Eyes and vision in Arion rufus and Deroceras agreste (Mollusca; Gastropoda; Pulmonata): What role does photoreception play in the orientation of these terrestrial slugs?

Acta Zoologica

(Stockholm)

90

: 189–204 (April 2009) doi: 10.1111/j.1463-6395.2008.00369.x

© 2008 The AuthorsJournal compilation © 2008 The Royal Swedish Academy of Sciences

189

Abstract

Zieger, M.V., Vakoliuk, I.A., Tuchina, O.P., Zhukov, V.V. and Meyer-Rochow,V.B. 2009. Eyes and vision in

Arion rufus

and

Deroceras agreste

(Mollusca;Gastropoda; Pulmonata): What role does photoreception play in theorientation of these terrestrial slugs? —

Acta Zoologica

(Stockholm)

90

: 189–204

This paper deals with the orientational behaviour in the two terrestrial slugs

Arion rufus

and

Deroceras agreste

. It presents anatomical details of their eyes andprovides an appraisal of the eyes’ optical system. In both species the retinaecontain two principal types of cell: photoreceptive and pigmented supportivecells. While only the eye of

A. rufus

apparently contains neurosecretoryneurones, that of

D. agreste

is the only one equipped with a small additionalretina with its own separate lens. Lens shapes vary between ovoid (

A. rufus

) andspherical (

D. agreste

). Our results demonstrate that the camera-type eyes in

A. rufus

and

D. agreste

have optical systems that do not allow the productionof a sharp image on the retina. The slugs demonstrate negative visuallymediated phototactic behaviour, but no polarization sensitivity. Only oneaspect of the visual environment, namely the overall distribution of light anddark, seems to be important for these slugs. As the main role of the slugs’photoreceptors is to monitor environmental brightness and to assist the animalin orientating towards dark places, we conclude that these slugs do not needto perceive sharp images.

Victor Benno Meyer-Rochow, International University Bremen (JacobsUniversity as of February 2007), School of Engineering and Science, CampusRing 6, Research II, Room 37, D-28759 Bremen, Germany. E-mail: [email protected] and [email protected]

Blackwell Publishing Ltd

Eyes and vision in

Arion rufus

and

Deroceras agreste

(Mollusca; Gastropoda; Pulmonata): What role does photoreception play in the orientation of these terrestrial slugs?

Marina V. Zieger,

1

Irina A. Vakoliuk,

2

Oksana P. Tuchina,

2

Valery V. Zhukov

3

and Victor Benno Meyer-Rochow

1,4

1

School of Engineering and Science, International University of Bremen ( Jacobs University as of February 2007), Research II, Campus Ring 6, D-28759 Bremen, Germany;

2

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

3

Department of Agricultural and Soil Ecology, Faculty of Bioresources and Natural Usage, Kaliningrad State Technical University, Sovetsky avenue, 1, 236000 Kaliningrad, Russia;

4

Department of Biology, Faculty of Sciences, University of Oulu, SF-90014, PO Box 3000, Oulu, Finland

Keywords:

retina, neurosecretion, optics, phototaxis, visual orientation, snails

Accepted for publication:

22 July 2007

Introduction

Slugs form a polyphyletic group of terrestrial molluscs thatarose by way of convergent evolution from shell-bearingsnails (Barker 2002). The absence of a shell has reduced theslugs’ dependence on calcium, enhanced their ability toaccess food and shelter in confined spaces and contributed totheir ability to occupy a wider range of habitats than mostother snails can tolerate. Slugs are most active at night andon cloudy or foggy days. On sunny days they seek shelters tohide away from bright light and heat (South 1992). A variety

of morphological, physiological and behavioural adaptationsthat prevent excessive water loss have assisted slugs incontrolling hydration and, thus, must have been critical tothe success of the Stylommatophora (Solem 1974).

Gastropods, generally, are known to exhibit two easilydistinguishable kinds of visual behaviour. They can use lightto orientate and will move either toward or away from regionsof high light intensity, and/or they can respond to a suddendecrease in light intensity with a reaction known as theshadow-response, that is, a retraction of their tentacles andwithdrawal into their shell (in case of shell-bearing species).

The role of eyes in slugs

•

Zieger

et al.

Acta Zoologica

(Stockholm)

90

: 189–204 (April 2009)

© 2008 The Authors

190

Journal compilation © 2008 The Royal Swedish Academy of Sciences

The first kind of behaviour is clearly concerned with habitatselection and the second with defence against thosepredators that cast a shadow prior to an attack (Land 1968).There are good reasons to believe that in molluscs funda-mentally different types of receptors are involved in themediation of the two kinds of behavioural response ( vanDuivenboden 1982; Meyer-Rochow and Moore 1988).Because in phototaxis the central nervous system needs to becontinuously kept informed about the relative intensities oflight coming from different directions, it would be reasonableto assume that the eyes contain the receptors that provide thebackground for the phototactic response (Stoll 1973;van Duivenboden 1982). The shadow response is consideredto be linked to dermal photoreceptors and, thus, quiteindependent from the eye (Stoll and Bijlma 1973).

Assuming that generally the eyes are matched to the visualneeds of their bearers (Nilsson and Pelger 1994), it followsthat in order to understand an animal’s behaviour, it isimperative to know the functional limitations of the animal’seye.

It has frequently been asserted that terrestrial snails andslugs have only rudimentary eyes and could detect no morethan the general light level and perhaps broad areas oflight and dark (Chase and Croll 1981). Based mainly onstructural studies of the eyes of various

Helix

species (e.g.Eakin and Brandenburger 1975a) and on investigations ofthe optical properties of the eye of the slug

Agriolimax reticulates

,it was concluded that these snails are adapted to detect onlychanges in light intensity, especially when active at night(Newell and Newell 1968).

This paper deals with the anatomy, optical physiology andorientation behaviour of two terrestrial slugs with overlappingdistribution:

Arion rufus

and

Deroceras agreste.

It expandsour knowledge of photoreception in gastropods and permitscomparisons with earlier results on the visual behaviour,photoreceptor organization and optics of other species ofterrestrial gastropods.

Materials and methods

Animals

Numerous, fully grown specimens of

Arion rufus

(Linnaeus,1758) and

Deroceras agreste

(Linnaeus, 1758), measuring

c

. 70 and 35 mm in extended condition, were used. Theanimals came from private gardens in the Kaliningrad regionand most of the behavioural observations were made in thesummer during the months of June–August in Kalinigrad.The histological examinations were carried out in Bremen.The slugs were maintained at a constant temperature of20–21

°

C and under the natural 14–17 h photoperiod of thesummer season in terrariums filled with humid soil and amixture of leaves and wood bark for shelter. The soil waschanged once a week and the animals were fed lettuce andcarrot pieces.

Light and electron microscopy

Preparations of the eyes for histology took about 5 min andwere made under a VEB Carl Zeiss Jena dissectingmicroscope, using a combination of heat-protecting infraredand red filters to avoid possible tissue alterations by brightlight during the dissection.

For light and electron microscopy, severed tips of thetentacles together with the eyes were first fixed for 2 h in2.5% glutaraldehyde, buffered with 0.1

m

cacodylate to a pHof 7.8 and then postfixed for 1 h at room temperature in 1%OsO

4

, also buffered by 0.1

m

cacodylate to a pH of 7.8.Following dehydration, the eyes were embedded in Epon812. Thick (1.5 µm) sections were then stained with a 1%solution of toluidine blue in 0.8% sodium borate or with a1 : 1 solution of 1% azure II and 1% methylene blue in 1%sodium borate. The sections were photographed with anAxiomat Carl Zeiss or Reichert–Jung Polyvar lightmicroscope. Ultrathin sections were cut on Reichert ultracutE or Reichert OMU3 ultramicrotomes and stained with leadcitrate and uranyl acetate for a few minutes each. Thesections were examined under either Jeol JEM 100CX II orPhilips EM 300 transmission electron microscopes.

Behavioural experiments

The behavioural tests were carried out in cylindrical glasstanks with a diameter of 31 cm and a wall height of 30 cm inthe case of

A. rufus

or a diameter of 18 cm and wall height of20 cm in experiments with

D. agreste

(Fig. 1). An incandescentlight source (100 W), providing an illumination with whitelight of

c.

40 lux intensity at the bottom of the tank, wasequipped with an infrared absorbing filter to exclude heat.

Fig. 1—Semi-schematic representation of set-up for behavioural experiments. —A. View from the side and —B. From above. (1) Light source. (2) Infrared filter. (3) Matt (scattering) filter. (4) Polarization filters. (4) Cylindrical and transparent glass tank. (5) Nontransparent box. (6) Base of the set-up. Tank (view from above) with one-half darkened (grey) and direction of the slug’s head (arrow), shown as in the experiments for light orientation (phototaxis).

Acta Zoologica

(Stockholm)

90

: 189–204 (April 2009)

Zieger

et al.

•

The role of eyes in slugs

© 2008 The AuthorsJournal compilation © 2008 The Royal Swedish Academy of Sciences

191

The temperature inside the tank during experiments was

c.

20

°

C. The tank was placed in a black nontransparent boxwith a window (15

×

30 mm) on one side to allow continuousvisual control. The window could be closed with a blackcover. Light reached the tank through a circular opening of120 mm in diameter at the top of the black box. The openingwas equipped with matt (= light scattering) and polarization(as an option) filters. The entire set-up was placed on a purelywhite and transparent surface. Prior to the experiments theanimals were kept in total darkness for 1 h. Any trace ofmucus left behind by a slug was carefully cleaned away aftereach experiment.

Four different experiments were carried out with eitherspecies in order to study: (1) the distribution of directionalmovements of each animal in total darkness (control); (2)light orientational (i.e. phototactic) behaviour; (3) sensitivityto polarized light; and (4) thresholds of visually mediatedbehavioural responses.

Two groups of 10 individuals of both species of slug wereused in the controls for the observations on phototacticbehaviour and for tests to reveal sensitivity to polarized light.These experiments involved intact and operated individuals(distal tips of optic tentacles amputated 5 days prior to theexperiments), but to determine the threshold of visuallymediated behavioural response (direction of movement) onlyintact slugs were used.

In each experiment a single slug from the control or testgroup was gently placed in the centre of the tank bottom. Thedirections of the movements were monitored and plotted ona co-ordinate gride, placed under the bottom of the tank,until the animals reached the wall of the tank.

The wall of the tank was covered with black paper beforethe start of a phototaxis test. During the latter, one half of thetank was surrounded with white and the other with blackpaper. Slugs were individually placed in the centre of thepartition line. To determine the absence of any extraneousstimuli other than illumination that could interfere with aslug’s orientation response (e.g. airborne odours, gravity,temperature gradient), the positions of the light and darkcompartments were reversed twice. Moreover, each

A. rufus

individual was tested two times, while

D. agreste

individualswere even tested three times. Black stripes of photographicpaper of the same height, but different widths correspondingto 5

°

, 10

°

, 15

°

, 20

°

, 25

°

, 26

°

, 27

°

, 45

°

, 90

°

and 180

°

of arc,were sandwiched between the outer surface of the tank anda sheet of white paper in order to determine thresholdwidths. Slugs were individually placed in the centre at thebottom of the tank with head pointing toward the black teststripe. Their movements were then registered. To determinesensitivity to polarized light, the outer wall of the tank wascovered with a sheet of black paper before the polarizationfilter was inserted into the light path. Four series of experimentswith the polarizing filter being turned four times by 90

°

inclockwise direction (0

°

, 90

°

, 180

°

and 270

°

) were carried outwith both species.

Data obtained were statistically processed by the ‘SignTest’ method for paired samples (Siegel and Castellan 1988),‘Rao’s Spacing Test’ as well as ‘Rayleigh Test’ (Batschelet1981). The mean vector of movement was determined as asegment of the straight line between the centre of the tankbottom and the point of the tank wall that the test animalreached. Values of ‘

U

’, describing the deviation of animalsfrom the predicted direction ‘

U

crit

’ were calculated accordingto Bobkova

et al

. (2004b) and to determine statisticalsignificance the ‘Table of Significance’ (

P =

0.05) in Batschelet(1972) was used. In total 490 and 460 experiments werecarried out with

A. rufus

with

D. agreste

, respectively.

Optical measurements

Using a combination of infrared (heat-absorbing) and redfilters (to execute the dissection under), lenses of both specieswere isolated during daytime under a VEB Carl Zeiss Jenadissecting microscope. Focal length measurements werecarried out on freshly isolated lenses immersed in a drop ofphysiological saline and exposed to air. Basically, the methodsdescribed by Nilsson

et al

. (1988), and by Seyer (1992)to determine focal lengths were followed. Images, given bythe isolated lenses, were observed with an Axiomat Carl Zeisslight microscope at

×

30 and numerical aperture 0.90. Toreduce manipulation times with the lenses (which were losingtheir transparency soon after dissection) standard bright-field objectives (but not of the water-immersion type) wereused for all measurements. The procedure for the determi-nation of the focal length was the following: diameters weremeasured of lenses immersed in physiological solution (

D

L

,water) (1); and exposed to air (

D

L

, air) (2). Then (3) theimage size given by the lens in physiological solution(

L

i

, water) was measured; and (4) the value (

κ

) of themagnification that arose due to the curvature of the drop ofthe physiological solution was given by:

κ

=

D

L

, water/

D

L

,air. (5) The radius (

R

L

) of the lens was

R

L

=

D

L

, air/2, and(6) the corrected image size in water (

L

i

) was:

L

i

= L

i

, inwater

/

κ

. Finally, (7) the principal focal length of the lens (

F

L

)was determined as:

F

L

=

L

i

/d

*(

l

+

R

L

), where

d

is the sizeof the object equal to 162 µm and

l

is the thickness of theobjective glass equal to 1 mm.

Angular resolution of neighbouring photoreceptors in objectspace

was calculated according to the equation:

Δϕ = 2 × arctg(d/2f )

The F-number was defined as f/A and relative aperture as A/f.The diameter of the Airy disc (µm) was calculated for the

wavelength of λ = 500 nm (a value close to the known photo-pigment absorption peaks in Lymnaea stagnalis and Planorbariuscorneus [490 nm and 495–500 nm, respectively: Zhukovand Gribakin 1990]), according to D = 2.44 f × λ/A.

Matthiessen’s ratio was determined as f/r, where r is theradius of the lens.

https://www.researchgate.net/publication/229981115_Restoration_of_morphological_and_functional_integrity_in_the_regenerating_eye_of_the_giant_African_land_snail_Achatina_fulica?el=1_x_8&enrichId=rgreq-c655f434-cca1-46c4-8ea8-371d600abc92&enrichSource=Y292ZXJQYWdlOzIyNzk3Mjc4MDtBUzoxMjEwMzk0OTMyNzU2NDhAMTQwNTg2OTQ3MjMxOA==

https://www.researchgate.net/publication/239809371_Resolution_and_Sensitivity_in_the_Eye_of_the_Winkle_Littorina_Littorea?el=1_x_8&enrichId=rgreq-c655f434-cca1-46c4-8ea8-371d600abc92&enrichSource=Y292ZXJQYWdlOzIyNzk3Mjc4MDtBUzoxMjEwMzk0OTMyNzU2NDhAMTQwNTg2OTQ3MjMxOA==

https://www.researchgate.net/publication/226181770_Optics_of_the_butterfly_eye?el=1_x_8&enrichId=rgreq-c655f434-cca1-46c4-8ea8-371d600abc92&enrichSource=Y292ZXJQYWdlOzIyNzk3Mjc4MDtBUzoxMjEwMzk0OTMyNzU2NDhAMTQwNTg2OTQ3MjMxOA==

The role of eyes in slugs • Zieger et al. Acta Zoologica (Stockholm) 90: 189–204 (April 2009)

© 2008 The Authors192 Journal compilation © 2008 The Royal Swedish Academy of Sciences

All the calculations described above were carried out usingMatLab computer software.

Results

General anatomy

Both A. rufus and D. agreste possess a pair of cephaliccamera-type eyes. The eyes are located at the ends of theoptic tentacles underneath a transparent area of integument(Fig. 2A,B). The optic nerve runs down the tentacleand ends in the cerebral ganglion. A retractor muscle isattached with its distal end to the tentacle wall. This musclesplits up and one branch leads into the eye capsule (butnot the area of the cornea), where it forms a circumferentiallayer.

The ovoid eyes of A. rufus, in specimens of c. 70 mmextended total body length, measure c. 240 and 290 µmacross the short and the long axis. The eyeball of D. agreste isasymmetrical in shape, on account of the accessory retina,which is situated on the ventro-lateral side of the main eyeand is contiguous with the eye’s cornea (Fig. 2C,D). In slugswith an extended length of c. 40 mm, the diameter of themain retina plus cornea is c. 220 µm, while the accessorycomponent measures c. 70 and 160 µm. The retina of the eyein both species is noninverted and monolayered with a widthalong the optical axis of c. 60 µm in A. rufus and c. 70 µm inD. agreste (the main retina). The retinae contain at least twocell types: photoreceptor cells (1); and pigmented (supportive)cells (2). Four distinct layers in the retina of the main eye canbe distinguished: microvillar, pigmented, somatic andplexiform. Functionally, the microvillar layer is the layer with

Fig. 2—Light micrographs of nearly longitudinal sections through the eye of A. rufus (A) and D. agreste (B–E) showing gross anatomical organization. Additional lens (AL) and retina (AR), cornea (Co), integument (Tnt), lens (L), light sensitive layer of microvilli (Mv), retina (R), tentacular ganglion (TG) and photoreceptor cells of additional retina (asterisks) are indicated.

Acta Zoologica (Stockholm) 90: 189–204 (April 2009) Zieger et al. • The role of eyes in slugs

© 2008 The AuthorsJournal compilation © 2008 The Royal Swedish Academy of Sciences 193

the light-sensitive membranes of the photoreceptor cells. Thepigmented layer is formed by an accumulation of screeningpigment granules in the supportive (pigmented) cells. Thesomatic layer consists of the nuclei of the photoreceptivecells, supportive (pigmented) cells and neurosecretoryneurones (the latter in A. rufus only). The plexiform layerincorporates both axons of photoreceptors of the main andadditional retinae (D. agreste) and axons of the photoreceptiveand neurosecretory cells (A. rufus).

Dioptric apparatus

Both species have a convex and monolayered cornea, com-posed of elongated, unpigmented and in vivo transparentcells (Fig. 2A,B). Corneal cells are characterized by electron-translucent cytoplasm and nuclei located in the basal portionof the cells (Fig. 3A). The cells are attached by multiple

hemi-desmosomes to the basal lamina and eye capsule(Fig. 3B). Cell organelles are abundant along the plasmamembrane, but rare in the remaining portion of the cells.However, the perinuclear area is rich in mitochondria, freeribosomes, rough endoplasmic reticulum and glycogengranules. The corneal cells are attached to each other byadhesion belts at their apical portions. The apicies bear shortmicrovilli (folds of membrane material) and are turnedtoward the vitreous body of the eyes. Large membrane-bound secretory bodies, filled with electron-dense granularmaterial similar to that of the vitreous body, are present(Fig. 3C). The thickness of the cornea is c. 40 µm along theoptic axis of the eye in D. agreste and c. 30 µm in A. rufus.

The eye of A. rufus contains an ovoid, hard lens, measuringc. 150 and 200 µm along the two axes. The long axis of thelens coincides with the optic axis of the eye (Fig. 2A). Theeye of D. agreste has a hard, spherical lens with a diameter of

Fig. 3—A. Light micrograph of longitudinal section through cornea in A. rufus. Cornea (Co), lens (L) and nuclei of corneal cells (asterisks) are discernible. —B, C. Electron micrographs of longitudinal section through cornea in D. agreste. Corneal cells (Co), eye capsule (EC), nucleus of corneal cell (Nco), part of vitreous body (Vb) and secretory vesicles (arrows) are indicated.



c. 110 µm (Fig. 2B). In vivo observations on isolated lenses,immersed in physiological solution and placed under amicroscope in the transmission mode within c. 10 s, revealedtotally transparent lenses in both A. rufus and in D. agrestethat slowly turn opaque. However, this process can be sloweddown if the lens is minimally exposed to air. The additionalretina is covered by a hard, bi-convex and in vivo transparentlens that can easily be isolated from the retinal cavity. Thislens measures c. 35 and 50 µm along its two axes (Fig. 2C,D).

The vitreous body of A. rufus and D. agreste occupies a verynarrow space between retina and the lens. It is of fine-granular, electron-dense material and in vivo appears,homogenous and transparent. The extremely thin vitreousbody in D. agreste that separates the additional retina and thelens, fuses with the vitreous body of the main eye through anopening in the main retinal cup (Fig. 4A–E).

Fine structure

Variety of photoreceptors. In A. rufus, photoreceptors (flareddistally) terminate in a broad plate that bears long microvilliin the direction of the lens (Fig. 5A). In D. agreste we couldclearly distinguish type I photoreceptors with columnar-likeapices, bearing long, well-organized light-sensitive microvilliand type II photoreceptors with short, whorled microvilli(Figs 2E, 6A–D). Photoreceptor cells in both species lackpigment granules. The flared apicies of photoreceptor cells inA. rufus and both types of photoreceptors in D. agreste arerich in mitochondria. The columnar-like apices in D. agrestehave a conspicuous cytoskeletal system of numerous,predominantly longitudinally orientated microtubules.Two kinds of nuclei can be found in the somatic layer ofA. rufus: round interphase nuclei that are noted for their

Fig. 4—Light micrographs of nearly longitudinal serial sections (A–E), showing fusion of additional vitreous body with vitreous body of the main eye in D. agreste. B. Enlargement of cornea-photoreceptor border, shown in (A). Additional retinae (AR), cornea (Co), integument (Int), main lens (L), photoreceptor (Ph), main retina (R) and vitreous body (asterisks) of the additional retina are discernible.



dominating chromatin, which stains diffusely (1); and ovalnuclei that contain condensed chromatin (2) (Fig. 5B).However, there is no clear structural difference in the apicalportion of the cells with these two kinds of nuclei. Typical forgastropods are the accumulations of uniformly sized photicvesicles of c. 0.06 µm in diameter in the photoreceptor cellsof both A. rufus and D. agreste. There is a marked differencein the distribution of the photic vesicles: they are aggregatedbasally near the nuclei and in the initial segment of photo-receptor axons in A. rufus, but massed distally in D. agreste(Figs 5B, 6D). No mass aggregations of photic vesicles(in spite of their presence) were found in the cytoplasm oftype II photoreceptor cells of D. agreste and in the cytoplasmof the cells with dense chromatin in A. rufus. Thus, due to thelack of mass aggregation of the clear vesicles, the cytoplasmof the latter appears electron-dense compared with that ofthe photoreceptor cells with spherical nuclei (Fig. 5B).Moreover, the clear vesicles in A. rufus are aggregated mainlyin the perinuclear area of the cells with the oval nuclei and thecondensed chromatin. The disorderly aggregations and thepresence of residual bodies as well as liposome-like organellesamongst the vesicles, are signs of degradation.

Features of the additional retina. There is no anatomical separa-tion between the main and the additional retina. The cellularcomposition of the additional retina in D. agreste is similar tothat of the main eye, but it does not contain neurosecretorycells. Moreover there are no screening pigment granules inthe supportive cells of the additional retina. The nuclei of thepigment-free supportive cells contain tightly packed hete-rochromatin and rings of rough endoplasmic reticulum aroundthe nuclei (Fig. 7A). The photoreceptor cells are large andtheir dome-shaped apices bear well-organized (regular) longmicrovilli (Figs 2D,E, 4B, 7A,B). Accumulations of photicvesicles are also present, but the latter are not as tightly packedas those of the photoreceptors of the main retina (Fig. 7A).The microvilli were observed to be aligned in perpendicularorientation (Fig. 7B), but we could not provide morphologicalevidence that the perpendicularly orientated microvilli belongto neighbouring (different) photoreceptor cells.

Pigmented supportive cells. Pigmented cells envelope the photo-receptors, but they never penetrate between the columnarapices in D. agreste (Fig. 6A–D). In A. rufus the pigmentedcells have deep, multiple projections into the photoreceptor

Fig. 5—A–C. Electron micrographs of longitudinal sections through (A) apical portion, (B) somatic layer and (C) perinuclear region of retina in A. rufus. Microvillar layer (Mv), round (Nph) and oval (Nrc) nuclei of photoreceptor cells (Ph), aggregation of photic vesicles (Pv), rough endoplasmic reticulum (R-EPR), neurosecretory cell (Sc) and pigmented supportive cells (Sp) are labelled.



cytoplasm (Fig. 5A). The nuclei of the supportive (pigmented)cells are located in the basal region of the somatic andplexiform layers. They are much smaller than the nuclei ofthe photoreceptors and are identifiable by their condensedheterochromatin (Fig. 8A,D). Multiple rings of roughendoplasmic reticulum together with large numbers ofmitochondria are present in the perinuclear area, suggestingthat the supportive, pigmented cells are synthetically active(Fig. 5C). The supportive cells (with heavily pigmentedextensions only distally) form multiple gap-junctionsbetween each other (Fig. 8B). It appears as if this type of cellhas a kind of syncytium-like intraretinal net.

Neurosecretory neurones. Neurosecretory cells were found toform a single cluster in a delimited region of the eye of

A. rufus. A group of three to five such cells is located justunder the cornea on one side of the eye (asymmetrically)close to the basal lamina. The location of the cells can clearlybe seen in representative tangential sections through the eye(Fig. 8C). The cluster looks like a chain of cells distributedamongst the other retinal cells. These oval cells have relativelylarge diameters of c. 13 and 19 µm and their nuclei onaccount of their large size (c. 10 and 15 µm) and conspicuousnucleolus are easily recognizable (Fig. 8D). The most visiblemarkers of the cell’s ultrastructure are the prominent roughendoplasmic reticulum, a labyrinth-like net of cytoskeletal fibresin the perinuclear area, very large pigment-like, mostly round,but also irregular, membrane-bound bodies and numerousdense core vesicles of c. 0.09 µm in diameter (Figs 8E, 9A).Clear (c. 0.06 µm), cored (c. 0.08 µm) and coated vesicles

Fig. 6—Light (A, B) and electron (C, D) micrographs, showing photoreceptor cells in the main retina of the eye in D. agreste. Additional retina (AR), cornea (Co), lens (L), mitochondria (M), light sensitive layer of microvilli (Mv), optic nerve (ON), photoreceptor cell of type I (PhI) and of type II (Phil), photic vesicles (Pv), retina (R), pigmented supportive cells (Sp), vitreous body (Vb) and apecies of photoreceptor cells (asterisks) are labelled.



are also present (Fig. 9A). Aggregations of dense-core vesi-cles are present close to the Golgi apparatus (Fig. 9B). Nosynaptic contacts were seen on the surface of the cell soma.

The axons of the neurosecretory cells can be identified inthe plexiform layer of the retina by their contents of densecore vesicles (Fig. 9C).

Axons and neuropile. In both species investigated, the plexiformlayer and neuropile of the eyes were composed of fibres of atleast two easily distinguishable morphological populations.The first population comprises axons of photoreceptors thatcontain mitochondria, granular material, clear vesicles ofc. 0.06 µm in diameter (measured in A. rufus) and numerous

longitudinally orientated microtubules. The second popula-tion of axons contains dense core vesicles of c. 0.09 µm indiameter (in A. rufus) and clear vesicles. Mitochondria, fila-ment bundles and microtubules are present as well.

Behavioural experiments

Control. In the controls, the distribution of both intact andblinded slugs in the darkened tank did not differ significantlyfrom random, that is, U < Ucrit (P = 0.05). This means thatthe slugs do not show preferential directions in the arena andthat, therefore, only vision could have been responsible forinducing the directional movement.

Fig. 7—A, B. Electron micrographs of additional retina in the eye periphery in (A) and receptors in (B) of D. agreste. The two insets show the area of photic vesicle accumulation (inset in A) and transverse section through light sensitive organelles, known as microvilli (inset in B). Cornea (Co), eye capsule (EC), microvilli (Mv), photoreceptor (Ph), plexiform layer (Px) of additional retina, supportive (pigment free) cells (Sp), neuropile of tentacular ganglion (TG) and vitreous body (Vb) are indicated.



Phototactic behaviour. The experiment showed that A. rufus(97.5%) and D. agreste (88%) significantly more frequently(P < 0.05) chose the dark compartment over the white one.Moreover, the distribution of the individual vectors of theslug’s movement revealed the same even when the positionsof the compartments were reversed (Fig. 10A). The‘Sigh Test’ and a P < 0.05 confirm that in the operated(eyeless) slugs there were no significant differences indistribution between light and dark tank compartments(Fig. 10B).

Threshold of visually mediated behavioural response. The resultsof this series of experiments indicated that the respectivethresholds of the behavioural response in A. rufus andD. agreste occurred to black stripes subtending 26° (level ofsignificance P < 0.05) and 90° of arc (level of significanceP < 0.05). A mere change from a black stripe width of 26° toone of 25° completely abolished the behavioural reaction inA. rufus (Fig. 11. Note that for both species the same set-upwas used, but that D. agreste could not recognize widths equalto or larger than 45°).

Sensitivity to polarized light. In A. rufus the calculated valuesof U, describing the deviation of animals from the predicted

direction, were much lower than Ucrit. However, we obtainedU-values greater than Ucrit in D. agreste (Table 1), which couldhave meant that D. agreste exhibited a behavioural response topolarized light. This seemed intriguing and we therefore con-ducted an additional series of experiments with intact slugs,but no polarization filter, and operated slugs, with polarizationfilter in place. Both controls (U = 135) and operated slugs(U0° = 100; U90° = 110), with a high level of statistical sig-nificance, showed a random distribution in the experimentaltank. The resultant data (Table 2), statistically processed byRayleigh Test caused us to reject the hypothesis that theD. agreste might be sensitive to the e-vector and had us acceptthe null hypothesis (i.e. absence of sensitivity to polarized light).

Optical measurements and calculations

A morphometric analysis of the components of the eyes ofthe slugs, summarized in Table 3, formed the basis for anevaluation of the eyes’ optical system.

Lens characteristics determined through observations ofimages formed by the isolated lenses in both physiologicalsolution and in air are given in Table 4. Parameters calculatedfor the optical systems in the eyes of A. rufus and D. agresteare shown in Table 5.

Fig. 8—Electron micrographs, showing in (A) nuclei of pigmented supportive cells (Nsp) and sites in (B) of gap junctions (arrows) between pigmented supportive cells (Sp) in A. rufus. C, D. Light micrographs of tangential section (C) through cluster of neurosecretory cells in the eye of A. rufus (framed) and (D) enlargement of the cluster. E. Electron micrograph of perinuclear region of neurosecretory cell rich in intracellular ducts and cytoskeletal elements (arrowheads). Eye capsule (EC), integument (Tnt), lens (L), nucleus of neurosecretory cells (Nsc), optic nerve (ON), photoreceptor (Ph) cells, retina (R), neurosecretory (Sc), tentacular ganglion (TG), vitreous body (Vb) and pigmented supportive (asterisks) cells are indicated.



Fig. 9—Electron micrographs of perinuclear region of neurosecretory cell (A, B) and fragment of plexiform layer (C) in A. rufus. The inset shows morphological diversity of membrane-bound bodies (Mb). Axons of neurosecretory (Ax) and photoreceptor (pAx) cells, cored (Cv), dense-core (Dv), coated (Ctv) and clear (V) vesicles, Golgi apparatus (GA), rough endoplasmic reticulum (R-EPR) are visible.

Table 1 Comparisons between calculated values of U and Ucrit in experiments with polarized light using the table of significance according to Batschelet (1981)

Species

Experimental data Tabled value

0° 90° 180° 270° P = 0.01 P = 0.05

Arion rufus 155 (10) 137 (10) 166 (10) 162 (10) 195.1 (9) 173.5 (9)Deroceras (series I) 145 (9) 46 (10) 198 (9) 192 (9) 192.2 (10) 172.1 (10)agreste (series II) 122 (10) 201 (10) 154 (10) 172 (10)

n-values are given in parentheses.



Discussion

Structural background

The retinae of both A. rufus and D. agreste contain at leasttwo cell types: rhabdomeric photoreceptor cells and pigmentedsupportive cells. Type I cells are characterized by microvilliand aggregations of ‘photic vesicles’ (Eakin 1990) and typeII photoreceptors, lacking the densely packed vesiclesbut bearing short microvilli, are structurally similar to the

photoreceptors described for many other snails and slugs(Eakin and Brandenburger 1975a; Kataoka 1975; Eakinet al. 1980; Jacklet and Colquhoun 1983; Herman andStrumwasser 1984; Katagiri et al. 1995; Bobkova 1998;Meyer-Rochow and Bobkova 2001; Bobkova et al. 2004a).Predictions on the sensitivity of these two rhabdomericreceptor types have largely been based on their anatomicaldifferences, but in the terrestrial slug Limax flavus, Suzukiet al. (1979) could show by electrophysiological means thatmost likely type I receptors operated in dim and type IIreceptors in bright light. Types I and II photoreceptors inA. rufus and D. agreste could function in similar ways.

Fig. 10—Phototaxis in the two species of slugs A. rufus and D. agreste. Numbers (A) of intact and (B) operated (eyeless) individuals choosing dark half (grey columns) or illuminated compartment (white columns) of the experimental tank. Dark and illuminated halves were exchanged for different series of experiments.

Fig. 11—Threshold of behavioural responses to visual stimuli (black stripe of different width) in A. rufus (A) and D. agreste (B). Level of significance (*P < 0.05; **P < 0.01).

Table 2 Means of movement vectors (r) and corresponding levels of significance (P) in the tests to investigate possible polarization sensitivity in Deroceras agreste

Series of the tests 0° 90° 180° 270°

Intact slugs (series I) r = 0.134 r = 0.442 r = 0.506 r = 0.256P > 0.846 P = 0.145 P > 0.085 P = 0.558

Intact slugs (series II) r = 0.482 r = 0.302 r = 0.198 r = 0.293P = 0.098 P = 0.417 P = 0.709 P > 0.417

Intact slugs, without polarizing filter

r = 0.415P > 0.12

Operated (eyeless) slugs

r = 0.184 r = 0.006 r = 0.161 r = 0.106P = 0.733 P > 0.900 P = 0.783 P > 0.897

Table 3 Eye parameters of slugs based on light and electron microscopy (in µm)

Parameter Arion rufusDeroceras agreste

Size of eyeball 240 × 290 220 × 220Size of lens 150 × 200 110 × 110Diameter of aperture 100 ± 12 (10) 80 ± 5 (10)Centre-to-centre separation of receptors 5.6 ± 0.3 (50) 17.0 ± 2.9 (50)*Receptor length 13.8 ± 0.6 (50) 27.8 ± 0.7 (50)

Values are means ± SD; n-values are given in parentheses. *Values based on thickness of microvillar layer.



However, we cannot exclude the possibility that the cellsbelong to the same functional type of photoreceptor andrepresent stages in the process of receptor cell renewal (deathand replacement) in the mature retina. Gap junctions, seenbetween pigmented supportive cells in A. rufus, have alsobeen described from the eye of Bulla sp., where they occurbetween photoreceptor cells, glial cells and neurones (Jackletand Colquhoun 1983) and Aplysia, where they occupy placesbetween neighbouring photoreceptors (Strumwasser et al.1979) and neurones (Luborsky-Moor and Jacklet 1977;Strumwasser et al. 1979).

It was shown earlier that the terrestrial snail Achatina fulicahas an additional eye, equipped with its own lens, structurallyseparate from the main eye (Tamamaki and Kawai 1983).The slugs L. maximus (Henchman 1897), A. reticulatus(Newell and Newell 1968) and L. flavus (Kataoka 1977;Tamamaki 1989) also have an additional retina but lack thelens. Deroceras agreste (this paper) possesses both, anadditional retina and an extra lens. However, its vitreousbody is continuous with that of the main eye. Thus, wesuggest the term ‘additional’ rather than ‘separate’ retina forD. agreste to underline this anatomical linkage between thetwo retinae. As for the functions of the additional retina, twotheories have been proposed: the perception of changes inthe intensity of the ambient light (Hesse 1902) and theperception of infrared radiation (Newell and Newell 1968).However, the latter could not be experimentally substantiated(Kataoka 1975). In fact it has not been possible to demonstratethat the additional retina is a visual organ at all. Lack ofscreening pigment permits light to reach the presumedphotoreceptors from any direction, making image formationimpossible. The hypothesis that in D. agreste the additionalstructure could play a role as a sensor of polarized light hadto be rejected on the basis of our behavioural tests. Thus, the

role of the additional retina remains an open question worthyof further investigation.

The retinal cells that we termed ‘neurosecretory’ inA. rufus, are likely to belong to cells that are known as ‘largeganglionic cells of the nervous system’ in L. maximus(Henchman 1897), secondary neurones (or basal retinalneurones) in Bulla (Jacklet and Colquhoun 1983) andAplysia (Jacklet et al. 1982), ganglion cells in Helix aspersa(Brandenburger 1975), Littorina littorea (Newell 1965),Lymnaea stagnalis (Stoll 1973; Bobkova 1998) and Ariolimaxcalifornicus (Eakin and Brandenburger 1975b) and ‘large-sized type II cells’ in L. flavus (Kataoka 1977). The cell typeslisted above are similar in appearance, contain a very largenucleus and are endowed with prominent rough endoplasmicreticulum, numerous granulated osmiophilic dense-core andclear vesicles, large liposomes or pigment granule like bodies,similar to the lipochondria of Aplysia ganglion cells describedby Baur et al. (1977). We have accepted the term ‘neurosecretoryneurones’ for A. rufus, because we are convinced that thecells control the secretory pathway by which proteins fromthe Golgi apparatus reach the plasma membrane and theaxon. The cytoskeletal web of microfilaments described inA. rufus probably provides cytoplasmic streaming and actsas a ‘passage’ along which a wide range of vesicles can betransported between organelles and toward the axon. Somevesicles carry many proteins or peptides and appear electron-dense, hence the term ‘dense-core vesicle’. But the majorityof the neuronal vesicles belong to the synaptic vesicles, whichcontain neurotransmitter substance for the presynapticterminals. We did not observe any chemical synaptic contactsin the retina and the neuropil, but axons in the plexiformlayer and in the neuropil contained both dense-core and clearvesicles, a feature, according to Geffen and Jarrott (1977),that usually characterizes catecholaminergic neurones.

Table 4 Lens characteristics determined through observations of images formed by the isolated lenses in water and in air

SpeciesLens diameter in water (µm)

Lens diameter in air (µm) Coefficient

Lens radius (µm)

Image size in water (µm)

Image size corrected (µm)

Arion rufus 211.5 ± 32.8 (12)* 192.3 ± 29.3 1.12 ± 0.05 101.2 ± 12.6 136.3 ± 23.2 123.8 ± 22.4Deroceras agreste 111.6 ± 8.2 (9) 100.5 ± 7.2 1.11 ± 0.05 55.2 ± 4.1 35.4 ± 3.1 31.8 ± 2.6

*Size of long axis only is given.Values are means ± SD; n-values are given in parentheses.

Table 5 Mathematically determined optical parameters

SpeciesPrincipal focal length of the lens (µm)

Relative aperture

Matthiessen’s ratio

Angular receptor spacing (°) F-number

Diameter of Airy-disc (µm)

Arion rufus 848.9 ± 97.3 (12) 0.1 ± 0.001 – 0.4 ± 0.06 – –Deroceras agreste 207.0 ± 12.0 (9) 0.4 ± 0.2 3.7 ± 0.4RL 4.7 ± 1.1 2.5 ± 0.4 3.0 ± 0.2

Values are means ± SD; n-values are given in parentheses.



Moreover, it was shown for Aplysia that the ratio of dense-core/clear vesicles changes with the release of hormones(Haskins et al. 1981) like, for instance, serotonin (Shkolnikand Schwartz 1980).

However, we cannot completely rule out the possibilitythat the neurosecretory cells in the eye of A. rufus are a kindof neuroendocrine cell that releases its vesicular contentdirectly into the body fluid to reach more distant effectororgans. As to the function of the retinal neurosecretory cells,they can be involved in various aspects of reproduction(gonadal development, sexual maturation, egg laying) ormetabolic regulation. It was earlier shown that the retinalneurones in the marine gastropods Bulla gouldiana (Michelet al. 1993) and Aplysia californica (Barnes and Jacklet 1997)are capable of generating a circadian periodicity. Actually,the cells are competent circadian retinal pacemakers and likeall circadian pacemakers, the oscillators entrain to cycles oflight and dark, especially the 24-h light/dark cycle of theenvironment (Whitmore and Block 1996). The labelling ofcytoplasmic structures of the basal retinal neurones withcone opsin antibodies in B. gouldiana reaffirms that thecircadian pacemaker neurones contain an opsin-basedphotopigment. Nevertheless, the intracellular site of photoncapture remains unknown (Geusz et al. 1997). Yet, inbehavioural experiments with A. rufus it was shown thatthese slugs have a circadian rhythm. The rhythm can beshifted after prolonged (2 weeks) exposures to light or darkness(Lewis 1969). That light is highly effective in controllingcircadian rhythmicity was shown for other gastropods(Newell and Newel 1968; Hodasi 1982; Ford and Cook1988, 1994; Flari and Lasaridou-Dimitriadou 1995;Hommay et al. 1998), but that the retinal neurones expresscircadian rhythmicity has yet to be proven.

Functional consequences

Arion rufus and D. agreste exhibit negative phototacticresponses. The responses are clearly visually mediated, asoperated (eyeless) slugs did not show any sign of phototacticresponse, while intact slugs clearly chose to move towards thedark compartment of the experimental arena. The resultsare, thus, fully compatible with the dim or dark environmentsthat slugs are most active in (Newell and Newell 1968; Rolloand Wellington 1981; Hommay et al. 1998; Cook 2001).

Although smaller and less active than A. rufus, D. agreste iseven more nocturnal in habit than A. rufus (Zieger, unpublishedobservations) and encounters lower light intensities duringits period of activity. Consequently it ought to require greatervisual sensitivity than A. rufus and for this reason has a widerrelative aperture, a more voluminous receptor (microvillar)layer, and larger centre-to-centre receptor separation thanA. rufus. A larger centre-to-centre receptor separation, ofcourse, leads to a decrease in acuity. Our theoreticallycalculated performance of the slugs’ eyes shows that acentrally placed receptor subtends about 0.4° in A. rufus and

4.7° in D. agreste, so that acuity on purely anatomical groundsshould be considered quite well in both species. However,the results of the calculations are not at all supported by ourbehavioural tests, which show that A. rufus and D. agrestehave resolution limits of c. 26° and 90°, respectively. Thereasons for such disparity between anatomically determinedestimates and behaviourally determined resolution limits areprobably related to features of either the visual processing orthe optical system of the eyes. We therefore direct our attentionto the optics of the eye now.

Obviously, we have to be mindful of the fact that the cornea,if in air, shortens the focal length of the dioptric system.However, it has earlier been shown that in the terrestrialsnails Cepaea nemoralis and Trichia hispida not the corneae,but only the lenses play a significant role as refractingsurfaces (Gál et al. 2004). We may therefore assume that the lensis the main element of the optical system also in A. rufus as wellas D. agreste. Although we did not evaluate the optical systemof the additional retina in D. agreste, for reasons explainedabove, we are convinced that it cannot form an image.

Assuming that a radial gradient of refractive index does notexist (anatomically the lenses appear homogeneous), oneoptical function of the spherical lens of D. agreste could be toprovide a certain amount of correction for the sphericalaberration inherent in reflections from spherical surfaces.The diameter of the Airy-disc in D. agreste is considerablysmaller than the smallest value of centre-to-centre receptorseparation. This means that the quality of the image wouldnot greatly suffer from diffraction.

Although the eyes in both slugs possess a lens, the focalpoint in both species lies far behind the light-sensitive retinallayer of microvilli. Any image that might be produced wouldtherefore be of no use. In fact the image would lie about850 µm in A. rufus and 200 µm in D. agreste behind the lenscentre, while the retinae would be present 110 µm in A. rufusand 60 µm in D. agreste behind the lens centre. As there isactually no space between lens and retina in both species, thecephalic eyes of these slugs would lack the optical requirementsfor image formation. In other words, a sharp image in theplane of the retina is not possible and light from distantsources cannot be brought to a focus on the retina by thedioptric system of the eye. The retinae can, however, monitorchanges of ambient light intensity in spite of their positionsimmediately behind the spherical lens.

The results of our research lead us to conclude that ana-tomically the eyes of the two species of slugs investigatedshould be able to form images, but images that would lie inthe wrong plane, well behind any retinal structures. The eyesof the slugs are designed to transmit spatially averaged intensitypatterns of light and dark to the central nervous systemfor orientational purposes and only one aspect of the visualenvironment, the overall pattern of light and dark, seems tobe important for the slugs. A sharp image is unnecessary, andmay even be a hindrance, when the aim is to provide nothingmore than orientational clues.



Acknowledgements

M. Z. wishes to thank the Centre of International Mobility(CIMO) and the University of Oulu (Finland) for the supportof this research through separate grants. V.B.M.-R. acknowl-edges the support received from the International UniversityBremen towards the completion of this research.

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Eyes and vision in Arion rufus and Deroceras agreste (Mollusca; Gastropoda; Pulmonata): What role does photoreception play in the orientation of these terrestrial slugs?