Sfakianakis Et Al. 2011. the Effect of Rearing Temperature on Body Shape and Meristic Count in Zebrafish (Danio Rerio) Juveniles

7/28/2019 Sfakianakis Et Al. 2011. the Effect of Rearing Temperature on Body Shape and Meristic Count in Zebrafish (Danio

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The effect of rearing temperature on body shape

and meristic characters in zebrafish(Danio rerio) juveniles

Dimitris G. Sfakianakis & Ioannis Leris &

Anastasia Laggis & Maroudio Kentouri

Received: 2 December 2010 /Accepted: 18 April 2011# Springer Science+Business Media B.V. 2011

Abstract Although zebrafish (Danio rerio) i s ahighly studied organism on many fields of research,many aspects of its basic biology still elude thescientific community. Its response to temperature -especially developmental one - has been very scarcelystudied and this is an important lack of knowledgesince the species is considered quite eurythermal innature. In the present study, zebrafish was subjectedto four different developmental temperatures (22, 25,28 and 31C) from the half-epiboly stage until after

metamorphosis in order to examine whether thetemperature can influence the juveniles phenotype.Morphometric and meristic characters were explored.Body shape and almost all of the meristic charactersstudied were significantly affected by the temperatureapplied during the first stages of development. Mostmeristic characters of the study, presented a signifi-cant differentiation in the extreme temperatures used(22 and/or 31C), whereas lower temperatures seemedto produce higher meristic counts in the majority ofthe characters. Zebrafish juveniles, as shown in this

study, exhibit highly variable phenotypes (phenotypicplasticity) induced by diverse thermal conditionsduring their early ontogenetic stages possibly in orderto successfully adjust to different environments.

Keywords Temperature . Zebrafish . Meristic . Bodyshape . Morphology . Larval rearing

Introduction

Temperature is known to be one of the most importantenvironmental factors that strongly affect all develop-mental processes in fishes. It influences the morphologyin general (Lindsey 1988; Wimberger 1992; Tudela1999; Koumoundouros et al. 2001; Pakkasmaa andPiironen 2001; Cabral et al. 2003; Turan 2004;Georgakopoulou et al. 2007), the muscle development(Johnston 1993, 2006; Wilkes et al. 2001; Johnston etal. 2009; Koumoundouros et al. 2009) and theappearance of morphoanatomical deformities (Wiegand

et al. 1989; Polo et al. 1991; Lein et al. 1997;Vgsholm and Djupvik 1998; Wang and Tsai 2000;Koumoundouros et al. 2001; Sfakianakis et al. 2004,2006; Abdel et al. 2005).

Although the studies on the topic are numerous, veryfew are those that directly correlate temporal conditions(during early ontogeny) and their effect on meristiccharacters in any fish species (Lindsey 1988; Murrayand Beacham 1989; Blaxter 1991; Georgakopoulouet al. 2007). On the other hand, only recently, it was

Environ Biol FishDOI 10.1007/s10641-011-9833-z

D. G. Sfakianakis (*) : I. Leris : A. Laggis : M. KentouriBiology Department, University of Crete,P.O. Box 2208, Vasilika Vouton,71409 Heraklion, Crete, Greecee-mail: [email protected]

Present Address:

I. LerisBehavioural Biology, Department of Biology andHelmholtz Institute, Utrecht University,P.O. Box: 800.86, 3508 TB Utrecht, The Netherlands


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reported for the first time that body shape differentiationin a fish species, sea bass (Dicentrarchus labrax, L.),was induced by the environmental temperature duringthe early life stages (Georgakopoulou et al. 2007). Theauthors used two different temperatures (15 or 20C)only in the larval rearing (from the egg stage until

metamorphosis) of the species to finally conclude thatdevelopmental temperature strongly affects both bodyshape and meristic characters at the subsequent

juvenile stage. In particular, they reported that fishbody shape at 15C tended to be more slender than at20C and that the final numbers of dorsal spines, softrays, pectoral lepidotrichia and caudal dermatotrichiawere significantly affected by temperature.

Zebrafish, Danio rerio, the case study of this work,is regarded as a valuable model-organism for all kindsof research (Lawrence 2007). For the past few

decades, it has been a very important model-organism in research fields such as Genetics, Neuro-

physiology, Developmental Biology and Biomedicine(Amsterdam and Hopkins 2006). In spite of its

popularity as a research tool, only recently did someintegrated studies about its biological and ecologicalcharacteristics emerge (Engeszer et al. 2007; Spenceet al. 2008) which however do not complete the

puzzle of knowledge regarding its early life condi-tions and especially the environmental temperature.Although the temperature of the species habitat

seems to be established at the range from 6C in thewinter up to 38C in the summer (Spence et al. 2008)there are still some contradictions concerning thetemperature range in which reproduction and earlydevelopment take place (Sfakianakis et al. 2011). Our

personal observations throughout many rearing trialsshowed that the rearing temperatures of 22C and 31Care the lower and upper limit values respectively forsuccessful rearing in the laboratory.

The purpose of the present work is to investigatewhether different rearing temperature conditions

result in differences on the body shape and themeristic characters of zebrafish.

Materials and methods

Rearing

Eggs of D. rerio were obtained from wild-typebroodstock (Aquaculture lab, Biology Department,

University of Crete) kept in 30 l tanks at 280.5Cand fed three times per day with industrial dry food(Sera Vipan, flakes for all ornamental fish, Germany).Two groups of about 80 and 100 individuals maintainedat a sex ratio of 2:1 females to males were used asspawners for the purposes of the study. After the

spawning, eggs were collected, separated (the live fromthe unfertilized ones) and submerged for 35 min inmethylene blue hydrate (0.001%) (Westerfield 1995).The eggs were then randomly separated into 4 groupsof 300 and each group was introduced in one of four120 l tanks with different water temperatures (22, 25,28 and 31C). The experiment was conducted in 2replicates and different parental fish were used eachtime. The experimental temperatures (22, 25, 28 and31C) were chosen in order to include the entire rangeof the suggested developmental temperatures that

zebrafish encounters in nature (Engeszer et al. 2007;Spence et al. 2008; Froese and Pauly 2009) and be atthe same time inside the range of the accomplishablesuccessful rearing in laboratory facilities.

The regulation of temperature was achieved with theuse of electrical heaters (Aquarium Systems, Visi-Therm, 100 W) and coolant device, when needed. Thefish were fed ad libitum initially with Paramecium sp.(Blades Biological CO, UK) and later with newlyhatched Artemia sp. nauplii (Instar I) (Westerfield1995).

Sampling and specimens handling

Rearing was ended well after the ending of the larvalstage, when the total length (TL) of a random sampleexceeded 12 mm. Specimens were collected andanaesthetised with ethylenglycol-monophenylether(Merck, 0.20.5 ml l1), individually photographed(left-side, Olympus Camedia C-5050 Zoom) andcollectively fixed in 5% buffered formalin (pH=7.2)(Taylor and van Dyke 1985). Staining for cartilage and

bone was performed based on the modified techniqueof Park and Kim (1984). The obtained digital pictureswere used to measure the TL (tpsDig, Rohlf, version5.0.3.32) and to perform the morphometric analysis.The stained specimens were used to count the meristiccharacters in study. All specimens with deformities inthe cephalic region, fins or vertebral column(Koumoundouros et al. 1997, 2001, 2002; Sfakianakiset al. 2003, 2004, 2006) as well as those with damaged

body parts due to sampling manipulations were

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excluded from both the shape analysis and themeristics study. A total of 386 specimens were finallyused in this study (approximately 50 per replicate).

Laboratory fish were reared and handled accordingto the policies and guidelines of the Greek nationallaw 2015/2001 which incorporates the convention

for the protection of vertebrate animals used forexperimental and other scientific purposes, of thecouncil of Europe.

Data analysis - morphometrics

For the morphometric analysis, truss measurementswere made on specimens by collecting XY co-ordinate data from morphological landmarks. Specificspots on the body, that were stable and visible at alldevelopmental stages of the fish, were selected for

landmark placement (Fig. 1), which was performedusing specific image processing software (tpsDig,Rohlf, version 5.0.3.32). The distances between thelandmarks corresponded to the morphometric charactersused for the analysis and 28 in total were finally used(Fig. 1).

The morphometric characters were significantlycorrelated with size, therefore each measurement wasadjusted by the following allometric equation Y=a *

Xb, such that the standardised value of this variable inthe case of an individual of size Xi would be:

Y

i YiX0

Xi

b

where i is the true value of the variable Y, i* the

standardised value, 0 an arbitrary reference size (inthe present study, the mean TL of all the examinedspecimens, 14.37 mm) and b the allometric exponent.This method normalizes the individuals in a sample toa single, arbitrary size, common to all samples and, at

the same time, maintains the individual variation(Tudela 1999). It has been successfully used by manyresearchers recently (Ibaez-Aguirre and Lleonart1996; Tudela 1999; Salini et al. 2004; Turan 2004;Turan et al. 2006).

The degree of similarity among samples and therelative importance of each measurement for groupseparation were assessed by stepwise discriminantfunction analysis (DFA) with cross-validation.Mahalanobis distances between groups and their asso-ciated probabilities were also evaluated. The signifi-

cance of differences among groups was verified bymultivariate analysis of variation (MANOVA) of themorphometric characters. The two replicates were

pooled before the analyses which were conducted bythe SPSS v.15 package.

Data analysis - meristic characters

Meristic characters such as the total number ofvertebrae and the rays of the fins were recorded onthe stained specimens. The fins used in this study

were the dorsal, anal, pectoral, pelvic and caudal. Thetotal number of rays of each fin was counted. Caudalrays were counted as upper and lower, lepidotrichia

Fig. 1 Landmarks collected on the in vivo photographed larvaeand morphometric characters (distances) used in the presentstudy. 1, anterior tip of upper jaw; 2, anterior margin of the eye,on the axis passing through the eye centre and the firstlandmark; 3, posterior margin of the eye, on the axis passingthrough the eye centre and the first landmark; 4, anterior base ofthe dorsal fin; 5, posterior base of the dorsal fin; 6, dorsal base ofthe caudal fin; 7, Ventral base of the caudal fin; 8, Distal tip of thehypural bones; 9, posterior central tip of the caudal fin; 10,posterior point of the body, on the vertical axis passing through the

posterior tips of the upper and lower caudal lobes (orientating theTL); 11, posterior base of the anal fin; 12, anterior base of the analfin; 13, anterior base of the left pelvic fin; 14, posterior tip of theoperculum; 15, ventral tip of cleithrum; 16, dorsal tip of the bodyon the vertical axis passing through the posterior margin of theeye; 17, dorsal tip of the body on the vertical axis passing throughthe 14th landmark. The 28 distances shown in the figure are thoseused in the morphometric analysis. The distance between land-marks 1 and 10 constitutes the TL. Landmark 9 was not used forextracting distances

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and dermatotrichia. Each fin meristic count wasperformed on the left side for paired fins. Skeletalterminology used is according to Harder (1975).

All 386 specimens that were used for the meristicsstudy had reached full completion of their skeletalelements (Table 1).

The effect of temperature on the meristic charactersof the zebrafish was studied by means of a MAN-OVA. The non-parametrical U test of MannWhitneyfor two samples was used (after the appropriatecorrection for tied measurements) to individuallycompare the meristic characters between the fourthermal treatments applied (Sokal and Rohlf 1995).All tests were performed at the significance level of0.05.

Results

Morphometric characters

Statistical analysis showed that morphometric charactersand therefore the body shape were significantly affected

by rearing temperature (Wilks =0.039, approximationF15.003, P


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et al. (2007) is the only one that proved somethingsimilar when they recorded that sea bass body shapetended to be more slender when reared at 15C asopposed to reared at 20C.

In the present study, a clear variation on the shapeof the body between the four populations wasobserved, showing a strong influence of the rearingtemperature. The results of the DFA showed that thedistances that played the most important role in thediscrimination were d45, d1213, d413, d78 andd56 (Table 2; Fig. 1) which are all located in the

posterior part of the body.The variance in fish body shape can be explained

by the general morphological plasticity that fishpresent in the different environments, due to alter-ations in muscle and bone developmental patterns(mechanical - functional approach; Wimberger 1992).In addition, physico-chemical water conditions (density,

viscosity etc.), are greatly affected by environmentalfactors, especially temperature. So, it is possible thatdifferent conditions of the media in which fish live andswim, require different locomotive responses that canlead to the observed morphological variance, during thedevelopmental period. Wimberger (1992) argued that if

plasticity is a result of the stress regime and the variousmechanical stimuli, behavioral plasticity is responsiblefor the morphological plasticity. Moreover, it is wellknown that temperature greatly affects fish metabo-lism, therefore the relative growth rate of some tissues

and organs of the myo-skeletal system of the fish,could be influenced by temperature during the develop-mental period (Lindsey 1988; Imre et al. 2002).

At the same time, temperature is known to havegreat effects on muscle development (Johnston 1993,2006; Nathanailides et al. 1995; Alami-Durante et al.2000; Ayala et al. 2001; Wilkes et al. 2001; Johnstonet al. 2009; Koumoundouros et al. 2009) with

possible further impacts on external morphology. Itis also proven that temperature affects the number andthe diameter of fast and slow muscle fibres(Johnston 2006; Koumoundouros et al. 2009). There-fore, considering that zebrafishs swimming type isthe subcarangiform (Plaut and Gordon 1994),achieved by the use of the posterior half of the body(1/22/3 of the overall body muscle tissue), and thatthe posterior part of the body was the most variant

between groups, it can be stated that differences in

body-shape, derive from differences in quantity and/or size of the muscle fibres, or the relative distributionof the muscle tissue of the fish reared in differenttemperatures.

Meristic characters

Many studies have compared populations of the samespecies (mostly wild ones) and have found variationof the meristic characters. Most of them hypothesize

Distances Wilks' Lambda F-remove (3.215) p-level Toler.

d412 0.053262 26.09458 0.000000 0.105658

d45 0.045830 12.45399 0.000000 0.108204

d1213 0.044712 10.40123 0.000002 0.165672

d413 0.043969 9.03674 0.000012 0.026775

d7

8 0.043614 8.38671 0.000027 0.539391d56 0.042537 6.40943 0.000354 0.161932

Table 2 The distances(Fig. 1) that variedsignificantly between the4 populations based on theMANOVA analysis

Table 4 Classification Matrix, (rows, observed; columns,predicted)

Percent Correct 22 25 28 31

22 88.88889 48 6 0 0

25 86.76471 7 59 2 0

28 92.64706 0 2 63 3

31 96.42857 0 0 2 54

Total 91.05691 55 67 67 57

Table 3 p-levels (***,


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that its due to the different temperature conditions ofthe living area populations (Tudela 1999; O'Reilly

and Horn 2004; Turan 2004; Turan et al. 2006),whereas there are only a few studies that directly

Fig. 3 Numbers of meristic counts (MeanS.E.) on zebrafish juveniles (N=89101) of the four different temperature groups (22, 25,28 and 31C). Same letters (e.g. a,b) indicate absence of statistical difference (MannWhitney U-test)

Fig. 2 Plot of zebrafishjuveniles (N=200) reared atfour different temperatureconditions (22, 25, 28 and31C) on the first 2 axes ofCanonical Analysis basedon their morphometriccharacters. Ellipses

represent 95% ofconfidence areas

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correlate early developmental temperature with meristicvariation (Lindsey 1988; Murray and Beacham 1989;Blaxter 1991; Georgakopoulou et al. 2007). In the

present study we concluded that early life temperatureinfluences the meristic characters of the developingfish. Variation of the meristic counts in the extreme

temperatures (22 and/or 31C) was observed in most ofthe cases.Zebrafish seems to be a highly variable (in respect

to its phenotype) fish species. Ferreri et al. (2000)studied the meristic counts in wild and reared zebra-fish and reported that the variability of the meristiccounts (in many of the tested anatomical areas)exhibited by the wild specimens was higher than theone exhibited by the reared ones. Reared specimensof the Ferreri et al. (2000) study developed at aconstant laboratory temperature and, based on the

results of the present study, it is logical to expect themto present less variable meristic counts than the wildones which probably encountered non-stable tempera-ture conditions in their early life.

When comparing the present data with otherstudies of the meristic counts in zebrafish (Dentryand Lindsey 1978; Axelrod and Schultz 1990; Ferreriet al. 2000; Bird and Mabee 2003; Parichy et al.2009) it is clear that they are in agreement as far asthe total number of the characters is concerned.However, the present study is the only one that

presents both the minimum and the maximum numberof counts previously reported for each character andthis is a result of the four different developmentaltemperatures used. It is therefore reasonable to arguethat the temperature spectrum used in the presentstudy covered sufficiently the observed morphological

plasticity of the species.It is common knowledge that fish developing at lower

water temperatures produce higher numbers of meristiccounts than those developing at higher temperatures(Murray and Beacham 1989). This is in accordance

with the present study, where with the exception ofthe caudal fin rays, all other meristic charactersexamined presented either higher numbers at lowertemperatures (vertebrae, dorsal and anal rays) orlower numbers at higher temperatures (pectoral and

pelvic rays). The caudal fin rays (upper and lowerlepidotrichia and dermatotrichia) did not present any

particular trend or in some cases any differentiation atall, except from the lower caudal dermatotrichia whichwere significantly fewer at the lower temperature used.

It has been stated before that meristic counts arecontinuously subjected to environmental influencesfrom fertilization up to the final count fixation orsimply during the entire larval period (Taning 1952;Fowler 1970). Georgakopoulou et al. (2007) reportedrecently that temperature effect from the half-epiboly

stage until metamorphosis is enough to permanentlyalter the meristic counts of many fins in sea bassjuveniles. All the above completely agree with thepresent findings since it was proven here that iftemperature is applied only between the egg stage andmetamorphosis (as opposed to during the fertilizationand spawning as well), it can have a dramatic effecton a fishs final meristic count.

As far as the number of the vertebral centra isconcerned, which is clearly a different case than thefin rays, Lindsey and Ali (1965) suggested in an

earlier period that it can be modified only duringearly embryogenesis. In a study conducted by Dentryand Lindsey (1978) in zebrafish, it was reported thatin some cases the vertebral counts of fish cultured atthe same water temperature were affected by thetemperature history of the parents before and duringfertilization. The present study showed clearly thatvertebral numbers are modified by temperature whenthe latter is applied only during the embryonic andlarval stages.

To further specify where exactly in the develop-

mental process does the effect of temperature act, weshould try and isolate the exacttime window wherethe meristic counts or the body shape are defined.Could it be early or later in the development (e.g.during the embryonic stage only) or are the entireembryonic and larval stages combined necessary forthe effect to present itself? In order to answer thesequestions, a narrower stage-targeted experimentshould be conducted, possibly with the same tempera-ture range as the one used in the present study.

The overall effect of temperature on zebrafish

phenotype exhibited in the present study is certainlyanother case of phenotypic plasticity. This fishesability of adapting successfully to all kinds ofvariations in their environmental conditions has long

been regarded as a requisite for their survival (Fuimanand Batty 1997). Zebrafish seems to be copeing wellin the different thermal treatments by adjustingsuccessfully both its body shape and its meristiccounts. The question that remains to be answeredhowever is whether those changes are adequate for

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the specimenssuccessful living and reproduction atsuch extreme temperature conditions.

Acknowledgements The authors thank two anonymousreviewers for their helpful comments in producing the finalmanuscript. The present study was f inanced by theEuropean Social Fund and National Resources (EPEAEK

II

PYTHAGORAS I) to M.K.

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Sfakianakis Et Al. 2011. the Effect of Rearing Temperature on Body Shape and Meristic Count in Zebrafish (Danio Rerio) Juveniles