Ephyra jellyfish as a new model for ecotoxicological bioassays

Accepted Manuscript Ephyra Jellyfish as a new model for ecotoxicological bioassays M. Faimali, F. Garaventa, V. Piazza, E. Costa, G. Greco, V. Mazzola, M. Beltrandi, E. Bongiovanni, S. Lavorano, G. Gnone PII:

S0141-1136(13)00119-0

DOI:

10.1016/j.marenvres.2013.07.004

Reference:

MERE 3766

To appear in:

Marine Environmental Research

Received Date: 23 April 2013 Revised Date:

8 July 2013

Accepted Date: 15 July 2013

Please cite this article as: Faimali, M., Garaventa, F., Piazza, V., Costa, E., Greco, G., Mazzola, V., Beltrandi, M., Bongiovanni, E., Lavorano, S., Gnone, G., Ephyra Jellyfish as a new model for ecotoxicological bioassays, Marine Environmental Research (2013), doi: 10.1016/ j.marenvres.2013.07.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

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Ephyra Jellyfish as a new model for ecotoxicological bioassays

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Faimali M.1, Garaventa F.1, Piazza V.1, Costa E.1,2, Greco G.1, Mazzola V.1,2, Beltrandi M.2,

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Bongiovanni E.2, Lavorano S.2, Gnone G. 2

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CNR - Institute of Marine Sciences (ISMAR), Genoa, Italy

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Acquario di Genova - Costa Edutainment S.p.A, Genova, Italy.

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Abstract

The aim of this study was a preliminary investigation on the possibility of using the ephyra of

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Scyphozoan jellyfish Aurelia aurita (Linnaeus, 1758), the common moon jellyfish, as an innovative

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model organism in marine ecotoxicology.

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A series of sequential experiments have been carried out in laboratory in order to investigate the

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influence of different culturing and methodological parameters (temperature, photoperiod, ephyrae

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density and age) on behavioural end-points (% of Frequency of Pulsations) and standardize a testing

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protocol. After that, the organism have been exposed to two well known reference toxic compounds

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(Cadmium Nitrate and SDS) in order to analyse the acute and behavioural responses during static

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exposure. Results of this work indicates that the proposed behavioural end-point, frequency of

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pulsations (Fp), is an easily measurable one and can be used coupled with an acute one

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(immobilization) and that ephyrae of jellyfish are very promising model organisms for

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ecotoxicological investigation.

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Keywords: Ecotoxicology, larval bioassay, sublethal effects, jellyfish, swimming speed, SDS,

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Cadmium nitrate, Aurelia aurita

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1. Introduction

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In ecotoxicology there is always a urgent need to identify new model organisms for their use in the

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development of sensitive and reliable test methods for laboratory testing (bioassays).

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Gelatinous zooplankton are not represented in routine ecotoxicology even though over the past few

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decades the importance of its role in marine ecosystems balance has become widely recognised.

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Gelatinous zooplankton comprise the phyla Cnidaria (including siphonophores, corals, hydrozoans,

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and scyphozoans), Ctenophora (‘comb jellies’) and Chordata (pyrosomes, appendicularia, doliolids

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and salps). Cnidarians occupy a key evolutionary position as basal metazoans and are ecologically

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important both as predators and preys in the aquatic ecosystems.

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Among the cnidarians only benthic organisms have been used recently in toxicity testing, including

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hydras, colonial hydroids, sea anemones, and scleractinian corals and sub-lethal endpoints including

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budding, regeneration, gametogenesis, mucus production and larval metamorphosis have been

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developed.

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The Hydra spp. are the most widely used cnidarians in toxicity testing (Wilby and Tesh, 1990;

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Blaise and Kusui 1997; Pollino and Holdway, 1999; Karntanut and Pascoe, 2000; Holdway et al.

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2001; Pascoe et al. 2003), other cnidarians are occasionally used (e.g., anemones, Mercier et al.

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1997; colonial hydroids, Chicu et al. 2000) in toxicity testing with dissolved contaminants.

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As regards hydras, the most commonly observed endpoints are sub-lethal, such as budding, polyp

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structure and polyp regeneration. Most laboratory strains of hydras reproduce primarily by budding,

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but sexually reproducing strains have also been used to test the effects of chemicals on

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gametogenesis (Segner et al. 2003; Fukuhori et al. 2005). However a protocol for toxicity testing

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with hydras has not yet been standardized.

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In addition to solitary hydras, colonial marine hydrozoans belonging to the genus Hydractinia are

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widely used in studies of development, metamorphosis and immune response (reviewed by Frank et

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al. 2001 and Muller and Leitz 2002) but they have not been fully exploited as a model for

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investigation of toxicity.

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Even anemones have not been used extensively in toxicity testing although in 2005 Harter and

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Matthews exposed the small burrowing anemone, Nematostella vectensis, to acute and chronic

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doses of cadmium chloride and measured mortality, change in weight and egg production.

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Scleractinian corals have been used in the past to evaluate the effects of chemicals including

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herbicides, petroleum products and dispersants (Epstein et al. 2000; Negri and Heyward 2001;

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Tarrant et al. 2004; Jones 2005) by measuring endpoints such as tissue thickness, skeletal growth,

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fecundity, fertilization, and larval metamorphosis. As test species, scleractinian corals have a clear

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ecological relevance, but are difficult to be maintained in laboratory and generally take several

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years to reach reproductive maturity.

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Data regarding the use of gelatinous plankton as ecotoxicological model are very scarce in

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literature, and that is a big lack because jellyfish may be useful proxies.

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In fact, jellyfish may be exposed to chemicals through uptake of dissolved compounds, through

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diffusion, ingestion of food, or contact with suspended solids and sediments. In addition to organic

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chemicals, heavy metals can also accumulate in tissues (Mitchelmore et al. 2003) and can be

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transferred up the food chain. For example, high levels of cadmium and other metals in leatherback

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turtles are thought to be derived from their jellyfish prey (Caurant et al. 1999).

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Among the cnidarians, jellyfish play a very important role in the ocean ecosystem. They prey on

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planktonic organisms like crustaceans, copepods, and fish larvae. In turn, some species of sea

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turtles, fishes (like sunfishes, for instance) and sea birds feed on jellyfish. As predators, gelatinous

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organisms can dramatically change the composition of plankton and may impair fisheries yield

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through dietary overlap and direct predation.

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Jellyfish aggregations or blooms are common episodic events caused and maintained by a

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combination of physical and poorly understood behavioural and physiological processes (Lotan et

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al. 1994; Purcell et al. 2000; Graham et al. 2003; Lucas 2001) that may also be aggravated by

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anthropogenic factors (Mills 2001) including overfishing of competitors (Brodeur et al. 2002),

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eutrophication and changes in estuarine circulation (Xian et al. 2005).

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These are the reasons that have led to a preliminary investigation on the possibility of using the

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ephyra stage of Scyphozoan jellyfish Aurelia aurita (Linnaeus, 1758), the common moon jellyfish,

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as an innovative model organism in marine ecotoxicology.

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A series of sequential experiments have been carried out in laboratory in order to, investigate the

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influence of different culturing and methodological parameters (temperature, photoperiod, ephyrae

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density and age) on considered end-points and to standardize a protocol for organisms’ maintenance

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and testing. After that, the organism have been exposed to two well known reference toxic

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compounds in order to assess the possibility of using ephyrae as a proxy in ecotoxicological testing

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Two end-points have been chosen: the number of pulsation in a time unit and the organism ability

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to perform any kind of movement (respectively measured as % of Frequency Pulsation Alteration

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and % of Immobilization). The end-points have been measured using an automatic recording system

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coupled with video graphics analyser (Swimming Behavioral Recorded - SBR) already used with

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other marine organisms (Faimali et al. 2006, Garaventa et al. 2010).

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2. Materials & Methods

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2.1 Model organism and End-points

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Aurelia aurita (Linnaeus, 1758) is a Scyphozoan jellyfish, commonly known as moon jellyfish. It is

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a cosmopolitan species with a worldwide distribution in neritic waters between 70 °N and 40 °S

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(Kramp, 1961; Russel, 1970). A. aurita has two main stages in its life cycle, the polyp stage

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(asexual reproduction) and the medusa stage (sexual reproduction). Polips produce additional

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ephyrae by strobilation. Ephyrae used in these experiments have been obtained directly from polyps

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bred at CNR - ISMAR laboratories.

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Two end-points have been evaluated, one sub-lethal and the second one acute. The sub-lethal is the

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frequency of pulsations (Fp), defined as the number of pulsations made by the ephyra within a

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defined time-unit (one minute), measured as % of Pulsation Alteration (compared to the Control).

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The acute one consists in the evaluation of the organism ability to perform any kind of movements

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(immobilization), measured as % of Immobilization (compared to the Control).

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Both the end-points have been measured using an automatic recording system coupled with a

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specifically designed video graphics analyser (Swimming Behavioral Recorded – SBR).

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The Swimming Behaviour Recorder system (SBR), developed at ISMAR-CNR, is a video camera-

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based system, coupled with an image analysis software, specifically designed to track and analyse

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linear swimming speed of aquatic invertebrates. The experimental set up consists of a video camera

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with a macro-objective, which records the paths of a sample of larvae swimming in a small

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recording chamber. The apparatus is caged inside a black box (60·60·100 cm3) to exclude external

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sources of light, and the recording chamber is monitored under infrared light. Swimming behaviour

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is digitally recorded by a frame grabber plugged into a PC. Images are analysed through an

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advanced image processing software (SBR System developed by e-magine IT, Genova, Italy) to

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obtain the reconstruction of individual path-tracks and the measurement of the average swimming

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speed (mm/ s) for each sample (Faimali et al., 2006; Garaventa et al., 2010). A specific software for

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the recording and analysis of swimming behaviour of ephyrae has not yet been developed, and the

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current SBR system has been adapted to be used for measuring their frequency of pulsation (Fp).

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In this work, experiments with ephyrae were performed using multi-well plates with round-shaped

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wells, in order to remove refractive effects of infrared light and to avoid the possibility of

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mechanical damages for the organisms. The recording time was 1 minute; the SBR may record both

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in light and dark conditions.

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2.2 Experimental activities

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2.2.1 Obtainment of ephyrae

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Colonies of polyps attached on PVC tubes have been received from the laboratories of the

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“Acquario di Genova, Costa Edutainment S.p.A.”, transported to CNR - ISMAR and placed in the

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thermostatic room at 20 °C in 1.5 L dark plastic tanks, covered with a lid in order to keep polyps in

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dark conditions. Tanks were filled with filtered natural sea water (FNSW, 37 ‰ salinity) and gently

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aerated. Polyps were fed daily with nauplii of Artemia salina (about 40 specimens/ml) and water

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was changed every two days. Strobilation has been induced by thermic shock: PVC tubes with

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polyps were moved into 1.5 L dark plastic tanks filled with natural sea water at 10 °C. During this

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period, in order to not cause stress, polyps were not fed and seawater was not changed. Once

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released by strobilation, ephyrae were poured into a beaker and immediately used for the assay or

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bred by keeping them in a 6 L plastic tank filled with gently aerated FNSW (salinity 32 ‰) at 20 °C

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(photoperiod 12:12 light:dark). During breeding, ephyrae were fed daily with nauplii of A. salina

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(about 2 ml with a density of 40 specimens/ml) and water was changed every two days.

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142 2.2.2 Toxicity test protocol standardization

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In order to standardize the protocol of the toxicity assay, it has been necessary to set some of the

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main test parameters: Density of exposed organisms (Experiment 1), Photoperiod (Experiment 2)

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and Temperature and ephyrae age (Experiment 3). To carefully identify the proper parameters,

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these bioassay have been performed considering the most sensitive end-point, the sub-lethal one

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namely the frequency of pulsations (Fp). The experiments were performed in sequence, for that the

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informations obtained from the results of the first one have been applied to the second and then to

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third one. All the tests have been carried out using filtered natural sea water (0.22 µm FNSW) and

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placing ephyrae, collected immediately after strobilation, in a polystyrene 4 ml multi-well plate.

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Test conditions of these preliminary experiments were the following:

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Experiment 1 – Organisms Density and Recording Light Conditions: different ephyrae densities

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have been selected: 2, 4, 8 and 12 specimens were placed into each well with 2 ml of FNSW, for

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each density 3 replicates were prepared. Plates were then sealed and kept for 24 hours in a

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thermostatic chamber at 20°C in dark conditions; then the frequency of pulsation (Fp) was recorded

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for 1 minute using the SBR both in light and dark conditions.

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Experiment 2 - Photoperiod: one ephyra was placed into a well with 2 ml of FNSW, five replica

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plates were prepared each sealed and kept in thermostatic room at 20°C for 24 hours in different

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photoperiods: totally dark (24h D), 12 hours light:12 hours :dark (12h L: 12h D) and totally light

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(24h L). After 24 hours the frequency of pulsation was recorded for all of them in dark conditions.

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Experiment 3 – Ephyrae age and Temperature: one ephyra was placed into a well with 2 ml of

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FNSW, ten replica wells have been prepared for each temperature series and then plates have been

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sealed and kept in thermostatic room at 10, 20 or 30°C for 24 hours. After 24 hours the frequency of

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pulsation was recorded in dark conditions. After 7, 14, 21 and 28 days of ephyrae breeding in the

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maintenance tanks, the experiment has been repeated.

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2.2.3 Toxicity tests

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After the first experimental phase (described in 2.2.2), where test parameters of the toxicity assay

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were defined, a toxicity test using the two different reference toxicants has been performed in order

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to assess the possibility of using ephyrae as a proxy in ecotoxicological testing.

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Experimental parameters adopted in this phase were established from results obtained in the first

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part of the work. Toxicity tests have been prepared using ephyrae collected immediately after

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strobilation (0 days old ephyrae), organisms were placed individually into a multi-well plate

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containing 2 ml of test solution (one individual for each well). For each concentration, three

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replicates have been prepared, each replicate consisting of 8 wells containing one ephyra. Plates

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were then sealed and kept in the thermostatic room at 20°C in dark conditions, after 24 and 48 hours

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both the acute end-point and the sub-lethal one were evaluated using the SBR set to record in dark

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condition for 1 minute.

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The selected reference toxic compounds were Cadmium nitrate (CdNO3)2 and sodium dodecyl

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sulphate (SDS). SDS (CH3(CH2)11OSO3Na) is an anionic surfactant capable of emulsifying lipids

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and low-surface tension aqueous solutions. SDS was purchased from Sigma Aldrich (St. Louis,

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MO)..

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Cadmium Nitrate Cd(NO3)2 was purchased from Sigma Aldrich (St.Louis, MO). It is commercially

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available in a 2% HNO3 standard solution for AA at a concentration of 1000 ppm. Stock solutions

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of SDS and Cadmium Nitrate were prepared in ASW (artificial sea water, Instant Ocean®, 37‰).

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Tested concentrations were: 0 (Ctr) - 0,01 - 0,05 - 0,1 - 0,5 – 1 - 5 mg·L-1 for Cd(NO3)2 and 0 (Ctr) -

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0,1 - 0,5 – 1 – 5 – 10 – 50 mg·L-1 for SDS.

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2.2.4 Data processing and statistical analysis

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Analysis of Variance (ANOVA) was employed to test the effects of experimental treatments on the

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end-points. Prior to analysis the assumption of the homogeneity of variances was tested by

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Cochran’s test. Student-Newman-Keuls test (SNK test) was performed for the a posteriori

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comparisons on the means (Underwood, 1997).

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ANOVA was applied to the following experimental designs:

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Experiment 1: 2 factors, Organisms Density (OD) fixed with 4 levels and Recording Light

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Condition (RLC) fixed and orthogonal with 2 levels.

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Experiment 2: 1 factor, Photoperiod with 3 levels

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Experiment 3: 2 factors. Ephyrae Age (EA) fixed with 5 levels and Temperature (T) fixed and

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orthogonal to factor 1 with 3 levels.

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Data from toxicity tests have been analysed as follow: the Median Effective Concentration

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(expected to produce a 50% effect both on the immobilization and on the alteration of Frequency of

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pulsations), EC50 and related 95% Confidence Limits (CL) were calculated using trimmed

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Spearman–Karber analysis (Finney, 1978) after 24 and 48 hours. One-way analysis of variance

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(ANOVA), followed by Student Newman–Keuls (SNK) test pair-wise comparison, was performed

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to calculate the Lowest Observed Effect Concentration (LOEC) for % of alteration of Frequency of

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pulsations (% Fp).

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3. Results

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Experiment 1 – Density and Recording Light Conditions

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Results of the first experiment, aimed to investigate the influence on the sub-lethal end-point

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(Frequency of Pulsation, Fp) of Organisms Density(OD) in the presence and absence of light during

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the recording phase (Recording Light Condition, RLC) with SBR, are reported in Figure 1. In order

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to satisfy the assumption of the homogeneity of variances data have been Ln (X+1) transformed.

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The 2-way ANOVA pointed out a significant effect of both factors but not of their interaction on

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the considered end-point (OD: F=9.11, p<0.01; RLC: F=56.49, p<0.01; OD×RLC: F=1.10,

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p=0.3786). The overall effect of Recording Light Conditions identify Dark Condition as the proper

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recording setting and the a posteriori comparison of the means identify 2 ephyrae for each well as

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the best Organisms Density; in fact, under these experimental conditions it is possible to observe a

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Frequency of Pulsation equal to 34.90 ± 1.12 puls/min, that is the higher observed value (p < 0.01).

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On the contrary, even if in light conditions Fp values are significantly lower than those recorded in

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dark ones, the number of ephyrae does not seem to affect the end-point, with the exception of Fp

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obtained considering a density of 8 ephyrae (p<0.05).

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#HERE FIGURE 1#

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Experiment 2 – Photoperiod

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The trend of Fp values obtained by maintaining organisms under different photoperiods prior to the

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analysis is reported in Figure 2. The one-way ANOVA highlighted a significant effect of the

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Photoperiod on the sub-lethal end-point (F=49.60, p<0.01). The Frequency of Pulsation

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significantly decreases (SNK test, p< 0.01) with the increase of the hours of light, thus identifying

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the maintenance of ephyrae at dark as the condition to obtain the highest frequency of pulsation (36

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± 1.41 puls/min).

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Experiment 3 – Ephyrae age and Temperature

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Results of the experiment performed to assess the influence of the Ephyrae age (EA) and of

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temperature (T) on the Frequency of Pulsation (Fp) are reported in Figure 3.

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The 2-way ANOVA pointed out a significant effect of both factors but not of their interaction on

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the considered end-point (EA: F=3.86 p<0.01; T: F=860.81 p<0.01; EA×T: F=1.28 p=0.26).

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Considering data obtained from T of 10 and 30°C, it is evident that only newly born ephyrae show a

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recordable pulsation even if very low (8.38 ± 2,12 and 2.75±1.64 puls/min, respectively). Among

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the investigated temperatures, 20°C seems to be the one able to produce a significant and well

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measurable Fp (from a value of 26.88±1.13 for 0 days old ephyrae to 27.25±1.13 for 28 days old

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organisms) that is consistent with the age of organisms.

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#HERE FIGURE 3#

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On the basis of the previous experiment it was possible to define the following parameters to be

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used in the following Toxicity tests (Table 1):

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#HERE TABLE 1#

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3.2 Toxicity tests

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As final part of this study, aimed to the validation of the effectiveness of the proposed

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methodological protocol, a toxicity screening with two different reference toxic substances

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(cadmium nitrate and SDS) was performed. Two end-points were considered: the frequency of

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pulsation (Fp) and the immobilization (I); the percentage of alteration of frequency of pulsation (%

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Fp) and the percentage of immobilization (% I) were calculated, compared to the control.

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The results obtained exposing 0 days old ephyrae to different concentrations of Cadmium Nitrate

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(CdNO3)2 are reported in Figure 4. This compound causes a significant effect on both the end-points

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(24 hours: % I - F = 102.04, p <0.001, % Fp - F = 51.77, p <0.001. 48 hours:% I - F = 260.11, p

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<0.001, Fp - F = 273.25, p <0.001). After 24 hours, a difference in sensitivity between the two end-

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points is observed: in fact the LOEC (Lowest Observed Effect Concentration), calculated by the a

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posteriori pair-wise comparison for the % of immobilization is 0.5 mg·l-1 while for % Fp is 0.1 mg·l-

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extent of the sub-lethal end-point is higher than the acute one (% I = 16.67 ± 4.17 and % FP = -

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40.66 ± 2.9).

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It is also evident how for both end-points from the concentration of 1 mg·l-1 a 100% response is

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obtained and in the range of concentrations between 0.01 and 0.05 mg·l-1 the frequency of pulsation

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is the most sensitive end-point (in terms of magnitude of the response) compared to immobility.

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. This difference is no more evident after 48 hours of exposure (LOEC =0.05 mg·l-1) even if the

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#HERE FIGURE 4#

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Results obtained exposing organisms to SDS are reported in Figure 5. Also this reference toxicant

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turns out to determine a significant effect on both the end-points (1 way ANOVA. 24 hours,% I: - F

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= 56.00 p <0.001; Fp - F = 39.23, p <0.001. 48 hours, % I - F = 69.57, p <0.001, Fp - F = 0.13, p

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<0.001). For both end-points, from the concentration of 10 mg·l-1, a 100% response is observed.

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After 24 hours, a slight hormetic effect on the frequency of pulsation (Fp) in organisms exposed to

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0.5 mg·l-1 of SDS is registered, although it is not significant from a statistical point of view. After

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24 hours of exposure, the LOEC value corresponds to 5 mg·l-1 for both end-points; on the contrary,

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after 48 h of exposure, the frequency of pulsation results to be more sensitive, showing a LOEC

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value of 0.1 mg·l-1.

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#HERE FIGURE 5#

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4. Discussion

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The first part of this work has been directed to characterize some experimental parameters that can

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influence the frequency of pulsation (Fp) of ephyrae of A. aurita, a behavioural end-point identified

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as potentially sub-lethal response of this innovative model for ecotoxicological testing.

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The proposed model organism, shows a marked swimming activity in accordance with what

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reported by several authors, confirming that ephyrae of this species do not just passively float but

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are able to carry out an extremely active swimming behaviour (Rakow and Graham, 2006; Frost et

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al., 2010). Jellyfishes belong to a group of aquatic animals that use periodic contractions of their

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own body to generate ring-vortices of the surrounding medium in which they swim (Peng et.al.,

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2012).

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In literature some studies on the swimming behaviour of adult Scifomeduse can be found; several

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authors have highlighted and characterized the complex patterns of interaction between some key

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parameters, such as the propulsion of swimming speed or trajectory, in order to understand their

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role in the behavioral strategies of these animals (Costello et.al 1994; Ford and Costlow, 1974 ).

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Due to the limited information regarding the ephyra stage of A. aurita, data obtained in this work

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have been in some cases compared to data reported by other authors on behavioural strategies

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observed in the adult stage.

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The first set of experiments performed allowed to understand the influence of different parameters

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and to standardize the bioassay protocol. In Fig. 1 it is possible to observe how the density of the

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organisms in the well significantly affect the number of pulsations. In fact, the frequency of

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pulsation (Fp) decreases proportionally to the increase of the number of ephyrae in the well; this

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result, leads to hypothesize that a decrease of the available space causes a negative larval-larval

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interaction, inhibiting their movement. Considering the effect of Recording Light Conditions

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with the with SBR system, the inverse correlation between density and Fp is observed when the

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recording is performed in dark conditions, whereas light seems to affect pulsation so that the density

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effect results to be covered up. This is confirmed by the SNK test that puts in evidence a significant

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difference only between the mean Fp obtained with 2 ephyrae and the one obtained with 8. For that

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light can be considered one of the key abiotic parameters in the modulation of the pulse frequency.

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The role of light has been deeply investigated in the further experiment, where the inhibition of Fp

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results to be strictly proportional to the increase of light hours exposure (Fig. 2). One hypothesis

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for this ephyrae swimming behaviour in response to light stimulus could be found in a positive

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response (increased propulsion and pulsation) induced by the sense organs responsible for the

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transduction of light stimuli (ropalia, sensitive organs of the medusa that control its position in the

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water and the rhythm of the umbrella contractions) (Purcell, 2008). In fact, a light decrease may be

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seen as a warning of sinking to the bottom. This behaviour could also be interpreted as a secondary

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response related to the search for food that is supposed to follow the movements of the zooplankton.

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Unfortunately, in literature no information about this stage of A. aurita life cycle are available, so it

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is not possible to compare our results and possibly confirm our hypothesis. Furthermore, we must

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also take into account that this species does not present symbiotic zooxanthellae and that therefore

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does not need particular light conditions (Arai, 1987). On the contrary, benthic phase of A. aurita

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appears to be strongly influenced by environmental parameters such as light intensity, temperature

339

and salinity (Purcell, 2008).

340

In any case, from a practical point of view and according to the purposes of this work, it is clear

341

that both light and organisms density play a key role in the modulation of the behavioural response

342

and therefore they must be kept under control during ecotoxicological testing . The results suggest

343

to use during the exposure a number of organisms not exceeding two individuals for each well,

344

keeping them in full dark condition both during the exposure period (24 and 48 hours) and the

345

video recording (the only light source during this phase is an infrared one). As regards the influence

346

of temperature during exposure, results obtained (Fig. 3) show that a decrease or an increase of 10

347

°C from the breeding temperature (20 °C) , lead to a drastic inhibition of the frequency of

348

pulsation (Fp). Further investigations should be carried out to investigate responses to softer

349

temperature enhancements or reductions, in order to precisely identify a range of no effect for this

350

key parameter.

351

It is still not clear how the ontogenetic changes, the movement and morphology of aquatic animals

352

can affect swimming performance, for example Matthew et. al (2003) published an interesting study

353

aimed to understand the hydrodynamic propulsion systems of A. aurita; using video recording and

354

simulation of swimming through computer modelling, the authors stated that, for this species, the

355

number of pulsations decreases with the growth of the individual, thus reducing the swimming

356

speed. Some authors have highlighted the importance of fluid/animal interactions during the

357

ontogenetic development, that appears to be influenced by a marked phenotypic plasticity able to

358

generate adaptation strategies against fluid dynamic alterations imposed by environmental

359

variables, such as in particular temperature (Nawroth et al., 2010), showing that a decrease of

360

temperature determines an energy storage within animal tissues, related to a decrease of propulsion.

361

The frequency of pulsation seems therefore temperature dependent.

362

In literature, the increase of ephyrae age (days of growth) is related to a decrease in Fp at 20°C

363

(Nawroth et.al., 2010), on the contrary our results suggest that, at 20 °C, there is no influence of

364

ephyrae age on the frequency of pulsation, that remains constant till an age equal to 28 days (Fig.

365

3). On the basis of our results, 20°C results to be the optimal temperature for the execution of the

366

bioassay.

367

To validate the bioassay with ephyrae of A. aurita and investigate the reliability and sensitivity of

368

the investigated end-points (frequency of pulsation and immobilization) the methodological

369

protocol, optimized after the first part of this work, was applied using two reference toxicants,

370

whose toxicity data with other model organisms, in particular marine invertebrates, are well known

371

(Cadmium nitrate and SDS).

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372 In this second part of the work, two end-points were simultaneously evaluated: an acute one,

374

immobilization, meaning a total lack of pulsations during the recording time-unit with the SBR

375

system (1 minute ) and a sub-lethal end-point: the alteration of the pulsation frequency (Fp).

376

For each tested reference toxicant, results will be discussed and compared to the toxicity data found

377

in literature. Due to the total absence of available ecotoxicological data for ephyrae of A. aurita ,

378

results will be compared with those obtained with other biological models (crustaceans, mollusks,

379

rotifers, fish etc.).

380

Cadmium nitrate shows, after 24 hours of exposure, an effect for both end-points (acute and sub-

381

lethal), already evident from the concentration of 0, 5 mg·L-1 for immobilization and 0,1 mg·L-1 for

382

the frequency of pulsation (Fp). After 48 hours, the same effects are caused by a lower

383

concentration (0.05 mg L-1) for both end-points. It should be highlighted that both end-points show

384

a 100% response at 1 mg L-1 already after 24 hours of exposure. In Table 1 literature data (LC50

385

and EC50) on the effect of Cadmium Nitrate on other marine organisms are reported.

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386 387 388

#HERE TABLE 2#

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The comparison of EC50 obtained with A. aurita shows that the new biological model appears to be

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the most sensitive among the considered model organisms.

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SDS is a classic reference compound used in toxicological tests, it is representative for the class of

396

surfactants used in oil spill remediation at sea (Singer et.al., 1996).

397

In Table 2, LC50 values of SDS for some of the most representative marine invertebrates are

398

reported and compared with those obtained with the ephyrae of A. aurita in this work.

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#HERE TABLE 3#

401 402 403

As reported in Table 2 e, the 48 hours EC50 values calculated for ephyrae of A. aurita are similar or

404

even lower to the LC50 values showed by some of the most sensitive model organisms found in

405

literature (i.e. A. amphitrite).

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In addition, it is interesting to observe that both with Cd and SDS (Fig. 4 and 5) it is possible to

407

point out the presence of an hormetic phenomenon at the lower tested concentrations Hormesis is

408

represented by a dose-dependent biphasic response, modulated by homeostatic control mechanisms

409

present in all biological systems, that respond independently to changes in state or alteration of the

410

mechanisms regulated and/or induced by exogenous agents (Amendola et.al., 2006). The hormetic

411

responses are characterized by a modest stimulation of the affected function (end-point) at low

412

doses and by an inhibition of the same at high doses. This phenomenon is now widely recognized in

413

the field of ecotoxicology (Calabrese and Baldwin 2001) and the hormetic responses have to be

414

taken into consideration when determining the minimum effect concentration (LOEC).

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415 5. Conclusion

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This experimental work has allowed us to characterize and optimize fundamental breeding and test

418

parameters in order to make a preliminary standardization of a new bioassay, that proposes the use

419

of the ephyrae of A. aurita as a very new and innovative model organism in ecotoxicological

420

studies. The experiments allowed to identify two end-points (sub-lethal, frequency of pulsation and

421

acute, immobilization) with different levels of sensitivity. Experiments have been performed using

422

an automatic recording system coupled with video graphics analyzer (Swimming Behavioral

423

Recorded - SBR), already employed with other marine invertebrates. The SBR has been properly

424

set to investigate the role of some methodological parameters on the ephyrae swimming

425

performance and then it has been used to analyse the acute and behavioural responses during static

426

exposure to two toxic reference substances: Cadmium Nitrate and SDS.

427

Results of this work pointed out that the proposed behavioural end-point, frequency of pulsations

428

(Fp), is an easily measurable one and can be used coupled with an acute one (immobilization).

429

The comparison of the EC50 values obtained in this work for the two reference toxicants, and those

430

from literature, obtained with other marine invertebrates, indicates that jellyfish are very promising

431

model organisms for ecotoxicological investigation.

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Acknowledgements

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The authors acknowledge RITMARE Flagship Project, a National Research Program funded by the

435

Italian Ministry of University and Research.

436 437

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570 FIGURE CAPTIONS Figure 1: Frequency of pulsation (puls/min) after 24 hours of exposition (M±SE, n=3) of an increasing number of ephyrae of A. aurita (organisms density: 2, 4, 8, 12) in dark and light conditions during the recording procedure with SBR (Swimming Behaviour Recorder). RLC= recording light conditions. * = p< 0.01; * = p< 0.05 (2-way ANOVA).

581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597

Figure 3: Frequency of pulsation (puls/min) of ephyrae of A. aurita of different ages (0, 7, 14, 21, 28 days) maintained in culture at three different temperatures 10, 20 and 30°C (M±SE, n=10). * = p< 0.05; * * = p< 0.01 (one-way ANOVA).

TABLE CAPTIONS Table 1: Test parameters to be used in the Toxicity test with ephyrae of A.aurita. Table 2: EC50 - LC50 values reported in literature for some marine model organisms exposed to Cadmium Nitrate. Table 3: EC50 - LC50 values reported in literature for some marine model organisms exposed to

598

SDS.

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Figure 2: Frequency of pulsation (puls/min) of ephyrae of A. aurita after 24 hours of exposure in three different conditions of illumination: dark, with photoperiod (12 h L : 12 h D) and continuous light (M±SE, n=5). * * = p< 0.01 (one-way ANOVA)

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Figure 4: Alteration of Frequency of pulsation (% Fp) and immobility ( % I) of ephyrae of A. aurita after 24 h (a) and 48 h (b) of exposition at increasing concentration of Cadmium Nitrate (M±SE, n=3). * * = p< 0.001 (one-way ANOVA).

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Figure 5: Alteration of Frequency of pulsation (% Fp) and immobility ( % I) of ephyrae of A. aurita after 24 h (a) and 48 h (b) of exposition at increasing concentration of SDS (M±SE, n=3). * * = p< 0.001 (one-way ANOVA).

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Species

Exposure

EC50-LC50

time

(mg/L)

24 h

4,52

48 h

1,55

Artemia salina

48 h

19,1

Greco et.al 2006

Amphibalanus

48 h

7,49

Greco et.al 2006

Americamysis bahia

96 h

6,6

Roberts et.al 1982

Daphnia magna

24 h

45,8

48 h

19,1

organisms Cnidarians

Crustaceans

Aurelia aurita

This study

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Test Parameters 2 Ephyrae for each well

Recording light conditions

Dark

Photoperiod

24h Dark

Ephyrae age

Not significant (at 20 °C)

Temperature

20 °C

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Exposure

EC50-LC50

time

(mg/L)

24 h

0.07

48 h

0.13

Tigriopus brevicornis

96 h

0.47

Forget et al.(1998)

Amphibalanus amphitrite

48 h

0.49

Piazza et al.(2012)

Homarus americanus

96 h

7.8

Johnson et al.(1979)

Mollusc

Anadara granosa

96 h

320

Pierce (2008)

Bacteria

Vibrio fisheri

22 h

0.22

Radix et al.(1999)

Fishes

Baleophthalmus dussumieri

96 h

640

Martins et al.(2007)

Rotifera

Brachionus plicatilis

Cnidarians

24 h

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organisms

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Snell et al. (1991)

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Highlights

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The use of ephyrae of A. aurita as a model organism in ecotoxicology is proposed. A series of preliminary tests was carried out to define a test protocol. Two reference chemicals were used to verify the integrity of the proposed assay. EC50 values point out jellyfish as a promising model organisms for ecotoxicological studies.

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