Air-breathing rhythm in<i style=""> Clarias batrachus </i>(Linn.): Modulatory role of eyes, pineal and exogenous melatonin

Air-breathing rhythm in Clarias batrachus (Linn.): Modulatory role of eyes, pineal and exogenous melatonin

Swati Sahu & Maya Shedpure*

Department of Zoology, Govt. D. B. Girls' P.G. College, near Kalibadi, Raipur 492001, India Received 4 August 2004; revised 23 August 2005

Effects of enucleation followed by pinealectomy and administration of exogenous melatonin on air-breathing activity rhythm in a fresh water catfish, C. batrachus maintained at LD 12:12 and laboratary temperature during its prepratory phase, were examined. Results of cosinor analysis clearly reveal that most of the intact individuals exhibited circadian rhythm in their air-breathing activity and such rhythm persists even after enucleation followed by pinealectomy and then melatonin administration. However, the period (τ) of the activity obtained by power spectrum analysis was prominent 24 hr in most of the intact individuals, but it was increased (τ > 24 hr) after enucleation in most of the individuals. In most of the enucleated + pinealectomized individuals τ was less than 24 hr, and after receiving melatonin treatment τ was shifted to prominent 24 hr in most of the individuals. In addition, visual analysis of the actograms depicted that in intact individuals air-breathing activity is entrained with the timings of lights on/off with elevation of activity during dark period and decreased activity during light hours. However, enucleated and enucleated + pinealectomized individuals showed free run in their activity rhythm. The treatment of melatonin reestablished the entrainment of activity atleast with the timing of lights off, in most of the studied individuals. Further, daily mean of the air-breathing activity was decreased in enucleated + pinealectomized individuals as compared with other studied groups (intact, enucleated, enucleated + pinealectomized + melatonin receiving).

It could be speculated that there may be existence of extraretinal and extrapineal photoreceptors in C. batrachus. However, eyes play an important role in regulating air-breathing activity rhythm in such species. In addition, exogenous melatonin may also have some modulatory effect on such rhythm.

Keywords: Air-breathing, Blinding, Catfish, Circadian rhythm, Enucleation, Pineal.

The cyclic changes in envirnomental parameters can entrain biological clock mechanisms, consisting of regulatory and modulatory, physiological and molecular parameters of cells or cell groups, within organ systems like retina, pineal gland or suprachiasmatic nucleus. As lower vertebrates lack a distinct regulatory structure like the suprachiasmatic nucleus, circadian rhythmicity in these animals is generally believed to be regulated by several loosely coupled oscillators, that are mainly situated in pineal gland and retina. In general, in lower vertebrates, entrainment by light works through the retina and photosensitive pineal gland

. The influence of rhythmic physiological events on teleost behaviour has been well documented especially for reproduction and locomotion

.

Circadian rhythmicity of locomotor activity has been described in a number of teleost fishes and was

related to pacemakers within the pineal gland

. A characteristic locomotory behaviour, riding up and down intermittently, that occurs in tropical fresh water teleostean fishes is called surfacing/air-gulping behaviour/activity

. The air breathing fishes show this as behavioural adaptation for air breathing

. Existence of 24 hr rhythm in air-breathing behaviour has been documented in some species, such as Clarias batrachus and Heteropneustus fossilis

. Further, light-dark cycle has been shown to play an important role in regulating such rhythm in C. batrachus, however, pinealectomy has been found to be ineffective in modulating the air-breathing activity rhythm

. In catfishes, pineal has been suggested to bear some photoneuroendocrine functions

, like positive or negative phototaxis

, change in pigmentation in response to background colour

, circadian rhythmic photoperiodism

etc. Further, in several piscine species pineal has been suggested as a modulator of locomotor activity rhythms

. Arrhythmicity has been caused by pinealectomy in locomotor activity in juvenile sockeye salmon,

_______________

* Correspondent author

Phone: 0771-2229248 (O); 2262402 (R) E-mail: Swati_bunda@yahoo.co.in

Oncorhynchus nerka

. However, melatonin, the principal secretion of pineal gland synchronizes the disrupted or free-running circadian rhythms and regulates a variety of daily and seasonal changes in the physiology and behaviour of animals

. Further, presence of melatonin in some nonpineal tissues particularly in the retina, harderian gland, gut and leucocytes has been demonstrated but the mechanism of its production and kinetics of its release may be different in these organs from that is known for pineal gland

. Data from catfish suggest that the eyes and the pineal contribute to synchronize the behavioural rhythms by a light:dark cycle, but that other photoreceptors and oscillators also contribute

.

At present, it remains obscure that the photoperiod dependent modulation of the air-breathing activity rhythm in some piscine species are mediated via retinal pathways and/or by extra retinal receptors.

Therefore, present study is aimed to examine the role of retinal and extraretinal photoreceptors in regulating the air-breathing activity rhythm in Indian freshwater cat fish, Clarias batrachus (Linn.).

Materials and Methods

Collection and care of animals ⎯ Clarias batrachus is a tropical freshwater catfish and has been suggested as a facultative air breather

.

Live Clarias batrachus of mixed sex (40-50 g body weight) were procured locally during the preparatory phase (February-March) of its annual reproductive cycle and kept in the stock aquaria under the laboratory conditions (10 days) for proper acclimation. During the period of acclimation, water inside the aquaria was renewed every alternate day.

Fishes were fed pieces of small dry fishes locally available in the market ad libitum.

Experimental design and surgical operation ⎯ Following 7 days of acclimation, animals (n=07) were randomly selected from the stock aquaria and were kept in specially designed glass aquaria inside the chronocubicles exposed under L:D 12:12 (lights on at 0600 hrs). All the animals remain intact (IG) for first 7 days and then bilateral ocular enucleation was performed with the help of fine surgical instrument.

After anesthetizing the animals by keeping them in ice-tray, an incision was made around the eyeball.

The eyeball was pulled out carefully from the socket and the connections were cut with scissors (EG)

. Subsequently, after 20 days of enucleation, the pineal was surgically removed from the enucleated fishes.

The method of Nayak and Singh

was slightly modified for performing the pinealectomy. These authors used a dental drill to make a hole (1 mm diameter) in the skull to remove the pineal. In the present study thus this step was skipped as it is invariably associated with profuse bleeding and chances of damage of the other vital brain areas. In C.

batrachus the pineal lies inside a shallow concavity called pineal window covered by a thin translucent skin having sparse melonophores

. The fish was anesthetized by keeping it on ice tray and then pineal was exposed by cutting the skin flap over the pineal window from three sides and folding it anteriorly.

Thereafter, the pineal was removed by using surgical forceps under the binocular microscope and left for next ten days (EPxG)

. Then in next 15 days animals were received daily late afternoon (1730-1800 hrs) treatment of melatonin (2 μg/fish) (EpxMG).

Experimental protocol was as follows:

Intact (n=07)

(C. batrachus, Lights on at 0600 hrs) (after 7 days)

↓ Surgical enucleation

(after 20 days)

↓

Surgical pineal extirpation (after 10 days)

↓

Daily late afternoon (1730-1800 hrs) treatment of melatonin (2 μg/fish) for 15 days

Activity recording and data collection ⎯ Each animal was kept in a specially designed glass aquaria inside the chronocubicles for recording of air- gulping/breathing activity. A porous plate was fixed just below the surface of water with a single large space, available in the centre from where the fish can come to gulp the atmospheric air. The infrared photoswithches were fixed in such way that a beam of infrared ray lies in the middle of this space. Air- gulping/breathing activity was monitored and recorded by using a PC-based event recorder (Vision Automation, Pune). This device employs the principal of IR beam interruptions to record air- gulping/breathing activity effectively. Whenever fish came to the surface for gulping air it intrupted the infrared beam. This signal was amplified and recorded by a microprocessor based event recorder.

The two types of data files were prepared, viz;

graphical (for obtaining actograms) and numerical (to

obtain number of gulps per hour for statistical

analysis) with the help of such event recorder. In addition, hourly recording of climatic variables such as water temperature, room temperature and humidity was also done simultaneously on daily time scale.

Light schedule was maintained with the help of automatic timers.

Statistical analysis ⎯ The data, expressed in number of gulps/hour and were subjected to single cosinor method at τ = 24 hr

. A rhythm was characterized by three parameters, viz., the mesor (M, rhythm adjusted mean), the amplitude (A, half of the difference between the minimum and maximum of the best fitting cosine function) and the acrophase (φ, the time of maximum of this cosine function with local midnight as φ reference). The φ was obtained with its 95% confidence limit if a rhythm was detected with regards to the considered τ in that the amplitude differed from zero (non null amplitude test) with P<0.05. A power spectrum method was also employed for detecting prominent period in air- gulping/breathing activity of the fishes under LD 12:12

. Student's t-test

, ANOVA

and Duncan’s Multiple range test

were applied whenever required.

Results

Results of cosinor analysis clearly reveal that most of the intact individuals exhibited circadian rhythm in their air-breathing activity and such rhythm persists even after enucleation followed by pinealectomy and then melatonin administration (Table 1) However, the period (τ) of the activity obtained by power spectrum analysis was prominent 24 hr in most of the intact individuals, but it was increased (τ > 24 hr) after enucleation in most of the individuals. In most of the enucleated + pinealectomized individuals τ was less than 24 hr, and after receiving melatonin treatment τ was shifted to prominent 24 hr in most of the individuals (Table 2) In addition, visual analysis of the actograms depicted that in intact individuals air- breathing activity is entrained with the timings of lights on/off with elevation of activity during dark period and decreased activity during light hours.

However, enucleated and enucleated + pinealectomized individuals showed free run in their activity rhythm. The treatment of melatonin, reestablished the entrainment of activity atleast with the timing of lights off, in most of the studied individuals (Fig.1). Daily mean of the air-breathing activity was decreased in enucleated + pinealectomized individuals as compared with other

studied groups (intact, enucleated, enucleated + pinealectomized + melatonin receiving) (Fig. 2).

Further, results emerged from peak map clearly depicted that most of the peaks of air-breathing activity were located during the dark hour of the LD regime in intact, enucleated, enucleated + pinealectomized groups (Fig. 3a-c) but in the enucleated + pinealectomized + melatonin treated group most of the peaks of air-breathing activity were located during the light hour of the LD regime (Fig. 3d).

Discussion

It has been unequivocally accepted that pineal organ acts as a neurochemical transducer of photoperiodic information and is linked to several physiologic processes in various vertebrate species including fishes

. In general, most of the rhythmic activities have been reported to be governed by the pineal in variety of vertebrate species

. Further, data obtained from fish, reptiles and birds

Fig. 1⎯ Double plot (48 hrs) of air-breathing activity of C.

batrachus under LD 12:12 regime during prepratory period. (En = Enucleation, Px = Pinealectomy, MT → = Melatonin treatment indicating the 1^st day of respective treatments).

support the hypothesis that within the circadian system of non-mammalian vertebrates the pineal acts as a circadian pacemaker or atleast as a coupling device of two self-sustaining circadian oscillators (or sets of oscillators) which are located outside the pineal

. It has also been suggested that there may be variations in the strength of the coupling among these extrapineal circadian oscillators or variations in the frequencies of the individual oscillators, resulting interspecies differences in the effect of pinealectomy

. In addition, the lateral eyes are also thought to be sites of melatonin synthesis in a

variety of vertebrates

. In general, melatonin has been known to regulate a variety of daily and seasonal changes in physiological and behavioural activities of animals

.

Like earlier findings

, present results also reveal the existence of circadian rhythm with prominent 24 hr period in air-breathing activity in intact C. batrachus. While enucleation and enucleation + pinealectomy caused alteration in period of activity rhythm i.e. not equal to 24 hr, but shifting of τ to prominent 24 hr has been noticed in the enucleated +

Table 1⎯ Results of Cosinor analysis of air-breathing activity at τ =24 hr in intact, enucleated, enucleated + pinealectomized and enucleated + pinealectomized +melatonin treated C. batrachus maintained under LD 12:12 at laboratory temperature during preparatory period.

Group P^a Mesor + SE^b Amplitude + SE^c Acrophase^d (95% CL)

IG 0.001 92.89 + 4.18 69.56 + 14.39 21.66 (22.47, 20.86)

0.001 74.35 + 2.94 30.95 + 10.23 2.15 (3.43, 0.86)

0.0357 64.99 + 3.75 13.62 + 12.91 22.01 (26.82, 17.26)

0.001 15.29 + 2.46 16.27 + 8.48 22.58 (24.71, 20.46)

0.001 3.69 + 0.12 1.14 + 0.45 22.68 (24.21, 21.14)

0.1336 10.55 + 1.23 3.44 18.66

0.004 14.57 + 2.15 10.19 + 7.58 11.30 (14.39, 8.08)

EG 0.0001 8.54 + 0.16 1.03 + 0.58 22.38 (24.66, 20.10)

0.001 4.80 + 0.13 0.96 + 0.47 18.95 (20.93, 16.95)

0.004 10.90 + 0.42 1.99 + 1.47 15.94 (19.14, 12.75)

0.001 164.81 + 3.26 27.80 + 11.36 22.87 (24.47, 21.28)

0.0001 35.95 + 0.67 3.98 + 2.29 18.34 (20.75, 15.98)

0.001 37.68 + 1.76 20.49 + 6.07 10.48 (11.62, 9.31)

0.001 69.13 + 2 23.60 + 6.94 16.55 (17.69, 15.42)

EPxG 0.004 6.18 + 0.17 0.76 + 0.57 20.59 (23.87,17.19)

0.0001 6.82 + 0.26 1.49 + 0.87 19.74 (22.26, 17.23)

0.001 157.26 + 8.15 64.53 + 27.98 5.12 (6.91, 3.44)

0.02 22.90 + 1.97 7.45 + 6.75 9.95 (14.11, 5.42)

0.02 6 + 0.26 1 + 0.90 21.78 (25.86, 17.36)

0.001 26.95 + 2.26 26.72 + 7.92 11.35 (12.46, 10.20)

0.0004 213.08 + 6.19 35.33 + 21.70 15.91 (18.47, 13.52)

EPxMG 0.001 14.14 + 0.75 10.56 + 2.58 18.50 (19.45, 17.55)

0.001 31.31 + 0.83 10.9 + 2.91 15.44 (16.45, 14.43)

0.001 75.21 + 2.98 37.62 + 10.26 11.42 (12.47, 10.37)

0.001 12.52 + 1.60 12.04 + 5.59 13.90 (15.70, 12.09)

0.001 8.92 + 0.38 2.77 + 1.34 16.69 (18.61, 14.77)

0.001 47.47 + 1.14 11.19 + 3.94 18.38 (19.76, 17.01)

0.001 175.27 + 4.28 73.19 + 14.73 11.23 (12, 10.46)

aFrom F test of null amplitude rejection hypothesis.

bRhythm-adjusted mean of best-fitting cosine function + 1 SE.

cHalf of the difference between maximum and minimum of best-fitting cosine function + 1 SE.

dTiming of maximum in best-fitting cosine function with 95% confidence limit.

IG = intact group EG = enucleated group

EPxG = enucleated + pinealectomized group

EPxMG = enucleated + pinealectomized + melatonin treated group

pinealectomized + melatonin treated animals. Morita et al.,

have suggested that pineal is essential for behavioural rhythmicity in lampreys but the eyes are not. In addition, it has also been reported that the pinealectomy does not abolish circadian rhythmicity in any teleost species tested so far, but it does alter the period, stability and/or amplitude of behavioural circadian rhythms in lake chub, burbot and white sucker

. Another study on larval zebrafish suggested no effect of pinealectomy or ocular enucleation on the period or amplitude of free running rhythmicity, or on entrainment

. However, in the present study, results emerged from actogram revealed the occurrence of the entrained air-breathing activity rhythm in the intact C. batrachus and after

Table 2 ⎯ Results of power spectrum analysis showing period (τ) of air-breathing activity in intact, enucleated, enucleated + pinealectomized and enucleated + pinealectomized + melatonin treated C. batrachus maintained under LD 12:12 at laboratory temperature during prepratory period

Individuals Groups

IG EG τ EPxG EPxMG

Fish1 Fish2 Fish3 Fish4 Fish5 Fish6 Fish7

24 24 16 24 24 21.81 16.61

18.46 34.28 22.85 21.66 22.9

24 29.64

24 24 26.4

12 20 24 20

24 24 27.42 21.33 24 24 24 Legends same as in Table 1.

Fig. 2⎯ Daily mean of the air-breathing activity of the C.

batrachus under LD 12:12 regime during prepratory period. Bars having dissimilar alphabets differ with statistical validation. IG = intact, EG = enucleated, EpxG = enucleated + pinealectomized, EPxMG = enucleated + pinealectomized + melatonin treated.

Fig. 3⎯ Peak map for air-breathing activity of C. batrachusunder LD 12:12 regime during preparatory period. (a) intact (b) enucleated (c) enucleated + pinealectomized (d) enucleated + pinealectomized + melatonin treated.

enucleation the rhythm is being free run which also persists in enucleated + pinealectomized individuals.

In melatonin receiving enucleated + pinealectomized individuals entrainment of rhythm reappeared atleast with the timings of lights off. Like present findings, in another teleost Couesius plumbeus, pineal extirpation significantly affected the circadian locomotor activity

. In addition, exposure under constant dark caused alteration in circadian period of locomotor activity rhythm in Couesius plumbeus

. In lizard, Sceloporus occidentalis melatonin has been found to entrain the locomotor activity rhythm to a periodicity of 24 hr

. It is accepted that light-dark cycle of the environment synchronizes the biological clock in almost all organisms which have been studied

. Further, like virtually any circadian rhythm, the cycle of melatonin synthesis and release, generated by the circadian pacemaking system is regulated by light

59

. Like present findings, an earlier report also documented that the peaks of air-breathing activity located during dark and light phase of the LD regime in intact and melatonin treated C. batrachus, respectively

.

There have been limited attempts to localize components of the circadian pacemaking and photoreceptive systems that regulate fish behavioural rhythms

. In lower vertebrates, rhythmic synthesis and secretion of pineal hormone, melatonin, is suggested as the mechanism by which the pineal controls circadian oscillators located elsewhere

. However, the data from teleost indicate that other oscillators (possibly in the hypothalamus) are primarily responsible for regulation of behavioural rhythmicity and that the pineal and retina play a modulatory role

.

On the basis of present findings it could be concluded that in C. batrachus, eyes play an important role in regulating air-breathing activity rhythm. The extra-retinal, extra-pineal photoreception may also be suggested in such species. In addition, exogenous melatonin may cause modulation in air- breathing activity rhythm in C. batrachus.

Acknowledgement

Authors are thankful to Department of Science and Technology, New Delhi for financial support.

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