Role of dietary photoprotective compounds on the performance of shrimp Pleoticus muelleri under UVR stress

Journal Pre-proof Role of dietary photoprotective compounds on the performance of shrimp Pleoticus muelleri under UVR stress M. Alejandra Marcoval, A. Cristina Díaz, M. Laura Espino, Natalia S. Arzoz, Susana M. Velurtas, Jorge L. Fenucci PII:

S0044-8486(19)30625-8

DOI:

https://doi.org/10.1016/j.aquaculture.2019.734564

Reference:

AQUA 734564

To appear in:

Aquaculture

Received Date: 14 March 2019 Revised Date:

30 September 2019

Accepted Date: 1 October 2019

Please cite this article as: Marcoval, M.A., Díaz, A.C., Espino, M.L., Arzoz, N.S., Velurtas, S.M., Fenucci, J.L., Role of dietary photoprotective compounds on the performance of shrimp Pleoticus muelleri under UVR stress, Aquaculture (2019), doi: https://doi.org/10.1016/j.aquaculture.2019.734564. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier B.V.

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Role of dietary photoprotective compounds on the performance of shrimp Pleoticus muelleri under UVR stress.

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Running head: UVR on the performance of Pleoticus muelleri

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* Corresponding author: [email protected]

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*M. Alejandra Marcoval (1,2), A. Cristina Díaz (1,2,3), M. Laura Espino (1,2), Natalia S. Arzoz(1,2), Susana M. Velurtas(2)& Jorge L. Fenucci (1,2) (1) Instituto de Investigaciones Marinas y Costeras (IIMyC), UNMdP/CONICET, Argentina (2) Departamento de Ciencias Marinas, Universidad Nacional de Mar del Plata (UNMdP), Argentina (3) Comisión de Investigaciones Científicas, Argentina

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ABSTRACT

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The aim of this study was to assess the effect of ultraviolet radiation (280–400

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nm) on survival, concentration of photoprotective compounds and total antioxidant

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capacity of Pleoticus muelleri. Two experimental diets were prepared: control diet and

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other supplemented with Undaria pinnatifida extract (3g/100g diet), a brown seaweed

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rich in photoprotective compounds. Animals were exposed for 7 days to two radiation

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treatments: a) PAR (Photosynthetically Active Radiation: 400-700 nm) b) PAR+UVR:

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total radiation spectrum (UVR 280-400 nm + PAR), under controlled conditions (20±0.8

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ºC, S=33, pH=7.5). Shrimp in UVR treatments without supplemented diet, showed the

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highest mortality (72%). Under UVR-stress, concentrations of UV absorbing compounds

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were highest in animals fed the diet supplemented with U. pinnatifida, while the

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carotenoid concentration decreased, and the total antioxidant activity was the highest.

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These results suggest that under in UVR-stress animals could bioaccumulate

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photoprotective compounds from a diet with added seaweed extract.

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1. INTRODUCTION

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Ultraviolet radiation (UVR, 280-400 nm) is a natural component of solar

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radiation, and therefore the marine environment has always been exposed to different

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amounts of UVR, varying historically with the composition of the atmosphere (Tang et

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al. 2011). However, anthropogenic influence via stratospheric ozone depletion has caused

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an increase in the UVR flux to the earth surface; although in recent years the ozone layer

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shows signs of recovery, this is not enough to reach the level of the ozone layer prior of

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1960s (Steinbrecht et al. 2018). In this way, the effects of enhanced UVR on the marine

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environment can be considered a problem (Whitehead et al. 2000, Häder et al. 2010).

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There is some evidence that UVB radiation and the shorter wavelengths of UVA (320–

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400 nm) can penetrate natural waters to the biologically significant depth of 20 m (Häder

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et al. 2003) and cause physiological adverse effects on aquatic organisms such as the

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inhibition of the photosynthetic rate, damage in the genetic material and elevated

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mortality (Worrest & Hader 1997, Halac et al. 2011). The shrimp Pleoticus muelleri is an

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important target species for aquaculture from the coasts of Southern Brazil to Patagonia

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(23-50ºS). In recent years, several works have developed culture techniques for this

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species, and studied important parameters such as nutritional and environmental

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requirements in the different stages of the biological cycle (Fernández Gimenez et al.

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2008, Diaz et al. 2014, Marcoval et al. 2018). These authors have obtained massive

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postlarvae crops from wild spawners or matured and impregnated females in captivity

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(Mallo & Fenucci 2004). However, a bottleneck for the complete development of culture

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technology has been detected: a low growth rate of juvenile in ponds. Several causes may

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induce depletion of juveniles growth in this species: nutrition, pond management, and

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environmental factors, like temperature, and solar radiation effects, considered most

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significant anthropogenic climate changes registered in the Southern Hemisphere

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(Gonçalvez et al. 2010, Williamson et al. 2014). Under culture conditions, individuals of

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shrimp species are kept in ponds at a depth of less than 2 m, so they are exposed to

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extreme environmental conditions.

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Early life stages of P. muelleri are highly vulnerable to UV radiation, but this can

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also be differentially modulated depending on the quality and previous conditioning of

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their diet (Marcoval et al. 2018). These authors showed that when the food had a low

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content of UV-absorbing compounds, the exposure of mysis to UVR resulted in 100%

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mortality rate. Conversely, larvae fed food rich in UV-absorbing compounds had survival

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rates of around 90% in the transition from mysis to postlarvae and bioaccumulated UV-

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absorbing compounds through the diet.

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Food additives are substances that are added to foods to improve them; the

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research is currently aimed toward additives acting not only on the quality of the food,

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but also on the health of the animals in culture. In this sense, seaweeds can be an

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interesting alternative as fed supplement in aquaculture by virtue of their properties. In

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their cell wall, seaweeds have bioactive substances such as sulfated polysaccharides and

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polyphenols with antioxidant activity that can prevent oxidative stress, and when exposed

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to high solar radiation prevent damage from exposure to UV radiation (Pavia et al. 2000,

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Rastogi et al. 2010, Li et al. 2011, Guinea et al. 2012). Undaria pinnatifida, known as

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"wakame", is a brown seaweed (Phaeophyceae) native to northeastern Asia (Akiyama &

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Kurogi 1982). In Argentina, this alien species was detected for the first time in 1992 in

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the Golfo Nuevo (North Patagonia), where its introduction was attributed to transport by

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means of ballast water from international vessels (Casas & Piriz 1996, Piriz & Casas

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2001). In Golfo Nuevo, U. pinnatifida is found at depths from 2 to 20 m, and has been

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able to invade all kinds of hard substrata with different dispersal strategies. Studies on

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this seaweed have revealed its phenolics composition, which includes isoflavones and

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phenolic acids (Klejdus et al. 2010, Onofrejova et al. 2010). Guinea et al. (2012)

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evaluated the potential source of phenolic compounds of 21 commercially available

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seaweed species of Ochrophyta and Rhodophyta to use them to obtain phenolic

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compounds, and/or photo protective agents; they concluded that U. pinnatifida showed an

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intermediate yield of polyphenolic compounds. It has been demonstrated that algal

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polysaccharides play an important role as free radical scavengers in vitro for the

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prevention of oxidative damage in living organisms. Shrimp Artemesia longinaris fed a

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diet supplemented with 2% aqueous extract of U. pinnatifida showed the best antioxidant

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protective capacity due to the greater number of sulfated polysaccharides, which act as

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natural antioxidants (Diaz et al. 2017).

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The aim of this research was to assess the photoprotective effect of the addition of

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an aqueous extract of seaweed U. pinnatifida in the diet on shrimp P. muelleri under

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UVR stress. For this purpose, the effects of this extract on survival, bioaccumulation of

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photoprotective compounds, and free radical scavenging were evaluated.

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2. MATERIALS & METHODS

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2.1. Preparation of water-soluble brown seaweeds extract

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Algae extract was prepared according to the methodology of Fujiki et al. (1992)

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from fine powder of Undaria pinnatifida milled fronds obtained from Soriano S.A

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(Gaiman, Chubut, Argentina). Ten grams of the milled fronds were added to 300 mL of

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deionized water and heated to boiling under reflux for three hours. The suspension was

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filtered through a sintered glass mesh (15 µm). The filtrate was concentrated under

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reduced pressure in a rotavapor. Hot-water extract with a high content of sugars, mainly

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mannitol and fucoidan (Díaz et al. 2017), was dried and stored at -20°C until use.

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2.2. Experimental animals

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Juveniles of Pleoticus muelleri collected with a trawling net from coastal waters

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(38º 02’ S, 57º 30’ W), were placed in 3,500-L cylindrical fiberglass tanks at J.J. Nágera

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Coastal Station, National University of Mar del Plata. Four groups of 45 individuals

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(2.89±0.22 g initial weight) were kept under controlled conditions (20±0.8 ºC, S=33,

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pH=7.5) and fed ad libitum once a day with two diets: two groups with C: Control diet

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and other two groups with U: Control diet supplemented with 3g/100g of U. pinnatifida

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extract (Table 1). The animals were maintained under the aforementioned dietary regime

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during at least two molting periods (30 days; Díaz et al. 2011) to determine the possible

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bioaccumulation of photoprotective compounds.

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At the end of this period, 160 of the surviving animals (randomly selecting 80

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individuals from each dietary treatment) were transferred to 20-L tanks, at a density of 5

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animals per tank (adding up a total of n=32 tanks).

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Shrimps were exposed during 7 days to two radiation treatments: a) PAR

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(photosynthetically active radiation) (400-700 nm), and b) PAR+ UVR (280-700 nm). In

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summary, there were four radiation/diet combinations considered in the experiment, each

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one comprising n=8 tanks and ntotal=40 shrimp. Accordingly, the experimental treatments

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were: PAR/C (i.e. control), PAR+UVR/C, PAR/U. pinnatifida (PAR/U, hereafter), and

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PAR+UVR/U. Light sources were 40 W cool-white fluorescent bulbs (Philips) (for

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PAR), and 2 Q-Pannel UVA-340 bulbs (for UVR). The light regime consisted of an

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average irradiance of 65.4 W m-2 for PAR, 20 W m-2 for UVR (Marcoval et al. 2016).

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A total of 5 tanks from each experimental treatment were allocated for the

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estimation of survival; while the remaining 3 tanks in each treatment were destined for

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the estimation of photoprotective compounds (PPC) in the integument of the animals.

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2.3 Photoprotective compounds (PPCs)

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UV-absorbing compounds (UACs) were measured in the U. pinnatifida extract,

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and PPCs (carotenoids and UACs) content was estimated from P. muelleri juveniles

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integument obtained from of two individuals. This samples was ground and from that,

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n=3 sub-samples were separated for analysis. The analysis was performed according to a

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modified method of Karsten et al. (1998). PPCs were extracted with 5 mL of absolute

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methanol during at least 4 h at 4ºC, after which all samples were ground, sonicated and

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centrifuged at 800g for 15 min and the spectral characteristics of the supernatant were

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measured from 250 to 750 nm using a diode array spectrophotometer (Shimadzu UV-

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2102 PC, UV-visible) (Marcoval et al. 2018).

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The UV absorbing compound contents (UACs) were estimated by peak heights at 330 nm

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of the spectral scans between 250–750 nm (Marcoval et al. 2016). The same spectra

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obtained to determine UACs were also used to estimate total carotenoid concentration

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calculated by using the formula: C = (D × V × 104)/(E × W), where C is the carotenoid

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concentration in the sample (in ug g−1 dry weight); D, absorbance at 472 nm ; V, volume

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of the extract (in mL); E, extinction coefficient (2,500), W, dry weight (in mg) of the

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sample (Alberte & Andersen 1986, Hernandez-Moresino et al. 2014, Meléndez- Martínez

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2017).

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The UACs are expressed as optical density (OD), while carotenoid contents as

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weight of total carotenoids. The number of PPCs was normalized per gram of dry weight,

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which was determined by drying animals in the oven at 40°C for 48 hours until constant

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weight.

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2.4 Antioxidant Activity

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After 7 days, antioxidant activity of the midgut gland homogenate is analyzed

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using DPPH (2, 2-diphenyl-2-picryhydrazyl) as a substrate, through the measuring of

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signal decay of DPPH radical with time and represents the radical tissue reaction.

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Electron paramagnetic resonance experiments were performed at 298 K with a Bruker

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ELEXSYS E500T spectrometer, operating at X band. Typical data acquisition parameters

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were the same methodology as in Díaz et al. (2004).

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2.5. Statistical analysis

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A one-way ANOVA was performed to detect significant differences among

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treatments. Data expressed as percentages (survival) were transformed to arcsine before

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ANOVA analysis considering a significance level of 0.05. The differences among

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antioxidant activity between treatments measured for 2, 2-diphenyl-2-picryhydrazyl

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(DPPH) remnant was estimated by means of analysis of covariance (ANCOVA).

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Statistical differences for survival were determined with the statistical package Vassar

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Stats (v. 2013). In cases of statistically significant differences, the mean values were

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compared with the multiple ranges Tukey’s test (Zar 1999). All the rest of analyses were

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performed with the Statistical Package Statistix 8 (version 2000), with a significance

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level of α = 0.05.

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3. RESULTS The presence of UV absorbing Compounds (UACs) was detected in U. pinnatifida extract (Fig. 1) at concentrations of 0.47±0.052 OD g -1.

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After 7 days of exposure under different radiation, mortality was higher for

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animals exposed to PAR+UVR treatments than in those kept under PAR (ANOVA p<

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0.05). The highest value was detected in PAR+UVR/C treatment (without

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supplementation of U. pinnatifida) which reaching mortality of 72%; in case of

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PAR+UVR/U treatment it was 58%. PAR/C and PAR/U treatments presented mortality

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of 20% and 15%, respectively.

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Concentration of photoprotective compounds during 7 days radiation treatments is

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shown in Figure 2. The highest values of UACs were observed in animals that were

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subjected to UV radiation stress and fed with U. pinnatifida supplemented diet (Fig. 2D)

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(15.5±0.67 OD g-1 ANOVA p< 0.05). In other treatments, its concentrations were similar,

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with values between 1.9±0.08 and 1.63±0.034 OD g-1 (ANOVA p< 0.05). Carotenoids

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concentration in animals under PAR+UVR/U was significantly lower (1.69±0.026) than

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values obtained for shrimp exposed to other treatments, which showed concentration

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values between 45.37±1.35 and 49.2 ±2.36 OD g-1 (Fig. 2 A-B-C). In animals exposed to

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PAR+UVR and fed with seaweed extract, UACs concentration increased significantly

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from 4 day trial (ANOVA p< 0.01) while carotenoid concentration decreased (Fig. 2D) .

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In Figure 3, the kinetics of the DPPH reaction of tissue homogenates of shrimp

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can be observed. Free radical activity was detected at all treatments. Significant

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differences among radiation treatments were detected (ANCOVA, P=0.004). Signal

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decayed drastically in all homogenates of animal’s integument under UVR treatments

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within 3 minutes (65%), and after one hour DPPH remnant was about 20%. In PAR

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treatments within 3 minutes, there are not significant differences among treatment, after

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one hour showed the lowest scavenging activity compared to UVR treatments (60%

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DPPH remnant).

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4. DISCUSSION

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Brown seaweeds are becoming of increasingly important because of their

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bioactive compounds and their potential application in several branches of industry. The

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most common compounds include terpenes, phlorotannins, phenols, polyphenols, and

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carotenoids (Miyashita et al. 2013, Pádua et al. 2015, Sanjeewa et al. 2016). These

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compounds are associated with several therapeutic applications for humans and animals.

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Furthermore, due to their low lipid content, high concentration of polysaccharides,

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natural minerals, polyunsaturated fatty acids, and vitamins, they are a source of functional

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food (Ganesan et al. 2008, Bajpai 2017, Díaz et al. 2017, Wells et al. 2017).

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In aquaculture there is a growing interest in exploring the potential of seaweeds as

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a feed ingredient, with promising results for shrimp (Cruz-Suárez et al. 2008, Niu et al.

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2015, Francis et al. 2008, Lee et al. 2016, Xia et al. 2012, Diaz et al. 2017). In the present

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work, we evaluate the utilization of U. pinnatifida extract as feed additive for P. muelleri

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and its role under UVR stress. This radiation is absorbed by nucleic acids and proteins

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and may damage cells by altering enzymes and membrane lipids, generating free radicals,

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interfering with the mitotic cycle and producing lesions in DNA (Hansson & Hylander

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2009). U. pinnatifida extract showed a high photoprotective capacity in shrimp fed a diet

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supplemented with this extract.

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Animals displayed 58% mortality after exposed to UV irradiation. In contrast, the

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mortality of animals fed a standard diet (C) and exposed to UVR was 72%. This

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protection conferred by U. pinnatifida extract was comparable to that found in mysis and

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postlarvae of P. muelleri fed microalgae Pavlova lutheri (to induce UV-absorbing

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compounds) with 75 % survival after UVR-stress (Marcoval et al. 2018).

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Algal carotenoids act as antioxidants and light-harvesting complexes to reduce

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oxidative stress caused by light exposure (Forster et al. 2005). The protective capacity of

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the endogenous antioxidant system of P. muelleri to neutralize the DPPH radical can be

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quantified and provides a useful index to measure the relative susceptibility of biological

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tissue damage caused by free radicals. A previous study of Diaz et al. (2013) determined

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the relationship between tissue carotenoid concentrations and free radical scavenging

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properties of different stages of P.muelleri. It was demonstrated that the postlarval stages

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(with higher carotenoid concentration) promoted a greater percentage of decay of DPPH

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over time, since radicals are consumed in the tissue at a speed that depends on the amount

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of protective substances. In the present work, significant differences in the capacity to

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react and quench DPPH radicals were found only between the animals under the two

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radiation treatments, with the lowest activity in animals under PAR treatment. The

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pronounced DPPH decay observed in UVR treatments could be attributed to the higher

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accumulation of PPCs that provided protection against oxidative stress caused by

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radiation. It has been previously documented that UV absorbing compounds as well as

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carotenoids can help organisms to cope with excessive radiation (Hernández-Moresino et

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al. 2014), but other authors suggested that UVR-stressed animals may switch from

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carotenoids accumulation to UACs accumulation when dietary UACs were made

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available (Moeller et al. 2005; Marcoval et al. 2018).

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Previous reports have documented that U. pinnatifida extract could promote

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growth and immunological response in postlarvae of Penaeus monodon and

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Marsupenaeus japonicus juvenile (Traifalgar et al. 2009, 2010, 2012), and improve

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antioxidant capacity in juveniles of Artemesia longinaris (Diaz et al. 2017). However, no

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information is available on the possible photoprotection of U. pinnatifida under UVR in

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penaeoid shrimp. In the present work was found that carotenoid concentrations decreased

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in treatments exposed to UVR while the concentrations of UACs increased. These results

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suggest that UVR-stressed animals metabolize protective compounds into UV absorbing

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compounds, when they are available through the diet. Probably, polyphenolic constituents

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of U. pinnatifida, are the one of the component that confers such photoprotection

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(Rozema et al. 2002, Holdt & Kraa 2011).

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P. muelleri is an important target species for aquaculture in the region; according

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to the photoprotective performance of this shrimp, it can be indicated that U. pinnatifida

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is interesting as food additive. The results obtained in this study provide new data on

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possible applications for commercially available seaweed biomass, particularly for some

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species that are traditionally used as food.

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ZAR JH. 1999. Biostatistical analysis, Fourth ed. Prentice Hall, Upper Saddle River, NJ. 929 pp.

474

Captions figures

475 476

Figure 1. Initial spectral absorption characteristics (O.D) of extract of Undaria

477

pinnatifida. 330: peak of absorbance of UV absorbing compounds.

478 479

Figure 2. Progression of concentration of Photoprotective Compounds (carotenoids and

480

UACs) during 7 days radiation treatments on P. muelleri juveniles fed control diet and

481

added U. pinnatiffida diet.

482 483

Figure 3. Free-radical 1, 1-diphenyl-2-picrylhydrazyl (DPPH) reaction kinetics estimated

484

in midgut gland homogenates after 7 days radiation treatment on Pleoticus muelleri

485

juveniles. C: control diet; U: control diet supplemented with 3g/100g of U.pinnatiffida

486

extract.

487

488 489

Table 1: Ingredient composition of diets Ingredient (g100g-1)

C

U

Fish meal (65% crude protein)a

48.0

48.0

Soy bean meal (42% crude protein)b

17.0

17.0

Corn starch

20.0

20.0

Squid protein (85% crude protein)

1.0

1.0

Wheat bran

8.5

5.5

0

3.0

Fish oil

2.0

2.0

Fish soluble

2.0

2.0

Soy bean lecithin

0.5

0.5

Cholesterol

0.5

0.5

Vitamins c

0.5

0.5

Moisture

5.5

5.5

Crude protein

41.0

41.0

Total lipid

11.8

11.8

Ash

9.4

9.4

Extract of Undaria pinnatifida

proximate composition (% dry matter)

C: control diet, U: Control diet supplemented with 3g/100g of U. pinnatifida extract. a

Agustinier S.A. Mar del Plata, Argentina.

b c

Melrico S.A. Argentina

g/kg: cholecalciferol 1.8, thiamin 8.2, riboflavin 7.8, pyridoxine 10.7,

calcium

panthothenate 12.5, biotin 12.5, niacin 25.0, folic acid 1.3, B12HCl 1.0, ascorbic acid (RovimixStay C)

39.1, menadione

1.7,

tocopherolacetate 75, vitamin A acetate 5.0.

inositol 0.3,

cholinechloride

0.2,

α-

0,05

U. pinnatifida extract

330

Optical Density

0,04

0,03

0,02

0,01

0,00 300

400

500

600

700

800

Wavelength (nm)

Figure 1. Initial spectral absorption characteristics (O.D) of extract of Undaria pinnatifida. 330: peak of absorbance of UV absorbing compounds.

Carotenoids

Carotenoids

PAR +UVR /C

UACS 25

UACS

60

25

15 10

20 5 0

20

Carotenoids (ug/g)

40

UACs ( OD/DW)

Carotenoids (ug/g)

20 40

2

3

4

5

6

10

20

5 0

0 1

15

0 1

7

2

3

4

5

6

7

Time (days)

Time (days) Carotenoids

PAR/U

Carotenoids

UACS

60

UACS

PAR+UVR /U

25

60

25

15 10

20

5 0

0 2

3

4

Time ( days)

5

6

7

20

Carotenoids (ug/g)

40

UACs ( OD/DW)

Carotenoids (ug/g)

20

1

UACs ( OD/DW)

60

40

15 10

20

5 0

UACs ( OD/DW)

PAR/C

0 1

2

3

4

5

6

7

Time (days)

Figure 2. Progression of concentration of Photoprotective Compounds (carotenoids and UACs) in integument of P. muelleri juveniles fed control diet and added U. pinnatiffida diet during 7 days radiation treatments. (values are expressed as mean ± SE n=3)

DPPH remaining (%)

1 PAR/C 0,8

PAR/U PAR + UVR/C

0,6

PAR + UVR/U

0,4 0,2 0 0

10

20

30

40

50

60

70

Time ( minutes)

Figure 3. Free-radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) reaction kinetics estimated in midgut gland homogenates after 7 days radiation treatment on Pleoticus muelleri juveniles. All tests were conducted in triplicate and the means are used. C: control diet; U: control diet supplemented with 3g/100g of U. pinnatiffida extract.

Highlights •

Under UVR-stressed conditions, shrimps Pleoticus muelleri could bioaccumulate photoprotective compounds from diets.

•

Seaweed Undaria pinnatifida could be used potential of as feed ingredient, with promising results for photoprotection of shrimps.

•

The results obtained in this study provide new data on possible applications for commercially available seaweed biomass.

Credit Author Statement M. Alejandra Marcoval : Investigation, conceptualization, formal anlysis, methodology, writing origin draft, review & editing A. Cristina Díaz: conceptualization, funding acquisition, supervisión, review & editing M. Laura Espino : Data curation; formal analysis, review & editing Natalia S. Arzoz: Software , review & editing Susana Velurtas: Data curation, writing, review & editing Jorge L. Fenucci : Funding acquisition, Project administration, review & editing

Declaration of interests X The authors declare that they have no known competing financialinterestsor personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: