Toward unraveling the mystery of how the unusual principal eyes of marmoratus larvae work – constructing a first functional model.

A dissertation submitted to the

Division of Research and Advanced Studies

of the University of Cincinnati

In partial fulfillment of the requirements for the degree of

Doctorate of Philosophy (Ph.D.)

In the department of Biological Sciences

of the College of Arts and Sciences

2013

by

Annette Stowasser

B.S., Xavier University, Cincinnati 2007

Committee Chair: Elke K. Buschbeck, Ph.D.

1 Abstract

One might ask what one could possibly gain from exploring the function of tiny larval eyes? And one might answer that an exploration of the structure of such eyes and of the mechanical properties that evolved under their taxing size constraints has already served to inspire engineers in devising novel methods of navigation and visual task performance in robots. Moreover, an exploration of their specializations has also served to facilitate the study of very particular aspects of vision that can be found across many other species, including and humans.

Thus far, attention has been largely focused on adult compound eyes, but also have chambered eyes, which should probably merit no less attention.

Contrary to what is generally thought about these eyes, some of these visual systems are highly developed and complex. They too may result in the discovery of novel visual mechanisms, which may also serve to inspire engineers or to provide us with a more complete understanding of vision as well as of the evolution and development of eyes in general.

Among the most interesting of these eyes are the principal eyes, E1 and

E2, of (Coleoptera:) larvae. In previous studies of the behavior of these larvae, the anatomy of their eyes, the spectral sensitivity of their and the ultrastructure of the of their principal eyes, their visual system have been shown to be unlike any other known eyes. They are unique in that they are tubular in shape and have a very complex layered retina in which the photoreceptor cells of the proximal retina (PR) are sensitive to UV light while those of the distal retina

(DR) are sensitive to green light. The DR consists of many layers of photoreceptor cells

2 whose rhabdomeric portions are oriented perpendicular to the light path. Additionally, the retina is linear with a visual field that is a horizontal stripe. Due to the highly unusual construction of these eyes, some of the results were hard to interpret and raised additional questions as to how these eyes actually functioned in the context of both the behavior of the larvae and the visual challenges with which they were faced.

For the purposes of this dissertation, a series of experiments was carried out to address the questions raised by the aforesaid past results and to elaborate a first conceptual model of how these eyes might function (Chapter 5). To that end, the optical properties of the of these eyes were measured in order to ascertain where images are focused within these eyes, based on object distance and the spectral sensitivity of the retina (Chapter 2 and 4). The polarization sensitivity of the proximal retina was also measured (Chapter 3) for confirmation of its polarization sensitivity as suggested by the ultrastructure of its cells.

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4 Acknowledgements

I very much wish to thank my advisor, Elke K. Buschbeck, and my committee members, Dr. Tiffany Cook, Dr. Edwin R. Griff, Dr. George W. Uetz and Dr. John E.

Layne, for their academic support in this project, which was so perfectly suited to my passion for puzzles – especially those in which it is a wonder how the pieces ever fit together. I also wish to thank my advisors, Dr. Elke K. Buschbeck and Dr. John E.

Layne, for their very special support in personal matters, support well in excess of anything that I had a right to expect.

I thank the Buschbeck and the Layne lab groups as well as Dr. Ilya Vilinsky for helpful discussion; and, again, Dr. John E. Layne, for having lent me various lab equipment that included a rotating arm, camera, goniometer and still other things too numerous to mention. I thank Marisano J. James for his help in editing the polarization sensitivity manuscript. I thank Randy Morgan and the Cincinnati Zoo & Botanical

Gardens for providing the original population of Sunburst Diving ; Shannon

Werner and Emily Jennings for their help in care. I thank Doug J. Kohls, and

Necati Kaval for providing technical assistance with scanning electron microscope imaging; Birgit Ehmer for her help with confocal and TEM imaging; and Peter Müller for helpful tips regarding the laser method. I also thank the anonymous reviewers who provided valuable comments that greatly improved the manuscripts.

I offer a very special and heartfelt thanks to the families Wolfley, Hunt and

Simerlink. This project would never have been completed without them as it would have been impossible for someone in my position—a single mother and graduate student with no extended family for support—to manage all my personal, professional

5 and academic obligations. They were there to help my children whenever I could not, knowing that I might never be able to repay them in kind.

Another person who helped in invaluable ways was Rick Johnson. He took an enormous load off my shoulders when he undertook, of his own unprompted accord, to make sure that my son stayed on track at school during the last, busiest and most critical half-year of my project.

Many thanks also go to Lauren Simerlink, Edwin Hunt, Nadja, and Heiko

Stowasser (high school students or soon to be college freshmen) for having spent countless hours proofreading and participating in helpful discussions. Their expertise and keenness for a real taste of what awaits them in the future was most impressive. In this context I also would like to thank John Galvin very much for a final proofreading of this document.

Finally, there is no way to thank my children Nadja and Heiko Stowasser enough for all their understanding, patience and help, and I apologize with all my heart for not having always been the mother I had originally so much wanted to be.

All scientists—but biologists in particular—should probably stop, from time to time, and remark on how rare is life in the universe. In that spirit, I wish to thank my larvae for their sacrifice. However small and uncomprehending they were, they were precious life and little heroes to my cause.

Funding

This work was supported by the National Science Foundation under grants IOS0545978 and IOS1050754 and a University Research Council summer graduate fellowship to AS.

6 Contents

List of Abbreviations ...... 10

Chapter 1: Introduction ...... 11 Introducing Sunburst diving beetles ...... 14 What we need to know to understand how an eye works ...... 15 The visual system of T. marmoratus – past results and the subjects of this dissertation .... 17 Anatomy ...... 18 Spectral sensitivity ...... 20 Ultrastructure of the retina and polarization sensitivity ...... 23 Hunting behavior ...... 27 Range finding mechanisms ...... 28 Optical properties of the lenses of E1 and E2 ...... 29

Chapter 2: Biological bifocal lenses with image separation ...... 33 Summary ...... 34 Results and Discussion ...... 35 Sunburst Diving Beetle Larvae Have Tubular Eyes with Two Retinas ...... 35 Two Independent Methods Reveal the Existence of a Bifocal ...... 37 The Bifocal Lenses Might Function as “Two Eyes in One”...... 42 Image Disparity Might Allow Larvae to See Prey Better ...... 42 Experimental Procedures ...... 45 Animals and Lenses ...... 45 Image Contrast Measurements ...... 46 Laser Measurements ...... 48

Chapter 3: Electrophysiological evidence for polarization sensitivity in the camera-type eyes of the aquatic predacious larvae Thermonectus marmoratus (Coleoptera:Dytiscidae) ...... 51 Summary ...... 52 Introduction ...... 53 Materials and Methods ...... 59 Animals ...... 59

7 preparation ...... 59 Intracellular recording and neurobiotin iontophoresis ...... 61 Optimal e-vector orientation ...... 63 Quantifying polarization sensitivity ...... 64 Histology ...... 65 Results ...... 66 Response to changing e-vector orientation ...... 69 Polarization sensitivity measurements ...... 71 Discussion ...... 74 Polarization sensitivity in ...... 74 Evidence for two cell types that are sensitive to vertically polarized light ...... 77 Functional considerations ...... 80

Chapter 4: Multitasking in a larval eye: How the unusual organization of the principal eyes of Thermonectus marmoratus allow far and near vision and might aid in depth perception...... 82 Summary ...... 83 Introduction ...... 84 Material and Methods ...... 88 Animals ...... 88 Histology and anatomical measurements ...... 88 Optical measurements ...... 90 Optical calculations and modeling ...... 93 Results ...... 98 Anatomical measurements: E1 is longer than E2 ...... 98 Optical measurements: E1 is also bifocal and has longer focal lengths than E2 ...... 100 Each retina receives its own image ...... 103 Discussion ...... 108 Challenges in establishing accurate image positions within each eye ...... 109 Eye organization allows for near and far vision ...... 112 Eye organization could allow unilateral rang finding ...... 113 Depth from defocus or image shift ...... 114 Depth from changes in image size ...... 116

8 Multitasking taken into ecological context ...... 117

Chapter 5: A conceptual model illustrating how the visual system of Thermonectus marmoratus larvae functions based on its anatomy, physiological properties of the retinas, optical properties of its lenses, and the behavior of the larvae ...... 119 Introduction ...... 120 Conceptual model illustrating how the visual system might assist in prey capture ...... 125 Initial prey detection ...... 125 Stalking and approaching prey ...... 130 Range finding and prey capture ...... 131 Possible additional functions of E1 and E2 in the context of the natural history of this larvae ...... 134 To conclude ...... 135

Glossary ...... 137

References ...... 141

9 List of Abbreviations

E1,2 principal eyes one and two of T. marmoratus larvae

PR proximal retina, further away from the lens

DR distal retina, close to the lens

T1,2,3 types of the PR f’ focal length, distance between the second principal plane and the

focal point of the lens b.f.l. back focal length, distance between the back surface and

the focal point of the lens h2 distance between the second principal plane and the back surface of

the lens do object distance measured from the front surface of the lens di image distance measured from the back surface of the lens si image distance measured from the first principal plane of the lens si image distance measured from the second principal plane of the lens yi image size yo object size c maximal allowed blur circle diameter

A aperture diameter

10 Chapter 1 Introduction

Vision is one of the most complex senses, and its complexity makes scientists wonder how visual perception is formed. However, it is precisely this enormous complexity that makes it difficult to study its underlying mechanisms, especially in . On the other hand, in invertebrates such as , spiders, or crustaceans, size constraints lead to visual systems that are relatively small, less complex, highly efficient and compact. In addition, visual systems are often, at least in some aspects, specialized, making them the ideal systems to investigate for specific aspects of vision.

Many times, investigations of invertebrate systems have greatly advanced our general knowledge of vision and opened up new avenues of design for engineers.

By way of example, one of the most fundamental visual processes found in vastly different organisms is the inhibition of photoreceptors by neighboring photoreceptors, which takes place at the level of the retina. This process, called lateral inhibition, was first defined in the crustacean Limulus and is the basis of fascinating optical illusions. It causes stripes, for example, to appear darker or lighter at their borders and can cause mesmerizing color misperceptions. This mechanism greatly improves our ability to recognize the brightness or color contrasts and results from inhibitory interactions among neighboring photoreceptors occurring within the retina (Wagner and Ratliff,

1956; Barlow, 1969).

In other instances, the stream-lined organization of specialized invertebrate visual systems has made them valuable models for engineers. For example, the way insects visually maneuver in flight gave rise to the development of autonomous visual

11 air-craft guidance systems (Wu, 2012; Srinivasan, 2004). The compound eyes of insects inspired the development of miniature cameras with a wide field of vision and wide depth of field that resembles the organization of these insect eyes (Duparré et al.,

2008; Kawano et al., 2012; Nakamura et al., 2012). Strepsiptera, an insect that has eyes composed of many individual eyelets, each of which contributes a little portion to the whole image perceived (Buschbeck et al., 1999), became the model for yet another type of camera with a wide field of vision (Druart et al., 2009). The visual navigation strategies of insects also became models for the navigation system of mobile robots

(Lambrinos et al., 2000; Srinivasan et al., 1999; Srinivasan et al., 2011). For good reason, then, Wolpert (2009) pointed out that biological visual systems were great sources of inspiration and that engineers would find it worth their while to pay even more attention to these systems than they already had.

In the past, attention was largely focused on adult invertebrate eyes like the compound eyes of insects. Yet there are other types of eyes that deserve attention as they may also help to solve visual tasks in very effective, though possibly very different, ways. One such group of eyes are the very diverse chambered eyes of holometabolic insect larvae, called stemmatae. These eyes are much understudied, most likely because they are generally thought to have only poor vision and to perform only basic visual tasks. Nevertheless, there are some stemmata that are highly developed

(Gilbert, 1994). Possibly some of the most complex stemmatae are the principal eyes of E1 and E2 of an aquatic visually-guided predator: Thermonectus marmoratus

(Coleoptera: Dytiscidae). The remarkable ability of the larvae to visually detect small prey, such as larvae, and to estimate striking distances even while swimming,

12 suggests that the larvae have complex visual perception (Buschbeck et al., 2007; Bland et al., under revision). The unusual construction of their eyes suggests the presence of novel visual mechanisms, which make them an intriguing system for extensive investigations into how these eyes might function.

As aquatic visually-guided predators with a small visual system, T. marmoratus larvae are faced with extraordinary challenges. Their eyes must have a resolution high enough to be able to clearly see miniscule prey like mosquito larvae. Additionally, visibility under water is often quite poor. Nevertheless, they must be able to correctly gauge the distances of their prey while both they and their prey may be subject to unpredictable water perturbations. The fact that T. marmoratus larvae are such successful hunters shows that their visual system masters all of these challenges. Their bizarre eye organization implies that they might master at least some of these challenges in very unusual ways. A knowledge of how these eyes function should give us a more complete understanding of vision in general and provide engineers with new approaches to visual tasks in computer vision. Through subsequent comparative studies, this intriguing and highly specialized visual system may also lead to a more complete understanding of the development and evolution of eyes and their specializations.

The goal of this dissertation is twofold: to add to our knowledge of these eyes through investigations intended to answer questions raised by previous work (Chapter

2, 3 and 4); and to synthesize the results into a first comprehensive conceptual model illustrating how these eyes function both in the context of the behavior of this animal and in the context of the visual challenges with which it is faced (Chapter 5).

13 Introducing Sunburst diving beetles

Thermonectus marmoratus, shown as adult and larvae in Figure 1, belongs to the family of predacious diving beetles (Coleroptera: Dytiscidae) and is native to the Southwestern

United States. Its primary habitat consists of small freshwater streams and ponds

(Larson et.al.,2009; Morgan, 1992a). Like all beetles, it is a holometabolous insect, meaning it goes through complete metamorphosis like a butterfly or moth.

Thermonectus marmoratus has three larval stages, followed by a pupation stage, from which it emerges as a fully developed beetle (Morgan, 1992a). Adults prefer to spend most of their life in the water, only leaving their aquatic habitat for oviposition near the water, for dispersal or for escape from unfavorable environmental conditions. In contrast, the larvae are only found outside the water shortly after hatching, while seeking water, and directly prior to their pupation near the water (Morgan, 1992a).

Figure 1 T. marmoratus. A: an adult beetle. B: a third instar (third larval stage). C: the head of a third instar larva with the principal eyes, E1 and E2, as the dominant eyes, pointing directly forward.

14 The brightly colored beetles are primarily scavengers, but might also prey on live animals, including their own offspring. Their larvae, on the other hand, are ferocious visually-guided predators that prey on animals, such as mosquito larvae, and even each other (Morgan, 1995, Morgan 1992a, Velasco and Millan, 1998). The larvae have six eyes on each side of their head. The eyes that are dominant for hunting are E1 and E2 and point directly forward (Buschbeck et al., 2007). It is the unusual organization of these eyes which first drew attention to their visual system (Mandapaka et al., 2006) and which initiated ongoing research into how these eyes might function.

What we need to know to understand how an eye works

Vision is the acquisition of spatial information about the environment through the processing of light. In chambered eyes, such as the eyes of T. marmoratus larvae, this is a matter of a lens projecting an image onto a retina, the light sensitive part of the eye.

The retina consists of photoreceptors, which are sensory cells that generate electrical signals in response to light. These electrical signals are subsequently relayed to the central nervous system, where they are processed to provide visual perceptions. The resulting visual perception is also influenced by eye movement (Figure 2). In fact, visual perception of the environment (vision) is dependent on five basic components:

1. Eye movement and the behavior of the animal, which determine what kind of visual

stimulus the animal processes.

2. The optical properties of the lens, which determine the magnification and quality of

the image that is projected onto the retina.

15 3. The overall organization of the eye and the position of the lens relative to the retina,

which is important in ensuring that the image projected by the lens is focused on the

retina.

4. The organization of the retina, including its spectral sensitivity, polarization sensitivity

and temporal resolution, which determines what kind of image information will be

sensed and passed on to the higher processing mechanisms of the brain.

5. The higher-order neuronal visual-processing mechanism, which mediates the

resulting visual perception.

In order to understand the mechanisms that form the resulting visual perception, we must examine the functions of all the components and their interactions.

Figure 2. Basic schematic of the components mediating visual perception in a chambered eye.

16 It is well known, especially in small specialized visual systems, that image information often undergoes rigorous filtering at peripheral levels. Frequently, only truly relevant image information is passed on to the central nervous system so that small brains are not overloaded with information (Wehner 1987; Land and Nilsson 2006).

Thus, to understand the visual mechanisms of such systems, it is important to begin by investigating peripheral visual mechanisms where a significant portion of visual processing may already take place.

The visual system of T. marmoratus larvae – past results and the subjects of this dissertation

The first description of the anatomy of the larval eyes of predacious diving beetles

(Coleoptera: Dytiscidae) dates back to 1887 and 1888 when Patten described the larval eyes of the (Coleoptera: Dytiscidae) species (Patten, 1887,1888). Later, Günther

(1912) described the larval eyes of Dytiscus (Coleoptera: Dytiscidae). Schöne (1951 and 1953) then undertook further investigation of the Dytiscus and Acilius larvae’s color sensitivity and orientation to light in behavioral experiments. Aside from these, there are only a very few other historical accounts of investigations into the visual system of

Dytiscidae larval eyes. Several decades later, however, Morgan (1992a, 1992b, 1995) discovered that T. marmoratus was an excellent candidate for public display due to their beauty, interesting adult and larval behavior, and easy maintenance in captivity. It was then that the astonishing visually-guided hunting behavior of these larvae finally drew significant attention to their most unusual eyes, E1 and E2. Soon after, comprehensive investigations into how this visual system might actually work began by first examining

17 the anatomy (Mandapaka et al., 2006), spectral sensitivity (Maskimovic et al., 2009;

Maksimovic et al., 2011) and ultrastructure of the retina (Stecher et al., 2010), as well as the hunting behavior of the larvae (Buschbeck el al., 2007; Bland et al., under revision).

The results of these investigations revealed that this visual system was very different from that of any other known eye. The following paragraphs briefly summarize past results and outline my contributions (Chapter 2, 3 and 4) to a proposed first conceptual model (Chapter 5) of how these eyes might function.

Anatomy

The anatomy of the visual system of first instar larvae of T. marmoratus was investigated by Mandapaka et al. (2006). Their findings revealed that the most unusual aspect of the system was to be found in the principal eyes, E1 and E2, which are chambered, elliptical and tubular in shape. Their organization is illustrated in Figure 3 after Mandapaka et al. (2006). Unlike most other known chambered larval eyes of holometabolous insects, where the photoreceptors are positioned directly beneath or very close to the lens (Gilbert, 1994), these eyes are characterized by the great distance between the back surface of the lens and the retina.

Additionally, the eyes of T. marmoratus larvae have retinas that are divided into at least two distinct areas: a distal retina (DR) and a proximal retina (PR). The distal retina has photoreceptor cells which form two horizontal rows of stacked cells. This is very different from the structure of any known eye, since the light sensitive parts of these cells (the rhabdomeres) are oriented perpendicular to the incoming light. The proximal retina lies directly beneath the distal retina and consists of two horizontal rows

18 of photoreceptors which are oriented parallel to the direction of the incoming light

(Mandapaka et al. 2006). The distinct layering of the retina and its great depth raised two important questions: 1) where is the image focused within the eye? and 2) more importantly, what is the function of the retina layering?

Figure 3. Schematic of the basic organization of E1 and E2 with the lens (L), the crystalline cone

(CC)—a transparent tissue that fills the space between the lens and the retina—the distal retina

(DR) and the proximal retina (PR). The white lines illustrate the approximate angular visual field of the retinas in the horizontal and vertical planes. a illustrates the eye organization and photoreceptor orientation in the horizontal plane (horizontal section). b illustrates the eye organization and photoreceptor cell orientation in the vertical plane (sagittal section) (after

Mandapaka et al. 2006).

19 Spectral sensitivity

One property of light is its wavelength, which can vary as it is reflected, transmitted or absorbed by different objects. To gain insight, then, into what an animal can “see” of its surroundings, it is important to determine the wavelengths to which its visual system is sensitive.

Photoreceptors cells are generally limited to sensing a specific range of wavelengths (spectrum of light), which depends on the kind of photosensitive receptor they express (Land and Nilsson, 2001). The wavelength-dependent sensitivity of a photoreceptor is called its spectral sensitivity. Examining the spectral sensitivity of the photoreceptors of a retina is important because it reveals what kind of spectral visual information the animal can process within its environment.

For eyes that have an extensively layered retina, such as those of T. marmoratus larvae, there is an additional reason for investigating the spectral sensitivity of the retina. Lenses generally have slightly different focal powers for different wavelengths of light. Typically, the shorter the wavelength, the higher the focal power of the lens, with the result that images of objects illuminated by light of short wavelengths (UV, violet, blue) are focused closer to the lens than images of objects illuminated by light of longer wavelengths (green, yellow, red), as illustrated in Figure 4. This wavelength dependency of the focal power of lenses is called chromatic aberration (Hecht, 2001).

20

Figure 4 Chromatic aberration. Typically images of objects illuminated with light of shorter wavelengths are focused closer to the lens than images of objects illuminated with light of longer wavelengths.

To allow focus over a wide range of wavelengths, photoreceptors could be positioned at different distances from the lens, depending on their spectral sensitivity, to correct for chromatic aberration.

In the size range of the eyes of T. marmoratus larvae, only one other animal is known to have eyes with an extensively layered retina: jumping spiders (Land, 1969). It is thought that one of the functions of retina layering in jumping spiders is to correct for chromatic aberration (Land, 1969; Blest et al., 1981) because, in these eyes, UV sensitive photoreceptors are positioned closer to the lens than green sensitive photoreceptors (Blest et al. 1981; Land 1969).

To determine which kind of spectral information T. marmoratus larvae can process and whether or not the retina layering might have a similar function to that of

21 jumping spiders, Maksimovic et al. (2009 and 2011) explored the spectral sensitivity of these larvae. Their findings revealed that the spectral sensitivity of the retina of the principal eyes, E1 and E2—in contrast to that of jumping spider—was the very opposite of what would be expected for the correction of chromatic aberration: photoreceptors of the proximal retina are UV sensitive, while the photoreceptors of the distal retina are green sensitive (Figure 5).

Figure 5. Spectral sensitivity of the principal eyes, E1 and E2, of T. marmoratus. The distal retina (DR) of T. marmoratus is most sensitive to green light, while the proximal retina (PR) is most sensitive to UV light (after Maksimovic et al. 2011; Maksimovic et al. 2009).

This finding was difficult to interpret. A focused image must be projected onto the retina for good vision and this system seemed, not to correct for chromatic aberration, but to exacerbate it.

In Chapter 4, I measured the chromatic aberration of the lenses of the principal eyes, E1 and E2, of T. marmoratus for wavelengths that match the spectral sensitivity of the retina (UV and green light), and I explored whether there might be an alternative functional advantage to the peculiar spectral-sensitivity organization, an advantage different from that which had been suggested in jumping spiders.

22 Ultrastructure of the retina and polarization sensitivity

Another property of light is its polarization. Light spreads as waves and its polarization is the direction in which the waves ‘swing’. If light waves swing randomly in all directions, then the light is said to be unpolarized, but when most or all of the waves swing in the same direction, the light is considered either partially or fully polarized

(Figure 6). While humans cannot see the polarization of light, there are many animals that are sensitive to this property and use it to gain information about their environment.

As light is reflected, filtered or scattered by the environment, its polarization changes; thus, this property of light can serve as a valuable visual cue (Horváth and Varjú, 2004).

Figure 6. Polarization property of light. If light waves swing in all directions, the light is said to be unpolarized. If most or all of the waves swing in one direction, the light is considered either partially or fully linearly polarized, and if the direction of the swing changes (rotates) over time, the light is deemed circular polarized. The polarization direction of the light is also called the e- vector.

The eyes of many invertebrates are particularly sensitive to polarization. Their photoreceptor cells can be organized so that they are most sensitive to light that is polarized in a certain direction. Invertebrate photoreceptor cells consist of a cell body

23 and a light sensitive rhabdomere. Polarization sensitivity is achieved through a parallel arrangement of the rhabdomeric membrane, made of actin-based microvilli, where the photosensitive receptors are located. A photoreceptor with microvilli arranged in parallel is most sensitive to light that has a polarization direction (e-vector) aligned with the orientation of the microvilli (Figure 7) (Cronin, 2006; Sabbah et.al., 2005; Horvath and Varju, 2004).

Figure 7 Side view schematic of a typical

invertebrate photoreceptor with its cell body and

microvilli, which contain the photosensitive

receptors. In this illustration, the microvilli have a

parallel organization such that this cell would be

polarization sensitive. It would respond most

strongly to light with a polarization direction (e-

vector) that is parallel to the microvillar

orientation as illustrated by the blue arrow.

24 Generally, polarization-sensitive invertebrates have several types of photoreceptors, all of which have a different microvillar orientation. This allows for comparison between the responses of the different types of photoreceptors in finding the polarization direction of the light (true polarization vision). This mechanism is similar to color vision. For example: if blue receptors respond more strongly than green receptors, the object must be more bluish than greenish; and similarly the polarization direction can be found by integrating the responses of photoreceptors that have different polarization sensitivity (Bernard and Wehner, 1977). However, polarization sensitivity can also have much less sophisticated, though nevertheless important, functions. In aquatic environments, for example, visibility and contrast can be enhanced by simply filtering out the mostly horizontally polarized haze, created by the scattering of sunlight in the water (Lythgoe and Hemmings, 1967; Cronin and Marshall, 2011;

Johnsen et al., 2011). Additionally, many otherwise transparent animals have polarization-active body parts that increase their visibility to a polarization-sensitive predator (Shashar et al., 1998; Shashar et al., 2000; Johnson et al., 2011). In fact, polarization sensitivity is widespread in aquatic animals, which demonstrates its importance to animals living in this environment. A more comprehensive review of polarization sensitivity and its functions is given in Chapter 3.

One function of the layering of the retina in T. marmoratus larvae could be that the various layers extract different kinds of image information from the polarization of the light. To ascertain whether one or both retinas in this aquatic predator are polarization sensitive, Stecher et al. (2010) examined the ultrastructure of the photoreceptors. Their findings indicated that the proximal retina of E1 and E2 should be

25 polarization sensitive (since the cells have microvilli organized in parallel), while the distal retina should not (since it has irregularly organized microvilli). Further analysis showed that the proximal retina of larval eyes, E1 and E2, consists of three types of cells arranged in alternating patterns. According to the microvillar organization, two of these cells should be most sensitive to vertically polarized light, while the third type should be most sensitive to horizontally polarized light, as is illustrated in Figure 8 after

Stecher et al. ( 2010).

Figure 8. A frontal view of the proximal retina with its dorsal and ventral row of photoreceptors.

Each row of photoreceptors has three types of cells (T1, T2, and T3) that are arranged in alternating patterns. The microvilli are oriented in such a way that, relative to the orientation of the larva’s head, T1 and T3 should be most sensitive to vertically polarized light, while T2 should be most sensitive to horizontally polarized light (after Stecher et al., 2010).

26 Nevertheless, the actual polarization sensitivity remained unclear since, prior to reaching the proximal retina, light passed through the distal retina, which had the potential to depolarize polarized light, to polarize unpolarized light or, even, to change the polarization direction of the light.

In Chapter 3, I measured the polarization sensitivity of individual photoreceptor cells of the proximal retina using intracellular electrophysiological methods.

Hunting behavior

Buschbeck et al. (2007) investigated the hunting behavior of T. marmoratus larvae and found that, as soon as a larva detected potential prey, it oriented itself so that the prey item was brought into the visual field of the two principal eyes, E1 and E2. Then, as the larva approached the prey, it performed dorso-ventral (vertical relative to their head orientation) scanning movements with its head, extending the visual field in this direction. Scanning movements would cease just before the larva performed a ballistic strike prior to prey capture. The hunting range of the larvae extended from a couple centimeters (initial prey detection) to only a few millimeters (striking distance), and their high success rate suggested that they must have been able to see their relatively small prey well throughout this range.

For many small insect eyes, all relevant object distances are at effective infinity, such that images of objects at all relevant object distances are projected onto the focal plane of the lens. However, the relatively long eye tubes of E1 and E2 in T. marmoratus larvae implied a relatively long focal length, which, in view of their hunting range, meant that images of objects at relevant close distances were no longer focused onto the focal

27 plane. As a result, object distance became an important factor in understanding how these eyes functioned.

In Chapter 4, I model how these lenses, in combination with the overall eye and retinal organization, could project sufficiently focused images onto the retinas throughout their hunting range.

Range finding mechanisms

One of the greatest challenges of small predators, such as T. marmoratus larvae, is the need for very adept range-finding mechanisms for prey capture. Bland et al. (under revision) investigated which range finding mechanisms might be important for these larvae. In insects, the best known range finding mechanism is motion parallax. This is based on the idea that, when the predator performs well-defined lateral movements, it causes object distance-dependent image movements across the retina. This cue, however, is not effective in an aquatic setting where the prey may move unpredictably and the predator may not be in full control of its movements.

Another known cue in insects is stereopsis, which involves the processing of distance-dependent differences between the images of the two eyes. There are some cases, moreover, in which image size itself is used as a distance cue (for review see

Collett and Harkness, 1982; Schwind, 1989). The latter only works, however, if the objects are of a predictable size. In their investigations, Bland et al. (under revision) succeeded in excluding all of these cues as required elements: a) binocular stereopsis was excluded by covering the eyes on one side of the head; b) motion parallax was excluded by presenting dummy prey as a moving target; and c) absolute image size as

28 a cue was excluded by presenting dummy prey of different sizes that were novel to the larvae. Nevertheless, the larvae were still able to gauge distance correctly. So, the question remained: which mechanism did they actually use?

In Chapter 4, I examine whether the layering of the retina could be involved in the extraordinary ability of these larvae to gauge the distance of their prey and whether there is any other visual information that could be used as a distance cue.

Optical Properties of the lenses of E1 and E2

Previous results suggested that the peculiar retinal layering of T. marmoratus larvae might function to provide range finding cues and to extract different kinds of image information from the light spectrum and its polarization. To function as such, a reasonably well focused image must be projected onto each of the retinas, a seemingly impossible phenomenon given the depth of the retina and its spectral organization.

Thus, the question was: “Where are images focused within the eye, depending on the wavelength of light and the object distance?” Answering this question is the subject of

Chapters 2 and 4.

The first step in answering this question was to measure the focal length of the lenses because the focal length would indicate at what distance from the lens an image is focused. Biconcave lenses such as those of E1 and E2 project images of an object at infinity onto the focal plane behind the lens. As an object moves closer to the lens, the image moves out of the focal plane, further away from the lens (Figure 9). The magnitude of this shift is dependent on the focal length.

Since biconcave lenses project an image of an object at infinity onto its focal

29 plane, a convenient method for measuring the back focal length (b.f.l) is simply to measure the distance from the back surface of the lens at which an image of an object at infinity is projected. For insects, this method was first described by Homann (1924).

Figure 9. Schematic of where a biconcave lens focuses an image depending on the object distance. F and F’ are the focal points of the lens. A: An image of an object at infinity is focused onto the focal plane of the lens. B: As the object distance decreases, the image distance and the image size increase.

Preliminary data from such measurements suggested that the lens of E2 projected not one image but two. This was a surprising find because the only other known animals that might have had similar, truly bifocal lenses were the trilobites (Gál et al., 2000b). Chapter 2 focuses on establishing whether or not the lens of E2 is indeed bifocal.

In Chapter 4, optical and anatomical measurements were expanded to establish where images were focused within E1 and E2, depending on the wavelength of light and the object distance, and what function retinal layering might have in this context.

The extensive optical calculations and modeling that were necessary for this required

30 both the focal length (f’) and the back focal length (b.f.l.). The distinction between these two measurements, as illustrated in Figure 10, is necessary because the lenses of E1 and E2 are quite thick.

In thick lenses, the comparatively long light path within the lens can have a significant influence on its overall optical properties (Hecht, 2001). For this reason, a thick lens is, for all mathematical purposes, represented by two imaginary principal planes, where the focal length (f’) as well as the object and image distances are measured from the principal planes (Figure 10). These imaginary planes are positioned so that the light path between them can be treated as parallel to the optical axis and the light refraction can be treated as if it were taking place at the principal planes and not the surfaces of the lens (Figure 10). The distance between the two principal planes, and the distance between the principal planes and the surfaces of the lens depend on the thickness of the lens and the refractive powers of its optically active surfaces (Hecht,

2001). All of the optical calculations in Chapter 4 take all of these relationships into account.

Finally, Chapter 5 presents a comprehensive model of how these eyes might aid in hunting as based on our current knowledge of the anatomy, physiology and optical properties of these eyes and the hunting behavior of the larvae.

31

Figure 10. Schematic of the light path through a thick lens. The solid blue lines represent the actual light path (given that the lens has a homogeneous refractive index). The dashed lines represent the imaginary light path, devised for the purposes of the optical calculations. f and f’ are the focal lengths measured from the principal planes; do and di are the object and image distances measured from the principal planes respectively; so and si are the object and image distances measured from the surfaces of the lens respectively; yo and yi are the object and image sizes respectively; b.f.l is the back focal length measured from the back surface of the lens; and h2 is the distance between the back surface of the lens and the second principal plane.

32 Chapter 2

Biological Bifocal Lenses with Image Separation

Annette Stowasser,1 Alexandra Rapaport,1 John E. Layne,1

Randy C. Morgan,2 and Elke K. Buschbeck1,*

1Department of Biological Sciences, University of Cincinnati,Cincinnati, OH 45221-0006

2Insectarium, Cincinnati Zoo and Botanical Garden, 3400 Vine Street, Cincinnati, OH

45220-1399

* Correspondence: [email protected]

Published in Current Biology, Volume 20, pp. 1482–1486, 2010 doi: 10.1016/j.cub.2010.07.012

The final publication is available at http://www.cell.com

33 Summary

Almost all animal eyes follow a few, relatively well-understood functional plans. Only rarely do researchers discover an eye that diverges fundamentally from known types.

The principal eye E2 of sunburst diving beetle (Thermonectus marmoratus) larvae clearly falls into the rarer category. On the basis of two different tests, we here report that it has truly bifocal lenses, something that has been previously suggested only for certain trilobites (Gál et al., 2000b). Our evidence comes from (1) the relative contrast in images of a square wave grating and (2) the refraction of a narrow laser beam projected through the lens. T. marmoratus larvae have two retinas at different depths behind the lens, and these are situated so that each can receive its own focused image.

This is consistent with a novel eye organization that possibly comprises ‘‘two eyes in one.’’ Moreover, we find that in contrast to most commercial bifocal lenses, the lens of

E2 exhibits asymmetry, which results in separation of the images both dorsoventrally and rostrocaudally within the layered retina. Visual contrast might thus be improved over conventional bifocal lenses because the unfocused version of one image is shifted away from the focused version of the other, an organization which could potentially be exploited in optical engineering.

34 Results and Discussion

Sunburst Diving Beetle Larvae Have Tubular Eyes with Two Retinas

Almost all animal eyes follow a few, relatively well-understood functional plans.

Nevertheless, there is considerable variation among larval eyes (stemmata) of holometabolous insects. Although many of them are quite simple (Gilbert, 1994) others are highly specialized camera-type eyes (Gilbert, 1994; Toh and Okamura, 2001; Toh and Okamura, 2007). On the basis of their anatomy (Mandapaka et al., 2006) and behavior (Buschbeck et al., 2007), the eyes of sunburst diving beetle (Thermonectus mamoratus) larvae fall in the latter category. However, their physical organization, and most likely their mode of function, is highly unusual even among specialized forms.

T. mamoratus larvae (Figure 1A) are aquatic, visually guided predators native to the southwest United States (Larson et al., 2000). The larvae have 12 eyes, 6 on each side of the head. Four of these eyes (E1 and E2 on either side) are tubular and look directly forward (Figure 1B). The larvae scan with these principal eyes by oscillating their heads dorso-ventrally as they approach potential prey (Buschbeck et al., 2007).

The anatomy of the retinas of these principal eyes is unusual, as has been described in detail for first-instar larvae (Mandapaka et al., 2006). We here report that a similar organization is also observed in third-instar larvae, although the size and proportions change somewhat. The retinas are divided into distinct distal and proximal portions.

Figure 1C schematically illustrates the shape of the two retinas in E2.

35

Figure 1. Illustration of the Third-Instar Larvae of Thermonectus marmoratus and Its Principal

Eyes (A) Picture of the entire animal. (B) Scanning electron micrograph of the larval head, showing the two large lenses of the principal eyes (E1 and E2) on each side of the head. (C)

The gross optical and neural organization of E2. Inserts a and b schematically illustrate the eye organization of the two sections indicated in the scanning-electron-micrograph image. White lines show the approximate visual fields of the retinas. Abbreviations are as follows: PR, proximal retina; DR, distal retina; P, pit of distal retina.

The horizontal extents of the oval visual fields of the distal and proximal retinas

36 are 40°–50° degree, whereas the vertical extents are about 14° and 3.5° degree, respectively. The distal retina consists of at least 12 tiers of photoreceptor cells, which are oriented approximately perpendicular to the light path. The proximal retina lies directly beneath and contains photoreceptor cells oriented parallel to the light path. The pit of the distal retina (Figure 1C) lies 424 µm (±10 µm STD, n = 10), and the top surface of the proximal retina lies 493 µm (±16 µm STD, n = 10) behind the back surface of the lens. The lens diameter is 228 µm (±10 µm STD, n = 10). The presence of the two anatomically separated retinas raises the question, ‘‘Which of the retinas receives a focused image from the lens?’’ Our present findings suggest that a bifocal lens provides a focused image for each of them. To the best of our knowledge, this is the first demonstration of truly bifocal lenses in the extant animal kingdom.

Two Independent Methods Reveal the Existence of a Bifocal Lens

To measure the optics of the lenses, we first used a modified version of the hanging drop method (Homann, 1924), in which a lens produces images of an object at effective infinity, and these images are observed through a microscope (Figure 2A). We consistently observed focused images at two different distances behind the larval lens, and these images were clearly separated by a region where no sharp focus was formed

(Figure 2B).

37

Figure 2. Measurements of Relative Edge Sharpness of Serial Photographs Show that the Lens

Forms Two Sharp Images (A) Schematic illustration of the method. (B) Representative series of photographs used for computing edge sharpness. Arabic numerals indicate the distance from the back surface of the lens to the photographed frame (in µm). Roman numerals indicate correspondence between photographs and their edge sharpness in graphs (C) and (D). (C)

Measurements of edge sharpness of the lens that produced the images illustrated in (B). Error bars show the standard deviation of ten computations of edge sharpness for each frame. (D)

Average edge sharpness of the peak and trough values as indicated in graph (C) from 15 photograph series; standard errors are included. The two peak values (B) were significantly different from the troughs (A) with a p < 0.003.

For this method, the lens was mounted on a goniometer, and its orientation was adjusted so that one of the resulting images remained approximately stationary to the

38 viewer while the focus of the microscope was changed (see Figure S1C). We objectively established the positions of these two focal planes by computing relative edge sharpness in each of 70–100 images serially photographed approximately along the optical axes of 15 different lenses from E2 (see Experimental Procedures for details). To obtain the actual distance between frames, we corrected the measured distance for the refractive index of insect Ringer’s solution (1.33) (Galbraith, 1955).

Figure 2C illustrates the results from one lens: peaks occur in edge sharpness around

372 and 499 µm behind the apex of the back surface of the lens. The combined results from E2 lenses (n = 15; Figure 2D) show that edge sharpness significantly decreases between the two peaks as well as 40 µm before the first image plane and 40 µm after the second image plane (p < 0.003).

We verified the existence of two focal planes with a second method (Campbell and Hughes, 1981; Sivak, 1982) modified from Kröger et al. (2009) and Toh and

Okamura (2007). This method visualized the paths of tiny, parallel light rays passing through the lens. Specifically, a narrow light beam was directed at 20 to 50 locations in a line across the face of the lens (so the line passed through the center), and the refracted rays were visualized in a saline solution containing micro beads (Figure 3A).

The refracted beams could be classified into two gropus according to their point of intersection (Figure 3B). We further confirmed this bisectioning of the beams by computing relative beam density (Figure 4B,D).

39

Figure 3. Laser Images Confirm the Presence of Two Focal Planes (A) Schematic illustration of the method. (B) Composite of five scan frames reveal two beams that intersect each other nearer to the lens than do the other three. These intersections occur in the two focal planes, the positions of which correspond well to the edge sharpness measurements, as indicated by the two arrows. The position of the lens is indicated schematically by an oval. Grayscale photographs are illustrated with false color.

Our findings can only be explained by the presence of a truly bifocal lens. For instance, if it were an astigmatic lens, as described for the ocelli in some bees, wasps, and blowflies (Warrant et al., 2006; Schuppe and Hengstenberg, 1993), it would have two focal planes, but it would not consistently produce two images of a square wave of arbitrary orientation as we observed. Furthermore, point objects resulted in two point images (Figures S1C and S1D) and not the streaks expected from astigmatism. In addition, our results cannot be an example of spherical aberration, which would lead to an extended region of poor focus (caused by a gradual increase of focal power toward the periphery of the lens). In contrast, T. marmoratus lenses produce two distinct and well-focused images (Figure 2, S1D). Likewise, our observations are distinctly different from multifocal lenses found in fish (Kröger et al., 1999; Malkki and Kröger, 2005) and

40 terrestrial vertebrates (Malström and Kröger, 2006). Those lenses have several zones with different focal lengths and are thought to correct for chromatic aberration by focusing all wavelengths onto the same plane in the retina (Kröger et al., 1999; Malkki and Kröger, 2005; Malström and Kröger, 2006). T. marmoratus lenses, in contrast, have two distinct focal planes that are substantially separated from each other. In some ways, the eyes appear to resemble the principal eyes of certain jumping spiders that have retinas with four distinct layers (Land, 1969). In the spider retina, the layers closer to the lens have photoreceptors sensitive to shorter wavelengths, whereas those further away are sensitive to longer wavelengths. The layers are spaced so that they can compensate for chromatic aberration (Blest et al., 1981). However, this is not the design of the T. marmoratus E2 eye, in which all the layers of the distal retina (closer to the lens) express putative green opsins, whereas those in the proximal retina (farther away) expresses putative UV opsins (Maksimovic et al., 2009). Thus, in contrast to jumping spiders, in T. marmoratus larvae opsins are expressed in the ‘‘wrong’’ layers to compensate for chromatic aberration.

Probably the most similar previously described lens system is that of schizochroal trilobites (Clarkson el al., 2006; Fordyce and Cronin, 1993). These long- extinct arthropods had compound eyes, each unit of which might have functioned as an image-forming eye (Fordyce and Cronin, 1993). Their corneal lenses are thought to have consisted of an outer unit of calcite (Towe, 1973) and an inner unit possibly composed of organic material (Horváth, 1989). The reconstruction of the optics from lens unit surfaces revealed the possibility of bifocal lenses. This arrangement might have allowed trilobites to see relatively far and near objects simultaneously (Gál et al.,

41 2000b), which is not possible with a rigid monofocal lens. The bifocal lens therefore is thought to have compensated for the absence of the kind of accommodation mechanisms that exist in vertebrate eyes.

The Bifocal Lenses Might Function as ‘‘Two Eyes in One’’

It is plausible that T. marmoratus larvae also benefit from being able to simultaneously focus far and near objects on individual retinas. In addition, we think that within the principal eyes, separate images of the same object could be focused on each of two retinas, allowing each eye to function as ‘‘two eyes in one.’’ Our optical measurements suggest that the two focal planes of each lens are separated by about 100 µm, which roughly matches the anatomical separation of these two retinas. In E2 the images are separated by 105 µm (±10 µm standard error of the mean [SEM], n = 15), and the retinas of their contralateral eyes are separated by 132 µm (±5 µm SEM, n = 13) if measured from the upper edge of the distal retina to the upper surface of the proximal retina. Determining the exact locations of the two images will require further investigation because in vivo the image distance depends on the actual distance to a viewed object and is influenced by the refractive index of the tissue behind the lens

(which potentially could differ from that of the saline that we used for our measurements).

Image Disparity Might Allow Larvae to See Prey Better

In contrast to what has been described in trilobites, and what is found in many commercial bifocal lenses (such as contact lenses or cataract replacement lenses), T.

42 marmoratus larval lenses appear to use an interesting optical ‘‘trick.’’ A major problem with comparable bifocal lenses is that their concentric and collinear design causes the contrast in both images to be reduced by the unfocused version of the other image

(Montés-Micó et al., 2004; Navarro et al., 1993; Artal et al., 1995). In T. mamoratus, the images are vertically displaced, presumably as a result of asymmetries in the lens. It has been reported that in bifocal systems (such as those used for intraocular lenses), the first image is more ‘‘contaminated’’ by the blurry second image (Montés-Micó et al.,

2004) than is the second image by the first. Therefore, we estimated to what extent the observed image disparity improves the contrast of the first sharp image. Specifically, we calculated contrast modulation of the resulting image column by column. This allows the portion of the focused image that overlaps with its unfocused counterpart to be compared with the portion of the image that does not overlap (see Experimental

Procedures for details). Figure S1B shows that the image contrast improves at least 3- fold.

It is unclear exactly what causes the asymmetries, but they must relate to the precise location of the two optical centers in relation to their respective apertures. The image separation is visible in the 3D reconstruction of a point image (Figure S1D) and of the square-wave image series (Figure 4A), as well as in the ray density plot (Figure

4B). The images are separated in the vertical plane, and their deviation is not visible if lenses are turned by 90° (Figures 4C and 4D).

43

Figure 4. Visualization of the Image Disparity. (A and C) The three-dimensional reconstruction of an edge-sharpness frame series visualizes a square-wave image that has been projected by the insect lens. Insets show best-focused images, and arrows show their location along the light path. (B and D) illustrate ray density plots. (A) The side view shows that two blur circles diverge within the dorso-ventral plane of the lens.

(B) The divergence also is visible in the ray density plot (arrows indicate the approximate directions of blur-circle movement). (C and D) Viewed from the dorsal direction, blur circles are relatively well aligned.

The divergence of images is probably an advantage for T. marmoratus. Because these larvae use dorso-ventral scanning movements (Buschbeck et al., 2007), corresponding focused images would reach the two retinas with only a small temporal delay resulting from the small dorsoventral separation between the two images. These larvae normally hunt small objects that would be relatively distinct when viewed against

44 a homogenous aquatic background. When some of the photoreceptor cells ‘‘see’’ the focused image of a small prey object, this image is not contaminated by the blurry image of the same object, but rather by a blurry image of the background. A blurry homogenous background would interfere relatively little with the perception of the sharp image. A similar mechanism could be exploited by commercial bi- or multi-focal systems.

Experimental Procedures

Animals and Lenses

T. marmoratus used were offspring of beetles provided by the Insectarium of the

Cincinnati Zoo and Botanical Garden or of beetles collected between 2004 and 2008 near Tucson, AZ, USA. After hatching, T. marmoratus larvae were reared at 37°C in separate containers on frozen bloodworms and live mosquito larvae. All data were obtained from third-instar larvae (which have the largest lenses) 24 hr after ecdysis.

During this time, larvae hunt very successfully, suggesting that the visual system is fully functional. Schematics are based on histological sections, prepared as described in

(Mandapaka et al., 2006). To prepare samples for imaging via scanning electron micrograph, we dried whole animals, mounted them on coverslips, and viewed them with an ESEM XL30 (FEI Company) microscope. For optical measurements, larvae were anesthetized via cooling and were decapitated, and a small piece of the head capsule containing lenses was excised. We cleaned the backs of the lenses with a fine brush and mounted them on a pinhole by using wax to attach the exoskeleton surrounding the lenses.

45 Image Contrast Measurements

As in the hanging drop method (Homann, 1924), we used a microscope to observe the images formed by the lens. We tested the efficacy of this method by performing it on T. mamoratus adult compound-eye facet lenses, and this resulted in one focal plane (data not shown). The lens was mounted with wax between two coverslips so that images were formed between the lens and upper coverslip (Figure 2A). The space between the coverslips was filled with a 50% dilution of insect Ringer’s solution (O’Shea and Adams,

1981). In contrast to 100% Ringer’s solution (which resulted in the presence of minor wrinkles) and distilled water (which led to noticeable bloating), this concentration produced no visible deformation of the lens. The coverslip sandwich was mounted on a goniometer that replaced the microscope stage. The back surface of the lenses faced the microscope objective lens. A square-wave grating (0.353 cycles/mm, USAF 1951 negative test target from Edmund Optics) served as the object and was placed 12.5 cm beneath the microscope stage—effective infinity for this small lens. The condenser was removed, and the object was aligned with the center of the microscope optics. The square wave was illuminated with monochromatic light (542 nm), and the rays refracted by the lens were photographed with a 3CCD camera (Hitachi HV-f22) with a pixel resolution of 1360 x 1024 and acquired with ImageJ 1.38 (U. S. National Institutes of

Health, Bethesda, MD, with plug-in QuickTime_Capture modified for highdefinition image acquisition). The frames were photographed at 5µm intervals from the back surface of the lens to well beyond the focal planes. We evaluated the photographs for the focus of the square-wave image by computing relative edge sharpness. To do so, we first removed shot noise by digitally convolving the frames with a 35 x 35 point 2D

46 Butterworth low-pass filter with a cutoff of ten times the square-wave frequency (Matlab

7.4, The Mathworks, Natick, MA). Then, images were cropped to the region of the square wave (plus some background). Relative edge sharpness of each frame was computed as the grand mean of grayscale intensity value of neighboring pixels, twice differentiated (Figure S1A) along the x, y axes and both diagonal dimensions. This resulted in a single metric for the slope of the change in intensity in the image; higher values indicated a steeper slope (sharper edges). The final relative edge-sharpness value was the average of ten such computations for each frame series, which accounted for variation due to image cropping. This method allowed an automated assessment of image quality of individual frames without involving assumptions about the image. The combined data (Figure 2D) show the average of 15 individuals. Each individual contributed to each of the five bars with three points, as shown in Figure 2C.

A Turkey’s test accounted for multiple comparisons. To visualize light rays, we performed 3D reconstructions of image stacks with the Vortex module of Amira, version

5.2.2 (see Figures 4A and 4C; see also Figure S1D). To estimate to what extent the image disparity improves contrast, we calculated the contrast modulation across the images of five lenses (Figure S1B). First, we rotated each image to orient the stripe direction horizontally. Next, we calculated contrast modulation (Equation 1) at four points for each image column (between maximum gray values of the three light stripes and minimum values of the two darker inter-stripe areas). To average the five images, we aligned them at the peak.

I  I (1) Modulation max min Imax  Imin

47 Laser Measurements

The pinhole, with the lenses, was mounted vertically with wax on one side of a glass container filled with 50% concentrated insect Ringer’s solution (O’Shea and Adams,

1981) and a dilute suspension of microbeads (0.1 μm, Fluka, from Sigma-Aldrich)

(Figure 3A). To ensure a flat refracting surface, we filled the container to the top and covered it with a coverslip. A horizontal laser beam (commercial 50 mW high-powered green laser pointer, 530 nm) was projected through the lens, and the rays refracted by the lens and scattered by the microbeads were photographed through a microscope with a MagnaFire camera (a 1300 X 1030 pixel digital camera from Optronics, Goleta,

CA). In order to achieve a narrow beam of no more than 10–20 µm diameter, we focused the laser at the front surface of the insect lens with a glass lens (f = 30 mm). If we assume a beam of Gaussian intensity distribution, this resulted in approximately parallel light for several 100 µm around the focus of the beam (the initial beam diameter was ~1 mm; thus, the Rayleigh range was ~600 µm). For each lens, 20–50 photographs/lens were taken as the laser was moved with a motorized micromanipulator in steps of 10 µm, so that each lens was scanned from one edge through its center to the opposite edge, approximately horizontally or vertically relative to the animal. The image in Figure 3B is a composite of five representative frames from the scan. To further establish the presence of two focal points, we computed the relative local ray density of all images in each scan. To do so, we converted the paths of the laser beams in all photographs to linear equations and solved their y coordinates at 1 pixel (1.1 μm) intervals along the x axis. The relative local density of all such x, y coordinates from a laser scan was computed by 3 pixel kernel density estimation

48 (MATLAB toolbox Version 1.0 09/13/05, developed by Joern Diedrichsen). We chose this method over the method developed by Malkki and Kröger (2005) to avoid making assumptions about the aperture(s) and optical center(s) of the lens.

Figure S1, related to Figure 2. A. Illustration of relative edge sharpness calculations of a “sharp”

(top) and “slightly blurry” (bottom) image. Pixel intensity values are at a scale from 0 (black) to

255 (white). The example illustrates that the sum of the second differentiation of the sharp image (600) is higher than the sum of the second differentiation of the less sharp image (360).

B. To quantify the contrast improvement that results from image disparity, we calculated contrast modulation of image columns across most of the first sharp image (see background illustration). The contrast modulation sharply improves near the right of the image, where the unfocused second image least interferes (graph shows the average of 5 lenses with STD). C.

Super-imposed video frames that resulted from projecting a point image through the lens. The

49 upper image (arrowhead) remains relatively stationary, whereas the lower image shifts upwards

(arrow) as the focus of the microscope moves away from the lens surface. D. The 3D reconstruction of the entire image stack of the point image allows visualizing light rays from the side. Here the upper trace is more or less horizontally oriented, whereas the lower trace is slanted. Inserts illustrate the two sharpest images at their approximate position along the light path.

50 Chapter 3

Electrophysiological evidence for polarization sensitivity in the camera-type eyes of the aquatic predacious insect larvae, Thermonectus marmoratus (Coleoptera: Dytiscidae)

Annette Stowasser and Elke K. Buschbeck*

Department of Biological Sciences, University of Cincinnati, Cincinnati, OH 45221-0006,

USA

* Author for correspondence (e-mail: [email protected])

Published in The Journal of Experimental Biology, Volume 215, pp. 3577-3586, 2010 doi: 10.1242/jeb.075028

The final publication is available at http://jeb.biologists.org

51 Summary

Polarization sensitivity has most often been studied in mature insects, yet it is likely that larvae also make use of this visual modality. The aquatic larvae of the predacious diving beetle Thermonectus marmoratus are highly successful visually guided predators, with a UV-sensitive proximal retina that, according to its ultrastructure, has three distinct cell types with anatomical attributes that are consistent with polarization sensitivity. In the present study we used electrophysiological methods and single-cell staining to confirm polarization sensitivity in the proximal retinas of both principal eyes of these larvae. As expected from their microvillar orientation, cells of type T1 are most sensitive to vertically polarized light, while cells of type T2 are most sensitive to horizontally polarized light. In addition, T3 cells probably constitute a second population of cells that are most sensitive to light with vertical e-vector orientation, characterized by shallower polarization modulations, and smaller polarization sensitivity (PS) values than are typical for T1 cells. The level of PS values found in this study suggests that polarization sensitivity probably plays an important role in the visual system of these larvae. Based on their natural history and behavior, possible functions are: (1) finding water after hatching, (2) finding the shore before pupation, and (3) making prey more visible, by filtering out horizontally polarized haze, and/or using polarization features for prey detection.

52 Introduction

Polarization cues are known to be important for many adult insects. Most commonly they are used for navigation and habitat or ovipositor site detection, as well as for finding mates. In aquatic habitats, animals such as certain fish, lobster, crabs, crayfish, mantis shrimp, and cephalopods have been found to use polarization sensitivity for communication, to improve the visual contrast of their surroundings, or to detect prey

(Horváth and Varjú, 2004; Shashar et al., 2011; Wehner, 2001). Although it has been suggested that polarization vision for contrast enhancement and prey detection could also play a role in insect visual systems (Horváth and Varjú, 2004; Schneider and

Langer, 1969; Trujillo-Cenóz and Bernard, 1972), to the best of our knowledge, this has never been demonstrated. Even less is known about polarization sensitivity in insect larvae. With regard to the latter we only know that some, such as gypsy moth larvae, sawfly larvae, mosquito larvae, and tent caterpillar larvae, show polarotaxis (Baylor and

Smith, 1953; Doane and Leonard, 1975; Gilbert, 1994; Meyer-Rochow, 1974; Sullivan and Wellington, 1953; Wellington, 1955; Wellington et al., 1951). Previously our group presented ultrastructural data that raised the possibility of the existence of polarization sensitivity in a specialized region of the complex principal eyes of Thermonectus marmoratus (Coleoptera: Dytiscidae) larvae (Stecher et al., 2010). These larvae are highly successful visually guided aquatic predators, which could potentially exploit polarization sensitivity to improve contrast and see prey better. We present electrophysiological data that confirms our anatomical predictions, both with regard to the existence of polarization sensitivity, and with regard to the e-vector orientation to which individual cell types are maximally sensitive.

53 There are two main sources of polarized light in natural environments: (1) the scattering of light in bulk media such as the atmosphere or water, and (2) the light reflected from shiny surfaces (Horváth and Varjú, 2004). In the air, polarized light comes from light scattering in the atmosphere with a predictable polarization pattern that changes slowly over time. It also comes from reflecting surfaces such as leaves or water. The polarization patterns of this light might change rapidly and unpredictably, especially as the orientation of reflecting surfaces changes with waves or wind. Most studies with regard to polarization sensitivity or polarization vision in air show utilization of this ability within three broad categories. First, polarization sensitivity is used to gain insights on compass directions. For example, insects such as bees, ants, and locusts exploit the polarization pattern of the sky for orientation and navigation (Fent, 1986;

Mappes and Homberg, 2004; Rossel, 1993; Wehner and Müller, 2006). Second, polarization cues are used to recognize specific habitats. For example, water beetles and bugs use the polarization pattern of reflecting surfaces as a visual cue to find habitats (Schwind, 1984; Schwind, 1991), and insects such as mayflies, midges and dragonflies use the pattern to find water surfaces to use as their oviposition sites (Kriska et al., 2007; Kriska et al., 1998; Lerner et al., 2008; Wildermuth, 1998). Finally, polarization sensitivity is used for communication and mate recognition. Some animals have polarization-active body parts. For example, polarization-sensitive butterflies have been shown to use this visual cue for finding mates in the rain forest where there is little interference from other polarized light sources due to the dense vegetation (Sweeney et al., 2003).

Some animals are also known to use underwater polarization cues. Due to its

54 higher refractive index, in water less polarized light is reflected from surfaces than in air. Instead, almost all polarization emerges from the scattering of light in bulk media, resulting in polarization patterns that are more predictable but also more complex than those found in air. The complexity arises from factors such as the depth, the line of view, the elevation of the sun, the wavelength of the light, the visibility of the bottom, the proximity of the shore, and water as well as weather conditions (Ivanoff and Waterman,

1958; Novales Flamarique and Hawryshyn, 1997; Waterman and Westell,

1956). However, it is precisely the predictability of polarization patterns that allows for exploitation of polarization sensitivity for orientation, contrast enhancement, and for using the polarization features of animals as reliable visual cues for communication or prey detection (Cronin, 2006; Shashar et al., 2011; Wehner and Labhart, 2006).

Generally there is relatively poor visibility in water as compared with air. This is primarily because the contrast of any scenery is drastically decreased due to the scattering of light within the water. However, scattered light is mainly polarized horizontally, so that a vertical polarization filter can increase the overall contrast by filtering out the haze (Cronin and Marshall, 2011; Johnsen et al., 2011; Lythgoe and

Hemmings, 1967). Additionally, muscle tissue and other body structures can influence the polarization of light, leading to a visual cue that can be used to detect prey or enhance communication. Specifically, tissue might polarize unpolarized light, or depolarize or change the e-vector orientation of existing polarized light (Cronin et al.,

2003; Johnsen et al., 2011; Sabbah and Shashar, 2006; Shashar et al., 2000). Such body parts can increase the visibility of prey to polarization-sensitive predators such as fish and cephalopods (Johnsen et al., 2011; Kamermans and Hawryshyn, 2011;

55 Shashar et al., 2000; Shashar et al., 1998), or might be used for communication as suggested in cephalopods and mantis shrimps (Marshall et al., 1999; Shashar et al.,

1996). So far, such use of polarization sensitivity has never been shown for any insect, even though some, such as T. marmoratus and other predacious aquatic insects, could clearly benefit from such mechanisms.

Thermonectus marmoratus larvae are aquatic visually-guided predators native to the southwest United States (Larson et al., 2009). The larvae are found in shallow ponds and small slow-flowing streams (Evans and Hogue, 2006; Velasco and Millan,

1998) and tend to swim with their principal eyes directed approximately horizontally. Thus, the polarization patterns that are formed relatively close to the surface in the horizontal line of view should be most important. In this line of view the polarization of light can be primarily explained by the refractive angle of the incident light. Additionally, it is influenced by weather and water conditions, the wavelength of the light, the albedo of a visible bottom, and the proximity to the shore (Ivanoff and

Waterman, 1958; Novales Flamarique and Hawryshyn, 1997; Waterman and Westell,

1956). Overall the percent polarization during the day might reach up to 40% and the e- vector of the polarized light during the day is approximately horizontal as long as the sun zenith angle is not too large (Novales Flamarique and Hawryshyn, 1997). In the presence of polarized light, zooplankton and many other small transparent organisms that possess polarization-active body parts are potentially more visible to a polarization- sensitive predator (Johnsen et al., 2011). For example, prey of T. marmoratus larvae, such as mosquito larvae, show clear polarization features (Stecher et al., 2010) which the larvae potentially could use as visual cues to better detect their prey, if adequate

56 polarization sensitivity exists in the principal eyes of these larvae.

Thermonectus marmoratus larvae have 12 eyes, six on each side of the head. Four of these eyes (E1 & E2 on each side) are tubular and look directly forward

(Fig. 1A). The larvae scan with these principal eyes by oscillating their heads dorsoventrally as they approach potential prey (Buschbeck et al., 2007). The anatomy of the retinas of these principal eyes is unusual (Maksimovic et al., 2011; Mandapaka et al., 2006).

Fig. 1. Schematic of the principal eye’s structure of Thermonectus marmoratus larvae. (A)

Horizontal (a) and sagittal (b) schematic of eye 2 (E2) indicating the position of the distal (DR) and proximal (PR) retinas. The white line marks the approximate position of B. (B)

Microstructure of the PR, containing three photoreceptor types: T1, T2, and T3. The insert

57 schematically illustrates the microvillar orientation for each of these cells. D, dorsal; V, ventral;

L, lateral; M, medial.

The retinas are divided into distinct distal and proximal portions. The distal retina consists of at least 12 tiers of photoreceptor cells with rhabdomes that are oriented approximately perpendicular to the light path. The microvillar orientation of these cells is irregular (Stecher et al., 2010). The proximal retina lies directly beneath and contains photoreceptor cells, the rhabdomes of which are oriented parallel to the light path as illustrated in the schematic of Fig. 1A. Based on an ultrastructural study (Stecher et al.,

2010), it has been suggested that the proximal retina could be polarization sensitive because it contains cell types that meet common key characteristics that leads to polarization sensitivity in invertebrates. Those include the presence of parallel microvilli within individual photoreceptors, perpendicular orientation of microvilli in neighboring photoreceptors, and the presence of identical spectral sensitivity (Wehner and Labhart,

2006). In T. marmoratus the proximal retina is sensitive to UV (Maksimovic et al., 2010) and composed of three cell types. Two of these types (T1 and T3) have a vertical

(dorsoventral) and one (T2) has a horizontal (mediolateral) microvillar orientation (Fig.

1B). Within the retina, the three types are situated in an alternating pattern so that cells with vertical microvillar orientation are adjacent to cells with horizontal microvillar orientation (Fig. 1B). However, before light reaches the proximal retina, it first travels through the rhabdomeric portion of the distal retina (Fig. 1A). Prior to this study, it was unclear if the polarization sensitivity might diverge from what was expected from the microvillar orientation, since rhabdomes potentially alter polarized light (Chiou et al.,

2008).

Based on electrophysiological measurements of third instar larvae, we present

58 data that clearly demonstrate that the proximal retina is indeed polarization sensitive.

Our data show that two of the three cell types have relatively high polarization sensitivity

(PS) and that the orientation of polarization sensitivity corresponds well with predictions from the anatomical data: T2 cells are most sensitive to horizontally polarized light and

T1 cells are most sensitive to vertically polarized light.

Material and Methods

Animals

Thermonectus marmoratus larvae were offspring of beetles provided by the Insectarium of the Cincinnati Zoo and Botanic Garden, or of beetles collected between 2004 and

2012 near Tuscon, Az, USA. A population of T. marmoratus is maintained in our laboratory throughout the year. Thermonectus marmoratus larvae were reared in isolation on previously frozen bloodworms and live mosquito larvae. All data were obtained from third instar larvae, 3 – 5 days after ecdysis.

Animal Preparation

The larvae were anesthetized on ice and placed, head downward, onto a 35 deg slope so that the eye tubes of E1 and E2 were oriented approximately horizontally (Fig. 2).

Apart from the head and the tip of the abdomen, larvae were immobilized in 2% agar gel. The head and mandibles were immobilized with dental wax (# 091-1578,

Patterson, St. Paul, MN, USA). In some trials, to specifically target photoreceptors of E1 or E2, the excluded eye was occluded with opaque nail polish. The animal was positioned with its eyes 1 cm behind the polarization filter (Fig. 2). Apart from the tip of

59 the abdomen, the animal was submerged in 50% insect Ringer (O'Shea and Adams,

1981) containing 0.01% trypsin (Fisher Science Education, Hanover Park, IL, USA) or

0.01% protease from Streptomyces griseus (Sigma-Aldrich Corp., St. Louis, MO, USA).

The protease inhibited the coagulation of the hemolymph, which otherwise formed a gelatinous mass that made it difficult to advance the electrode.

Fig. 2. Schematic of setup which contains a rotating arm with the light stimulus (that could be moved freely during recordings), a polarization filter that can be rotated, and a sloped specimen holder within a small glass container (filled with saline solution) onto which the larvae was mounted so that the principal eyes were oriented horizontally. During experiments a sharp glass electrode was inserted near the back of each eye tube, and the indifferent electrode was placed into the saline solution.

To gain access to the photoreceptors of E2, the lens of E6 was removed. To access the photoreceptors of E1, either the lens of E6 or E5 was removed. Immediately thereafter a microelectrode was advanced into the tissue with a motorized manipulator,

60 and from then on manipulations where performed under dim red light, to which the photoreceptors showed no response. In total we recorded from 38 animals (14 E1 and

24 E2). Although we most often only recorded from one cell per eye, in few instances we recorded from two cells: one most sensitive to vertical e-vector orientation and one most sensitive to horizontal e-vector orientation.

Intracellular recording and neurobiotin iontophoresis

The electrophysiological setup was composed of standard equipment including an

Axoclamp-2A amplifier with a HS-2A gain x1 headstage (Molecular Devices, Sunnyvale,

CA, USA), iWorks AD board 118 (iWorks Systems, Inc., Dover, NH, USA), A-M systems audio monitor 330 (A-M Systems, Sequin, WA, USA), Tektronix 5103N oscilloscope

(Tektronix, Beaverton, OR, USA), a vibration isolation platform (TMC-66-501, Technica

Manufacturing, Peabody, MA, USA) and a Faraday cage. A silver wire that was inserted into the insect Ringer solution served as a reference electrode.

The experimental setup also included a UV transmissive polarization filter (BVO

UV Polarizer RAW film, Bolder Vision Optics, Boulder, CO, USA) that was mounted onto a rotary optic mount (Edmund Optics, Barrington, NJ, USA). The light stimulus consisted of a UV LED with a peak wavelength of 383 nm and a half width of 10 nm (30 mW/15, RL5-UV0315-380, Super Bright LEDs, Inc., St. Louis, MO, USA) that was mounted onto a rotating arm. The peak emission of the LED was close to the peak sensitivity of the photoreceptor cells of the proximal retina, which were previously reported to be 375 nm with a half-width of 75 nm (Maksimovic et al., 2011). The LED was positioned a couple of millimeters behind the polarization filter. Both the

61 polarization filter orientation as well as the stimulus position could be freely adjusted throughout the recording, as they were mechanically uncoupled from the vibration isolation table. The light intensity of the LED was controlled through the AD board with

LabScribe2 (vs 2.301, iWorxSystems). The light intensity, measured with a cosine corrector (Ocean optics, Dunedin, FL, USA), ranged from 7.97 x 1015 to 1.18 x 1019 photons cm-2 s-1 at the position of the eye. The intensity was measured with a calibrated spectrometer (USB2000+; Ocean Optics).

To establish the response-stimulus intensity (V-logI) relationship, 20 ms light pulses (with 2s intervals) were presented for 12 light intensities over 3 log units. Driving the LED with the chosen voltages yielded stable and reproducible light intensities and a stable emission spectrum. Our LED stimulus tended to truncate the flatter upper and lower portions of the V-logI curve; however, all critical measurements, as well as the PS value calculations, were performed within its confirmed linear range. A 20 ms stimulus yielded a clean response that did not overlap with the stimulus artifact. Intracellular recordings were performed with high impedance glass microelectrodes (A-M systems; catalog no. 601000) with a resistance of 70 – 120 MΩ, which were pulled with a horizontal puller (P97, Sutter Instrument, Novato, CA, USA). The tips of the electrodes were filled with 2% neurobiotin in 3 mol l-1 KCl (Vector Laboratories, Burlingame, CA,

USA), and the remainder with 3M KCl (separated by a small air bubble).

After a photoreceptor cell was impaled, the stimulus was positioned to maximize the response. Measurements were only taken from cells with stable resting potentials and response strengths of at least 20 mV, even when the polarization filter was turned perpendicular to the optimal e-vector orientation. After successful recordings, cells

62 were iontophoretically injected with neurobiotin for ~15 minutes by either passing a constant or pulsing current (150 ms, 2-3 nA pulses at 3 Hz). Thereafter, intact animals were placed in 50% insect ringer for 10-30 min at room temperature to allow neurobiotin to distribute throughout the cell. The data were recorded and stored, a moving average

(10 points; 1ms) was calculated using LabScribe software (LabScribe2, v. 2.301) and data were analyzed with customized MATLAB (MathWorks, Natick, MA, USA) programs. For each stimulus, the stimulus intensity was calculated from the average resting potential (over 200 µs prior to the stimulus onset) and the maximum response.

Optimal e-vector orientation

Light intensities were chosen that fell in the linear range of the V- logI response curves.

Stimulus intensities were slightly adjusted for individual cells. To determine how well each cell responded to polarized light of different orientation, the polarization filter was turned in 5 deg steps over 180 deg. This was repeated up to five times per cell, and the e-vector direction for which a cell showed minimal and maximal responses was determined from these data. To achieve this, for each individual cell, the cycles were normalized to the maximum response magnitude of the cell and fitted to a sinusoidal curve f(x)=asin(bx+c)+d using the cftool() function of MATLAB’s curve fitting toolbox.

The e-vector direction with respect to the head position (taken from frontal images of the head) that yielded minimum and maximum response was obtained from this fit. To visualize the response magnitude dependency on e-vector direction, for each cell, the response magnitudes were averaged and normalized (maximum = 1; minimum = 0).

After rounding the e-vector directions to the nearest 5 deg, the average of all cells was

63 calculated.

Quantifying Polarization sensitivity

V-logI relationships were determined for e-vector orientations that yielded minimum responses (min V-logI), as well as to perpendicular e-vector directions (max V-logI). For each stimulus intensity the response was measured three to five times. For each cell the response magnitudes of both e-vector orientations were fitted to the hyperbolic

Naka-Rushton function (Menzel et al., 1986; Naka and Rushton, 1966; Skorupski et al.,

n n n 2007), V=(I Vmax)/(I +K ), where V is the response magnitude (in mV), I is the stimulus

-2 -1 intensity, K is the stimulus intensity at Vmax/2 (measured in photons cm s ) and n is the slope of the curve. From this fit, the PS was calculated from the shift of the V-logI

∆i response curves at Vmax/2. Specifically, polarization sensitivity is defined as PS = 10 where ∆i is the difference in log I units between the two V-logI curves at K (Dacke et al.,

2002; Kleinlogel and Marshall, 2006). To visualize the normalized V-logI curves (Fig.

6), we first determined the maximum and minimum responses of the max V-logI of each cell. Subsequently, max V-logI and min V-logI curves were normalized to these values

(max=1; min=0). Cells of E1 (Fig. 6A,B) and E2 (Fig 6C,D) were considered separately.

In order to visualize relative response differences between cell types (Fig. 7), we pooled data from E1 and E2 for cells for which we had V-logI curves and therefore could confirm that measurements were indeed within the linear range of these curves. To normalize measurements without affecting the magnitude of the modulation, for each data point we calculated the difference to the maximum response magnitude of the cell

(∆ to max response, in mV).

64 Histology

After completion of the recordings and injection of neurobiotin, the animal was decapitated and processed as previously described (Maksimovic et al., 2011). In brief, animals were fixed in 4% paraformaldehyde solution (Electron Microscopy Sciences,

Hatfield, PA, USA) in 0.2 mol l-1 Sorensen’s buffer (Electron Microscopy Sciences,

Hatfield, PA, USA) for 14 to 16h at 4°C. After thorough washing in Sorensen’s buffer the tissue was dehydrated, washed in propylene oxide for ~15 min to improve penetration, and rehydrated. Subsequently, the tissue was incubated with streptavidin conjugated with Alexa Fluor 568 (Life Technologies Corporation, Carlsbad, CA, USA) diluted 1:200 (working concentration 0.5 μg ml-1) in Sorensen’s buffer with 1% Triton X-

100 for 14-16h at room temperature, washed, dehydrated and embedded in Ultra-Low

Viscosity Embedding Medium (Polysciences, Warrington, PA, USA). Finally, the tissue was serially sectioned at 15 µm, mounted and imaged with an Olympus 60806 digital camera (Olympus America, Center Valley, PA, USA) or a Zeiss LSM 510 laser scanning confocal microscope (Carl Zeiss AG, Oberkochen, Germany). For transmission electron microscopy, tissue was processed as described by Wolff (Wolff, 2011), with the following modifications: Sorensen’s buffer was used instead of sodium cacodylate, the heads were incubated in the fixative in the refrigerator overnight, and tissue was embedded in Ultra-Low Viscosity Embedding Medium. Ultrathin sections of the proximal retina were taken with an Ultracut E Microtome (Reichert-Jung), visualized with a transmission electron microscope (JOEL JEM-1230) and digital images were taken with a Megaplus ES 4.0 camera. The brightness and contrast of all final images was adjusted with Adobe Photoshop CS3 (Adobe Systems, San Jose, CA, USA).

65 Results

Based on transmission electron microscopy, the proximal retina of the principal eyes of first instar larvae of T. marmoratus is composed of three distinct cell types (Stecher et al., 2010). To evaluate if a similar organization also exists in third instar larvae, we first examined ultrathin sections of both principal eyes. As illustrated for E2 in Figure 3A, this is indeed the case: three distinct cell types are discernable. T1 and T2 are somewhat larger, and have vertically and horizontally aligned microvilli, respectively. T3 is organized similarly to T1, but its rhabdomeric portion is much smaller. Next, we used intracellular recordings to measure the polarization sensitivity of individual proximal photoreceptors. We found two physiologically distinct cell types in both eyes: one is most sensitive to horizontally polarized light, and the other is most sensitive to vertically polarized light. Comparable data were obtained for E1 and E2. Neurobiotin staining allowed us to link our physiological findings to two (T1 and T2) of the three anatomically distinct cell types (Table 1). In many cases multiple cells were stained, making it impossible to identify the cell that was recorded from. In some cases such staining was used to confirm the eye from which we recorded. If only one cell was stained, without exception, this was cell type T2 for cells most sensitive to horizontally polarized light

(see Figure 3B for example) and T1 for cells that were most sensitive to vertically polarized light (see Figure 3C for example). Although there is some indication in the physiological data that we may have recorded from two different populations of cells that are most sensitive to vertically polarized light (see below), none of the stained cells were of cell type T3.

66

67 Fig 3. Histological images. (A) Transmission electron micrograph of a cross section of the proximal retina of E2 of a third instar larva. As has been the case for first instar larvae, three distinct cell types are discernible: T1 and T3 have vertically oriented microvilli; T2 is situated between T1 and T3 and has horizontally oriented microvilli that are immediately adjacent to the microvilli of T1 and T3 (with two sets of microvilli for each cell). MT1, MT2 and MT3 indicate the position of microvilli for each cell. (B) Example of a neurobiotin-stained T2 cell.

The bright staining of the cell is visible between the unstained rhabdomeric portions of T1 and

T3, which is specific to T2 cells. (C) Example of a T1 cell, which is characterized by bright staining of the center of one of the large rhabdomes. V, vertical; D, dorsal; L, lateral; M, medial.

Table 1. Overview of recordings and results

Polarization Number of cells e-vector angle (deg.) sensitivity stained (type) Min response Max response N PS N

Eye 1 4 (T1) 179.6±6.8 268.5±5.7 8 11.1±8.2 7

271±6.5 182.2±5.2 6 8.8±3.2 5

Eye 2 3 (T1) 178.3±5.4 269.2±4.3 12 12.2±8.0 13 4 (T2) 268.7±7.4 181.9±6.3 11 9.5±3.4 9 Values are means ± s.d.

68 Response to changing e-vector orientation

An example of a recording from a cell that was most sensitive to horizontally polarized light is illustrated in Figure 4. The cell’s response is modulated by about 44% while the e-vector orientation is rotated through 180 deg (Fig 4A). In addition the shape of individual voltage responses was slightly different between recordings. Specifically, a cell’s maximum response was characterized by a fast initial peak, followed by a slightly slower maximum (Fig 4B) similar to what has been reported in sawflies (Meyer-Rochow,

1974). Weaker responses did not show the fast initial peak (Fig 4C).

Fig. 4. Example recording of a cell that was most sensitive to horizontally polarized light. (A)

During stimulation with light pulses the e-vector orientation was turned through 180 deg in 5 deg steps. (B) Response of the cell to a single 20 ms stimulus at the e-vector orientation (185 deg) that yielded the maximum response. (C) Response of the cell to the e-vector orientation (265 deg) that yielded the minimum response.

69 To visualize the response magnitude modulation (Fig. 5), the data were normalized and averaged. Three cells that showed a maximum and minimum response to e-vector orientations that deviated by more than 3s.d. from the average were excluded from this and further analysis. These outliers likely were probably the result of tissue distortion from excessive gut movement that sometimes occurs during recordings.

Fig. 5. Average relative response magnitude at different e-vector directions. The response magnitude of each cell was normalized to minimum response=0 and maximum response=1. (A)

Average relative response magnitude with s.d. for E1. Cells most sensitive to horizontally (H,

N=6) and vertically (V, N=8) polarized light. (B) Average relative response magnitude, with s.d. for E2 (H, N=11; V, N=12).

On average, cells that were most sensitive to horizontally polarized light had a maximum response to polarized light with an e-vector direction of 182.2±5.2 deg

(mean±s.d., N=6) in E1 (Fig. 5A) and 181.9±6.3 deg (N=11) in E2 (Fig. 5B) and a minimum response to polarized light with an e-vector direction of 271±6.5 deg (N=6) in

70 E1 and 268.7±7.4 deg (N=11) in E2. There was no significant difference between measurements from E1 and E2 (two-tailed Student’s t-test, min response P=0.539, max response P=0.957).

Cells most sensitive to vertically polarized light had a maximum response to an e-vector direction of 268.5±5.7 deg (N=8) in E1 (Fig. 5A) and 269.2±4.3 deg (N=12) in

E2 (Fig. 5B), and a minimum response to an e-vector direction of 179.6±6.8 deg (N=8) in E1 and 178.3±5.4 deg (N=12) in E2. No significant difference between the two eyes was observed (two-tailed Student’s t-test, min response P=0.642, max response

P=0.769).

Polarization sensitivity measurements

Trials were excluded when they a) had an unstable baseline (three recordings) or b) the response magnitude could not be recovered to within 10% of the initial response (three recordings). Fig. 6 illustrates (separately for E1 and E2) the average of the normalized

V-logI curves of cells most sensitive to horizontally and vertically polarized light.

Normalized V-logI curves are illustrated for both maximum (max V-logI) and minimum

(min V-logI) response e-vector orientations. To calculate the PS values we first measured the V-logI relationship for each cell at the maximal and minimal sensitive e- vector orientation (Fig. 6A-D). However, at the time of the recordings no exact measurements of these directions were available. Therefore they were estimated by slowly turning the polarization filter while observing the response magnitude. These estimations were on average within 5.2±4.9 deg (mean±s.d., N=30) of the measured value (based on subsequent data analysis). This small diversion from the optimal angle

71 probably leads to a small underestimate of the PS for some of the cells.

The PS was calculated from the shift of the V-logI curves along the intensity axis

(Fig. 6A). The range of PS values, especially for the cells that were most sensitive to vertically polarized light (of both eyes), was very large (as illustrated in Fig. 6E,F). Cells of E1, which were most sensitive to vertically polarized light, had a PS of 11.1±8.2 (N=7) and cells that were most sensitive to horizontally polarized light had a PS of 8.8±3.2

(N=5). For E2 the PS of cells most sensitive to vertically polarized light was 12.2±8.0

(N=13), and of those most sensitive to horizontally polarized light the PS was 9.5±3.4

(N=9). From these data we could detect neither a significant difference in PS values between eyes, nor between cells that were most sensitive to vertically or horizontally polarized light within each eye (Student’s t-test, P>0.05).

72

Fig. 6. Average normalized V-logI curves with s.d. and PS values of cells most sensitive to vertically (V) and horizontally (H) polarized light. The PS was calculated from the shift of the V- logI curves. (A) Cells of eye 1 most sensitive to horizontally polarized light (N=5). (B) Cells of eye 2 most sensitive to horizontally polarized light (N=9). (C) Cells of eye 1 most sensitive to vertically polarized light (N=7). (D) Cells of eye 2 most sensitive to vertically polarized light

(N=13). E and F. PS values of eye 1 and eye 2 respectively.

73 Discussion

Although polarization sensitivity has been studied fairly well in adult insects, little is known about it in larvae. Nevertheless, it is likely that at least some larvae, such as those of T. marmoratus, could substantially benefit from it. In previous work, the possibility of polarization sensitivity in these larvae has been raised based on the ultrastructure of their eyes (Stecher et al., 2010). Here we used electrophysiological methods to confirm that the proximal retinas of the principal eyes E1 and E2 are indeed polarization sensitive. As expected from the ultrastructure, cells of the type T1 are most sensitive to vertically polarized light, and cells of the type T2 are most sensitive to horizontally polarized light.

Polarization sensitivity in arthropods

To the best of our knowledge, there has only been one other physiological study

(Meyer-Rochow, 1974) of polarization sensitivity within holometabolous insect larvae.

In that study the PS values of the sawfly larval eye had a mean of 6.1, with a maximum of 10. Much more is known about polarization sensitivity in adult insects and crustaceans. For the former, the highest PS values are generally found in the dorsal rim area, an area of the that is known to be specialized for polarization vision. The PS values of T. marmoratus larvae are comparable to values commonly found in the dorsal rim area (for example, those of crickets, locust, and ants; Table 2).

Moreover, they are clearly higher than the typically low PS values found in other areas of insect eyes. Specifically, our values are most similar to those of bees, scarab beetles, and some flies (Musca, Calliphora). Similarly, when compared to crustaceans,

74 our values are similar to the higher PS values in the literature. In some of these species behavioral relevance has been demonstrated (Chiou et al., 2008). Taken together, these comparisons make clear it that PS values in the visual system of T. marmoratus larval eyes are fairly high, making it likely that polarization sensitivity plays an important role for them.

PS values are often quite variable in invertebrates (Stove, 1983).

Correspondingly, the range of the measured PS values in T. marmoratus was large, ranging from 4.5 to 14.2 for cells most sensitive to horizontal e-vector orientation, and from 2.7 to 24.9 for cells most sensitive to vertical e-vector orientation. Some, but probably not all of the variability might be due to measurement inaccuracies (Stowe,

1983). Another previously discussed source of the typically large range in PS values is natural variability in microvillar orientation, as well as distortions that might be caused by the microelectrode penetration. Nilsson et al. (Nilsson et al., 1987) modeled the effects of microvillar misalignment on PS values and found that relatively minor misalignments can strongly affect PS values. In addition, in fused rhabdomes neighboring cells can act as lateral filters for one another, adding further variability (Nilsson et al., 1987; Shaw,

1969; Stowe, 1983).

75 Table 2. Comparison of polarization sensitivity (PS) values in different arthropods, from dorsal

rim area (DR) or other areas of the eye, and with spectral sensitivity as indicated

Organism Species PS values Reference

Insects

Beetle Pachysoma striatum 12.8 ± 1.2 (DR, UV) Dacke et al., 2002* 6.5 ± 1.1 (DR, UV/green) Scarabacus zambesianus 7.7 (DR, UV) 12.9 (DR, UV/green) Dacke et al., 2004* 1.5 - 1.6 T. marmoratus larvae 10.8 ± 6.4 (UV) Current Study

Bee Megalopta genalis 21.2 ± 7.5 (DR, UV) Greiner et al., 2007* 1.4 (green) Apis mallifera mallifera 5.6 ± 2.1 (DR, UV/green) <2 (UV) Labhart, 1980 3.8 ± 2.4 (DR, UV/green) 13 ± 4.5 (DR, UV) Ant Cataglyphis bicolor 6.3 ± 2.4 (DR, UV) Labhart, 1986 2.9 ± 1.6 Fly Musca domestica 6 - 19 (DR, UV) Hardie, 1984* Calliphora erythrocephala Pega affinis larvae larval eye 6.1 ± 2.1 Meyer-Rochow, 1974

Bug Gerris lacustris 6.7 ± 1.7 (blue) Bartsch, 1995 6.9 ± 0.63 (green) Gryllus campestris 6.2 Blum & Labhart, 2000

Gallus campestris 2.6 ± 0.8 (DR, green) Labhart et al., 1984 8.3 ± 5.9 (DR, blue) Crustacea

Stomatopod Odontodactylus scyllarus 2.75 ± 0.42 (row5&6, UV) Kleinlogel and Marshall, 2009* Gonodactylus chiragra 3.8 ± 1.6 Kleinlogel and 6.2 ± 1.3 Marshall, 2006* Crab Leptograpsus variegatus 5 Doujak, 1984

Leptograpus variegatus 10 Stowe, 1980

Carcinus maenas, 4.5 Mote, 1974* Callinectes sapidus crayfish Procambarus clarkii 1.3 - 12.5 Glantz, 2007

*These studies derived PS values from the shift of maximum and minimum V-logI curves.

76 In addition to sensitivity to linearly polarized light, animals can be sensitive to circularly polarized light. In the mantis shrimp compound eye distally situated photoreceptors act as a retarder that converts circularly polarized light into linearly polarized light (and vice versa), allowing them to be sensitive to circularly polarized light instead of linearly polarized light (Chiou et al., 2008). In E1 and E2 of T. marmoratus, light that enters the polarization sensitive proximal retina also first has to cross the microvilli of distally situated photoreceptor cells (Fig. 1A), an organization that potentially could alter the incoming light. However, in contrast to the mantis shrimp organization, the microvilli of the distally located photoreceptor cells of T. marmoratus are relatively irregular (Stecher et al., 2010). Moreover, cells typically are most sensitive to either linearly or circularly polarized light (Chiou et al., 2008). Therefore it is unlikely that T1-T3 cells are sensitive to circularly polarized light, though we did not directly test for this possibility.

Although PS values generally are highly variable, the range of values for those cells that were most sensitive to vertical e-vector orientations was particularly large. In the next section we discuss evidence that this may be due to the presence of two distinct groups of cells.

Evidence for two cell types that are sensitive to vertically polarized light

The proximal retina is composed of three cell types (T1, T2 and T3) that are arranged in an alternating pattern (Fig. 1B). All three cell types have the same spectral sensitivity in the UV range (Maksimovic et al., 2011), there is no obvious optical barrier between cells, and the microvilli are directly adjacent. Based on our transmission electron

77 micrographs (Fig. 3A), in third instar larvae two of these cells (T1 and T3) have microvilli that are oriented vertically, whereas only one cell type (T2) has microvilli that are oriented horizontally. From post-recording staining of cells we could confirm that, as expected from their microvillar orientation, T2 cells indeed are most sensitive to horizontally polarized light, and that T1 cells are also most sensitive to vertically polarized light. However, we were not successful in staining any of the much smaller T3 cells. Considering that post-recording injection of neurobiotin only succeeded in single cell staining in less than a third of the experiments, it is conceivable that some of our physiology data are nevertheless from T3 cells. Based on the confirmed directional sensitivity of the T1 cells and the more or less identical microvillar orientation of T3, it is highly likely that these cells too are most sensitive to vertically polarized light. However, the large structural difference between these cells (including the sizes of adjacent rhabdomeres) could result in differences in PS values. As modeled by Nilsson et al.

(Nilsson et al., 1987), an unequal light absorbance ratio between neighboring cells (that act as lateral filters for one another) leads to different modulation strengths and hence unequal PS values for these cells. Specifically, the model shows that a cell with a relatively large rhabdomere, next to a cell with a smaller, orthogonal rhabdomere would result in less modulation and lower PS values. Conversely, the cell with the smaller rhabdomere is expected to have increased modulation and a higher PS value. As is apparent in Fig. 3a, the rhabdomere of T3 cells (labeled MT3) indeed might be surrounded by very small T2 cell rhabdomeres (MT2). Accordingly, from the anatomy it might be expected that T3 cells have relatively low PS values.

Based on our combined physiological data, cells most sensitive to vertical e-

78 vectors appear to fall into two distinct populations (Fig. 7): one showing shallower modulation (lower ∆ response magnitude) than does the other group of cells, some of which have been identified as T1 cells.

Fig. 7. Change in response magnitude of E1 and E2 cells most sensitive to vertically and horizontally polarized light. (A) Data of all cells. Triangles indicate a population of cells that is most sensitive to vertically polarized light with a relatively low modulation, when compared to other cells with equivalent e-vector orientation sensitivity (squares). Diamonds indicate cells that are most sensitive to horizontally polarized light. (B) Average of all cells (with s.d.) after separating vertical sensitive cells into shallow and large modulation groups.

In addition, when we recalculated the average PS values according to these groupings, we found that the PS values of the cells most sensitive to horizontal e-vector orientations (9.3±3.2, mean±s.d., N=14) tend to fall in between the values of the low

(3.1±0.4, N=4) and high (13.9±7.5, N=16) modulated cells most sensitive to vertical e- vector orientations. The shallower population potentially could represent T3 cells. No separation into two groups could be observed for cells most sensitive to horizontally

79 polarized light, neither anatomically nor based on physiology. Interestingly though, the shape of the polarization modulation for all cells, with a broadened range around the peak and a narrow range around the trough, corresponded well with theoretical curves

(Nilsson et al., 1987).

Despite the relatively large literature on polarization sensitivity, few studies have evaluated the modulation strength of neighboring cells in the light of rhabdomere anatomy. The unequal rhabdomere organization of T. marmoratus makes it well suited to test existing theoretical models, and we are excited that our data are conceptually consistent with theoretical considerations (Nilsson et al.,1987). It would be interesting to empirically investigate the reciprocal influence of neighboring cells in greater depth by examining other comparable systems.

Functional considerations

The high PS makes it likely that polarized light plays an important role in the vision of T. marmoratus. The larval eyes nearly completely degenerate during pupation while the adult compound eye develops de novo (Sbita et al., 2007). Thus, the polarization sensitivity of the larval eyes can only benefit their vision in the larval phase. In order to discuss possible functions, we need to first consider the natural history and behavior of these beetle larvae. They are highly successful visual predators: once a prey item is detected, they stalk and follow it using their principal eyes E1 and E2. While slowly approaching the prey, larvae scan their visual field with dorsoventral head movements and finally strike to catch the prey (Buschbeck et al., 2007). It has been shown in other aquatic animals that polarization sensitivity can be used to either enhance visual

80 contrast by filtering out horizontally polarized haze, or to use its prey’s polarization features for detection (Shashar et al., 1998). It is conceivable that polarization sensitivity in T. marmoratus has similar functions. However, there are other ways in which polarization sensitivity could be beneficial. For example, T. marmoratus develop on land, near water. After hatching young larvae need to find the nearby water, a behavior for which the use of polarization cues has been demonstrated in a variety of insects (Schwind, 1991; Schwind, 1999). Moreover, late instar T. marmoratus larvae need to return to land to pupate and therefore need to find the shore. Within a pond, horizontal background polarization is expected to be highest away from the sore, and it has been shown that such cues can be used to find open water (Schwind, 1999). It is conceivable that T. marmoratus uses similar visual cues for the opposite purpose, namely to find shore when it is time to pupate. Behavioral experiments will be necessary to determine for which of these behaviors polarization sensitivity might be important.

81 Chapter 4

Multitasking in a larval eye: How the unusual organization of the principal eyes of Thermonectus marmoratus allow far and near vision and might aid in depth perception.

82 Summary

Only a few visual systems fundamentally diverge from the basic plans of a relatively few well-studied animal eyes. Nevertheless, the study of these unusual visual systems promises new insight into vision and may provide engineers with new ideas for executing visual tasks. One such system are the unusually constructed principal eyes of a visually-guided aquatic predator, the Sunburst diving beetle larva, Thermonectus marmoratus (Coleoptera: Dystiscidae). We previously reported that their principal eye

E2 has a bifocal lens (Stowasser et al., 2010); that both principal eyes are characterized by a complex layered retina that has distinct distal and proximal portions (Mandapaka et al. 2006, Maksimovic et al., 2011); and that behavioral experiments suggested that these larvae has an unilateral range finding mechanism that might be the product of their bizarre eye organization (Bland et al., under revision). In our present study, we have expanded our optical measurements and found that: 1) E2 also has a bifocal lens;

2) the retina organization, in combination with the bifocal lenses supports the extraction of distinct image information with the two retinas; 3) the tiering of the retina, and differences between E1 and E2 in regard to object distance dependent image positions appear to function in place of an accommodation mechanism, where E1 appears most suited for far vision while E2 appears most suited for near vision; and 4) the peculiar eye organization could allow unilateral range finding, which could explain the extraordinary hunting abilities of these larvae.

83 Introduction

The functional organization of small visual systems such as those found in insects is often optimized to perform specific visual tasks, because their small size does not allow for an all-purpose system. In such systems, specialized filtering of visual information often already takes place at peripheral levels, involving the optics or the retina of the system, such that only selective visual information is passed on for further processing at higher levels (Wehner, 1987). While much is known about the compound eye systems of adult insects, the highly diverse small chambered-eye systems of holometabolous insect larvae are greatly understudied. This is likely because the larval eyes are generally thought to be very simple, performing only basic visual tasks and providing poor vision.

However, some larval eyes are well developed and seem to have relatively complex visual functions. Among the most complex, and presumably highly specialized larval eye systems, are the principal eyes of a successful visually-guided predator, the

Sunburst diving beetle larvae. T. marmoratus larvae have 12 eyes, six on each side of the head. Four of these eyes (E1 & E2 on each side), are tubular and face directly forward (Fig. 1a). The retinas of these principal eyes are divided into distinct distal and proximal portions. The distal retina consists of at least 12 tiers of green-sensitive photoreceptor cells, with rhabdomeres oriented approximately perpendicular to the light path. The proximal retina consists of UV and polarization sensitive photoreceptor cells

(Maksimovic et al., 2009; Maksimovic et al., 2011; Stowasser and Buschbeck, 2012).

The rhabdomeres of these photoreceptor cells are oriented parallel to the light path (Fig.

1b).

84

Figure 1 Thermonectus marmoratus third instar

larvae. (a) the entire head with principal eyes,

E1 and E2, pointing directly forward. The inset

shows a larva, in its entirety, swimming. (b)

Schematic of the principal eyes, sagittal section

(S) and horizontal section (H) with the proximal

retina (PR), distal retina (DR), lens (L) and the

crystalline cone-like structure (CC). The black

lines indicate the approximate horizontal and

vertical visual fields of the retinas. The visual

field is a horizontal stripe relative to the head:

horizontally, it is very wide, while, vertically, it is

very narrow.

Thermonectus marmoratus is an aquatic predator that lives in small streams and ponds in the Southwestern United States (Evans, 2006, Morgan 1992a, Larson, 2009).

The larvae move freely through the water as they hunt, track and slowly approach prey items, which they then strike from a few millimeters distance. The typical striking distance of second instar larvae is only about 4-5 mm (Bland et al., under review).

Because each layer of the retina consists of only two horizontal rows of photoreceptors, the visual field of their eyes is a horizontal stripe. Accordingly, as the larvae approach potential prey, they scan with their principal eyes by oscillating their heads dorso-ventrally (Buschbeck et al., 2007). When only a few millimeters from their

85 prey, they stop their scanning movements and initiate body flexion and ballistic prey capture. This behavior, in combination with their high hunting success, suggests that these larvae have a superb range finding mechanism. We recently hypothesized that this mechanism is related to their bizarre eye organization because—even after severely compromising commonly known range finding mechanisms in insects such as image size, stereopsis, and motion parallax—these larvae could still successfully gauge prey distance (Bland et al., under review). However, the question remained: “what specific range-finding cues could their eye organization provide?” The only well-studied tubular visual system that has a tiered retina and a monocular range finding mechanism that appears to involve the layering of the retina is that of the jumping spiders (Nagata et al., 2012). We investigated whether T. marmoratus larvae could have a comparable range finding mechanisms that involves the retina tiering, a mechanism that is so far un- described in insects.

The size of the chambered eyes of small invertebrates, moreover, greatly limits their resolution due to their short focal length. Under the size constraints of such animals, resolution could be enhanced theoretically with tubular eyes of a relatively long focal length. Such an arrangement would result in a larger image size, which would then serve to enhance spatial resolution. In that case, however, the animal might also need an accommodation mechanism in order to have both near and far vision. The very short striking distance of T. marmoratus larvae, in combination with their tubular eyes, suggested that this system might operate in its near range for prey capture. This, however, raised another question: “how could the larvae also have far vision, as implicated by their overall hunting range, despite their presumed inability to change the

86 focal length of their lenses?” The only well-studied visual system within the size range of T. marmoratus that possibly had near and far vision is that of the trilobites. Some of these long-extinct arthropods had compound eyes, each unit of which might have functioned as an image-forming eye. Reconstructions of the optical properties of their lenses suggest that these lenses were bifocal (Egri and Horváth, 2012; Gál et al.,

2000a; Gál et al., 2000b). It has been proposed that these bifocal lenses might have allowed trilobites near and far vision, which would not be possible with a rigid monofocal lens (Egri and Horváth, 2012; Gál et al., 2000a; Gál et al., 2000b). We investigated whether in T. marmoratus the bifocal lenses in combination with the tiering of the retina could have a similar function.

Finally, the difference between the proximal and distal retinas, in regard to their organization and sensitivity, suggests that they extract different kinds of image information. However, for this to be the case, a reasonably sharp image must be projected onto each of the retinas. Our previous investigation showed that, in E2, the lens projected two sharp images (Stowasser et al., 2010), but we had still to ascertain whether the lens of E1 had similar properties and where the images were focused within the eyes, particularly in response to changing object distances.

Compared to known visual systems, the principal eyes of T. marmoratus have a very unusual organization, which, in conjunction with their optical properties and the larva’s ability to correctly gauge distances unilaterally, hints at the presence of visual mechanisms that are poorly understood or have even yet to be described. These mechanisms likely involve the tiering of the retina. To address the question of how these eyes might function, we expanded the optical and anatomical measurements. As

87 a result, we are now able to present a more complete model of how images of prey may be focused within the two eyes and to postulate what kind of range finding cues the bizarre eye organization might provide.

Materials and Methods

Animals:

The Thermonectus marmoratus larvae were the offspring of beetles provided by the

Insectarium of the Cincinnati Zoo and Botanical Gardens and beetles collected between

2004 and 2012 near Tuscon, Az, USA. A population of T. marmoratus, previously provided by the Insectarium and collected near Tuscon, AZ was maintained in our laboratory throughout the year. The larvae were reared in isolation on previously frozen bloodworms and live mosquito larvae. All data was obtained from third-instar larvae, 1

– 3 days after ecdysis. Animals were anesthetized on ice and decapitated. The heads were dissected in 50% insect Ringer’s solution (O'Shea and Adams, 1981) as previously reported (Stowasser et al., 2010). The lenses of the eyes from one side of the head were prepared for optical measurements and the eyes from the other side of the head were prepared for histology.

Histology and Anatomical Measurements:

Tissue preparation was as previously described (Mandapaka, 2006; Sarah, 2007). In brief, after overnight fixation in a 4% paraformaldehyde solution (EM grade; Electron

Microscopy Sciences, Fort Washington, PA, USA) in Sorenson’s phosphate buffer at pH

7.4 (Electron Microscopy Sciences, Fort Washington, PA, USA), the tissue was washed in buffer, followed by a 1% osmium tetroxide (OsO4) (Electron Microscopy Sciences)

88 post-fixation for 1.5 h at 4 °C and for 1.5 h at room temperature. It was then washed, and stained with a saturated ethyl gallate (Fluka, Buchs, Switzerland) solution for 1 h at

4 °C and 1 h at room temperature. Afterwards, the tissue was washed and dehydrated through a series of graded alcohol solutions, and embedded in Ultra-Low Viscosity

Embedding Media (Polysciences, Warrington, PA, USA). Specimens were sectioned at

7 or 8 mm sagittally. Images were taken from these sections with an Olympus Magna

Fire digital camera, and analyzed with ImageJ 1.38 (U. S. National Institutes of Health,

Bethesda, MD).

For each individual, we measured the following distances along the long axis of the eye (Fig. 3): a) the distance between the back surface of the lens and the pit of the distal retina; b) the distance between the back surface of the lens and the surface of the proximal retina; c) the length of the proximal retina; and d) the distance between the pit of the distal retina and its ventral and dorsal rim. We also measured the diameter of the lens. With the exception of the lens diameter, per individual measurements from 3 - 10 sections from the mid-region of the eye were averaged. The lens diameter was assessed from the section that showed the largest diameter. To correct anatomical measurements for shrinkage, we assessed eye tissue shrinkage during tissue preparation. Sagittal photographs were taken of the heads of newly molted third instars, which had yet to exhibit exoskeleton pigmentation, so that the eye tubes could be seen through their cuticle. These heads were then processed for histology. Shrinkage was determined from the difference in the total eye tube length of E2 between the measurements taken from photographs through the cuticle and the average of 3-5 sagittal sections of the mid-region of the eye. E2 was chosen because its full length

89 was most readily distinguishable through the cuticle.

Optical measurements

As previously described (Stowasser et al., 2010), similar to the hanging drop method

(Homann, 1924), we used a microscope to observe the images formed by the lens. In brief, the lens was mounted with wax between two coverslips so that images were formed between the lens and upper cover slip. The space between the coverslips was filled with a 50% dilution of insect Ringer’s solution (O'Shea and Adams, 1981), which was determined to best conserve lens structure over time and which corresponds well to haemolymph measurements (Stowasser, unpublished data). The coverslip sandwich was mounted on a goniometer, which replaced the microscope stage. The back surface of the lenses faced the microscope objective lens. A square-wave grating (0.353 cycles/mm, USAF 1951 negative test target from Edmund Optics) served as the object and was placed 12.5 cm beneath the microscope stage—effective infinity for these small lenses. The condenser was removed, and the object was aligned with the center of the microscope optics. The square wave was illuminated with green light (542 nm, half width 47 nm) or UV light (396 nm, half width 78 nm), and the rays refracted by the lens were photographed with a Olympus Magna Fire digital camera or with a 3CCD camera (Hitachi HV-f22) and image acquisition with ImageJ 1.38 (U. S. National

Institutes of Health, Bethesda, MD, with QuickTime_Capture plug-in modified for high- definition image acquisition with a pixel resolution of 1360 x 1024). The frames were photographed at 5 μm intervals from the back surface of the lens to well beyond the focal planes. Photographs were evaluated for optimal focus of the square-wave image

90 by computing image contrast using a customized Matlab program. We first digitally rotated the images so that the depiction of the square wave was oriented horizontally.

Then, images were cropped to the region of the square wave. For each cropped image, an average gray scale value was computed for each row of the image matrix and the values were plotted (Fig. 2a). In plots that resolved the three bars of the object, the three peak and two trough gray-scale values of the wave were found, as illustrated in

Fig. 2d. Four contrast values were computed from these three maximal (Imax) and two minimal (Imin) gray-scale values (Equation 1) (Hecht, 2001), namely the contrast between: a) Imax1 and Imin1, b) Imin1 and Imax2, c) Imax2 and Imin2, and d) Imin2 and Imax3. The average of these four values was the contrast value of the image.

I  I (1) Contrast  max min I  I max min

If plots did not show a wave with three bright bars, the contrast value was set to zero.

Generally, the graph of the contrast values across an image series showed two peak areas with a trough in-between (Fig. 2b). For each of the two peak areas, the computation was repeated separately to obtain the final position of the two focal planes.

For the purposes of reducing interference between the in-focus and the fuzzy second image in the background, the image was cropped to the portion that only showed the corresponding image of the square wave (Fig. 2c). The image that had the highest contrast value in this computation was accepted as the best focused image and represented the position of the focal plane.

91

92 Fig. 2. Image analysis example. (a) sample of the image series with corresponding gray-scale value profiles. The values in the images are the distances from the back surface of the lens at which that image was taken in µm. (b) Image contrast across the entire image series. The two peaks indicate the locations of the two best-focused images. (c) Determination of the best focused image. To avoid background interferences, the contrast computation as illustrated in (a) was repeated for each peak area separately with a portion of the image that only showed the corresponding square wave image. The final best focused images were the images that had the highest contrast values in these computations. (d) Image contrast was calculated from the three peak (Imax) and two trough (Imin) gray-scale values when a wave was discernible. Otherwise, the contrast value was set to zero. (e) Image size calculation. The dark wave is the Gaussian wave that was fitted to the gray scale profile of the best focused image. The image size was the distance between the two outer peaks of this fit (resolution: 1 pixel was 0.5 µm).

Optical calculations and modeling

Since the lenses of these eyes are relatively thick, we treated this optical system as a thick lens system. To obtain the focal lengths (f’), measured from the second principal plane of the lens to the focal plane, first a Gaussian wave was fitted to the average gray scale profile of the best-focused image as illustrated in Fig. 2e (Model: gauss3, curve fitting toolbox, Matlab). The image size was calculated from the distance between the two outer peaks of the fitted curve, see Fig. 2e (an image row was 0.5 µm wide). The focal lengths (f’) of the lenses were calculated from the size of the image (Equation 2)

(Hecht, 2001).

yi (2) f ' * so yo

93 In this equation, yi is the image size, yo is the object size and so is the object distance, which was corrected with the refractive index of the insect Ringer’s solution (1.33) to correct for an object being in air while the lens was in solution (Hecht, 2001).

Additionally, the back focal length (b.f.l.) of the lens, measured from the back surface of the lens, was calculated from the distance between the image frames that showed the best-focused back surface and the best-focused images. To obtain the actual distance between frames, the measured distance was corrected with the refractive index of insect Ringer’s solution (1.33) (Galbraith, 1955). Finally, the distance between the second principal planes and the back surface (h2) was calculated from the focal length

(f’) and the back focal length (b.f.l.) with h2=f–b.f.l.

Although larvae approach prey from a few centimeters, the usual striking distance of the larvae is only a few millimeters and therefore within the near field of these lenses. For this reason, to access image positions within the eye for near objects, we modeled the image distances (di), from the back surface of the lens, within a distance range of 5 cm and 2 mm. Since this lens is relatively thick, we first calculated the image distance (si) measured from the second principal plane of the lens (Equation

3), and then calculated di, where di=si–h2 (Hecht, 2001).

f '*so (3) si  so  f '

The putative image positions relative to the retinas within each eye were determined for those individuals for which both optical and anatomical data was available to account for overall size variations between individuals, as suggested by Toh

94 and Okamura (2001). The anatomical measurements were corrected for average shrinkage. To better understand where images might be in-focus within the retina layers, we further assessed the depth of focus. In any optical system, there is a range within which images are conceived as “in focus”. Depending on the properties of the system, this range is either determined by physical or geometrical optics. In a system with a relatively small aperture, the depth of focus might be determined by physical optics, namely by the diffraction due to the aperture (Wolf and Born, 1965). In a system with a relatively large aperture, depth of focus might be determined by geometrical optics (Collett and Harkness, 1982) as illustrated in Fig. 3. To determine which of these two criteria was most relevant for the visual system of T. maromoratus, similar to what had been done for jumping spiders (Land, 1969), we calculated depth of focus for physical optics (Equation 4) and geometrical optics (Equation 5) for relevant object distances and wavelengths of light. 2 2  R  (4) d     i n a  

In this equation, Δdi is the depth of focus according to physical optics for an image at distance di; λ is the wavelength of the light (green: λ=540nm, UV: λ=396nm); n is the refractive index of the medium behind the lens (n=1.33); R is the radius of the wave front that converges at the optimal image plane; and a is the radius of the exit aperture

(Land, 1969; Wolf and Born, 1965). The equation is based on the Rayleigh limit. In the principal eyes of T.marmoratus, the exit aperture is the pigment ring that surrounds the lens, so that R is the image distance measured from the second principal plane (R=si),

95 and a is ½ the lens diameter.

c * s c*si (5) i dif   d ic  A  c A c

Equation 5 calculates the depth of focus according to geometrical optics, and is based on the relationships illustrated in Fig. 3 and described by Collett and Harkness (Collett and Harkness, 1982). These are calculations of the depth of focus for image distances

(si) of object distances that are closer than the hyperfocal distance H=A*f’/c (the closest distance that is deemed in focus when an object at infinity is focused onto the retina):

Δdif is the depth of focus for object distances that are further away and Δdic is the depth of focus for object distances that are closer, see Fig. 3. A is the lens diameter and c is the diameter of the maximal allowed blur circle (7 μm).

Fig. 3 The geometrical optics of depth of focus. Depth of focus of a given optical system is dependent on the size of the aperture A, allowed blur circle c and the object distance so. The image of an object O appears to be in focus as long as its blur circle is not larger than maximally allowed.

96 The maximal allowed blur circle was based on the receptor or receptor unit spacing of the DR and PR as measured from frontal sections. For the DR, which consists of only one histologically distinguishable cell type (Stecher et al., 2010), we measured the distance between neighboring photoreceptors (E1: 5.7±0.7μm; E2:

3.0±0.2μm, mean±s.d,.N=4). The proximal retina, on the other hand, consists of three photoreceptor types (T1, T2, and T3) that alternate along the horizontal axis (Stecher et al., 2010; Stowasser and Buschbeck, 2012). T1 and T3 are sensitive to vertically polarized light, while T2 is most sensitive to horizontally polarized light (Stowasser and

Buschbeck, 2012). T2 cells have two rhabdomeric regions, one bordering a T1 cell and the other a T3 cell, such that the repeated pattern is: T1 – T2 – T3 – T2 – T1. Each T1

–T3 group was considered as one functional unit, and the distance between such units was assumed as the distance between photoreceptor units (E1: 6.3±0.47μm; E2:

6.7±0.2μm, mean±s.d,.N=8). We assumed a maximal allowed blur circle with a diameter of 7μm as the limit for all theoretical considerations.

Finally, we determined whether it was theoretically possible for these animals to use the change in image size that resulted from changes in object distance (for example, while the larvae approache its prey) as a range finding cue. For this, we modeled image sizes for finite object distances between 1 cm and 2 mm (Equation 6;

Hecht, 2001) for object sizes (0.25 and 0.5 mm) that correspond well to natural prey.

si (6) yi  * yo so

97 Results

To answer the question as to where within the eyes images are focused, particularly in response to changing object distances, we expanded our optical and anatomical measurements. Since the proximal retina in both eyes is UV sensitive while the distal retina is green sensitive (Maksimovic et al., 2011), chromatic aberration had to be considered. We here report results from our optical measurements of both eyes for both wavelengths, green and UV, and bring them into direct relation to the anatomy of the contralateral principal eyes of T. marmoratus larvae.

Anatomical measurements: E1 is longer than E2

To compare E1 and E2, we took the following anatomical measurements: (a) the distance between the back surface of the lens and the pit of the distal retina; (b) the distance between the back surface of the lens and the surface of the proximal retina; (c) the length of the proximal retina; and (d) the lens diameter. The dimension of the cup formed by the distal retina was quantified from the distance between the pit of the distal retina and the dorsal and ventral rims of the distal retina, as illustrated in Fig. 4.

Measurements were taken from E1 and E2 of 19 individuals. The results are summarized in Table 1, and Fig. 4 illustrates the average dimensions of the eyes. All measurements were corrected for an average shrinkage of 5.9%. This tissue shrinkage

(5.9% ±3.1 s.d., N=21) was assessed from freshly molted third instars (see Materials and Methods, and Conclusions for discussion). This value corresponded well with values reported in the literature for comparable tissue preparations (Brunschwig and

Salt, 1997; Denef et al., 1979; Lim, 1980). If not otherwise stated, all following P-values

98 (two tailed paired Student’s t-tests) are corrected for multiple comparisons with

Bonferroni correction for seven comparisons.

Fig. 4 Average anatomical dimensions of E1 and E2. The sections exampled were morphed and the back surfaces of the lenses were aligned to illustrate the average dimensions and differences between the eyes. Abbreviations: distal retina (DR), proximal retina (PR), lens (L), dorsal (Do), ventral (Ve), distal (Di), and proximal (Pr).

Table 1 Anatomical measurements of E1 and E2, corrected for 5.9% tissue shrinkage.

E1 E2 P-value Average in µm Average in µm DR Do tip 59±19 66±18 DR Ve tip 57±15 37±11 DR pit 536±26 453±29 <0.0007 PR surface 609±30 527±33 <0.0007 PR length 110±23 98±15 0.23 L diameter 278±22 262±19 <0.0007 Values are mean ± s.d., N=19, two tailed paired Student’s t-test, all P-values are corrected with Bonferroni correction for 7 comparisons

99 The distance between the lens and the pit of the distal retina in E1 was

82.9±4.7µm (mean±s.e., P<0.0007, N=19) longer than that in E2. Likewise, the distance between the lens and the surface of the proximal retina in E1 was 82.1±4.3µm

(mean±s.e., P<0.0007, N=19 ) longer than that in E2. Even though the overall length of these eyes is different, there was no significant discrepancy between them in regard to the depth of the distal retina (E1: 73.5±14s.d., E2: 74.4±10.6s.d., P=0.71 without

Bonferroni correction, N=19) and the length of the proximal retina (see Table 1). This suggested that the main difference in eye length resulted from differences in the crystalline cone-like structure that determined the spacing between the lens and retina.

A second difference between E1 and E2 involves the shape of the retinal cup.

Only in E2 was the cup that was formed by the distal retina significantly asymmetrical, as illustrated in Figure 4, where the cup was dorsally 29.0±3.0μm (mean±s.e.,

P<0.0007, N=19) deeper than it was ventrally. In E1, there was no significant difference between the dorsal and ventral depth of the cup with P=0.61 without Bonferroni correction (see Table 1). Another anatomical difference was the dimension of the lens.

The lens diameter in E1 was 16.3±3.3μm (mean±s.e., P<0.0007, N=19) larger than in

E2.

Optical measurements: E1 is also bifocal and has longer focal lengths than E2

Previously, we reported that E2 had a bifocal lens (Stowasser et al., 2010). We expanded the optical measurements to E1, and confirmed that this eye also had a bifocal lens similar to the previously reported optical properties of the lens of E2 (Figure

5). We next compared the optical properties of these two eyes. Since the distal retina

100 is green-sensitive while the proximal retina is UV-sensitive (Maksimovic et al., 2011), we measured the optical properties of these eyes with green and UV light. We obtained both the back focal length (b.f.l.), which is the distance between the back surface of the lens and the focal plane, and the focal length (f’), which is the distance between the second principal plane of the lens and the focal plane (see Material and Method section). The measured back focal lengths (b.f.l.)—obtained from the distance between the surface of the lens and the location of the in-focus image of an object at effective infinity—and the focal lengths (f’) calculated from the image size are summarized in

Table 2.

Differences between the eyes were determined from the directly measured back focal lengths (b.f.l.). Since not all trials for E1 and E2 had data for green and UV light, we assessed differences (two tailed paired Student’s t-tests) on subsets of the data shown in Table 2 in order to account for variability among individuals.

Table 2 All back focal lengths (b.f.l.) and focal length (f’) measurements of E1 and E2

E1 E2 Back focal length Focal length Back focal length Focal length Average Average Average Average N N N N in µm in µm in µm in µm Green light First 451±51 494±57 381±36 426±48 22 20 26 22 Second 590±25 627±47 485±28 530±32 UV light First 422±47 457±62 360±29 404±39 12 12 11 11 Second 567±23 577±38 463±22 495±37 Values are mean ± s.d.

101

Fig. 5 Average normalized image contrast of the square wave image with s.d. of E1 and E2 for green light. The image contrast of the image series (graphs illustrated in Fig. 2b) was normalized to max=1 and aligned to the midpoint between the two focal planes. Distance was measured from the midpoint between the two focal planes in µm. The two peaks illustrate the relative position of the two best-focused images according to the average of these graphs. For both eyes, the contrast values across all image series at the peaks of the graphs are significantly higher than the contrast values at the midpoint between the two focal planes (paired t-tests P≤0.0087, E1: N=22, E2: N=26).

Both back focal lengths of E1 are significantly longer than those of E2 (P<0.0001, green: N=21, UV: N=8). Additionally, the focal planes in E1 are significantly farther apart than in E2 with an average difference of 33±9.8 µm (mean±s.e., P=0.0031, N=21) for green light and 32.4±11.1 µm (P=0.0226, N=8) for UV light. Lastly, UV light in both eyes is focused distal to green light. In E1 (N=12), the chromatic aberration between

UV and green light was 29.4±8.0 µm (P=0.0037) for the first focal plane and 16.6±7.0

µm (P=0.038) for the second. The results for E2 (N=11) were similar. The chromatic

102 aberration was 26.6±5.9 µm (P=0.0012) for the first focal plane and 24.8±4.6 µm

(P=0.0003) for the second. We did not observe a significant difference in chromatic aberration between or within the eyes.

Each retina receives its own image

The unusual construction of E1 and E2, combined with their bifocal lenses, raises questions as to where the two images are focused within the eyes and as to whether there is a functional difference between the two eyes on either side. Consideration must also be given to the fact that their behaviorally relevant object distance ranges from a couple centimeters to a couple millimeters: i.e., the image plane changes as the larva approaches its prey during hunting. This is because images of objects at effective infinity are focused onto the focal plane of a lens while images of near objects are focused onto planes that are further away from the lens. Based on behavioral observations, larvae are probably able to see far and near objects despite their presumed inability to adjust the focal length of their lenses.

To determine the image positions of an object at infinity relative to the retinas, we used the back focal lengths (b.f.l.) of one side of the head and contrasted them to the anatomical measurements of the contra-lateral eyes (Fig. 6).

103 Fig. 6 Position of the two images relative to the retinas of the eyes for green (green circle) and

UV (purple square) light with standard deviation. Illustrated data is the average from trials of which anatomical and optical for green and UV light was available (E1: N=10, E2: N=11).

Retina schematic and dashed lines illustrate the anatomical dimensions of the eyes with standard deviation, and the arrow shows the eye orientation: dorsal (D), ventral (V). The anatomical dimensions were corrected for 5.9% shrinkage.

To better understand which object distances are at effective infinity for these lenses, we calculated the hyperfocal distance, which is the closest object distance perceived in focus while an object at infinity is focused onto the retina (see Materials and Methods section). For E1 this distance is 19.8 mm for the first image in green light and 22.5 mm for the second image in UV light. For E2, the hyperfocal distance is 14.6

104 mm for the first image in green light, and 17.5 mm for the second image in UV light. For these object distances, for green light, in both eyes, the first image falls near the rim of the DR, providing the DR of both eyes with a focused image. For E2, however, this is only the case for the ventral photoreceptors. For UV light, the second image in E1 is focused near the surface of the proximal retina, providing the PR of this eye with a focused image, while the image in E2 is clearly focused distal to the surface of the PR.

For a subset of 13 individuals, we obtained anatomical and optical data for green light in both eyes, allowing us to specifically calculate whether there was indeed a statistically significant difference between the eyes. The data from these individuals was analyzed with two tailed paired Student’s t-tests. Despite the overall large difference in size between the two eyes, there was no significant difference in regard to the position of the first image relative to the dorsal tip of the distal retina. The first image, relative to the dorsal rim of the DR in E2, was only 9±15 μm (mean±s.e.,

P=0.543) proximal to the image in E1. However, the second image in E1, relative to the surface of the proximal retina, was 26±9 μm (P=0.0134) significantly proximal to the image position in E2. For UV light, although the number of individuals for which we had sufficient data was smaller (N=7), we calculated that the second image in E1 was still on average 42±17.3 μm (P=0.049) proximal to the image position in E2. Most interestingly, this data suggests that the proximal retina of E1 is adapted to viewing objects at infinity, whereas the proximal retina of E2 is designed to focus on objects within striking distance (Fig. 7).

In regard to the green-sensitive distal retina, consisting of many retinal tiers in both eyes, the image is focused near the top of the photoreceptor stack when the object

105 is at infinity. As the object moves closer, the focused image passes through the retinal tiers such that the first few layers are passed when objects move between infinity and

~7mm. As the object moves even closer, the image passes through the retinal layers at a progressively faster rate such that objects in typical striking range are best-focused near the pit of the distal retina in both eyes. The images pass through the entire stack at an object distance of 2-3mm (Fig. 7). To better understand where an image is perceived in focus, we calculated depth of focus for physical and geometrical optics under the assumptions described in the materials and methods section. Under these rather conservative assumptions, the depth of focus for all relevant object distances was greater for geometrical than for physical optics (Table 3); so, the depth of focus according to geometrical optics was deemed the most relevant measurement for this system. Figure 7 illustrates these values. When integrated, this visual system appears to allow far and near vision with both retinas within the hunting range of the animal.

Table 3 Depth of focus according to physical and geometrical optics.

Physical optics Geometrical optics depth of focus (µm) depth of focus (µm) Green light UV light Green light UV light so (mm) 1st 2nd 1st 2nd 1st 2nd 1st 2nd

Δdi Δdi Δdi Δdi Δdif Δdic Δdif Δdic Δdif Δdic Δdif Δdic E1 infinity 3.7 5.8 2.3 3.6 13.6 17.0 12.4 15.6 10 ±4.1 ±6.6 ±2.5 ±4.1 -13.6 14.3 -17.2 18.2 -12.4 13.0 -15.7 16.6

6 ±4.4 ±7.2 ±2.7 ±4.4 -14.1 14.8 -18.0 19.0 -12.8 13.5 -16.4 17.3 4 ±4.8 ±8.1 ±2.9 ±5.0 -14.7 15.5 -19.1 20.2 -13.3 14.1 -17.3 18.3 2 ±6.6 ±12.2 ±3.9 ±7.2 -17.2 18.1 -23.5 24.7 -15.4 16.2 -20.9 22.0 E2 infinity ±3.1 ±4.9 ±2.1 ±3.1 12.5 15.7 11.8 14.5 10 ±3.4 ±5.5 ±2.2 ±3.4 -12.3 13.0 -15.7 16.6 -11.7 12.3 -14.4 15.3 6 ±3.6 ±5.9 ±2.4 ±3.7 -12.7 13.4 -16.3 17.3 -12.0 12.7 -15.0 15.8 4 ±3.9 ±6.5 ±2.6 ±4.1 -13.2 14.0 -17.1 21.4 -12.4 13.2 -15.7 16.6 2 ±5.0 ±9.1 ±3.2 ±5.5 -14.9 15.8 -20.2 18.1 -14.0 14.8 -18.2 19.3

106

Fig. 7 Average calculated image positions relative to the retina for objects viewed within the near range of the lenses, as close as 2 mm, with standard error. The green and purple lines indicate the depth of focus for an allowed blur circle of 7 µm. The black bars show the position of the dorsal edge of the distal retina (DR Do rim), the distal retina pit (DR pit), and the proximal retina surface (PR) with standard error. The anatomical measurements were corrected for 5.9% shrinkage. Only trials for which anatomical and optical data was available were included. E1: green N=13 and UV N=10; E2: green N=18 and UV N=13.

107 Discussion

Here, we confirmed that E1, like E2, had a bifocal lens. However, we identified differences between the two eyes in regard to the object distance-dependent image positions relative to the proximal retina, which suggested that each eye was specialized for vision at different distances, allowing near and far vision with both retinas.

Additionally we found that sharp images were projected on specific, but different, retinal layers in each eye for the behaviorally most relevant distances. According to our theoretical considerations, this could potentially provide cues for range finding.

Compared to other known eyes, these eyes are most similar to the median eyes of the jumping spider, which are also tubular and have a layered retina (Land, 1969).

However, the differences between these two visual systems suggest that they might function differently. One of the most interesting differences involves the organization of the retina in regard to its spectral sensitivity, which, in T. marmoratus, is the very opposite of that in jumping spiders (Blest et al., 1981; Maksimovic et al., 2011). In jumping spiders, it is thought that one function of spectral organization is to correct for chromatic aberration (Blest et al., 1981; Land, 1969). However, this is not the case in T. marmoratus. On the contrary, the configuration is such that UV sensitivity is proximal to green sensitivity, while UV light is focused distal to green light. This spectral organization, in combination with the chromatic aberration of the lenses, allows the two images to be farther apart than they would be if the retinal organization were the opposite. This is because the two images would then have to be closer together for focus at the same retina levels. Consequently, this particular retinal organization results in low interference between the two images and supports extraction of different kinds of

108 image information by means of its two different retinas.

Challenges in establishing accurate image positions within each eye

To establish where images may be focused in the two eyes during relevant behaviors, we first measured where images of an object at infinity were focused within the eyes.

Then we modeled the image position for behaviorally relevant near-range object distances. For the most accurate results, we conducted paired measurements of the lens optics with histological measurements of the contralateral eye tubes. However, there were several factors that could have led to inaccuracies. First, we assumed that the cone had a similar refractive index to the saline solution since we did not have measurements of the cone’s refractive index. This assumption was based on our observation that, when dissecting the tissue, the crystalline cone appeared soft and jelly-like and blended in well, optically, with the surrounding saline solution. Its function is presumably similar to that of the vitreous humor in humans, which has a refractive index nearly identical to that of water (1.336 vs. 1.333; Pedrotti and Pedrotti, 1998).

A second possible complication for our measurement derived from our circumstantial observations that the back surface of T. marmoratus lenses might be susceptible to osmotic changes. To minimize such effects, measurements were completed swiftly post dissection (within 30-45 min), and performed in a saline solution at the concentration that least effected the lens (see Materials and Methods section).

A third possible source of error was related to the suspicion that the tissue might shrink during tissue preparation. To best account for this, we compared eye tube lengths from animals that were first imaged live through the cuticle, and then measured

109 again post-histology. Our histological measurements led to an observed average shrinkage of 5.9% (±3.1s.d, ±0.7s.e, N=21). This compares well to literature values.

Denef et al. (1979) reported that a 2.5% glutaraldehyde fixation, followed by 1%

Osmium post-fixation, alcohol dehydration and Epoxy resin embedding led to a final shrinkage of thyroid glands of about 6% that mainly occurred during the alcohol dehydration. Lim (1980) found that a 2% glutaraldehyde fixation, followed by an alcohol dehydration, caused a shrinkage of cochlea tectorial membranes of less than 5%, which again occurred during the final steps of the alcohol dehydration. Finally, Brunschwig and Salt (1997) compared the effects of glutaraldehyde and formaldehyde fixation alone on the tissue size of Reissner’s membrane and found that, in 4% formaldehyde, the tissue shrank only slightly more than in 3.1% glutaraldehyde (2.8% and 0.33% respectively).

Lastly, the curved surface of the cone-DR interface could potentially act as a negative lens, because the refractive index of rhabdomeres is, in general, slightly higher than that of water (Stavenga, 1975). In jumping spiders, a similarly curved pit is thought to have a significant telephoto image-enlarging effect with a magnification of about 1.5 times, and does in fact influence the final image position (Williams and McIntyre, 1980).

However, this is unlikely in the case of T. marmoratus for two reasons. First, the curved pit would act as a nearly plan-cylindrical lens, with negligible refractive power along the horizontal axis. Only the image of a horizontal edge might be affected. However, there are only two horizontal rows of photoreceptors at any retina level. Therefore the image information is very limited in the vertical aspects unless there is adequate temporal integration of a scan. This, however, would be difficult because of the unpredictability of

110 the larvae’s movements due to water perturbations. Additionally, in jumping spiders, the radius of the curvature (~5 μm) is very small (Williams and McIntyre, 1980), while in T. marmoratus, it is relatively large. In E1, it is 49.1±11.4 μm (mean±s.d, N=16) and, in

E2, it is 49.8±12.6 (N=10). Assuming the rhabdomers of the distal retina of E2 and E1 have the same refractive index as that of jumping spiders (n=1.4, Williams and

McIntyre, 1980)—which is at the high end of indices found in invertebrates (Stavenga,

1975)—there is very little expected effect on the image size and the image position.

The maximal magnification is only 1.1, and an image that is projected between the pit of the DR and the surface of the PR would be projected at the most 7 μm farther back.

Since even this maximal image shift is well within the limits of the depth of focus of this system (Fig. 7), there should be no perceivable effect on the overall quality of the image1.

Finally, while some of these complications likely contributed to the relatively high variability in our measurements, and may even lead to minor systematic errors, such inaccuracies would affect both eyes equally, and thus could not explain qualitative differences between them. Nevertheless, as outlined in the following section, the optics of the two eyes appeared different, which likely contributes to the larvae’s ability to simultaneously use near and far vision and may aid in range finding.

1 The focal length of the pit was calculated with f’= r/(nC-nDR), where r is the radius of the pit, nC is the assumed refractive index of the cone (1.33), and nDR is the assumed refractive index of the

DR (1.4). The final image distance, di, was calculated using the thin lens equation di=f’do/(do-f’), and the magnification M was calculated with M=-di/do (Hecht, 2001).

111 Eye organization allows for near and far vision

The very short striking distance of T. marmoratus larvae, in combination with their tubular eyes, suggested that this system might operate in its near range for prey capture. However, this then raised the question, “how could the larvae also have far vision—as was implied by their overall hunting range—despite their presumed inability to change the focal length of their lenses?”

Thorough evaluation of the optics and histology of both tubular eyes, E1 and E2, of T. marmoratus larvae determined where images of objects of different distances were focused in each eye. The UV-sensitive PR has photoreceptors with more or less typically organized rhabdoms, which can presumably act as light guides (Miller, 1974;

Menzel and Snyder, 1975). To be maximally excited, a sharp image must be focused on top of the rhabdoms. According to our results, this is the case for the PR of E1 for

UV-illuminated objects at infinity, while the PR of E2, at that distance, receives a blurry image. As an object moves into the preferred striking distance (around 4-5 mm), the image becomes focused on the PR of E2, while the PR of E1 receives a burry image.

Therefore, our data suggests that, synergistically, the PR of E1 and E2 appear to be adapted for near and far vision, which would not be possible with just one eye.

Object distance-dependent dynamics are also apparent in regards to focused green images at the level of the DR. The DR of both eyes consists of at least 12 tiers of shallow cells, the rhabdomeric portions of which are oriented perpendicular to the light path. The position of the focused image would therefore result in the highest contrast for the cells that are found in that specific layer. For both eyes, when the object is at infinity, the image is focused near the top of the DR stack. As the object moves closer,

112 the best-focused image moves through the retinal tiers such that, for all relevant object distances, some layers of the DR receive a focused image. Altogether, our optical data suggests that these eyes are adapted to function over a wide range of object distances, which explains the larvae’s ability to track prey within their relatively wide hunting range.

Eye organization could allow unilateral range finding.

T. maromoratus larvae are highly successful visually-guided predators that strike their prey with precision from a distance and therefore must have a very efficient range- finding mechanism. Only a few range-finding mechanisms are well described in insects

(see for review Collett and Harkness, 1982). The most common and best known mechanism is motion parallax. Many insects, such as mantids (see for review Kral,

2012), wasps (Zeil, 1993), locusts (Collet, 1978; Sobel, 1990), crickets (Goulet et al.,

1981) and bees (Srinivasan et al., 1989) are known to employ this mechanism. It is based on the idea that, as the insect performs well-controlled translational (peering) movements, the movement of the image across the retina is strictly distance-dependent.

However, this mechanism only works well if the object is stationary (or, at least, moves predictably) and the animal has tight control over its own translational movements.

Even animals that are known to use this cue frequently, such as mantids, do not use it when these criteria are absent (see for review Kral, 2012). Another range-finding mechanism, in mantids, not as common, but at least as well described (Rossel, 1983,

1986), is stereopsis, which is based on binocular distance-dependent differences between the images. Finally, insects such as the hoverfly (Collett and Land, 1975), use absolute image size as a distance cue. However, this only works well if the object size

113 is predictable. By means of a behavioral test, we recently discovered that none of these mechanisms could fully explain the extraordinary range-finding abilities of T. marmoratus larvae (Bland et al., under revision) because they were still able to gauge distances even when none of these cues were available to them. This then posed the question: “What cue do they use?”

Theoretically, tiering could provide distance cues in several ways, namely as an object moves closer:

a) the animal could use the defocus (blurriness) of the image or the shift of the

focused image as a range finding cue.

b) the animal could use the magnitude of the changes in image size as the

focused image moves through the layers

In the following section, we provide evidence that, in T.marmoratus, sufficient image information is available for both of these possibilities.

Depth from defocus or image shift

The relatively large image shifts across photoreceptor tiers, especially around the striking distance (Fig. 7), could provide a variety of distance cues. However, this is only feasible if the depth of focus is narrow enough. To better understand this possibility, we calculated depth of focus. Our data suggests that the images are focused (Fig. 7) narrowly enough, in both eyes, for the object distant-dependent specific layers of the

DR to perceive a focused image while other layers perceive a blurred image. The differences in defocus between layers, or the contrast gradient of the image across all layers, could potentially be used as a range-finding cue. The absolute blurriness of the

114 image could also be used as a distance cue. Yet other possibilities include that of using the differences in image size that result from image blur and that of using the specific locations within the eyes of the two best-focused images as a cue.

It has recently been suggested that jumping spiders employ a range finding mechanism that involves the layering of their retina (Nagata et al., 2012), but thus far, no such mechanism has been reported in insects. However, the proposed mechanism in spiders involves utilizing differences in defocus between two somewhat distinctly organized retinal layers; while, in T. marmoratus, it would involve using differences in defocus across many equally organized layers within the DR, or between the equally organized PR of E1 and E2. Our calculations demonstrated that such a mechanism was feasible. This is due to the a) relatively large depth of the DR; b) the differences between E1 and E2 in regard to the image position relative to the PR; c) the relatively small depth of focus; and d) because the system operated in its near range such that the shift of the image and, therefore, the object distance-dependent differences in blurriness around the striking distance were relatively large.

In humans, it has been shown that blurriness is an important cue for depth perception (Pentland, 1987; Held, 2012; Mather, 1997) that complements stereopsis

(Held, 2012). Following the human model, gaining depth information from defocus is widely applied in computer vision (see for review Chaudhuri and Rajagopalan, 1999).

Interestingly though, in humans as well as in computer vision, mechanisms are based on the blurriness received by a single-layered retina, not by a stack of receptors as it is the case in T. marmoratus. Held (2012) argued that one great advantage of gaining depth information from defocus is that it requires much less complex neurological

115 processing mechanisms than stereopsis, making it suitable not only for computer vision, but also for animals that have relatively simple nervous systems. This is because distance information from blurriness can be extracted peripherally, while stereopsis requires higher order visual processing mechanisms that integrate image information from two eyes. Following this notion, Emde et al. (1998) discovered that electrical fish use the blurriness of the electrical image of their surroundings to gain distance information, and Lewis and Maler (2002) subsequently suggested that using blurriness of sensations in general might be used for gaining distance information much more than we think.

Depth from changes in image size

We have recently found that T. marmoratus larvae do not use absolute image size as a major distance cue (Bland et al., under revision), but this cue might play a minor role in vision; even though the larvae approached their prey from nearly constant distances, there was a small tendency to strike at larger targets from slightly further distances

(Bland et al., under revision). It is conceivable that after initial distance estimation, they use the change in the image size to track changes in distance. Around the typical striking distance of 4-5 mm (personal observation), the distance-dependent changes in image size were relatively large and could therefore provide a relatively reliable depth cue (Fig. 8). Specifically, for a known object distance, the percent change in the image size correlates directly with a specific change in object distance. The percent change is independent of the absolute size of the object. Since the focused images of objects over a wide range of distances are projected onto the retinas of both eyes (proximal

116 retina synergistically) both retinas could potentially provide such a cue.

Fig. 8 Image size for near object distances of two object sizes that are well within the range of the width of mosquito larvae. a. first image size calculated for green light. a. [?] second image size calculated for UV light.

Multitasking taken into an ecological context

In general, the relatively small image-size of small visual systems greatly limits spatial and distance resolution. As a result, small predators such as T. marmoratus are faced with the challenge of having to see their small prey and accurately assess its distance.

Additionally, aquatic environments such as the habitat of T. marmoratus larvae impose still other challenges. For example, overall visibility is often poor due to the scattering of light in water. Of course, visibility can be improved by polarization sensitivity but that requires specialized photoreceptors (Cronin, 2006). It is also the case that both objects and predators move in semi-unpredictable ways, preventing motion parallax—the most common range-finding mechanism in insects—from providing a reliable distance cue.

Taken together, the visual challenges faced by T. marmoratus larvae are

117 extraordinary. Yet these tiny creatures seem to have evolved eyes that meet all of these challenges. Our data suggests that their success involves their retinal layering and bifocal lenses. In this context it should be noted that the function of bifocal lenses was previously discussed for trilobites, but only near and far vision was considered (Egri and

Horváth, 2012; Gál et al., 2000a; Gál et al., 2000b),. Our data suggests that bifocal lenses could also aid in the extraction of distinct image information and range finding, a finding that might inspire engineers.

Interestingly, within the very diverse family of diving beetles (Coleoptera

Dytiscidae), there are other species faced with similar visual challenges. Preliminary investigations (unpublished data from Buschbeck and Stowasser) suggest that at least some of these species have similarly organized eyes but show some variation in regard to the specifics of eye organization such as the depth of the distal retina, the shape of the cup that is formed by the distal retina, and the overall eye length. Thus, it would be intriguing to compare the functional similarities and differences between these and the eyes of closely related species in the context of environmental and behavioral challenges in order to gain insight into how these eyes evolved. This could lead us to a more complete understanding of the evolution and development of eyes in general.

118 Chapter 5

A conceptual model illustrating how the visual system of

Thermonectus marmoratus larvae functions, based on its anatomy, the physiological properties of the retinas, the optical properties of its

lenses and the behavior of the larvae.

119 Introduction

As aquatic predators with such small visual systems, Thermonectus marmoratus larvae face extraordinary visual challenges, as discussed in earlier chapters. In brief, the overall visibility in water is often poor due to the scattering of light, the contrast is generally low, and prey may even be transparent (reviewed in Stowasser and

Buschbeck, 2012, Ch. 3, pp. 54-56). The larvae must have relatively good resolution, must be able to obtain sufficient contrast to see their small prey and must be able to gauge distances very well (Ch. 1, pp. 27-29). The extraordinary hunting abilities of these larvae suggest that their visual system is sufficient to meet all of these challenges.

The question is, how?

Thermonectus marmoratus larvae have six eyes on each side of their head (Mandapaka et al., 2006). Behavioral experiments showed that T. marmoratus larvae mainly use their unusually organized and forward pointing principal eyes, E1 and

E2, for hunting (Buschbeck et al., 2007; Bland et al., under revision). These eyes have extraordinary features: a) they are tubular in shape and, compared to many other stemmata, have a relatively large distance between the lens and the retina (reviewed in

Ch. 1, pp. 18-19); b) they have a very complex, layered retina, in which the proximal retina (PR) is sensitive to UV light while the distal retina (DR) is sensitive to green light

(reviewed in Ch. 1, pp. 20-22 ); c) the PR is polarization sensitive (Stowasser and

Buschbeck, 2012, Chapter 3); d) the DR consists of many layers of photoreceptor cells that have rhabdomeric portions oriented perpendicular to the light path (reviewed in Ch.

1, pp. 18-19); e) the retina is linear with a visual field shaped like a horizontal stripe

(reviewed in Ch. 1, pp. 18-19); and f) the eyes have bifocal lenses (Stowasser et al.,

120 2010, Ch. 2; Ch. 4, pp. 100-103) whose chromatic aberration causes the UV light to be focused distal to green light (Ch. 4, pp. 100-103).

There are very few known eye systems similar to that of the principal eyes of T. marmoratus. The only well-studied tubular visual system with a layered retina is that of the jumping spider. One of the greatest challenges facing both of these predators is one of correctly gauging striking distances despite their very small visual systems.

Even though jumping spiders seem to use range finding mechanisms that involve synergistic interactions between many of their eyes (Forster, 1982), it has been shown that they can also estimate distances using monocular vision (Nagata et al., 2012). It has been suggested that this ability involves the layering of the retina. In jumping spiders, as in T. marmoratus, their largest eyes (called median eyes), which have a layered retina and point directly forward, are involved in distance estimation (Forster,

1982; Nagata et al., 2012).

These resemblances suggest that there could be functional similarities between the eyes of these two predators. However, distinctive characteristics in retinal organization indicate that there are differences in the mechanisms by virtue of which similar visual tasks might be accomplished. For instance, the retinas of jumping spiders have four distinct layers of photoreceptor cells (Land, 1969). Electrophysiological measurements have revealed that, in jumping spiders, the most distal layer is UV sensitive, while the most proximal layer is green sensitive (Blest et al., 1981). Since green sensitivity in jumping spiders is proximal to UV sensitivity, it has been suggested that one of the functions of this organization is to correct for chromatic aberration (Blest et al., 1981; Land, 1969). However, it is unlikely that this is the case in T. marmoratus

121 because the retinas of these beetles are organized in the exact opposite manner

(Maksimovic et al., 2011).

Another difference is that, in jumping spiders, the distal layers are relatively thin, while the most proximal layer is comparatively thick, forming a staircase such that the surfaces of some photoreceptors are farther away from the lens than others. This staircase-like organization is thought to allow both near and far vision because close and distant objects can be focused on different levels of the staircase. It was considered that this could also provide distance cues because image size and blur are object-distance dependent at the different staircase levels. However, theoretical calculations suggest that this alone cannot allow for distance-range estimations in jumping spiders because the depth of field is too long (Blest et al., 1981).

Another well-studied chambered eye system, which is thought to have some monocular range-finding ability, is that of the tiger beetle larvae. The tiger beetle larva is a terrestrial predator that waits at the exit of its burrow to attack prey coming within striking range. These animals have one pair of enlarged eyes, which are used predominantly for hunting. However, differing from the visual system of jumping spiders and T. marmoratus, their retinas consist of only one layer of photoreceptor cells that are positioned directly beneath a relatively large and thick lens (Toh and Okamura, 2007).

It has been proposed that the relative focus of an image, projected onto the single retina layer, plays a role in their ability to judge distances monocularly (Toh and Okamura,

2001). However, the exact mechanism of this visual system is still unknown.

The one group of species that may have had lenses with optical properties comparable to T. marmoratus is that of the trilobites. Some of these long-extinct

122 arthropods had compound eyes, each unit of which might have functioned as an image- forming eye. Their corneal lenses are thought to have consisted of two units, an outer unit of calcite and an inner unit possibly composed of organic material. A reconstruction of the optical properties of the lenses from their unit surface geometry suggests that the lenses were bifocal (Egri and Horváth, 2012; Gál et al., 2000a; Gál et al., 2000b). It has been proposed that these bifocal lenses might have provided trilobites with near and far vision, which would not have been possible with a rigid, monofocal lens (Egri and

Horváth, 2012; Gál et al., 2000a; Gál et al., 2000b). Unfortunately, we know nothing about the cellular organization of the retina in these eyes.

Compared to other known eyes, the visual system of T. marmoratus larvae are highly unusual and their organization suggests the existence of visual mechanisms that are poorly understood or even un-described. The goal of this Chapter is to present the first conceptual model of how these eyes might function so as to enable such successful visually-guided . This model is based on most of the known features of these eyes and accounts for the behavior of the larvae as well as their visual challenges.

Figure 1 will be referred to many times throughout this chapter. It illustrates the core findings of the model: the object distance-dependent dynamics of the image position relative to the retinas during the hunting behavior of the larvae. Even though the model leaves many questions open, it will hopefully serve as a valuable guide and inspiration for further experiments. It is also intended to illustrate the potential complexity of the function of these eyes. In this context, it shows that some stemmata are not as simple as they seem and no doubt merit greater attention as they may lead us to a more complete understanding of vision.

123

Figure 1. Illustration of the sharpest-image positions relative to the retinas as the larvae approach prey during hunting. A typical striking distance of second instar larvae is ~ 4 mm

(Bland et al., under revision). The positions of the first image relative to the DR (green light) and the position of the second image relative to the PR (UV light) are illustrated. Dark bars are the depth of focus according to geometrical optics as described in Chapter 4.

124 Conceptual model illustrating how the visual system might assist in prey capture

First, it should be noted that, in a lab setting, larvae that were accustomed to hunting mosquito larvae, which tend to hang vertically from the surface of the water, hunted novel dummy prey configured as a dark vertical stripe (Bland et al., under revision).

This suggests that a very large portion of their hunting strategy is based on visual cues, and that olfaction is not necessary.

Furthermore, behavioral experiments showed that, as soon as the larvae detect potential prey, the larvae orient themselves so that the prey is brought into the visual field of E1 and E2 (Buschbeck et al., 2007). It seems then that E1 and E2 are the most important eyes for hunting. Therefore, our focus will be on the function of these eyes although there will be brief mention below of possible functions for the remaining four eyes (E3 – E6).

Initial prey detection

In addition to principal eyes, E1 and E2, T. marmoratus larvae have four other eyes (E3

– E6) on each side of their head. These are arranged such that their visual field includes an almost panoramic view of their environment (Mandapaka et al., 2006). These eyes also have layered retinas (Mandapaka et al., 2006; Maksimovic et al., 2009) and so, may also need bifocal lenses in order to project focused images onto both the distal and proximal portions of their retinas. However, these eyes are much smaller and seem less developed than E1 and E2. In these eyes, the retinas are positioned directly beneath or very close to the back surface of the lens (Mandapaka et al., 2006) such that their spatial resolution is probably poor in comparison with that of E1 and E2. However,

125 these eyes (E3 – E6) are likely involved in the initial prey detection through motion or possibly rough-shape detection, which probably initiates the turning of the larvae toward potential prey to bring it into the visual fields of E1 and E2. This initial turning behavior has been observed many times, but has yet to be quantified.

As soon as potential prey is brought into the visual fields of E1 and E2, the larvae perform dorso-ventral scanning movements to widen the visual fields of the horizontally- oriented linear retinas (Buschbeck et al., 2007). When potential prey is initially brought into view of E1 and E2, the distance to the prey is frequently still at effective infinity for these eyes such that the image of the prey is projected onto, or is indistinguishably near, the focal planes of the bifocal lenses of these eyes (Ch. 4, pp. 112-113). This applies to all object distances that are no closer than the hyperfocal distance of a visual system (see Ch. 4, pp. 104-105).

As described in Chapter 4, p. 104-105, the hyperfocal distance (the closest object distance perceived as in focus when an object at infinity is focused onto the retina) for E1 is ~20 mm and for E2 ~15 mm. For these object distances, the bifocal lenses in both eyes focus the first image near the rim of the green sensitive DR, and the lens in E1 focuses the second image near the surface of the UV-sensitive proximal retina (Ch. 4, pp. 103-106), see Figure 1. Thus, the visual system of these larvae allows visual information of objects at effective infinity to be obtained with the DR of both eyes in the green spectrum. It also allows image information to be extracted in the

UV spectrum with the PR of E1. As the PR is polarization sensitive, this system also allows the extraction of polarization information at this relatively far object distance. It should be noted that bifocal lenses are the mechanisms that make the simultaneous

126 extraction of image information in the green and UV spectrums possible.

Even so, the contrast of the focused images projected by a bifocal lens might be compromised. This is because in both image planes, the out-of-focus image interferes with the in-focus image, decreasing the contrast of the in-focus image (discussed in

Stowasser et al., 2010, Ch. 2, pp. 42-45). Regardless, it seems that the visual system of these larvae is able to minimize the interference through two novel functional characteristics. First, the two images are projected in such a way that the out-of-focus image is slightly to the side of the focused image (Stowasser et al., 2010, Ch. 2, pp. 42-

45). Secondly, in contrast with the retinal layering in jumping spiders, which corrects for chromatic aberration (discussed earlier in this Chapter and in Ch. 1, pp. 20-22), the spectral sensitivity organization of the retina in T. marmoratus larvae, together with its chromatic aberration, maximally separates the images while maintaining a compact retinal organization (see Figure 2). Together, these reduce interference between the two images because the more an image is out of focus, the less it interferes with the in- focus image.

Since information in both green and UV spectrums can be extracted, the existence of color vision should be considered. Color vision generally requires photoreceptors that have different spectral sensitivities but, in order to avoid ambiguities between spectral and polarization information, they should not be polarization sensitive

(Wehner and Bernard 1993). Since the PR is polarization sensitive, the nervous system of these larvae has probably not evolved color vision to avoid ambiguities between polarization sensitivity and color vision. Although this hypothesis has yet to be tested, it is still conceivable that, even without color vision, the larvae benefit from gathering

127 image information in both green and UV spectrums. Since objects reflect, absorb, and transmit different wavelengths of light, the mere existence or non-existence of light within a specific spectral range can be informative.

Figure 2. Illustration of the relationships between spectral sensitivity organization of the retina, image position and chromatic aberration. (A) Spectral sensitivity and average image position of an object at infinity after Figure 6 of Ch. 4, p 104. (B and C) If the retina organization were the opposite: (B) Hypothetical image positions while preserving the retina dimensions as well as the position of the first image relative to the DR and the position of the second image relative to the PR; (C) Hypothetically necessary retina dimensions while preserving the distance between the two images and the positions of both images relative to the corresponding retinas. Squares

() indicate the image position for UV light and circles () indicate the image position for green light.

Another feature of these eyes that needs to be considered is the polarization sensitivity of the PR. During hunting, this could conceivably benefit larvae by a) enhancing overall contrast through the filtering out of mostly horizontally polarized haze

(reviewed in Stowasser and Buschbeck, 2012, Ch. 3, pp. 54-56); b) improving the

128 visibility of transparent prey items via their polarization features (reviewed in Stowasser and Buschbeck, 2012, Ch. 3, pp. 55-56); and c) identifying prey based on the specifics of their polarization features. Behavioral experiments demonstrated that the larvae would hunt novel dummy prey, which were characterized by high contrast but did not have the typical polarization features of the prey that these larvae were accustomed to hunting (Bland et al., under revision). Thus, polarization sensitivity was not—at least under our laboratory conditions where the water was very clear—necessary for prey identification. However, it is conceivable that these larvae benefit from an overall enhancement in contrast and an improvement in the visibility of otherwise transparent prey under more natural conditions where the overall visibility and contrast is decreased due to water turbidity. Regrettably, despite several attempts, there is not enough experimental data to confirm this hypothesis at this time. However, personal observations suggest that not only T. marmoratus larvae, but also other related species, may benefit from enhancing prey contrast through polarization sensitivity. Specifically, I was observing the hunting behavior of diving beetle larvae of a related species in their natural habitat, a small pond in the vicinity of Cincinnati, OH. Watching these larvae hunt was mesmerizing because they were hunting something that was, to my eyes, invisible in the turbid water of their aquatic habitat. As it turned out, they were hunting almost completely transparent midge larvae. Subsequent tests with T. marmoratus larvae in the lab demonstrated that they too spontaneously hunted this nearly transparent prey, and had absolutely no problem with prey capture despite its novelty

(my own observations).

129 Stalking and approaching prey

After initial prey detection, the larva stalks, follows and slowly approaches potential prey while performing scanning movements with its head (Buschbeck et al., 2007). Because of the generally short focal lengths of insect eyes, all relevant object distances are effectively at infinity for many small image-forming visual systems (discussed in Chapter

4, pp. 112-113). However, this is not the case for T. marmoratus larvae. These larvae move closer to the prey than the theoretical hyperfocal distance of their visual system before they strike (discussed in Chapter 4, pp. 112-113). Once the distance to the prey is closer than the hyperfocal distance, their visual system starts to operate in its near range. This is possible in these eyes because of the layering of the DR and the differences between E1 and E2 (discussed in Chapter 4, pp. 112-113). As the object distance decreases, the focused first image moves through the DR layers in both eyes such that, for all relevant object distances, some layers of the DR receive a focused image. See Figure 1 for depiction. Thus, the basis for extracting image information by the DR in the green spectrum is maintained throughout their hunting range. The scenario is a little different in regard to the PR. As the object distance decreases, the image becomes blurry (out of focus) in E1, but is focused in E2 (discussed in Chapter 4, pp. 112-113) near the surface of the PR (Figure 1). Thus, the PR of E1 and E2 appears to be set up to extract image information in the far and near range of this visual system synergistically. As a result, the layering of the DR, as well as the difference between E1 and E2 in regard to the position of the second image relative to the PR, appears to function in place of an accommodation mechanism, allowing far and near vision simultaneously.

130 In this context, it seems that one advantage and function of the tubular eye- organization is to provide a comparably high resolving power despite the size constraints of these small animals. This is because these eyes have a relatively large focal length. Generally, the larger the focal length, the larger the image is that is projected by the lens, and the better the potential resolving power of the system.

However, a larger focal length generally comes at the price of a larger hyperfocal distance. Systems that must perceive an in-focus image of objects that are closer than the hyperfocal distance and of objects at infinity require an accommodation mechanism that allows for near and far vision. Thus, the closest the object distance for which an animal needs to perceive in-focus images, the shorter must be the focal length in the absence of an accommodation mechanism. In the case of T. marmoratus—assuming a hyperfocal distance of 4 mm (typical striking distance of second instar larvae), a maximally allowed blur circle of 7 µm (the receptor unit spacing of the PR) and the same

F number (ratio between the focal length and the lens diameter)—the focal length could not be longer than ~230 µm, which is about ½ of the actual focal lengths of their lenses.

The relationship between the image size and focal length is directly proportional, such that, in this system, the image is twice the size of what it would be if the system were not in need of an accommodation-like mechanism to allow for near and far vision.

However, in the context of their hunting behavior, these larvae can only take advantage of their enhanced focal length because they are capable of near and far vision.

Range finding and prey capture

The larva approaches the prey until the prey is no more than a few millimeters away, at

131 which time it performs a ballistic strike to capture the prey. At the preferred striking distance, which for second instars is about 4 mm (Bland et al., under revision), the first image in green light is focused near the pit of the DR in both eyes, and, in E2, the second image in UV light is focused near the surface of the PR. This enables extraction of image information about the striking distance of the larvae from both retinas (Fig. 1).

Video recordings from second instars suggest that, just before they strike after termination of the scanning behavior, larvae are able to keep the distance to the prey constant. Even if the prey moves during that time, larvae follow the movement of the prey, giving the appearance of having locked onto a specific distance (Bland et al., under revision). Given their high hunting success, larvae are probably able to gauge distances very well. We recently performed a behavioral experiment to test which of the commonly known range-finding cues in insects T. marmoratus larvae might employ.

The experiment examined, in particular, whether binocular stereopsis, motion parallax and image size were involved in range finding. Results showed that the larvae could still gauge distances correctly when all of these cues were strongly compromised (Bland et al., under revision). This suggests that the larvae employ other range finding mechanisms, which may involve their bizarre eye organization. As discussed in

Chapter 4, several distance cues could be provided by the layering of the retina.

Namely the larvae could a) use the defocus or the shift of the focused image as a range finding cue with the DR of both eyes (Ch. 4, pp. 113-114); b) use the defocus synergistically with the PR of both eyes (Ch. 4, pp. 114-116); and/or c) use the change in image size as the object distance changes after initial distance estimation as a distance cue (Ch. 4, pp. 116-117). Possible interactions between multiple cues could

132 result in better acuity than any individual cue alone. Exactly which range-finding mechanisms the animal employs needs to be further investigated by experiments that consider all of these possibilities. One conceivable approach could be to evaluate their hunting behavior, striking distance and success while selectively covering appropriate combinations of eyes – an approach similar to our previous behavioral range-finding experiment (Bland et al., under revision), but more comprehensive, including the separation of E1 and E2.

All of the discussed potential range-finding mechanisms require that the system operate in its near range (discussed in Chapter 4, pp. 114-117). It seems that here lies the second advantage and function of the tubular organization of these eyes with their relatively long focal length in combination with accommodation-like mechanisms. This is because without near vision and operating in the near range around their striking distance, the visual system would not produce any of these distance cues.

In this context, interaction between the scanning behavior and the DR organization deserves attention. The DR has many layers of shallow photoreceptor cells that have rhabdomeres oriented perpendicular to the light path. The tiering of the light sensitive rhabdomeres of these cells seems to be the basis for near and far vision and might be involved in range finding. To achieve such a layering, the cell bodies must be positioned peripheral to the rhabdomeric tiering. The only conceivable way to accomplish this is via the organization seen in T. marmoratus larvae: a retina with only two rows of stacked cells forming central rhabdomeric tiers with cell bodies positioned on either side. Scanning would then be necessary to expand the visual field of such a linear retina.

133 Another interesting point is that the potential function of a bifocal lens was previously discussed in great detail for certain trilobites. However, only near and far vision were considered (Egri and Horváth, 2012; Gál et al., 2000a; Gál et al., 2000b).

Our data suggests that, in addition to this, a bifocal lens could also be beneficial for extracting a variety of image information and might aid in range-finding, a discovery that could inspire engineers to solve visual tasks in novel ways.

Possible additional functions of E1 and E2 in the context of the natural history of these larvae

Besides aiding in prey capture, the eyes, E1 and E2, might also be involved in other important visual tasks. In this context, their polarization sensitivity could be very beneficial. These larvae hatch on land and must find water to survive. However, they must also find the shore for pupation (Morgan, 1992a). For these tasks, polarization sensitivity could be useful because of the specifics of the polarization pattern of the environment: a) the polarization pattern of the reflecting water surface could be used to find water (reviewed in Chapter 3, pp. 54-56, 80-81) and b) the differences between the underwater polarization pattern that occurs toward shore and out in open water could be used to find shore (reviewed in Chapter 3, pp. 80-81). What exact function the polarization sensitivity has still needs to be determined in appropriate behavioral experiments. Carefully designed behavioral experiments with transparent prey could test whether the larvae benefit from using polarization features of their prey to enhance their visibility, or use their polarization sensitivity for overall contrast enhancement by filtering out horizontally polarized haze, particularly in turbid water. Behavioral

134 experiments that involve the simulation of polarization patterns of reflective water surfaces could test whether freshly hatched larvae use this cue to find water. Finally, behavioral experiments in which the polarization pattern of shore vs. open water is simulated could determine whether or not third instar larvae that are about to pupate use this cue to find shore.

In the context of navigation and orientation, one additional function of these eyes could be the exploitation of image information extracted in UV and green spectrums. By processing differences in the background illumination in either UV or green spectrums, or both, they could navigate their way to preferred areas. However, there is not yet enough spectral data regarding their natural aquatic habitat to recommend this hypothesis.

To Conclude

The function of this visual system is potentially highly complex, and clearly contradicts the assumption that stemmata are simple. The complexity of this system would suggest that the diverse group of small chambered eyes in invertebrates merits greater attention as that group might very well lead us to a deeper understanding of vision. To answer the questions that remain, further experiments will have to consider the potentially extraordinary complexity of this system for correct and comprehensive interpretations of the results. Additionally, the complexity of this system is intriguing for comparative studies. The family of predacious diving beetles (Coleoptera: Dytiscidae) is highly diverse. Preliminary examinations and existing literature show that, within this group, stemmata organization ranges from very simple chambered eyes to highly complex

135 eyes like those of T. marmoratus larvae. Thus, this system appears to be ideal for gaining greater insight into eye evolution and development. Such studies, however, must be careful to remember that other similar systems might also be less simple than they appear. In this context, the proposed conceptual model for how these eyes might function will hopefully serve as a valuable guide and inspiration despite those questions that have yet to be answered.

136 Glossary

Aperture: In the context of optical systems, it is the opening of the system that limits

how much light can enter or exit the system (entrance or exit aperture

respectively).

Blur circle: In the context of this text, it is the blurred image (blurred image disk) of a

point object in any plane that is not the plane where the image is focused.

Corneal lenses: In the context of insect eyes, corneal lenses are generally attached to

the cuticle and are the outermost optically active parts of the eye. Their front

surface is the interface to the environment, while the back surface is the interface

to inside of the animal.

Contra-lateral eyes: In the context of this text these are the corresponding eyes on the

other side of the head.

Distal: In anatomy, it is a position or direction that is outward from the center of the

body. depth of field: It is the range of distances within which an image of an object is in

focus. The depth of field is dependent on the focal length of a visual system, the

aperture of the system, and the resolving power of the system. depth of focus: It is the depth of field projected into the image space. Meaning, it is the

range of image distances that are perceived as in focus.

Effective infinity: Optical systems project images of objects at infinity into the focal

plane of the system. Effective infinity is all object distances that are not truly

infinity, but lead to the projection of the image indistinguishable near the focal

137 plane. It is dependent on the resolving power of the system, the size of the

aperture, and the focal length. In this context also see hyperfocal distance.

Electrophysiological methods: Methods that allow measuring the electrophysiological

properties of cells. This is accomplished by positioning a microelectrode (probe)

either near cells (extracellular), or inserting a microelectrode into a cell

(intracellular). Measured is the electrical potential between the probe and a

reference electrode. Changes in this potential arise from changes of the

membrane potential in response to a stimulation of cells. The membrane

potential arises from the ion distribution between the inside vs. the outside of a

cell.

F number: It is the ration between the focal length (f’) and the size of the aperture of the

system (D), F = f’/D. It is informative in regard to the light gathering abilities of a

system and the depth of focus. As smaller the F number, as more light can be

gathered, and as smaller is the depth of focus. A typical F number in diurnal (day

active) insects is around 2, while nocturnal (night active) insects might have F

numbers as low as 0.5 (Land and Nilsson, 2001).

Holometabolous insects: Insects that undergo complete metamorphosis (complete

change their body plan as they develop). They have four live stages: ,

larva, pupae, and imago (adult). Groups that contain holometabolous insects are

for example: beetles, butterflies and moths, flies, ants, bees, wasps, and fleas.

Hyperfocal distance: In the contest of eyes, if an object at infinity is focused onto the

retina, it is the closest object distance that results in an image that is perceived

138 as in-focus. It depends on the focal length, the aperture of the system, and the

resolving power of the system.

Lenses

Bifocal: they have two focal planes

Cylindrical lenses: In two perpendicular directions, they have two different focal

planes, so that the image of a point object are two stripes that are perpendicular

to one another, projected into two different image planes.

Plan-Cylindrical lens: A cylindrical lens that only has a refractive power in one

direction, but not the other.

Plus lens: Collecting lens, converging lens, convex lens

Minus lens: diverging lens, divergent lens, concave lens

Microvilli: In the context of insect photoreceptors, these are the cellular membrane

protrusions that express the photoreceptors. Collectively, this region is called the

rhabdomere. motion parallaxes: A visual mechanisms for depth perception. It is based on that the

magnitude and speed of the image movement across the retina is dependent on

the speed and magnitude of the movement of the object relative to the eye and

the object distance. The mechanism requires knowing the magnitude and speed

of the movement of the object relative to the eye. Otherwise, distance information

is ambiguous.

Oviposition: deposition of eggs

Optics

Geometrical: Ray optics, it is an abstraction and describes how light

139 propagates as “rays”, following geometrical rules.

Physical: It is based on the wave property of light and describes how

the light waves propagate and interact.

Proximal: In Anatomy, it is a position or direction that is toward the center of the body.

Rhabdomere: It is the light sensitive portion of an insect photoreceptor cell

Rayleigh limit: Rayleigh criterion, it is based on the wave property of light and on the

“Rayleigh’s quarter wavelength rule” which assumes that the image quality is not

significantly affected when the wave front that converges on the optimal point is

no more distorted than ¼ of the wavelength of the light (Wolf and Born, 1965).

Stereopsis: A visual mechanisms for depth perception. It is based on that the

differences between the images of two eyes are object distance dependent.

Spatial resolution: Or the resolving power of an eye. It is the quantification of how

close two object points can be so that they still are perceived as two separate

points.

Spectral sensitivity: In the context of eyes, it quantifies its sensitivity to the different

wavelengths of light.

Temporal resolution: It is the quantification of how fast the system responses to

stimulation: how fast (one after the other) can flashes of light be so that the

system still can distinguish them as separate flashes of light.

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