How do Stemmata Grow? The Pursuit of

Emmetropia in the Face of Stepwise Growth

A thesis submitted to the

Graduate School of the University of Cincinnati

(Division of Research and Advanced Studies of the University of Cincinnati)

In partial fulfillment of the requirements for the degree of

Master of Science

In the Department of Biological Sciences

of the College of Arts and Sciences

2014

By

Shannon Werner

B.A. Theater, Northern Kentucky University (2005)

B.S. Neuroscience, University of Cincinnati (2011)

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

i Abstract

However complex or atypical the visual system, all of its components the lens, eye size and shape, retina and nervous system - must be well tuned to one another. As organisms grow, their eyes must be able to achieve and maintain emmetropia, a state where there is a good fit between the optical and receptor portions of the eye. Based on vertebrate studies, emmetropia is accomplished through a combination of initial regulation by genes, followed by homeostatic visual input from the environment for fine-tuning. How quickly and effectively emmetropia can be achieved will influence the ability of an animal to execute visually guided behavior. While there has been ample research into how vertebrates manage eye growth toward emmetropia, this has never been addressed in arthropods, which develop in a stepwise fashion through ecdysis. Of particular interest are larval holometabolous arthropods with camera type eyes, called stemmata in larval arthropods. Stemmata, such as those of the larval eyes of the Sunburst Diving Beetle

(Thermonectus marmoratus, Coleoptera, Dytiscidae) must recover emmetropia after ecdysis, developing a well formed lens and receptor components that are well tuned to the lens’ optical properties. Here I use this system as a model for measuring when and where eye growth toward emmetropia occurs around different molting periods. Histological and optical techniques were used to investigate growth of the principal larval eyes of T. marmoratus. These studies revealed that larval eye tubes grew significantly within the first hour after molt, with the crystalline cone contributing the most to overall growth. Lenses were slower than the eye tubes to reform, regaining the ability to project sharp, distinct images eight hours after molt. Further osmotic experiments using immersion in hypotonic (water) or hypertonic (100% insect Ringer’s) solutions explored water uptake as a potential mechanism of growth. Preliminary data showed significant differences in the change of eye tube length for eye two between individuals exposed to water and

ii Ringer’s solution. These results are the first demonstration of how holometabolous arthropod stemmata grow and change after a molt. Specifically notable is that the eye tube and optical components grow very rapidly, within the first hour, before the lens is capable of image formation. Results also show that within the first day after molting, larval eyes already regain functional emmetropia, and that water uptake within the eye via osmosis may be vital to the rapid growth of these larval eyes. I anticipate that this first exploration of how stemmata grow after molting will open new avenues of investigation in other arthropod camera type eyes or investigations into comparisons between post-ecdysal camera type eye development in aquatic arthropods land based land based arthropods. Additionally, preliminary evidence of osmosis as a potential mechanism of growth may provide a basis for a thus far unknown mechanism for invertebrate eye growth. Further investigation into water regulation in vertebrate and invertebrate eyes as a mechanism for growth and fine-tuning are warranted.

Keywords: Emmetropia, Eye growth, Arthropod, Vision, Stemmata

iii

iv Acknowledgements

I would like to thank my advisor Elke Buschbeck for her enthusiasm for science and teaching, her unsurpassable energy for any and all things we might learn, her willingness to allow students take on unusual projects with potential for opening new avenues into exploration of invertebrate eyes. Elke has gone above and beyond, and shown me compassion in times of stress and injury. I would like to thank my committee members: Dr John Layne, whose Sensory Physiology course kicked my brain into motion, where I found a niche no one else had yet explored for this project. For Dr Tiffany Cook, for her incredibly sharp eye for improvement and all the knowledge she brings, which pushed me to work harder.

Thanks to Ron Debry extending GAC support and funding for the final semester stretch of my thesis work, with consideration for the major back injury I sustained and the long process of recovery. Special thanks to Randy Morgan and the Insectarium of the Cincinnati Zoo & Botanical Garden provided the initial culture of Sunburst diving beetles, and to Brian Hodge for his willingness to lend his expertise on all matters diving beetle.

This research was funded by the National Science Foundation under grants IOS0545978 and IOS1050754, and in part by the University of Cincinnati’s Weiman Wendel Benedict Student Award in 2012 and 2013.

Special thanks go out to my friends and Prem Rajkumar, Sri Pratima Nandamuri, Jessie Ebie, Srdjan Maksimovic, Aaron Stahl and Jeremy Didion, and Luke Hong. Having these wonderful, and intelligent people in my life really helped me through grad school, happy to talk science and brainstorm, or just relax and enjoy the sort of life it is hard to explain to people who are not in your field. They made me feel as though I belonged somewhere, my “science family.”

I am eternally indebted to my father Richard and stepmother Mary for their unconditional love and support. Of all my relatives I owe my sister, Ashleigh, the most. We were the closest of friends and sisters growing up as Marine Corps Brats, and carried that into adulthood. Then there is Matthew, my beacon of sanity and calm, who listens and provides a safe place to fall when I stumble and who is the best co-caretaker I could ask for when it comes to our wonderful, weirdo dogs, Ari and Tina.

v Table of Contents

Introduction – 1

Materials and Methods – 7 Initial behavioral experiments - 7 Establishing an appropriate timeline – 7 Animal rearing and surveillance – 8 Sampling for histology and optics – 9 Histology processing and staining – 9 Histological imaging and measurements - 10 Optical imaging and image contrast measurement – 11 Histological and optical comparisons and statistical analysis - 12 Point of molt osmotic comparisons – 13

Results - 15

Histological observations – 15 Optical observations – 19 Optical and retina fit – 21 Post molt osmotic observations – 22 Discussion - 23 Changes in eye size - 23 Lens reformation and fit of images with retinas – 28 Preliminary evidence for an osmotic method of growth – 30 Summary - 32

Figures – 34-45

Works Cited – 46-49

vi List of Figures and Tables

Figure 1) A- An electron microscopy image of the whole larval head. B- Schematic of a sagittal section of the larval eyes showing the positions of the primary eyes 1 and 2 and their retinal layout. C- Diagram of the life cycle of T. marmoratus, from egg to adulthood, all phases are shown to scale. D, - sagittal section of a first instar with back of the lens to end of eye measurements for eyes 1 and 2, D’- sagittal section of a second instar with measurements for eye 1 and 2 , D’’- sagittal section of a third instar with measurements for eyes 1 and 2. Pp - 34

Table 1) For an example second instar molt time of 9 AM, this outlines sampling times, their time points, and notations for each time point. Pp - 35

Figure 2) Chart of third instars average total number of larvae captured by day. Pp - 35

Figure 3) Histological measurements taken for each eye. A- Back surface of the lens to start of distal retina, B- Back surface of the lens to the distal retina pit, C- Back surface of the lens to the end of the distal retina/start of the proximal retina, D- Start of the proximal retina to the end of the proximal retina, E- Back surface of the lens to the end of the eye tube. Pp - 36

Figure 4) Scatter plot of mean values for major Eye 1 histology points. Symbols correspond to the major landmarks that are shown on the diagram of the eye. Diamond shows the end of the crystalline cone and start of the distal retina, squares show the end of the distal retina and the start of the proximal retina, triangle shows the end of the proximal retina, and circles indicates the end of the entire eye capsule. Pp - 37

Figure 5) Scatter plot of mean values for major Eye 2 histology points. Symbols correspond to the major landmarks that are shown on the diagram of the eye. Diamond shows the end of the crystalline cone and start of the distal retina, squares show the end of the distal retina and the start of the proximal retina, triangle shows the end of the proximal retina, and circles indicates the end of the entire eye capsule. Pp - 38

Figure 6) Mean values for the length of the crystalline cone regions. A. Eye 1. B,Eye 2,. Significant differences between time points are marked with *. Pp - 38

Figure 7) Mean values for the total retinal area (distal retinal length and proximal retina length). A. Eye 1. B,Eye 2,. Significant differences between time points are marked with *. Pp - 39

Figure 8) Diagram showing a comparison of average major landmark sizes for eye 1 of final day of Second Instar (day 5), and first day of third Instar (at twenty hours). It also shows growth values for each portion of the eye (between these time points), and the percent of the total growth of the eye that each area represents. Pp - 40

Figure 9) Diagram showing a comparison of average major landmark sizes for eye 2 of final day of Second Instar (day 5), and first day of third Instar (at twenty hours). It also shows growth

vii values for each portion of the eye (between these time points), and the percent of the total growth of the eye that each area represents. Pp - 41

Figure 10) A-A’’ Example images of back lens surfaces. A. Second Instar Day 5 individual, A’- Third Instar Day 1 individual, A’’- Third Instar Day 2 individual. B & C. Example graphs of image contrasts that are observed at different time points. - The shift of focal planes, in Eye 1 before (2ID5@M12h, violet), at (3ID1@M, aqua), and after molt (3ID2@M, orange). C- The shift of focal planes in Eye 2 before, at and after molt. Pp - 42

Figure 11) Locations of the first focal plane generated by the lens, relative to the back surface of the lens. A- Eye 1. B- Eye 2. Significant differences between time points are marked with *. Pp - 43

Figure 12) Locations of the second focal plane generated by the lens, relative to the back surface of the lens. A- Eye 1. B- Eye 2. Significant differences between time points are marked with *. Pp – 43

Figure 13) Diagrams showing A- Eye 1 fit between the average crystalline cone length (shown in orange), and average focal plane 1 length (shown in green), and B – Eye 1 average distal end length (shown in orange) fit with average focal plane 2 (shown in violet). All distances are shown relative to their distance from the back surface of the lens. Pp - 44

Figure 14) Eye 2 diagrams showing A- the fit between the average crystalline cone end (shown in blue), and average focal plane 1 (shown in green), and B – the fit between average distal retina end (shown in blue) and average focal plane 2 (shown in violet). All distances are shown relative to their distance from the back surface of the lens. Pp - 44

Figure 15) Example images of individuals the eye tube length of which were monitored while emerged in water (A) or Ringer’s solution (B). A- Individual at 20 minutes, immersed in water, A’- The same water individual at 90 minutes. B- Individual at 20 minutes, immersed in 100% insect Ringer’s solution, B’- The same individual at 90 minutes. Pp - 45

Figure 16) A- A comparison of Eye 1 lengths in water and Ringer’s solution individuals at 20 minutes (shown in blue) and 90 minutes (shown in purple) A’’ – A comparison of Eye 2 lengths in Ringer’s solution and water at 20 and 90 minutes. B&B’- Percent change between eye length at 20min and 90 min post molt. B. Eye 1 for water and Ringer’s solution, B’- Eye 2 for water and Ringer’s solution. Pp - 45

viii Introduction

To date the vast majority of information on how eyes achieve emmetropia is drawn from vertebrate eyes. Emmetropia is defined as the state of vision where an object at infinity for the eye is in sharp focus on the receptor elements when the lens is in a relaxed or neutral state. In the present study I refer to it as an optimal match between all components of the eye from optical to neural. This is the desired state of an eye as opposed to those states of myopia, short sightedness where the optical components project a focused image too far in front of the retina and hyperopia, where the focused image is projected behind the retina.

As of this study, there has been little to no research as to how camera type eyes of larval arthropods, also referred to as stemmata, or adult arthropod camera type eyes attain emmetropia.

The only investigations of molting eyes have focused on changes in the external diameter of lenses after ecdysis (Kawaguchi et al. 1996, Lin et al. 2002) as a reliable marker of instar phase.

Ecdysis is the molting of the previous exoskeleton and reformation of a new one, after an increase in overall body size, and marks the transition between the “instar” periods of a holometabolous arthropod’s larval growth. Ecdysis is a “stepwise” method of growth where the animal is forced to grow in large bursts from a smaller size to a much larger one in a period as little as a day. The growth requirements of an animal undergoing stepwise growth will be very different from that of an animal which may grow “gradually’ as vertebrates do.

The gradual growth of vertebrates leaves ample time for the various components of the eye to develop and attain emmetropia with the aide of visual input. Arthropods, on the other hand, are constrained by their stepwise growth where their bodies grow in size very quickly after the old exoskeleton is shed. They must likewise rapidly correct the refractive state of the lens, as well as whatever specific growth in the eye is necessary to achieve emmetropia. Rapid

1 reformation of the eyes is vital for those arthropods that are active visual hunters, and may also rely on vision to avoid predation.

The attainment of emmetropia by vertebrate eyes thus far investigated has suggested two mechanisms for the acquisition of emmetropia: passive regulation and active regulation. Passive regulation proposes that the growth of an eye toward emmetropia relies primarily on gene expression and regulation in combination with the physical constraints of eye growth (Mark

1972, Sorsby 1979), where as active regulation suggests that some sort of feedback from the environment, such as visual input, is crucial to obtain emmetropia (Young, 1977, Banks 1980,

Trolio 1991). An example of the latter has been demonstrated in the eyes of chicks subjected to incorrect visual inputs develop non-emmetropic states of vision, and ultimately recover by actively adjusting a variety of different areas of the eye, depending on whether hyperopia or myopia was induced (Troilo 1991).

In regards to arthropods, while there is some information regarding the exterior changes in lens diameter in spiders (Fenk 2010), nearly nothing is known about the changes in the internal areas of the eye during the ecdysal periods of growth. Specifically, it is unknown if invertebrate larval camera coordinate eye growth passive or to match the new focal properties of the post-ecdysal lens, whether it occurs prior to the molt, directly after, or some combination of both of these, and in what area of the eye growth occurs. Understanding when and how arthropod eyes achieve coordinated growth will form the basis of knowledge on how these animals cope with the rapid “stepwise” growth process that occurs around ecdysis.

This stepwise mechanism of growth may present a particular challenge to those arthropods with camera-type eyes who are active visual hunters, when only a short period of time

2 may be available to allow the lens to reform, and for receptors to grow to match the lens, especially when good vision is required for food acquisition. The number of arthropods possessing camera type eyes at the time of their stepwise growth is significant. They include arachnids, some of which are visually complex predators (Arachnidae, Saltacidae), and certain types of insects such as predatory beetle larvae (Order Coleoptera, family Dytiscidae and

Hydrophilidae and subfamily Cicindelinae), antlion larvae (Order Neuroptera, family

Myrmeleontidae), and the larvae of butterflies and moths (Insecta Order Lepidodoptera). With such a wide variety of species with camera type eyes, the question of eye growth is of relatively broad importance.

The basic organization of the invertebrate camera type eye follows a basic plan: a lens of varying thickness, a clear “crystalline cone” structure that is analogous to the vitreous chamber and aqueous humor of a vertebrate eye, followed by a retinal area that is composed of a layer or multiple layers of light sensitive photoreceptor tiers (Mandapaka et al 2006). If growth of the eye and changes to the lens accompanies the molt, each of these elements would need to grow and change accordingly to remain well tuned to one another in order to maintain the eye’s function.

Some eye-growth related studies have been performed on invertebrates, namely the eyes of cephalopods, which are remarkably similar to those of humans. Specifically, an experiment was performed similar in design to that of Trolio (1991), which relied on the induction of artificial myopia and hyperopia in chicks. In experiments by Phillip Turnbull of the University of Auckland, cephalopods (Sepioteuthis australis) were exposed to exclusively shorter (blue light) or longer (orange light) wavelength light environments as a means of inducing myopia or hyperopia in the cephalopod eyes. Short wavelengths are refracted more, and thus resulted in images that were focused nearer to the lens than those that were formed under long wavelength,

3 illumination.

Consequently measurements (lens diameter, distance from center of the lens to retina, and retina diameter) were taken to determine if these treatments had an effect on the refractive states of the eyes. By day 45 post hatch, cephalopods developing in a blue light environment consequentially had smaller Matthiessen’s ratios (the ratio of the lens diameter compared to the retinal diameter) than those in an orange environment, making cephalopods raised in blue light more hyperopic while those raised under orange light were slightly myopic and had larger

Matthiessen’s ratios. Cephalopods raised under the aforementioned abnormal light conditions were able to compensate for the induced myopic and hyperopic conditions via regulation of the size of the lens and the retina, and were shown to be able to re-adapt to normal light conditions after exposure to abnormal light environments (Turnbull 2014).

In the current study, I investigated eye growth of the arthropod camera-type eye of

Thermonectus marmoratus (Coleoptera, Dytiscidae) larvae. These larvae are primarily visually guided hunters. The bulk of their diet in a laboratory setting is comprised of mosquito larvae.

Their larval phase is comprised of three larval instars separated by two molting periods. Their visual system consists of twelve “simple”, or camera-type eyes, six on each side of the head, which are replaced by compound eyes during metamorphosis (Sbita et al. 2006). Four larval eyes (E1 and E2 on both sides of the head) are tubular in shape (Mandapaka et al. 2006).

Particularly notable are their large bi-focal lenses (Stowasser et al. 2010) and the presence of two distinct primary retinas: distal and proximal. The distal retina is located most closely to the lens, is deeply tiered, and sensitive to green light between the wavelengths 520 and 540nm. The proximal retina, consisting of two fan-shaped layers, lies directly proximal to the distal retinal, and is sensitive to ultraviolet light (between the wavelengths of 340 and 360 (Maksimovik et al

4 2009). The bi-focal lens has been shown to project two separate and distinct images, with the areas of the lens closest to the cuticle producing the first focal plane which is projected closest to the lens, and the inner ring producing the second focal plane which is projected furthest from the lens (Stowasster 2010 Figure 3B).

Preliminary studies suggest that for Eye 1, images at effective infinity are focused at two depths behind the lens: the first image at the start of the distal retina for both green and violet light ranges, while second image falls and just on top of the proximal retina for green and ultra violet light ranges. Eye 2 is slightly myopic for both focal planes, in both eye ranges compared to eye one. (Stowasser et al 2014, Figure 4). As images move closer to the lens, they will be projected more deeply into the proximal and distal retinas respectively, and this shift in image position may provide T. marmoratus larvae with a depth perception mechanism useful for prey capture. If this change in image location on the retina as larvae move relative to their target or is important to their hunting capability, then would also be vital for larvae to regain this relationship between focal plane and retina location after molt for optimal prey capture.

Based on my preliminary histological observations, the two principal eyes change dramatically in size after molts (Figure 1), beginning with a length of approximately 224 µm for

Eye 1 and 175 µm for Eye 2 in the first instar phase, to a length of around 756 µm for Eye 1 and

658 µm for Eye 2 in the third and final instar phase. This change in eye size reflects the change in overall body size for the larvae across these molts, which changes from roughly 0.9 cm body length at first instar to 1.3 cm body length at third instar. It is thought that due to the bi-focal nature of their lenses and the layered retinas, that these eyes must be able to precisely focus in order to function thus making it an excellent initial study organism for a study of this type.

5 T. marmoratus has three instars, because optical measurements are more reliable in larger instars, the second to third instar molt period was studied. Histological measurements were taken at different developmental stages to gauge which area underwent the most change during eye growth. Specifically I measured the total length of the crystalline cone region, the total eye length relative to the back surface of the lens, and the length of the two retinas.

Additionally, these freshwater larvae would normally be relatively hypertonic to their pond water surroundings, and exhibit a “pumping” behavior of their mandibles and bodies to take on water after their molt. It is possible that water uptake facilitated by aquaporins surrounding the eye tube makes growth of the eye at least partially osmotic in nature. While there has not yet been much, if any, investigation in invertebrates on how osmosis via aquaporins contributes to body or other vital organ growth after molt, some inquiries have been made in vertebrate eyes as to how certain types of aquaporin may contribute to fluctuations in intraocular pressure or general eye function and intraocular pressure fluctuations (Verkman 2003), and intraocular and retinal injury (Dibas et al 2008) and retinal signal transduction (Karwoski et al

1989, Newman 1996, Nagelhus et al 1998). Similar mechanisms may occur in invertebrate eyes, both compound and camera type, however no studies have yet been performed on invertebrates to begin to test this idea.

To evaluate how hunting might be influenced by eye growth I evaluated prey capture behavior throughout T. marmoratus larvae’s third instar period. These preliminary observations suggest that larval prey capture is fairly low on day 1 of third instar, rising sharply to peak hunting ability between days 3-5, and finally falling below day 1 levels of capture ability during the final two days of the third, and last, instar phase (figure 2). Based on these observations I

6 hypothesize that these eyes will have regained emmetropia around the time their hunting becomes optimized, around day three.

Materials and Methods

Initial behavioral experiments

To obtain an idea of how well larvae hunt on each day of their third instar phase a 5- compartment arena was built from Plexiglas and waterproofed with aquarium silicone. The sides and back were frosted with sand paper to provide a homogenous background, while the front panel was left clear for filming and observation. Five freshly molted third instar larvae were selected from the general lab population for the 6-7 day trial. At the onset of the experiments larvae had recently molted and had not eaten since their second instar stage. Each was placed in an individual compartment with 5 large late instar mosquitoes and given twenty minutes to hunt, during which their hunting activities were recorded. After each daily trial, larvae were allowed to freely eat mosquito larvae and bloodworms for an additional 2 hours, at which point all remaining food was removed to prepare them for the trial the following day. The data in Figure

2 is based on three such trials.

Establishing an appropriate timeline

As a first step towards measuring optical and histological parameters pre-and post molting, I first established how fast larvae developed under consistent rearing conditions. In an incubator at 24° C, 14 hours light, 10 hours dark, the second instar phase typically lasts 5 days, and the third instar phase 6 days. To achieve a complete picture of growth activity, I sampled optical and histological data starting with day three of second instars and continued to the third day of third instars. After initial daily samples, sampling intervals were increased to every 4

7 hours on the first day of the third instar phase to better capture the window of change for lens and receptor component development (Table 1).

Animal rearing and surveillance

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. Sibling egg clutches were selected from the general rearing population tanks and allowed to hatch in a separate tank to ensure that all larvae were of uniform age. Once hatched, larvae were placed into individual plastic cups to prevent cannibalism. To ensure uniform rearing conditions, they were kept in a dedicated incubator that was set to 24 °C with a light cycle of 14 hours light, 10 hours dark. First instar larvae were fed approximately 15 mosquito larvae and 5 bloodworms (Ocean Nutrition Brand) and second instars were given 25 mosquito larvae, and 15 bloodworms. Third instar larvae were given 35-40 mosquito larvae and

20 bloodworms daily.

In order to observe individual molt times accurately, and thus determine the individual sampling schedule for each individual, larvae were monitored 24 hours a day. To do so, the incubator was equipped with a stand-alone fluorescent fixture for day lighting, and two infra-red

LED arrays for illumination during dark cycles. Images were captured every 15 minutes with a

Microsoft Life Cam Studio 1080p, modified for light and infra-red capture by removal of the infra-red cut-off filter. This modification did not disrupt the normal function of the camera, but allowed for the capture of high-resolution images during dark conditions. To capture time lapse imagery, Booru Webcam 2.0 (Lumai software) was installed on a dedicated computer and high resolution still images (1080p) were taken every 15 minutes and transmitted to a Dropbox

8 account where they could be reviewed remotely to establishing when each larvae had molted. In order to minimize bias of individual clutches, no more than two individuals in each of the time course categories (Chart 1) were sampled from a single sibling group of larvae.

Sampling for histology and optics

The age for each larvae was determined by their second instar or third instar molt time.

Prior to dissection larvae were anesthetized on ice for several minutes, and decapitated. To allow for normalization of the data, the head was photographed against a background with a scale. This image was then used to establish a normalization score based on head size. The head was then bisected, with the left half was preserved in 2.5% Glutaraldehyde (EMS) in

Sorensens Phosphate Buffer for histology, and the right half was used for measurements of the lens optics following a modified hanging drop technique as described in Stowasser et al.

(Stowasser 2010).

Histology processing and staining

This procedure was modified from (Strausfeld and Seyan, 1985). The left side of the head capsule was fixed in a solution of 2% gultaraldehyde (Electron Microscopy Sciences) in

Sørensen’s phosphate buffer at pH 7.4 (Electron Microscopy Sciences) at 4° C. After fixation, the tissue was washed twice in Sørensen’s phosphate buffer, then postfixed in 1% osmium tetroxide (OsO4) solution (Electron Microscopy Sciences), and held for 1h at 4° C followed by

1h at room temperature. Thereafter, the tissue was thoroughly washed in RO water and then dehydrated through a graded series of alcohol and acetone, and embedded in Ultra-Low

Viscosity Embedding Medium (Electron Microscopy Sciences). All sections were sectioned sagittally, with a thickness of 6-7 µm on a microtome. Sections were transferred to slides and

9 sealed with Permount (EMS).

Histological imaging and measurements

All photographs were taken with a camera (Olympus BKH022083 or Q-imaging 01-RGB-HM-

S-IR), which was mounted on a dissection microscope (Olympus SZX16). All sections were viewed and photographed at 4x and 10x, and then processed in ImageJ (without further modifications) to determine pixel distances between structures of interest. Images of a microscale (Nikon) at 4x and 10x were made in order to convert pixel measurements (that were generated by ImageJ) to actual micron lengths. To ensure consistency in image selection, and that all images measured were from central sections of each eye, only sections in which eye 5 was also visible (in addition to eyes 1 and 2) were evaluated. To minimize sampling errors, five measurements were taken of each eye.

In each case the following structures were measured: the length of the crystalline cone

(Figure 3, A), the distal retina start point (Figure 3, A) the total length of the distal retina (Figure

3, C-A), the proximal retina start point (Figure 3, C), the end of the eye (Figure 3 E), the length of the proximal retina (Figure 3, D) and the overall thickness of both retinas (Figure 3, C-A+D).

Histological data was compiled in Excel and converted from pixels to microns. To compensate for variation in size, data then was normalized to the head size. From these measurements it was possible to calculate the total length of the crystalline cone region, the total eye length relative to the back surface of the lens, and the length of the two retinas. For both primary eyes, means of crystalline cone length and eye length relative to the back surface of the lens, total distal retina length, and proximal retina length by time point were evaluated to determine which area of the eye contributed most to eye growth and between which time points the eye growth occurred.

10 Measurements were analyzed in JMP (JMP 9.0.1) for means and standard deviation, and a Tukey

Test was used to compare all sample pairs

Optical imaging and image contrast measurement

In order to accurately determine the stages pre and post molt at which the lenses were able to form images, and to determine where these images were placed with respect to the lens, the right half of the head capsule was analyzed using imaging methods previously established by

Stowasser et al. (2010). Specifically, the lenses of the two primary eyes and some of the surrounding exoskeleton were excised from the head capsule and the back surfaces of the lenses were gently cleaned with a small brush to remove any excess tissue. Lenses were then secured to the edge of an 800 µm diameter pinhole (Edmund Optics) by a small segment of exoskeleton that was attached to wax. To ensure complete submergence in solution, the pinhole was then mounted to a wax ring that was sandwiched between two glass cover slips, and filled with a solution of 50% Insect Ringer’s solution (O’Shea and Adams 1986). This Insect Ringer’s solution concentration has previously been established as optimal in regards to preventing bloating or shrinkage of the lens (Stowasser et al. 2010).

The coverslip stack was then placed on a goniometer microscope stage, and the back surfaces of the lenses were positioned to face a 10x objective lens. The microscope was further modified by removal of the condenser, to insure that an object could be placed (see below) so that it was at effective infinity for the insect lenses. In addition, a green filter and diffuser was placed between the light source and microscope to produce monochromatic light (542 nm).

Image series were acquired with a high-resolution camera (Moticam 3.0) and time-lapse software

(Motic Images Plus 2.0.25). An initial series of images of the lens was taken from approximately the front surface to the back surface in intervals of 5 µm. Following the lens

11 image series, a square-wave grating (0.353 cycles/mm, USAF 1951 negative test target from

Edmund Optics) was used as an object and positioned approximately 12.5 cm from the microscope stage, in effective infinity for larval lenses. Images of the object projected through both primary lenses were then taken in 5 µm intervals from well before to well after the focal planes for each lens. Analysis of the object image stacks were then performed by using a custom-made Matlab (Matlab 2014, The Mathworks, Natick, MA) program that evaluated images in regards to their edge-sharpness (Stowasser et al. 2010). In addition, image stacks were manually evaluated in regards to the quality of the images that were produced by the lens. Those image stacks generated by lenses that were visibly damaged during dissection, or improperly positioned during the early phases of this experiment were excluded from evaluation.

To determine how far behind the lenses images were focused, lens image series were used to locate the position of the back surface of each of the two primary eyes. All distances were corrected by multiplying by a factor of 1.33 to account for the refractive index of the

Ringer’s solution. Finally data were compiled in Excel for further analysis.

Histological and optical comparisons and statistical analysis

For both primary eyes, normalized histological data points for all landmarks (by time points), and optical focal plane locations (by time point) were analyzed in JMP (JMP 9.0.1). Analysis determined means and standard deviations, and a Tukey Test was used to compare all sample pairs. Means for distal retina start position were compared against means for focal plane 1, and means for proximal retina start position were compared for fit against means for focal plane 2, as these histological landmarks should fall very close in relation to the location of each respective focal plane to be considered a good emmetropia fit.

12 These histological positions selected for good fit evaluation compared to each respective focal plane based on findings that show each retina receives its own image of the two generated by the bifocal lens. For green illumination (542 nm) in eye 1, the first focal plane falls roughly at the start of the distal retina, and the second focal plane falls roughly at the start of the proximal retina. In eye 2 the first focal plane also falls roughly at the start of the distal retina, while the second focal plane falls within the end of the distal retina, roughly 50 µm above the start of the proximal retina (Stowasser et al 2014, Figure 4).

Point of molt osmotic comparisons

To test whether an osmotic mechanism could underlie rapid eye growth, I tested initial eye tube growth under normal and hyperosmotic conditions. During most of their larval stages a pigment patch on the exoskeleton occludes the visualization of larval eye tubes in-vivo.

However, this pigmentation is shed with the old exoskeleton and adequate visualization of eye tube length is possible for roughly an hour and a half after molting. Based on these parameters, I developed a protocol to measure eye-tube length of freshly-molted larvae immersed in fresh water (as typical in nature) or in 100% insect Ringer’s solution (which, as noted in Stowasser et al. 2010, is hypertonic relative to the larvae’s haemolymph).

For these experiments, second instar larvae were kept as previously outlined and monitored closely on day 5 of their second instar phase. Upon molting, they were anesthetized on ice for at least two minutes and transferred to a silicone dissection dish with a special trench cut in to the silicone, that allowed the larvae’s head to be held in place and visualized from the side, which optimally exposes the eye tubes. Larvae were further secured in the dish by application of a 1.5% agarose gel around the dorsal side of the head, and across their thorax and

13 a portion of their upper abdomen, leaving as much of the lower abdomen, and the mandibular area of the head free to make contact with the fluid they would be immersed in for the trial. The dish was then positioned under a dissection microscope (Olympus) and fixed in place with wax.

The total processing time from molt to the beginning of photography did not exceed 20 minutes in order to assure an early and uniform start point for comparison.

Using a high-resolution camera (Moticam 3.0) and time-lapse software (Motic

Images Plus 2.0.25), larvae were imaged every minute over a period of 90 minutes after mounting, while immersed in either water or 100% insect Ringer’s solution (O’Shea and Adams) which was refreshed as needed to keep the larvae well submerged. Images at twenty minutes post molt were used as a starting measurement point, and those at 90 minutes post molt were the end measurement point. Any individual that shifted during filming due to inadequate amounts of agarose, or that were found to have agarose gel obscuring any part of the head capsule around the eye tube, were excluded from analysis.

The 20 minute and 90 minute images from the time-lapse sequence were taken for analysis. In order to convert these eye tube lengths from pixels to microns, a conversion factor was established based on the primary eye’s lens diameters. Images were re-labeled for blind analysis, and an Excel spreadsheet was prepared with the pixel to micron conversion factors to allow pixel measurements to automatically convert for each image set. To assure that measurements were not biased, blind measurements of eye-tube lengths were made by Madeline

Owens, using Image J (ImageJ 1.45s NIH) from the front surface of the lens to the posterior end of the pigmented eye tube. In order to compensate for variation in total eye length, percent change relative to each eye’s original length was calculated for eye one and eye two for both

Ringer’s solution and water individuals. Finally mean percent change in Ringer’s solution and

14 water groups was analyzed with a Student T test.

Results

Histological Observations

To investigate eye growth in T. marmoratus, I first used general histological staining to evaluate the size of the eye and its main components at different time points. For each eye, I evaluated the total length of the eye tube, the length of the crystalline cone, the starting positions of the distal and proximal retinas, the total length of the distal and proximal retinas independently, and both retinas together (Figure 3). In both Eye 1 (Figure 4) and Eye 2 (Figure

5), the eye tubes grew significantly after the second to third instar molt, by the first sampling period of the third instar (0H). The overall size of Eye 1 from an average of 619.42 µm on second instar day five (-24H) to 761.16 µm at third instar day one at molt (0H), a change of

141.7 µm. The overall size of Eye 2 grew from an average of 515.68 µm on day five to 692.92

µm at third instar, day one at molt (0h), a change of 177.2 µm.

The greatest area of growth in both eyes was the crystalline cone. In Eye 1, the crystalline cone grew from an average of 360.3 µm on second instar day five (-24H) to 466.2 µm after molt (0H), a change of 105.9 µm. In Eye 2, the crystalline cone grew from an average of

278.5 µm on second instar day five to 379 µm after molt (0H), a change of 100 µm. Some growth was observed in the distal and proximal retinas as well. While the length of the proximal retina was not significantly different after the molt period, the distal retina did become significantly longer on the second day of third instar and onward (24H-48H). The length of both

15 retinas also became significantly longer after the second day of third instar and onward (24H-

48H).

Histology: Eye 1

In Eye 1, the crystalline cone region extended on average over 354.75 µm during the second instar phase, and lengthened by 111 µm (to 466.22 µm) by the third instar day 1 (0h) time point. The total length of this structure averaged at 477 µm across all third instar sample points.

An ANOVA for the eye one crystalline cone region [F(11,61)=24.20, MSE=952.6, P<0.0001] demonstrates that there were significant differences between some of the crystalline cone lengths. Further post hoc Tukey tests showed that there was no significant difference between the crystalline cone lengths at all second instar points (-72H, -48H, -24H) compared against themselves, nor any significant differences between crystalline cone lengths at all third instar points (0H-48H) compared against themselves. However all third instar crystalline cone lengths were significantly longer than all second instar crystalline cones (P=<0.0001 for all comparisons;

Figure 6 A).

The ANOVA results for the distal retina thickness of Eye 1 are: F(11,61)=27.49,

MSE=1388.8, P=0.0001. This demonstrates that there were significant differences among some of the sampled points. A post hoc Tukey HSD tests showed that for the distal retina thickness the only time points at which their length differed significantly were the second instar points (-

72H, -48H, -24H) compared to the last three sampling points within third instars (24H, 36H,

48H). The distal retina thickness for third instar day two at molt (24h) differed from all second instar time points significantly with P=0.0001 for all pairwise comparisons. The distal retina

16 thickness at third instar day two at twelve hours (36 h) differed from all second instar time points by P=≤0.0066. The distal retina thickness at third instar day three (48h) differed from all second instar time points by P=≤0.0175. The distal retinal thicknesses for third instar points 0H-20H did not differ significantly from the distal retinal thickness at second instar time points (-72H, -48H,

-24H), or the final three third instar points (24-48H). ANOVA results for the proximal retina length in eye 1 are F(11,61)=1.6195, MSE=250, P=0.1158 and post hoc testing showed no significant difference in size across all time points tested.

The ANOVA results for the combined retina thickness (total distal retinal depth plus total proximal retina length) of Eye 1 are F(11,61)=5.7893, MSE=733.05, P=0.0001 suggesting that there is significant difference between some of the sampled points. A post hoc Tukey HSD test showed that the combined retinal thickness at third instar day 2 at molt (24H) differed significantly from all second instar time points (-72,H -36H, -24H) P≤ 0.0002. Third instar day two at 12 hours (36H) differed from all second instar time points P≤0.039, and third instar day three (48H) differed from all second instar time points P≤0.0223. Otherwise there were no significant differences between the combined retina thickness between any third instar time points, or second instar time points when compared to one another (Figure 7 A)

Histology: Eye 2

In Eye 2 the crystalline cone region ends on average at 278.72 µm during the second instar phase, and lengthens by 110 µm to a total of 379.09 µm by the third instar day 1 molt (0h) time point, and averages 393.74 µm in length across third instar sample points. ANOVA results for the eye two crystalline cone region (F(11,61)=32.62, MSE=625.7, P<0.0001) demonstrate that there were significant difference between some of the sampled points. Further post hoc

17 Tukey tests showed that there were no significant difference between crystalline cone lengths of all second instar points (-72H, -48H, -24H), and that there were no significant differences between the crystalline cone lengths of third instar points (0H-48H). However all third instar crystalline cone lengths differed significantly from all second instar crystalline cone lengths

P=<0.0001 (Figure 6 B).

The ANOVA results for the distal retina thickness of Eye 2 are F(11,61)=3.3521,

MSE=677.26, P<0.0012, demonstrating that there was significant difference among some of the points. Further post hoc Tukey tests showed that only the distal retina thickness on third instar day two (24H) differed significantly from the distal retinal thickness for all second instar groups

P=≤0.0129. The distal retinal thicknesses for 24H are not significantly different from distal retinal thickness at second instar time points (-72H, -48H, -24H), or distal retinal thickness at any other third instar time point. The proximal retina ANOVA (F(11,61)=0.7051, MSE=259.882

P<0.7289) shows that there is no significant difference in size across any of the time points tested.

The ANOVA results for the combined retina thickness (total distal retinal depth plus total proximal retina length) of eye two (F(11,61)=2.7191, MSE=1183.64, P<0.0064) demonstrate that there was a significant difference between some of the points. Post hoc testing revealed that for combined retinal thickness only second instar day 5 (-24H) larvae differed significantly from third instar day two larvae (36H) P=0.0050 (Figure 7 B). There was no significant difference between the combined retinal thickness of any other points.

Optical measurements

18 To evaluate when lenses were able to begin forming sharp images, and how far away from the lens those images were focused at various time points (in third instar compared to second instars) the modified hanging drop technique, outlined in Stowasser 2010, was used. In this technique lenses are dissected, and images that are produced by insect lenses are evaluated.

Lenses during second instar show no degradation even on second instar day five, the final day

(Figure 10 A), and are capable of forming good, high contrast bifocal images with two distinct focal planes. On average for eye 1, the first focal plane was on average 357 µm behind the back surface of the lens, and the second around 431 µm. For eye 2, the first focal plane was on average 290 µm behind the back surface of the lens, and the second around 345 µm (Examples:

Figure 10 B and C, violet plots).

Upon ecdysis a portion of the lens is shed with the exoskeleton, leaving behind a relatively thin section of remaining lens tissue with a distinct “doughnut” like shape and rough surface (Figure 10 A’). In contrast lenses sampled at molt (0H), and four hours later (4H) are not capable of forming images (Examples: Figure 10 B and C, blue plots). Accordingly image processing at these time points did not reveal focal peaks.

Once lenses have passed the 12 hour point on third instar day 1, lenses are larger in diameter than they were during second instar phase, once again have a smooth back surface

(Figure 10 A’’), and are capable of good image formation. The images formed by third instar lenses are projected deeper into the eye than are those of second instars. Within eye one of third instars the first focal plane fell at an average depth of 503 µm behind the back surface of the lens and the second focal plane at 603 µm behind the back surface of the lens (for time points 12H-

48H). In eye two the average distance for the first focal plane was 402 µm and for the second

19 focal plane it was 476 µm. Taken together, between instars in eye 1, focal plane one shifts on average 145 µm deeper into the eye tube and focal plane two shifts 173 µm deeper into the eye.

For eye 2, the shift is 112 µm for focal plane one, and 130 µm for focal plane two. (Examples:

Figure 8 B and C, orange plots).

Optics: Eye 1

ANOVA results for the focal length one in Eye 1 (F(9,47)=26.21, MSE= 1071.8, p <

0.0001) demonstrate that there was a significant difference among some of the focal lengths.

Further post hoc Tukey tests showed that there was no significant difference in focal plane one lengths between all second instar points (-72H, -48H, -24H) when compared against themselves, and that there were no significant difference between third instar focal plane one lengths compared against themselves. However, all third instar time point’s focal length one differed significantly from all second instar time points focal one lengths P≤0.0055 (Figure 9 A).

For Eye 1 focal length two, ANOVA results (F(9,47)=18.06, MSE= 1969.2, p < 0.0001) demonstrate that there was significant difference among some of the points. Further post hoc

Tukey tests showed there was no significant difference between all second instar focal lengths (-

72H, -48H, -24H) focal plane two lengths compared against themselves, nor any significant difference between third instar focal length two compared against themselves. However all third instar categories focal plane two lengths differed significantly from all second instar points focal plane two lengths by P=≤ 0.0008. (Figure 9 B)

Optics: Eye 2

ANOVA results for the focal length one in eye two (F(9,47)=30.1261, MSE= 681, p <

20 0.0001) demonstrate that there was significant difference among some of the points. Further post hoc Tukey tests showed that there was no significant difference between all second instar (-72H,

-48H, -24H) focal length one compared against themselves, nor any significant difference between third instar focal plane one lengths compared against themselves. All third instar focal length one differed significantly from all second instar point’s focal one length by P=<0.0001

(Figure 10 A).

For Eye 1 focal length two, ANOVA results (F(9,47)=22.2354, MSE = 1323.0, P=<

0.000) demonstrate that there was significant difference among some of the. Further post hoc

Tukey tests showed that there was no significant difference between all second instar points (-

72H, -48H, -24H) focal length two compared against themselves, nor any significant difference between third instar points focal plane two lengths compared against themselves. All third instar time point’s focal plane one lengths differed significantly from all second instar point’s focal one lengths by P=≤0.0005 (Figure 10 B).

Optical and Retina Fit

To evaluate whether the fit between focal plane one and the start of the distal retina, and focal plane two with the start of the proximal retina established in Stowasser et al

(2014) was applicable during the second instar time period, and whether this fit was recovered during third instar, means for focal planes and histological locations were overlaid on graphs and evaluated. For Eye 1 focal length one, during second instar the images fall just at the end of the crystalline cone, once the third instar day one twelve hour time point (12H) is reached, the images again fall just at the end of the crystalline cone or slightly into the distal retina. At the day one eight hour time point (8H) in eye 1, the third

21 instar focal length one is somewhat shorter than at later stages. Given my sample sizes this difference however is not significant (P=0.057; Figure 11A). For focal length two, focal planes consistently fall near the transition of proximal and distal retinas (Figure 11B).

For Eye 2 focal length one, during second instar the images fall near the proximal end of the crystalline cone. Once the 12H time point is reached, the images again fall just at the end of the crystalline cone or slightly into the distal retina (Figure 12A). For focal plane two, images within the same times post-molt consistently fall near the distal margin of the distal retina (Figure 12B).

Post Molt Osmotic Observations

Initial observations suggest that larvae may be swallowing pond water while transitioning to larger instars, suggesting that an osmotic mechanism could underlie rapid eye expansion. In order to test if an osmotic mechanism does play a role in the expansion of eye tubes, larvae were immersed in either water (hypotonic relative to the animal’s haemolymph; Figure 13 A,A’) or 100% insect Ringer’s solution (hypertonic compared to the animal’s haemolymph; Figure 13 B,B’) and their eye tubes where filmed via time lapse.

Measurements of eye tube length for both water immersed individuals (Figure 13 A, A’) and Ringer’s solution immersed individuals (Figure 13 B, B’) were made as quickly as possible after molting (at twenty minutes; 20M), and then again at ninety minutes post molt (90M). Means for all categories were examined, as well as the percent change between the 20 and 90 minute time slots for Ringer’s solution and water trials in eye one and eye two. Since individuals showed substantial variation in eye-tube length, eye tube length were also analyzed in terms of a percent change to its original value. When

22 analyzing percent change in eye one water individuals compared to Ringer’s solution individuals, water individuals had a mean percent change of 4.43 percent while Ringer’s solution individuals showed a mean percent change of -8.28percent. When analyzed using a two tailed student’s T-test, these values were not significantly different at P=0.27 (Figure

16B). In Eye 2, water individuals had a mean percent change of 21.46 percent, while

Ringer’s solution individuals had a mean percent change of 13.48. Using a two tailed student’s T-test, eye two values were found to be significantly different at P= 0.0125

(Figure 16 B’)

Discussion

This study provides the first analysis of arthropod camera type eye growth, investigating the process of return to functional emmetropia after molting. Key findings are that the eye tubes grow only after molt and do so very rapidly, with the most significant area of growth in both primary eyes being the crystalline cone region that is situated between the lens and the start point of the distal retina. Compared to the eye tube length the lenses transform more slowly, unable to reform sharp, bifocal images until approximately 8h after the molting process. Additionally I have explored a potential mechanism for the rapid growth of the eye tube via osmotic regulation. I initially hypothesized that eyes would regain emmetropia around day three, when their hunting behavior becomes optimized. However, current data shows that larval eyes achieve emmetropia as early as 12h after molting.

Changes in eye size

23 The rapid growth of the eye tube is particularly notable, as it is already significantly longer than that of second instars within the first hour after molt. This is relatively rapid if compared to the development of post-natal vertebrate eyes, many of which begin in some state of ametropia, growing and changing in particular regions toward emmetropia. In guinea pigs, emmetropia is achieved within the first month of life, though their initial refractive error is already halved after the first week (Howlett and McFadden 2007). Mice are much slower, taking 70 days, and passing the onset of sexual maturity before refractive errors present at birth are eliminated (Schmucker and Schaffel 2004). In humans overall eye length (axial length) will grow about 25% for the first three years of life, and then about 1% every year for the following ten years (Gordon & Donzis, 1985; Larsen, 1971d;

Sorsby & Leary, 1970). Various components of the vertebrate eye will change continuously over the course of the life of the animal in question.

While I cannot exclude that there is some fine-tuning of eye-length at a later time, it is notable that all major changes have occurred within one hour after the molt (0H), and that I did not observe any significant growth in any of the measured areas of the eyes thereafter, with the exception of the distal retina on the second and third days of third instar. The distal retina of Eye 1 is the only area that correlates with the previously observed hunt behavioral patterns (Figure 2). The greatest contributor to overall growth is the crystalline cone region, which is functionally analogous to the vertebrate vitreous.

Specifically, it contributes 67 % of the overall growth in eye one, and 68 % in Eye 2 The data make it clear that the primary way larvae are able to so rapidly expand their eye tubes so quickly after molt is the expansion of the crystalline cone region with smaller adjustments in the distal and proximal retina and the most proximal region of the eye.

24

With such rapid growth after molt, there is potentially room for adjustment of the eye tubes for optimal emmetropic conditions. Further examination at more closely spaced time points after lens reformation (8h), or under different developmental conditions may reveal fine-tuning of the eye tubes once active visual input is present. However there are limitations to the histological and focal length methodology used in this study, as they are not sufficiently sensitive to pick up on small or very rapid changes, and cannot be used to monitor a single individual over time. The development of either better staining methods to reveal eye tissue for in-vivo scanning via micro-CT or use of an opthalmascope may make more finely tuned study of the eye possible in future.

In vertebrates areas of greatest growth as the eye progresses toward emmetropia vary depend on species. In guinea pigs the vitreous chamber depth (roughly analogous to the crystalline cone) grew an average of 10 μm a day over the course of day 2-30 from birth, which was only half the rate of thickening of the lens (Howlett and McFadden 2007).

In marmosets vitreous chamber depth increases 2.5 mm over 10 months from birth, while the axial length (the distance between the anterior surface of the cornea to the fovea area of the retina) increased from an average of ~7.75 mm to 10.5 mm over the same time period. This shows that the vitreous chamber’s growth contributes roughly 90% to the overall growth of the eye (Graham and Judge 1999). Adult eyes of this species have an axial length of ~11mm (Trolio and Howland 1993). However, the vitreous dos not always expand. For example, in tree shrews and mice the vitreous chamber depth shrinks

(McBrien and Norton 1991, Schmucker and Schaeffel 2004) as the animal grows.

25

In T. marmoratus larvae the retina region contributes roughly 25% of the overall growth of the eye tubes, a degree of growth not demonstrated by vertebrate eyes.

However in some vertebrate eyes additional growth toward emmetropia can occur in the choroid region, a layer of vascular and connective tissues which lies between the retina and sclera. In chicks exposed to spectacles of varying corrective lens powers (negative powers for induced hyperopia, positive power for induced myopia), the retinal and choroid layers were able to thin to compensate for induced hyperopia and thicken to compensate for induced myopia (Wallman et al 1995, Wildsoet and Wallman 1995). The effective thickening or thinning of the choroid and retinal layer in concert with previously studied emmetropic change mechanisms (changes to the corneal curvature, anterior chamber depth and vitreal chamber depth (Troilo and Wallman 1991, Wallman and Adams 1987,

McBrien and Norton 1992), allowed the developing eyes to achieve emmetropia.

In several vertebrate species, the choroid region has also been shown to fluxuate in thickness in concert with circadian rhythm, with chicks (Nickla, Wildosoet, and Wallman

1997) and marmosets (Nickla, Wildosoet, and Troilo 2002) showing increase in axial length and thinning of the choroid layer during the day, and a decrease in axial length and thickening of the choroid layer at night. Rabbits display an opposite pattern, where axial lengths are longer at night and shorter during the day (Rowland Potter and Reiter 1981).

Under experimental conditions, even brief exposure to a myopic induction method can cause transient thickening of the choroid region (Nickla 2007).

26 Invertebrate retinas also demonstrate dynamic changes within the eye. For example Camponotus species of ants in a light adapted state the rhabdom is 10 μm shorter than in its dark adapted state (Menzi 1986). Similar rhabdomeric ultrastructure changes have been observed in hemipterans (Ltidtke 1953, Walcott 1971), coleopterans (Meyer-

Rochow 1972, Wada and Schneider 1967, 1968; Eckert 1968, Horridge et al. 1983), lepidopterans (Horridge and Giddings 1971), and dipterans (Sato et al. 1957, Williams

1980). This rhabdomeric adjustment, or retinomotoric response, allows for good adaptation to longer term light changes (such as diurnal and nocturnal phases), by lengthening or shortening the rhabdom to best cope with the light intensity of each phase.

Such changes however appear not to occur in response to rapid changes in light intensity, but instead might be regulated by the circadian clock.

While, to the best of my knowledge, there are no other emmetropia related studies on arthropods, there has been one other (albeit apart from two abstracts unpublished) study on emmetropization in an invertebrate. This study on cephalopods shows that when exposed for 45 days, post hatching, to an environmental stimulus that induces hyperopia

(through exposure to blue light) or myopia (through exposure to orange light) cephalopods eye development lead to compensating eye changes. Specifically, blue light exposed animals showed smaller Matthiessen’s ratios (the ratio between the distance from the center of the lens to the retina versus the lens radius, MR) indicating hyperopia, while orange reared animals had larger MRs. When light environments were switched after the initial 45 days, blue reared animals were exposed to orange light and vice versa, the eyes of each respective group of animals switched. Orange reared animals now exposed to blue light displayed smaller MRs and

27 blue reared animals exposed to orange light acquired larger MRs. There were indications that the primary areas of change were the location of the center of the lens and the location (in this case the thickness of photoreceptor area) of the retina, but in this pilot study there was no significant difference in changes to either of these areas with the exception of the center of the lens becoming significantly posterior in cephalopods reared under blue light conditions. At this point it remains unclear whether the refractive power of the lenses, or the position of the retina most strongly contributed to emmetropization, or how much other factors such as accommodation played a part. However this study provided evidence that in this invertebrate

(with eyes that are similar to those of vertebrates) visual control is an important regulator of emmetropization.

Lens reformation and fit of images with retinas

Compared to the lengthening of the eye tubes, the reformation of the lens is relatively slower. As previously stated, a portion of the old lens shed with the old exoskeleton, and the bi-convex lens expands both in diameter and thickness. Lenses do not regain a smooth appearance and capacity to project two sharp bifocal images until hour eight of the first day of third instars. My preliminary behavioral data indicates that third instar hunt behavior shows that day one individuals capture fewer larvae on average than larvae in the peak hunting area between day three and five (Figure 2), and this delay in lens reformation may relate to the observed delayed onset of maximum feeding rates.

28 At 8H images for focal plane one are an average of 30 μm above the locations of all other third instar points in eye one, and 12 μm above the focal plane average in eye two.

Focal plane two for eye one falls about 14 μm above following all other third instar time point locations, and 27 μm for eye two. The images move a bit further back into the eye by

12H and remain fairly stable from that point on. These data suggests that even though clear bifocal images are present at this time, there are additional minor adjustments in the following 4h.

It’s important to note that ultraviolet light is refracted more than green light, and thus images in ultra violet would be projected closer to the lens than images in green light.

This is important because the distal retina is sensitive to green light whereas the proximal retina is sensitive to ultraviolet light. Eye one falls under the working definition for emmetropia established for the purpose of this study, which was conducted entirely in green light conditions. For focal plane one, images in green light are projected within the first few layers of the distal retina. For focal plane images in green light are projected at the start of the proximal retina. By contrast, eye two is slightly myopic with regard to the proximal retina, with images for focal plane two falling within the end of the distal retina, instead of at the start of the proximal retina (Stowasser 2014). The pattern previously established in Stowasser 2014, where green light is projected at the start of the distal retina and start of the proximal retina for eye one, and at the start of the distal retina and around the end of the distal retina for eye two, holds true for first and second instar individuals at all points measured (-72H, -48H, -24H), and third instars after the twelve hour time point on third instar day one (12H).

29

Where the focal planes fall when the objects projected are at effective infinity is important, as object distance influences where exactly within the eye images are projected.

As objects move closer to the lens, resulting images move deeper into the eye. I the case of

T. marmoratus larvae further into the distal or proximal retina respectively. I has been proposed (REF to Annette’s paper) that this basic optical principle, together with the layered retina organization may allow T. marmoratus larvae to achieve a mechanism of depth perception useful for prey capture. The optics is set up so that images of move deeper into the retinas as larvae approach optimal striking distances. My data demonstrates that images are projected onto similar retinal layers in second instars as they are in third instars. This further supports the importance of images being precisely focused onto specific layers. The fact that this relationship recovered so quickly post-molting reinforces the notion that this retina and focal plane relationship is important for the animal’s visual hunting strategy.

Preliminary evidence for an osmotic method of growth

One of my most surprising initial findings has been the speed by which the eye- tubes regain its new lengths. To investigate this further this study explored the possibility that this rapid expansion could be mediated by an osmotic mechanism. Currently unpublished preliminary data from our lab supports this possibility, by revealing the presence of at least one water-channel (Aquaporin 6, also known as drip) in the cells that make up the crystalline cone and retina of both eyes in larvae that had just molted.

Together with circumstantial observations that larvae tend to swallow water as part of

30 their molting process, these findings led to the idea of testing larval eye growth in normal hypotonic conditions as well as abnormal hypertonic conditions to test if these treatments would lead to differential eye growth. Water uptake may be vital for expanding not only their eyes but their entire bodies.

To eliminate eye-tube length variation between individuals, I analyzed these data in regards to the percent change between starting size (at twenty minutes post molt) and the final size that could be imaged through the cuticle (at ninety minutes). The eyes of water immersed individuals grew on average. On the other hand, eyes of Ringer’s solution immersed individuals shrank on average. In regards to eye one there was a clear trend, however at this point differences were not statistically different (p=0.276). Data on eye two indeed supports the presence of an osmosis-based mechanism, as eye two showed significant percent change differences between water and Ringer’s solution immersed individuals (p=0.0125). There were some challenges in measuring these eye tube lengths by observing them through the head capsule in a very short window of time. There is a notable differences in eye-tube length between water and Ringer’s solution treated individuals already at the twenty minute start point, which could suggests that the eyes had already begun to grow even in the relatively short time that it took to mount them for photography. In addition sample sizes for these experiments were relatively small, with only 7 individuals in each category, and significance for eye one may emerge upon the addition of more individuals to the sample pool.

31 The rapid expansion of the eye tubes in T. marmoratus may also be fostered by larvae being entirely aquatic. This unimpeded access to water for body expansion may potentially give them an advantage over non-aquatic arthropods, which do need adequate levels of humidity in order to survive a molt. Investigations into how terrestrial arthropods cope with molting and humidity with regard to eye expansion may reveal if this osmotic mechanism is unique to aquatic arthropods, or a common feature in arthropod molting growth in general.

In summary

The findings that both the histological and focal components are so quickly and precisely recovered suggest that the invertebrate eye system is relatively dynamic, perhaps more though than previously thought. However, much remains to be investigated in these eyes to determine exactly how dynamic they are. Perhaps the main value of the present study is that it establishes a baseline of when histological and optical components adjust to their new size under normal conditions. These data are important in the light of current investigations that gear towards establishing the role of specific lens components under normal and abnormal conditions.

Rearing larvae under abnormal conditions, similar to those which have been performed on vertebrates to test active eye growth versus passive eye growth (Troilo 1991

, Norton 1992, Wallman et al 1995, Wildsoet and Wallman 1995), may also reveal if regulatory mechanisms are present that would allow the larval eye to actively adapt to the specific conditions that are present after molt. For example, it would be important to

32 determine if visual input is necessary to regulate eye length, and if so, if eyes are able to recover normal vision when returned to normal conditions. Since this topic has not been investigated beyond this study, it is also be possible that the growth of arthropod larval eyes is strictly passively guided.

The possibility of an osmotic mechanism for growth also presents the possibility that osmotic mechanisms are present in other types of invertebrate eyes, possibly underlying previously discovered processes such as the retinomotoric function in compound eyes that assists those animals in adapting optimally to their light environment.

Aquaporins are also being investigated in vertebrates to demonstrate their role in the general functioning of the eye (Verkman 2003, Dibas 2008) and more specifically their role has been noted in regards to the presence of intraocular pressure and the ability to produce aqueous fluid (Zhang 2002). Further investigation into osmotic mechanisms of growth in arthropods and vertebrates may reveal the importance of osmosis as a regulatory factor in eye growth and maintenance.

33 Figures

Figure 1. A- An electron microscopy image of the whole larval head. B- Schematic of a sagittal section of the larval eyes showing the positions of the primary eyes 1 and 2 and their retinal layout. C- Diagram of the life cycle of T. marmoratus, from egg to adulthood, all phases are shown to scale. D, - sagittal section of a first instar with back of the lens to end of eye measurements for eyes 1 and 2, D’- sagittal section of a second instar with measurements for eye 1 and 2 , D’’- sagittal section of a third instar with measurements for eyes 1 and 2.

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Table 1: For an example second instar molt time of 9 AM, this outlines sampling times, their time points, and notations for each time point

Figure 2 – Third instar, average total number of larvae captured by day

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Figure 3 – Histological measurements taken for each eye. A- Back surface of the lens to start of distal retina, B- Back surface of the lens to the distal retina pit, C- Back surface of the lens to the end of the distal retina/start of the proximal retina, D- Start of the proximal retina to the end of the proximal retina, E- Back surface of the lens to the end of the eye tube.

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Figure 4 – Scatter plot of mean values for major Eye 1 histology points. Symbols correspond to the major landmarks that are shown on the diagram of the eye. Diamond shows the end of the crystalline cone and start of the distal retina, squares show the end of the distal retina and the start of the proximal retina, triangle shows the end of the proximal retina, and circles indicates the end of the entire eye capsule.

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Figure 5 – Scatter plot of mean values for major Eye 2 histology points. Symbols correspond to the major landmarks that are shown on the diagram of the eye. Diamond shows the end of the crystalline cone and start of the distal retina, squares show the end of the distal retina and the start of the proximal retina, triangle shows the end of the proximal retina, and circles indicates the end of the entire eye capsule.

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Figure 6 – Mean values for the length of the crystalline cone regions. A. Eye 1. B, Eye 2,. Significant differences between time points are marked with *

Figure 7 – Mean values for the total retinal area (distal retinal length and proximal retina length). A. Eye 1. B, Eye 2,. Significant differences between time points are marked with *.

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Figure 8 – Diagram showing a comparison of average major landmark sizes for eye 1 of final day of Second Instar (day 5), and first day of third Instar (at twenty hours). It also shows growth values for each portion of the eye (between these time points), and the percent to the total growth of the eye that each area represents.

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Figure 9 – Diagram showing a comparison of average major landmark sizes for eye 2 of final day of Second Instar (day 5), and first day of third Instar (at twenty hours). It also shows growth values for each portion of the eye (between these time points), and the percent to the total growth of the eye that each area represents

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Figure 10 – A-A’’ Example images of back lens surfaces. A. Second Instar Day 5 individual, A’- Third Instar Day 1 individual, A’’- Third Instar Day 2 individual. B & C. Example graphs of image contrasts that are observed at different time points. - The shift of focal planes, in Eye 1 before (2ID5@M12h, violet), at (3ID1@M, aqua), and after molt (3ID2@M, orange). C- The shift of focal planes in Eye 2 before, at and after molt.

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Figure 11 – Locations of the first focal plane generated by the lens, relative to the back surface of the lens. A- Eye 1. B- Eye 2. Significant differences between time points are marked with *.

Figure 12 – Locations of the second focal plane generated by the lens, relative to the back surface of the lens. A- Eye 1. B- Eye 2. Significant differences between time points are marked with *.

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Figure 13– Diagrams showing A- Eye 1 fit between the average crystalline cone length (shown in orange), and average focal plane 1 length (shown in green), and B – Eye 1 average distal end length (shown in orange) fit with average focal plane 2 (shown in violet). All distances are shown relative to their distance from the back surface of the lens.

Figure 14– Eye 2 diagrams showing A- the fit between the average crystalline cone end (shown in blue), and average focal plane 1 (shown in green), and B – the fit between average distal retina end (shown in blue) and average focal plane 2 (shown in violet). All distances are shown relative to their distance from the back surface of the lens.

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Figure 15 – Example images of individuals the eye tube length of which were monitored while emerged in water (A) or Ringer’s solution (B). A- Individual at 20 minutes, immersed in water, A’- The same water individual at 90 minutes. B- Individual at 20 minutes, immersed in 100% insect Ringer’s solution, B’- The same individual at 90 minutes.

Figure 16 – A- A comparison of Eye 1 lengths in water and Ringer’s solution individuals at 20 minutes (shown in blue) and 90 minutes (shown in purple) A’’ – A comparison of Eye 2 lengths in Ringer’s solution and water at 20 and 90 minutes. B&B’- Percent change between eye length at 20min and 90 min post molt. B. Eye 1 for water and Ringer’s solution, B’- Eye 2 for water and Ringer’s solution.

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