A RED BLOOM OF EUGLENA SHAFIQII , A NEW SPECIES, IN DALLAKE, SRINAGAR, KASHMIR

SHAFIQ-UR-REHMAN∗Division of Environmental Sciences, Sher-e-Kashmir University of Agricultural Sciences and

Technology, Shalimar Campus, Srinagar 191 121, Jammu and Kashmir, India

(Received 18 June, 1996; accepted in final form 18 July, 1997)

Abstract. A bloom ofEuglenawas observed at the surface of Dal Lake, Kashmir, in the last week ofJune, 1991. The species exhibited a circadian colour change, from green at dawn to blood-red duringthe day and again green at dusk. The present new species is, therefore, namedEuglena shafiqii. Itis distinguished from the cogenus by the structure and occurrence of Z-tubules, chromatophores,nucleus shape and by a coloured pulsating structure at the posterior end. A stress induced transfor-mation from an elongated egg to a round shape is also peculiar. The light absorption spectra of thepigment showedλmaxima at 416, 441, 467 and 485 nm. The red bloom was found in the part of thelake basins which receives waste effluents.

Keywords: colour change, Dal lake, environmental stress,Euglena shafiqii, pigment spectra, red-bloom, shape transformation

1. Introduction

Dal Lake (34◦04′–34◦11′ N and 74◦48′–74◦53′ E) is situated north-east of Srinagar,in the heart of Kashmir Valley, Himalaya, at 1583 m.a.s.l. It is shallow (maximumdepth of 3 m) and has a total area of ca. 20 km2 of which approximately 12 km2

is open water. In the north, a small stream (Telbal Nalah) flows in, carrying hugequantities of silt-clay particles, and drains into the Jhelum river in the south. Inrecent years, the quality of Dal Lake water has suffered from rapid urbanization,agricultural activities, deforestation, siltation and municipal sewage.

A brick-red bloom appeared for the first time at the water surface of the Dalduring the last week of June, 1991. The bloom turned blood-red within 35 days.The red bloom in the Dal Lake was ascribed to anEuglenoid(Shafiq-ur-Rehman,1991). During close examination of the organism it has been found that the presentspecies is a new one and is distinguished from the cogenus by the structure, stress-induced body transformation and pigmentation.

∗ Address for correspondence: Dr. Shafiq-ur-Rehman, P.O. Box 56 G.P.O., Srinagar – 190 001,J&K, India

Water, Air, and Soil Pollution108: 69–82, 1998.© 1998Kluwer Academic Publishers. Printed in the Netherlands.

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Figure 1.Map of Dal Lake and location of sampling sites.

A RED BLOOM OFEUGLENA SHAFIQII 71

2. Materials and Methods

Samples of the bloom were collected during sunny days (10.00 – 12.00 h) in Julyand August from the sites S-1 and S-2 in Dal Lake (Figure 1). The samples ofthe bloom were fixed immediately in 4% formalin and stored until observation.Also, other sets of intact bloom samples were taken for the colour and behaviouralstudies. During the examination of both the formalin-fixed and the immediatelycollected fresh samples, we found most of the cells in round-shape while few inelongated egg-shape, which tempted us to study the behavioural changes of theshape and the colour of the organism following stressful environment created byshaking of the water samples or keeping the samples away from direct sunlight.Some of the samples were also supplied with nutrient broth (Sigma, U.S.A.) andstudied for the behavioural changes. The pigment from fresh samples was extractedin methanol containing 5% KOH, as described elsewhere (Davies, 1976). The sepa-ration of pigment was carried out on silica gel column and the fractions were elutedwith solvents having increasing polarity. The individual fractions (I–IX) of pigmentwere dried and redissolved in petroleum ether for spectrophotometric examination.The solvents used in the study were of chromatography grade.

3. Results

3.1. STRUCTURE

The body ofEuglena shafiqiiis elongated, ca. 50µm long and 10µm broad, withblunt anterior and round posterior ends. The pellicle is distinct and flexible, theanterior portion has a reservoir. A large flagellum, about the length of the body, isattached to the bottom of the reservoir. A paraflagellar body is found near the cy-tostome. The second flagellum is short and restricted to the reservoir. A yellowishstigma is found at the left side and a star-like reddish-brown contractile vacuole onthe right side of the reservoir.

Two pairs of highly elastic and flexible Z-shaped light-yellowish tubules arefound at the opposite side of the pellicle. Several yellowish brown myonemes arestretched in the middle of the body. There are two paired chromatophores of dark-red colour with dark-brown pyrenoid; each situated at the above and at the right sideof the nucleus. Third large almond-shaped chromatophore of pinkish-red colour isfound below the nucleus.

A large blood-red continuous pulsating chromatophore, surrounded with greenpigment, is situated at the posterior end of the body. The whole structure is irregularand spontaneously pulsating, i.e. exhibiting a rhythmic motion of expansion andcontraction. A red-coloured nucleus, with two reddish-brown nucleoli, is locatedat the centre. The whole structure is localized in a dark-brown granular mass(Figure 2).

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Figure 2. Euglena shafiqii, Shafiq-ur-Rehman. Normal shape of blood-red waterbloom causing or-ganism in the surface of Dal Lake. bp: blepharoplast; cp: chromatophore; cpcp: continuous pulsatingchromatophore (Coloured contractile structure); cv: contractile vacuole; fl: large flagellum; fs: smallflagellum; m: myonemes; n: nucleus; ns: nucleolus; pcp: paired chromatophore; ps: photostome; r:reservoir; st: stigma; zt: Z-tubules.

A RED BLOOM OFEUGLENA SHAFIQII 73

3.2. BEHAVIOUR

The colour of the bloom was green at the dawn and at the dusk, when the sunrays were oblique and soft. During the day, when the sun radiation was straightand intense, the colour of the bloom was changed to a brick-red. A month afterthe appearance of the bloom, the pigment was intensified to blood-red. The or-ganisms, when kept away from direct sunlight, exhibited significant changes incolour. The colouration of the organisms was changed to green after disappearanceof the red pigment. Subsequently, the green colour was lost leaving the organismscolourless. Besides, the organisms remained less active and latter most of themchanged the body to a round shape. Supplement of nutrients reactivated about 30%of the organisms. Restoration of the green pigmentation in the body and brick-redpigmentation in vital structures such as chromatophores, nucleus and continuouslypulsating chromatophores were also observed.

E. shafiqii used its flagellum for locomotion and changing the direction ofmovement. Normal locomotion, performed by undulations of tip of the flagellum,was slow. Rapid movement, however, was achieved by sinus undulation of the en-tire flagellum. Spiral undulation of the whole flagellum caused rotation of the bodyand change in the direction of movement. Normally, the organism does not performeuglenoid movements. However, quick and rapid movements were performed un-der stressful environment. The organisms performed saprozoic (and saprophytic)and holozoic (and halophytic) nutrition. The organism could bend one third ofits body for catching food by the help of the flagellum.E. shafiqii is extremelysensitive to environmental stress. The organism could rapidly transform its bodyinto a round shape when water is shaken. The highly flexible pellicle and otherbody structures appeared to help in body transformation. The structural anomalieswere achieved through I-transition and II-transition phases (Figure 3).

3.3. ABSORPTION SPECTRA OF PIGMENT

The light absorption spectra obtained in the preliminary study are presented inTable I and, Figures 4 and 5. Fractions I and II showed a single absorption peak467 and 485 nm, respectively. The spectra of fraction III exhibitedλ maxima at485 nm with a shoulder at 416 nm. The spectra of 416 nm in fraction IV to IXappeared as strong band. In fraction V, however, the 485 nm band depressed andbroadened. In subsequent fractions, VI to IX, peaks of 416, 441 and 459 nm (exceptin IX) were prominent, while spectra of 485 nm appeared as shoulder. Hence, lightabsorption spectra of the pigment isolated fromE. shafiqiiexhibitedλ maxima atthe regions of 416, 441, 459, 467 and 485 nm (Figure 4). In reverse phase silicagel chromatography, however, the spectral patterns of the pigment were shown tobe 416, 468 and 505 nm (Figure 5).

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Figure 3. Euglena shafiqii. Shape change from normal elongated egg-shape to round-shape in stress-ful environment. These shape changes attain two transition phases. The I-transition phase is reversiblewhere the organism can return to normal if favourable environment persists. The II-transition phaseis irreversible where the organism can not transform back to normal (see text).

A RED BLOOM OFEUGLENA SHAFIQII 75

Figure 4.The light absorption spectra of the pigment isolated fromEuglena shafiqii. The pigment ischromatographed through silica gel column. The nine fractions (I–IX) were obtained and analysed.

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Figure 5. The light absorption spectra of the pigment isolated fromEuglena shafiqii, afterreverse-phase silica gel chromatography.

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TABLE I

Light absorption spectra of pigment extracted fromE.shafiqii

Fraction Absorption (λ) maxima (nm)

1 2 3 4 5

I – – – 467 –

II – – – – 485

III 416 (S) – – – 485

IV 416 – – – 485

V 416 – – – 485 (B)

VI 416 441 459 – 485 (S)

VII 416 441 459 – 485 (S)

VIII 416 441 459 – 485 (S)

IX 416 441 449 (S) – 485 (S)

S = shoulder (peak appeared as shoulder).B = broad (peak depressed and broadened).

3.4. METEOROLOGY AND APPEARANCE OF THE BLOOM

Annual periodicity and frequency of the bloom formation appeared to be influencedby nutrient load, high temperature, increased sunlight and prior heavy rain falls.The annual maximum day temperature ranged 24–34◦C in the air and 17–32◦Cof the water surface of the lake. The annual periodicity (i.e. starting) of the bloomappearance and atmospheric temperature are presented in Figure 6.

3.5. CLASSIFICATION

The present new species ofEuglenaexhibited a typical characteristic of colourchange from green to blood-red in sunlight and is distinguished from the cogenusby the structure, body transformation in stressful environment and pigmentation,hence it is classified as under:

Phylum Protozoa

Sub-phylum Plasmodroma

Class Mastigophora (Flagellata)

Sub-class Phytomastigina

Order Euglenoidina

Family Euglenidae

Genus Euglena

Species shafiqii (Shafiq-ur-Rehman)

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Figure 6.A five-years maximum and minimum temperature trends of standard meteorological week.The circles with year show periodicity ofEuglena shafiqiiblood-red bloom appearance in the surfaceof Dal Lake.

4. Discussion

The rapid growth and excessive aggregation of a particular microbe in a formof scum at the water surface of lakes and reservoirs are referred as the water-bloom. The phenomenon is world-wide and known under various names, such as‘flowering of water’, ‘wasser-blüthe’, ‘flos-aquae’, ‘Tsvetenie vody’ and ‘red-tide’(Reynolds and Walsby, 1975). One of the earliest documented event of water-blooming occurred at Llangorse Lake of Breconshire in 1188. The another exampleof the blooming ofOscillatoria rubescensDC ex Gom appeared as red-brownscum on the water surface of Murten See in Switzerland during late 1500 AD. Outbreak of the ‘red-tide’ due to blooming ofdianoflagellates (Gonyaulax polyedra,Nictiluca) often occures in the oceans. The mass occurrence ofchlamydomonasnivalis in polar regions colours snow red. The ‘red-water’ phenomenon is first ofits kind in the Dal Lake. It appeared due to the blooming of a new species ofEuglena(Shafiq-ur-Rehman, 1991). The organism exhibited circadian changes incolour. The colour of the cell remained green in the morning. And, during the day

A RED BLOOM OFEUGLENA SHAFIQII 79

when the sun rays fell straight with stronger intensity, the colour of the cell turnedblood-red. Further, in the evening when the run rays fell obique and soft, the redcell again turned green or yellowish green. Besides the circadian changes in colour,the new species differed from the cogenus in various aspects such as structure,body transformation and pigmentation and, therefore, was namedEuglena shafiqii,Shafiq-ur-Rehman.

The E. shafiqiidiffered structurally and behaviourally from known species ofEuglenasuch as,E. viridis, E. spirogyra, E. gracilis, E. sanguinea, E. oxyuris, E.ehrenbergiandE. rubra. The body ofE. shafiqii is elongated egg-shaped. WhileE. spirogyraandE. rubraare elongated spindle-shaped with pointed posterior enddrawn out like a tail.E. gracilishas round anterior end whereasE. oxyurisinheritsa large cylindrical body. The cell surface ofE. ehrenbergiis spirally striped.

There is a morphological peculiarity with respect to the construction of thenucleus inE. shafiqii, showing two reddish-brown nucleoli embedded in fibrillarmass. However,E. veridishas a large andE. oxyurishas a small central nucleolus.E. gracilishas three whileE. spirogyraandE. rubrahave several nucleoli.

The chromatophores inE. shafiqii are three. A bigger almond-shaped chro-matophore is located behind the nucleus. The other two chromatophores, how-ever, are paired each located at the anterior and right sides of the nucleus. Inother species, the chromatophores are present as fusiform inE. gracilis, disc-likein E. spirogyraand, long rod-like inE. viridis. A peculiar coloured contractilestructure, situated at the posterior end ofE. shafiqii, is named as continuous pul-sating chromatophore due to its typical characteristic of rhythmic contraction andexpansion.

The present species undergoes typical behaviour of the shape change in stress-ful environment. The impact of environmental stress, due to physical or chemicalstimuli, has been shown to be associated with cell rounding inEuglena(Murry,1981; Bovee, 1982). As in skeletal muscle, the cell energy metabolism seemsto be fundamentally the same in the rounding ofE. shafiqii. The stress-inducedbehavioural changes in shape are typical as the organism swiftly transforms itselongated egg-shaped body to a round-shape. This rapid transformation of thebody shape seems to take place through two transition phases. During I-transitionphase, the elongated egg-shape (Figure 3a) changes to a pear-shape with a pointedposterior end (Figure 3b), and subsequently to an egg-shape (Figure 3c) body. Inthe II-transition phase, however, the organism assumes a round shape and passesthrough different internal structural changes (Figures 3d, e and f). Moreover, theorganism also attains a bell-shaped body between I and II transition phases (figurenot shown). The organism passing through I-transition phase may return to thenormal shape from shape b within a couple of hours while, from shape c it maytake several hours to a couple of days to return to the normal shape a, if favourableenvironment persists (Figure 3). The behavioural shape changes that took placeduring I-transition phase seemed to be necessary for protecting and avoiding the se-rious damages due to environmental stress. Nevertheless, in the II-transition phase

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the organism (Figures 3d, e and f) could not return to the normal shape. Thus instressful environment, the organismper seperforms cell rounding which seemsto serve two purposes of, i) self protection and survival and, ii) self destructionwhen the chances of survival are blocked by severe and prolonged stress. Hence,the events occuring during the II-transition phase could prove to be an importantphenomenon which could well be applied for the management of the growth andaggregation of the bloom through agitation (and aeration) of the upper most surfaceof the affected lake waters (Shafiq-ur-Rehman, 1995).

The light-induced directional motion or change of motion is well establishedin Spp. of Euglena. Light stimulated additional carotene synthesis is found incertain microorganisms through photoinduction mechanism when it is needed toprotect the organism against the harmfull effects of excessive light or oxygen. Inmost recent experiments, the stress in the form of UV-B irradiation and simulatedsolar radiation have been found responsible to impair photoorientation and motility,cell shape, and photosynthesis in flagellates likeEuglena gracilisand Euglenasanguinea(Eklund, 1993; Gerber and Haeder, 1994, 1995). The experiment onhyperphoto-bleaching effects were not performed in the present study, however,hypophoto-bleaching effects onE. shafiqii have been noticed when maintained,in the laboratory, away from direct sunlight. Furthermore, a close association ofthe appearance of the bloom, its aggregation at the surface of the Dal Lake andthe change of colour (of the bloom) from greenish-yellow to brick-red to blood-redhave been observed with the increasing sun radiation and atmospheric temperature.

Pigmentation inEuglenophytawas found to be due to the presence of carotenoidssuch asβ-carotene, zeaxanthin, neoxanthin and diadinoxanthin (Goodwin, 1976;Kirk and Tilney-Bassett, 1978). Rosowski and Parker (1982), however, reportedthe presence of zeaxanthin, lutein, violaxanthin and neoxanthin in euglenophaceae.Kumar (1982) has described the presence ofβ-carotene, lutein, neoxanthin and as-taxanthin. Previously, secondary carotenoids (i.e., ketocarotenoids of xynthophyls)were associated with chlorophyceae (e.g., green algae), but has now been encoun-tered in euglenophyceae, wherein diesters of astaxanthin are predominantly presentin E. sanguinea(Grung and Liaasen-Jensen, 1993). In recent experiment, Gerberand Haeder (1994) reported the red colour of the algaeE. sanguineadue to oc-currence of haematochrome, a mixture of carotenoids, the main component beingastaxanthin diester. In the present preliminary findings, the light absorption spectraof the pigment isolated fromE. shafiqiiwere determined at 416, 441, 459, 467 and485 nm in the fractions of silica-gel chromatography. A similar pattern of the lightabsorption spectra was also obtained in a reverse-phase silica-gel chromatography.These spectral patterns showed the presence ofβ-carotene and a mixture of xyn-thophyls, possibly astaxanthin as one of the components. Further studies, however,could discernate the possible profile of the pigment ofE. shafiqii.

In the recent years, the rapid urbanization, unsustained agricultural practices,deforestation, siltation and disposal of sewage into the Dal Lake have contributed toits deterioration. These activities are considered to be responsible for the increase

A RED BLOOM OFEUGLENA SHAFIQII 81

in the cultural eutrofication which ultimately posed a threat to the valley’s mostbeautiful natural water body. The adversities in the biodiversity of the lake aremainly due to the increased availability of the nutrients. High temperature, heavyrainfalls before the blooming, high pH and increased nutrients in summer, tendto be in favour of the periodicity of the bloom appearance. (Shafiq-ur-Rehmanet al., 1995). Gonzalves and Joshi (1946) regarded high temperature, great lightintensity and large amount of organic matter favourable for the growth of manyspecies ofEuglena. The sun radiation as shown in the present study was responsiblefor the colour change from green to red and the growth ofE. shafiqii. More-over, the decrease in the dissolved oxygen level during bloom period indicates afavourable condition for the growth of Euglenineae (Singh, 1960; Munawar, 1970).During the bloom period the concentrations of ammonium-N, nitrate-N, chlorideand phosphate-P were reduced in the lake water which seemed to be utilised byE.shafiqii for the growth (Shafiq-ur-Rehmanet al., 1995).

Acknowledgements

I thank to Professor Dr. R. A. Khan, Department of Pathology, Government Med-ical College, Professor G.N. Mir, Director Research, Professor M. A. Masoodi, Di-vision of Entomology, Professor, A. R. Talib, Division of Soil Sciences, Dr. BadrulHassan, Division of Agronomy of SKUAST and Dr. K. Z. Khan, Departmentof Chemistry, Miss Tabassum Rashid, Department of Pharmaceutical Sciences,Dr. Nooruddin and Mr. A. M. Parray, Instrumentation centre (USIC) of KashmirUniversity, for providing necessary facilities. I am also thankful to Mr. Bashir. A.Shah, Department of Pathology, Govt. Medical College and Ms. Shaheen Rehman,New Style Pathological Laboratory, Srinagar, for excellent technical assistance. Iam grateful to Professor S. U. Mir, Head, Division of Environmental Sciences ofSKUAST, for his support and interest.Euglena haematosolariswas named by theauthor, Shafiq-ur-Rehman, and is now renamed asEuglena shafiqii.

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