Acta Zoologica

(Stockholm)

85

: 1–14 (January 2004)

© 2004 The Royal Swedish Academy of Sciences

Abstract

Bobkova, M.V., Tartakovskaya, O.S., Borissenko, S.L., Zhukov, V.V. & Meyer-Rochow, V.B. 2004. Restoration of morphological and functional integrity inthe regenerating eye of the giant African land snail

Achatina fulica

. –

ActaZoologica

(Stockholm)

85

: 1–14

To determine whether vision returns to its original state following eye removalin

Achatina fulica

, light and electron microscope examinations, electrophysiologicalrecordings and behavioural tests were carried out on the regenerating snails.Reparative morphogenesis can result in the restoration of the peripheral senseorgan even in the absence of complete regrowth of the tentacle, but it can alsolead to the formation of aberrant regenerates. We found that anatomically andultrastructurally the eyes of the ‘most normal’ regenerates were basically thesame as the original eyes. Under normal conditions each eye is composed of aprincipal and an accessory eye, both sharing a common cornea. The onlydifference between regenerated and native eyes is the smaller size of the former,as a result of a reduced number of retinal cells. Electroretinographic responsesrevealed that the molecular mechanism of phototransduction is restored, inprinciple, but that flicker fusion frequency in the regenerated eye is significantlylower than in the normal eye. The directional movement to a visual stimulus(a black stripe of 45

°

width) had not completely recovered even 6 months afteramputation. This suggests that the central projections of the optic nerve hadnot become fully re-established at the time of testing.

Dr V. B. Meyer-Rochow, International University Bremen, School of Engineering and Science, Campus Ring 6, Research II, room 37, D-28759 Bremen, Germany. E-mail: b.meyer-rochow@iu-bremen.de

Blackwell Publishing, Ltd.

Restoration of morphological and functional integrity in the regenerating eye of the giant African land snail

Achatina fulica

Marina V. Bobkova,

1

Olga S. Tartakovskaya,

2

Serguei L. Borissenko,

2

Valery V. Zhukov

2

and Victor B. Meyer-Rochow

1

1

International University Bremen, School of Engineering and Science, Campus Ring 6, Research II, D-28759 Bremen, Germany and Department of Biology, Oulu University, SF-90014 Oulu, Finland

2

Kaliningrad State University, Department of General and Ecological Physiology of Human and Animals, Faculty of Bioecology, Universitetskaya ulitsa 2, 236040 Kaliningrad, Russia

Keywords:

gastropoda, photoreceptor, behaviour, ERG, development, regeneration

Accepted for publication:

6 October 2003

Introduction

The ability of snails to restore tentacles following injury orloss was described more than 200 years ago by Spallanzani(see: Chetail 1963). Complete regeneration of amputatedoptic tentacles, accompanied by a restoration of the structureof the eye, has been observed in a number of terrestrialspecies, e.g.

Helix

spp. (Eakin and Ferlatte 1973; Scarssoand Pellegrino de Iraldi 1973),

Melampus bidentatus

(Moffettand Austin 1981),

Cryptomphallus aspersa

(Flores

et al

. 1983),

Arion rufus

and

Agriolimax agrestis

(Chetail 1963), and inmarine forms, e.g.

Strombus

spp. (Hughes 1976; Gillary1983),

Ilyanassa obsoleta

(Gibson 1984),

Pomacea

sp. (Beverand Borgens 1988) and

Hydrobia ulvae

(Gorbushin

et al

.

2001). The newly generated eye usually possesses the samestructure as the native one, differing principally only in size(Scarsso and Pellegrino de Iraldi 1973). Although adults canregenerate an organ in an environment that differs greatlyfrom that in which the embryo formed the organ originally,the events of eye regeneration appear to recapitulate embry-onic eye formation, i.e. the sequence of invagination, pig-mentation and differentiation of lens, optic nerve and retinalpatterns (Chetail 1963; Eakin and Brandenburger 1967;Eakin and Ferlatte 1973; Gibson 1984). Electroretinographicresponses of regenerated snail eyes are thought to parallel therestoration processes of synthesis and photopigment storage(Flores

et al

. 1983). Behavioural tests with regenerated eyesof

I. obsoleta

demonstrate recovery of the snail’s reaction to

Eye regeneration in

Achatina fulica

•

Bobkova

et al.

Acta Zoologica

(Stockholm)

85

: 1–14 (January 2004)

© 2004 The Royal Swedish Academy of Sciences

light (Gibson 1984). However,

Achatina fulica

, the largestland snail in the world, seems to be exceptional in this regard.Eyes either do not re-appear at all or several eyes are formedin one and the same tentacle (Chase and Kamil 1983;Sidelnikov 1991). The anatomical diversity of the regeneratingeyes in

A. fulica

begs the question of their functional capabil-ities. Previous studies on eye regeneration in this species didnot reveal the formation of an optic nerve (Sidelnikov 1991)and it has been suggested that the optic nerve is either notformed as a separate entity or it joins the tentacle nerve(Chase and Kamil 1983).

Observations on the behaviour of snails with regeneratedeyes demonstrate that phototactic responses do not re-appear (Sidelnikov and Stepanov 1998). This provided someresearchers with an argument to assume that there was nomorphological connection between the newly formed eyesand the central nervous system. Nevertheless, until theultrastructure and electrophysiological characteristics of theregenerated eye in

A. fulica

have been studied in detail, onecannot decide if the regenerated eye is able to function as anorgan of vision or not. This supplied a reason for the moredetailed investigation reported in this paper and for thefollowing set of questions.

Does the postoperative structural restoration allow the eyeof

A. fulica

to fulfil its function? Do the optic system andcellular components of the retina recover completely as is thecase in other investigated gastropods (cf. above)? Can regen-erated eyes give electric responses to stimulation with a flashof light? Is the formation of the eye accomplished through there-growth of the optic nerve? Finally, is there a physical con-nection between the regenerated eye and the central nervoussystem and does the structural restoration of the visualsystem also lead to a restoration of the preoperative light-induced behaviour?

These questions were examined through morphological,physiological and ethological observations on both intact andregenerated eyes.

Materials and Methods

Light and electron microscopy

Young adults of

Achatina fulica

(Linné 1758) were maintainedin the laboratory at a constant temperature of 26–28

°

Cin a terrarium containing 5–8 cm of wet sand. The terrariumwas covered with glass to create a humid atmosphere.The animals were fed cabbage and dandelion leaves,carrot pieces and chicken eggshell. Twenty-four animalswith shell heights of 6–9 cm (corresponding to an age of5–7 months) comprised the controls and 32 snails ofsimilar sizes and ages made up the experimental group.Each snail’s shell was labelled with nail polish for easyidentification.

To study reparative eye morphogenesis, the distal ends ofthe tentacles were amputated. The severed tentacle pieces,

both those containing the original (native) eyes, which servedas controls, and those with regenerating eyes were processedfor light and electron microscopy. Control eyes and regener-ating eyes were also obtained from amputated tentacles forelectrophysiological recordings.

The first samples were those in which a tiny black spot wasonly just visible with the naked eye under the regeneratingtentacular surface epithelium. Tips of eye tentacles from dif-ferent individuals were, thus, sampled 50 and 86 days, as well as6 months after amputation of the original eye-bearing tentacles.

For light microscopy, parts of the amputated tentacleswere fixed either for 24 h in Bouin’s solution or for 2 h in2.5% glutaraldehyde buffered with 0.2

cacodylate bufferto pH 7.5. Standard paraffin-embedding methods were used.Serial 15–20-

µ

m paraffin sections, cut with a rotary micro-tome (steel knife) MPS-2, were stained with Delafield’shaematoxylin & eosin (Humanson 1979). Serial, 1.5-

µ

mthick sections were prepared from specimens embeddedin Epon 812 with a glass knife on a Reichert Ultracut Eultramicrotome and were stained with a mixture of toluidineblue and azur-II (1 : 1) dye. Micrographs of specimens exam-ined under a light microscope were taken on a Reichert–JungPolyvar. For electron microscopy, amputated tentacles werepostfixed for 1 h at room temperature in 1% aqueous solu-tion of osmium tetroxide, buffered with 0.1

cacodylatebuffer to pH 7.5. Ultrathin sections, cut with a diamondknife on Reichert Ultracut E or Leica ultracut UCT ultra-microtomes, were prepared from Epon 812-embeddedspecimens. The sections were then stained with uranyl acetateand lead citrate for a few minutes each and examined andphotographed under a Scanning Transmission ElectronMicroscope JEM-100 CX II and Energy-Filtered ScanningTransmission Electron Microscope LEO 912 OMEGA.

For planes of sectioning and corresponding micrographs1–11, please see Fig. 14.

Behavioural experiments

To verify whether the visual stimuli (black stripes of differentwidths) significantly influenced the direction of movement inthe snails, an experimental procedure, first described byHamilton and Winter (1982) and then modified by Zhukovand Baikova (2001), was used. The experimental set-up wasconstructed from three parts:1. An incandescent light source (100 W), providing an

illumination of

c

. 740 lux and equipped with matt (

=

lightscattering) and green filters (range: 480–660 nm;

λ

max

=

540 nm), was positioned 40 cm away from the snail eye.2. A white, but nontransparent cylinder with an inner

diameter of 21.2 cm and a height of 25 cm was placed ona glass bottom, covered by a semitransparent foil, dividedinto 16 numbered sectors.

3. Black stripes of photographic paper of the same height butdifferent widths corresponding to 5

°

, 10

°

, 22.5

°

and 45

°

ofarc were attached to the inner surface of the white cylinder.

Acta Zoologica

(Stockholm)

85

: 1–14 (January 2004)

Bobkova

et al.

•

Eye regeneration in

Achatina fulica

© 2004 The Royal Swedish Academy of Sciences

To determine the directions (movement vectors) of eachanimal, the latter were placed one by one in the middle of theglass bottom. The number of the sector on the inner surfaceof the cylinder reached by the snail was registered. The move-ment vector was defined as the bisector of the central angleof the corresponding sector. At the beginning of successiveexperiments, the orientation of the animal was changedmanually by approximately 22.5

°

shifts in a clockwise oranticlockwise direction. A single experiment lasted no longerthan 30 min. The trace of mucus left behind was carefullywashed away after each experiment.

Three series of experiments were carried out. The firstrequired two kinds of control experiments. In one control, noblack stripe whatsoever was used in the presence of light.This was done to determine the absence of stimuli other thanthe black stripe; stimuli that could interfere with the snail’smotivated movement. The second control was performed inthe presence of a black stripe of 45

°

, but in the absence oflight to exclude any influence of the photographic paper’sodour. The second series of experiments aimed to determinethe threshold of visually mediated behavioural responses(direction of the movement) in the control snails. Blackstripes of 5

°

, 10

°

, 22.5

°

and 45

°

were presented to these snailsand their movements were registered as described above.Both series of experiments were carried out on 24 snails.The distal tips of the optical tentacles were then amputatedand examined by light and electron microscopy, repre-senting the morphological controls. The third series of experi-ments was performed on 32 snails with amputated tentaclesto study the behavioural responses of these animals to ablack stripe of 45

°

width (well above the threshold of thereaction). It was performed 7, 14, 30, 60 and 180 days aftersurgery.

The data obtained were statistically processed by

V

-tests(Batschelet 1972).

The mean vector of movement was determined as

,

where and , and

n

is the number

of observations (

=

the number of snails), whereas

α

i

is theangle (grad) between the movement vector of each individualsnail and the axis X.

The projection of the mean vector on the predicted directionof the movement was calculated as

,

where

Θ

0

is the angle of the predicted direction relative toaxis X.

Values of

U

, describing the deviation of animals from thepredicted direction, were calculated as

.

Statistical significance was determined through compari-sons between the calculated value

U

and

U

crit

(using the Tableof Significance,

P

=

0.05, in Batschelet 1972).

Electrophysiology

Preparation.

The most distal region of a tentacle, with eye(s)present, was carefully dissected to expose the snail’s eyeball.The preparation was then pinned to the wax bottom of theexperimental bath with tungsten needles. Any surround-ing tissue of the eyeball was removed. All procedures wereperformed under the red light of a lamp equipped with aheat filter.

Electrophysiological recordings.

Electroretinographic responses(ERG) of native and regenerating eyes were carried out withglass suction pipettes of approximately 70-

µ

m tip diameter andan Ag–AgCl electrode. A DC-amplifier and two-dimensionalpen recorder were used.

Light stimulation.

The light of a halogen lamp, passing throughgreen and heat filters, was delivered to the eye through 1.5-mm diameter light guides. An electromagnetic shutter,controlled electronically, restricted the duration of a singlestimulus to 0.2 s. The intensity of the light beam was adjustedusing neutral grey filters and was measured by quantumphotometer.

Experimental procedure.

Before the start of an experimentthe preparation was left in complete darkness for 30 min.ERGs evoked by light stimuli of increasing intensity withdark interstimulus-intervals of 3 min were recorded. In orderto record flicker fusion frequencies (FFF), light flashes from0.1 Hz up to the frequency at which FFF occurred wereused. All experiments were carried out at 22–24

°

C.

Physiological solution.

The physiological saline consistedof a mixture of NaCl (61 m

), KCl (3.2 m

), CaCl

2

(10 m

),MgCl

2

(13 m

) and glucose (5 m

) and the pH was adjustedto 7.6–7.8 with NaHCO

3

, following Suzuki

et al

. (1979).

Results

Native (control) eye: anatomy and ultrastructure

Achatina fulica

possesses a pair of cephalic camera-type eyes.The eyes are located on the dorsal side of the optic tentaclesbeneath the monolayered transparent epithelium and consistof a ‘main’ and ‘accessory’ component. The optic nerve,which serves both main and accessory eye, runs down thetentacle and ends in the cerebral ganglion. A powerful retrac-tor muscle is attached by its distal end to the tentacle wall andthe eye capsule (Fig. 1A).

The eyeball is of asymmetric shape because of the accessoryeye, which is situated on the ventral–lateral side of the main eye

r x y ) ( )= +( 2 2

xn i

n

i cos=1

1Σ α=

yn i

n

i=1

1Σ α=

sin

′ = ⋅ + ⋅∑ ∑V ı ı cos cos sin sinα Θ α Θ0 0

U Vn

= ′ ⋅2

Eye regeneration in

Achatina fulica

•

Bobkova

et al.

Acta Zoologica

(Stockholm)

85

: 1–14 (January 2004)

© 2004 The Royal Swedish Academy of Sciences

under the common cornea (Fig. 1B). In molluscs with shellheights of

c

. 8 cm the diameters of the main eye are

c

. 250and 320

µ

m, while the accessory eye measures

c

. 55 and110

µ

m.Under each cornea are two vitreous bodies, two lenses, and

two noninverted retinas. A single eye capsule is present andone optic nerve exists for both main and accessory eyes.

The convex and monolayered cornea of the eye is composedof elongated, unpigmented and

in vivo transparent cells. The thick-ness of the cornea is c. 40 µm along the optic axis of the main eye.

The aperture of the eye is c. 115 µm. Corneal cells arecharacterized by flaky cytoplasm and nuclei located in thebasal portion of the cells (Fig. 2A). The perinuclear area isrich in mitochondria, rough endoplasmic reticulum, and

granules of glycogen. Cell organelles are abundant near theplasma membrane but are rare in the cytosol of the remain-ing portion of the cell. The basal surface of the corneal cellsis anchored by hemidesmosomes to the underlying basallamina of the eye (Fig. 2B). The apical portions of thecorneal cells bear short microvilli and are turned towardthe vitreous body. The apices and their cytoskeletons areattached to each other by adhesion belts.

The lens of the main eye is bean-shaped and of seeminglyharder material than the surrounding tissue. Its lens is c. 135and 145 µm along its two axes. The short axis of the lenscoincides with the optic axis of the main eye. The accessorylens is c. 30 and 40 µm, when it is ovoid in shape. The vitreousbodies of main and accessory eyes are isolated from eachother by their respective retinae.

The retina of the main eye (noninverted and monolayered)measures c. 64 µm in thickness and contains at least threecell types: type I photoreceptors with dome-like apices, bear-ing long light-sensitive microvilli (1), type II photoreceptorswith short microvilli and a much rarer presence (2), and (3)pigmented (supportive) cells (Figs 3A, 4A–C). The cellularcomposition of the accessory eye is similar to that of themain eye but there are no screening pigment granules inboth photoreceptor and supportive (so-called ‘pigmented’)cells.

Four distinct layers in the retina of the main eye can bedistinguished: microvillar, pigmented, somatic and plexi-form. Functionally, the microvillar layer is the layer with thegreatest amount of light-sensitive membranes of the photore-ceptor cells. The average diameter of the tube-like microvilliis c. 0.1 µm. The thickness of the layer (under completelight adaptation) is c. 15 µm when measured along the opticaxis.

The pigmented layer is formed by an accumulation ofscreening pigment granules in both supportive and type Iphotoreceptor cells. Under extreme light adaptation, thethickness of the pigmented layer is c. 26 µm. Screeningpigment granules measure c. 1 µm in diameter.

The somatic layer consists of the large (c. 6.0 and 7.0 µm),but slightly oval, nuclei of type I photoreceptor cells and thesmaller (c. 4.0 and 7.0 µm) elongated nuclei of type IIphotoreceptor cells, as well as the nuclei (c. 4.5 and 5.0 µm)of the supporting (pigmented) cells (Figs 3B and 4D). Thethickness of this layer is c. 16 µm. The interphase nuclei ofthe type I photoreceptors are noted for their dominatingeuchromatin, which stains diffusely, while the nuclei of typeII photoreceptors and supportive cells have more condensedheterochromatin. Usually the nuclei of type II photorecep-tors and of supportive (pigmented) cells are located morebasally than the nuclei of type I photoreceptors.

Typical for gastropods is that type I photoreceptor cellspossess accumulations of uniformly sized photic vesicles,measuring c. 0.05 µm in diameter, while no aggregations ofphotic vesicles were found in the cytoplasm of type IIphotoreceptor cells. Longitudinally orientated numerous

Fig. 1—A. Light micrograph of longitudinal section of partially retracted tentacle with intact eye. Cornea (C), muscular retractor (MR), tentacle epithelium (Ep), sensory epithelium (Sep), and haemolymph lacunae (Hm) are discernible. —B. Light micrograph of longitudinal section through the eye with surrounding interstitial tissue (IT) are indicated. Cornea (C), lens of main eye (LM), lens of accessory eye (LA), and eye capsule (CA) are visible.

Fig. 2—A. Electron micrograph of longitudinal section through cornea. Eye capsule (CA), muscle cell (MC), and nucleus of corneal cell (NC) are visible. —B. Enlargement indicates, how hemi-desmosome anchors corneal cell (CC) and eye capsule (CA) to basal lamina (BL).

Acta Zoologica (Stockholm) 85: 1–14 (January 2004) Bobkova et al. • Eye regeneration in Achatina fulica

© 2004 The Royal Swedish Academy of Sciences

microtubules (being part of the cytoskeleton), snakelikemitochondria, and a few clear vesicles are the main organellesto be found regularly in the distal portions of type II photo-receptor cells (Fig. 4). The perinuclear area of the cytoplasm isfilled with glycogen granules, rough endoplasmic reticulum,mitochondria, and cisternae of Golgi. However the cytosol oftype II photoreceptor cells remains electron-lucid. Theplexiform layer of the main eye is composed of fibres of twomorphological populations (Fig. 5). The first populationcomprises axons of type I and II photoreceptors that containmitochondria, granular material (which may be glycogenand ribosomes), clear vesicles of c. 0.1 µm in diameter, andnumerous longitudinally orientated microtubules. Thesecond population of axons contains vesicles of c. 0.08 µm indiameter with an electron-dense core, surrounded by a paleand very narrow halo, and clear vesicles of c. 0.04 µm indiameter. Mitochondria, filament bundles and microtubulesare present as well.

Axons of photoreceptors belonging to the accessory eyecontribute to the plexiform layer of the main eye. Fibres thatleave and penetrate the eye capsule as separate bundles, jointogether and form the optic nerve. The optic nerve measuresc. 30 µm in diameter and is surrounded by glia. The basallamina is c. 0.04 µm thick and continues along the basalsurfaces of corneal and retinal cells, separating the eye fromconnective (interstitial) tissue and its capsule. The eye capsuleconsists mainly of striated collagen fibrils and a layer ofmuscle cells located circumferentially (except the area of thecornea) (Fig. 6).

Reparative morphogenesis: general observations

Studies on the stumps of the amputated tentacles reveal thatthe start of reparative eye morphogenesis requires, first ofall, a certain reorganization of the interstitial tissue and theformation of a wound-covering epithelium. Complete re-growth of the optic tentacle, however, is not essential. At thesame time the surface of the tentacle stumps can be badlydeformed, i.e. thickened or bifurcated.

At the early stages of regeneration, the eye appears as aninvagination of the new epithelium into a mass of uniformcells of interstitial tissue precursor material (Fig. 7A).

Further redifferentiation and specialization of the inter-stitial tissue cells is accompanied by a progressive redeve-lopment of both eye and sensory epithelium (Fig. 7B,C).

The first small, darkly pigmented spots, visible to thenaked eye, were seen 50 days after tentacle amputations.Twenty-five per cent of the experimental animals exhibiteda normal reparative eye morphogenesis, comparable toembryonic development (Fig. 7D,E). However, 29% of theanimals displayed abnormal formations: (i) multiple, butgrouped eyes (eyes with two or three optic cavities, separatedor not separated by collagen capsules: Fig. 8); (ii) rudimen-tary eyes (eyes without a lens and optic nerve, or just separatecornea with accessory eye and its optic nerve, but no maineye: Fig. 9); (iii) supernumerary eyes (two to four independ-ent eyes, not grouped, in one and the same tentacle). Of theexpected regenerates, 46% did not show any regeneration6 months after the amputation of the tentacles had occurred.

Fig. 3—A. Electron micrograph of tangential section through apical portion of retina. Massive amounts of microvilli (MV) of type I photoreceptors (PhI), pigmented (= supportive) cells (PC), screening pigment granules (Pg), vitreous body (VB), and points of adhesion belts of cytoskeletal filaments (arrows) are clearly discernible. —B. Electron micrograph of somatic layer of retina with nucleus of photoreceptor cell type I (NPhI), and nuclei of pigmented (= supportive) cells (NPC) are clearly distinguishable.

Eye regeneration in Achatina fulica • Bobkova et al. Acta Zoologica (Stockholm) 85: 1–14 (January 2004)

© 2004 The Royal Swedish Academy of Sciences

Structural features of the normally regenerated eyes

Eyes that regenerated normally have the same anatomyand ultrastructure as the original ones (Figs 7D,E, 10).Newly formed axons were observed in the plexiform layer ofthe retina (Fig. 11A,B) and the distal part of the regeneratedoptic nerve (Fig. 11C). However, in the majority of the sam-ples all trace of an optic nerve was lost and we assume thatthe optic nerve could have joined the tentacle nerve shortly

after leaving the eye. Nevertheless, in vivo the regeneratedoptic nerve was followed up to the cerebral ganglion. In fact,the only clear difference between normally regenerated andnative (control) eyes was the smaller size of the eyeball, andcorrespondingly smaller linear proportions of the main struc-tures in the regenerates. One of the peculiarities of regener-ation is the uneven speed of the process. Thus, even 50 daysafter the lesion, the majority of the animals possessed no eyesat all, but some allowed a distinction to be made between

Fig. 4—Electron micrographs of longitudinal section through retina of main eye, showing type II photoreceptor (PhII) in Fig. A and additional part-enlargements in Figs. B and C. Pigmented (= supportive) cell (PC), lens of main eye (LM), microvilli (MV), mitochondria (M), microtubules (arrowheads), and clear vesicles (arrow) are present. —D. Somatic layer with nuclei of type II photoreceptor (NPhII) and nucleus of a pigmented cell (NPC) as well as a conglomeration of pigmented granules (PG) and part of the eye capsule (CA) are visible.

Acta Zoologica (Stockholm) 85: 1–14 (January 2004) Bobkova et al. • Eye regeneration in Achatina fulica

© 2004 The Royal Swedish Academy of Sciences

cells of the retina and those involved in forming the vitreousbody, lens and microvillar apparatus. There are even some ofthe regenerating eyes that possess all the main structures likecornea, vitreous body, lens, retina and eye capsule.

A summary of the results and the measured parameters ofthe main eyes is given in Table 1.

Electrophysiological recordings

Light stimulation evokes electric responses in both nativeand regenerating eyes. The ERG of the native eye, recordedfrom the middle part of the eye cup, consists of two phases,a quick depolarization and a slow repolarization. Well-formedregenerating eyes responded to light stimuli in a similarway, but possessed lower amplitudes (Fig. 12A,B). However,incompletely formed eyes, resembling pigmented spots, did

not respond to stimuli of even the highest intensity. Theslopes of normalized V/ log I-curves for native and regener-ated eyes are almost identical, suggesting that sensitivity tolight is the same in both kinds of eye. Individual electricalresponses of regenerated eyes tend to fuse at lower frequencies(0.41 ± 0.08 s−1, n = 9) than those of native ones (0.57 ± 0.08 s−1,n = 9) (Fig 12C,D).

Behavioural experiments

In the first series of our control experiments, the distributionof the individual vectors of a snail’s movement did not differsignificantly from random, i.e. obtained value U < Ucrit. Inpractice this means that there are no significant effects (heat,smell, etc.) other than the visual one (black stripe) involvedin inducing voluntary movement.

Fig. 5—Electron micrograph through plexiform layer of retina. —A. Photoreceptor axons (Ax), microtubules (Mt), mitochondria (M), and eye capsule (CA) are typical structural elements of this layer. —B. Axons filled with electron-dense core vesicles (DV). Furthermore, mitochondria (M), clear vesicles (CV), a process of a pigmented cell (PPC), and part of the eye capsule (CA) are visible.

Fig. 6—Electron micrograph of most basal portion of retina. —A. Retina of the main eye (Rm) with rough endoplasmic reticulum (R-EPR), eye capsule (CA), and collagen fibrils (arrowheads) clearly visible. —B. Strands of muscle (Ms), embedded in the eye capsule (CA), and the retina of the main eye (ME) are discernible.

Eye regeneration in Achatina fulica • Bobkova et al. Acta Zoologica (Stockholm) 85: 1–14 (January 2004)

© 2004 The Royal Swedish Academy of Sciences

Fig. 7—A–C. Light micrographs of sections through the regenerated eye 50 days post-operation. —A. Location of pigmented pit (arrow). —B. Eye with small lens (arrow). —C. Enlargement of Fig. B, showing retina (ME) and lens of main eye (LM) as well as cornea (Co) and interstitial tissue of the optic tentacle (IT). —D–E. Light micrographs of longitudinal section through regenerated eye, 86 days post-operation: all the structural elements are restored. —D. Main eye with cornea (C), lens (LM), retina (R), microvillar (ML), pigment (PL), somatic (SL), plexiform (PxL) layers and eye capsule (CA). —E. Accessory eye with cornea (C), retina of the accessory eye (AE), lens (LA), and nuclei of the corneal cells (NC).

Table 1 Morphometrical comparison between parameters of the main control eyes and normal regenerates (values in µm)

Parameters Native eye Normal regenerate

Long axis of eyeball 329 ± 47 (N = 9) 246 ± 77 (N = 9)Short axis of eyeball 248 ± 20 (N = 9) 186 ± 76 (N = 9)Lens diameter 133 ± 27 and 143 ± 23 (N = 8) 81 ± 24 and 114 ± 44 (N = 6)Aperture 115 ± 17 (N = 8) 95 ± 12 (N = 6)Thickness of cornea 37 ± 8 (N = 8) 36 ± 3 (N = 6)Thickness of microvillar layer 15 ± 7 (N = 7) 15 ± 5 (N = 9)Thickness of pigmented layer 26 ± 6 (N = 9) 24 ± 6 (N = 9)Thickness of somatic layer 16 ± 7 (N = 9) 13 ± 4 (N = 9)Thickness of eye capsule 2.0 ± 0.3 and 5.0 ± 0.5 (N = 9) 2.0 ± 0.4 and 5.2 ± 0.4 (N = 9)(areas of cornea and of retina)Thickness of basal lamina 0.04 ± 0.001 (n = 25) 0.04 ± 0.001 (n = 25)Distance between centres of type I cells 15.0 ± 0.6 (n = 80) 20.4 ± 0.3 (n = 60)Distance between centres of type II cells very rare very rareSize of nuclei of type I cell 6.3 ± 0.3 and 7.1 ± 0.4 (n = 80) 6.0 ± 0.4 and 7.2 ± 0.6 (n = 60)Size of nuclei of type II cells 3.5 ± 0.7 and 6.5 ± 0.2 (n = 25) 3.9 ± 0.7 and 6.7 ± 0.1 (n = 25)Size of nuclei of pigmented cells 4.4 ± 0.7 and 5.1 ± 0.2 (n = 90) 4.5 ± 0.6 and 5.0 ± 0.2 (n = 70)Diameter of photic vesicles 0.05 ± 0.001 (n = 100) 0.05 ± 0.001 (n = 100)Diameter of dense-core vesicles in axons of one population 0.08 ± 0.002 (n = 50) 0.08 ± 0.002 (n = 50)Diameter of clear vesicles in axons of other population 0.04 ± 0.004 (n = 50) 0.04 ± 0.003 (n = 50)Diameter of clear vesicles in axons of type I cells 0.1 ± 0.03 (n = 50) 0.1 ± 0.03 (n = 50)Diameter of light-sensitive microvilli of type I cells 0.1 ± 0.02 (n = 100) 0.1 ± 0.03 (n = 100)Diameter of pigment granules 0.5–1.5 (n = 100) 0.1–1.5 (n = 100)

Data are expressed as mean ± standard deviation (N is the number of snails used, n is the total number of measurements).

Acta Zoologica (Stockholm) 85: 1–14 (January 2004) Bobkova et al. • Eye regeneration in Achatina fulica

© 2004 The Royal Swedish Academy of Sciences

Data obtained in the second series of experiments indicatethat the attractiveness of the black stripe increases with itswidth. The threshold of the behavioural response evoked by theblack stripe was close to 22.5° of arc (level of significance P =0.05) and a stripe of 45° was most attractive for the snails (Fig.13A). Therefore, in all further experiments designed to revealvisually mediated behaviour at the earliest stages of visualrestoration, a black stripe of 45° width was used. This third seriesof experiments showed that the distribution of the vectors ofmovement did not differ significantly from random even180 days after tentacle amputation, although it was markedlyhigher than that seen 60 days postoperation (Fig. 13B).

Discussion

An important component of this research has been theanatomical and ultrastructural comparison of native andregenerated eyes. To help in navigating through the anatomical

part of the discussion and to facilitate understanding our con-clusions, a semischematic drawing of the snail eye (Fig. 14)has been included on which the various planes of sectioningand corresponding micrographs have been indicated.

Our observations confirm an earlier report by Chase andKamil (1983) that the eyes of Achatina fulica regenerate, butsometimes contain abnormalities. In our study, only 54% ofthe eyes could be considered to have regenerated, but we areunable to say whether the percentage of successfully regen-erated eyes depended on where amputation took place in thedissection. However, according to Gibson (1984; p. 147) atleast in the marine gastropod Ilyanassa obsoleta ‘the averageregeneration rate was the same, whether the amputation wasmade just below the eye, through the eye and tentacle, or atthe base of the tentacle’. In A. fulica eyes can even be formedif the tentacle itself is absent (present study). On the otherhand, it was demonstrated that axonal regeneration in Heli-soma trivolvis neurones depended on the site of the axotomy(Kruk and Bulloch 1992).

Another interesting phenomenon deals with the formationof eyes inside several optic cavities. We believe that in theprocess of regeneration, the distribution of eye parts into sev-eral separate cavities can occur. Although we did not comeacross any detailed description of such a phenomenon in theliterature, one paper (Hughes 1976) contains a picture withtwo closely apposed eyes in Strombus pugilis. The process thatcan lead to the formation of such separated eyes is unknownand whether such abnormalities can also be found inother species is equally obscure. Multiple (grouped) andsupernumerary (not grouped) eyes in A. fulica probably cannot

Fig. 8—Light micrographs of abnormal formations during eye regeneration. —A. Two closely apposed eyes, separated by the eye capsule. One regenerated optic nerve (ON) is visible. —B. Eye with three optic cavities (I, II, III). Cornea (C), cells of the accessory retina (asterisk), lens (LM), and retina (ME) of the main eye, vitreous body (VB), eye capsule (arrows), and interstitial tissue (IT) are labelled.

Fig. 9—Light micrograph of tangential section through regenerate, showing cornea and accessory eye with optic nerve, regenerated separately from main eye. Cornea (C), some cells of the accessory eye (asterisk), optic nerve of the accessory eye (ON), and interstitial tissue (IT) are discernible.

Eye regeneration in Achatina fulica • Bobkova et al. Acta Zoologica (Stockholm) 85: 1–14 (January 2004)

© 2004 The Royal Swedish Academy of Sciences

Fig. 10—Electron micrographs of longitudinal sections through regenerated retina of main eye, providing morphological evidence for photosensitivity restoration. —A. Most apical portion of main retina. Vitreous body (VB), microvilli (MV) of type I photoreceptors (PhI), and pigmented (= supportive) cells (PC) are labelled. —B. Fragment of somatic layer (perinuclear region) with aggregations of photic vesicles (PV), the nucleus of a pigmented cell (NPC), and dense core vesicles (arrows) are indicated.

Fig. 11—A–B. Electron micrographs, showing two types of axon in the plexiform layer of the regenerate. —A. Axons of photoreceptors with clear vesicles (arrow). —B. Axon with electron-dense core vesicles (arrowheads), clear vesicles (arrow) and pigmented (= supportive) cell (PC) are indicated. —C. Electron micrograph of serial sections that provide evidence for the origin of the nerve branch (ON) from the regenerated retina of the main eye (ME): the place of penetration is marked by an asterisk. Eye capsule (CA), process of type I photoreceptor filled with photic vesicles (PPhI), muscle cell (MC), and interstitial tissue (IT) are labelled.

Acta Zoologica (Stockholm) 85: 1–14 (January 2004) Bobkova et al. • Eye regeneration in Achatina fulica

© 2004 The Royal Swedish Academy of Sciences

be explained by a mechanism of compensatory regeneration,because the removal of only one or both native eyes leads tothe same degree of abnormality. More probably, multipleeyes can appear as a result of abnormal gene regulationduring the process of regeneration (Gehring 1998). Animportant problem is the question of what controls the actualprocesses of regeneration.

It is well known that regeneration in animals generally, notonly in snails (Asashima et al. 1987), is brought about by thestimulation of nerve endings upon amputation of body parts.Nerve impulses in sensory neurones, in turn, stimulateneurosecretory activities in the central ganglia, and the neuro-secretory materials initiate regenerative processes. In Melam-pus bidentatus, for example, implanted cerebral ganglia can

develop an eye within the host snail haemocoele (Moffettand Austin 1981), while normal regeneration of eyes involvesthe formation of an ectodermal invagination (Eakin andBrandenburger 1967). Thus, regenerative phenomena andprocesses of embryonic development are similar in manyways – both involve cell division, cell movement, tissuedifferentiation and morphogenesis (development of form). InAplysia sp. it was shown that axon growth during develop-ment and following injury also shares a common process butdiffers in that regeneration alone requires the participation ofcells of the immune system (Farr et al. 2001).

Tentacles of young, premature A. fulica have greaterregenerative abilities than those of adult individuals since thespeed of the process is much higher and the regeneration

Fig. 12—Electrical responses of control and regenerated eyes to stimulation with green light of 0.2 s duration and of 17 lux illumination. —A. Electrical response of the control eye. —B. Examples of electrical responses of the regenerated eyes. —C, D. Electrical responses of control (C) and regenerated (D) eyes to stimulation with flashes of light of 0.2 s duration, delivered at different flash frequencies (marked as s−1).

Fig. 13—A. Behavioural responses of intact (control) animals to visual stimuli (black stripes of different widths). —B. Behavioural responses of operated animals with regenerating eyes to black stripe of 45°.

Eye regeneration in Achatina fulica • Bobkova et al. Acta Zoologica (Stockholm) 85: 1–14 (January 2004)

© 2004 The Royal Swedish Academy of Sciences

ends without structural abnormalities in the former(Tartakovskaya et al. 2003). In the mollusc Lymnaea stagnalis,recovery of the tentacle withdrawal reflex after peripheralnerve damage is considerably affected by the period of sensorydeprivation following nerve injury (Janse et al. 1979) as wellas the snail’s age, because of an impairment of the recoveryof some central synaptic connections (Janse et al. 1986). Inmature specimens of Strombus luhuanus restoration of the eyeand tentacle movements in response to stimulation with lightoccur in regenerating optic tentacles (Gillary 1983), butwithout a quantitative assessment one cannot decide if therestoration was complete and, in particular, if the observedreflexes were perhaps based on peripheral mechanisms notinvolving the central nervous system.

The reasons for the abnormal eye regeneration are stillunclear. Our experiments involved young mature specimens,but A. fulica is a long-lived species (Heller 1990) and theprocess of regeneration itself may take more time than it doesin gastropods with shorter lifespans. Moreover pulmonateeyes generally require a much longer regeneration time thando those of, for example, I. obsoleta (Gibson 1984), otherneogastropods (Hughes 1976), or the mystery snail Pomaceasp. (Bever and Borgens 1988).

In the snails S. luhuanus (Gillary 1983) and Cryptomphal-lus aspersa (Flores et al. 1983), the structural regeneration ofthe eye is accompanied, at a certain stage of the process, by

the restoration of the electrical responsiveness to light. Theform of the ERG of the eye and the electrical responses of theretinal cells of S. luhuanus are changing during the course ofthe regeneration and approach those of the native eye of themature individual. These changes show up first as additionalcomponents of ERG and as off-responses from the opticnerve. It seems that the evolution of the electric responseparallels the process of differentiation and morphogenesis ofthe retinal cells, and the development of intracellular connec-tions as well as the growth of axons along the optic nerve. Onreaching an eye size of 1 mm, the ERGs of the regeneratedeyes become indistinguishable from the signals of the nativeeye. Under rhythmic stimulation the responses of the regen-erated eyes of C. aspersa are identical to those of native eyeswith the exception of the faster decrease of the ERGsamplitude (Flores et al. 1983).

Our observations have shown that the regenerating eye ofA. fulica also recovers its bio-electrical capacity, whichsuggests that transduction mechanisms are operational. Dif-ferences in the flicker fusion frequencies between native andregenerated eyes may be the consequence of differences inthe ways intercellular connections were formed in the retina.Validation of such an assumption does not seem possible,because of our lack of understanding of the relative contribu-tions of the various retinal cell types to the ERG.

Most gastropods respond behaviourally to light withpositively or negatively phototactic reactions (Willem 1892).Such behaviour is generally mediated by ocular rather thannonocular photoreception (Stoll 1973). One may supposethat their well-developed, camera-like eyes supply snails witha certain capacity for visual discrimination at least of simplegeometrical objects. Visually mediated behaviour was dem-onstrated in several species, e.g. Littorina irrotata (Hamiltonand Winter 1982), Helix aspersa (Hamilton and Winter 1984)and Lymnaea stagnalis (Vakolyuk & Zhukov 2000). Recentobservations by Zhukov and Baikova (2001) have further-more shown that A. fulica also discriminates some visualstimuli, namely vertical and horizontal black stripes, as wellas grating patterns of different frequencies. Being a terrestrialsnail A. fulica, exhibits nocturnal activities (Takeda andOzaki 1986) and avoids direct light, perhaps partly becausein its natural environment solar radiation leads to an increasein temperature and a decrease in humidity. Probably forthe same reason A. fulica prefers a black stripe on a whitesurround over a white stripe on a black background. Thispreference clearly depends on ocular photoreception, becauseof the dramatic decrease of such behavioural choice aftertentacle amputation. Several months postoperation, how-ever, some animals (seven out of 32) had regained the originalbehavioural pattern typical of the intact snails (presentstudy). The reaction could serve as an indication of therestoration of the connection between regenerated eye andcentral nervous system, although U-values calculated forsnails after 180 days of regeneration, remained lower thanthose of the control animals.

Fig. 14—Semi-schematic drawing of snail eye with planes of serial sectioning indicated by Roman numerals I–III. Plane I served for figures 2, 5–7, and 11; plane II for figures 1, 3, and 4; plane III for figures 8–10. The abbreviations used stand for: cornea (C), lens of accessory eye (LA), lens of main eye (LM), optic nerve (ON), screening pigment (PG), retina of accessory eye (Ra), retina of main eye (Rm), and vitreous body (Vb). Note that in some regenerates more than one main eye was developed.

Acta Zoologica (Stockholm) 85: 1–14 (January 2004) Bobkova et al. • Eye regeneration in Achatina fulica

© 2004 The Royal Swedish Academy of Sciences

Two reasons can be advanced for this discrepancy. First,the tentacles of the studied snails represented different stagesof regeneration, ranging from total absence of any eye struc-ture to well-developed eyes similar in size to those of intactanimals. If one considers only snails with well-restored eyes,the discrepancy is much reduced. Second, restoration of visionfollows the anatomical recovery. The difference between thedevelopment of an eye’s structure and the appearance ofthe snail’s visual behaviour may be the result of the processesof morphogenesis in the central nervous system and thesmaller number of photoreceptors in the regenerates couldbe responsible for the higher threshold of the behaviouralreaction. Indirect evidence that regenerated eyes possess asmaller number of receptor cells comes from the muchreduced amplitudes of the ERG in the regenerated eyes.

In contrast to the visual behaviour of A. fulica, as shown byChase and Kamil (1983), the olfactory system regenerateswithout any abnormality. This underscores our conclusionthat the restoration of the morphological and functionalintegrity of the regenerated eye, and not olfaction or anyother unknown sensory modality, plays the crucial role in thedescribed behavioural pattern.

The main results of the regeneration of the optic tentaclein 5–7-month-old A. fulica are the restoration of the opticsystem of the eye and the basic features of the cell’scomposition of the retina as well as the process of photo-reception. The restoration of the entirety of the elementsof visual function, as our behavioural data show, isage-limited.

Initially, structures involved in the formation of the visualimage are restored. The dimensions of these structures (e.g.retinal layers) are the same in the regenerating and thenormal eye. Regenerated and native eyes differ only in theabsolute size of eye and lens. Our inference is that the size ofthe whole eye is determined by the number, but not thevolume, of sensory, supportive and corneal cells, as well as byan increase in the size of the lens. This view supports anearlier suggestion made by Scarsso and Pellegrino de Iraldi(1973) and agrees with studies on ontogenesis in Aporrhaispespelecani (Blumer 1996).

We also determined that, although the optic nerve is notrestored in every case, its formation is clearly evident. Towhat extent total behavioural recovery depends on a com-plete restoration of the optic nerve remains to be seen, but itis now clear that the regenerating eye is able to recover itsfunction and that there are no dramatic differences betweenthe regeneration processes of Achatina fulica and othergastropods.

Acknowledgements

M.V.B. and V.Zh. wish to thank the Centre of InternationalMobility (CIMO), University of Oulu (Finland) and theRussian Foundation for Basic Research (02-04-48261),respectively, for support through grants.

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