UNIVERSITY OF CINCINNATI

Date:______

I, ______, hereby submit this work as part of the requirements for the degree of: in:

It is entitled:

This work and its defense approved by:

Chair: ______

Structural and Functional Organization of The of The Larvae of The Sunburst Diving , marmoratus

A Thesis submitted to the

Division of Research and Advanced Studies of the University of Cincinnati

in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE

in the Department of Biological Sciences

of the College of Arts and Sciences

2005

by

Karunyakanth Mandapaka

M.Sc; Pondicherry University, 2002

Committee Chair: Dr. Elke K. Buschbeck

Abstract:

The visual system of the aquatic and predatory diving beetle larvae, consists of 12 stemmata and two patches. Chapter one presents evidence that: (1) these stemmata are unique with features like multiple in each stemma and some ‘perpendicular to the axis of light’ photoreceptors and (2) each eye monitors a unique visual field. Chapter two presents evidence that: (1) the visual fields of the large tubular one and two are extremely narrow, while those of the other eyes are wide;

(2) optic nerve from each stemma projects majorly to a unique neuropil; (3) each optic lobe has six distinct neuropils and (4) giant neurons are richer in the neuropils three though six than in one and two. From these studies I suggest that eyes one and two are the principal hunting eyes and that the other eyes are important in detecting the prey.

2 Acknowledgements:

I am thankful to Dr. Elke Buschbeck for being a wonderful advisor. Her guidance, kind concern, and ever inspiring enthusiasm have been of enormous help, at every step. Her intention to help has been a resource that I could always be assured of. My research would not have been possible without her support.

I am thankful to my Research Advisory Committee members, Dr. Edwin Griff, Dr. John Layne and Dr. Dennis Grogan for their guidance, kind concern, suggestions, discussions, and encouragement. Their help has been valuable and essential for the successful completion of my research.

I am thankful to the Cincinnati Zoo Insectarium for kindly providing the adult Sunburst Diving . I am thankful to Randy Morgan, the Conservation Program Manager, Cincinnati Zoo, for his help in rearing the adults and larvae in the lab conditions and much advice. I am also thankful to Dr. Dan-Eric Nilsson of the Lund University for his helpful advice.

I am thankful to Heather Hoy, Srdjan Maksimovic, and Kelly Hagen of the Buschbeck lab for their technical, moral and intellectual support; and for their amicability.

I am thankful to all the faculty members of the department for excellently teaching the courses and for guiding me to do well with the TA responsibilities. I am thankful to Dr. Carl Huether and Dr. Dennis Grogan for their moral support and encouragement on times that I most needed.

I am thankful to all the fellow graduate students of the department for being very friendly and interactive; for sharing ideas and study materials and for all the fun we had together.

I am thankful to Dr. Guy Cameron, the head of the department and Dr. Brian Kinkle, and Dr. Charlotte Paquin, directors of the graduate affairs for their support and kind concern. I am thankful to all the staff members in the Biological Sciences department for all their help and support with my academic and TA responsibilities.

I am thankful to Dr. Elke Buschbeck, the department of Biological Sciences and the University of Cincinnati for the financial support and for providing me with an opportunity to learn. I am also thankful to NSF for the grant awarded to Dr. Elke Buschbeck, IBN- 0423963, from which I was funded.

3

4 Contents:

List of Tables and Figures …………………………………………………………6

General Introduction………………………………………………………………..8

Chapter One: “Twenty eight retinas but only twelve eyes; an anatomical analysis of the larval visual system of Thermonectus marmoratus ()”

Abstract…………………………………………………………………………………13

Introduction…………………………………………………………………………….14

Materials and Methods…………………………………………………………………17

Results……………………………………………………………………………….....20

Discussion……………………………………………………………………………...39

References……………………………………………………………………………..46

Chapter Two: “Measurement of the visual fields for eyes four, five and six, anatomical description of the optic lobes and the functional organization of the visual system in T. marmoratus larvae”

Introduction………………………………………………………………………………51

Materials and Methods…………………………………………………………………...53

Results……………………………………………………………………………………58

Discussion………………………………………………………………………………..73

General Discussion………………………………………………………………………77

References………………………………………………………………………………..81

5 List of Tables and Figures

Table 1 Acceptance angles of eyes one, two and three- measured by Dr. Buschbeck…59

Table 2 Average BFDs; and focal lengths of the eyes four, five and six……………….63

Table 3 Acceptance angles of eyes four, five and six in different sectional planes……..63

Figure 1 First instar T. marmoratus external features……………………………....9

Figure 2 Anatomical features of the large tubular eyes one and two……………………21

Figure 3 Three dimensional reconstructions of eyes one and two viewed from medial

(left) and dorsal (right)…………………………………………………………………...23

Figure 4 Detailed anatomical features of the crystalline cone and the lateral in eye one………………………………………………………………………………………..25

Figure 5 Anatomical features of the horizontal and vertical retinas in eye one…………28

Figure 6 Organization of the rhabdoms of the vertical retina of eye one……………….30

Figure 7 Anatomical features of the eyes three and four………………………………..32

Figure 8 Anatomical features of the eyes five and six; and the eye patch………………36

Figure 9 An illustration of the procedure of measurement of optical parameters………56

Figure 10 An illustration depicting various optical parameters measured……………...58

Figure 11 Acceptance angles of eyes four, five and six in different sectional planes…..60

Figure 12 An overview of the visual fields of the stemmata in sagittal plane…………..61

Figure 13 An overview the visual fields of the stemmata in the horizontal plane………62

Figure 14 External and anatomical features of the optic lobes………………………….65

Figure 15 Three dimensional reconstructions of the optic lobe…………………………66

6 Figure 16 Fluorescent stained neuronal projections from eyes one, three, five and six onto the respective optic neuropils………………………………………………………68

Figure 17 Fluorescent stained projections of nerve terminals in the neuropils two and four, from backfill studies of eye four…………………………………………………...70

Figure 18 Detailed anatomical features of the optic neuropils and fluorescent stained neuronal projections onto to the optic lobe neuropils from eyes two and four…………..71

7 General Introduction:

Though visual systems in the adult holometabolous have been extensively studied, those in larvae still remain poorly investigated. Many holometabolous larvae have single eyes, generally referred to as stemmata, which vary to a large degree in their number, location, size and functional capability (Gilbert, 1994); among their wide range of species. For example, they can be highly reduced, as they are in the maggot larvae of higher flies (Paulus, 1989) or they can be sophisticated image forming lens eyes with extended cup-shaped retinas such as in the largest stemmata of the tiger beetle larvae (Toh and Mizutani, 1987). This variation makes the studies of the holometabolous insect larval visual systems interesting.

In the current study, “Structural And Functional Organization of The Visual System of The

Larvae of The Sunburst Diving Beetle, Thermonectus marmoratus”, I investigate an interesting visual system of the larvae in the Sunburst diving beetle (also referred to as the

Spotted water beetle), T. marmoratus.

There are three larval instars in T. marmoratus and all of them are almost exclusively aquatic. Larvae are aggressive predators, feeding on soft bodied insects and other larvae.

They have six stemmata (single lens eyes in many holometabolous insect larvae) and an eye patch on either side. They also have two optic lobes for processing visual inputs.

8 Figure 1 First instar T. marmoratus larva external features

(A) A first instar T.marmoratus larva, about 1cm in length. (B) Fronto-dorsal view of the larva showing E1 and E2 on both sides; the eye patch (EP) and the pincer shaped mandibles (M). (C) Right lateral view of the right larval head showing the disposition of E1-6.

9 The objectives of this study are to investigate the structural organization of specific components of the visual system (stemmata, optic lobes and neuronal connections between them) and to derive hypothesis about the mode of function of the visual system. This study is divided into two chapters. In chapter one, “Twenty eight retinas but only twelve eyes; an anatomical analysis of the larval visual system of the diving beetle, Thermonectus marmoratus

(Dytiscidae)”, I describe the structural details of each of the six stemmata and the eye patch on one side of the larva. My preliminary anatomical studies on all the three larval instars showed no major differences in the visual systems among the three larval instars. So, in chapter one all the studies were performed in first instars, due to their greater availability. I have performed various staining techniques to investigate the structural details of the eyes as well as the eye patch. I have also performed three dimensional reconstructions of eyes one and two, to better illustrate their complex structural composition.

In chapter two, “Measurement of the Acceptance Angles for Eyes Four, Five and Six;

Structural Description of the Optic Lobes; and the Functional Organization of the Visual

System in T. marmoratus Larvae”, I present the acceptance angles of each of the stemmata.

These determine the visual fields that are sampled by the respective stemmata, which are in direct relevance to the larva’s behavior.

Acceptance angles for the eyes one, two and three has been measured by Dr. Buschbeck previously. In this study I measured various optical parameters like the back focal distances and focal lengths (explained in the introduction to chapter two) for stemmata four, five and six to calculate their acceptance angles.

10 In addition to optical measurements I investigate the anatomical organization of the optic lobes using various staining techniques and find that there are six distinct neuropils. This organization of optic neuropils in the optic lobes is different from other optic lobes studied and therefore, it was not obvious how optic nerves project onto the neuropils. So, I performed fluorescent dye backfill studies which allow tracing the optic nerve projections from each of the stemma to the respective neuropils. For these studies in eye two I used second instars, because of the convenience afforded by their larger size. Having traced the axonal projections from each eye onto the respective neuropil, I studied the detailed structure of each of the neuropils using various staining techniques. These studies are important because understanding the complexity of the visual input processing system can be useful for deriving hypotheses about the mode of function of the visual system. For example, presence of large optic lobes might indicate that the organism depends a lot on the visual inputs, presence of giant neurons can indicate the processing of movement detection and consequential reflex actions and presence of large and densely packed complex optic neuropils might suggest rigorous processing of the visual stimuli from the environment.

I have also performed 3D reconstructions of the optic lobes to better understand the structures and location of the individual neuropils.

11

Chapter one

Twenty eight retinas but only twelve eyes; an anatomical analysis of the larval visual

system of diving beetle Thermonectus marmoratus (Dytiscidae; Coleoptera).

Karunyakanth Mandapaka (1), Randy C. Morgan (2) and Elke K Buschbeck (1) +

(1) Department of Biological Sciences; University of Cincinnati

Cincinnati, OH 45221-0006. Tel: (513)-556-9794, Fax (513) 556-5299

(2) Insectarium; Cincinnati Zoo and Botanical Garden; 3400 Vine Street; Cincinnati,

OH 45220-1399

+ Corresponding author: [email protected];

Submitted to the Journal of Comparative Neurology, July 2005

12 Abstract:

The larvae of the sunburst diving beetle, Thermonectus marmoratus (Dytiscidae;

Coleoptera) are highly efficient visually-guided predators. Their visual system consists of

a cluster of six stemmata and one eye patch on each side of the head capsule.

Histological investigations show that the organization of individual stemmata differs

strongly from any eye that previously has been described. Based on a general

morphology and the presence of actin-rich areas that are typical for rhabdoms, we find that each eye is characterized by several retinas. The most dorsal eye of each side is tubular and we have identified three spatially distinct areas with retinula cells. These are:

(1) a band of two rows of rhabdoms along the medial side of the long tubular eye; (2) a cone-shaped region towards the bottom of the tube which is formed by the accumulative rhabdomeric regions of a large number of retinula cells that are oriented perpendicular to the light path, and (3) two rows of long rhabdoms at the base of the long tube. A second large eye is organized similarly but lacks the medial band. The remaining four eyes are nearly spherical in shape, and each has two distinct retinas. The twelve eyes hence account for a total of 26 retinas, and two further retinas are present in eye patches. Our anatomical findings suggest that this is an example of a visual system in which specific visual tasks are distributed, and which relies on a variety of presumably highly specialized retinas.

13 Introduction:

Although considerable effort has been put into studying the compound eyes of adult insects, few studies have considered the function and organization of the eyes of holometabolous insect larvae. Each side of the larval head bears several (most commonly six) eyes (stemmata) that range from simple visual organs to complex camera-type eyes.

The larval visual system is of great interest since eyes collectively process all visual information, and therefore have the potential to specialize in different tasks. In this paper we describe the anatomical organization of the stemmata of the sunburst diving beetle

Thermonectus marmoratus (Dytiscidae; Coleoptera) which is a predatory water beetle which, on each side of its had has six stemmata. Those stemmata vary greatly and indeed appear to be specialized for specific tasks.

Among holometabolous insects the number, location, size and functional capability of stemmata differs to a great degree (Gilbert, 1994). This is not surprising, since holometabolic insects are a large and diverse group comprising more than three quarters of all described insect species (Gullan and Cranston, 2005). The larval stemmata are thought to have originally evolved from compound eyes (Liu and Friedrich, 2004;

Paulus, 1979), but have since greatly diverged. They can be highly reduced, as they are in the maggot larvae of higher flies (Paulus, 1989) or they can represent sophisticated image-forming lens eyes with extended cup-shaped retinae, as in the largest stemmata of tiger beetle larvae (Toh and Mizutani, 1987). The discovery of stemmata dates back to the year 1669 when Malpighi mentioned stemmata in a saturniid moth

(Gilbert, 1994). Since then, the presence of stemmata has been established in most holometabolic insects, and there have been a few thorough studies on specific aspects of

14 stemmata. For example, neurophysiological mechanisms of trichromatic color vision have been particularly well-studied in the caterpillar of the swallowtail butterfly, Papilio xuthus (Ichikawa, 1990; Ichikawa, 1991). Other studies in the same species illustrate how neurons of larval optic neuropils can be incorporated into the adult optic neuropils

(Ichikawa, 1994a; Ichikawa, 1994b). The best studied, and probably one of the most sophisticated larval visual systems, is that of the tiger beetle, Cicindela chinensis, which shows some superficial similarities with T.marmoratus in our study. For example, in both the larvae of diving beetles and tiger beetles, two of the six stemmata are particularly large and are important during prey capture (Friederichs, 1931; Toh and

Mizutani, 1987; Toh and Okamura, 2001). In tiger beetle larvae they also mediate escape behavior (Gilbert, 1989; Toh and Okamura, 2001). The largest stemmata represent camera-type eyes with extended cup-shaped retinas, and behavioral studies suggest that binocular visual inputs of these stemmata are used for distance estimation (Toh and

Okamura, 2001).

Here we present the stemmata of a visual system that may be equally elaborate as that of the tiger beetle, but differs greatly in its organization. According to illustrations in a recently published book on diving beetles (Larson et al., 2000), the eyes of

Thermonectus larvae are among the most sophisticated within the dytiscids. Both the adults and larvae of T. marmoratus are highly predatory, feeding on small insects and other soft-bodied aquatic organisms. They are found in shallow fresh water streams in extreme southern California and (Larson et al., 2000; Powell and Hougue, 1979),

New Mexico and southwestern (Morgan, 1995). The adults show a beautiful pattern of yellow spots on a black background, which is probably an aposematic

15 coloration (Meinwald et al., 1998; Morgan, 1992a). There are three larval instars, all of which are predatory and almost exclusively aquatic. Only the first instars are briefly on land immediately after they hatch, and the third instars return to the land for pupation.

All instars have flattened heads with pincer-like mandibles that are used in prey capture.

Under laboratory conditions they also exhibit cannibalism (Morgan, 1992a; Morgan,

1995). It has previously been suggested that the larvae of T. marmoratus track their prey by using visual clues (Morgan, 1992b; Morgan, 1995). Our own observations of prey capture events confirm this notion. Considering that dytiscid larvae are generally know as sophisticated predators (Larson et al., 2000; Schöne, 1951), it is surprising that the only detailed publications on the eyes of dytiscid beetle larvae that we were able to find date back to 1889 (Patten) and 1912 (Günther) studies on and Dytiscus respectively. In addition, Hermann Schöne conducted a behavioral study of a phototaxic response (Schöne, 1951) and found no evidence for color discrimination (Schöne, 1953).

Since then the visual system of dytiscids seems to have been forgotten.

Externally, each side of the larval head capsule of T. marmoratus houses six stemmata and one peculiar eye patch. Two of the stemmata are very large, and even from outside it can be seen that these form deep tubes, rather than the spherical shape of typical camera eyes. In the first part of this paper we focus on these tubular eyes, as they greatly diverge from the organization of stemmata of other insects: our anatomical findings suggest the presence of three physically separated and anatomically distinct retinas within the most dorsal tubular eyes. The other tubular eye is organized in a similar way, but lacks one of the retinas. The second part of this paper describes the remaining four eyes, which are closer to resembling a spherical eye, but nevertheless

16 diverge from the organization of other stemmata and typical camera eyes. Finally, we

describe a seventh visual organ, which appears to be an eye patch without a lens.

Ultimately we hope to discover how these peculiar eyes function. This paper focuses on

the first step towards this goal, which is an anatomical investigation of the eyes. Since

there are six eyes on each side that together form the visual system, there is the potential

for any one of these eyes to specialize for specific visual tasks. The unusual and variable

organization of the individual eyes and the presence of multiple retinas in individual eyes

suggest that such specialization is indeed the case.

Materials and Methods:

Specimens: Adult specimens of the sunburst diving beetle, Thermonectus marmoratus were kindly provided by the Insectarium of the Cincinnati Zoo and Botanical Garden, or were collected in August 2004 in the vicinity of Tucson, AZ. They were kept in a fresh water aquarium at 28˚C on a diet of freshly-killed or frozen crickets at a 16L:8D

photoperiod . Larvae of T. marmoratus were separated from the adults and raised on live

or freshly thawed brine shrimp and live larvae. All histological preparations

were made on first instar larvae.

Bodian Staining:

Larvae were anesthetized by chilling, decapitated and part of the head cuticle removed.

Heads were fixed in AAF (100% ethanol, 37% formaldehyde and glacial acetic acid in

the ratio of 17:2:1), dehydrated, embedded in Paraplast plus (Tyco Healthcare Group LP,

17 MA., USA), and serially sectioned at 8µm. Dewaxed sections were incubated for 24h at

40oC in 1% silver proteinate (Alfa, Ward Hill, Mass., USA) with the addition of 0.3g pure copper per 50ml of the solution. Afterwards, sections were conventionally stained after Bodian's (1937) original method with the following small modifications: step 3, only 2% sodium sulfite; step 4a, gold chloride without acetic acid for 10 min.; and step

4b, oxalic acid bath for 8 mins.

Ethyl gallate staining:

This procedure was a minor modification of Strausfeld and Seyan (1985). Larvae were anesthetized by chilling, decapitated and part of the head cuticle was removed. Heads were fixed in 4% paraformaldehyde solution (EM grade; Electron Microscopy Sciences,

Fort Washington, PA., USA) in Sorensen’s phosphate buffer pH 7.4 (Electron

Microscopy Sciences, Fort Washington, PA., USA). After several washes in buffer, heads were transferred into 1% Osmium tetroxide (OsO4) solution (Electron Microscopy

Sciences, Fort Washington, PA., USA) in distilled water for 1 hr on ice followed by 1 hr at 20oC. Tissue was washed several times in distilled water, and finally treated with

saturated ethyl gallate (1hr at 0°C and 1hr at 20°C). After staining the heads were

dehydrated, embedded in Ultra-Low Viscosity Embedding Media (Polysciences,

Warrington, PA) and serially sectioned at 8 µm.

Phalloidin staining:

Larvae were anesthetized by chilling, decapitated and some of the head cuticle was

removed. Heads were fixed in 4% paraformaldehyde solution (EM grade; Electron

Microscopy Sciences, Fort Washington, PA., USA) in Phosphate Buffered Saline (PBS)

18 pH 7.4 for two hours. After fixation and thorough washing, the heads were cryoprotected overnight in a 30% sucrose solution in distilled water. Finally, heads were mounted in

Neg -50 Frozen Section Medium (Richard-Allan Scientific, Kalamazoo, MI , USA), frozen in liquid nitrogen and cryo-sectioned at 10 µm. After several washes in PBS, the sections were blocked with 1% Bovine Serum Albumin (BSA; Fisher scientific Fair

Lawn, New Jersey., USA) in PBS for two hours. Phalloidin staining solution (Molecular

Probes, Eugene, Oregon., USA) was prepared by adding a single 50µl aliquot (200 units/ml, in methanol) into 200µl 1% BSA in PBS. Slides were incubated for 30 minutes, washed, mounted in Vectashield (Vector laboratories Inc, Burlingame, CA., USA) and observed and photographed within a day under a fluorescent microscope.

Phalloidin and DAPI double staining:

After the Phalloidin staining (see above), the slides were incubated in a 0.2% solution of

4'-6-diamidino-2-phenylindole (DAPI; Sigma, St. Louise, MO; D-9564) for 30 minutes.

Slides were then washed and mounted as described for Phalloidin staining.

3D reconstruction

All of the 8µm thick Ethyl Gallate stained sections of a given area were photographed

(with an Olympus Magna Fire digital camera) and imported into Amira (Mercury

Computer Systems, Berlin), where they were pre-aligned manually and automatically optimized within the align slices module. To increase the number of sections additional sections were interpolated using Amira's interpolation algorithm, specific areas were labeled in the Image Segmentation Editor and surfaces were smoothed by using three dimensional filters.

19 Results:

Larvae of the water beetle Thermonectus marmoratus have several prominent eyes are well visible from the outside (Fig. 1A-C). In the fronto-dorsal view, the of the two largest eyes are readily visible (Fig. 1B). Both these eyes point forward and upward and we refer to them as eye one (E1) and two (E2). All eyes are visible in the side view (Fig.

1C). E1 is the most dorsal of the three anterior eyes, E2 is situated just ventral and slightly anterior to it, and the somewhat smaller eye three (E3) is situated ventral, pointing laterally. The remaining three eyes are smaller and are situated posterior to the first three eyes. Eye four (E4) points ventrally, eye five (E5) points latero-posteriorly and eye six (E6) points dorsally. In addition to these six stemmata the larvae of T. marmoratus have a seventh visual organ on each side. This is an eye patch with retinula cells that are situated directly beneath the cuticular layer. There is no lens associated with the eye patch. In the following section we provide a detailed anatomical description for all of these photosensitive structures.

20

Figure 2 Anatomical features of the large tubular eyes one and two. (A) An ethyl gallate stained sagittal section showing lens (L), the crystalline cone (CC), the

21 rhabdomeric portion (rhabdomeric aggregation RA) of the horizonal retina (HRe) and the two rows of rhabdoms of the vertical retina (VRe) in E1 and E2. (B) An ethyl gallate stained section along the horizontal axis of E1 showing the lens (L), the crystalline cone (CC), the lateral retina (LRe) and the vertical retina (VRe) with long rod-shaped rhabdoms. (C) An ethyl gallate stained section along the horizontal axis of

E2 showing the lens (L), the crystalline cone (CC) and the vertical retina (VRe) with long rod-shaped rhabdoms.

The large tubular eyes: E1 and E2

E1 and E2 are the largest eyes, and they share many anatomical characteristics.

Both eyes point directly forward and upward at an angle of about 35o from the horizontal

body axis (Fig. 2A). Each eye is characterized by a large cuticular lens, and by a

relatively long, cylindrically-shaped cup that extends throughout the larval head to the

ventral side (Fig. 2A). E1 is slightly longer than E2, measuring about 500µm in length

from the surface of the lens to the base of the retina (Fig. 2B). E2 has a slightly larger

diameter but only measures 454µm in length (Fig. 2C). The general organization of the

two eyes differs greatly if viewed in a sagittal section (Fig. 2A) or in a nearly horizontal

section (tilted so that the plane of section corresponds to the long axis of the eyes; Fig

2B,C). We have reconstructed the main components of E1 and E2 to better illustrate

their spatial relationships (Fig. 3). Fig 3A illustrates E1 viewed from medial (left) and

from dorsal (right). The lens is depicted in purple and the underlying crystalline cone is

illustrated in magenta. Along the median border of the crystalline cone lies a narrow

22 band which we believe to be photosensitive tissue (see below), entitled lateral retina

(illustrated in orange). The lateral retina is largest closest to the lens and becomes a

narrow band towards the base of the cylindrical crystalline cone, where it merges into

another area, the rhabdomeric aggregation (marked in yellow), which is in the shape of a

flattened cone which is situated medially, directly adjacent to the crystalline cone.

Figure 3 Three dimensional reconstructions of eyes one and two viewed from medial

(left) and dorsal (right). (A) 3D reconstructions of E1 showing the lens (L; purple), the lateral retina (LRe; organge), the crystalline cone (CC; magenta), the rhabdomeric aggregation (RA; yellow) and the vertical retina (VRe) with two rows of

23 long rod-shaped rhabdoms (green). (B) 3D reconstructions of E2 showing the lens (L: purple), the crystalline cone (CC; magenta), the rhabdomeric aggregation (RA; yellow) and the vertical retina (VRe) with two rows of long rod-shaped rhabdoms

(green).

Although the rhabdomeric aggregation has an atypical shape and organization for

photoreceptive tissue, we have accumulated evidence for its rhabdomeric nature (see

below). Medial to the rhabdomeric aggregation two rows of vertically-oriented rhabdoms

are visible (illustrated in green), the vertical retina. We here follow the terminology that

has been introduced by Günther (1912). Note that the terms vertical- and later horizontal

retina refer to the axis of the eye, with the lens on top, rather than the axis of the structure

within the organism. As each eye is situated at a different angle, the actual orientation of

the retinas differ in E1-E6. E2 (Fig. 3B) is organized in a fairly similar way as E1, except

that it generally appears shorter and stouter, and the proportions of some of the tissues are

slightly altered. For example, the crystalline cone is longer and narrower in E1 whereas

it is shorter and wider in E2. The most apparent difference is that the lateral retina is

absent in E2. In its place, at the base of the crystalline cone, the rhabdomeric aggregation

reaches further distally than it does in E1.

24

Figure 4 Detailed anatomical features of the crystalline cone and the lateral retina in eye one. (A) A Bodian stained horizontal section of E1 showing the lens (L) and the long and narrow cells of the crystalline cone (CC). (B) A DAPI stained sagittal section of E1 showing the nuclei of crystalline cone cells (N) which are arranged in a single row near the base. (C) An ethyl gallate stained frontal section of E1 showing a dense layer of strongly pigmented cells (PC) that surround the crystalline cone (CC), undefined small

25 globular structures (G) between the cells of crystalline cone, the surrounding pigmented cells, and the lateral retina (LRe). (D) An ethyl gallate stained frontal section of E1 showing the lateral retina at larger magnification (LRe) and lightly stained cells that are associated with it (CLRe). It also shows the surrounding strongly pigmented cells (PC) and undefined small globular structures (G) towards the crystalline cone. (E) An ethyl gallate stained parallel-to-eye section of E1 showing the horse-shoe shaped rhabdoms of the lateral retina (LRe) and the lightly stained cells (CLRe) associated with the lateral retina. (F) A phalloidin stained sagittal section of E1 showing the horse-shoe shaped rhabdoms of the lateral retina (LRe).

In the following sections we describe the cellular composition and anatomical

organization of the individual components. The most distal part of each of the two eyes

consists of a circular, biconvex lens that is formed by the outer cuticle and has a diameter

of approximately 115µm in E1, and 108µm in E2 (Fig. 2B&C). Underneath the lens lies

the crystalline cone which is a loose tissue which is about 250µm long in E1 and 203µm

long in E2. The crystalline cone consists of long and narrow cells that extend throughout

its length (Fig. 4A). DAPI staining shows that all nuclei of the crystalline cone are

situated in a single layer at the side and near the base of the crystalline cone (Fig. 4B).

No nuclei are present in the central areas of the structure which is filled with the lumen of

large cells as well as with extracellular matrix. The crystalline cone is surrounded by a

dense layer of strongly pigmented cells (Fig. 4C; this component is not illustrated in the

reconstruction of Fig. 3). In a cross section (frontal section of the ), each of these

26 cells is narrow and elongated, and the majority of cells are oriented perpendicular to the axis of the crystalline cone. Towards the center of the eye, cells are surrounded by a large number of small globular structures that stain lightly in ethyl gallate staining (Fig.

4C,D). It is currently unclear if these are part of the pigment cells or represent extracellular secretions. Exclusive to E1, embedded within the pigmented surrounding layer, lays the lateral retina (Fig 4C.D). In a cross section, the core of the lateral retina is a small, membrane-dense area that stains relatively dark in ethyl gallate staining and is surrounded by a group of lighter cells (Fig. 4D&E). The dark area is composed of horse- shoe shaped rhabdomeric units, that are oriented approximately perpendicular to the axis of the crystalline cone, and stain strongly with phalloidin (Fig. 4F). Phalloidin binds to f- actin and stains strongly in particularly actin-rich areas such as muscle and microvilli. In insect eyes the presence of abundant microvilli typically indicates the presence of rhabdoms (Baumann, 1992; Hafner et al., 1992) which are the photoreceptive portion of retinula cells. Because of its position to the side of the crystalline cone we refer to this region as lateral retina. Between the rhabdoms of the lateral retina and the crystalline cone lies a region that appears as a homogeneous dark grey in ethyl gallate staining (Fig

4E). The significance of this are so far remains unclear.

27

Figure 5 Anatomical features of the horizontal and vertical retinas in eye one. (A) An ethyl gallate stained sagittal section showing the location of the rhabdomeric aggregation (RA) between the crystalline cone (CC) and the rhabdoms (Rh) of the vertical retina. Also seen are the cells of the horizontal retina (HRe), with rhabdomeric regions congregated to form the rhabdomeric aggregation. (B) A phalloidin and DAPI double stained sagittal section showing the rhabdomeric aggregation (C), the rhabdoms of the vertical retina (Rh), and the nuclei of the horizontal retina (NHRe). Note that the rhabdomeric aggregation shows layering.

(C) A Phalloidin and DAPI double-stained frontal section showing the segregation of rhabdomeric aggregation into dorsal (DC) and ventral (VC) regions, both of which are layered. (D) A Phalloidin and DAPI double-stained frontal section showing two rows of rhabdoms (Rh) of the vertical retina. In each row, horse-shoe shaped (HRh) and an oval (Orh) rhabdoms alternate.

28 Below the crystalline cone lies the rhabdomeric aggregation (Fig. 3). This area appears to be formed by the densely compacted rhabdomeric regions of retinula cells that lie dorsal and ventral to it. In contrast to retinula cells in other insects, and to deeper layers of retinula cells within E1 and E2 (the vertical retina), these retinula cells are oriented perpendicular to the path of light. In accordance with Günther (1912), who labeled corresponding cells in Dytiscus horizontal rods (“horizontale Stäbchen”) we refer to these retinula cells as horizontal retina. Evidence for the rhabdomeric nature of the structure derives from (a) the color and appearance in ethyl gallate staining that is comparable with that of other rhabdomeric regions, (2) the presence of high levels of actin (Fig 5B, C), and (3) transmission electron microscopical data that shows stacks of microvilli (unpublished observation). Although the rhabdomeric aggregation appears as homogeneous structure in ethyl gallate staining (Fig. 5A), some striation and layering is revealed in phalloidin staining (Fig 5B, C). A frontal section illustrates how it segregates into a dorsal and a ventral group (Fig. 5C). The absence of cell bodies (Fig 5B,C) in the rhabdomeric region confirms that the rhabdomeric aggregation is part of the surrounding retinula cells, and not a separate cellular structure.

29

Figure 6 Organization of the rhabdoms of the vertical retina of eye one. (A) An ethyl gallate stained preparation sectioned along the horizontal axis of the eye showing the vertical retina with the rhabdoms (Rh) of one of the two rows. Also seen are nuclei

(N) of the cells that separate the two rows of rhabdoms. (B) An ethyl gallate stained sagittal section showing two of the long, rod-shaped rhabdoms of the vertical retina

(VRe). (C) An ethyl gallate stained frontal section showing the vertical retina (VRe) with two rows of long rhabdoms. Also seen is the formation of each row by alternating horse-shoe shaped (HRh) and oval (ORh) rhabdoms.

Deeper in the eye, below the rhabdomeric aggregation lies a retina which is formed of

more typical vertically-oriented retinula cells (in axis with the path of light; Fig 5D,

30 6A,B). Approximately 180 rhabdoms are arranged in two parallel rows (Fig 5D, 6C).

Each rhabdom is around 120µm long, but it is unclear if it is formed by a single or multiple retinula cells. In the latter case, the rhabdom could be a composite structure and could be tiered. A group of nuclei which surrounds the rhabdoms dorsally and ventrally is visible in DAPI staining (Fig. 5D). It is possible that these are the nuclei of the vertical retinula cells. Note that these nuclei are larger and more globular than the narrow nuclei of the horizontal retinula cells. In ethyl gallate staining, the rhabdoms of the vertical retinula cells stain relatively darkly. If sectioned parallel to the eye, a sheath of alternating thick and thin rhabdoms are visible (Fig. 6A). Those nuclei that are visible at the posterior margin of the eye belong to cells that separate the two layers of rhabdoms.

The alternation of thick and thin rhabdoms is also visible in high resolution images of cross sections (Fig. 5D, 6C). The rhabdoms in each of the two rows alternate between a relatively large, horse-shoe shaped and smaller oval rhabdom.

31

Figure 7 Anatomical features of the eyes three and four. (A) An ethyl gallate stained horizontal section of E3 showing the lens (L), horizontal retina (HRe) with its layered rhabdomeric structure (HRh) and the rod-shaped vertical rhabdoms

32 (VRh) of the vertical retina (VRe). (B) A phalloidin stained horizontal section of E3

showing the rhabdoms (HRh) of the layered horizontal retina and the vertical

rhabdoms (VRh). (C) An ethyl gallate stained section of E3 at larger magnification

showing how the lens (L) is separated from the layered horizontal rhabdoms (HRh)

and the rod-shaped vertical rhabdoms (VRb). Note how the central rhabdom of the

vertical retina is enlarged (arrow) and the distal ends of all other vertical rhabdoms

curve outwards (*). (D) An ethyl gallate stained sagittal section of E3 showing the

rhabdomeric grid (RG) formed by the out-curving vertical rhabdoms. (E) An ethyl

gallate stained frontal section of E4 showing the lens (L), retinula cells of the

horizontal retina (HRe) with its layered rhabdom (HRh) and the short rod-shaped

vertical rhabdoms (VRh). (F) An ethyl gallate stained horizontal section of E4

showing the rhabdomeric grid (RG) formed by the vertical rhabdoms.

The nearly spherical eyes: E3 and E4.

E3 is located anteriolaterally and its axis points primarily laterally at 63o from the front. It also points slightly downwards at an angle of 10o from the horizontal body plane

(Fig. 1 C). This eye is smaller and much shorter than E1 and E2. The shape is almost spherical, but slightly wider (200µm) than deep (190µm; Fig. 7A). At its outer surface, the eye consists of a thick, spherical, cuticular lens about 50-55µm in diameter. Posterior to the lens, a thin layer of tissue is visible, which stains darkly in ethyl gallate staining

(Fig.7A,C). Below this layer, a thicker, layered structure is visible, which seems to be contributed to by cells that are dorsal and ventral to the structure. Similar to the rhabdomeric aggregation area in E1 and E2, it stains strongly with fluorescently labeled

33 phalloidin, indicating high levels of actin and possibly photoreactivity (Fig. 7 B). Several elongated cell bodies are visible on both sides. These long and thin cells generally look similar to retinula cells that are situated at a deeper level (Fig 7A). As in E1 and E2, we consider these cells to be horizontal retinula cells. Although the layering of the rhabdom- like structure is clearly more pronounced than the layering of the rhabdomeric aggregation of E1 and E2, individual layers are in close physical proximity. The layers are closest to each other beneath the middle of the lens, and gradually become more widely spaced towards the periphery, such that the bottom layer appears flat rather than following the curvature of the lens (Fig. 7C).

Posterior to the giant rhabdomeric structure that is formed by horizontally- oriented retinula cells, are several rows of relatively short vertically-oriented rhabdoms, which stain dark in ethyl gallate staining. The vertical rhabdoms are also visible in phalloidin staining (Fig. 7B). Approximately 25 rhabdoms are visible in horizontal cross sections. A comparable number of nuclei situated well below the rhabdoms suggest that each rhabdom may be formed by only a single cell. The rhabdom of each cell is narrow, and the distal end of all but the very central rhabdoms curve outward (Fig. 7C). The rhabdoms of the most central cells in each row are slightly larger than the other rhabdoms and remain relatively straight throughout their length. The outward curvature of the remaining rhabdoms leads to a network at the most distal region, which is evident from the darkly stained grid in an ethyl gallate stained sagittal section (Fig. 7 D).

E4 is similar to E3 though smaller and located posterior and slightly ventral to E1-

3 (Fig. 1 C). Its central axis points side ways (at an angle of 90o from the front of the

head) and downwards (at an angle of 85o from the horizontal body axis). In a sagittal

34 section E4 is about 175µm wide and around 115µm deep. It too is covered by a nearly spherical cuticular lens (with a diameter of 50-55µm) and it has two regions of presumably photoreceptive cells (Fig. 7E). Beneath the lens is a narrow layer of horizontally-oriented cells, with a relatively dark staining striated structure in their middle (horizontal retina). Below, a slightly asymmetric, nearly cup-shaped retina

(vertical retina) is visible, which contains about 25 vertically-oriented rhabdoms within a single shallow row. Each of the rhabdoms is short and curves sideways at the most distal end, leading to a slightly irregular array in a horizontal section (Fig. 7F). In contrast to

E3, we do not observe enlarged rhabdoms in the middle of E4.

35

Figure 8 Anatomical features of the eyes five and six; and the eye patch. (A) An ethyl gallate stained sagittal section of E5 showing the lens (L), the retinula cells of the horizontal retina (HRe), the cone-shaped horizontal rhabdomeric structure (HRh), and the rod-shaped vertical rhabdoms (VRh). (B) A phalloidin stained sagittal section of E5 showing the actin rich cone-shaped horizontal rhabdomeric structure (HRh)

36 and vertical-rod shaped rhabdoms (VRh). (C) An ethyl gallate stained frontal section of E6 showing the lens (L), the retinula cells of the horizontal retina (HRe), the cone- shaped horizontal rhabdomeric structure (HRh) and the rod-shaped vertical rhabdoms (VRh). (D) An ethyl gallate stained cross section of E6 showing the horse shoe shaped rhabdoms (Rh). (E) An ethyl gallate stained cross section of the eye patch showing the rod- shaped rhabdoms (Rh) of the retina (Re). (F) A phalloidin stained cross section of the eye patch showing the rod-shaped rhabdoms (Rh).

The intermediate eyes: E5 and E6

E5 and E6 are more asymmetric than E3 and E4, and hence somewhat more like

the long tubular eyes E1 and E2. E5 is the most posterior eye, and its axis points upwards

at an angle of 15o (from the axis of the body) and slightly backwards (110o from the

front; Fig 1C). E6 is located slightly anterior and dorsal to E5, and its axis points

upwards at an angle of 65o from the back and 20o from the side. Both eyes are covered

by cuticular lenses, each with a diameter of about 45-50µm. Based on sagittal sections,

E5 is about 120µm wide and 130µm deep (Fig 8A). E6 is around 140µm wide in sagittal

but only about 100µm wide in frontal sections, and it is130µm deep (Fig 8C). As in E3

and E4, groups of horizontally-oriented cells are visible beneath the lens (Fig. 8A,C).

The striated middle region, however, is relatively thick, resulting in a darkly stained

structure in the shape of a flattened cone. On both sides of the cone (dorsal and ventral),

elongated cells are visible projecting to the periphery of the eye, where nuclei are located.

These cells are reminiscent of the horizontal retinula cells of E1 and E2, which form the

37 rhabdomeric aggregation. In both eyes phalloidin staining (illustrated for eye 5; Fig. 8B) results in bright staining of the rhabdomeric aggregation-like region, which further supports the notion that the region is constructed of the rhabdomeric segments of horizontally-arranged retinula cells. Underneath the horizontal retina, there is a cup- shaped retina that is formed by vertically-oriented retinula cells. In E5 this retina is more symmetric than in E6; in the latter, it is close to three times as long as wide (Fig. 8D). In contrast to E3 and E4 (in which rhabdoms of the vertical retina are rod shaped), rhabdoms of E5 and E6 are horse-shoe shaped in the cross section. The shape of individual rhabdoms remind most strongly of the rhabdoms of the lateral retina of E1

(Fig. 4F). In fact, at the most distal region of the cross section of E6 (Fig 8D), only two rows of rhabdoms are visible. As is the case for the lateral retina of E1, in the cross section of rhabdoms the center of two horse-shaped structures are adjacent to each other.

The rhabdoms of the vertical retinas of E6 and E6 remain straight throughout their length, and do not show the characteristic bend that is seen in E3 and E4.

Eye Patch:

In addition to the six eyes, the larvae of T. marmoratus are characterized by a patch of presumably photoreceptive tissue. The eye patch is localized below the dorsal cuticle, medially of E1. In contrast to the six eyes it is not covered by any lens, are now eand there are no changes in the cuticular that suggests its presence. A retina that lacks screening pigment is present immediately below the cuticle. The eye patch is around

240µm long and 120µm wide. In a cross section long and narrow cells are visible (Fig

38 8E). At their distal ends a darker region is evident, which presumably correspond to

rhabdoms. The rhabdoms also can be visualized with phalloidin (Fig 8F).

Discussion:

Our study focuses on the stemmata of T.marmoratus larvae which greatly diverge from other known eyes. For this paper we focused exclusively on first instar larvae. Later instars have similar eyes, though cell numbers and structural proportions are somewhat different. Major characteristics include: (a) a segregation of stemmata into three anatomically distinct categories, (b) the presence of two to three retinas in each stemma and (c) the presence of rhaboms that are oriented perpendicular to the axis of light. T. marmoratus larvae have six stemmata and one eye patch on each side of the head capsule. The two anterior-most stemmata are noteably large, tubular in shape, and associated with up to three distinct, highly asymmetric retinas. These eyes are the most atypical stemmata of T. marmoratus larvae, as they differ greatly from those of other described insects (Gilbert, 1994), and in fact from any eye thus far described. In the following paragraph we will discuss how they differ from known systems and what functional implications may result from their unique organization.

If the large eyes of T. marmoratus are compared to other described stemmata, the closest parallel can likely be found in the larvae of tiger beetles (Toh and Mizutani, 1987,

1994). Both species are characterized by two of the six eyes being much larger than the remaining four eyes, and by their ability to use visual guidance for prey capture (Morgan,

1992b; Toh and Okamura, 2001). However, tiger beetle larvae have only one type of retina, which is cup-shaped and nearly symmetrical. Furthermore, these eyes are

39 basically spherical and do not have long tubes. There is one described example for

tubular eyes outside the stemmata which shows some similarities. These are the anterior

medial eyes of jumping spiders, which consist of a relatively long tube and a narrow retina with four distinct strata (Land, 1969b). In jumping spiders, the retina is also narrow and its long axis extends dorso-ventrally, and can be moved laterally in order to scan a wider visual field (Land, 1969a). The similarity between the large T. marmoratus eyes and jumping spider eyes hence extends beyond the organization into long tubes, but

also includes the presence of a narrow retina. For T. marmoratus, this is the most

proximal retina of the tube (referred to as vertical retina), which consists of only two

rows of rhabdoms. In contrast to jumping spiders the long axis of the T. marmoratus

retina however runs medio-laterally. Other examples of narrow retinas are found in the

eyes of the heteropod mollusk Oxygyrus (Land, 1982), and in the exquisite color vision

system of the shrimp (Cheroske et al., 2003; Chiao et al., 2000; Cronin et al.,

1996; Cronin et al., 1994; Land et al., 1990). It is noteworthy that in each of these

systems scanning motions have been observed. The linear retina thus functions to scan a

larger visual field, rather than representing a highly narrowed visual field. Therefore it is

likely that the vertical retina of T. marmoratus is also specialized for scanning. However,

we have not seen movements of the tubular eyes of T. marmoratus larvae (which are well

visible through the cuticle shortly after molting) and we have not found any muscles

within the larvae that could account for the movement of eyes within the head. If the

vertical retina indeed is optimized for scanning (as are all known examples of one-

dimensional retinas), then we would predict that the larvae exhibit dorso-ventral flexions

of the entire head, during prey capture. We have indeed observed such head movements

40 at several occasions, but a detailed behavioral study will be necessary to confirm this.

Such movements would allow both, scanning of the vertical retina in the back of the tubes, and dorso-ventral movements of the narrow lateral retina of E1. The shape and position of the lateral retina along the medial border of the crystalline cone could potentially extend the visual field laterally. However, the shape of the lens and its close proximity to the retina makes it questionable if the lateral retina falls anywhere near the image plane.

In the jumping spider anterior-median eyes there are four discrete layers of retinula cells (Land, 1969b). In light-microscopical preparations the vertical rhabdoms of

T. marmoratus in contrast appear long and uninterrupted, alternating as oval or horse- shoe shaped structures in cross section. There are a large number of cell bodies visible dorsally and ventrally to the retina, which likely belong to the retinula cells that contribute to the rhabdoms. It therefore is conceivable that what appears as a long vertical rhabdom at the light microscopical level really represents a tiered structure of multiple cells, similar to the rhabdoms of R7 and R8 in flies (Nilsson, 1989). The rhabdom furthermore could be fused and contain contributions of several neighboring cells.

A clearer case of a tiered organization is found in the rhabdomeric aggregation region of E1 and E2. However, the retina of the rhabdomeric aggregation differs from other tiered retinas in that the photoreceptive region of each cell is oriented perpendicularly to the axis of light, instead of in line with the axis of light. The rhabdomeric aggregation appears to be formed from the rhabdomeric region of at least 10 cell layers of which are situated dorsally and ventrally to it. The layering of the

41 rhabdomeric aggregation itself is not apparent from general light microscopical

techniques in E1 or E2, though it can be seen in the apparently homologous structure in

E4-6. However, there is a slight stratification in the actin-density (Fig 5B) even in E1

and E2 that indicates layering of microvilli. Transmission electron microscopy will be

necessary to confirm the presence of microvilli and to clarify their layering throughout

the depth of the rhabdomeric aggregation. If the rhabdomeric aggregation in fact is

formed as one giant photoactive rhabdom, the organization is highly unusual. We were

unable to find any comparable organizations within the literature except for old

anatomical descriptions of other dytiscid larvae (Günther, 1912; Patten, 1888) where

many rows of horizontally oriented rhabdoms are indicated. The function of such an

organization remains unclear, but it could relate to optical properties of the lens and the tube as a whole. A key issue yet to be resolved is where within the eye the image is focused. Preliminary measurements suggests that the level of focus for an object at infinity may be close to the distal margin of the rhabdomeric aggregation, and that it shifts into the rhabdomeric aggregation at object distances of few millimeters. It therefore could be possible that the rhabdomeric aggregation functions as a distance detector. Optical and behavioral tests will be necessary to clarify this notion.

Slightly less extraordinary, yet still quite unusual are the remaining four larval eyes of T. marmoratus. Although each of these eyes is much closer to the typical spherical shape of a camera eye, they are slightly asymmetrical and differ from other described camera eyes (see Land and Nilsson, 2002 for a detailed review) in that there are two layers of retinas. Scallops of the genus Pecten, are one of the few examples where eyes have two layers of receptor cells (Land, 1965). However, whereas in Pecten they

42 are oriented in the same direction, they differ in T. marmoratus. As in E1 and E2, the

more conventional vertical retina lies along the back of the eye. In E3 and E4 the

rhabdoms of the vertical retina are rod shaped, fairly short, and it is unlikely that they are

tiered (based on the distribution of surrounding nuclei). At the distal end they bend

towards a neighboring rhabdom. This is most apparent in E3 where there is a single row

of straight rhabdoms in the middle; all other rhabdoms bend outward. It is unclear if

these rhabdoms act as light guides (see Warrant and McIntyre, 1993), in which case this

organization could enhance light capture. In E5 and E6, the rhabdoms of the vertical

retina are also short and presumably not tiered, but they are horse shoe shaped in cross

section. The retinas in the latter two eyes show stronger asymmetries, presumably

leading to asymmetric visual fields. The strongest asymmetry is visible in E6, which has

only two rows of vertical rhabdoms at its most anterior end, but about six rows more

posterior. The longest rows consist of more than 20 rhabdoms. The vertical retina of E6

hence also represents a narrowed retina which could function as a scanning eye.

However, in contrast to E1 and E2 (in which dorso-ventral head flexion would be

necessary for scanning) scanning would be achieved by roll movements. The most

anterior end is slightly more lateral than the posterior one. A scanning motion of this

retina would not result from dorso-ventral flexions, but would result from roll or yaw

movements of the head. A behavioral analysis will be necessary in order to asses if

larvae indeed perform such movements.

In E 3-6, proximal to the vertical retina is a layer of cells that (based on its light

microscopical organization and actin distribution), also has a retina-like organization. As in E1 and E2 this closer retina is unusual in that its rhabdoms are horizontally- oriented,

43 lying perpendicular to the axis of light. In our description of individual eyes we refer to

the orientation of the retina as horizontal or vertical based on the orientation of rhabdoms

in regards to the main axis of the eye. In terms of eye specific coordinate systems within

in each eye, the more distal retina lies horizontally, and the more proximal retina lies

vertically. Since the anatomical position of each eye differs in regards to the animal as a

whole, the actual position and orientation of the retinula cells varies from eye to eye: in

E3, E4 and E6 the retinula cells of the horizontal retinas are situated laterally and

medially to their rhabdomeric area, and in E5 they are situated anteriorly and posteriorly.

In contrast to E1 and E2, these horizontal retinas are situated closely below the lens. The

functional role of these retinas is unclear, especially since these eyes may be

underfocused in regards to the horizontal retina (personal observation). In addition to the many retinas in the twelve eyes of T. maromratus, separate retinas are present in the form

of an eye patches. Considering the absence of a lens and of screening pigment it is

unlikely that any directional information can be resolved by each eye patch. However, its

position covers the most dorsal region of the head, which is an area that is poorly covered

by visual fields of the stemmata. Only the visual field of E6 receives dorsal visual inputs,

and could overlap with the eye patch at the posterior region of the head. It is possible

that the eye patch evolved to fill into this gap, and serves to detect sudden changes in

light level above the head, such as would result from the approach of a predatory bird.

Are individual eyes task-specialized ?

We describe a variety of different eyes that together form a highly functional and

apparently specialized visual system. The larvae of T. marmoratus are active predators

44 (feeding on mosquito larvae and other small aquatic organisms) and exhibit a well

defined escape response. At least the predatory behavior is primarily visually guided. It

appears that the success of this predator lies in different but coordinated tasks of individual eyes. For example, the smaller eyes with larger visual fields could be geared towards large-field movement detection, and may be used for the initial detection of prey.

The large tubular eyes, on the other hand, may allow a larva to optimally approach and orient itself towards a prey item, and hence would be specialized for the actual attack.

Such a division of visual tasks has previously been described for jumping spiders (Land,

1985), and for Thermonectus it is supported by our own circumstantial observations.

Within each eye there are at least two retinas that are organized in very different fashions and therefore are expected to underlie different functions as well. The exact function of the horizontally oriented retinula cells remains unclear. Considering that there are three distinctly separate retinas in E1, two in each of E2-6, and one eye patch, there are a total of 14 retinas on each side. Therefore the animal could act upon the visual input of 28 distinctly different systems. Presumably each of these systems is specialized for its own specific task. A division of tasks is not unusual in systems with multiple eyes and recently has been found even in some of the most ancestral forms of life (Coates and

Nilsson, 2002; Nilsson et al., 2005). Such systems can provide important insights into visual processing, as there is a physical segregation of different functions not only in the peripheral eye but also in the nervous system that makes them relatively easier to study than unsegregated systems. It will be a challenge for the future to determine how this strange visual systems adaptively functions, and how it evolved from simpler, more typical visual systems found in other dytiscids.

45

Acknowledgements

We would like to thank Drs. John Layne and Ilya Vilinsky for helpful discussions and Drs. John Layne and Birgit Ehmer for providing valuable comments on the manuscript. We are grateful to Heather Hoy for her technical assistance and to the

Cincinnati Zoo and Botanical Garden for providing us with the initial culture of dytiscid beetles. This project was funded by the National Science Foundation (IBN-0423963).

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marginalis, Ascilius sulcatus und ihren Larven. Z Vergl Physiol 35:27-35.

Strausfeld NJ, Seyan HS. 1985. Convergence of visual, haltere, and prosternal inputs at

neck motor neurons of Calliphora erythrocephala. Cell Tissue Res 247:5-10.

Toh Y, Mizutani A. 1987. Visual-System of the Tiger Beetle (Cicindela-Chinensis) Larva

.1. Structure. Zool Sci 4(6):974-974.

Toh Y, Mizutani A. 1994. Structure of the visual system of the larva of the tiger beetle

(Cicindela chinensis). Cell Tissue Res 278(1):125-134.

Toh Y, Okamura J. 2001. Behavioural responses of the tiger beetle larva to moving

objects: Role of binocular and monocular vision. J Exp Biol 204(4):615-625.

Warrant E, McIntyre PD. 1993. design and the physical limits to spatial

resolving power. Prog Neurobiol 40:413-461.

50 Chapter Two

Measurement of The Acceptance Angles for Eyes Four, Five and Six, Structural Description of The Optic Lobes, and The Functional Organization of The Visual System in T. marmoratus Larvae

Introduction:

After studying the structural composition and disposition of the stemmata in chapter one,

I wanted to pursue studies to explain the possible functions of different components of visual systems. For example, what are the visual fields sampled by each of the stemmata?

The large tubular eyes one and two point forwards and upwards and seem to be monitoring the area of pincer like mandibles with which the larva captures the prey. So, do they function as the principle hunting eyes? Do the smaller eyes three through six, which are located in such a way that they could sample visual information from the ventro-lateral and posterior-dorsal directions, function as the movement detectors, detecting the approach of prey or predators in those directions? To determine the visual fields of each of the stemmata I have measured the acceptance angles of the stemmata four, five and six in this chapter. The acceptance angles of the stemmata one, two and three have been previously measured by Dr. Buschbeck.

To understand how visual information of individual stemmata is integrated, I wanted to study the structural composition of the visual input processing organs, the optic lobes.

The presence of distinct optic lobes in holometabolous insect larval visual system is typical for sophisticated visual systems, facilitating higher processing of visual inputs

(Toh and Mizutani, 1994b). In some other studies on different larvae, axons from the

51 retinular photoreceptor cells have been shown to be bundled into a stemmatal nerve that projects directly into the protocerebrum (Yamamoto and Toh, 1975 and Toh and Iwasaki,

1982). Distinct optic neuropils have also been shown in other well developed visual systems with single lens eyes like the wandering spider, Cupiennius salei (Strausfeld and

Barth, 1993).

Generally, in the adult insects belonging to the order Coleoptera, the optic lobes are made of four retinotopic neuropils. They are lamina, medulla, lobula and lobula plate respectively (Strausfeld, 2003). Anatomical studies on the optic lobes of the coleopteran larvae are limited. In one of the most thoroughly described studies in Cicindela chinensis larva, each of the optic lobes is made of eight neuropils, two laminas (one each for the two large stemmata), two medullas (one each for the two large stemmata) and four small neuropils one each for the four smaller stemmata (Toh and Mizutani, 1994a).

In the larvae of T. marmoratus there are two optic lobes, one on each side. General staining reveals that each optic lobe is composed of six distinct neuropils. This opens up the following question: Do optic nerves from an eye project onto an individual neuropil or different neuropils? I performed fluorescent stain backfills for each of the stemmata to trace the optic nerve projections from each of the stemmata.

Knowing that the optic nerves from each of the stemmata majorly project onto a unique neuropil, and possibly onto other neuropils too, I went on to study the detailed structural composition of each of the neuropils, using various staining techniques. This study is to understand the organization of each of the neuropils. One of the questions I address is: Are the neuropils one and two, onto which the optic nerves from eyes one and two project respectively, more complex in structure than the rest of the neuropils? One and two are the

52 most complex eyes in terms of structural composition and are connected to neuropils one and two. So, it would not be surprising that the neuropils one and two are more complex than the

other neuropils as well. This would provide evidence for the hypothesis that the eyes one and

two are principal hunting eyes because to process the essential visual inputs more accurately,

complex neuropils are required.

The other question I wanted to answer was: Are there giant neurons in abundant number in

the neuropils three though six? This would provide evidence for the hypothesis that

movement detection is an important function of the eyes three through six. Giant neurons are

often found in neuropils that process movement detection.

I have also performed 3D reconstructions of the optic lobes to better understand the

structures and location of the individual neuropils. For this purpose, I have used ethyl gallate

stained horizontal sections of a third instar larva. Again, due to their larger size, structural

details were more conspicuous.

Materials and Methods:

Bodian Staining:

Larvae were anesthetized by cooling, decapitated and some of the head cuticle was

removed. Heads were fixed in AAF (100% ethanol, 37% formaldehyde and glacial acetic

acid in the ratio 17:2:1), dehydrated, embedded in Paraplast plus (Tyco healthcare group

LP, MA., USA), and serially sectioned at 8µm. Dewaxed sections were incubated for 24h

at 40oC in 1% silver proteinate (Alfa, Ward Hill, Mass., USA) with the addition of 0.3g pure copper per solution of 50ml. Afterwards, sections were conventionally stained after

53 Bodian's (1937) original method with the following small modifications: step 3, only 2%

sodium sulfite; step 4a, gold chloride without acetic acid for 10 min.; and step 4b, oxalic

acid bath for 8 mins.

Ethyl gallate staining:

This procedure was a minor modification of Strausfeld and Seyan, (1985). Larvae were

anesthetized by cooling, decapitated and some of the head cuticle was removed. Heads

were fixed in 4% paraformaldehyde solution (EM grade; Electron Microscopy Sciences,

Fort Washington, PA., USA) in Sorensen’s phosphate buffer (Electron Microscopy

Sciences, Fort Washington, PA., USA), for about two hours. After several washes in

buffer, heads were transferred into 1% Osmium tetroxide (OsO4) solution (Electron

Microscopy Sciences, Fort Washington, PA., USA) in distilled water for one hour on ice followed by 1 hr at 20oC. Tissue was washed several times in distilled water, and finally treated with saturated ethyl gallate (1hr at 0°C and 1hr at 20°C). After staining the heads were dehydrated, embedded in Ultra-Low Viscosity Embedding Media (Polysciences,

Warrington, PA) and serially sectioned at 8 µm.

Fluorescent Dye Back Fills:

The larvae were anesthetized by cooling. The lens of the eye to be filled with fluorescent dye was detached using a minucie pin (size 00). Dextran beads, conjugated to Texas Red

(3000 MW, lysine fixable; Molecular Probes, Eugene, OR) were introduced into the eye as crystals or saturated solution. The larvae were kept in moist chambers overnight at 4˚C and the dye was allowed to be taken up by neurons. The larvae were then decapitated; some of the head cuticle removed and the heads were fixed in 4% paraformaldehyde solution in Sorensen’s phosphate buffer. After several washes in the phosphate buffer,

54 heads were dehydrated embedded in Ultra-Low Viscosity Embedding Media and serially sectioned at 8 or 10 µm.

3D reconstruction All of the 8µm thick Ethyl gallate stained sections of a given area were photographed (with an Olympus Magna Fire digital camera) and imported into Amira (Mercury computer systems, Berlin), where they were pre-aligned manually and automatically optimized within the align slices module. The number of sections was up-sampled using Amira's interpolation algorithm, specific areas were labeled manually section by section in the Image Segmentation Editor and surfaces were smoothed by using three dimensional filters.

55

Figure 9 An illustration of the procedure of measurement of optical parameters.

An illustration of the procedure of measurement of the back focal distance and image size for an isolated eye lens of T. marmoratus. Object size and object distance are known parameters, so after measuring the image size the focal length can be calculated (see text).

Measurements of the Acceptance Angles/Visual Fields:

The lens of each of the eyes four, five and six were isolated and was mounted in such a

way that it was surrounded by water on either side, using microscope cover slips (Gold

Seal Products, Portsmouth, NH; USA), red utility wax (Patterson Dental Company, St.

Paul, MN; USA) and tap water. See Fig 9 for details on the experimental set up. An

56 image (the distance between two markings on a microscope slide, measuring 2cm in length) was projected through the lens using a Nikon light microscope at 10X magnification. In the first step, the Back Focal Distance (BFD), which is the distance between the focal plane and the back of the lens, was measured. The BFD for each of the lens was measured using the difference in the heights of the stage of the microscope when the image and the back of the lens were in sharp focus respectively. (Wilson 1978).

Then, the focal lengths for each of the lenses were calculated using the formula O/U = I/f

(Land 1981). Where O is the Object size (here 2 cm, as mentioned before), U is the object distance from the lens (here 13 cm), I is the image size (here measured at 40X magnification) and f is the focal length. The image size was measured for each of the lenses using an ocular micrometer and then corrected for lens magnification using a stage micrometer. When all the three parameters O, U and I were known, the equation was solved for f. Using the focal length, the nodal points for each of the lenses were determined. The Nodal point is the point in single lens eyes that the light rays pass through without being bent by the lens (Land and Nilsson, 2002) and can be calculated using BFD and focal length. Focal length starts where BFD ends and typically extends to a point into the lens, the nodal point (Fig 10). Digital images of five ethyl gallate stained

40X magnified histology preps for the eyes four five and six were taken and the average length for each of those eyes was determined. This was repeated for different sectional planes studied. Then the values of the BFD and focal length were scaled relative to the average length for each of the eyes in different sectional planes and the acceptance angle for each is measured. The nodal points form a triangle together with the extents of the

57 retinas. The acute angles of these triangles are equivalent to the acceptance angles of the respective eyes (Buschbeck et al, 2003)

Figure 10 An illustration depicting various optical parameters of individual eyes such as the back focal length (BFD), the focal length (f) and the nodal point (N).

Results:

Visual Field/ Acceptance angle measurements in the eyes Four, Five and Six:

58 Acceptance angles for each of the six eyes were measured in relation to the vertical retinas because the focal planes for eyes three through six (and for objects closer than

3mm in the case of eyes one and two) appear to be on the vertical retinas and therefore under focused for the horizontal retinas. Acceptance angles for eyes one, two and three have been previously measured by Dr. Buschbeck. In the horizontal plane eyes one and two have acceptance angles of 32º and 52º respectively. Eye three has an acceptance angle of 85º. In the Sagittal plane the acceptance angle for eyes one and two are very narrow, 1º for each. The values are summarized in table one:

Table 1: Acceptance angles of eyes one, two and three in different sectional planes

Acceptance angle in Acceptance angle in Eye Number Horizontal plane in ºs Sagittal plane in ºs

One 32 1

Two 52 1

Three 85 ____

Here, I measured acceptance angles for eyes four five and six in frontal and sagittal planes (also in horizontal plane for eye five). BFDs were measured for five lenses each for the eyes four, five and six respectively. Image sizes, used to calculate the focal length

(see procedure), were measured at 40X for only one lens each. The average length of each eye in the respective orientation was calculated from five samples. Then, position of

BFDs and focal lengths was indicated for each eye. Nodal points form triangles with the extents of retinas, whose acute angles are equivalent to the acceptance angles.

59

Figure 11 Acceptance angles of eyes four, five and six in different sectional planes.

(A) An ethyl gallate stained frontal section of eye four is used to illustrate the acceptance angle of 115º in that plane. (B) An ethyl gallate stained sagittal section of eye four is used to illustrate the acceptance angle of 145º º in that plane. (C) An ethyl

60 gallate stained frontal section of eye five is used to illustrate the acceptance angle of

90º º in that plane. (D) An ethyl gallate stained horizontal section of eye five is used

to illustrate the acceptance angle of 50º º in that plane. (E) An ethyl gallate stained

frontal section of eye six is used to illustrate the acceptance angle of 30º º in that

plane. (F) An ethyl gallate stained sagittal section of eye six is used to illustrate the

acceptance angle of 100º º in that plane.

Figure 12 An overview of the visual fields of the stemmata in sagittal plane. The visual field of: eye one (1) is 1º, eye two (2) is 1º, eye four (4) is 145º, eye five (5) is 50º and of eye six (6) is 100º.

61

Figure 13 An overview the visual fields of the stemmata in the horizontal plane

The visual field of: eye one (1) is 32º, eye two (2) is 52º, eye three (3) is 85º and eye five (5) is 50º.

As mentioned in the procedure, the visual fields of the small eyes four, five and six are measured and the results are tabulated below. Eye four had the widest acceptance angles,

115º in the frontal plane and 145º in the sagittal plane. Eye five had acceptance angles of

90º in the frontal plane, 50º in the sagittal plane and 50º in the horizontal plane. Eye six had acceptance angles of 30º in the frontal plane and 100º in the sagittal plane. Ethyl gallate stained sections showing the acceptance angles of the eyes four, five and six in the sectional planes as mentioned in Table two are illustrated (Fig 11 A, B, C, D, E and F). I have summarized these acceptance angles in the digital photographs of the larva to

62 illustrate the visual fields in lateral plane (sagittal; Fig 12) and from above in the horizontal plane (Fig 13).

Table 2 Average BFDs; and focal lengths of the eyes four, five and six

Standard deviation for Image size in Focal Length in Eye Average BFD in BFD µm µm Number µm n=5 n=1

Eye Four 33.00 2.54 8.125 52.81

Eye Five 33.20 5.80 7.5 48.75

Eye Six 29.80 2.17 8.125 52.81

Table 3 Acceptance angles of eyes four, five and six in different sectional planes

Eye Number Sectional Plane Acceptance Angle

Eye Four Frontal 115º

Sagittal 145º

Eye Five Frontal 90º

Sagittal 50º

Horizontal 50º

Eye Six Frontal 30º

Sagittal 100º

63 Optic Lobes and Neuronal connections:

There are two optic lobes, one on each side of the larva (Fig 14 A). Firstly, the basic structure of the optic lobes is studied using ethyl gallate and bodian stained preps. Ethyl gallate staining stains the membrane dense regions and therefore exceptionally large neuronal fibers can be visualized as light colored fibers on a dark background. Bodian staining stains selected neuronal fibers with a reddish color. From basic structure studies it is found that each optic lobe has six neuropils, two of them (marked one and two in Fig

14) are larger and the other four (marked three through six in Fig 14) are smaller, as seen in the ethyl gallate stained horizontal section.

64

Figure 14 External and anatomical features of the optic lobes. (A) A second instar

T. marmoratus larva, with a more transparent external cuticle than usual, showing the optic lobes (OL; indicated with an arrow) associated with the central brain (CB).

Also seen are the eye patches (EP; indicated with an arrow). (B) An ethyl gallate stained horizontal section showing the disposition of the neuropils one though six in an optic lobe. (C) An ethyl gallate stained frontal section showing the arrangement

65 of the optic lobes (OL) on the medial side of eye one (E1). (D) An ethyl gallate

stained frontal section showing the neuronal bundles (NB), probably originating

from the cells associated with the vertical rhabdoms (VRh) of eye one, to be

projecting onto the neuropil one (1).

Figure 15 Three dimensional reconstructions of the optic lobe. (A) Dorsal view of the three dimensional reconstruction of the optic lobe. Distinct optic neuropils, one through six, are indicated by numbers 1-6. (B) A mirror image of the Ventral view of the three dimensional reconstruction of the optic lobe. Distinct optic neuropils, one through six, are indicated by numbers 1-6.

I have performed a 3D reconstruction of the optic lobes to better illustrate the size, location and structure of individual neuropils. Each of the individual neuropils are indicated by different colors in the 3D reconstruction (Fig 15) as follows: neuropils one

(blue), two (green), three (orange), four (red), five (magenta) and six (yellow).

66 Neuropil one is large, elongated and is located in the periphery, at the lateral side of the optic lobe. Neuropil two, like neuropil one, is also large, elongated and is located on the medial side of the neuropil one, adjacent to it. Neuropil three is much smaller and cylindrical in structure and is located slightly anterior on the medial side of the larger neuropils. Neuropil four is also much smaller and cylindrical in structure and is located on the medial side of the large neuropils and between the neuropils three and five.

Neuropil five is also much smaller and cylindrical and is located slightly posterior on the medial side of the larger neuropils. Neuropil six is located on the most lateral side, posterior to neuropil one and is flat and elongated in structure. It is the smallest neuropil in terms of dorso-ventral thickness.

From my initial studies, in horizontal ethyl gallate sections, a bundle of neurons from eye one seemed to be projecting onto the first neuropil (Fig 14 D). Following the serial horizontal sections of ethyl gallate stained preparations it appears that neural tracts from eyes five and six project onto the smaller neuropils that are medially located. However, this staining did not allow us to identify which of the eye’s neural bundles project onto which neuropil. Therefore, I performed fluorescent stained backfills to trace the neural projections from individual stemmata to their respective neuropils, the results of which is presented below.

67

Figure 16 Fluorescent stained neuronal projections onto to the optic lobe neuropils from eyes one, three, five and six. (A) Fluorescent stained optic lobe section, showing the optic nerve projections and terminals from eye one onto the first neuropil of the optic lobe. (B) Fluorescent stained optic lobe section, showing the optic nerve projections and terminals from eye three onto the third neuropil of the optic lobe.

(C) Fluorescent stained optic lobe section, showing the optic nerve projections and

68 terminals from eye five onto the fifth neuropil of the optic lobe. (D) Fluorescent stained optic lobe section, showing the optic nerve projections and terminals from eye six onto the sixth neuropil of the optic lobe.

Fluorescent dye back fill studies using dextran red (see methods) is helpful in tracing the neuronal connections from the stemmata onto the neuropils and also in tracing the shapes of individual fibers, including their terminals. I have performed fluorescent dye back fills in all the eyes, one through six. Preliminary studies (at least two successful backfills for each eye) confirm observations of neural tracts in ethyl gallate staining and to identify the photoreceptor axons of which eye project to which neuropils. Neuropils in Fig 16 and 18 are numbered accordingly. Individual backfill studies indicate that neural projections: from eye one project onto the neuropil one (Fig 16 A), from eye two project onto the neuropil two (Fig 18 D), from eye three project onto the neuropil three (Fig 16 B), from eye four project onto the neuropil four (Fig 18 C), from eye five project onto the neuropil five (Fig 16 C) and from eye six projects onto the sixth neuropil (Fig 16 D).

However, some giant bundles of neurons from eyes three and four seem to be projecting onto the first and second neuropils (Fig 18 C & 16 B). So, these studies suggest that neurons from eyes three and four may project onto the neuropils three and four respectively, as well as onto the large neuropils one and two. But I have only two successful preps showing this data and more studies have to be done to confirm this notion.

69

Figure 17 Fluorescent stained projections of nerve terminals in the neuropils two and four. Neuronal terminals from eye four (A) A fluorescent dye stained horizontal section of the neuropil four showing neuronal terminals (indicated by an arrow). (B) and (C) are the same as (A) except that they are photographed at different focal planes and show different neuronal terminals. (D) A fluorescent dye stained horizontal section of the neuropil two, showing neuronal terminals

(indicated by an arrow) from eye four.

70 The receptor terminals from eye four onto the fourth neuropil appear to be projecting straight. There appears that there is no branching of the receptor terminals at the tip, but there appears to be some branching before they terminate. This can be observed in the fluorescent dye stained horizontal sections of the fourth neuropil (Fig 17 A, B and C).

The receptor terminals from eye four onto the second neuropil appear as bright circle (Fig

17 D). This indicates that they may project perpendicular to the plane of the section.

71 Figure 18 Detailed anatomical features of the optic neuropils and fluorescent stained neuronal projections onto to the optic lobe neuropils from eyes two and four. (A)

An ethyl gallate stained horizontal section showing the internal organization of the neuropils (as numbered). Giant neurons (GN; pointed by arrows) that project perpendicular to the axis of the section and smaller/more typical neurons (SN; pointed by arrows) are shown. (B) A bodian stained horizontal section showing the internal organization of the neuropils (as numbered). Giant neuronal fibers that project parallel to the axis of the section are seen (indicated by arrows). (C) A fluorescent stained horizontal section showing the traced of the neuronal projections

(bright areas in the neuropils) from eye four, projecting onto the neuropils one (1), two (2) and four (4). (D) A fluorescent stained horizontal section showing the traces of neuronal projections (bright areas in the neuropil) from eye two, projecting onto the second neuropil (2).

Several details are observed within individual optic neuropils. In the horizontal ethyl gallate stained sections, neuropil one shows some giant neurons that are profusely branched in the most dorsal layer. They run parallel to the long axis of the neuropil as they can be seen as neuronal fibers in the ethyl gallate stained horizontal sections.

Towards the centre and in a ventral layer of neuropil one, there are several small neurons arranged in three layers. Their main axis projects perpendicular to the neuropil, as they lead to the appearance of many circles in the ethyl gallate stained horizontal sections (Fig

18 A). Neuropil two, in the ethyl gallate stained horizontal sections shows some

72 profusely branched giant and small neurons on the dorsal layer. They primarily project

parallel to the neuropils as they appear as fibers in the ethyl gallate stained horizontal

sections. Towards the centre and ventral surface in neuropil two, there are several small

neurons arranged in three layers (Fig 18 A). They project perpendicular to the neuropils

as they appear as circles in the ethyl gallate stained horizontal sections (Fig 18 A). There

are giant neurons in the neuropil two in all the three layers, most in the central layer, lesser in the layer on the lateral side and least in the layer on the medial side. They also project perpendicular to the neuropils, as they are circular in ethyl gallate stained horizontal sections (Fig 18 A). Ethyl gallate stained horizontal sections show giant neurons in the neuropils three, four and five. Most of them project perpendicular to the long axis of the respective neuropils, since they are circular in shape in the ethyl gallate stained horizontal sections (Fig 15 B and 18A). In the neuropil six, branched giant

neurons project parallel to the neuropil and can be seen as fibers in the ethyl gallate

stained horizontal sections. A similar general neural architecture can be seen in Bodian

staining. These giant neurons project parallel to the neuropils as they appear as fibers in

the bodian stained horizontal sections (Fig 18 B). From both bodian and ethyl gallate

stained horizontal sections, it appears that neuropil one has fewer giant neurons, when

compared to the neuropils two, three, four and five (Fig 18 A & B).

Discussion:

The visual fields measured in this study (eyes four, five and six; Fig 12) and by Dr.

Buschbeck previously (eyes one, two and three) indicate that in sagittal plane (which

demarks the dorso-ventral visual field) smaller eyes three through six have much wider

73 visual fields than the larger eyes one and two. As summarized in figure 12, the larger eyes one and two have very narrow visual fields. This is because in each of these eyes there are only two rows of rhabdoms that are part of the vertical retina. .Eye four has the largest visual field and it monitors the ventral side. Because of its large visual field, it can function as a detector for approaching prey or a predator that approaches from ventral. It can be seen that the visual fields of eyes five and six are also wide and they slightly overlap. They monitor the posterior-dorsal directions and their large visual fields could allow to detect preys or predators from that direction.

Because the visual fields of the larger eyes one and two are extremely narrow in the dorso-ventral plane (Fig 12) and there are no scanning movements observed within those eyes, it can be expected that the larva moves its entire head in dorso-ventral direction to scan the environment with the eyes one and two. This is evident from the repeated dorso- ventral flexions that the larva shows before capturing the prey as observed from preliminary behavioral studies.

As summarized in Figure 13, in horizontal plane the visual fields of eye one and eye two overlap greatly and they monitor the area above the mandibles, with which the larva captures the prey. Eyes one and two appear to be the principal hunting eyes; this is supported by my personal observations of hunting behavior which indicates that before prey capture, the larva orients itself such that the prey is in the visual field of the large tubular eyes.

The visual field of eye three in the horizontal plane is larger than those of eyes one, two and five. It also overlaps with the visual fields of eyes one and two. These facts suggest

74 that eye three might play a role in the strike process of the hunting behavior and as well in detect prey or a predator, by monitoring areas immediately adjacent to the large tubular eyes in the anterio-lateral direction. The visual fields of eyes one and two overlap also

overlap in the horizontal plane, suggesting that they may work together in the prey

capture. The wide visual field of eye five monitors the posterio-dorsal area, suggesting

that it can play a role in detection of prey or a predator approaching in those directions.

From the optical parameters measured and summarized in Figure 11, it can be noticed

that for eyes four, five and six; the images are under focused for the horizontal retinas.

Acceptance angles would be wider, if measured in terms of the horizontal retinas because

of their wider extension than the vertical retinas. In eye four, the image is slightly under

focused even for the vertical retina. This suggests that it may not capture a high

resolution image, but it still could detect coarse images with high contrast. Because it has

the largest visual field it monitors a large area on the ventral side and could play a key

role in detection of prey or a predator in that direction. Eyes five and six also have wider

visual fields, suggesting that they can monitor larger areas in the posterior-dorsal

directions.

In T. marmoratus larva, the presence of multiple stemmata, with each stemmata

consisting of more than one retina, and an eye patch on each side indicates that visual

information from different directions are processed by separate units. This is evident

from the fluorescent stained backfill studies which suggest that nerves projecting from

stemmata one through six project onto the optic neuropils one through six respectively

75 (Fig 16 A, 18D, 16 B, 18 C, 16 C and 16 D). But the large neuropils one and two may be

composed of neuronal projections from other eyes as well, as observed in the case of

backfills of eyes three and four (Fig 16 B and 18 C), whose neural bundles project onto

the neuropils three and four respectively; in addition to the neuropils one and two. But I have limited number of the preps (only two), and the experiment has to be repeated for a definitive answer.

The neuropils one and two are much larger than the rest of the neuropils and have many neural projections arranged in three rows (Fig 18 A). Since neural projections from eyes one and two project onto the neuropils one and two respectively, they may process visual information majorly from those eyes. But since the fluorescent backfill studies in eyes three and four indicate that eye three might have some neural terminals projected onto the neuropils one and two; and eye four might have some giant nerve terminals projected onto the same neuropils, they might also process visual information from the smaller eyes

(Fig 16 B and 18 C). The smaller neuropils three though six have giant neurons throughout their thickness (Fig 18 A & B) and backfill studies show that neural bundles from eyes three through six project on to them respectively. This suggests that those eyes may play an important role in movement detection because giant neurons are essential for movement detection and consequential quick reflexes (Mizutani and Toh. 1995).

To summarize, eyes one and two have smaller visual fields, but in horizontal plane, they monitor the area above the mandibles, they are connected to larger and more complex neuropils, than the eyes three, four, five and six. It indicates that they may function as the principal hunting eyes. This is also supported by some of my preliminary behavioral observations. Large visual fields of eyes three, four, five and six suggest that they may

76 play an important role in movement detection. This is supported by the presence of giant neurons in the neuropils three through six (Fig 18 A & B). The visual field of eye three overlaps with that of eye two (Fig 13) and hence eye three may play a role in hunting behavior as well.

General Discussion:

The structural details of the stemmata in T. marmoratus larvae greatly diverge from other described eyes. The most distinguishing features are (a) the classification of stemmata into three anatomically different categories, (b) the presence of three retinas in eye one and two each in the rest of the eyes, (c) the presence of photoreceptor cells orienting perpendicular to the axis of light and (d) the existence of an unusually large crystalline cone in the large tubular eyes one and two, as compared to other stemmata studied, separating the lens from the photoreceptive rhabdomeric aggregation region and the vertical retina.

The large tubular eyes are more unique than the rest of the eyes. They have some similarities with other described eyes like the two largest stemmata in the larvae of tiger beetles, in being the two largest among the six eyes on each side, (Toh and Mizutani,

1987; Toh and Mizutani 1994) and the anterior median (AM) eyes of jumping spiders

(Land, 1969b; other comparative eyes are described in the discussion of chapter one). But they are unique because the former compared eyes are more or less typical symmetric stemmata, each with just one cup shaped retina and the latter also have just one retina each, though more comparable because the retinas are narrow. Based on the position and

77 general morphology, I predict that the eyes one and two in T. marmoratus are the

principal hunting eyes just like the AM eyes in the jumping spiders. To compensate for

the narrow visual fields caused by narrow retinas, AM eyes in jumping spiders show

lateral scanning movement of the retinas (Land, 1969a). In T. marmoratus larva, eyes one

and two also have extremely narrow visual fields in the dorso-ventral direction because

of narrow vertical retinas. So, I pursued to investigate for scanning movements of the

retinas (as in AM eyes of the jumping spiders) and such movements are not observed in

T. marmoratus (personal observation). If the retinas are steady in the head, one way to

increase the visual fields would be to move the entire head to visually scan the

environment. So, one can expect that the larvae would exhibit dorso ventral flexions of

the entire head. In fact, I have actually observed such movements from my preliminary

behavioral observations.

The other eyes three through six also have two retinas each. Eyes three and four appear to

be with more or less symmetric retinas, whereas the retinas of eyes five and six are more asymmetric. A narrow retina in eye six (like vertical retinas in eyes one and two) indicates that it may also be involved in some scanning of the environment, which could be achieved by roll movements or side to side movements of the head. This possibility needs to be confirmed by behavioral analysis.

Though the eye patch is without lens, its position covering the dorsal region of the head along with eye six, I suggest that it may play an important role in detecting the change in light intensity like, for example, during the approach of a predatory bird.

78 The position and orientation of the eyes can also play an important role in determining

the specific function of each. The large tubular eyes one and two are located anterior- dorsally and point upwards, monitoring the area above the mandibles, with which the larva captures the prey, in the horizontal plane. This suggests that they can function as the principal hunting eyes and is supported by my preliminary behavioral observation that before the prey capture, the larva orients itself such that the prey is in the visual field of the large eyes one and two.

Eye three is located laterally (slightly ventral) on the anterior side, suggesting that eye three may also play a role in the prey capture. This is supported by the fact that the visual field of eye three overlaps with the visual fields of eyes one and two, to an extent. But because of its wide visual field which allows to monitor a large area of the environment, eye three can play a role in movement detection. Eye four is located ventro-laterally and optical measurements suggest that the image in eye four might be focused slightly behind the vertical retina, leading to blurred vision even though a much clearer image in projected by the lens. But eye four has the biggest visual field, suggesting that it might play a key role in movement detection. Eyes five and six are posterior-dorsally located and have large visual fields and therefore, can play a role in movement detection. The visual fields of the large eyes one and two are very narrow in the dorso-ventral plane, and in fact the dorso-ventral flexions they exhibit could be to scan the environment with the eyes one and two. This possibility should also be confirmed by behavioral analysis.

As discussed before, three retinas in eye one, and two each in eyes two through six and an eye patch on each side, together forms fourteen photoreceptive areas on each side. So,

79 with 28, in total, sources to bring in visual inputs, it is not surprising that the larvae would

possess complex optic lobes to process the visual stimuli. Based on the fluorescent dye

backfill studies I suggest that optic nerves from eyes one through six project on to the

neuropils one through six respectively. Since the eyes one and two are much larger than

the rest, and the neuropils one and two are much larger than the rest, it can be suggested that eyes one and two might capture a lot of visual inputs which require complex neuropils for processing. This supports the idea that the large eyes one and two could be the principal hunting eyes. Larger neuropils one and two, may also process some visual inputs from eye three and four, as suggested by the fluorescent backfill studies.

Distribution of giant neurons throughout the thickness of small neuropils suggest that the eyes three though six could be important for movement detection, since giant neurons are essential to detect movement and also to elicit quick reflexes (Mizutani and Toh, 1995).

To summarize, the idea that the large tubular eyes one and two in T. marmoratus could be the principal hunting eyes is supported by: (a) their anterior disposition monitoring the area of the mandibles (b) preliminary behavioral observation that the larva orients to bring the prey into their visual field (c) connections with large complex neuropils and (d) comparative equals, the AM eyes in the jumping spiders. The idea that the smaller eyes three through six could be important in movement detection is supported by: (a) their ventral, lateral and dorso posterior location, monitoring various directions for an approaching prey or predator (b) their wide visual fields and preliminary behavioral observations that if the prey is in the their visual fields, the larvae orient to bring the prey

80 into the visual field of eyes one and two (c) presence of giant neurons in the neuropils

three through six, all along their thickness.

Such a division of labor is not uncommon in visual systems with multiple eyes, as has

been recently discussed in the case of upper and lower lens eyes on each of the

very ancestral box jellies (Nilsson et al; 2005). Since resolution and sensitivity are two

important functions to determine the performance of an eye, (Land and Nilsson, 2002)

some of the eyes in multiple eye visual systems may contribute more to resolution (like

the large tubular eyes one and two in T. marmotatus with long rhabdoms in ventral retina

and narrow visual fields) and the other could contribute more to the sensitivity (like the

eyes three through six in T. marmoratus with large visual fields) and therefore, multiple

eyes on each side could compensate for the short comings of each other and

synergistically be well adapted for visually guided prey capture.

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