Can starvation influence cellular and biochemical parameters in the crab ? Valerio Matozzo, Chiara Gallo, Maria Gabriella Marin To cite this version: Valerio Matozzo, Chiara Gallo, Maria Gabriella Marin. Can starvation influence cellular and bio- chemical parameters in the crab ?. Marine Environmental Research, Elsevier, 2011, 71 (3), pp.207. 10.1016/j.marenvres.2011.01.004. hal-00673196 HAL Id: hal-00673196 https://hal.archives-ouvertes.fr/hal-00673196 Submitted on 23 Feb 2012 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Accepted Manuscript Title: Can starvation influence cellular and biochemical parameters in the crab Carcinus aestuarii? Authors: Valerio Matozzo, Chiara Gallo, Maria Gabriella Marin PII: S0141-1136(11)00015-8 DOI: 10.1016/j.marenvres.2011.01.004 Reference: MERE 3500 To appear in: Marine Environmental Research Received Date: 3 December 2010 Revised Date: 17 January 2011 Accepted Date: 20 January 2011 Please cite this article as: Matozzo, V., Gallo, C., Marin, M.G. Can starvation influence cellular and biochemical parameters in the crab Carcinus aestuarii?, Marine Environmental Research (2011), doi: 10.1016/j.marenvres.2011.01.004 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. 1 1 Can starvation influence cellular and biochemical parameters in the crab 2 Carcinus aestuarii? 3 4 Valerio Matozzo*, Chiara Gallo, Maria Gabriella Marin 5 6 Department of Biology, University of Padova, Via Ugo Bassi 58/B, 35131 Padova (Italy) 7 8 9 10 *Corresponding author: Dr. Valerio Matozzo 11 Department of Biology 12 University of Padova 13 Via Ugo Bassi, 58/B 14 35131 Padova, ITALY 15 Phone: +39-049-8276201 16 Fax: +39-049-8276199 17 e-mail: [email protected] 18 19 20 21 22 23 24 25 2 26 Abstract 27 Crustacea experience periods of starvation during moulting or when limited food availability 28 occurs. The effects of starvation on Crustacea physiological responses have been 29 demonstrated, whereas the effects of starvation on Crustacea immune parameters remain to be 30 more fully studied. In the present study the effects of starvation on immune parameters and 31 antioxidant enzyme activities of the crab Carcinus aestuarii were evaluated for the first time. 32 Treated crabs were starved for 7 days, whereas control crabs were fed daily with mussels. 33 Total haemocyte count (THC), haemocyte diameter and volume, haemocyte proliferation, 34 cell-free haemolymph (CFH) glucose and total protein levels, and phenoloxidase (PO) activity 35 in both haemocyte lysate (HL) and CFH were measured in crabs. In addition, superoxide 36 dismutase (SOD) and catalase (CAT) activities were evaluated in both gills and digestive 37 gland from crabs, in order to evaluate whether starvation induced oxidative stress in C. 38 aestuarii. THC increased significantly in starved crabs, with respect to controls, whereas no 39 significant variations were observed in haemocyte diameter, volume and proliferation. In 40 CFH of starved animals glucose concentration significantly increased, whereas total protein 41 concentration significantly reduced. A significantly higher PO activity was recorded in HL 42 from starved crabs, than in control crabs. Conversely, PO activity did not vary significantly in 43 CFH. Starvation did not cause significant alterations in antioxidant enzyme activities in both 44 gills and digestive gland. Results obtained demonstrated that starvation influenced crab 45 immune parameters, but did not induce oxidative stress. Results also indicated that C. 46 aestuarii can modulate its cellular and biochemical parameters in order to cope with 47 starvation. 48 49 Keywords: crabs; Carcinus aestuarii; starvation; immune parameters; haemocytes; glucose; 50 antioxidant enzymes 51 3 1. Introduction 52 The green crab Carcinus aestuarii (Crustacea, Decapoda) is native of the Mediterranean 53 Sea, and it is very similar to the Atlantic species Carcinus maenas. The two species are 54 mainly distinguishable by morphological traits, such as the shape of the copulatory 55 appendages (pleopods) in males, the shape of the frontal area between eyes, and the carapace 56 width to length ratio (Yamada and Hauck, 2001). Owing to natural dispersal, maritime 57 commerce and ballast transport, Carcinus species have colonised several regions outside their 58 native areas, showing high tolerance to air exposure, starvation, and variations in temperature 59 and salinity (Yamada and Hauck, 2001). In particular, C. aestuarii has also colonised 60 estuarine areas along the Italian coasts, such as the Lagoon of Venice. 61 Crustacea have an open vascular system in which numerous haemocytes circulate in 62 haemolymph. It has been demonstrated that crustacean haemocytes are involved in important 63 functions, such as wound repair and defence mechanisms against parasites, viruses and 64 bacteria (Bauchau, 1981). Haemocyte-mediated immune defence includes phagocytosis, 65 encapsulation, nodule formation, clotting, agglutination, melanisation and microbicidal 66 activity (Bauchau, 1981; Smith and Söderhäll, 1986; Söderhäll and Cerenius, 1992). Three 67 types of circulating haemocytes are generally recognised in Crustacea: hyalinocytes, the 68 smallest cells without evident granules; semigranulocytes, which contain small granules, and 69 granulocytes, with abundant cytoplasmic granules (Bauchau, 1981). The three haemocyte 70 types were also identified in C. aestuarii (Matozzo et al., 2010a). Only hyalinocytes were able 71 to phagocytose yeast cells or Zymosan. All haemocyte types produced superoxide anion, 72 whereas only granulocytes were positive to some hydrolytic and oxidative enzyme activities 73 (Matozzo et al., 2010b). 74 In the last few years, efforts have been addressed to the evaluation of stress effects in 75 Crustacea, considering that many species are commercially-important and disease outbreaks 76 may cause a decline in both natural and farmed populations. In this context, it has been 4 77 demonstrated that environmental factors can cause immunemodulation in Crustacea 78 (LeMoullac and Haffner, 2000). Among stressors, starvation is worthy of consideration. 79 Crabs experience periods of starvation during moulting (Lipcius and Herrnkind, 1982; 80 Sanchez-Paz et al., 2006) or when limited food availability occurs (Crothers, 1967). Feeding 81 generally resumes postmoult, when the exoskeleton hardens (Sanchez-Paz et al., 2006). 82 Although it has previously been demonstrated that starvation can cause several physiological, 83 metabolic and behavioural changes in Crustacea (Sanchez-Paz et al., 2007), little attention has 84 been addressed to the evaluation of the effects of starvation on immune responses of 85 Crustacea. To fill this gap, in the present study the effects of 7 days’ starvation on immune 86 parameters and antioxidant enzyme activities in gills and digestive gland from the crab C. 87 aestuarii were evaluated for the first time. Considering that a good nutritional status is 88 generally associated with the maintenance of an efficient immune system in animals (Biao et 89 al., 2008), we posed two questions: 90 i. can starvation influence immune parameters and antioxidant status in C. aestuarii? 91 ii. can C. aestuarii modulate its immune parameters to cope with starvation? 92 93 2. Materials and methods 94 2.1. Crabs 95 Intermoult adult male crabs (4 cm mean carapace length) were collected by handmade 96 traps in the Lagoon of Venice and kept in the laboratory in large aquaria containing seawater 97 (salinity of 35 ± 1 psu and temperature of 17 ± 0.5 °C) and a sandy bottom. The crabs were 98 fed on alternate days with mussels (Mytilus galloprovincialis) and acclimatised in the 99 laboratory for 5 days before the experiments. Only crabs without obvious injuries or infection 100 were used for experiments. 101 102 5 103 2.2. Experimental setup 104 Treated and control crabs (12 starved and 12 fed crabs, respectively) were kept for 7 days 105 in two distinct aquaria at the same experimental conditions described above. Every 24 h, both 106 treated and control crabs were transferred to two distinct aquaria fitted with partition boards 107 forming 12 individual boxes each (Fig. 1A). Half a mussel (4 cm mean shell length, 1.5 g 108 mean fresh weight) was supplied daily to each control crab, whereas treated crabs did not 109 receive food (Fig. 1B, C). The maintenance of each control crab in separate boxes allowed 110 animals to eat quietly, avoiding assault by the other crabs during feeding. After one hour, both 111 fed and starved crabs were moved again to the two unpartitioned aquaria. 112 113 2.3. Haemolymph and tissue collection 114 Crabs were anaesthetised on ice for 10 min, and the haemolymph (at least 500 µL per crab) 115 was collected from the unsclerotised membrane of the walking legs using a 1 mL plastic 116 syringe, placed in Eppendorf tubes on ice and diluted (except for glucose determination) 1:2 117 in an anticoagulant solution of citrate buffer/EDTA (NaCl 0.45 M, glucose 0.1 M, sodium 118 citrate 30 mM, citric acid 26 mM, EDTA 10 mM, pH 4.6, stored at 4 °C) (Söderhäll and 119 Smith, 1983). Six pools of haemolymph from two crabs each were prepared. Pooling was 120 necessary because individuals did not provide enough haemolymph for analyses.