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Opsin and Actin in Fly Photoreceptors 395 flies are therefore suitable to study the cellularevents associ- newlyemerged ated with the rhabdomere morphogenesis. Second, we de- MVB m 10dold scribed opsin-containing large complex body (LCB) in the rERmass. The opsinsin the LCB maybe incorporatedinto the rhabdomeres. Finally, electron microscopic histochem- LCB istry of opsin and actin suggested that the actin filaments serve as a route foropsin transport towardsthe rhabdomere. LB Double labeling Two double labeling methods employed here both func- 2 4 6 8 10 tioned properly, and gave similar results. The labeling Area / Rl-6 (uW) densitywasconsistentexceptforthe antiactin labelingon the Fig. 4. Areas occupied by multivesicular body (MVB), large com- rhabdomereinbothmethods. Theobservedvariationinthe plexbody(LCB),andlysosomalbody(LB)inRl-6. Measure- density of antiactin labeling is probably attributed to the mentwasdoneon23ommatidiafrom5individualsforeachage arrangement ofactin in the rhabdomere: actin contributes to (n=5). **; P<0.01, *; P<0.05 (Student's f-test). the slender core of the microvilli [2, 20]. We cut the hexagonallypacked microvilli, eachofwhich is about 100nm in diameter, roughly along their longitudinal axes. In the ultrathin sections, about 70nm in thickness, the number of cores contained in a section would vary depending on the cutting angle and/orthe degreeofdistortionofthe microvilli arrangement. The numberofcoresin thesection affectsthe frequencyofepitopesexposedonthesectionsurface,whichis a determinant ofthe labeling density. Opsin synthesis The photoreceptors of newly emerged flies actively Hr synthesize proteins, which is indicated by the rich content of rER in the cell body (Figs. 1 and 2). The proteins should include opsin, for the rER was occasionally labeled with antiopsin (Fig. 6b, f, Table 1). The newly emerged flies appeared to have large opsin- Fig.5. Control labeling. The L. R. White sections were first bearingstructures ofirregularprofile, whichcontainvesicles, labeled either with antiactin (a) or antiopsin (b), and were furthertreatedwiththemixtureof15nmgold-conjugatedGAM ribosomes and/or rER, making the cross sectional appear- IgGand5nmgold-conjugatedGAMIgM. Scalebar=0.2^m. ance complex (Figs. 1 and 3). We therefore termed the structure as large complex body (LCB). Some LCBs were enclosedbyseverallayersofmembranesdenselylabeledwith Figures6canddshowSl-decoratedactinfilamentslining antiopsin (Fig. 3f, h). Although some of these resemble theplasma membrane. The actinfilamentspointawayfrom autophagicvacuoles, there is also apossibilitythattheopsins therhabdomere(arrowheadtriplets): wecouldnotdetectany in the LCBs, in the lamellated membranes in particular, are actinfilamentswiththeoppositepolarityinthisregion. The newly synthesized rather than removed from the rhabdo- antiactin labeling around the plasma membrane most likely mere. Our preliminary observation indicates that the LCBs corresponds to these actin filaments (arrows in Fig. 6g). are abundant even in the late pupal stage and then mostly Antiactin also labeled the filaments in the space between the disappearwithintwodaysafteremergence: theappearanceof rhabdomere and the cell body (Fig. 6g, h, see also [2]). the LCBs coincides with the activity of the rhabdomere morphogenesis [15]. The functional significance of the DISCUSSION LCBs is remained for further investigation. MVBs and LBs are both involved in the membrane The results reported here are the following. First, the degradation [8, 11]. Although not frequent, the MVBs and rhabdomeres ofnewly emerged flies are developing. These LBs, both contain opsin, were also found in the newly Fig. 3. Opsin-bearing structures embedded in the rER (arrows) in the photoreceptor cell body. The left column (a,c,e,g) represents the images of conventional EM, whereas the right column (b,d,f,h) represents the L. R. White sections double labeled with antiopsin and antiactin. (a) (b) Multivesicular body, (c) (d) Lysosomal body, (e) (f) Large complex body enclosed by the lamellated membranes (arrowheads). ThemembranesenclosesomerER(arrows), (g) (h)Largecomplexbodywithoutthelamellatedmembranes. In(h), an LCB enclosed by the lamellated membranes (arrowheads) is also seen. I; intraommatidialspace, LB; lysosomal body, M; multivesicular body, R; rhabdomere, r; ribosome, v; vesicles. Scale bar=0.5/im. , 396 K. Arikawa and A. Matsushita « 1 \ - \ -s» . » % &*n - .13. ** '%' 6'! -•• #i •;>'* '. •' ', '*{*, -V _ 3 * — a ' " ^ R . ii ::.:. Fig. 6. Histochemistryofopsinandactininthephotoreceptorsofnewlyemergedflies, (a) (b)Antiopsinlabeling(arrowheads)ontheplasma membranefacingtotheintraommatidialspace. Arrowsin(b)indicateantiopsinlabelingontherER. (c) (d)Sl-decoratedactinfilaments lining the plasma membrane (arrows). The polarity ofthe filaments was indicated on the left side ofthe filaments by arrowhead triplets, (e) Opsin-bearingvesicles(arrowheads)foundclosetotheplasmamembrane (arrowheads),whichwaspartiallylinedwithantiactinlabeling (arrows), (f) Some portionsofrER masswere labeledwith antiopsin (arrowheads), (g) (h) Antiopsin labelingscattered in thecellbody appeared to be associated with some endomembranes (arrowheads), arrows; antiactin labeling. I; intraommatidial space, R; rhabdomere. Scale bar=0.2,um. emerged flies (Fig. 4), suggesting that the rhabdomere de- There are twopossible routesfor newopsins toreach the gradation was not inhibited during the stage where the rhabdomere: via the plasma membrane facingtothe intraom- rhabdomere morphogenesis is actively taking place. matidial space [21], and through the subrhabdomeral cisternae(SRC) [14,24]. Actin and transport ofopsin Here the antiopsin clearly labeled the plasma membrane Transverse sections of the retina clearly show that the (Fig. 6a, b, Table 1). The opsins in the plasma membrane is photoreceptors have two distinct domains: the rhabdomere probably being transported towards the rhabdomere rather and the cell body (Fig. 1). The opsinssynthesized in thecell than removed from there. One reason for this is that the body should be transported to the site of function, the rhabdomere volume is increasing in this stage (Fig. 2): the rhabdomere, through the gap between the domains. flies retain high activity of rhabdomere morphogenesis that Opsin and Actin in Fly Photoreceptors 397 Table 1. Distributionof15nmgoldparticles (antiopsin labeling) onthephotoreceptorsofnewly emerged and lOd old flies mean particle number+se (%) newly emerged, n=4 10 d old n=4 , rhabdomere 116.7+18.1 (54.7) 90.1+ 8.9 (61.3) plasma membrane 4.9+ 1.0 (2.5) 1.3+ 0.1 (0.8)** MVB 5.6± 2.5 (2.5) 12.1+ 1.7 (7.8) LB 8.4+ 4.1 (3.6) 33.1+ 2.3 (22.4) LCB 65.9+ 5.4 (33.1) LCB not found other regions 6.4+ 1.2 (3.5) 11.2+ 1.3 (7.7) Total 2.7.9+21.4 (100.0) 152.1+11.3 (100.0) "Other regions" include endomembranes and rER. LCBs were not found in the measured micrographsof10doldflies. Theparticlenumberswerecomparedwiththecorrespondingregionsin the different developmental stage. Particle counting was done on 57 photoreceptors (ranging 2-14 photoreceptors per individual) from 4 individuals for each age (n=4). **; P<0.05, Student's /-test should require opsin incorporation into the rhabdomere. between the rhabdomere and the cell body. The labeling Secondly, the labeling density on the plasma membrane is appeared to be associated with the vesicles or pieces of significantlyhigherinthenewlyemergedthanin 10doldflies membranes (Fig. 6g, h). These endomembranes are most (Table 1). Thirdly, the plasma membrane is lined with the likelypartsofthetubules,forthetubulesappearintransverse uniformlyoriented actinfilamentswiththeplusendstowards sections as elongated or swollen vesicles [14], or even as the rhabdomere (Fig. 6c, d) and anti-NINAC labeling [13]. fragmented membranes ifone side ofthe tubule was tangen- As suggested by Adams and Pollard [1] in Acanthamaeba tiallysectioned. TheantiopsinlabelingontherERprobably myosin I, the myosin-like NINAC could move the mem- representsnewopsinsthatwillbetransferredintothetubules brane-embedded opsin towards the plus end of the actin (Fig. 6b, f). filaments, i.e., towards the rhabdomere. The regionbetween the rhabdomere andthecellbodyis In fact, actin is probably involved in the rhabdomere furnished also with the NINAC [13]. By the presumptive morphogenesis. In a crab, Hemigrapsus sanguineus, the interactionbetween actin and NINAC, the opsins embedded rhabdom volume increases at dusk and decreases around in the endomembranes can be transported towards the dawn [3]. Thevolumeincreasewasinhibitedbytreatingthe rhabdomere. isolatedeye at duskwithcytochalasinD, whichdisruptsactin filaments. As the cytochalasin D treatment had no effect at ACKNOWLEDGMENTS night on the enlarged rhabdom, the inhibition ofthe volume increaseshouldbeattributednottothedisintegrationofonce We thank Dr. T. Tanimura for providing the monoclonal established rhabdom but to the disruption of some proces- anti-Drosophila Rhl opsin antibody. Drs. T. Tanimura, S. Stowe, ses) in the rhabdom morphogenesis [16]. The most plausi- waonrdkE.. EWguechailsporotvhiadnekdthweolpfaunlocnoymmmoeunstrsefienrteheesifnoirtiamlasntyagevsaloufabtlhee ble process mediated by the presumptive actin-myosin suggestions on the manuscript. The work was supported by the interaction is the transport of opsin. When applied also at Grants to K. Arikawa from Whitehall Foundation (Florida, USA), dusk, colchicine, a microtubule inhibitor, failed to stop the Kihara Foundation for Life Sciences (Yokohama), and the Ministry volume increase, suggesting that the actin was more directly ofEducation, Science, and Culture ofJapan. involved in the rhabdomere morphogenesis than the micro- tubules [16]. Further analyses using other inhibitory drugs REFERENCES are in progress. The actin-NINAC interaction also explains another 1 Adams RJ, Pollard TD (1989) Binding of myosin I to membrane lipids. Nature 340: 565-568 route of opsin transport through the SRC, which is an 2 Arikawa K, Hicks JL, Williams DS (1990) Identification of opsin-containing network that wraps the entire base of the actin filaments in the rhabdomeral microvilli of Drosophila rhabdomere[14,24]. TheSRCisconnectedtotherERwith photoreceptors. J Cell Biol 110: 1993-1998 membrane tubules forming an extensive endomembrane sys- 3 Arikawa K, Kawamata K, Suzuki T, Eguchi E (1987) Daily tem. The opsins in the SRC are probably originated from changes of structure, function and rhodopsin content in the the rER, the site of opsin synthesis, through the membrane PchoymspioolunAd1e6y1:e 1of61t-h1e74crab Hemigrapsus sanguineus. J Comp tubules, although no opsin had been so far localized in the 4 Arikawa K, Williams DS (1989) Organization of actin fila- tubules. 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Invest Ophthalmol Vis Sci 26: 1659-1694 20 SaibilHR(1982) Anorderedmembrane-cytoskeletonnetwork 10 de Couet HG, Tanimura T (1987) Monoclonal antibodies in squid photoreceptor microvilli. J Mol Biol 158: 435-456 provide evidence that rhodopsin in the outer rhabdomeres of 21 SchwemerJ (1986) Turnoverofphotoreceptor membrane and Drosophila melanogaster is not glycosylated. Eur J Cell Biol visualpigmentininvertebrates. In "The molecularmechanism 44: 50-56 of photoreception" Ed by H Stieve, Springer-Verlag, Berlin 11 EguchiE,WatermanTH(1976) Freeze-etchandhistochemical Heidelberg New York Tokyo, pp303-326 evidenceforcyclingincrayfishphotoreceptormembranes. Cell 22 StarkWS,SappR,SchillyD(1988) Rhabdomereturnoverand Tissue Res 169: 419-434 rhodopsin cycle: Maintenance of retinula cells in Drosophila 12 Hafner GS, Tokarski TR, Kipp J (1992) Localization ofactin melanogaster. J Neurocytol 17: 499-509 in the retina ofthe crayfish Procambarusclarkii. J Neurocytol 23 Stowe S (1980) Rapid synthesis of photoreceptor membrane 21: 94-104 and assembly ofnew microvilli in a crab at dusk. Cell Tissue 13 Hicks JL, Williams DS (1992) Distribution of myosin Hike Res 211: 419-440 ninaC proteins in the Drosophila retina and ultrastructural 24 Suzuki E, Hirosawa K (1991) Immunoelectron microscopic analysis ofmutant phenotypes. J Cell Sci 101: 247-254 study of the opsin distribution in the photoreceptor cells of 14 Matsumoto-Suzuki E, Hirosawa K, Hotta Y (1989) Structure Drosophila melanogaster. J Electron Microsc 40: 187-192 of the subrhabdomeric cisternae in the photoreceptor cells of 25 Williams DS, Blest AD (1980) Extracellular shedding of Drosophila melanogaster. J Neurocytol 18: 87-93 photoreceptor membrane in the open rhabdom of a tipulid fly. 15 Matsushita A, Arikawa K, Eguchi E (1992) Distribution of Cell Tissue Res 205: 423-438 opsinin thephotoreceptorsinthelatepupalandnewlyemerged ZOOLOGICAL SCIENCE 11: 399-406 (1994) 1994Zoological SocietyofJapan Experimental Perturbations of the Litonotus-Euplotes Predator-Prey System Nicola Ricci and Franco Verni Dipartimento di Scienze dell'Ambiente e Territorio, via A. Volta 6 56100 Pisa, Italy ABSTRACT—A model previouslyproposedtodemonstrate the interactions between Litonotus (predator) and Euplotes (prey), ledtoanewroundofexperiments. Thedifferentexperimentalapproachesusedtosolvethesequestions(starved cells;killedcells;enzymes;lectins;ions;inhibitors)resultedinquiteanewmodelofthecellinteractionswhichaccountsfor thedifferentstepsofthephenomenon:themainpointdemonstratedbytheseexperimentsisthatthecellularcortexofboth predator and prey is involved in many ofthe successive steps ofthe cascade reactions enabling Litonotus to prey upon Euplotes experiments: (a) how is the toxicyst-discharging system trig- INTRODUCTION gered and controlled? (b) which are the spatio-temporal Effortsspent in attempting to deepen ourunderstanding sequences of a toxicyst-discharge phenomenon? of predation among protozoa are completely justified by the The unique nature of our pet-organism protozoa (i.e. basicimportanceoftheprocess. Protozoa,indeed,werenot indeed perfect eukaryotic cells and complete organisms, at only the first primary consumers in the primeval Oceans, but the same time) offers a double advantage: (a) it enables us the first predators as well [12]. Such a new trophic niche is to use all those techniques typical ofexperimental studies on quite an important one, due to the two consequences it leads cell interactions to investigate also the relationships between to: (a)itcreatesnewemptyspacesforneworganismstosettle entire organisms and (b) it allows us to transfer any results in, (b) it triggers a sort ofevolutionary competition between obtainedfor these truly sophisticate organisms to the general preys and predators (to escape and to strike each other, field of cell biology. respectively) as to their morpho-functional acquisitions. Manyexampleshavebeenalreadystudiedandtheknowledge MATERIALS AND METHODS of the Didinium-Paramecium [1], Dileptus-Colpidium [34], Enchelys-Tetrahymena [5], Chaenea-Uronema [5]; Homalo- Both L. lamella and E. crassus were grown, collected and used zoon-Paramecium [4] predator-preysystems, cannotbuthelp asalreadydescribedbyRicciandVerni[24]. Theobservationswere us to complete our overall picture of this phenomenon made with a Wild M5 (20-60X) stereomicroscope, and a Leitz consideredfromamorespecificsinecologicalpointofview: in Orthoplan (400X) microscope (together with its Nomarsky inter- this perspective, indeed, the study of predator-prey interac- ferential contrast), coupled to a PanasonicTVC camera and a VHS tionsalsolendsitselftobeusedinanattempttopenetratethe videorecorder. Unless otherwise indicated, the prey organism was adaptive strategies, conditioning the reciprocal (co)evolution E. crassus. Thefollowingspecificprocedureswerefollowedforthe of predators and preys. Let us recall the example of the different kinds ofexperiments: bat-moth relationships, as atrulyparadigmaticone, toclarify Expt. 1 The effects of starvation on preys and on predators were studiedusingnormal andstarvedLitonotus, exposedtobothnormal our idea [28]. andstarvedEuplotes. Normalpopulationsofpredatorswereused4 In this context we studied another predatory model, daysafterthelastfeeding,whilethestarvedonesweretestedafter11 namely that of Litonotus lamella-Euplotes crassus, focusing days. Normal Euplotes were not fed for 24hr, while the starved our attention successively on (a) the ultrastructure of the ones were used 7 days after the last feeding. toxicystsofthepredators [11], (b) theultrastructural analysis Expt. 2 To study the role played by the body itself, of both the of the consequences to Euplotes of toxicyst discharge by predator and the prey, in the specific toxicyst discharge (TD) Litonotus [32]; (c) the peculiar digestion process [33] and, processes at the very moment when the two organisms come into finally, (d) the behavioural patterns following each other direct contact with each other and the TD itself is triggered and along a path characterised by the succession of several basic actuallyoccurs, bothLitonotusand E. crassuswerefrozen, andthen steps, namely casual encounter (CE); toxicyst discharge thawed at room temperature (the experimental populations were (TD); research (R); engulfment of the prey (PE)[8]. The immersed in liquid nitrogen for 2min): in this way the structurally predator-prey interaction model proposed in the previous andchemicallypreserved,butphysicallyinertbodiesofbothLitono- tusandEuplotesweretestedwithlivingpreyorpredatorrespectively. paper focused our attention on several closely related prob- InsomeoftheseexperimentshomogenizedEuploteswerealsoused. lems, which represented the targets of the next round of Expt. 3 Whenever a predator contacts a prey TD occurs: does TD affectthemicroenviromentwhereitoccurred? HowfarforTDarea Accepted May 31, 1994 is such an effect perceived? How longdoesitpersist? How is the Received December 16, 1993 behaviourofEuplotesaffected? Tosolvetheseproblems,manyTD 400 N. RlCCI AND F. Verni eventswerevideorecordedandthevideotapesscoredframebyframe RESULTS accordingtothestandardtechniqueforbehaviouralstudiesreported elsewerebyRicci [20]. In thiswaywequantified: a)thesubcircular Expt. 1 The effects of different starvation of Litonotus and area where the TD effects are perceived by Euplotes; b) their Euplotes. duration; c) the changes in behaviour ofthe prey. Previous microscope observations (Verni, unpublished Expt. 4Toassesstherolepossiblyplayedbycalciumconcentrationin results) had shown that the longer the starvation, the more the sea water, 3 different standard set ups (cf. Expt. 5) were caudal the distribution of toxicysts: the effects were statisti- prepared: mthMe first contained standard marine water (control), the cally significant. On the basis of these findings, the conse- second 15 calciumchloride, the third the same concentration of PCraeCvli2oupsluesxp0e.r1immeMntsE,DcTaArr,iedtoouitnhiwbiittht5h,e 1e0f,fec1t5s, o2f0,thaendca2l5cimumM. quencTehseorfessutlatrsvaotbitonaiwneerdeinstuthdiisedromuonrdeosfpeceixfpicearlilmy.ents de- Ca++ andwith0.01, 0.1 and 10mMEDTAhadshownthatthebest monstrated that (a) severe starvation affects the efficiency of results were obtained with 15mM Ca++ and with 0.1 mM EDTA. predator's TD: Table 1, the III and the IV columns vs the I In other words these two concentrations were the lowest capable of and the II; (b) the starvation ofthe preys affects, to a limited inducing clearcut results. Five experiments were then carried out extent, the ingestioncapabilityofLitonotus (Table 1, 1At, the and microvideorecorded, to measure: (a) the time lag between the IIvsthe I column andtheIVcolumnvsthe III), while itdoes introduction ofLitonotus andthefirst instance ofTD (thisperiod of not affect the corresponding TD At. time will be referred to asTD Atthroughout thispaper, itsomehow Expt. 2 The predatory interactions between Litonotus and measurestheefficiencytoLitonotusininterceptingtheprey);(b)the frozen-thawed Euplotes. time lag between the introduction of Litonotus and the actual Litonotus cannot be frozen and then thawed, without engulfmentofthe killed prey (thisperiod oftimewill be referredto asI Atinthispaper, itsomehowmeasurestheefficiencyofLitonotus being disrupted: no result could be obtained, except that the feeding on the prey); (c) the length of the backward motion of areawhere adisrupted Litonotus lies is avoided bythe preys. Litonotus followingTD; (d) the numberofTD per Litonotus. Normal Litonotus exposed to a population offrozen-thawed Expt. 5 The effects of trypsin (Sigma, T8253; concentration 2.5, 2, Euplotes demonstrated that: (a) Litonotus can ingest them 1.5, 1 and 0.5%) were also studied. About 50 Litonotus were without discharging any toxicysts (Fig. IB); (b)Izlt is longer incubatedinthedifferentconcentrationforvarioustimeperiods(15, than twice as much as thatofthe control (Fig. 1: B vs A); (c) 30,60,90, 120, 180,210and240min);theywerewashed3timesand when freshly prepared homogenate of Euplotes is added to then used in a50/AdropletwithconcentratedEuplotes, tostudythe the system, ID At is strikingly reduced (Fig. 1C). TD At, I At and the percentage of inhibited (namely not-toxicyst Expt. 3 The TD-affected area. discharging)Litonotus. When100%TDinhibitionwasinduced,the Litonotus still incubated by trypsin were washed free ofthe enzyme fresh water and then used in Euplotes populations to monitor their recovery period. Expt. 6-A The effects of concanavalin-A (Con-A, Sigma C2010; concentration2, 1.5, 1,0.5and0.25%;treatmenttime 15,30,60,90, 400 120, 150, 180and210min)onLitonotuswerestudiedbywashingthe treatedcellsafterdifferentperiodsoftimeandmeasuringTDAt, IAt and the percentage of inhibited cells, when these experimental Litonotusweretransferredintoa50p\dropletofconcentratedpreys. 300 ThesametreatmentswerealsocarriedoutonincubatingLitonotusin the same dosages ofCon-A andforthe same times as before, in the mM presence of 40 of a-methyl-D-mannoside, well-known as a specific competitor of Con-A. The same parameters were measured. 200 Expt. 6-B Concentrations of2 and 1% of Con-A were also used to incubate Euplotesfor 15,30,60,90, 120, 150, 180,210,270,330,and 390min: these populations were washed three times and then ex- posedtoLitonotus, tomeasuretheTD At,theI At, thepercentageof non-discharging Litonotus. 100 Expt. 7In a finalexperimental approach, Litonotuswastreatedwith different concentrations (0.5, 0.25, 0.125, 0.06, 0.03 and0.015%) of IAt cycloheximide (Chx)(Sigma C-6255) for different times (1 to 9hr); ABC theywereusedsinglywithpopulationsofEuplotestomeasureTDAt, \AlandthepercentageofTDinhibition. When 100%TDinhibition L+E L+ftE L+ftE+hE was obtained the still incubated cells were washed free of Chx and then used with Euplotes to study the kinetics of their recovery in Fig 1. The IDAt (white bars) and the \At (shadowed bars) of terms of the percentage ofTD inhibited cells. Litonotus (L) in presence of [A] normal Euplotes (E). [B] frozen-thawed Euplotes (ftE) and [C) ftE and homogenated Euplotes (hE). On the ordinates the times in seconds are reported. Litonotus-Euplotes Predator-Prey System 401 Table 1. The effects of starvation, on the predation of Litonotus lamella on Euplotes crassus, measured by the durations of the TD^t and of the \At (described in Materials and Methods section). normal Litonotus starved Litonotus + normal + starved + normal + starved Euplotes Euplotes Euplotes Euplotes X 1'25" 1'20" 2'21" 2'15" TD/lt s 55" 50" ri2 ri8" n 35 26 29 32 X 2'30" 3' 3'42" 4'9" Izlt s r 1'5" 1'15" 1'18" n 35 26 29 32 % %ofEavoiding Table2. The effects of Ca++ (15mM) on the 4 parameters D astruckE indicatedonthe left;BM=thelengthofthebackwardmotion of a Litonotus lamella after discharging its toxicysts: it is 80 f%roozfeEn-atvhoaiwdeidngL lmeenagstuhreodfinLiRteolnaottiuvse Ulnaimteslla(RU—):2510^RmU.=1Thsepecniuesm-bspeercifoicf observations for each parameter was of 50 parameters population Control 15mM Ca" 01.51mmMMCEaD+T+A& studied 60 TD/lt (sec; 43+17 41.5+18 42+26 BM (RU) 1.1+0.7 2.5+0.7 1.2+0.6 lAt (sec) 132+30 186+80 139+33 40 Lni?toonfotTuDs per 1.2+0.4 2.4+0.5 1.2+0.5 ++ Expt. 4 The role of Ca in the environment. 20 The results given in Table 2 clearly show that calcium ions affect the general physiology of the Litonotus-Euplotes system at least at three different levels: (a) TD At is not affected; (b) the length of the backward motion is doubled; (c) lAt increases by about 50%; (d) the number of TD per EDTA 30 60 90 120 150 180 210 Litonotus is doubled. inhibits calcium effects, as times (sec) expected. As considered in the Materials and Methods Fig. 2. The temporal trend of avoidance of the "toxic" area by section, the differential effects of calcium ions on TD At (no Euplotes, expressedintermsofindividuals(ordinate: %)creep- effect) and on \At (strong inhibition), actually demonstrate ing away from it, in the time (abscissa: sec). The shadowed the importance of both parameters in interpreting correctly bars show the same trend of Euplotes avoiding frozen-thawed the effects ofcalcium ions on the Litonotus-Euplotes system. Litonotus Expt. 5 The effect oftrypsin on Litonotus The enzyme was already known to affect the physiology The resultsweobtained are the following: (a) the effects of Litonotus; progressively higher concentrations and longer ofaTD eventextendaroundtheTD pointoverasub-circular treatments, indeed, induce (a) aprogressive darkeningofthe area of about 300,«m in diameter, (b) the same effects cytoplasm, (b) a rounding of the body shape, (c) reduced increase up to their maximum for 90sec after the TD event locomotion, (d) immobilization and (e) lysisofthe cell (Ricci and last for about 3 min; (c) the behaviour of the preys is and Verni, unpublished results). It was found (Fig. 3) that clearly affectedbytheTD event as demonstratedbythe high the different concentrations used for the different times frequency of avoidances induced in Euplotes by TD im- indicated in Material and Methods, clearly affect TD At (the mediately afterwards (Fig. 2, shadowed areas); the avoi- higher the concentration, the longer TD At) but not IAt (the dances correspond to the behavioural pattern called Side difference between TD At and IAt is indeed rather constant). Stepping Reaction [19] and indicated as SSR; (d) these SSR For a certain concentration a longer treatment affects the occur according to a temporal pattern parallel to that of percentage of cells not discharging their toxicysts (Fig. 4), Euplotes avoiding the area where a frozen-thawed disrupted while TD At tends to be more or less constant, the different Litonotus is placed (Fig. 2, white areas). values depending solely upon trypsin concentration. Once — 402 N. RlCCI AND F. Verni 30 25 20 c Il5 90 120 150 180 210 240 270 10 treatment (min) Fig 4. The progressive action of the trypsin in the time: for a certain concentration the longer the treatment (abscissa) the smaller the percentage of discharging Litonotus. The figure closetoeachofthedifferentpointsalongthecurves, represents theaverageTDAtobservedforthatpoint;thefiguresinthesmall panels represent the trypsin concentration for the different _i curves and the relative mean values ofthe TDAt. 0.5 1.0 1.5 within 60-120min. trypsin (%) Similar treatments with trypsin were also conducted on Fig.t3.rypsTihn,egaivveernagoenTtDh/eltabascnidsslaA;t0in%duocfedtrbypysitnhe=ccoonncternotlrsationsof Euplotes; theenzymeeitherhasnoeffect alallonbothTD At or lAt, and it kills the preys. Expt. 6A The effects of Con-A on Litonotus. 100% inhibition is induced (namely, when no Litonotus can The results ofthe experiments are the following: (a) the discharge its toxicysts) they still behave quite normally and higher the Con-A concentration, the stronger the effect, on theirphysiology seems to be unaffected: afterwashing, these TD At, but not on lAt (Table 3) (b) for the same concentra- completely inhibited populations recover 80% of their TD tion, the longer the treatment, the stronger the effect on TD activity within 30-60min, and 100% of their potentialities At but not on lAt (Table 3) (c) as the Con-A concentration Table3. TheTDAt andthe IAt ofdifferent populationsofLitonotustreatedbydifferentconcentrationsofCon A (shown in the first line) for different times, given in the left column Con A 2% 1.5% 1% 0.5% 0.25% control TDAt lAt TDAt lAt TDAt lAt TDAt lAt TDAt lAt TDAt IAt n 15 15 10 10 10 10 10 10 8 8 15' x >30' >35' 3' 4' 2' 3'20' 1'31" 2'20" ND ND T 3' — — s 1'20" 1'46" 50" 1'30" 1' 1'17" 1'05' ITS' n 14 14 14 14 12 12 11 11 12 12 8 8 30' X >30' >35' 10'50" 15'30" 6'23" 7'30" 2T0" 3'30" 2T5" 3'24" 1'20" 2'30" — — s 2'19" 3' 3'55" 3'27" r 1'27" r 1'21" 1 1'50' n n 16 16 9 9 10 10 11 8 8 60' X t t —>30 —>35' 8T0" 11'50" 5'20" T 2'27" 3'40" 1'30" 2'55" s 5'15" 5' 2'20" T 1'12" 1'25" 1*15" 1'20" n 10 10 7 7 8 8 90' X t t 11'15" 13'30" 10'35" 12T5" ND ND 2T0" 3'15" s 5'18" 7'47" 2'20" 2'30" 050" 1'35" n 18 18 13 13 8 8 120' X >30' >35' 12'35" 13'43" ND ND 1'20" 2'20" — — s 1'24" 1'21" ro5" 1'50" n 6 6 10 10 8 8 150' X t t 12'40" 13'26" 5' 6'42" 1'50" 3' s 1'25" rio" L'36" 1'40" L'10" 1'50"