BlOCKIUllCAL Cl.OCit Mf!CHAHlSMS

Rhythtd.c oscillation has bi.!en unequivocally re<;arded

as a funda.aantal propertt of life {:.icheving et al., 1978 J

3cheving, 1976). Hhythms of varying fl'-EF;u::.ncies ( circacilan

ar.Kl/or circannual ate:.) nave oeen d,eteet.ed in several

physiolo.Jical and met.a~'olic varid~Jles, viz., cholesterol,

glucose, triglycerides, phospholipids, free fatty acids,

corticost:Jrone, tostost,,rone, insulin, thyroxiM,

5-hydroxytryptaraina, _Sialic .Jcid, uric acid, urea, phosphate,

sodium, potassium, c.1lcium, ~iliriWin, protein, JlJUJ:U.n, globulin,

.Jlk.J!ine phos,>hatase, transaminases, lactic dehyJroJenasa,

80

GPT, ate.,. in plaSH1a and/or serum 1 glycogen, Got, GPT,. e~-Hal:lH,

acid pho&i)h-:ttasa.LDH, tyrosine trzmsamtnase, lJN,~ RN~ etc:., in

liver and rtmny other tissues (H~intxu:<J. 1974 ; Halberg and

i\hl.Jr(ln, 1980 ; ltalL;.;~r o at <d .•• 1911 ; Halb~!rg, 1974 1 Sf:b9v1ng

et al., 1978 ; iJ,aye:rsb>lch, 1976 J Halberg et at •• l900h

it()'mW,c;:t J il!Ost of the :H.udies are <:onfined to flla:mmls itu:luding

h\lman bein<;;J&. In notlllla!m;:~al..ian and other lower fl!rtebrates

re;.}orta on rhytl\J:5ic variations in the physiolo~:i.cal and

:~tabolic correlat·1S are r<H.atively limited (Sctw..as&t~Va.nn,. 1911,.

Urown. Jr., 1•:;73). However, rhytrodc l!lQtor activities in

fishes have been studied extensively (Thorp~t, 1918).. Rhytbml

(circadian and/or circannual etc.) in carhohydrate, lip~d.

erythropoiesis, oxidativ~, endocrinological, and reproductive

parawet.;rs have bQen I'<"Jll documented in f.ishes (Fernandez

cmd nan<•s, 198·J ; D'Jbhunty at al., 1980 .J .:iimpson, 1976 i

Ya;ldshit.a, 1969 ; Billard and Greton, 1978 • Jonas and

uilinski, 1965 ; Leach and Taylor, 1977 ; Valtonen, 1974 a Larsson and rrzmga, 1977). 3ut the mannC1r in which the

periodici tie a of these cycles have been de! tett:.ined is based

m.ostly on visuc.~l reauing of the macros~opic chronogrilms.

Furthermore, not a sin.Jlu piece of report is avail:lLl!! on

rllyth .. ls of cave fishes (Da.rr, 1976).

In the present chapter, ,lfl attempt has .:nan made to

Jn.:JerstJnd prope.cti(!S of S)me of the biochemic<ll clocks of

a cave fish. The author has Oiapl()yl.ld the lat,Jst .w.Jilable

81

mathenutical modal to validate citCf.idian rhythms fo:r glycogen

and lact<l ta eont·~nts in mU$Clc, lac:t.l te in livor, alkaline

phosph<l tase dnd acety lcholinestorase enzyme aeti vi ties 1n

gill, and acetylcholinesterase activity in brain tissue.

1wenty four fishas, approximately of same body weight

and len9th, were seh<:tGd from the stock. a'lU<&riUlll ( 40 l.i ~e

capacity} and wa·re transf<.'lrred to unother smaller aquarium.

They w.::re accli::;;ated to UJht-dark phot<;)pceriod,with 12 h~tur.s

o£ li']ht. alternating with 1. hf'urs of darkness (light awitclled

on at oa.oo and off at 20.00 h) fox twant·1 one dar·s. rhe

temperature of the experimental rOO>;;~ remained near constant

(26 ,t. 4°C). Four fishes w<&re sacrificed t>y docap:itation

at each of the following time points, viz., 09.00, l3.ou~

l.7.oo, Ll.OJ, 0l.,Xl, and 05.00 h. Muscla• liver, gill and.

brain :;ere recoverea froo autopsied fishes, froz<>n at -200c

in a deeP-frigidaire until utilis2d in biochemi~l assay.

~evels of ,;lyCOJOO and lactate in muscle, lactate in liver;

..1Cti vi ty of alkaline phosphatase & acetyl.ebolinestcrase in

'Jill 01nd acetylcholinBster;J.ae in brain were assaJed according

Glycogen

;lyco y'n content ,Jf tha ::tu!lclo t13:>ue \':JS d'~t !rmi,ned

!1y th~ mathxl of Jeiftar ?t ·ll. (1950) lft•H' hot 1l<:tl1

ax truction.

82

Principle

Concent.rat<;ld sulphuric .acid hydrolyses ()lycosidt.e

bond to give mon•:.~saccharides which are fu.:rthar dE~hydrnted

to f~furnl ~nd its derivatives. the furfural reacts with

antlu::.me (lO...kato, 9-10 dihytiroanthracene) to give a blue

<;~reen coruplex ..

30 a of pot...s<>ium {l<;.oH} pGll~ts dissolve>d in 100 ml.

of distilled water.

2 l Y. $odium sJlphate solution

l 9 of sodium sulph,Tte (Na2:>o4 ) dissolvad in 100 ml

of distillud water.

3 95 i; :c. tha nol

95 ml of absolut: et:1an.:>l ilkld& to 100 ml with

J.2 'J of ;J:lt::r,Jne disS:JlV·"'-' in lw ml. of 95 ~~ sul;)hUX'ic

acid {H2 ~:.}4 ). This ro.:~'}Unt \l.lS fresnly pre:'·'red.

~it.andard glucose solution

20 mg of glucose d1ssolvad in lvJ ml nf Jistilled

IYJter .:md diluti!d to 1 litre. ~ ml of thia solution

cont.1ins !.JJ ~J of •Jlucoso.

83

.ieighed samples of muscl&& we:n~ hlten in test tubes

containin9 3 ml of 3J ;?. Kc»i solution and ctiguted in boiling

water bath. After cooling the tubes, o.5 mJ. of l ~ Na-2 so4 and 3.5 ml of 95 ;< ethanol <tere added to eacll tube to

prc:ipi tate cylyeog m.

Th~ cont<lnts in ec"iC:h tuno were stirrad ~tith a glass

rod and the rod WJS washed ir. tll:? tube itself with a small

quantity of <Jthan;>l. Th;: tubas ,·,rn:.] jently heated, cooled

and .1Umv d to "t:.1.1 r>V~rai·,:;ht for cmaplate precipitation of

•JlYCQ·Jon ;,tt room t >:.'lpa.r:.rttl.'t~. !hli! Bext ~Y th<i tu.bo$ l'i~u·e

c .ntl.'ifuvud dt 3·JV0 .rpm for lf> c:linuws., The suj)llrn~tilint was

disqa.r;.t ;d .11'ld th.a r"'siduu 'NilS dissolv<o:d in al.>out 3 ml of

distilled •~<~tar :md .r;:pr;;c;i.pitatod >dth 2.0. mJ. of 9;:J _.;

ethanol. It wc1s c•.:mtrifu..J\Od a.Jain ;.;oo supernatant we~& discarded,

The pr. c;;.~;i L:. \;.ls fin,:jJ.ly dissol vc•d in 5·J ul of distill~

,:ctt ·r. ·..;c;ti;:ntion of ~;lycor;en 1'/.::lS c<Jrri"'d out as follows a

o r.Jl of ;.Jli ,uot W•lS transfdrr;;td to a wide mouth boiling tuoo.

This served as a tost s.:~r:1ple. 5 ml of standard glucose solution,

cont:Jining lJ.J ~;J of glucose; vns t::~k;:n in another boiling tube.

This .-;.on·eJ c;s st.md~1.rd. 5 ml of .Jlass distilled ::~ater was

taken in a thi.:d t~st tube. This served i1S a bl;mt.

fhe t<ves containin,;; t .•3t 1nd stHld 1rd solutions and

olallk .. :re pL:~c.1J in ic·~ cold water. Jry ~nJ cla:Jn JlJss beads

.:.JrJ l13 :d t.> ,>J:>JV ~nt. i).JmpillJ f Jll uillg 3ddi tion Of ·'lllthron~

r9ag$nt. 10 ml of freshly prapa~ud an~1rone reagvnt (4) was

adJod to each tube with a iast flowing p.ip&tte. The contents

1rJere mix;:ed. by sl1<lkin;J. The tubes were then transferred t.o

:j tnilini;J wator !;!CI.th for <~x:C~ctly 30 minut•e and th~;~n cooled

to room tcmr:.>erature. The optical density (o.u.} of the teet

dnd sbndard solutions were read agoinst blank at 6::-:~; nm in

S:}G\tal.

'tho amount of the- tJJ.yco-;cn '""'s calcc~latad from

the ecpJu tion

1).9 of ')lyco-)en in 5 ml of .1liquot = ----loll X S

;.) = o.D. of standard solution

l.ll = ,;onverd:.m factor of •Jluco-~ , to Jlycogen.

Lactic acid

K.nown ·.vei,;hts of ~:~UScle tissue N<;re horno<Jeniz•~d in

lJ ;-~ trlch.loroac-)tic >cid (tc::,) <~nd the homo:r n.~t.es ,•,ere

C3ntrifUJ0d ;;.t 3JJJ r;>r.l for 1:':> r:linutes. The supern:1t~nts in

vach •.. ,:;e >Nre used for the estimation ~~ lactic acid by the

me thvd 'J f i3 J rk;l r ,1nd .j\.lltr,IO ra :>n ( 1941) •

Principle

->lucose .1nd other interfering llU':eriah of ;1rotein

fr~oJ ·xt"'·.c;t dre .r.amoved ;:;y Van .:>lyke-.ialkowsi !119thod of

treatment 11i th copper sulphate and calcium hydroxide. The

solution io than he;~ teu with cmlcQntra t'"d sulphuric acid.

Lactic ~:•ci.d is thus converted tv acetald\>lhtde, the lattez

is then <ktcrn.i:>i!d by relcti::>n with p.-hyd£oxydipllenyl in

1 20 I'· Co;:;;Jer ~\llPhate solution

~.

6

2011 g of Cu:Jo4 • oo2o ctisnolvtH.i in 150 ml of •.-nt'!l'r

witll gentle heal;in~n ttuil solution wm~ cooled .:<lt..l later

diluto;J to 1 Utre ·•itn distilled ;·.rater.

4 ;.-: C:>;J(:Ja:C sulph3toii solutl.un :

l vol'-.1•:1_,. of 2.; '' copper s..~l,)ilata solution (1) was

•.til ;bi t.:> ) volu;'la5['Jith d:i.wtil!~ l•nter nnd th~rouJhly

-, ·JyJr"~··.rJ'p''·'n •1 ~-- · , .... · '~ Vf-.1 ..L th.. • .lo\.::'<.:Jt.'Hioo 1

5 Y. NaOH s:>lution ; 2 ml di<Jtilled ~~;)t·3l!' ud.:bd >·lith

sliqht w.:1rming ·11'\d stirrin9; the solution then made

to l..J,J ml ,fith distillod \·Htar and ntorod in brown

bottle •

. ~.215 :;; oi 1m:::e: l~t!liULil 1-~ct t<.~ d;!.ssolved in l.OO ml

of :.li;:;'tillod .:;:. ter !>1 1 li u·c voll..tt:<otric flask J l m1

of concantl.\1ted suJ.phi.Xic .:LCid <.!OOGd ; tJw whole uade

t,.; O!W lit.i:>? .1i t!\ t.li.;.; t.ille!.\ l<<:~tol:' GlllU tl'lttl:OU\,lillV mixed.

i !.is .,,av>:;; l m;~ lnct.ic ~,:;iu in ? ml. of soluti;;~n and was

ut.:..i..<lo for :1 lon;;; tir,J~ ;vlwn k"pt in .lZ<!fl'iger;:;tor.

5 ml of s to eli: s t:m,br'l soL; tion (1) C:ilutoo to lO'.J m1

in t;L .. sc sto; .. ·pu.rcc,; vol.Jti•£:tr1c fLwk witi. distilled

;~atc•r .'lnd r.d.xod tho.~:ou;Jhly J tl)iS solution (}3Ve o.Jl mg

ldc tic .otc:id p-er r.tl and ,v-;s peer a red fre~h claily.

Procedure

2 nl of prot::<in fr~::c s:..~pcrnat.Jnt, 5 ml of fr,:s•1ly

;:.r . .,:· •. ;r<~<1 li thiu:n Ltct .• te standard solution .:md a :re:~,ymt

cl.lnl< (5 rul of ni~till:!'<J ·.J::tor) .mre taken in three separate

;:;.;,;; t..- H >.~ 1 1i t . .L .JS ; .L ol of 2U 1• Cr':>;)er sulph.• tc solution

,, .~ J J~a·.< to .J .. l ;:11 ·"lJ ttun r~'le to lJ tal ··1ith dis tilled •~ater •

.Jne 'J of po;'Jdere<.i ~ll( Jd) 2 cus 1 .. J ·<.i to dc.~Cll tu·.,o ·.;i ~h 'l

spatula. They v.~era stoppered :tnd shaken vigorously until

uniformly dispersad. The tubes \'lore kept for an hour with

frequent shakiwJ and latar e:ertrifuged at 3000 rpm for

15 minutes. 1 mt of supernatant vla& taken from eacb of

aoov& tubes in separuw tube& with an internal diameter

of 18 to 20 •• <l.05 ml of 4 ;1. CuSo4 (2) was added to

a.:~cb tube. .;}ubsaquently 6 ml of <:billed analax' f'2S04 was

added• initially drop i.>y drop, with uniform mbin9 of the

contents, to each tube. t~l the tubes ~re kept in a

boiling water botn for 5 minute& and cooled to 2;)0r; ~

below in running cold vtrlte:r:. ;J.l m1 of P-bydroxydi.phenyl

na>Jent was the-n added drop by drop \o eaeh tooe.

Precipitating reagent was dispersed again throughout the

solution quickly and uniformly by shaking. All the tubes

were incubated at 30° :1:. 1°c for 90 seeonds and were allowed

to cool to room ~mpera tare. The opti<:al density was

measured against blank at 570 nm in Spekol. The wlues

are exprees;:d as i.19 lactate per 100 mg fresh weight o~ muscle.

Non-specific pbosphosaoDOe$'teraaee (i\J.kaline phospatase il.C.

No. 3.1.3.1 and Acid phosphatase ~.c. No. 3.1.3.2)

Homo Jenates of fresh tissues were prepared in u.25 l'1

ice cold sucrose solution to approximately 5 i~ w/v 1

eertritu,Jed 3 t 300J rpm at 4°C for 1'.) rlinutes ;-~nd supernatant

w~s used for enzyme assay.

Pd.MJ.J)l•

Enzyme extract.. on 1ncubatic.n w1 th buffered aodiua

p-glycero:phoaphate, :t:llleaa~a iru>rganic pnoaph.at<J by

ena·~t1c hodrolyds., The inor.Janic phosphate reacts Vlith

an acid nolybdate solution to form phosphomolybdic acid.

The phosph:lmol)'bd.f.c acid is X''ilduc&d 'oy addition of 1. 2. 4

amino...noptholsulp!lonic acid reagent to produce blue colour

(Fiske and Subba aao. 1925). The intensity of the colour

developed is directly propotional to the amount of i~ganic

phosphate. which in tw.'n indicates the enzyl!liltiC activity

of phosphatase.

H.aa;enta

1 AlkaUne phosphatase aubetrate (pH lO.o); o.5 g of

sodium p-Jlyc~rophosphata and 0.424 g sodium diethyl

barbituri.lte dissolwd in 10J ml of distilled \Vater

and stored under a layer of petroleum ether in a

refrigerator.

2 Acid 11hosphatase subs tra t:: (pH 5.0) :

o.5 g of sodium P-glycerophosphate. 0.242 g sodium

dietnyl barl.liturate and 5 Ill of 1 N aceUc acid

dissolved in 100 all of distilled water 3lld stored

undar:"l.:.1er of petroleum ether in a refri']erator.

69

3 30 1- T:ric:hloroac:&Uc add (TCA) :

30 g .of !CA dissolved in 100 1111 of distilled water.

4 5 ;.~ !richloroac:otic acid ;

5 9 of TCA dissolved in 100 ml of distilled ~t.er.

5 10 N Sulphuric acid

4"50 ml of con;;:,}"ltr:.toaaulphuric ~ctd added to 1300 ml

of distill~ v1ater.

6 ;;>tock standard phosphate st:~lution :

o.35l g of pure dry monopotassium phosphate dissolved

in tiistille<i wa te:t in a l Ut:te volumetric: flask J

10 ral of 10 N ~so4 added and tho whole diluted to

l litre .vith distilled water. Thb solution contains

J.4 m,; of phosphate in 5.0 ml.

7 ,Jorking st..ltlddrd phosphate solution·.

6.25 ml of stock phosph:~te solution (6) tak.un in a

100 ml volumetric flask J 16.7 ml of 3C 1~ r..;.\ a<.ided

and the whole diluted ~vith distilled wat•Jr to 100 ml.

This solution contains 0.04 ~J of phosphate in 8 ml

8 ,\::T.JOnium oolyW<.~ tc :>olution ·.

!>.a g of a;:unonium molyndate dis3olv.'CI in 40 ml of

distilled 1vat·!r ; 60 ml of lu u ~~04 add :d to 1t

and the vlhole diluted to 200 ml with distillfrd water.

9 15 ;-:. &oJium bleulphite so1ut1o11:

15 g of sodium bisulpili~ dissolved in 100 ml of

distilhd w.:.~t·n:·.

10 2u Y. Sodium su1phi te solutbn :

20 g of sodium sulphite dissolved in 100 ml of

distilled water.

11 .\!llinonaphtholsulphonic acid re:1gent :

97.5 ml of 15 ;~ socii wn bi-!Zulphi te solution taken

in a ';Jlass st.:>ppered me::~su:ein;;; cylinder ' 0.25 g of

1.2.4 aminonaphtolsulphonlc ac~d and 2.5 ml of 20 Y.

sodium sulphite added and shalutn until the powder

dissolved. The solution was then transferred to a

brown 9l1ss iJottle and stored in cold.

Test·.

9.00 ml of buff~red sut,str.:te of r.:-:!Jir.~d pH and

1.0 ml of axtrac:t \ll·:.!re shak::n in a t,st tuu.c. The

ra:~ction r.tiJCturG w..1s incu:;.ltad for \>J r:U.nutes at

J~C. Th•:.! reaction \V<lS stoj)j)Od , 1 addin J .: 1nl of

3; t' TCA.

91

Conuol:

Control s3t11ple was prepared with 9.00 ml of buffered

.substrate. 2.0 llll of 30 :;{ TCA and 1.0 ml of ext.ract

l3oth tas t and control sarnples wel'e centrifuged

at 3000 rpm for 15 minute$. a.o ntl each of t'o?st sample,

control !lample, v~!d.ng at.Jttdard phosphate solution (7),

and 5 it. TI:A as blank Vtrere tak.,n in fou;,. t1!lst tubes ~aduated

to 10 ml ; 1.0 ml of anuoniu~lybdate added to •Jadl tube

and mi~ed tnornu\ihlY ; o.4 ml of a."!l!ncmaphthol~ulphonic

acid :te.Hj .~nt them added to e:'leh tube 100 mixc:d thoroughly.

tut:es '.F~rcJ left for ::; minutvs and optical density was read

ilc}aine t '1 H\k i:.l t (>(,;) nm in 3pekol. The values are expressed

Protein content of the oxtr.:1ct t•tas dot~~rmined

by the method of Lowry t?t a1. (1951) as described 1n Chapter 1\1

Acetylcholinesterase (a.c. 3.1.1.7):

0.25 m sucroGo iiulution to a,J[JJ:Oximdt.:1!( :.> ~~ w/v; centrifugoo

at 31XJO rpr:~ at 4<t for lj minutes 5 supe.rnutant \lc>a used for

'?nZ:YtU•.1 a!h.l:f ·l :.hu colo.~:itOOtric floJ\.hod of .. lotc<~lf (1951),

as :<lodifiod ; 1 .• urali .J-i:;hnati:.lss {1967).

PJ:i.nclple

CH.., - 0 - C\l;l. CH20H ...

I

Clia c~

I

u:;1 .W ·(CH3)3 + H:;tl iCl .. N-(Cf~s)3 + HCQI.ii

I

OH UH corresponding acid

Acetylcholine chloride Choline

;l.emoval of acetylcholine p~r unit time is measured

by comp.'lrison of its initial concentration in a ref~rence

tube .. i th the finul conce-ntra t!.on in exp<:rimental tube •

.. ~cetylcholine reacts vtith hydroxylamine to form correspc;nding

acylhydroxc•nic acid, ':lh:i.ch forms ~ stroo<Jlj'C. coloured farric

hydr:>xar!u te ,,1 th ferric salts.

Reagents

1 ;.i/15 phosphate ouf:hr(nH 7.4)

16.72 g of disodil.lll hy<Jro 1 ·n ph0sph.?te (i~a.Ll!P04 ) and

93

2.12 g of l'ilOnopotasai\lm dihyd:cog<m phosphate (~a4)

dissolved tn 100 ml of double distilled water.

2 J.004 rt ,\c;•tylcholine solution,

::itock solution (0.04 Ml was prc:pared by dissolVing

o.7466 g of ac«atyleboline chloride in 100 ml of HCl

of pH 4.o. ->toc:k solution >liaS diluted vdth 9 volU!Il9s

of phosphata buf'0.r (l) before uaut.

13.9 g of hydroxylamine hydrochloride dissolved in

lChJ ml of distilled ~vater.

4 3. 5 N sodium hydroxide :

of J.istiliad wator.

5 Alkalin0 hydl:oxybmine solution;

•iqual volumes of hydrolCyl:Jmine hydrochl.Jr~de SJlution(3)

c~nd 3.5 ~~ H.aOII solution (4) were milC~d to.pther before

use.

Hydrochloric acid (ltl):

'l:>lU:.10 :Jf cii.;tlll J 1\tlt.r.

7 0.37 ;,t Ferric: c:hl;.>rida solution

10 g of forric chlor:tde dissolved in 10\J ml of

O.l H HCl.

a 0.2~ M sucros~ solution

l ml of extr"ct vr\$ udded to 1 ml of buffered substrab

solution ; incubated :fo;.: 3i) :;Uay·;aa ;;. t; 37°t,l°C ; reactlon

was sto,Jp\ld by tiN ciddi tion of 2 ml of ulk.--:1lina hydroxylamine

b~'drochl.;ri.:l.?. Tho t:h<Js wJra sba~;.en thorou]hl.y .:md l m1 of

HCl (l:l l1Gl:ll2o) w..;s ~Jd,?d with further shaking lnd the whole

>'1·1S cantl:ifugod. 2. 5 ml of suptlrnatant was hken in •'l test

tube an~ 0.5 ml of ferric chloride solution was added. The

contents •wre a')ain thorou;;hly lllixed. Zero tilu0 controls

vnre m<>.intained ';::y adding 2 ml of alkaline hydroxylamine

hydroclll-.~ride prior to the oddition of the en~ylllO extrac:t.

Colour of the hydroY.ami.c acid fornod .os read at 54~) nm

a::pinst the bl:mk. The bl:mk cont1in·1d 1 >ll of \)uff :r

insta:1d of ~;:..~£fer subs tr.>ta :aixtur '•

The activity of tho enzyme 1'/,js d :t·.:rminod u1 the amount

of unreacted acetylcholine ct\loride left. aft~r 3.; ::dnutes of

95

incubation. 'fhe intensity of the coloUJ." at the end of the

incui:>;;,Uon ,Jives the nmount acotylcholine left unhydrolysed.

concentration and subst:c~te concentr .s tion left unhydrolysed,

the act\Hl l~v<)l of the '30ZJll'lO activity >m& G:;;tiwat'd and

e:<;rr-i.Hl<)'~d 5.n :" ~:101es of acet1•lcbolina chloride hydrolysl#d

p>:J: mg l)rotein per il:Jur. Protein content of th.:~ extract was

dot0rrainect~LO'>JI'Y ct ;:,l. ( 1951).

Statistical methods

uata (m<::an :J:. 1 St:) wc~re pbtted by the convent!.onal

way :.,Jainst clock tulur. i.liHter .. n>t fe..ttures of tinto-:iependent

v,,;;L.iJJ..,,s ,,,ere 2<;;$,;sseti. ..;t..:;U.,\.ical analysis :for comparison

of J:Coups of uuta •l~t:Ci.:: ct~rriod out by one W<lY analysis of

anll :::.intz, h/77). vill,Jla co;;inor was employt.>d to dctortaned

rhythm parar:Je ~;,_,;rs, viz.., HK!Sor, :;::Jpli twJe • ,md acroph.lse

(Halberg :t ,;1., um, 1972 ; !lelson ct al., 1979).

Results

Mu•cle glycogen

tioa sc1lc. ;; 't' ,~,>r;J 3U :j 'Ct<)d to 1!1-llJsis of v •ri ,mea.

fable lJ).

The muscle glycogen content was found to be highest

at ul • ..;u h. rne muscle had lowost .%lount. of glyco;Jen

stor.lS -1t. 13 • ..); h (Tal,le 17). Joth the time points are

exactly l~ hours dp.u·t :fr.Jm ~;ell other. dhen both h:l.ql'utat

.md lowvst mt#ans Wc!t:J coii<p<.u::'.!d~ u statistically s1•;:;nif1cant

•i.lif:. :ronco . .~~,s witnessed (l.i:.mc .. m• s .Jul tiple-range test,

'fabla l7). 1:110 pce.tc.:•r,t ci·:~m-Je fl:'u'll low,)st to hi:Jhest. mean

,iasul ts of tho cosinor <::Ml_isis of d~ta :t<;v;wl detection

of statistically si.;rrificant circ.'ldhn rhythra in the lmlSClet

glycog. n content of cave fishes (Table 19 1 fiql..l.t'~ J.8). Tbe

zero-a1:1pli tude hypothesis W-lS rc}Jct.:d at 2.9 r~ .l.ovcl. The

acro;;hccse w.J.s lo..:at d <lt ... 16° {01.07). with 9':> Y. con~id,.;.nc.e

intorVcllS between -339° {22.6 h) and -53° (03.;)3 h)(Tavle !9).

The m;,sor vns of 0.31 (mr.J/100 mg), with <lO amplitude o.04

(lll<J/hU mg).

Muscle lactate

rlesults of analysis of variJnce revaal st~tistically

signific:.nt time .~ff?ct (P < o.o!'>) on the content of lactate

in t.ha ouscle ti~sue (Tabla 18).

The max1.mum conc<1ntration of lacta tu in the r.l'Jscle

WdS ca:ord<:d 1t l3.uv h and ;;linimum ;:~t lv.uO h (Table 17).

Both the m-~.:.10 v.J!ues diff,)red st;tisticJ.lly significantly

97

...t stJ tis tic<Hly llliynifici:lnt ciJ.'..;.:~dian rhythm in Inuscle

lact<:~te cc;me<~ntrations Wd& ct~tl1!cted (Taole 19 ; r'i'JI.U:e la).

i!u prol:J.Jbili ty t:n t. tita .~;a?li '"v..l.<;~ -..£ tnis t:hftltJ\ is not

'ihiildJ. t.o zero ~~"'a :~;;.1 '' (Jaal~ 19). The ilcroph~\$0 •Nas

Liver lact.lte

,;;e.1n ;nlue at J5 •. JJ h :nd that of J.o,J;::st .1t 11 • .;: h (;~i·;ure 15).

uoth the tin:.? ;)olnt::; •·J.!:CO .1 0 ) •• rt from e,1cll -.~t.l<Jl: '.Jy 13:.;;".

·' :> \.·J tis ticJ.lly SiJnificant cir;; dian rnythm in the

l.•ct.1t.• conc·nt.r1tions of livor ti~suo .'/JS not v.,licJ.,ted

with the help of coSinor rhytlll!lom(:try. lbwover, the

acx·o!Jh;;se 1'1<.13 :litn>?ssed at -48° (03.2 h) with a meso:.:

18.2 (Jlg/lW mg), iJnd an amplitude 2.56 (ilg/l•JO mg)

('raLle 19).

Gill alkaline phosph.::~tase

iJcJta .wura')as <t. l .:;,~) n:.ve i;c~en ;'lotbd as o fut11::tion

of ti;;1e (Pi Jt.l:t<l l~). ·.,hul ts of atMlysis of v.u:l.;n<;o; did

hi·]hes t rocordad r.1van w~•s 42 ;.; •

th0 Jill (l:::.,le 22). 'io.vev:?t', t:·~~ .lC.i.';ipi·us.; :1.1S '.i ·:ectad

:~t -167° (11.13 h), ,;ith •l :J so.r of 2.~~4 (y_i )i/:nJ/h), alld

Fl . •'">)li tud~ J. 56 (.t] •Ji/n:J/h).

Gill dcetylcholinestarase

.... -> .'i"f ..~ .... . d on -.:, h tt,.J. nld

99

Maxi.11Uill enzyme activity was noticed at 17.00 h and

1~1inimum .1t u9.·)) h. ihc lo·ln>t ra.:an value signiiic.:Jntly

diffar:•cl fron that of tha ni jhc)St (P ( 0.01 J fable 20).

'l'he p·!l:'Cc·mt <:iunqa from tho lowest to highest .r:)corded

1\. .,t 1 t.iot.ic<<llY oi•Jnificant circadion rhythm w ... s ;~lso

" Lin·.'d ··1lt.io tb1 J;1lp .CJf til 1 cosincr anill·rds of the data

te;t ·:ns 1'c1j:·c~J:d .. t 1.7 ;.• lovul. The ci.rcadi.m acr'.>i>llase

vns rL t·~ : t: · .. t. -·''"'·p ( 1-:P •. J7 hj, with 9~ 1:. confidence

Bruin ~catylchblinesteras&

(Tacle 21).

to .31 . : •

100

i\ statistically significant circadian rhythm was

valid,.'lted for the~ acetylcholinesterase activity in the

b:nin tissue with the help of codnor a""'llysis of the

data (I<tble 22 ; Figure 19). The assumption of &ero­

amplitude hypothesis was r~jected at 3.8 t. level. The

eirc,ldian acrophase was d(~te>cted at -271° (16.07 h). with

95 ;t. confidonce intorvals between ..:J30° and •312° (T.tnle 22).

The muor Willi of 2.9 (jlll!Ol ;:.t;h/UifJ protein/h) with an ati'IPlitude

o.312 {~l ;i;h/rJKJ protein/b).

Dlscuss1oa

Present results clearly demonstrate the presence

of circadian rhythm in the conwnts of muscle :;~lyco;en,

muscle lactate, and in the acetylcholineaterasa enzyme

activities of ;ill and train tissues of cave fish.

Nemas;h•4U!Q gwz.ardi.when subjected to LD 12tl2 photoperiod.

Since liter3ture on biological clocks of cave fishes is

completely lacking, it is probably the first information

of such a kind. However, a statistically si:mificant circadiaJ1

rhythm -!1 ther in the content of liver lact::~ te or in the

alkaline phosphatJse activity of gill tissue was not detected.

This clearly indicates th1t oven though fishes live inside

the CJ vc!rn under eompl•.:> te darknoss their biochJ:nic 11 clocks

could be synchronis-.!d ll:r .JrtHieial photoperiods. ,\t presont

101

1t b not possible to explain why rhythms 1n livJr lactate

and alkaline phosphatase activity in gill tissue did not

appe'lr following exposure of the fishes to UJ 12c 12

photopedod for 3 week1:r. Could 1t be that still longer

period of accliraat.i<m is required for the expression of

rhythm in the above two biochamical paramet&rll 1 furthel'mOre,

invrssti\;l;at.iona ;)f rhythm in all tne above parameters of cave

fishes under natural condi ti()ns would be of great physiolQ<Jte.al

importance.

l'be aerophase of tha circadian muscle glycogen rhythla

appeared at -16° (;)J..o7 h), •vitll 9!> ;4 confidence inteJ:val

ran;;ing between -339° and -53°. Qv.lar the single transverse

circ:,dian time scale there Wi1S a 38 ;4 varianility from

lowest to hi·Jh<.!st recorded means. Although glycogen is

found in almost all tissues, muscle and liver tissues are

met.) -olic,,lly more imPOrtant in this re ;ard. Usually

glycogen func ti:ms as a res"rva of glucose and, dGp•~nding

upon the requir:a.Jnts, it is readily broken down into glucose

to meet energy demands of th~ ~ntire organism{Newsholme and

.:;tart, 1973). Present results, ther.Jfore, St.IJ':)est thilt

probably thare is less ;lucose utiliz:.~tion in the cave fishes

during mid-night 1Vhich in turn allows maximum accumul'l tion

in the form of musclo glyco-Jen during this time of the <:ircadl:u.

tlrae scala. .>ince we do not have J.ny d,J td/informa tion on

circadian rhythm'> -11ther in Jlyco;Jen syntn.t.ase or in glyco<;Jen

102

phosphoryhse aetiviti<?S of this cave fish, or .any other

fish, it is difficult to make any suqljlostion at present.

Possibly, a similar machanism(s), as in I!II!lnwah. might

be operlting l0Z~ding to exhibition of circ<~dian rhythm

in the contont of ;rruscl,~ ~~lycogan,

~xistence of circadian rhythm in ~he content of

;nusch lac to< te has bll<~t!l r'Jpor tc1d in snak0s ( Gra t& and

autchison. l'J17). How-~ver, too above repOrt eo:nsist5of

obaervutions a"t two tima points only over a ci.rcadian

scale. t\dde this, tlla authors h<tVe not utiltzod inferential

st,>tistics to validate circadian rhythm for u1U#Hile lactate

concentrations. rn tile pr-:~sent study• however, lactate

C.)ntent of rausclu has been determined at six time points

and a rhythm was detect·Jd by cosinor analysis of the data.

rhe hi;;llest lactate accu.nulation in the muscle 0cf cave fishes

was found to be at -162° (lO.a h). Conversely, the circadian

bathyph:Jse wi tl'l respect to lactate accumulation in the muscle

w<.~s <.iGtected at -342° (22.8 h), with 95 y. confidence intervals

ran,JinJ .l:!t•~-><:ln -313° (22.87 h) and -11° {0.73 h).

surprisin _;ly, there is a higher de ;re:e of ov.crla;>ping bet~ve~n

the acrophase timings of muscle Jl yco,;en :Uld ba thyphase

timings of muscle lact.l te. This SUJ'JuSts that th"re is an

invc~rse r ;1,• tionship between the ,1ccu -,ul.l tion of Jl yeo jen

.11\d lact.>te in the :-:1uscle tissue, i.e., ··men ooscle JlYCoJen

(;ontent of the cuve fish is lll.:lximum, lact.1to concentr,•tion

103

1s lowest. At tho .nonent H is difficult to correlate

the rallil of livJr lactJt·J in th& ,JICCltrtlul .. 3tion of muscle

glyco;,•m since dJta on liver Jl)"co;;~<!Jn have not been

collectl.td.

Circ&Jlan rhtthrn tn the concentr.:•tion of beta te

in the li\l'lU~ t:Lssue of a nu;:Jber of v&rto>.>rctt;:~s has boen

known (.>Llyers;J:lch., 1976 ~ .Jratz and Hutchison.. 1971}.

Ine absence of rhythm in tim livvr bctata in the

present case is surprising. HoW9v~z. thlll acropi'h:uu.t ;MS

cl~teet~d iilt -4~0 (oo .. ·.; h). i.~ •• appl'oxl.llhltely dJBing the

mid-scotophase •

. \lkdline ph:Jsp!utaso is an enzy!A$ which hydrolyses

v ;::bus )hcJSphate t?sters and rela tod comrrJunds (Long. 1971).

\l tluu ;h, in ·n:J:-::::~-lls, seru . .tu allalin<? phosphatase hns be'ln

kno:~:m to under ;o hi ih "lmpli tude circadian rhyth.'3 ( ;>cheving

-:Jt 11., 1')78). in the p:r;;;s:·,nt st.1d·;, a stati:>tically

siJaific·mt circ.Jdian ri<,tho in gill alkaline phosph:lta

CJpious urina. osmotic r.? ; !l-< ti)n is ef: · ct·~-1 throagh

u)t.1ke of ions t.:r Y.!Jh Jills. d1Jhar .. 1ctivi t1 of 'll~oline

phos;>lnt.Jsil, to promot~ actlv-· .;}take of ions from 1mbient

:aediu:n, is the rut-lral corollary.

,\el.J 1:.1 Vc>ly tlUCh hi:Jher activit; of .:tl.-<~line

phos.Jhat.1ae in the pres.~nt C>VO fiahas, ilS comp.lr~ from

104

thaiJ:" ep.io<)e<.m relative::~, (Chapter IV). l~ncts testimony

to incrH·3~,.o m><.1Jtil;: stress in c;.~v" environment and is

suncs·~ive uf c. tooch hi;bar r,:tte of active uptake of ions

to malnta.ln lllfi>.arosnotic ra;ul;d;.i,.n. :'ibsence of a cU:eadian

rhythm in the .1ctivit)' of alkalino phosphabse is again

>~<ltWi.ll in that l:.h<.t f~<;ht~~s are almost ~ontinuously ~nder

osribtic etr~ss in 1;iw cave Gnvirofl!llet\t.

Tho il.1J)Ol:i:.::.ne~ of z;cfitt.ylchoUne-a.cetylchclinestorase

in nouro tl:w•smiosion iS too well known. Circadian rhythlll

in th(• ac,)tylcholinest ·rase a{;:tivity in the brain tissue of

a nur.Jl.Jer of .:m.'i..t:k1ls, viz •• inVJrte.:·.t'at.0s (Pavan KU\llaJZ

et at •• 19'79). liz,>:t'ds (;~ubra(ili)!ny~'ll vt al •• l\#01 J l?aV/lfl

t(umu~ et al •• 1979) ma,::;nals (:,tuy.Jrs -~ch, 1976 ; ;;ichJ.ebaler

and Mayerst:~ach• 1974) has been well documont·ad. In the

pr<J•Jent stuJy also. a:;sumption of zero-at:lplltude hypothesis

for brain ac~tylcholinesterase of cave fishes was r~tjected,

sugjestive of occur:r,mc;, of .:acadian rhvthm.

Ch:>linc5tcr .. \'H) is an t.tnzym· . .: tha:t hydroly,~s ac~tyl­

choline to for:.t ~&-~tic "cid .:~nd cnoline (,;Ultinson, 1')7 6).

ln otil()J.; .mi·1 ls ii:. h.~:> b~i~<:n SIJJ..,e; ;;ad that tho ,lmplitud.J

of wGetilCholl.n .:::,ytru is greater in CiJ.::~ th:m in other

ti::;suas (..:u' .:.·· :~c:.ny:n.., e? ;..tl •• l9Ul). Ho;revcr, in t;Jw prasent

study, tho Npli ~Jdo of th~ rhytho in ;Jill is found to btt

greu ter th:1n \..!:: t of tiL· a~.v11 t:J.Jo in the bl:ain. This

definitely su;,•J.:;sts the gr~Z~t.or im.>ortanc• of

acettlCi1olinente:r:d>t in active uptake tiHln in neuro­

trans;nission s{sta:.l in this c«ve <iwoJ..U.ng apecies of n.an. /H.:rot)h:~ses .:~£ cir:adbn rhythm in Jill and brain

tissues !.Ui'pt'is:l.rt;ly overl..:l;>pect ;,u\1:1 wll*re .:let.ected dur1ng

tile lc~tc- li ;!'It i:c1ction of the W l2U.2 pboto~dod.

Similar li;nt-im:l.uced incrc..~sa in the acetylcholine ctsterase

enzyr,1e acti •li ty l'k<s baan rap;:>rted for the other <u'limnls

incl•Jding inV'artebrates as well as ~ls (Mayer&bllcn.l976J

~u.bramanyam et al •• 19Sl). Could it be t1·1at the light ind~

response of this enzytne is ~ tllroughout the animal

kingd001 1 Even tissut~f> of !>ighly specialized caw adupted

fishes are not sparad. Pur·thel!' studies on this a$pe<:t

would be rewarding.

Concluding, it is su;t;i<~~sl;ed that cave adapted fishes

retain tne ability of synehroniZd tion by the photoperiod,

whicn is SU0)c;O<Wd to lJe the strongest • • Zei t.Jei>e:t •' of the

environraent. ilovreV·Jr• this is true only for this

troglophilic fish. c~xtension of tnis study in a specialized

troglobite ~vould be revealing in clearing several existing

controversies in the field of chronob1o1ogy.

Ta

ble

17 1

Circad

ian v

aria

tion

s in th

e g

lyco

gen

and lactat~

con

tent o

f tissues o

f cave

fish

, !:!• evezard~

Clock h

ou

r T

iaa

ue

Variab

le (u

nit)

-------"--

-----------

Muscle

09

.00

* a

Glycogen

o.2S±O.O

J (m

g/1

00

mg)

Lactate

88

.JQ±

8.5

0

( ..,g/l.JO m

g)

l3.W

1

7.0

0

2l.u

u

Ol.~J

a-0-~----------

· -·--------------

o.2~v.o3 o.3Q

t0.04 u.32~o.o1

o.J~u.oe

95

. OO

;t;.17. oo 7

8. O

O;t.J.4. oo

66

. 50;t;,5. ·ro e,d

51.00:1:.4.50

o~.oo

o.J4

;t;,0.0

2 c,d

5

3.2

5;,!;.5

.00

Liv

er

Lactate

l8.5

Qt3

.oo

l7

.Qq

t3.4

l (..,g/100 m

g) l6.0J~2.16

17.5Qt3.77

19.~1.70

20

.50

,tl.26

------------------------·--------------------------------------------

* M

ean ;t;. l

SE

(m

ean valu

e of

fou

r ob

servatio

ns)

a,b

,c,d

Differ from

mean v

alue o

btain

ed a

t 01

.00

, ~.oo,

13

.00

, and 09.00 re

spectiv

ely

: P <

o.o

5 in

each case (based on D

uncan•o mu

ltiple-ran

ge te

st)

Ta

blo

1a

~ ~

""~l"·~i'

r.~ V

' ,•L ·•tC

•' "U

''l'l .·~,{ u

•-.i y:,;,

~

.,..A.

'·•.J..

~4• \01

-.:;J '"

,, ~•

fo:r

stu<.iy

of

t:ioe

i.'oo:;t$ o

n

tiu:;.;ua JlY"~.

and

la

ctJ

to

con

tcJ:J.t o

f c.lV

G

fish

, J.

a'\/'.};!\:U:\li

""'" ·~-"'""'""'''''~

-•·----.,.,. _

__

,.,.._

.. -~ .. -

-·-.., ............. ,

._...,_.,.,,...,--.------...,-"""'---'-~-"---·-"'-'•'"...,.....,..__ _

_ ~,.,._..-,.,.-..,_

I"'''"

f is~t.JO

: ;ri :·')1

&

.:.·y~:rce _;j1jj~)

{) f

, ·~--lun ..

t-" ...

(un

it) o

f n

,u·u~oG

df

;l(jiUJ:e

v

.1ri ·1 ti•.;!n

--------~---· ___ ..... ....._, .... "

-·--·~"""' ---......----·-·--·"'-'· .... ~

"""-·-·-··"""""--""""'"-·'--' Y#~---.. ~ .... •-t~.~<~,.,.,,_ .....

V ...... ~ ~-

... ------·

\ 1!/CO

JOn

T-;>

t:ll (J.~:ti4

2.3 ....

--

\ r.lJ/ .i. j..J[;JJ) .1 th.i:l

'J.l:'OU

fl'\ .~ ~ ;4:;;. lC

:~.· ,1},1.

.. -

!' itne

U.d~~3

5 J.:..; .>6

:J.:}J

<

;. )':) .,u~cle

-· ,_,. ____ ,,,, ........ "'

·---->~<-*$• .,

-~-

·-"·---..,.,.~,-

T ;ta

l 14l·J4.9~~·

23

-

-l..i.l\,; t.n

.a .;i th

in g

rou

p,

15

2:). 1

4

10

4

16

.32

:'

'· . / t . JlllJ) T

i 657~.2:1

5 J.3

1.5

.A

., .... m

e -

-;'3.1.t4

<

' ';','• ,_ .... """

-----

f'()t,il-· ,~,.,...,._.__""'""'-''"'58.3::f3"""".....,

2~ • ...... ,, . .,.

W _

__

_ ,....~--,._...,. _

__

._

_:._

,.

~--"""'""'*"'

w••

Lt.(t..:t.Jt..e

"i th

in gro1..1: "~.o

~;;~~-l." J iS

'''9

"'::! ..

-;..l.v<Jr

",r;. •

..;.,.._

( -J/l <'..J!Jl:J)

Tim

e 5:'•.33

:> ll.u

7

~'·3'17 }

,).,;_.:6

-------------~ ... -

.,,._, "

·-~------

__ ........_... .......... ....,. ,~,.~---"'-'""''"~-

.... ,-... ,, _____________ _

* '

' \..

• •..I ~' ••

Wd.S

p•.:.Cfo.~:uuJ '.m

tiat;l :i .u.u;;t:.rT

t:•d in

Tab

la l7

Ta

ble

19

1

ahy

thm

om

etric su:nmary

basad

on least-squ.:~re fittin

g o

f 24 h cnsin

e fun

ction

(2

4 h •

36

0°) to d:Jta illustr~ted

in T

able 1

7

Tissu

e

Muscle

I.1vo

r

Variab

le (u

nit)

No.

.Vat a lim

it o

ba.lo

w

hig

h

Plla

Glycogen

24

0

.19

o

.39

2

9

(mg/lJU

!lg)

d p

b M

<:t, SE

tt.•

in d

egrees

(9:> Y. \...L

.) 9

0.0

29

0

.3lt.O

o0

l 0

.04

•16(-339.~53)

Lacta

te

24

4

1.0

0

14

0.0

0 43

0.0

03

7J..9t¢'4.00

22

.40

•1

62

(-13

3.-1

91

} ( j.1 <J/ J. .. .:.om

g) ---·-

Lacta

te

24

8

.00

2

6.0

0

13

( llg

/l'JOm

g)

Legend aa in

Table 7

o.21s 1a.2~.oo

2.56 ~a

¢If in h

ou

rs

01

.07

10

.80

03

.20

Tab.la

20

1

Circ

ad

hn

V-JrJ.,:ltio

ns o

f th

o enzym

e activ

ities in

thsu

ee o

f cav

e fisht

l:!• ey

nard

i

Tissu

e

Gill

Brain

Clo

ck n

ou

r v

ariable

(un

it) ------------------------------------

Alk

aline

ph

osp

hatase

(~gpi/mg/hl

09

.00

1

3.0

J

* 2.Q

Q,to.a

2.1~0.2

17

o'J'J

1. 90;t.O

. 7

21

.00

U

lo-!0

os.vo

2.3

0;t_

0.4

2

.2o

.t_J. 7

2.3

0;t.0

.2

----------------------·------,---------------------------------------.-'\cetyl c

ho

line-

a,b

ater3

se l.4

7z0

.2l

( 1-' m

ol

ilCh

/mg

/h)

Acety

lch

olin

e-

a l.01:t;P

.2l

stera

se

2.7~0.12

3.0~0.36

(;.! mol

fC.h/m

g/h)

2.5

t,0.3

2

3.2

;a.0.3

l

2.15:t;.0.09 a

l.~U;!:.0.20 a

l.S'>

.I:;,J.lO

2.~0.04 2.86j;,O

.OS

2

.45

j;0.0

6a

--------------------------------------------------------------------------------* M

ean ;t. l 3E

(m

ean ValiA&

of

fou

r obae.rvationa)

a,b

D

iffer fro

m me~n v

alue o

utain

ed a

t 17

.00

h t

P < o.05.

and P

< 0

.01

resp

ectiv

ely

(b

aa

ed

o

n U

un

can

•a m

ultip

le-rang

e tE

Hi>t)

~

Ta

b!&

2

1

: :\n

aly

sis o

f y,lrian•::<

a sum.:~a.t:"/

for

stud

/ o

f tim

<f

ofi:',Jcts

on

on;&fillll!

activ

ity

in tissu

es o

f cav

e fish,

r.J. ovaz:1rdi

--

--""~" "-·---"~""-''""--~-""

------T

is3u

e

Varia

ule

Gill

dra

in

(un

it)

Alk

ali no p

ho

sp

ha

tase

( tl9

p1

/mg

/h)

Acety

lcho

line-

ate rase

( 1-l

mo

l N

:;.h/mg/h)

:\cety

lch

olin

e­

ster3

se

( j.Unol

i'Ch

/mg

/h)

~ource o

f Sum

of

varia

tion

sq

uares

-

To

te! 25.31

Ji th

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