cyanobacterium that is typically present in estuaries and brackish waters [START_REF] Rinehart | Nodularin, microcystin and the configuration of ADDA[END_REF]). To date, seven naturally occurring variants of NOD have been identified [START_REF] Pearson | On the chemistry, toxicology and genetics of the cyanobacterial toxins, microcystin, nodularin, saxitoxin and cylindrospermopsin[END_REF]. NOD are 824 Da cyclic pentapeptides that are composed of (2S,3S,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid (Adda), D-glutamic acid (D-Glu), L-arginine (L-Arg), D-erythro-b-methylaspartic acid (D-MeAsp) and 2-methylamino-2-dehydrobutyric acid (Mdhb; Fig. 3).

These cyanotoxins are only produced by the genera Nodularia and are mainly measured in the brackish water of the Baltic Sea, where the cyanobacteria form

Gavage and intraperitoneal injection with 200 lg kg -1 and sacrificed at 24 h and 5 days Histological damages more pronounced for fish that were injected (excepted for intestinal tract) and for fish that were sampled at 5 days Liver = increase hepatocyte nuclear diameter, glycogen accumulation, steatosis, cytoplasmic vacuolisation, scarce cytoplasmic organelles Kidneys = atrophic and hyalinised glomeruli, hyperaemia, decreased width of the proximal and distal convoluted tubules, dilated Bowman's capsule, elongated podocyte primary foot processes, tubular cells with necrotic nuclei Heart = myofibrolysis, oedema, loss muscular fibres, pleomorphic fibres, scarce mitochondria Intestine = catarrhal enteritis process, oedema, hyperaemia, loss of microvilli, cytoplasmic vacuolisation, necrotic enterocytes, increased calceiform cells Gills = tumefaction processes, hyperaemia, inflammatory foci, desquamation of lamellae, haemorrhages regular summer blooms [START_REF] Buynder | Nodularin uptake by seafood during a cyanobacterial Bloom[END_REF][START_REF] Karjalainen | Fate and effects of Nodularia spumigena and its toxin, nodularin, in Baltic Sea planktonic food webs[END_REF][START_REF] Karjalainen | Ecosystem consequences of cyanobacteria in the northern Baltic Sea[END_REF]). These toxins are mostly found in intracellular spaces, and experimentally, less than 10-20 % of the total toxin amount is released in the surrounding water (Chorus and Bartram 1999).

The toxicity of NOD comes from its capacity to inhibit the catalytic subunits of the serine/threoninespecific protein phosphatises (PPs) 1 and 2A (PP1 and PP2A), leading to the hyperphosphorylation of cells, causing cytoskeleton disintegration, the loss of cell junctions, and disturbances of cell metabolism and cell cycle control [START_REF] Campos | Molecular mechanisms of microcystin toxicity in animal cells[END_REF]. However, contrary to microcystines (MCs), which can covalently bind to PPs, NOD cannot interact covalently with PPs due to the lack of a methyldehydroalanine (Mdha) residue [START_REF] Mackintosh | The cyanobacterial toxin microcystin binds covalently to cysteine-273 on protein phosphatase 1[END_REF][START_REF] Martins | Microcystin dynamics in aquatic organisms[END_REF]. NOD is also considered a tumour-promoter-possessing carcinogen [START_REF] Karjalainen | Fate and effects of Nodularia spumigena and its toxin, nodularin, in Baltic Sea planktonic food webs[END_REF][START_REF] Van Apeldoorn | Toxins of cyanobacteria[END_REF].

At a macroscopic scale, NOD have been implicated in various disturbances of both primary and secondary consumers of aquatic ecosystems. In this way, the negative impacts of NOD have been observed in both the zooplankton and fish compartments [START_REF] Van Apeldoorn | Toxins of cyanobacteria[END_REF]). In fish, information about the physiological effects of NOD is still scarce, but several papers investigating the bioaccumulation of this toxin in various species of the Baltic Sea have already been published. Most studies have focused on the Baltic Sea because this area provides large amounts of fish for human consumption. As a consequence, the effects of NOD on fish were first investigated in terms of human food safety.

Accumulation of NOD in fish

The accumulation processes of NOD were investigated in different fish species from the Baltic Sea (Table 5). In flounder (Platichthys flesus), NOD concentrations were observed in different organs. The liver was the main organ of NOD accumulation in this fish species, with concentrations varying widely between specimens (Table 5) and ranging from nondetection (ND) to 1,100 ng g -1 DW (Sipia ¨et al. 2001a(Sipia ¨et al. , b, 2002[START_REF] Sipia | Analysis of nodularin-R in eider (Somateria mollissima), roach (Rutilus rutilus L.), and flounder (Platichthys flesus L.) liver and muscle samples from the western Gulf of Finland, northern Baltic Sea[END_REF][START_REF] Karlsson | Mass spectrometric detection and quantification of nodularin-R in flounder livers[END_REF][START_REF] Kankaanpa ¨a | Heterogeneity of nodularin bioaccumulation in northern Baltic Sea flounders in 2002[END_REF][START_REF] Mazur-Marzec | Accumulation of nodularin in sediments, mussels, and fish from the gulf of Gdansk, southern Baltic Sea[END_REF][START_REF] Persson | Detection of nodularin in European flounder (Platichthys flesus) in the west coast of Sweden: evidence of nodularin mediated oxidative stress[END_REF]. Interestingly, Kankaanpa ¨a ¨et al. ( 2005) investigated the NOD accumulation in the liver of flounder using two different measurement tools, LC/ MS to evaluate the concentrations of pure NOD in fish samples and the ELISA method, which gives the total extractable hepatotoxin (TEH) concentration in the same samples. Concentrations ranging from ND to 390 ng g -1 DW of pure NOD and from 20 to 2,230 ng g -1 DW TEH were observed for the LC/ MS and ELISA measurements, respectively. This result suggests the importance of the measurement method (1) in terms of the concentration relevance and the consequences that this relevance may have in making comparisons between distinct studies and (2) in terms of toxicity evaluation. In addition, NOD were observed in various organs of flounder, indicating the presence of toxins in the muscle (from 1 to 100 ng g -1 DW), guts (20.7 ± 3.3 ng g -1 DW), and gonads (21.3 ± 2.4 ng g -1 DW; [START_REF] Sipia | Analysis of nodularin-R in eider (Somateria mollissima), roach (Rutilus rutilus L.), and flounder (Platichthys flesus L.) liver and muscle samples from the western Gulf of Finland, northern Baltic Sea[END_REF][START_REF] Mazur-Marzec | Accumulation of nodularin in sediments, mussels, and fish from the gulf of Gdansk, southern Baltic Sea[END_REF]). As a function of the toxicity of NOD and the presence of these toxins in the gonads of fish, it hypothesised that NOD could negatively interfere in reproduction processes and thus could lead to fish population disturbances. NOD were also found in the liver of cod (Gadus morhua), in the liver and muscle of roach (Rutilus rutilus), in the visceral tissues of the three-spined stickleback (Gasterosteus aculeatus), in the liver and stomach of herring (Clupea harengus membras), in the liver of salmon (Salmo salar) and in sprat (Sprattus sprattus), with a high degree of variation in the NOD concentrations according to the species (Sipia ¨et al. 2001a(Sipia ¨et al. , b, 2006[START_REF] Sipia | Transfer of nodularin to three-spined stickleback (Gasterosteus aculeatus L.), herring (Clupea harengus L.), and salmon (Salmo salar L.) in the northern Baltic Sea[END_REF][START_REF] Karjalainen | Nodularin concentrations in Baltic Sea zooplankton and fish during a cyanobacterial bloom[END_REF]. Australian ecosystems are also affected by blooms of Nodularia spumigena (Table 5). In Lake Victoria (New South Wales), [START_REF] Buynder | Nodularin uptake by seafood during a cyanobacterial Bloom[END_REF] observed the presence of NOD in the viscera (152 ng g -1 FW) and muscle (2.5 ng g -1 FW) of finfish that were caught during a bloom of Nodularia spumigena. More recently, Stewart et al. ( 2012) measured the NOD concentration in sea mullet (Mugil cephalus) that were caught in a recreational lake in south-east Queensland and found concentrations reaching 47.8 lg g -1 DW in the liver and 56.8 ng g -1 DW in the muscle. These results show that NOD can accumulate in various organs of fish during Nodularia spumigena blooms. However, most of these studies used ELISA measurements that quantify not only pure NOD but also other metabolites and detoxification products that are potentially toxic. In terms of safety food, this measurement method is most likely the most suited because it reports the worst case of contamination. In terms of toxicity evaluation, the concentrations of pure NOD are overestimated with this technique, thereby leading to the potential misinterpretation concerning the actual toxicity of NOD in tissues. Future studies are needed regarding the detoxification capacities and the effects of NOD on the reproduction processes of fish that are exposed to toxic Nodularia blooms both for short and long durations to better understand the potential risk of NOD contamination on fish populations. Furthermore, other ontogenic stages than adults must be investigated in terms of NOD accumulation in ecosystems.

Biological effects of NOD on fish

In vitro effects of NOD on fish cells

The first study assessing the effects of NOD on fish was conducted in vitro on the hepatic cells of juvenile Atlantic salmon (Salmo salar). [START_REF] Fladmark | Sensitive detection of apoptogenic toxins in suspension cultures of rat and salmon hepatocytes[END_REF] exposed salmon hepatocytes to a medium containing pure NOD (824 lg l -1 ). After incubation, many signs of an apoptotic response were observed with surface budding being particularly important, as well as cell shrinkage and chromatin condensation. Only recently have other authors investigated more precisely the in vitro effects of NOD on fish cells [START_REF] Zhang | Apoptotic responses of Carassius auratus lymphocytes to nodularin exposure in vitro[END_REF]. The lymphocytes of Carassius auratus were exposed to different concentrations (1, 5, 10 and 100 lg l -1 ) of NOD to reveal the apoptotic reaction of lymphocytes in a dose-dependent manner, leading to intracellular changes, such as a condensed cytoplasm, nuclear chromatin agglutination and marginalisation. The DNA also presented fragmentation, and both the Ca 2? and ROS levels increased in a dose-dependent manner, indicating an important role of Ca 2? in the mechanism of NOD-induced programmed death. Furthermore, the mitochondrial membrane potential (MMP) decreased, suggesting an early apoptotic event. The levels of caspases (CAP) 3 and 8, which play an important role in the execution phase of apoptosis, increased in a dose-dependent manner, while such an increase was not observed for CAP 8. This result, together with other observations, suggests that NOD-induced apoptosis involves a mitochondrial pathway. At a biomolecular scale, the expression and protein detection of the apoptosis-related genes Bcl-2 and Bax changed, specifically in a dose-dependent manner for Bcl-2, while Bax increased at both the mRNA and protein levels. These findings suggest that the mitochondria-mediated pathway is involved in fish cell apoptosis after NOD exposure and thus that fish that are exposed to NOD-producing blooms could be rapidly and strongly affected by this cyanotoxin.

Biological effects of NOD on larvae and juvenile fish

To our knowledge, no study has investigated the effects of NOD on fish embryos, but a few studies have 123 investigated older stages. [START_REF] Karjalainen | Trophic transfer of cyanobacterial toxins from zooplankton to planktivores: consequences for pike larvae and mysid shrimps[END_REF] studied the effects of NOD (and more widely of cyanobacterial extracts) on Esox lucius larvae that were fed daily (for 11 days) copepods containing NOD or cyanobacterial extracts. These authors observed that, when in contact with contaminated copepods, the ingestion rate, faeces production and growth rate decreased in pike larvae. The results showed significant differences with the control, but only for pike larvae that were exposed to cyanobacterial extracts, suggesting that the negative effects of these extracts were stronger than those of pure NOD, most likely due to the synergistic effects of NOD with other unidentified toxic metabolites that are produced by cyanobacteria. In the same way, based on a 15-daylong experiment, young-of-year (YOY) three-spined stickleback were exposed to Nodularia-exposed plankton either with (B-NOD) or without (N-NOD) Nodularia in water as well as to plankton that were unexposed to cyanobacteria (i.e., control; Pa ¨a ¨kko ¨nen et al. 2008). Interestingly, the specific growth rates were lower in B-NOD than in the other treatments despite the fact that the food consumption was higher in the B-NOD treatment. First, this result suggests that the turbidity that was caused by Nodularia did not affect the food consumption of the fish. Second, the highest consumption in this treatment was not correlated with the growth rate, and the decrease in protein synthesis was most likely due to the consumption of the energy intake for the detoxification of NOD and toxic metabolites. In this study, the most significant effects were observed in the B-NOD treatment, whereas for the N-NOD treatment, trends were observed but were generally not significant. This result is explained by the fact that in the B-NOD treatment, zooplankton and fish were chronically exposed to NOD and other toxic metabolites that are produced by Nodularia; the fish and zooplankton were thus more affected than in the N-NOD treatment where the NOD equivalent concentrations in both the zooplankton and fish decreased during the experiment (Pa ¨a ¨kko ¨nen et al. 2008). According to these results, the actual effects of NOD on young-stage fish seem too complex to highlight. However, the crude extract of Nodularia has an unequivocal effect on fish physiology. The exposure of fish to cyanobacterial extracts is relevant because in ecosystems, fish are exposed to Nodularia blooms that produce NOD and other toxic metabolites with potential interaction between these compounds, leading to stronger effects on fish than that of exposure to only one toxin. Future work should therefore consider all of the toxic substances that are produced by the cyanobacteria to precisely comprehend the potential effects of NODproducing blooms on fish populations.

Biological effects of NOD on adult fish

Experimental accumulation and detoxification of NOD Few studies have investigated the biological effects of NOD on adult fish. Some investigations have focused on the accumulation process of NOD in adult fish using an experimental approach to better understand the detoxification process of fish to counteract the negative effects of NOD (Table 6). The first study was conducted by Kankaanpa ¨a ¨et al. (2002) on Baltic Sea trout (Salmo trutta m. trutta L.) that were orally exposed to Nodularia (Table 6). In this study, no fish mortality was observed, and an ELISA analysis showed an increasing concentration of NOD and NOD-like compounds in the liver of sea trout at the end of experiment (approximately 1,200 ng g -1 DW after 8 days). However, the HPLC analysis did not detect NOD or NOD metabolites in the liver. The authors suggested that the discrepancy between the two measurement methods could be due to the transformation of NOD via conjugation with thiolcontaining proteins, peptides and amino acids, such as GSH, methionine or cysteine, associated with the lack of NOD conjugate standards for the HPLC identification of the toxins and their metabolites. The lowest concentrations were found in the muscle, in which the concentration that was measured on the first day (125 ng g -1 DW) decreased and then stabilised at days 2 and 4 (35 and 43 ng g -1 DW, respectively). Furthermore, no modification in the swimming activity or behaviour of the fish were revealed under the effects of food containing NOD [START_REF] Kankaanpa ¨a | Acute effects and bioaccumulation of nodularin in sea trout (Salmo trutta m. trutta L.) exposed orally to Nodularia spumigena under laboratory conditions[END_REF]. A similar approach was used by Engstro ¨m-O ¨st et al. ( 2002) using adult three-spined sticklebacks that were fed daily for 10 days NOD-contaminated copepods (Table 6). The results showed a peak but without a continuous increase in the NOD equivalent concentration in the liver of fish that were exposed. This result suggests the presence of a detoxification process capable of rapidly metabolising and excreting the toxins that were taken by the fish. More recently, [START_REF] Vuorinen | Accumulation and effects of nodularin from a single and repeated oral doses of cyanobacterium Nodularia spumigena on flounder (Platichthys flesus L.)[END_REF] exposed adult flounder by force-feeding with either a single dose or three repeated doses of a Nodularia slurry (Table 6). In the liver of flounder that were exposed to a single dose and repeated doses, NOD was found at mean concentrations of 220 ± 70 ng g -1 DW and 60 ± 5 ng g -1 DW, respectively. No GSH conjugates were observed in the liver of fish that were exposed, suggesting a rapid excretion/ disintegration of NOD detoxification products in the fish. In parallel, an ELISA analysis was performed on the bile samples of fish and revealed the presence of NOD-like compounds at concentrations reaching 49 ± 5 and 37 ± 13 ng ml -1 for single and repeated exposures, respectively. A study of GST showed a significantly higher activity of the enzyme in the repeated treatment than in the single treatment and control (Table 6). In agreement with the decrease in the NOD concentrations in the liver, this result suggests that fish that were repeatedly exposed to NOD, such as during bloom periods, can more rapidly and efficiently detoxify and excrete the toxin than can fish that were exposed to a single but higher concentration of the toxin.

Oxidative parameters Cyanotoxins in organs are responsible for the appearance of an oxidative stress state in exposed fish (Table 6). [START_REF] Persson | Detection of nodularin in European flounder (Platichthys flesus) in the west coast of Sweden: evidence of nodularin mediated oxidative stress[END_REF] studied the effects of different concentrations of NOD on the oxidative stress state of flounder using intraperitoneal injections of NOD at different concentrations (Table 6). The fish were observed on the fourth day, and some of the fish that were injected with the highest dose (50 lg kg -1 BW) were sacrificed on the fourteenth day to evaluate the potential recovery of the oxidative stress enzymes. The lowest CAT and GST activities were observed in flounder that were exposed to the highest dose, but the activity levels of both of the enzymes returned to the control level after the recovery period. The authors suggested that the decrease in the GST activity could be explained by the reduction of GST protein synthesis at the molecular level or by the modification of the enzyme via the NOD-induced hyperphosphorylation state. The decrease in the CAT activity suggests a NOD-induced oxidative stress, leading to an increase in ROS that are directly scavenged by CAT. The authors also suggested that the CAT activity could decrease in response to direct damages to the enzyme that are caused by ROS. In parallel with these enzymatic measurements, the authors quantified the concentration of malondialdehyde (MDA), an oxidation product of polyunsaturated fatty acids in lipoproteins, in flounder livers, and observed no change, suggesting an efficient oxidative defence against ROS (Table 6). To our knowledge, there are no other publications investigating the effects of NOD on oxidative stress in fish. Again, most studies have investigated microcystins (MCs), which have similar cellular effects as NOD, and thus potentially the same effects at a larger biological scale. In this way, at a larger scale, histological analyses are widely used in terms of the evaluation of cyanotoxins effects on fish species.

Histological parameters We found two papers that investigated the effects of NOD on the histological integrity of the organs of exposed fish (Kankaanpa ¨a ët al. 2002; [START_REF] Vuorinen | Accumulation and effects of nodularin from a single and repeated oral doses of cyanobacterium Nodularia spumigena on flounder (Platichthys flesus L.)[END_REF]. Following a single oral dose of Nodularia (Table 6), the livers of Baltic Sea trout were severely affected after 1 and 2 days [START_REF] Kankaanpa ¨a | Acute effects and bioaccumulation of nodularin in sea trout (Salmo trutta m. trutta L.) exposed orally to Nodularia spumigena under laboratory conditions[END_REF]. The authors observed a loss of the normal liver architecture with pyknosis, no visible nucleoli and some disintegrated hepatocytes with an unclear cell shape. However, on the 8th day of the experiment, the authors observed healthier livers but inflamed and swollen intestines [START_REF] Kankaanpa ¨a | Acute effects and bioaccumulation of nodularin in sea trout (Salmo trutta m. trutta L.) exposed orally to Nodularia spumigena under laboratory conditions[END_REF]. These results suggest that exposure to NOD through food can induce histological damages to the liver of fish but with a progressive recovery of the organ after a single NOD exposure. The exposure of flounder by the force-feeding of a single or repeated dose of NOD (Table 6) gave similar results [START_REF] Vuorinen | Accumulation and effects of nodularin from a single and repeated oral doses of cyanobacterium Nodularia spumigena on flounder (Platichthys flesus L.)[END_REF]. Indeed, for both treatments, the liver architecture of the fish was incoherent but not totally destroyed. The cell shape was not strict, and the presence of round lipid vacuoles was highlighted. Furthermore, the focal area showed degenerative cells, cytoplasm shrinkage, and a loss of the nucleolus and pyknotic nuclei, leading to the same conclusions as in the first study (Table 6).

An overview of these results shows that NOD can accumulate in various organs of fish that are exposed to Nodularia blooms. However, the widespread use of the ELISA method to measure the NOD cannot discriminate between the different toxins that are produced by Nodularia, that can accumulate in fish and thus that may play a toxic role in fish physiology. NOD produce some cellular effects, such as oxidative stress, leading to apoptotic processes and, at a larger scale, causing physiological disturbances. However, fish can detoxify NOD when exposed to the toxins and thus to counteract the negative effects of NOD. Interestingly, few studies have investigated the biological effects of NOD on fish, most likely because most investigations have focused on the biological effects of MCs that have the same cellular effects as NOD. One way to contribute to the knowledge of the effects of NOD on fish would be to identify all of the toxic compounds that are produced by Nodularia blooms and which could accumulate in fish and affect their physiology.

Conclusions

Both CYN and NOD accumulate in the organs of fish that are exposed to cyanobacterial blooms. This accumulation leads to negative effects in individual fish, with major effects being observed during the youngest ontogenic stages. To better comprehend the potential negative effects due to toxin accumulation, more accurate analytical methods need to be used to discriminate the pure toxins from the less toxic detoxification metabolites that accumulate in the organs of fish. Currently, it is difficult to determine the actual long-term potential impact of these toxic effects on fish populations that are exposed to cyanobacterial blooms. The different cyanotoxin effects that have been observed between fish species depend most likely on the recurrence of blooms in each ecosystem. In this way, in an ecosystem where blooms are recurring, fish adaptation (at the genetic scale) and/or accommodation (behavioural and physiological scales) phenomena could take place to avoid or counteract the deleterious effects of cyanotoxins. In fact, the available studies on the topic have only investigated the effects of a given toxin on a given fish species for a short duration. It is important to keep in mind that during cyanobacterial blooms, fish are exposed to a mixture of toxic compounds that can interact and increase the toxic effects on fish individuals. Furthermore, different fish species exhibit different detoxification capacities, which could explain the different toxic effects. Future studies need to integrate the synergistic and/or cumulative effects of toxic cyanobacterial metabolites on various fish species to better understand and anticipate their potential effects on fish populations after both short-and longterm exposure in ecosystems where blooms occur repeatedly.

Fig. 1

 1 Fig. 1 Number of publications dealing with the effects of nodularin (black) and cylindrospermopsin (white) on fish

Fig. 3

 3 Fig. 3 Chemical structure of NOD and its variants

Table 2

 2

	uptake (NR) and tetrazolium salt reduction (MST) was
	observed (Gutie ´rrez-Praena et al. 2011b; Liebel et al.

). A decrease in cell viability reflected by a decrease (in a time-and concentrationdependent manner) in the protein content, neutral red

Table 1

 1 Cylindrospermopsin (CYN) accumulation in fish tissues as observed in field conditions

	References	Sampling	Cyanobacterial	CYN concentration	CYN concentration in	Measurement
		location	concentration	in water	fish tissues	method
	Saker and Eaglesham	Aquaculture	3.2 9 10 5 cell ml -1	589 lg l -1	Salmo Trutta:	HPLC-MS/MS
	(1999)	pond			1.2 lg g -1 DW in
					viscera
	Messineo et al.	Lake Albano 30 9 10 6 cell ml -1	10-110 lg l -1	Melanotaenia	ELISA
	(2009)				eachamensis:
					2.7 ng g -1 FW in viscera
					0.8 ng g -1 FW in muscle
					0.07 ng g -1 FW in
					ovaries
	Berry et al. (2012)	Lake	NS	ND	Rhamdia sp.:	ELISA
		Catemaco			0.24 lg kg -1 FW
					Oreochromis aureus:
					0.09 lg kg -1 FW
					Vieja sp.:
					0.42 lg kg -1 FW
					Vieja fenestratus:
					0.81 lg kg -1 FW
					Heterandria jonesii:
					1.26 lg kg -1 FW
					Bramocharax cabelleroi:
					0.81 lg kg -1 FW
					Cichlasoma
					urophthalmus:
					0.26 lg kg -1 FW
					Cichlasoma helleri:
					0.15 lg kg -1 FW
					Dorosoma Mexicana:
					0.80 lg kg -1 FW
					In the muscle of these
					species
	HPLC high-performance liquid chromatography, MS mass spectrometry, ELISA enzyme-linked immunosorbent assay, DW dry
	weight, FW fresh weight, NS not specified, ND not detected

Table 2

 2 In vitro effects of CYN on the hepatic cells of fish

	References	Species	Mode, concentration and time exposure	Observable effects
			duration
	Liebel et al.	Prochilodus	Immersion of hepatocytes in 0.1, 1 and	; cell viability (0.1 and 1 lg l -1 ), MRX (all
	(2011)	lineatus	10 lg l -1 CYN during 72 h	treatments)
				: RONS (all treatments), LPO (10 lg l -1 )
				$ GST, G6PDH, GSH/GSSG ratio, protein and
				DNA damages
	Gutie ´rrez-	Poeciliopsis	Immersion of hepatocytes in 0.3, 0.6, 1.2, 2.5,
	Praena et al.	lucida	5, 10, 20 and 40 lg ml -1 CYN for 24 or
	(2011b)		48 h

Table 3

 3 Effects of CYN on the oxidative parameters of fish (Oreochromis niloticus)

	References	Mode, concentration and	Observable effects on O. niloticus
		exposure duration
	Gutie ´rrez-Praena
	et al. (2011a)

  ; Gutie ´rrez-Praena et al. 2013b; Guzma ´n-Guille ´n et al.

Table 4

 4 Effects of CYN on the histological parameters of fish (Oreochromis niloticus)

	References	Mode, concentration and exposure duration	Observable effects on O. niloticus
	Puerto et al.	Gavage with 200 and 400 lg kg -1 of CYN	Histopathological changes more pronounced for the highest dose of
	(2012b)	and observation at 24 h	exposure:
			Liver = disorganised parenchymal architecture, large hepatocytes
			with central nuclei and cytoplasmic vacuolisation and
			hyalinisation, cytoplasmic glycogen accumulation, dense nuclei
			surrounded by scarce cytoplasm with abundant granular glycogen
			and lipid accumulation
			Kidneys = glomerular atrophy, dilated Bowman's capsule,
			haemorrhage, dilated capillary lumen, congestion, irregular
			intramembranous electron-dense deposits, thickening of the
			basement membrane, elongation of podocytes
			Heart = myofibrolysis, absence of contractile material, scarce
			mitochondria
			Intestine = small size of enterocytes lining villi, basophil and
			irregular nuclei, necrotic intestinal cells, dilated lacteal vessels,
			oedema, pleomorphism of the villi and partial loss of microvilli
			Gills = loss of the lamellar structure, swelling, presence of
			inflammatory cells, thickening of the connective tissue,
			congestion, catarrhal inflammation, haemorrhages, destruction of
			branchial arches, tumefaction, cell infiltration
	Gutie ´rrez-
	Praena et al.

Table 6

 6 Experimental effects of NOD on adult fish

	Reference	Species	Mode, concentration and time of exposure	Observable effects
	Kankaanpa ¨a ët Salmo trutta	Sea trout exposed orally to a single dose of food	: NOD-like compounds concentrations in liver of
	al.	m. trutta L.	containing 440 lg kg -1 BW of NOD sampled	fish during time with average concentrations of
	(2002)		at 1, 2, 4 and 8 days	19 and 1,200 lg kg -1 DW at day 1 and 8
				respectively
				Lowest NOD-like compounds concentrations in
				muscle with 125 lg kg -1 DW, 35 lg kg -1
				DW, 43 lg kg -1 DW at day 1, 2 and 4
				respectively
				No mortalities
				No effects of the treatment on swimming activity
				and other behaviour
				Histology of liver severely affected after
				1-2 days of exposition = Normal liver
				architecture lost, pyknosis, no visible nucleoli,
				disintegrated hepatocytes
				Healthier liver at day 8 but intestine inflamed and
				swollen
	Engstro ¨m-	Gasterosteus	Fish fed with 40 copepods per day containing	Peak of toxin in total fish at day 5 (approximately
	O ¨st et al.	aculeatus	1.251 9 10 -5 ng NOD equivalent copepod -1	0.15 lg g -1 DW)
	(2002)		during 10 days	No increase of concentrations with time
	Vuorinen	Platichtys	Fish force-fed with (1) a single dose of NOD	NOD concentrations in liver reaching 220 ± 70
	et al.	flesus	corresponding to 277 lg kg -1 BW and (2) 3	and 60 ± 5 ng g -1 DW in treatment (1) and
	(2009)		repeated doses of NOD corresponding to a total	(2) respectively. No GSH conjugates
			of 379 lg kg -1 BW and sampled 4 days after the last repeated dose or the single dose	NOD-like compounds in bile with concentrations reaching 49 ± 5 and 37 ± 13 ng ml -1 in the
				treatment (1) and (2) respectively
				GST activity significantly highest in treatment
				(2)
				Architecture of liver incoherent in both treatment
				but not totally destroyed, irregular cell shape
				and round lipid vacuoles, degenerative
				hepatocytes, shrinkage of cytoplasm, loss of
				nucleolus and pyknosis
	Persson et al.	Platichtys	Fish exposed to NOD at concentration of 0, 2, 10	No difference in NOD accumulation between
	(2009)	flesus	and 50 lg kg -1 BW by an intraperitoneal	treatments and no detection of NOD in muscle
			injection and sampled at 7 days or 14 days (7 days of recovery)	; CAT and GST activities for fish exposed to 50 lg kg -1 BW but same activity levels as
				control after recovery
				No difference in MDA content between
				treatments

: = increase, ; = decrease, GSH glutathione, GST glutathione-S transferase, CAT catalase, MDA malondialdehyde Rev Fish Biol Fisheries (2015) 25:1-19

Rev Fish Biol Fisheries (2015) 25:1-19

Rev Fish Biol Fisheries (2015) 25:1-19


H Velly

P Renault
email: pierre.renault@jouy.inra.fr.

A.-L Abraham

V Loux

A Delacroix-Buchet

F Fonseca

M Bouix

Genome Sequence of the Lactic Acid Bacterium Lactococcus lactis subsp. lactis TOMSC161, Isolated from a Nonscalded Curd Pressed Cheese

Lactococcus lactis is a lactic acid bacterium used in the production of many fermented foods, such as dairy products. Here, we report the genome sequence of L. lactis subsp. lactis TOMSC161, isolated from nonscalded curd pressed cheese. This genome sequence provides information in relation to dairy environment adaptation.

L actococcus lactis belongs to the group of lactic acid bacteria and is encountered in a wide range of environments, such as animals and plant materials, and even in a great variety of traditional food products (as spontaneous growth) [START_REF] Cogan | Characterization of the lactic acid bacteria in artisanal dairy products[END_REF][START_REF] Salama | An ecological study of lactic acid bacteria: isolation of new strains of Lactococcus including Lactococcus lactis subspecies cremoris[END_REF]. Industrially, this mesophilic and homofermentative bacterium is extensively used as a starter in the production of many fermented foods, and it is particularly used in the dairy industry for cheesemaking. L. lactis, which has generally regarded as safe (GRAS) status, is involved in milk acidification by the formation of lactate, proteolysis, and in the synthesis of aromatic compounds, polysaccharides, and bacteriocins, contributing therefore to the taste, texture, and safety of dairy products [START_REF] Ayad | Flavour forming abilities and amino acid requirements of Lactococcus lactis strains isolated from artisanal and non-dairy origin[END_REF][START_REF] Ayala-Hernandez | Production, isolation and characterization of exopolysaccharides produced by Lactococcus lactis subsp. cremoris JFR1 and their interaction with milk proteins: effect of pH and media composition[END_REF][START_REF] Cocaign-Bousquet | Physiology of pyruvate metabolism in Lactococcus lactis[END_REF][START_REF] Daly | Technological and health benefits of dairy starter cultures[END_REF][START_REF] Piard | Purification and partial characterization of lacticin 481, a lanthionine containing bacteriocin produced by Lactococcus lactis subsp. lactis CNRZ 481[END_REF].

L. lactis, which is by far the best characterized lactic acid bacterium (LAB) with respect to its physiology, metabolic pathways, and regulatory mechanisms, has become the model bacterium for most of the LAB research in biotechnology. The genomes of several L. lactis strains have thus been determined, including strains of L. lactis subsp. lactis, such as IL1403 (8), KF147 [START_REF] Siezen | Complete genome sequence of Lactococcus lactis subsp. lactis KF147, a plant associated lactic acid bacterium[END_REF], and CV56 [START_REF] Gao | Complete genome sequence of Lactococcus lactis subsp. lactis CV56, a probiotic strain isolated from the vaginas of healthy women[END_REF], as well as strains of L. lactis subsp. cremoris, such as MG1363 [START_REF] Wegmann | Complete genome sequence of the prototype lactic acid bacterium Lactococcus lactis subsp. cremoris MG1363[END_REF] and SK11 [START_REF] Makarova | Comparative genomics of the lactic acid bacteria[END_REF]. We report here the genome of L. lactis subsp. lactis TOMSC161, a strain isolated from the dairy environment and more specifically from a scaled nonscalded curd pressed cheese, and presenting very interesting technological properties (acidifying, proteolytic, and texturing activities).

Sequencing was performed by Imagif (CNRS, Gif-sur-Yvette, France) using HiSeq paired-end sequencing. A total of 20,391,131 reads were used for assembly by using the Velvet and VelvetOptimiser softwares, leading to an average coverage of 730ϫ. A total of 56 large contigs ranging from 1,013 to 431,322 bp in size were generated and annotated using AGMIAL, an integrated bacterial genome annotation platform [START_REF] Bryson | AGMIAL: implementing an annotation strategy for prokaryote genomes as a distributed system[END_REF].

The genome of L. lactis subsp. lactis TOMSC161 consists of 2,603,548 bp, with an average GϩC content of 35.09%. It contains 2,686 protein-coding sequences covering 86% of the genome, of which 1,991 were annotated with known biological functions and 695 encode hypothetical proteins. It also harbors 53 tRNA-coding genes and 5 rRNA-coding operons.

Analysis of the genome of L. lactis TOMSC161 revealed an adaptation to milk, a nutrient-rich environment. The presence of the complete set of genes involved in lactose and galactose metabolism, as well as the presence of a wide variety of substrate transporters (carbohydrates, peptides, and amino acids) and inversely, the reduction of several amino acid biosynthetic capacities (auxotrophic for glutamate and aspartate) demonstrated this adaptation. However, the genome of L. lactis TOMSC161 still contains genes involved in the utilization of certain plant sugars (mannose, mannitol, arabinose, ribose, and xylose) related to the original habitat of L. lactis. Furthermore, the analysis showed the presence of genes involved in exopolysaccharide and bacteriocin (e.g., lactococcin) production, which are interesting technological features.

The availability of the genome sequence of L. lactis TOMSC161 will allow the powerful application of transcriptomics and proteomics to investigate the metabolic responses and thus the physiological processes involved in the adaptation to different environmental conditions.

Nucleotide sequence accession numbers. The genome sequence and annotation of L. lactis subsp. lactis TOMSC161 have been deposited in the EMBL database under accession numbers CBUJ010000001 to CBUJ010000062.

Genome AnnouncementsNovember/December 2014 Volume 2 Issue 6 e01121-14 genomea.asm.org 1

Genome Announcements 2 genomea.asm.org November/December 2014 Volume 2 Issue 6 e01121-14

ACKNOWLEDGMENTS

This work was supported by the Centre National Interprofessionnel de l'Économie Laitière (Paris, France).

We thank Savoîcime for providing the reference strain and the MIGALE bioinformatics platform for providing computational resources (INRA, Jouy-en-Josas, France)


Rémi Gaudin
email: remi.gaudin@supagro.fr

Berthe Rasoamampionona

Lilia Rabeharisoa

Field-specific difficulties for transplanted rice to cope with ammonia from a localized enriched environment

Keywords: Lowland, urea super-granule, Madagascar, nutritional disorder, ferrous toxicity

The objective of the study was to find some relationship between the location of rice fields in a typical valley of central Madagascar and the response of transplanted rice to nitrogen fertilizer. Comparison was made between lowland locations and plain location. High rainfalls are typically occurring during the periods of soil puddling and rice transplantation. As a consequence, it is difficult to manage nitrogen fertilization of the crop. To avoid nitrogen losses by runoff, urea deep placement (2 g urea-supergranules at 10 cm depth) was practiced. Probes attached to urea super-granules enabled to observe the dynamics of the ammonia derived from the fertilizer. These observations were performed in three fields (different location). The response of transplanted rice to nitrogen was weak in the lowlands, and rice performed badly in ammonia uptake. In the adjacent plain, the response was better and ammonia correctly assimilated. Ferrous ion toxicity and low phosphorus status are discussed as causes for the rice difficulty to benefit from an ammonia enriched environment in the lowland fields.

INTRODUCTION

Transplanted rice is the dominant crop in Madagascar, well before upland rice, cassava, yam, potato and corn. It is cultivated in alluvial plains and in numerous lowlands of the central highland. The alluvial plains are fertile and can even support two crops per year in favorable climate (Marovoay in the western part of the country). But the dominant region for rice cropping is the central high plateau. Under the pressure of the central kingdom regime (period of time spanning several centuries), farmers transformed the landscape of inland valleys, marshes and alluvial plains [START_REF] Randrianja | Madagascar. A Short History[END_REF] in order to obtain soil conditions suited for wetland rice cultivation. Field bunding is very ancient in contrast to its recent introduction in West African countries such as Ivory Coast [START_REF] Touré | Response of lowland rice to agronomic management under different hydrological regimes in an inland valley of Ivory Coast[END_REF]. The mean yield of wetland rice is low, around 2 Mg.ha -1 [START_REF] Minten | Agricultural technology, productivity, and poverty in Madagascar[END_REF]. Most of the farmers grow biological rice because they have no access to fertilizers (or very limited access to fertilizers). Moreover, the island which has been at the origin of the "system of rice intensification" does not succeed in its development because the organic manure it requires [START_REF] Tsujimoto | Soil management: The key factors for higher productivity in the fields utilizing the system of rice intensification (SRI) in the central highland of Madagascar[END_REF] is not produced in sufficient quantity for large areas.

Principal constraints of the central high plateau are the rainfall regime as well as the location of the fields within valleys arranged as fingers in the landscape. Very intense and numerous rain events are observed between November and February. Water circulates directly (over the field bunds) from higher to lower fields of the lowlands impeding any control of the paddy soil humidity. It is collective adjustment of the floodwater level which enables wetland rice cultivation.

To insure food security in the future, the paddy yield has to rise. A seemingly easy solution should be the use of fertilizers by farmers. This investigation aimed at defining the response of transplanted rice to fertilizer in relation with the field situation in the landscape. The lowland situations were compared to the nearest alluvial plain situation.

MATERIALS AND METHODS

Agronomic practices

Three sites of the Ambohitrakoho lowland (18°46'34"S, 47°25'17"E towards 18°47'S, 47°26'06"E) were chosen for the installation of a trial comparing the following NPK treatments: 0-0-0 and 58-90-45. The site positions were selected according to the degree of wetness during the dry season (Figure 1): up and down positions totally or partly saturated (water table depth at less than 20 cm from the soil surface), and plain position with good drainage (water table descending 50 to 100 cm below the soil surface). The trial covered an area of 343 m 2 (up position) or 365 m 2 , representing 49 and 63% of the field in the up and down positions, and only 15% of the field in the plain.

Wetland rice (local 1632 variety) was transplanted in 20 cm x 20 cm geometry. Three seedlings (three weeks old) were used per hill. Transplanting was at the same date (13 November) for the up and down situations; it occurred one month later for the plain situation because it takes a certain time for the first rains to deliver sufficient water to permit harrowing and puddling after the plowing operation that has taken place some weeks before. Harvest was practiced at 120 DAT (days after transplanting). In the fertilizer treatment, nitrogen was deep placed because it is the only way to avoid nitrogen loss through runoff during the rainy season in central Madagascar [START_REF] Dupuy | Etude à l'aide d'urée enrichie en 15N de la fertilisation du riz inondé à Madagascar. I-Comparaison entre supergranules d'urée, perlurée et solution d'urée placés à 1 cm ou 10 cm de profondeur[END_REF]. N was applied at transplanting as 2 g urea supergranules (USG) placed by hand 10 cm deep between four hills. P was brought as hydroxy apatite and K as potassium chloride, mixed with the soil at the end of puddling operation. Each trial had four replications. The number of plots was 8. Bunds 30 cm large separated the plots and enabled passage for soil solution sampling.

Analytical measurements

The disappearance of ammonia from deep placed USG was monitored by measuring the ammonia content of soil solution. This soil solution was drawn by vacuum action (manual pump) acting on small ceramic cup attached to USG, as described in [START_REF] Gaudin | Ammoniacal nutrition of transplanted rice fertilized with large urea granules[END_REF]. In this position, the disappearance of ammonia is driven first by diffusion then by root uptake, with a transition at around 10 mM ammoniacal-N [START_REF] Gaudin | The kinetics of ammonia disappearance from deep-placed urea supergranules (USG) in transplanted rice: The effects of split USG application and PK fertiliser[END_REF][START_REF] Gaudin | Is the sourcesink relationship in transplanted rice receiving deep-placed urea supergranules dependent upon the geometry of transplanting? Paddy and[END_REF]. The urea content of soil solution was also monitored in order to verify full urea hydrolysis. To this effect, a mean sample was built with the volumes left by ammonia determination. Urea and ammonia were determined by colorimetric methods, respectively diacetylmonoximthiosemicarbazide [START_REF] Douglas | Colorimetric determination of microgram quantities of urea[END_REF] and indophenol-blue [START_REF] Novozamsky | Total nitrogen determination in plant material by means of the indophenol-blue method[END_REF] methods.

Statistical analysis

An analysis of variance (ANOVA) was performed on the yield. The analysis was performed according to the procedure described in [START_REF] Little | Agricultural Experimentation. Design and Analysis[END_REF]. The test of Snedecor-Fisher was applied. The observed F value (F for Fisher), defined as the ratio of the mean square of factor (position, fertilizer) to the mean square of error, was calculated and compared with the required F values for significance at the 5% and 1% probability levels.

RESULTS

Grain yields

The yield of control was 1.7 and 1.8 Mg.ha -1 in the lowland situation and 2.4 Mg.ha -1 in the plain. The fertilizer a pronounced effect in the plain (Table 1) with an increase of 1.5 Mg.ha -1 in paddy yield. The ANOVA indicates very significant effects of position and fertilizer with observed F larger than required F at the 1% probability level (Table 2).

Urea hydrolysis

The hydrolysis was rapid in the first few days. In the following weeks, residual urea-N was observed (Figure 2). In this respect, the three sites presented the same kinetics.

Ammonia disappearance

During six weeks, the kinetics of ammonia disappearance was in agreement with point-like positioning of ammonia and its spherical diffusion. The diffusion coefficient, calculated from the ammonia concentration in the plain situation (0.2845 M at 4 DAT) and assuming 0.6 cm 3 .cm -3 porosity was 1.06 cm 2 .d -1 . This value is similar to ammonia nitrogen diffusion coefficient values obtained in other paddy fields of Madagascar.

Around 45 DAT and beyond, it was possible to draw a straight line to distinguish between kinetics of plain and lowland fields (Figure 2). Ammonia disappearance occurred quickly in the plain and with less rapidity (and with more heterogeneity) in the two lowland situations. Figure 2: Time-course of ammonia-N and urea-N concentrations at the placement sites of 2 g USG for three field positions: up, down and plain. The theoretical curve is for an un-transplanted situation with a diffusion coefficient for ammonia at 1.06 cm 2 d -1 . The broken line separates data in the up and down situations (points above the line) from data in plain situation (points below the line).

DISCUSSION

Rice soils of the highlands are derived mostly from highly weathered ferrallitic clay and peat materials. Important constraints are low available soil P and/or high P fixation, N deficit in mineral soils, slow organic matter decomposition and poor nutrient uptake due to low temperature, and iron toxicity [START_REF] Balasubramanian | Rice soil constraints and fertility management in the highlands of Madagascar, in Fragile lives in fragile ecosystems[END_REF]. In the present case, no evident symptom of iron toxicity such as bronzing was found.

Iron toxicity is a complex phenomenon [START_REF] Becker | Iron toxicity in riceconditions and management concepts[END_REF] and it is plausible that differences in the hydrological regime between fields can be sufficiently important as to interfere with this phenomenon. In an inland valley of Ivory Coast, Touré et al. (2010) has observed difference of rice yields in relation with field hydrological regime and position. The hydrological regime is clearly identified by [START_REF] Fageria | Iron toxicity in lowland rice[END_REF] as one factor on which the rice grower can intervene to limit the occurrence of iron toxicity: good drainage enables elimination of part of the ferrous iron and transformations of the noneliminated iron between its various chemical forms. Some of these transformations are influenced by the rhizosphere.

The present study shows that the coupling between the ammonia enriched environment and the rice roots was correct in the plain when in the lowland, things occurred as if rice roots were unable to provide the carbon for ammonia assimilation. [START_REF] Becker | Iron toxicity in riceconditions and management concepts[END_REF] noted that in paddy soils producing high concentrations of ferrous ion, the roots may partly avoid iron uptake by oxidizing the proximate environment (formation of iron plaques around rice roots).

In our case, it is possible that a large part of the photosynthates was dedicated to this mechanism, allowing insufficient carbon for ammonia assimilation. This could explain the weak response of transplanted rice in the lowland situations. In the plain, the response of rice to fertilizer is correct. Moreover, it is possible to increase the yield with increased PK application and split application of USG [START_REF] Gaudin | The kinetics of ammonia disappearance from deep-placed urea supergranules (USG) in transplanted rice: The effects of split USG application and PK fertiliser[END_REF]. The influence of P was already demonstrated by [START_REF] Rabeharisoa | Larger bioavailability of soil phosphorus for irrigated rice compared with rainfed rice in Madagascar: results from a soil and plant survey[END_REF].

Conclusion

Comparing lowland situations and plain situation, we found that transplanted rice fertilized with deep-placed urea supergranules did not uptake ammonia at the same rate. Ammonia was badly assimilated in the lowland. In the plain, ammonia was correctly assimilated and the grain yield difference with control was very significant. This dual response shows that the paddy soils are complex, with interaction between hydrological factors and parental material of the soil. Progress can be made by a better coordination of drainage in lowland fields during the dry season: it should permit counter-season crops such as potatoes or leguminous crops and it should place these fields in a better trajectory to avoid ferrous ion toxicity in the following wet season.

COMPETING INTERESTS

The authors declare they have no competing interests.

AUTHORS' CONTRIBUTIONS

RG supervised manuscript preparation and contributed to data collection and analysis. LR contributed to fields' selection and data collection. BR contributed to soil solution sampling and analysis.

Figure 1 :

 1 Figure 1: Map of the lowland of Ambohitrakoho with the three localizations (up, low and plain) of the trials led by the team of Antananarivo University. Shade of blue related to the degree of wetness in dry season (full blue for permanent saturation of the soil). The plain is extending several km to Mahitsy, receiving waters from a dozen lowlands.

Table 1 :

 1 Grain yield (Mg.ha -1 ).

	Position	Control	USG (N=58)
		(NPK = 0-0-0)	with PK = 90-45
	Up	1.73	1.96
	Low	1.81	2.10
	Plain	2.38	3.94

Table 2 :

 2 Analysis of variance of yield with position and fertilizer treatments

	Source of	Degree of	Sum of	Mean	Observed	Required F
	variation	freedom	squares	square	F	5%	1%
	Total	23	15.050
	Position	2	8.571	4.2855	23.71	3.49	5.85
	Fertilizer	1	2.864	2.864	15.85	4.35	8.10
	Error	20	3.615	0.18075

ACKNOWLEDGEMENTS

The authors thank Samuel Randrianarison for monitoring soil solution extraction in the fields.