Indian Jou rna l o f Experimenta l Bio logy Vol. 41. August 2003. pp. 880-884

Interrenal responses to high ambient temperature in soft-shelled turtle, Lissemys punctata punctata

Prajna Paramita Ray & B R Maiti

Hi sto phys io logy Laborato ry. Departme nt of Zoology. U ni v~ rs i ty o f Ca lcutta (Bal lygunge Campus). 35 Ba ll ygunge Ci rcular Road. Calcull a 700 0 19. India

I?eceil'ed 2 Jllly 2002; revised 25 February 2003

An ex posu re to ambie nt temperature o f 25°C had no pe rcept ible e llec t o n interre nal func ti o n but funher inc rease of

temperature to 35°C eau set! nuc lear hypenrophy w ith inc reasc of nuc lea r diamete r. RNA concentration, aeid phosphatase ,lIld alka line phosphatase ac tiv iti es , accompa nied by qU<llllitati ve deple tion s o f cho les te rol ( free. es te ri fied and lOtal ) and ascorbic ac id leve ls in the inte rrenal g land of the soft -she lled tunic Lisselllvs p . IJIII/c/O /ll. Sim il a r man ifesta ti o ns o f

, timulati on. except in the nuc leus. we re marked after ex posure to 38°C. but the degree of response in respect of es tcrified

and free c ho l es t ~ro l levcls was highe r at 38°C than at 35°C. Moreover, w ithdrawa l o f 38°C tc mperature and ;,ubsequcntl y

mainta ining a t 25°C for 15 days showed reverse man ifestations to those of 35°C138°C. lead ing to a tendeilcy towards

norma lcy. It is sugges ted that hi gh a ambient t e mperatur~ o f 35°C s ig nificant ly s timula tes inte rre na l fun c ti on of Lisselllys

tun les. hut fun her i n creas~ of 38°C does not cause funh er ove ra ll stimul a ti on, and wi thd rawa l o f hi ghe r temperat ure (38°C) shows a tendency towards no rmalcy. It is a lso suggested tha t (a) high ambient temperature cau ses therma l stress , (b) it is reversibl e and (c) it ac ts on inte rrenal activity presumabl y via C RF-ACT H-axis in tunl es.

Keywords : Int e rrena l response. LisselllYs pllClaw PIlI/C/OW ,

Hi gh ambient temperature is known to stimulate interrenal acti vity in li zards l

. In Anolis interrenal responses to ACTH is decreased at 2 1°C compared to that of 30°C;. The magnitude and rate of interrenal acti vities have been reported to be altered in Dipsosau rtls dorsalis when exposed to environmental temperature between 10°-40°C for 7 daysJ. Corticosterone sy nthesis is also influenced by temperature in the turt le ChryselllYs p icta l

. In many reptiles, interrenal ti ssue is know n to regress in winter and proliferate in summer4

. Thus, in format ion relating to the influence of amb ient temperature on interrenal ac ti vity is inadequate in reptiles and negli gible in turtles. Therefore, the present study has been undertaken to ascertain the role of high ambient temperature on interrenal function in so ft-shelled turtles, Lissel1lYs puncta/a punctata.

Materials and Methods Adu lt female soft-shelled turtl es, Lissemys p.

punctata were collected from local natural populations near Calcutta during the nonbreeding

*Correspondent author : Phone: 09 1-033 -2475368 1 Fax: 091 -033-24764419

ue lear hypertro ph y. Te mpe rat ure, Turtle

phase (January ) of the reproductive cycle. They were kept in the controlled laboratory condi [ions (30°C and II L + 130 photoperiod) In the thermostatica lly controll ed aq uaria ( 150 x 90 x 90 em) with food (tubifex and shrimps) access ible ad libinll1l for 5 days prior to study. Water temperature was checked regularly throughout the period of experi ments. An imals (30) were eq uall y divided into 6 groups (1-VI) of 5 each. Each group was kept in separate aquarium. Group I served as control and was maintained at 30°C. Groups II , III and IV were ex posed to 25°, 35° and 38°C respectively for 15 days. Group V served as control fo r Group VI which was fi rst exposed to 38°C fo r 15 days and subsequently to 25°C for a further period of 15 days. Turtles belonging to Groups I, II , III and IV were sacrificed on day 16 of the experiments. Groups V and VI were sacrificed on day 3 1 of the experiments. All the animals were killed by deca~itation at a particular time of the day, i.e, 1000 hrs to avoid effects due to diurnal rhythmS. The interrenal gland of each specimen was quickly dissected out and the left one was placed in the Bouin 's fixative and processed for hi stological studies. Following routine microtomy, 5 11m thick paraffin sec ti ons were stained by

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RA Y & MAITI :INTERRENAL RESPONSE & HIGH AMB lE T TEMPERATU RE IN TURTLE 88 1

Masson's trichrome technique. Diameters (Jim ) of the interrenal cel l nuclei (means of short and long axes) were measured from the subcapsular and central zones of the interrenal gland by an ocul ar micrometer. At least 200 cortica l nuclei ( 100 each from the subcapsular and central zones) were cou nted from 10 widely separated random secti ons of the interrenal gland of each specimen . Only those nuclei th at had a compl ete nuclear membrane outline were consideredo. Ascorbic ac id7

, cholestero l (esteri fied, free and total frac tions)H, acid and alkaline phosphatasesl) and RNA 10 concentrations were measured from the right interrenal gland of each spec imen. All co lorimetric sampl es were measured on a PERKIN-ELM ER spectrophotometer (550 S, West Germany). Data were analysed stati stica ll y by Students' t test ll .

Results Contro l animals belonging to Groups I and V did

not show any perceptible difference in the findings and hence the data from both the Groups (I and V) were pooled together and treated as a single contro l group fo r all the experiments.

-+- I. Behavioural change

Animals when ex posed to 25°C temperature showed no perceptible behavioural change from th at of cont ro l. But they showed increased running ac ti vity when the temperature was raised to 35°C and furth er increase of temperature to 38°C caused certai n unusual behavioural manifestations, such as ki ck ing of legs, stretching of necks with buccopharyngeal movements, gasping and eventually restlessness. Add iti onally. some of the turtles showed oedema in

i.... the eye lids, hyperemia of the buccal cav ity (evaluated fro m reddish appearance of buccal cav ity due to profuse blood supply) and enhanced mucus secretion d ~ipped out of the mouth (frothing) after exposure with 38°C temperature which were no longer observed after withdrawa l of 38°C temperature and subsequently maintaining at 25°C.

2. Interrenal gland

(aJ Histological change

Co ntrol: The interrenal gland is encapsulated by a thin tough transparent membrane. The cortica l ti ssue remains intermingled with chromaffin elements and forms nunierous strands of variable size, consi sti ng of columnar cell s with centrally located nuclei (Fig. I).

Treated: Exposure to 25°C temperature had no perceptible effect on cortical hi stology, but furth er increase of temperature to 35°C caused stimulati on of the interrenal gland . The interrenal cords were enl arged and their cell outline became conspicuoll s. The interrenal cell s with their nuclei were hypertrophied and the nuclear di ameter was also increased (Fig. 2). Further increase of temperature to

Fig. I - A scc tion through thc inte rrenal g land o f control tunIc. Note irregular o rie ntati on of small interrenal ce ll s and their nucle i. Fig. 2 - A section through interrenal g land o f turtl e ex posed to

35°C for 15 days. Note wide inte rrenal cords with regular o ri entations of hype rtrophied cell s and nucle i Fig. 3 - FUl1her hypert rophy of inte rrenal ce ll s of tu rtle cxposecl to 38°C fo r 15 days . Masson' s trichrome stain . (X 500).

882 INDIA J EX P SIOL. AUGUST 2003

38°C showed cellu,l ar hypertrophy without affect ing nuclear size or diameter (Fi g. 3), but these manifestations were decreased with a tendency of returning towards normalcy following withdrawal of 38°C and subsequently maintaining at 25°C (Fig. 4).

(b) Biochemical change

(i) Choles terol and ascorbic acid : Cho lesterol (free. es teri ri ed or total ) and ascorbi c ac id co ncentrati ons of the interrenal gland were not altered at 25°C, but were signifi cantly depleted at 35° and 38°C, with a hi gher degree of depleti ons of esterified

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and free cholesterol fraction s at 38°C th an that of 35°C, But withdrawal of 38°C temperature and subsequent ex posure of 25°C for 15 days still showed depletions of a corbic acid and cholesterol levels co mpared to control s, but definitely to a less cx tent than those of 38°C alone (Fig. 4).

(ii) RNA and phosphatases: Ribonu cleic acid leve l or acid phosphatase and alk aline phosphatase ac ti vities of the interrenal gland remained unaltered at 25°C, but signifi cantl y increased at 35°C and 38°C, without showing much difference hetween 35° and 38°C or aner withdrawa l of temperature (38°C) (Fig. 4) .

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Fig. 4 - Changes in nuclear diameter (11m) of (a) subcapsular and (b) centra l zones of the interrenal gland and concentrati ons of (c) ascorbic ac id. (d) choles terol. (e) acid phosphatase. (f) alkaline phosphatase and (g) RNA following exposure to ambient temperature in turtles. rValues are M ean±SE. Unidirectional verti ca l bars represent standard error values . P values for each parameter refers to the com­p;]ri son between contro l and lre;]ted groups. *p < 0.025 ; ** p < 0.01 ; *"* P < 0.005 ; **** P < 0.001; C: control. NS : Not signifi cant] .

Discussion Hi gh ambient temperature stimulates interrenal

acti vity in so ft-shell ed turtles. Exposure to 25°C temperature had no perceptible effect on interrenal function , but further increase of temperature to 35° or 38°C for 15 days caused signi ficant interrenal stimulation by increasing cell and nuclear sizes (diameter) (barring nuclear size after 38°C), RNA concentration , ac id phosphatase and alkaline phosphatase activities accompanied by depletions of cholestero l (free, esterifi ed and total) and ascorbic ac id levels from the interrenal gland. The degree of manifestations was hi gher at least in respect of esteri fied and free cholesterol levels at 38°C than at 35°C. But the magnitude of such manifestations of interrenal function was no longer observed after withdrawal of high ambient temperature of 38°C. It is known that an increased nuclear diameter of interrenal ce ll s l2

, accompanied by an elevation of RNA level13

and higher ac id phosphatase and alkaline phosphatase act iviti es '4 reflect hi gher interrenal ac tivity. The depletions of cholesterol I) and ascorbic acidi C> levels from the adrenal gland are indices of enhanced steroidogenes is. Several stero idogenic enzy mes are also know n to be stimulated at hi gher temperature in lower vertebrates I 7

.I R

. Elevati ons of RNA, ac id phosphatase and alk aline phosphatase followed by depletions of cholesterol and ascorbic ac id have also been reported to occur following ACTH treatment in h I · L' 19 t e same turt e spec ies Issemys p. pune/ala .

Therefore, in the present study, parallel changes in the interrenal gland of turtl e may refl ect its stimulation including steroidogenesis. Catecholamines are also known to influence adrenocortical steroidogenesis4

.

Additionally, both norepinephrine and epinephrine level s were increased following ex posure to high ambient temperature in turtles20

. Therefore, an in volvement of catecholamines in inducing corticoidogenesis via steroidogenic enzy me follow ing high ambient temperature cannot be ruled out. Nevertheless, the current findings corroborate the results of earlier works in another turtle species, Chrysemys pie/a2 and in other reptili an species, Ano/is earolinensis l and Dipsosaurus dorsalis3

• It is known that adrenocortical activity is regulated by CRF­ACTH axis22

. Therefore, in the present ex periment high ambient temperature may have stimulated interrenal activity presumably through CRF-ACTH pathway in turtles, but it has to be confirmed in turtles.

Though exposure of both the higher ambient temperatures (35° and 38°C) caused interrenal

stimulation in turtles, the latter temperature (38°C) showed certain unusual behav ioural manifestati ons, such as frothing (dripping of saliva) , gasping, hyperemia and restl essness in ex perimental turtles. Temperature, whether low or hi gh, acts as stress that primaril y affects the interrenal gland and hence interrenal hyperacti vity may be considered as all index of stress, because stress in general incl uding water depri vati on, os tomi c stress, etc. are known to cause elevati ons of both corti costerone in birds2

.l and ad renomedull ary hormones in mice24

. Though low or hi gh ambient temperature acts as stress25

, temperature lower than 35°C is less stressful than 38°C since no abnormal behaviour was marked with the former temperature, whereas an exposure to 38°C temperature is more stressful than that of 35°C since abnormal behavioural manifes tations were encountered only with 38°C temperature and that mi ght eventually lead to ex hausti on of animals, and withdrawal of 38°C temperature no longer showed abnormal behaviour thereby indicating that the effect of temperature on interrenal function is reversible in turtles. , Acknowledgment

This work was supported by a Specia l Assistance grant (No. UGC/496/SPNZoo/81) from UGC, ew Delhi , to the Department of Zoology, Uni versity of Calcutta, with Junior and Senior Research Fellowships awarded to PPR. A minor research grant (No. F/PSW-045/99/00/ERO) from UGC was also awarded to PPR.

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