Bulgarian Journal of Veterinary Medicine, 2017 ONLINE FIRST ISSN ; DOI: /bjvm.2021

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1 Bulgrin Journl of Veterinry Medicine, 2017 ONLINE FIRST ISSN ; DOI: /bjvm.2021 Originl rticle HAEMATOLOGICAL, BLOOD BIOCHEMICAL AND IMMU- NOLOGICAL RESPONSES TO GRADUAL ACCLIMATION TO LOW-SALINITY WATER IN WALTON S MUDSKIPPER PERIOPH- THALMUS WALTONI KOUMANS, 1941 (PERCIFORMES: GOBIIDAE) S. SOLTANIAN & M. S. FEREIDOUNI Aqutic Animl Helth nd Diseses Deprtment, School of Veterinry Medicine, Shirz University, Shirz, Irn Summry Soltnin, S. & M. S. Fereidouni, Hemtologicl, blood biochemicl nd immunologicl responses to grdul cclimtion to low-slinity wter in Wlton s mudskipper Periophthlmus wltoni Koumns, 1941 (Perciformes: Gobiide). Bulg. J. Vet. Med. (online first). The present study investigtes nd reports the effects of grdul cclimtion to low slinity wter on some hemtologicl, biochemicl nd immunologicl responses in Wlton s mudskipper, Periophthlmus wltoni. For this purpose, mudskippers cught from Persin Gulf costl re (Bndr Khmir, Hormozgn Province, Irn) were mintined in lbortory quri with hlf sewter (50% SW, 17 ppt) nd fed dily with frozen blood worms (Chironomus spp.) for one month prior to the strt of experiments. After cclimtion, groups of 18 individuls were either directly trnsferred to 50% SW (control), or cclimted to low slinity wter during two sub-periods. In the first sub-period, fish were exposed to low slinity wter nmely to grdul wter slinity decrese of 1 ppt per dy (during 17 dys) until the finl slinity of 0.4 ppt ws reched. Afterwrds, fish continued to mintin in this point of slinity (0.4 ppt), for further 15 dys until dy 32 (second sub-period). Fish were smpled on dy 0, 17 nd 32. Sttisticl nlysis showed significnt influence of reduced slinity on erythrocytes, hemoglobin, hemtocrit, leukocytes, lymphocytes, neutrophils, monocytes nd on ll biochemicl nd immunologicl prmeters tested on dy 17. However, these indices returned to the control level on dy 32. Bsed on results, the extremely euryhline P. wltoni cn be cclimted to freshwter medium without showing ny helth disturbnce if grdul decrese in slinity is crried out for long period of time. Key words: cclimtion, euryhline, oxudercine gobies, slinity, stress INTRODUCTION Environmentl slinity is very importnt fctor for qutic orgnisms nd ny chnge in slinity seriously ffects the osmotic pressure nd metbolism (Mustfyev & Mekhtiev, 2008; Fzio et l., 2013), cuses chnges to the ctivity, structure, nd physiologicl function of fish digestive enzymes; nd ffects fish

2 Hemtologicl, blood biochemicl nd immunologicl responses to grdul cclimtion to. development, hbits nd survivl (Wng & Zhu, 2002; Ruscoe et l., 2004). In fish, studies on the effect of slinity hve minly looked t chnges in osmoregultory orgns nd their hormonl control, plsm prmeters, energy metbolism, growth etc. (McCormick, 2001). However, little is known bout the chnges in the fish immune system fter slinity disturbnce despite the fct tht slinity is one of the most importnt environmentl fctors in the qutic medium. On the other hnd, the hemtologicl profile is good indictor of physiologicl dysfunctions since there is close ssocition of circultory system with the externl environment (Elhee & Bhgwnt, 2007). It provides informtion not only bout the helth sttus of fish nd the physicl nd chemicl prmeters of wter in which they live, but lso llows ssessing the reltionship between these fctors nd the susceptibility of orgnisms to chnges in environmentl conditions (Elhee & Bhgwnt, 2007; Mced-Veig et l., 2010; Ayool et l., 2011). There re some fish species tht cn tolerte vrious slinities nd few tht cn survive extended exposure in wter with different slinities (Nordlie & Hney, 1998). According to Frncis et l. (2007), slinity is one of the most fluctuting wter qulity prmeters in brckish wter environment. Growth of euryhline species is often ffected by slinity becuse the energy used for osmoregultion is not vilble for growth (Wootton, 1990). Consequently, mny of these species hve n optimum slinity level t which the growth rte is highest nd the cost of osmoregultion lowest, which my ffect fish distribution in the wild (Blber, 1997). Mudskippers (Teleostei: Gobiide: Oxudercine: Periophthlmini) re gobies tht re fully terrestril for some portion of the dily cycle (Murdy, 1989). They re subject of biologicl nd ecotoxicologicl studies, to determine their potentil use s bio-indictor in environmentl ssessments of costl wters, tropicl or subtropicl soft bottom intertidl systems (Aligen & Mngo, 2011). They could be lso relible qurium or ornmentl fish (Mleczko, 2003). The mudskippers re euryhline nd cn withstnd rpid nd drstic chnges in slinity (Aligen & Mngo, 2011). The Wlton's mudskipper (Periophthlmus wltoni, Koumns, 1941), is one of the three species of oxudercine gobies (Gobiide) (Agorret et l., 2013), living in the Persin Gulf nd Gulf of Omn (Murdy, 1989; Ghnbrifrdi et l., 2014,b,c; 2016). Irnin mudskippers re differentilly distributed from more qutic to more terrestril hbitts, respectively, from Scrtelos tenuis to Boleophthlmus dussumieri to P. wltoni (Clyton, 1985). P. wltoni hs been recorded in wide rnge of intertidl hbitts, including tidl mudflts nd mngrove forests (Clyton, 1985; Ghnbrifrdi & Mlek, 2007). Such environments, re prticulrly hrsh during low tide, including rpid nd wide fluctutions of temperture (Tytler & Vughn, 1983) nd slinity (Ssekumr, 1994). Therefore, the nturl hbitt of P. wltoni lies usully in dynmic stte of fluctuting slinities. Movement between fresh wter nd brckish or sewter cuses physiologicl chnges, including blood composition fluctutions. Limited informtion exists with respect to the effects of slinity on physiology of P. wltoni. Hence, the present work imed to exmine the possible vritions of blood profile nd immune responses in P. wltoni s result of grdul cclimtion to reduced slinity. 2 BJVM,, No

3 S. Soltnin & M. S. Fereidouni MATERIALS AND METHODS Collection nd mintennce of mudskippers Live specimens of Wlton's mudskipper, (n=36; 10.6±2.8 cm totl length; 7.2±1.9 g weight) were collected by hnd from the mudflts of the costl re of the Persin Gulf in Bndr Khmir (Hormuzgn Province, Irn; 26 56'40''N, 55 35'55''E). Fish were morphologiclly identified to species level by mens of the vilble morphologicl keys (Murdy, 1989). The nimls were brought to the lbortory in plstic troughs contining only se wter to 3 cm level. They were mintined under lbortory conditions in 50% slinity wter (SW) or 17 ppt in smll quri ( cm, wter depth pproximtely 3 cm) t 25 C, providing pproprite terrestril res to the fish. Different slinities were provided by dilution of costl Persin Gulf sline wter (pproximtely 35 ppt) with dechlorinted municipl freshwter. Slinity ws determined by using hnd-held refrctometer (Model HRN-2N Atgo Product Jpn). Fish were fed frozen blood worms (Chironomus spp.) d libitum once dily in the morning, nd the wter ws chnged every other dy. They were llowed to dpt to such environment for month before experiments were performed. Grdul cclimtion of mudskippers to freshwter After the holding period, the fish were rndomly divided into two equl tretments. Ech tretment ws replicted 3 times with 6 fish in ech qurium. The first group of fish ws directly trnsferred to 50% SW nd served s control. The second group of fish ws cclimted to low slinity wter during two sub-periods. In the first sub-period, fish were exposed to low slinity wter grdul wter slinity decrese of 1 ppt per dy (over period of 17 dys) until the finl slinity of 0.4 ppt (the slinity of freshwter) ws reched. Fish were mintined t this slinity (0.4 ppt), for nother 15 dys until dy 32 (second sub-period). Slinity ws checked every dy nd djusted when necessry. In both tretments, fish were free to be in or out of wter using pproprite terrestril res inside quri. They were fed frozen blood worms (Chironomus spp.) once dily in the morning, nd the wter ws chnged every other dy. During the tril, ll fish were mintined under nturl photoperiod (sunrise t 05:55 h, sunset t 20:00 h) nd temperture, slinity, dissolved oxygen levels nd mmoni concentrtion were mesured bsed on the methods described by APHA (1985). Blood collection Six fish were smpled t rndom from ech experimentl nd respective control group t dys 0, 17 nd 32. On the smpling points, fish were rndomly collected in the tnks with nets nd nesthetised in MS-222 (Sigm-Aldrich) (150 mg/l). Blood smples were collected from cudl vein using 2 ml syringe nd 24 guge needle. Hemtologicl prmeters were ssessed within one hour of blood collection. One prt of ech smple ws trnsferred into preheprinised plstic Eppendorf tubes while the other prt into blood collecting tubes or Eppendorf tubes without nticogulnt nd ws llowed to clot for 2 h t room temperture in slnting position. The clot ws then cut with glss rod nd cre ws tken not to hemolyse it. The tubes were kept t 4 ºC overnight nd then centrifuged t 2500 g for 15 min nd the superntnt serum ws collected. The serum ws stored t 80 ºC in screw cp glss vils until use. BJVM,, No 3

4 Hemtologicl, blood biochemicl nd immunologicl responses to grdul cclimtion to. Hemtologicl nd blood biochemicl prmeters Blood smples were immeditely nlysed for estimtion of numbers of erythrocytes (RBC) nd leukocytes (WBC), hemoglobin (Hb) nd hemtocrit (Hct). RBC nd WBC counts were determined s described in Schperclus et l. (1991). Hemoglobin content (Hb) ws ssyed using the cynmethemoglobin method with Drbkin s solution (Goldenfrb, 1971). Hemtocrit ws determined by the microhemtocrit method (Fox, 1997). The men corpusculr volume (MCV), the men corpusculr hemoglobin (MCH) nd the men corpusculr hemoglobin concentrtion (MCHC) were clculted using the formuls (Jin, 1993.: For differentil WBC counts, whole blood ws smered on glss microscope slides, dried in ir, nd stined with My- Grunwld/Giems. Leukogrms were ssessed for ech fish under n oil immersion lens. One hundred white blood cells from ech smer were ssessed nd the percentges of different types of leukocytes were clculted (Schperclus et l., 1991). The quntittive determintion of glucose ws crried out using commercilly vilble dignostic kit (Prs Azmun, Irn, ) (Hosseini et l., 2011), t 546 nm nd 37 C ccording to the glucose oxidse method suggested by Trinder (1969). Lctte ws mesured with n enzymtic method by lctte kit (Prs Azmoon, Tehrn, Irn). Totl serum protein ws mesured by the Biuret method (Kwpinski, 1965). Immunologicl prmeters Phgocytic ctivity ex vivo. The phgocytic ctivity of whole blood cells ws nlysed using commercil bker s yests, Scchromyces cerevisie, s indictor ccording to Zhou et l. (2002). Dried live yests were incubted in 2% sucrose solution (ph 3 4) for 2 h t 30 C nd boiled for 30 min. The yest ws then centrifuged nd the pellet wshed twice nd re-suspended in 0.85% sline (t cell/ml). A 20 μl liquot of suspension s well s 40 μl heprinised whole blood ws then dded to 0.1 ml Eppendorf tube nd the mixture incubted t 30 C for 30 min with gentle shking. After this period, smers were prepred nd the ir-dried slides then stined with Wright-Geims stin. Phgocytic ctivity (PA) ws determined by evluting 100 phgocytes per slide using light microscope. A minimum of 3 slides/fish ws evluted. The men PA ws clculted s 100 number of phgocytic cells with engulfed yest cells/number of phgocytes counted. Respirtory burst ctivity. Oxidtive rdicl production by neutrophils during respirtory burst ws mesured by the NBT (nitroblue tetrzolium) ssy s described by Anderson & Siwicki (1995). Briefly, blood nd 0.2% NBT were mixed in equl proportion (1:1), incubted for 30 min t 25 C, nd then 50 μl ws tken out nd dispensed in glss tubes. For solubilistion of reduced formzn product, 1 ml of dimethylformmide (Sigm, USA) ws dded nd centrifuged t 2000 g for 5 min. Finlly, the superntnt ws tken nd the extent of NBT reduced ws mesured t n opticl density of 540 nm. Dimethylformmide ws used s blnk. Lysozyme ssy. Serum lysozyme ctivity ws determined using the method described by Jin & Wu (2003). Briefly, suspension of n overnight grown Micrococcus lysodeikticus ws prepred by dissolving 20 mg of M. lysodeikticus into 100 ml of mol/l sodium phosphte buffer, ph 6.4 nd then, 100 μl of fish 4 BJVM,, No

5 S. Soltnin & M. S. Fereidouni Tble 1. Hemtologicl profile of P. wltoni smpled t different time points of grdul cclimtion to lowered wter slinity. Dt re presented s men ± SD (n=6). Vlues in the sme row showing the sme superscript letter re not significntly different (P>0.05). Control group (17 ppt) Low slinity group (0.4 ppt) Dy 0 Dy 17 Dy 32 Dy 0 Dy 17 Dy 32 RBC ( /L) 1.38±0.10 b 1.47±0.23 b 1.51±0.18 b 1.42±0.11 b 1.11± ±0.13 b Hct (%) 16.5±2.34 b 15.3±2.58 b 17.5±2.50 b 18.3±2.16 b 12.0± ±1.86 b Hb (g/l) 53.0±06.9 b 60.1±7.1 b 59.0±6.4 b 51.6±5.9 b 41.1± ±5.9 b MCV (fl) 120.5± ± ± ± ± ±12.2 MCH (pg) 38.62± ± ± ± ± ±6.12 MCHC (g/l) 329.8± ± ± ± ± ±60.3 WBC ( 10 9 /L) 8.32± ± ± ± ±0.82 b 9.0±0.73 Lymphocyte (%) 66.0± ± ± ± ±5.6 b 71.83±5.91 Neutrophil (%) 5.5± ± ±1.36 b 5.0± ±1.36 b 7.16±0.98 b Monocyte (%) 10.33± ± ± ±1.63 b 15.0±1.78 b 12.33±2.58 b Tble 2. Blood biochemicl profile of P. wltoni smpled t different time points of grdul cclimtion to lowered wter slinity. Dt re presented s men ± SD (n=6). Vlues in the sme row showing the sme superscript letter re not significntly different (P>0.05). Control group (17 ppt) Low slinity group (0.4 ppt) Dy 0 Dy 17 Dy 32 Dy 0 Dy 17 Dy 32 Glucose (mmol/l) 3.23±0.33 b 3.47±0.36 b 3.28±0.20 b 2.87± ±0.42 c 3.49±0.30 b Lctte (mmol/l) 0.65± ± ± ± ±0.22 b 0.87±0.18 Totl protein (g/l) 32.0±1.8 30±3.6 29±2.9 30±2.3 40±3.7 b 30±2.6 BJVM,, No 5

6 Hemtologicl, blood biochemicl nd immunologicl responses to grdul cclimtion to. serum ws dded to 3-mL suspension of M. lysodeikticus. The rection ws crried out t 25 C nd n bsorbnce t 540 nm ws mesured fter 0.5 nd 4.5 min. One unit of lysozyme ctivity ws defined s the mount of lysozyme producing decrese in bsorbnce of 0.001/min. Sttisticl nlysis Dt re presented s men±sd. Hemtologicl, biochemicl nd immunologicl prmeters were nlysed by one wy nlysis of vrince (ANOVA) nd Tukey s multiple comprison test. All sttisticl nlyses were tested t the 0.05 level of probbility, using the softwre SPSS 16.0 for Windows. RESULTS No bnorml behviour or mortlity ws seen in the medium during the entire period of cclimtion to lowered slinity. All fish were considered helthy on the bsis of n externl exmintion for ny signs of bnormlities. MCV, MCH nd MCHC remined unchnged during the study, however, the number of erythrocytes, Hb nd Hct levels were significntly diminished in fish smpled from lowered slinity tretment on dy 17 but returned to the control level on dy 32 (P<0.05) (Tble 1). In contrst, the opposite of the bove discussed pttern ws found for the number of WBC nd the percentge of lymphocytes, neutrophils nd monocytes s well s totl protein nd lctte levels which were significntly incresed on dy 17 nd then declined to the control level on dy 32 (P<0.05) (Tbles 1 nd 2). In ddition, vrition in levels of ctivities ws pronounced for phgocytosis, lysozyme nd respirtory burst ctivity, such tht fish smpled from lowered slinity tretment on dy 17, hd significntly phgocytic ctivity (%) b b Control b Low slinity dy 0 dy 17 dy 32 Fig. 1. Phgocytic ctivity of peripherl blood leukocytes of P. wltoni smpled t different time points of grdul cclimtion to lowered wter slinity (men ± SD; n=6). Different letters over brs indicte sttisticlly significnt difference (P<0.05). Lysozyme (U/mL) Control b b Low slinity b dy 0 dy 17 dy 32 Fig. 2. Serum lysozyme ctivity of P. wltoni smpled t different time points of grdul cclimtion to lowered wter slinity (men ± SD; n=6). Different letters over brs indicte sttisticlly significnt difference (P<0.05). NBT (OD540) 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0 Control Low slinity dy 0 dy 17 dy 32 Fig. 3. NBT ctivity of peripherl blood leukocytes of P. wltoni smpled t different time points of grdul cclimtion to lowered wter slinity. Brs represent men ± SD (n=6). Different letters over brs indicte sttisticlly significnt difference (P<0.05). b 6 BJVM,, No

7 S. Soltnin & M. S. Fereidouni lower phgocytosis vlue but showed higher lysozyme nd respirtory burst ctivity thn respective control fish. However, these indices returned to the control level on dy 32 (P<0.05) (Fig. 1, 2 nd 3). DISCUSSION Fish hemtologicl prmeters re closely correlted to the response to the environmentl nd biologicl fctors such s ge, weight, sex, food, bcteri, prsite nd wter qulity prmeters, including wter temperture, slinity, oxygen vilbility nd ph (Hider, 1973; Steinhgen et l., 1990; Fernndes & Mzon, 2003). Our results for P. wltoni showed significnt influence of lowered slinity on most of the prmeters studied on dy 17. However, in ccordnce with the previous studies (Gbriel et l., 2007; Akinrotimi et l., 2010; 2012), no chnges occurred in MCV, MCH nd MCHC vlues between tretments on ny smpling dte. Nevertheless, significnt reduction in the level of RBC, Hct nd Hb ws observed on dy 17 compred to the control group. These reductions my be ttributed to slinity-induced osmoregultory dysfunction which led to erythrocyte frgility (Girling et l., 2003). In fct, osmoticlly obliged wter movement due to incresed blood osmollity reduced blood hemtocrit nd hemoglobin concentrtions t lower slinities. Furthermore, the observed hemodilution my be consequence of impired erythropoiesis (production of erythrocytes) cused by disrupted osmoregultion (Gbriel et l., 2007). Hyperglycemi is n expected result of stress or exhustive exercise in fishes (Hrubec et l., 1997). It is known tht the degree of hyperglycemi my chnge depending on the type of stress nd the smpling times (Rollnd et l., 1997). Lctte is produced by nerobic metbolism in the muscle under stressful conditions of hypoxi or strenuous exercise nd relesed to the plsm (Begg & Pnkhurst, 2004). In ddition, lctte cn provide energy for brin, gills nd kidney (Mommsen, 1984). In our study, increse in the lctte levels on dy 17 observed in fish exposed to lowered slinity, suggests tht this metbolite is presumbly used s n energy source by osmoregultory orgns. Similr results were obtined in common crp nd Seneglese sole (Sole seneglensis) when fish were exposed to sudden chnge of slinity (Slti et l., 2010; Herrer et l., 2012). Totl plsm protein concentrtion reltive to reference intervl is used s brod clinicl indictor of helth, stress, nd well-being of qutic orgnisms (Riche, 2007). In the current study, protein level incresed in fish kept t low slinity level. In fct, our dt re in greement with the findings of Fzio et l. (2013), who ddressed the possible importnce of incresed serum protein s fuel for tissues during osmotic cclimtion once crbohydrte stores hve been mobilised. Amino cids seem to ply n importnt role in llowing fish to djust to the different environmentl slinities, either s energy sources or s importnt osmolytes for cell volume regultion (Argão et l., 2010). Elevted serum or plsm protein levels hd previously been reported in strved red se brem (Chryrosphrys mjor), blck se brem (Mylio mcrocephlus) nd red grouper (Epinephelus kr) exposed to low slinity environments (Woo & Murt, 1981; Woo & Wu, 1982). Furthermore, in ccordnce with our study, serum protein concentrtions were significntly elevted in silver se brem (Spms srb) s the mbient slinity diminished (Chun-yin, 2001). BJVM,, No 7

8 Hemtologicl, blood biochemicl nd immunologicl responses to grdul cclimtion to. Understnding chnges in innte immunity is relevnt for evluting chnges in the generl helth of qutic environment inhbitnts (Skours, 2002). Nevertheless, the effects of slinity re not well documented despite its importnce in fish immunity nd disese resistnce. In fct, few studies reveled tht slinity chnge cn modulte the immune response in fish species (Jing et l., 2008; Birrer et l., 2012; Schmitz et l., 2016). Therefore, in the current study, the effects of slinity disturbnce on mudskipper immune system were evluted for the first time. Some studies hve investigted the chnges in numbers nd composition of circulting leukocytes following cute nd chronic stress (Dvis et l., 2008). Indeed, leukocytes re good physiologicl stress indictors in fish (Svobodová et l., 2001). The present study demonstrted significnt increse in the totl number of WBC s well s in lymphocytes, neutrophils nd monocytes in fish exposed to lowered slinity on dy 17. Conversely, no chnges in leukocyte numbers in dolphin fish, Coryphen hippurus (L.) ws observed fter sudden drop in slinity occurred. However, comprble findings were observed in green bck flounder Rhombosule tpirin (Girling et l., 2003) nd in Tilpi guineensis (Akinrotimi et l., 2010) exposed to fresh wter. Furthermore, pipefish (Syngnthus typhle), kept under low slinity (6 ppt) reveled higher monocyte counts in the blood compred to the mbient slinity (18 ppt) nd the high slinity (30 ppt) tretments (Birrer et l., 2012). However, the mount of monocytes correlted negtively with the mount of lymphocytes (Birrer et l., 2012) which is in contrst with the pttern observed in our findings. On the other hnd, n increment in the number of monocytes nd neutrophils ws lso noticed in stripped ctfish (Pngsinodon hypophthlmus) exposed to chronic hyperosmotic environment (Schmitz et l., 2016). This increment might be due to non-specific immune response to stress s result of interction of prolctin nd cortisol hormone to restore ion blnce in hyposmotic environment (McCormick, 2015). An increse in the count of WBC my lso be cused by relese of cells ccumulted in the spleen, to combt the stressor (Ajni et l., 2007). Chnges occurring in innte immune prmeters cn be useful indictors in ssessing the condition of chronic stress induced by vrious stressors (Cruso et l., 2005). It ws reported erlier tht stress ffects the lysozyme level or ctivity, phgocytic nd/or respirtory burst ctivity of blood leukocytes (Thompson et l., 1993; Pulsford et l., 1994; Vzzn et l., 2002; Liebert & Shreck, 2006). In the current study, significntly higher lysozyme nd respirtory burst ctivity but lower phgocytic ctivity ws observed in fish smpled from lowered slinity tretment on dy 17. A comprble finding ws observed in phgocytosis vlue in Nile tilpi, which significntly incresed in response to decrese in slinity from 20 g/l to 5 g/l (Choi et l., 2013). In ddition, exposure of tilpi (Oreochromis mossmbicus) to hyperosmotic conditions significntly incresed phgocytic bility s well s serum lysozyme nd respirtory burst ctivities (Jing et l., 2008). Increse in lysozyme ctivity nd phgocytosis were lso found in slmon during freshwter to se wter trnsfer (Mrc et l., 1995). In fct, following stress in teleosts, both enhncement nd suppression of plsm lysozyme ctivity hve been reported, depending on the type, intensity nd durtion of the stressor (Fevolden et 8 BJVM,, No

9 S. Soltnin & M. S. Fereidouni l., 2003). For exmple, higher lysozyme ctivities during both cute nd chronic hyperosmotic stress hve been described in euryhline species (Mrc et l., 1995; Yd et l., 2001; Jing et l., 2008). Therefore, the level of serum lysozyme ctivity is recognised s stress indictor of fish (Fevolden et l., 1999). To conclude, cclimtion of mudskipper to freshwter ltered blood profiles nd immune prmeters ccording to the cclimtion time. These results might be explined by the effects of osmoregultory hormones nd the involvement of different orgns in the immune nd osmoregultory responses. Bsed on our results, the extremely euryhline P. wltoni hs been found to be very tolernt to low slinities nd ble to withstnd grdul trnsfer from brckish wter to slinities s low s 0.4 ppt without showing ny mortlities or even ny bnorml behviour during the cclimtion period. Further, there re no indictions tht low slinities hve detrimentl effects on blood profile nd/or immunity-relted prmeters fter long-term cclimtion. Thus, the study points t n economic dvntge nd esier rering of P. wltoni s n ornmentl fish in freshwter rther thn in brckish or sewter. Abrupt slinity chnge would hve pprently long-term negtive effects on growth nd helth conditions. Therefore, mudskippers rered in slinities lower thn brckish or sewter should be given grdul cclimtion to reduced slinity if the husbndry prctice clls for long-term rising in freshwter. ACKNOWLEDGEMENTS This study ws supported by the Aqutic Animl Helth & Diseses Deprtment, School of Veterinry Medicine t Shirz University through reserch grnt to the first uthor. REFERENCES Agorret, A., D. Sn Muro, U. Schliewen, J. L. Vn Tssell, M. Kovčić, R. Zrdoy & L. Rüber, Moleculr phylogenetics of Gobioidei nd phylogenetic plcement of Europen gobies. Moleculr Phylogenetics nd Evolution, 69, Ajni, F., O. A. Olukunle & S. A. Agbede, Hormonl nd hemtologicl response of Clris griepinus (Burchell 1822) to nitrite toxicity. Journl of Fish Interntionl, 2, Akinrotimi, O. A., B. Uedeme-N & E. O. Agokei, Effects of cclimtion on hemtologicl prmeters of Tilpi guineensis. Science World Journl, 5, 1 4. Akinrotimi, O. A., E. O. Agokei & A. A. Arnyo, Chnges in blood prmeters of Tilpi guineensis exposed to different slinity levels. Journl of Environmentl Engineering nd Technology, 1, Aligen, J. C. & D. D. Mngo, Climte Chnge Integrted eduction: A Model for Lifelong Lerning System SEAMEO RECSAM Penng, Mlysi, A report. Anderson, D. P. & A. K. Siwicki, Bsic hemtology nd serology for fish helth progrms. In: Diseses in Asin Aquculture II, eds M. Shri, J. R. Arthur & R. P. Subsinghe, Mnil, Philippines: Philippines Fish Helth Section, Asin Fisheries Society, p APHA, Stndrd Methods for the Exmintion of Wter nd Wste Wters, 16 th edn, Americn Public Helth Assocition, Wshington, USA. Argão, C., B. Costs, L. Vrgs-Chcoff, I. Ruiz-Jrbo, M. T. Dinis, & J. M. Mncer, Chnges in plsm mino cid levels in euryhline fish exposed to different environmentl slinities. Amino Acids, 38, Ayool, S. O., M. P. Kuton, A. A. Idowu & A. B. Adelekun, Acute toxicity of Nile tilpi (Orechromis niloticus) juveniles exposed to queous nd ethnolic extrcts BJVM,, No 9

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11 S. Soltnin & M. S. Fereidouni nd Gulf of Omn, Irn. Irnin Journl of Animl Biosystemtics, 3, Ghnbrifrdi, M., S. Ghsemin, M. Alibdin & A. Pehpuri, Length-weight reltionships for three species of mudskippers (Gobiiformes: Gobionellide) in the costl res of the Persin Gulf nd Gulf of Omn, Irn. Irnin Journl of Ichthyology, 1, Ghnbrifrdi, M., M. Alibdin, H. R. Esmeili & G. Polgr, 2014b. Morphologicl divergence in the Wlton's Mudskipper, Periophthlmus wltoni Koumns, 1941, from the Persin Gulf nd Gulf of Omn (Gobioidei: Gobiide). Zoology in the Middle Est, 60, Ghnbrifrdi, M., M. Alibdin & H. R. Esmeili, 2014c. Morphometric vrition of Periophthlmus wltoni Koumns, 1941 (Teleostei: Gobiide) in the Persin Gulf nd Gulf of Omn. Irnin Journl of Animl Biosystemtics, 10, Ghnbrifrdi, M., H. R. Esmeili, Z. Gholmi, M. Alibdin, & B. Reichenbcher, Moleculr phylogeny of three mudskippers (Gobiide) from the Persin Gulf nd Gulf of Omn (Mkrn) Se. Journl of Applied Ichthyology, 32, Girling, P., J. Pupper & B. Nowl, Effect of cute slinity nd wter qulity chnge on juvenile green bck flounder, Rhombosole tprin. Act Ichthyologi Pistoric, 22, Goldenfrb, P. B., F. P. Bowyer, T. Hll & E. Brosious, Reproducibility in the hemtology lbortory: The microhemtocrit determintion. Americn Journl of Clinicl Pthology, 56, Fox, H. E., S. A. White, M. F. Ko & R. D. Fernld, Stress nd dominnce in socil fish. The Journl of Neuroscience, 16, Hider, G., Comprtive studies of blood morphology nd hemopoiesis of some teleost. observtions on cells of the red series, Journl of Zoology, 179, Herrer, M., C. Argão, I. Hchero, I. Ruiz- Jrbo, L. Vrgs-Chcoff, J. M. Mncer & L. E.Conceição, Physiologicl short-term response to sudden slinity chnge in the Seneglese sole (Sole seneglensis). Fish Physiology nd Biochemistry, 38, Hosseini, S. M., S. A. Hosseini & A. Jfr Nodeh, Serum biochemicl chrcteristics of Belug, Huso huso (L.), in response to blood smpling fter clove powder solution exposure. Fish Physiology nd Biochemistry, 37, Hrubec, T. C., J. L. Robertson & S. A. Smith, Effects of temperture on hemtologic nd serum biochemicl profiles of hybrid striped bss (Morone chrysops x Morone sxtilis). The Americn Journl of Veterinry Reserch, 58, Jing, I. F., V. Bhrth Kumr, D. N. Lee & C. F. Weng, Acute osmotic stress ffects tilpi (Oreochromis mossmbicus) innte immune responses. Fish & Shellfish Immunology, 25, Jin, N. C., Essentils of Veterinry Hemtology, Le nd Febiger, Phildelphi, p Jin, J. & Z. Wu, Effects of trditionl Chinese medicine on non-specific immunity nd disese resistnce of lrge yellow croker, Pseudoscien croce (Richrdson). Aquculture, 218, 1 9. King, R. P. & M. T. Udo, Vegettion succession-medited sptil heterogeneity in theenvironmentl biology of Periophthlmus brbrous (Gobiide) in the esturine swmps of Imo River, Nigeri. Interntionl Journl of Surfce Mining, Reclmtion nd Environment, 11, Kwpinski, J. B., Methods of Serologicl Reserch. John Wiley nd Sons, New York. Liebert, A. M. & C. B. Shreck, Effect of cute stess on osmoregultion, feed intke, IGF-I, nd cortisol in yerling steelhed trout (Oncorhynchus mykiss) during se- BJVM,, No 11

12 Hemtologicl, blood biochemicl nd immunologicl responses to grdul cclimtion to. wter dpttion. Generl nd Comprtive Endocrinology, 148, Chun-yin, L., Influence of slinity on ure nd mmoni metbolism in silver sebrem (Spms srb). Mster thesis. The Chinese University of Hong Kong. Hong Kong. Mced-Veig, A., M. Monroy, G. Viscor & A. De Sosto, Chnges in nonspecific biomrkers in the Mediterrnen brbel (Brbus meridionlis) exposed to sewge effluents in Mediterrnen strem (Ctloni, NE Spin). Aqutic Toxicology, 100, Mrc, A. M., C. Quentel, A. Severe, P. Y. Le Bil & G. Boeuf, Chnges in some endocrinologicl nd non-specific immunologicl prmeters during sewter exposure in the brown trout. Journl of Fish Biology, 46, McCormick, S. D., Endocrine control of osmoregultion in fish. Americn Zoologist, 282, McCormick, S. D., Endocrine control of osmoregultion in teleost fish. Integrtive nd Comprtive Biology, 41, Mleczko, R., Mudskippers: The Periophthlmus species, Prt 3. Journl of Interntionl Goby Society, 2, 9. Mommsen, T. P., Metbolism of the fish gill. In: Fish Physiology, vol XB, eds W. S. Hor nd D. J. Rndll, Acdemic Press, New York, USA. pp Murdy, E. O., A txonomic revision nd cldistic nlysis of the Oxudercine gobies (Gobiide: Oxudercine). Records of the Austrlin Museum, 11, Mustfyev, N. J. & A. A. Mekhtiev, Chnges of the serotonergic system ctivity in fish tissues during n increse of wter slinity. Journl of Evolutionry Biochemistry nd Physiology, 44, Nordlie, F. G. & D. C. Hney, Adpttions in slt mrsh teleosts to life in wters of vrying slinity. Itlin Journl of Zoology, 65, Pulsford, A. L., S. L. Gony, M. Tomlinson, N. Collingwood & P. J. Glynn, Effects of cute stress on the immune system of the db (Limnd limnd). Comprtive Biochemistry Physiology, 9, Rhimin, H. & A. Pehpuri, Intertidl fishes of Qeshm islnd, the Persin Gulf. I. Gobiide (Pisces: Perciformes). Journl of Science, 33, Riche, M., Anlysis of refrctometry for determining totl plsm protein in hybrid striped bss (Morone chrysops M. sxtilis) t vrious slinities. Aquculture, 264, Rollnd, R. M., M. Gilbertson & R. E. Peterson, Chemiclly induced ltertions in functionl development nd reproduction in fishes, SETAC Press, Penscol, FL pp Ruscoe, I. M., C. C. Shelley & G. R. Willims The combined effects of temperture nd slinity on growth nd survivl of juvenile mud crbs (Scyll serrt). Aquculture, 238, Slti, A. P., A. Bghbnzdeh, M. Soltni, R. Peyghn & G. Rizi, The response of plsm glucose, lctte, protein nd hemtologicl prmeters to osmotic chllenge in common crp (Cyprinus crpio). Interntionl Journl of Veterinry Reserch, 4, Schmitz, M., J. Douxfils, S. N. M. Mndiki, C. Morn, S. Bekelndt & P. Kestemont, Chronic hyperosmotic stress interferes with immune homeostsis in striped ctfish (Pngsinodon hypophthlmus, S.) nd leds to excessive inflmmtory response during bcteril infection. Fish & Shellfish Immunology, 55, Schperclus, W., H. Kulow & K. Schreckenbch, Hemtologicl nd serologicl technique. In: Fish Disese vol. 1, 2 nd edn, ed V. S. Kothekr, Oxonin press New Delhi: Gulbprimlni, pp Skours, A., The use of piscine innte immune responses s indictors for environmentl pollution in mrine ecosystems. PhD Thesis. 12 BJVM,, No

13 S. Soltnin & M. S. Fereidouni Steinhgen, D., P. Kruse, & W. Körting, Some hemtologicl observtions on crp Cyprinus crpio L. experimentlly infected with Trypnoplsm borelli Lvern nd Mesnil (Protozo: Kitenoplstid). Journl of Fish Diseses, 14, Svobodová, Z., M. Fljshns, H. Modrá, M. Svobod & V. Vjcová, Leukocytes profiles of diploid nd triploid tench, Tinc tinc L. Aquculture, 198, Thompson, I., A. White, T. C. Fletcher, D. F. Houlihn & C.J. Secombes, The effect of stress on the immune response of Atlntic slmon (Slmo slr L.) fed diets contining different mounts of vitmin C. Aquculture, 114, Trinder, P., Determintion of glucose in blood using glucose oxidse with n lterntive oxygen cceptor. Annls of Clinicl Biochemistry, 6, Tytler, P. & T. Vughn, 1983.Therml ecology of the mudskippers Periophthlmus koelreuteri (Plls) nd Boleophthlmus bodderti (Plls) of Kuwit By. Journl of Fish Biology, 23, Ssekumr, A., Meiofun of mngrove shore on the west cost of peninsulr Mlysi. Rffles Bulletin of Zoology, 42, Vzzn, M., M. Cmmrt, E. L. Cooper & N. Prrinello, Confinement stress in se bss (Dicentrrchus lbrx) depresses peritonel leukocyte cytotoxicity. Aquculture, 210, Woo, N. Y. S. & J. C. Murt, Studies on the biology of red se brem Chrysophyrys mjor III. Metbolic response to strvtion in different slinities. Mrine Biology, 6, Wootton, R. J., Ecology of Teleost Fishes. Chpmn nd Hll, London, p Wng, Y. F. & X. H. Zhu, A review on impct of slinity on ptterns of fish ecophysiology. Studi Mrin Sinic, 44, Woo, N. Y. S. & R. S. S. Wu, Metbolic nd osmoregultory chnges in response to reduced slinities in the red grouper, Epinephelus kr (Temminck nd Schlegel), nd the blck se brem Mylio mcrocephlus (Bsilewsky). Journl of Experimentl Mrine Biology nd Ecology, 65, Yd, T., T. Azum & Y. Tkji, Stimultion of non-specific immune functions in sewter-cclimted rinbow trout Oncorhynchus mykiss, with reference to the role of growth hormone. Comprtive Biochemistry nd Physiology Prt B, 129, Zhou X., C. Niu, R. Sun & Q. Li, The effect of vitmin C on the non-specific immune response of the juvenile softshelled turtle, Trionyx sinensis. Comprtive Biochemistry nd Physiology, 131A, Pper received ; ccepted for publiction Correspondence: Siyvsh Soltnin, Aqutic Animl Helth & Diseses Dept, School of Veterinry Medicine, Shirz University, Shirz, Irn, tel: , , fx: , e-mil: siyvshsoltnin@yhoo.com BJVM,, No 13