Emmetropization in Arthropods: a New Vision Test in Several

Emmetropization in arthropods: A new vision test in several

arthropods suggests visual input may not be necessary to

establish correct focusing

A thesis submitted to the

Graduate School of The University of Cincinnati

(Department of Biological Sciences)

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

July 2019

Madeline Owens

B.S. Biological Sciences, University of Cincinnati (2016)

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

This work will be submitted to The Journal of Experimental Biology

Authors: Madeline Owens, Elke K. Buschbeck ABSTRACT

The visual systems of vertebrates and invertebrates alike must be able to achieve and maintain emmetropia, a state of correct focusing, in order to effectively execute visually guided behavior. Vertebrate studies, as well as a study in squid, have revealed that for them correct focusing is accomplished through a combination of gene regulation during early development, and homeostatic visual input from the environment that fine-tunes the eye. While eye growth towards emmetropia has been long researched in vertebrates, it is largely unknown how it is established in arthropods. To address this question, we built a micro-ophthalmoscope that allows us to directly measure how a lens projects an image onto the retina in the eyes of small, live arthropods, and to compare the eyes of different light-reared and dark-reared arthropods.

Surprisingly, and in sharp contrast to vertebrates, our data on a diverse set of arthropods suggests that visual input in arthropods may not be necessary at least for the initial development of emmetropic eyes. First, we measured the image-forming larval eyes of diving beetles

(Thermonectus marmoratus), the eyes of which are known to rapidly and dramatically grow between larval instars. Then we compared jumping spiders (Phidippus audax) after their emergence from their egg case and finally we compared measurements of individual ommatidia of the compound eyes of flesh flies (Sarcophaga bullata) that had developed and emerged either under light or dark conditions. The refractive error of these arthropods was comparable between rearing conditions, suggesting that visual input plays less of a role in developing arthropod eyes than it does in other eyes that develop emmetropia. Although it remains unclear if visual input that is received after the initial development can further improve focusing, these results suggest that in arthropods the initial coordination between the lens refractive power and eye size may be more strongly pre-determined than typically is the case in vertebrates. This research opens new

i questions regarding how the growth of arthropod eyes might be regulated and highlights arthropod eyes as a particularly powerful model system for organogenesis.

Madeline Owens

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ACKNOWLEDGEMENTS

I would like to thank my advisor, Elke Buschbeck for her guidance, encouragement, and patience.

Elke fosters a genuine excitement and passion for learning that will stay with me throughout my life.

Throughout my education she has been role model as someone who constantly displays a moral character, work ethic, and general attitude toward unexpected challenges that I strive to emanate in my daily life. I would like to thank my committee members: Annette Stowasser, who spent the time to teach me many of the technical skills that I apply in this paper. Discussing science and working with Annette has been a privilege. Also, Nathan Morehouse, whose enthusiasm for biology and ways to effectively share research inspires me.

Thanks to Hailey Tobler for maintaining the labs insect colony with a level of care and attention to detail that few have been able to do. Also, a thank you to Isaiah Giordullo for performing his blind data analysis for this work and to John T Gote and Patrick M. Buttler for collecting jumping spiders in the vicinity of Pittsburgh. A special thanks to Jennifer Hassert, Rose Conley, and Jeremy Didion for always being there for me throughout my graduate experience for support and to talk or brainstorm. Having such wonderful lab peers boosted me up and helped keep me going. Also, a special thanks to Rebecca Meurer in the Biology Department office who helped get me prepared for graduation and constantly goes above and beyond with her willingness to help answer any questions I have.

This research was funded by the National Science Foundation under grant IOS1456757 to EKB.

TABLE OF CONTENTS

Introduction – 1

Materials and Methods – 4

Animal rearing Thermonectus marmoratus – 4 Phidippus audax – 5 Sarcophaga bullata – 5 Micro ophthalmoscope measurements – 6 Focal length – 7 Optical assessment-method – 7 Histological assessment – 8

Results – 9

Thermonectus marmoratus – 9 Phidippus audax – 10 Sarcophaga bullata – 12

Discussion – 14

References – 19

Figures – 24 Thermonectus marmoratus – 24 Phidippus audax – 25 Sarcophaga bullata – 26

LIST OF FIGURES

Figure 1) Thermonectus marmoratus optical analysis. (A) External image of T. marmoratus larva in water. Note the principal front facing eyes E1 and E2. (B) Ophthalmoscope image of proximal retina showing two rows of rhabdoms. (C-E) Analysis of E1. (C) Ophthalmoscope measurements showing the object distances (in mm) for the range (represented by bars) over which the E1 photoreceptors are optimally focused for each individual (Dark N=10; Control

N=10) as well as their average. (D) Measured focal lengths for dark and control individuals from isolated lenses. (E) Distance between the surface of the retina and the focal plane of the lens of

E1 (in μm) based on the measured micro-ophthalmoscope measurements in combination with focal length measurements of each individual. (F-H) The same optical analysis as in C-E but on

E2. (F) Object distance for E2 (N=10; Control N=10). (G) Focal length for E2. (H) The distance between the surface of the retina and the focal plane of the lens for E2 based on the focal lengths of the individual’s lenses.

Figure 2) Phidippus audax optical analysis. (A) Schematic of the tiered jumping spider retina in principal AM eyes (modified from Stowasser et al., 2017). (B) Ophthalmoscope image of the boomerang shaped AM retina, layer 1. (C) Micro-ophthalmoscope measurements showing the object distances (in mm) including the range for which the photoreceptors of layer 2 are focused for each individual (Dark N=16; Control N=9). The average is indicated as a line. (D)

Measurements of focal lengths for AM eye lenses. E) Calculation of the distance between the surface of the retina and the focal plane (and the range of focused images) of the lens for layer 2 based on the micro-ophthalmoscope and focal length measurements for each individual. (F)

Object distance for layer 1 as established with the micro-ophthalmoscope, (G) Image of

Phidippus audax spiderling, illustrating the prominent principal anterior median eyes. (H) The distance between the surface of the retina and the focal plane of the lens for layer 1 based on individual micro-ophthalmoscope and focal lengths measurements.

Figure 3) Sarcophaga bullata optical analysis. (A) Ophthalmoscope post-measurement image of the surface of a S. bullata compound eye lens array illustrating bleaching of a single ommatidium (left) which was illuminated by a small square light beam (right). (B)

Ophthalmoscope image showing photoreceptors of a single ommatidium. (C) Histological section of S. bullata photoreceptors; scale bar is 50 μm. (D) Ophthalmoscope measurements showing the object distances (in mm) ) for the range (represented by bars) for which the photoreceptors are focused for each individual (Dark N=10; Control daytime N=10; Control nighttime N=10). (E) Measured focal lengths for each individual calculated from the image magnification between the histological and optical images. (Dark N=7; Control daytime N=6;

Control nighttime N=9). (F) The calculated distance between the surface of the retina and the focal plane of the lens, based on the micro-ophthalmoscope and lens focal measurements of each individual.

vii

INTRODUCTION

Eyes are among the most complex organs (Land and Nilsson, 2012) and many animals depend on an optimized visual system for survival (Cronin et al., 2014). As such, the visual systems of vertebrates and invertebrates alike must be able to achieve and maintain a state of correct focusing, in order to effectively execute visually guided behavior. Correct focusing relies on the tight coordination between lens refraction and retina position and needs to be established during development and then maintained even if the eye grows. Correctly focused eyes therefore are characterized by a precise match between the focal length of its optics, and the distance between the lens and the retina. Emmetrope eyes are eyes for which images of objects at infinity fall directly onto the retina. If images are positioned in front of the retina eyes are considered to be near-sighted or myopic, and if they are positioned behind the retina, we considered them to be far-sighted, or hyperopic. Typically such deviations are considered to be refractive errors, but note that some invertebrate eyes may have evolved to focus at distances other than infinity

(Stowasser et al., 2017)

An important question is how eyes develop their optics correctly. This has been investigated intensely in vertebrates (Flitcroft, 2013; McBrien and Barnes, 1984; Troilo, 1992;

Wallman and Winawer, 2004; Fledelius et. al., 2014) and has become a particularly important question considering that myopia has been found to be drastically on the rise (Dolgin, 2015).

Many arthropods also have sophisticated image-forming eyes (Land and Nilsson 2012) that require precise focusing and therefore likewise must have mechanisms that tightly control eye growth. However, the mechanisms that coordinate lens refraction and the spacing between the lens and retina in developing arthropod eyes remains elusive. Towards gaining a better

1 understanding we here investigate how several arthropod eyes develop with and without access to visual input.

Studies in vertebrates and in squid (Sivak and Sivak, 2018; Turnbull et al., 2015) have suggested that the coordination of eye growth towards emmetropia is accomplished through a combination of gene regulation during early development (passive regulation), and homeostatic feedback in the form of visual input from the environment (light) that fine-tunes the eye (active regulation). Vertebrate eyes are typically born as either myopic or hyperopic (Wallman and

Winawer, 2004) and later grow to establish emmetropia (Hofstetter, 1969). Passive emmetropization is understood as a nonvisual execution of a genetic plan, that often continues postnatally and acts in combination with the physical constraints that are imposed on a growing eye (Mark, 1972) allowing eye growth to diminish refractive errors. There is also strong evidence for the role of visual feedback in controlling eye growth from studies in numerous vertebrates. For example, tree shrews were able to recover from visual deprivation (Norton

2007). Other support derives from studies in fish (Kroger and Wagner, 1996), guinea pigs

(McFadden et al., 2004), primates (Hung et al., 1995), and chicks (Schaeffel et al., 1988; Troilo and Wallman, 1991; Wahl et al., 2015; Wahl et al., 2016). In the Troilo and Wallman (1991) study a refractive error was induced in chicks via visual manipulations including dark-rearing.

The chicks were ultimately able to recover emmetropia by actively adjusting the growth of their vitreous chambers. Specifically, growth stopped in eyes that were recovering from myopia and continued in eyes that suffered from hyperopia. In recent studies it has become clear that light is a critical regulator (Wahl et al., 2015; Wahl et al., 2016). To our knowledge, the only study that addresses a similar mechanism in invertebrates has been performed on squid (Turnbull 2014) in which a refractive error was introduced by raising them under abnormal lighting conditions

(orange or blue light) and the squid subsequently were able to compensate for introduced refractive errors.

Correctly focused eyes within arthropods are important for those with high-resolution eyes, some of which are camera-type eyes. It is especially important for visually guided predators, such as jumping spiders (Jakob et al., 2018; Land, 1969; Land, 1972) and predatory holometabolous insect larvae (Buschbeck, 2014; Gilbert, 1994; Toh and Mizutani, 1987; Toh and Okamura, 2007), both of which have species that are characterized by high acuity vision.

The importance of correctly focused eyes also applies to arthropods with compound eyes

(Warrant and McIntyre, 1993), such as flies, who rely on their visual system for fast flight control for example during mating flights or predatory behavior (Wardill et al., 2017), for animals with compound eyes having incorrect focus would be detrimental to their fitness and survival. To address the question of how arthropods develop emmetropic eyes we therefore examined species with both, fundamentally different eye designs, and diverse phylogenetic backgrounds. Specifically, we investigated the visual systems of the diving beetle larva

Thermonectus marmoratus, the jumping spider Phidippus audax, and the flesh fly Sarcophaga bullata.

Larvae of Sunburst Diving Beetles (T. marmoratus) have sophisticated camera-type image-forming eyes with elaborate optics (Stowasser et al., 2010). To catch prey these larvae perform a scanning behavior (Buschbeck et al., 2007) and their precise optics likely are important for the assessment of distance (Bland et al., 2014). T. marmoratus has three larval instars that differ dramatically not only in body size but also in eye size. At each molt larvae not only shed their exoskeleton, but also a small portion of the lens, which undergoes dramatic reformation to produce larger focal lengths for larger eyes (Werner and Buschbeck, 2015).

Proper focusing of the eye is reestablished approximately 8 hours post-molt. Since dramatic changes at this developmental transition are already documented, they represent a particularly good point of investigation for this study. The precise organization of the principal eyes

(anterior-median or AM eyes) of jumping spiders into multiple layers also is known to be important for distance vision (Nagata et al., 2012). Since it is unclear how the optics changes between instars in P. audax, we here focus on their initial development. Upon emergence from their egg cases, jumping spiders rely on their visual systems to capture prey, so their eyes are expected to be fully functional at that time point. Finally, we assessed the refractive state of individual ommatidia of the compound eye of Sarcophaga bullata. Here too we focused on their eye development immediately after emerging from a developmental stage.

To our knowledge this represents the first investigation into whether light might foster active regulation in developing arthropod eyes possibly due to the technical challenge of measuring such small visual systems precisely. This challenge has been overcome by a micro- ophthalmoscope imaging technique that allows directly measurements of refractive errors in small, live arthropod photoreceptors on the basis of autofluorescence (Stowasser et al., 2017). In combination with lens focal-lengths measurements we could further quantify the position of images within the eye, relative to its retina, or specific retina layers.

MATERIALS AND METHODS

Animal rearing

Thermonectus marmoratus:

T. marmoratus larvae used were offspring of our beetle colony which were originally collected in Arizona and obtained through Bugs of America (Portal, AZ). The colony was maintained in

4 freshwater aquaria and kept at approximately 28°C. Larvae were reared individually and fed a daily diet of bloodworms and mosquitoes. All larvae were raised under a light/dark cycle (14hr light, 10hr dark) and fed daily until the day they were expected to molt into their 3rd larval stage.

At this point larvae were divided into controls or dark reared larvae and deprived of food.

Controls were kept under their regular light/dark conditions while dark reared larvae were deprived of light until measurements were taken. Measurements for all individuals took place 1- day post-molt into 3rd instars to allow time for their lens to reform (Werner and Buschbeck,

2015).

Phidippus audax:

P. audax spiderlings were obtained by mating individuals from our lab population that were originally either wild caught in Cincinnati, OH, USA, purchased from Phids.net (West Palm

Beach, FL, USA), or collected in the vicinity of Pittsburgh, PA. For both, controls and tests, spiderlings came from 2 egg clutches each. Spiders were kept in our animal husbandry room at

28°C with a 14hr light and 10hr dark cycle. For the dark treatment group, once the egg clutches were laid (before spiderlings could develop) they were deprived of light, while the control egg clutches were kept under regular light/dark conditions. Upon emergence spiderlings were separated into individual vials with access to water but no food to control for any potential role diet could play on their visual development as the test animals are unable to hunt without light.

All spiderlings remained in their respective light or dark conditions till measured, within 10 days of emergence.

Sarcophaga bullata:

S. bullata flies were obtained as pupae from Carolina Biological Supply Company (Burlington,

NC, USA). The fly pupae were separated into 3 groups: a control group that was measured

5 during daytime, a control group that was measured during their nighttime, and a dark–reared group. The nighttime control group was added to this experiment to account for possible effects of retinomotor movements that previously have been described in insect compound eyes

(Walcott, 1971; Warrant and McIntyre, 1993; Narendra et. al., 2016). All flies were raised in incubators at 25°C with access to sugar and water. The control daytime flies were raised under a

12hr light and 12hr dark cycle and after emergence, measurements were taken during the fly’s daytime hours. Control nighttime flies were raised under the same light/dark cycle, but measurements were taken during the fly’s nighttime hours. Dark reared flies were deprived of light until measurements were taken. Measurements for all flies were taken within 10 days after emergence into adults from their pupal stage.

Micro-ophthalmoscope measurements

Our micro-ophthalmoscope allowed us to measure the refractive error of arthropod photoreceptors by taking advantage of their autofluorescence as described by Stowasser et al.

(2017). In brief, an animal is mounted with the eyes of interest looking up vertically and positioned under an objective so that the lens of the eye in question is in sharp focus for a camera. Thereafter an accessory lens is added which then allows to image the eye’s retina. The accessory lens can be moved along a rail with the refractive state of the eye determining the accessory lens position that yields a focused retinal image. The direction the lens is moved in reference to the zero position (indicating emmetropia) is informative in regard to the eye being myopic (near-sighted) or hyperopic (far-sighted). T. marmoratus’ principal eyes (E1 and E2) were imaged with the 20x water objective, the anterior median (AM) eyes of P. audax were imaged with the 10xUPlanFL objective, and the individual ommatidium of S. bullata were imaged with the 40xUPlanFL objective. For each objective we established a calibration curve for

6 the ophthalmoscope, that allowed us to determine the relationship between the accessory lens position and the corresponding in-focus object distance. After imaging the eyes were either dissected or fixed for histology to determine the lens’s focal length, which is necessary to obtain in order to determine refractive errors quantitatively.

Focal Length Measurements

Focal length measurements were taken individually to determine specifically where an image is focused within the eye relative the animal’s retina. Focal length measurements are needed to quantitatively evaluate an animal’s focus with the micro-ophthalmoscope. Two different techniques were applied to determine focal length. The hanging drop method was applied for the jumping spider and beetle larvae, which have large enough lenses to do so reliably. For flesh flies, for which clean measurements through the hanging drop method are more difficult, we assessed the focal length through the image magnification which is based on obtaining the absolute size of the retina through histology.

Optical assessment-method

We followed a previously described ‘hanging-drop’ method that was modified after (Homann,

1924; Stowasser et al., 2017) and is described in detail in Stowasser et al. (2017). In brief, the focal length of the animal’s lens was calculated from the image magnification (Land, 1969; Land and Nilsson, 2012). To do so dissected spider lenses were mounted upside-down, floating on a drop of Ringer solution (Oshea and Adams, 1981), and T. marmoratus larval lenses were submerged within Ringer solution. To determine the image magnification, we took images at

5μm intervals through the animal’s lenses (using a 10x objective) of an object (USAF 1951 negative test target from Edmund Optics, Barrington, NJ, USA) that was placed at effective infinity for the animal’s lens (at either 4.3cm, 12.1cm or 13.4cm distance). First the best focused

7 image was found, by using a custom-made MATLAB program that evaluated the images based on their edge-sharpness. Then the focal length of the dissected lens was calculated based on the magnification of the best focused image in comparison with the size of the used object.

Histological assessment

To obtain histological images of photoreceptors, fly heads were fixed at 4 °C in a solution of 2 % glutaraldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) in Sørensen’s phosphate buffer at pH 7.4 for up to 24hrs. The tissue was then rinsed and fixed in a 1% osmium tetroxide

(OsO4) solution for 1hr on ice then 1hr at room temperature. Next, the tissue was rinsed with water and dehydrated in increments with alcohol and acetone. The tissue was embedded in Ultra-

Low Viscosity Embedding Medium (Electron Microscopy Sciences, Hatfield, PA, USA) and then sectioned at 8um and mounted on slides. Images were taken at 40x and evaluated with

Adobe Photoshop CC 2018 to determine the dimensions of photoreceptors in pixels. An image of a microscale at 40x allowed conversion of pixel to microns. Histological measurements in combination with the images from ophthalmoscope allowed us to calculate the animal’s focal length using the relationship between the object size (photoreceptors from histology), image size

(image of photoreceptors, from images made with ophthalmoscope), and image and object distance, calculated from the principal plane of the animal’s lens. Using our calibration files for the 40xUPlan FL objective (which we used to image S. bullata) then allowed us to determine a relationship between the object distance, and the ophthalmoscope image size, which then, in comparison with the histologically-determined photoreceptor size, allowed us to calculate the lenses’ focal lengths based on image magnification.

RESULTS

To investigate arthropod eye growth towards emmetropia we assessed the refractive state of visually deprived and control animals using measurements from the micro-ophthalmoscope. For all individuals measured, the refractive error (relative to emmetropia) was evaluated based on object distance—the distance for which the animal’s corresponding photoreceptor array is in focus. This is calculated based on the micro-ophthalmoscope accessory lens position for the clearest focused image of the retina in combination with a previously established calibration for the objective. Specifically, the calibration established the correlation between accessory positions and in-focus object distances. Using the independently measured focal lengths for each individual in combination with object distance, we were able to also evaluate the refractive state of all dark and control animals quantitively in terms of where an image of an object is focused within the eye relative to the retina. This can also be thought of as the distance between the surface of the retina and the focal plane of the eye’s lens.

Thermonectus marmoratus

The proximal retina of Eye 1 (E1) and Eye 2 (E2) (Fig. 1A, B) of 10 dark-reared and 10 control

T. marmoratus larval individuals were imaged in the ophthalmoscope on one side of the head for all individuals. The range of object distances (in mm) where the animal’s retina is in focus were plotted for each individual. The object distances for E1 and E2 indicate that both, E1 (Fig. 1C) and E2 (Fig. 1F) are focused close to emmetropia, with the majority of individuals exhibiting slight myopia (short-sightedness). The differences between dark and control individuals were not significant for E1 (p=0.193) or E2 (p=0.282) based on a two-sample t-test assuming unequal variance, because the variation between the two groups was significantly different. The variance

9 of ophthalmoscope measurements for the average of the range of focus was significantly lower for test animals than the controls (F-test for variance; p= 8.7E-05 for E1, p= 3.2E-05 for E2).

Focal lengths for E1 ranged from ~452.4μm to 595.2μm for control animals (514± 46.9 μm) and from ~520.4μm to 608.8μm for test animals (567± 31.3 μm) (Fig. 1D). For E2 focal lengths ranged from ~397.9μm to 581.6μm for control animals (508.8± 55.3 μm) and from ~445.6μm to

602.06μm for test animals (514.6± 45.7 μm) (Fig. 1G), with E2 having a shorter focal length than E1 on average. There was no significant difference in focal lengths between the control and test animals for E2 (p=0.802) but the differences were significant for E1 (p=0.008) based on a two-sample t-test assuming equal variance because the variance was not significantly different here. The object distances and focal lengths then were used to determine where the image was focused relative to the proximal retina for each individual. For E1 values ranged from -20.8μm to 19.4μm (15.4± 19 μm) for control animals and from -16.03μm to 12.8μm (6.3± 12.8 μm) for test animals (Fig.1E). For E2 values ranged from -17.5μm to 40.9μm (14.9± 19 μm) for control animals and -15.6μm to 25.2μm (5.7± 13.7 μm) for test animals (Fig.1H). The measurements between dark and control animals here were also not significantly different for E1 (p=0.182) or

E2 (p=0.233) with the variance being insignificantly different as well. Although there was no significant difference, it is noteworthy that control larvae, on average, focused slightly more myopic than the dark-reared larvae (Fig. 1C, F, E, H). For example, the average object distance for E2 in dark-reared larvae was 0.901mm while for controls the average was 10.79mm (with more positive values focusing more towards myopia).

Phidippus audax

The anterior-median eyes of jumping spiders are known to be organized into multiple layers

(Land, 1969) with the lateral portion of the boomerang coming into focus before the medial

10 portion as the accessory lens is adjusted (Fig. 2A). The micro-ophthalmoscope allowed us to image the peripheral retina in regard to layer 1 (which exhibits a staircase organization) and layer

2 (Stowasser et al., 2017), whereas the central part of the retina is imaged as a generally bright area (Fig. 2B), possibly because of its high resolution or the presence of layers 3 and 4 that were not discernable in the ophthalmoscope. A single anterior-median eye was imaged for each individual for N=16 dark-reared spiderlings and N=9 control spiderlings. The object distance was plotted showing the range of object distance for where the animal’s retina was in focus (Fig. 2C,

F). Layer 2 values range from -10.26mm to -1.9mm for control animals and -9.9mm to 13.5mm for test animals (Fig. 2C), while layer 1 values range from -30.3mm to 146.7mm for control animals and -20.6 to 153.5mm for test animals. The individual object distances for dark-reared and control individuals show no significant differences for layer 2 (p=0.681) or layer 1 (p=0.647) based on a two-sample t-test assuming equal variance. The average object distances for the dark- reared groups are very slightly less hyperope than the controls for both, layers 2 and 1. For example, the average object distance for layer 1 dark-reared spiderlings is 11.5± 43mm and -

6.2E+13 ± 1.8E+14mm for controls. Focal lengths from the AM eye measured for each induvial ranged from ~395-460.6μm (418.6± 22 μm) for control animals and from 377.7-463.7μm

(423.5± 19.7 μm) for test animals (Fig. 2D). There was no significant difference between the dark and control spiderlings measured focal lengths (p=0.569). Each individual was also evaluated for where the image was focused relative to the retina based on the individual’s focal length; for layer 2 values ranged from -67.3μm to -16.6μm (-38.1± 15.8 μm) for control animals and from -67.2μm to 15.2μm for test animals (36.1± 19.1 μm) (Fig. 2E). For layer 1 values ranged from -34μm to 24.9μm (3.5± 17.9 μm) for control animals and from -48.54μm to 50.5μm

(-0.76± 21.9 μm) for test animals (Fig.2H). The differences between the test and control animals

11 were not significant for layer 2 (p=0.797) or for layer 1 (p=0.638). The graphs of how the image is focused on the retina is only an estimate, because it does not account for the effects of a negative lens that is formed by the surface between the proximal end of the lens tube and the retina layers. This lens, that in adult spiders is an important component of their telescopic function (Land, 1969), could potentially shift the focus slightly deeper into the photoreceptor array. Taken together, our measurements of jumping spider AM eyes show no significant difference between control animals and dark-reared animals. As has been previously noted for adult jumping spiders (Stowasser et al., 2017) layer 1 is focused very close to emmetropia.

Layer 2 on the other hand is hyperope, with the image focused approximately 37μm below its top surface.

Sarcophaga bullata

To test if light exposure influences the development of insect compound eyes, we measured individual ommatidia of S. bullata flies which were forced to emerge in the dark (N=10) or were kept under normal rearing conditions (control daytime; N=10). In addition, to account for possible circadian related rhabdom shifts (Walcott, 1971; Warrant and McIntyre, 1993; Narendra et. al., 2016) an additional control group was measured during their nighttime (“nighttime flies;

N=10). For each individual we imaged a single ommatidium (Fig. 3B) on one side of the fly’s head, which was ~9 lenses out from the frontal center border of the eye, by closing an aperture around an induvial lens of the fly’s compound eye (Fig. 3A; right). The fly’s lens array exhibits one clearly bleached lens after measurements are taken (Fig. 3A; left). S. bullata object distances were also plotted as the average object distance for the range of clearest images of the retina for each individual (Fig. 3D). Based on a one-way ANOVA there is a significant difference

(p=0.033) for object distance. Calculations used the reciprocal of the object distance to better

12 match the optics. Based on a post-hoc Tukey Kramer test there was a significant difference between two of the groups: the control daytime and control nighttime (q= 4.178 with critical value of 3.53). There was not a significant difference between the control daytime and dark- reared flies (q=1.98 with a critical value of 3.53) or the control nighttime and dark-reared flies

(q= 2.19 with critical value 3.53). Because the optical assessment of focal length, based on dissected lenses is relatively difficult and at risk for error, we instead determined the focal length through histology; an example of a frontal cross section through an S. bullata compound eye displaying individual stained photoreceptors used to determine focal length can be seen in figure

3C. Focal length ranged between 52μm and 64.4μm for the dark-reared individuals (59.7±4.6

μm), 57.2μm to 70.3μm (64.8±5 μm) for the daytime-control individuals and 53.3μm to 70μm

(62.6±6.6 μm) for the nighttime-controls (Fig. 3E). Based on a one-way ANOVA there is not a significant difference (p=0.2937) for focal length. As histological assessment was not successful for every individual, our calculations of where an image is focused relative to the retina had an

N=7 for dark-reared flies, N=6 for daytime-control flies and N=9 for nighttime control flies (Fig.

3F). These values ranged from -2.7μm to 1.1μm for the dark-reared individuals, -3.5μm to

0.27μm for the daytime-control individuals and -2.3μm to 2.4μm for the nighttime-control flies.

Here too, based on a one-way ANOVA, there is a significant difference (p=0.0418). Based on a post-hoc Tukey Kramer test there was a significant difference between two of the groups: the control daytime and control nighttime flies (q= 3.87 with critical value of 3.59). There was not a significant difference between the control daytime and dark-reared flies (q=2.41 with a critical value of 3.59) or the control nighttime and dark-reared flies (q= 1.38 with critical value 3.59).

The daytime-control flies were focused slightly deeper into the retina (-2.07±1.38 μm on average; towards hyperopia) than the dark-reared flies (-0.66±1.5 μm average) or the nighttime-

13 control flies (0.07±1.5 μm), which were essentially exactly emmetrope. Taken together, as was the case for the beetle larvae and the spider eyes, we observed no significant differences between the dark reared flies and the control groups.

DISCUSSION

To the best of our knowledge, this study is the first to directly address if arthropods need visual input for the initial coordination of eye growth and lens refractive development that is necessary to establish correctly focused eyes. To begin investigating the mechanism for arthropod emmetropization we assessed the refractive state of dark-reared and control individuals of three phylogenetically distant arthropods that are characterized by optically diverse eye types.

Unexpectedly, the arthropods used for this study developed relatively well-focused eyes with comparable levels of focus regardless of if they were deprived of visual feedback or not. These results suggest that visual input likely is not required at least for the initial refractive development of these arthropods.

Our investigation of T. marmoratus larva showed that for both E1 and E2 on average the range of clearest focused images (the second focus of the bifocal image, Stowasser et al., 2010) focused close to emmetropia (with a touch of myopia) in relation to the proximal retina. This finding in regard to E1, and our focal length measurements, correspond well with a previous study (Stowasser and Buschbeck, 2014) that used a less direct method. However, the previous study had suggested that E2 might be more myopic than what we found. It is noteworthy that the

Stowasser and Buschbeck (Stowasser and Buschbeck, 2014) study was performed on slightly older individuals that were allowed to hunt for several days, so it is conceivable and subject to further investigation that E2 becomes more near-sighted later in development. Regardless, our

14 data shows that the focus of both principal larval eyes is comparable between individuals that molted in the dark and control animals. Interestingly, although not statistically significant, dark reared larva showed slightly less variation than the controls and were on average focused slightly more towards emmetropia. A potential hypothesis for this could be that while a passive regulatory mechanism is the more dominant process at play here, that visual feedback in the light could have started to initiate a change in a few individuals. The dark larvae molting without any visual feedback, on the other hand, would be relying fully on their developmental program, potentially resulting in less variation. Also, in addition to generally having a lot of variation, the

T. marmoratus data also appear bimodal. A possible explanation for this involves the use an active regulatory mechanism. While insect lenses are usually considered to have a fixed focal length, some of our circumstantial observations have led us to wonder if there could be any way that the larvae are able to shift their focus slightly during the process of taking ophthalmoscope measurements. Such a mechanism could assist in distance vision, which these larvae already have been established to be able to perform well without requiring input from both sides of the head (Bland et al., 2014). Needless to say, this hypothesis would have to be explored further.

The AM eyes of the P. audax jumping spider’s also have a retina that is organized in layers. For spiders too our experiments found no significant difference between the dark-reared and control individuals in regard to their refractive error. Specifically, our data shows a clear difference in focus between layer 2 and layer 1, with layer 1 being focused more toward emmetropia than layer 2. These findings are consistent with what previously has been described for Metaphiddipus aeneolu (Land, 1969) and what has been found, following the same methods, for late juvenile P. audax (Stowasser et al., 2017). The latter is interesting in that it suggests that these young spiderlings already are focused in the same way as older spiders, and hence that the

15 eyes of the very young spiderlings are already similar, in regard to their refractive states.

Although the focus in regard to the layering seems to stay constant, it is noteworthy that the range of focus is larger, as expected for these spiderlings that necessarily have a much shorter focal distance. Our data in regard to how an image would be focused on the retina based on the focal length of the main lens also puts emmetropia to the top of layer 1, just like the object distance data itself. These findings suggest that, in these young spiderlings, the image shift from the negative lens that is formed by the retina’s surface must be relatively minor. Among other optical features, it recently has also been noted that the AL eyes of P. audax spiderlings already have a full complement of photoreceptors (Goté et al., 2019).

Mike Land (1969) described that layer 1, the deepest layer, focuses images which are very close for blue-green light, while distant objects are best focused onto layer 2, the next deepest layer and that the focal plane of the retina falls at the highest point of layer 1’s staircase, which our data reflects (Fig. 2F). Preliminary experiments of focal length measurements for the jumping spiders under red and green light demonstrated that there was no difference regardless of which wavelength was used. It is noteworthy that when imaging these layers with the ophthalmoscope we excite their receptors with TxRed (green excitation, red emission), with red light required to exploit the animals autofluorescence.

The larger variation seen in the beetle larva is also notable with the jumping spiders when compared to the fly data series. This likely is due to the beetle larvae and jumping spiders both having camera type eyes with relatively large lenses that have much longer focal distances than fly lenses. Another difference is that they undergo molting during their life span, which requires frequent readjustments. The compound eyes of S. bullata flies, on the other hand, are characterized by many much smaller lenses that have shorter focal length with a much smaller

16 range of focus and less variation. As was the case for the two other arthropods, no significant difference in focus was observed between dark-reared and both, the daytime and nighttime control animals of S. bullata.

Interestingly, the control daytime flies were significantly more hyperopic than the nighttime-control flies. Circadian and light induced differences in rhabdom position also have been observed in other insects (Narendra et al., 2016; Walcott, 1971; Warrant and McIntyre,

1993), although the results we found are opposite to what was observed in previous literature.

The high level of precision that was already present in the fly’s initial development further demonstrates the value of these flies as a model system for many developmental processes.

Taken together, measurements from all three arthropods show that they have good optics right from the start of their lifespan, or post-molt. These findings raise the possibility that in arthropods the coordination between lens refraction and retina position could be relatively stronger genetically pre-determined when compared to vertebrates and cephalopods (Schaeffel et al., 1988; Troilo and Wallman, 1991). While arthropods and cephalopods are both invertebrates, and species from both groups have camera type eyes, the study in squid demonstrates that there is an active mechanism dominating the regulation of their eyes—while our findings suggest that the primary mechanism for arthropods is passive regulation. This difference highlights a key evolutionary separation between the two groups of invertebrates. Small invertebrates might rely more on their genetic developmental pathways than vertebrates due to the selection pressures many face the start of their lifespan. For example, P. audax spiderlings must be able to capture food when they emerge from their egg case. There is more pressure for many arthropods to have correct vision to start with, especially taking into consideration that many have a far shorter lifespan than most vertebrates giving them less time to fine-tune their visual systems over

17 developmental time. It is important to note however that this study exclusively focused on the initial eye development, and it is conceivable that their optics could be further improved over time with visual input potentially fine tuning the eye, so this remains an open question that should be subject of further investigations.

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FIGURES

Fig. 1. Thermonectus marmoratus optical analysis. (A) External image of T. marmoratus larva in water. Note the principal front facing eyes E1 and E2. (B) Ophthalmoscope image of proximal retina showing two rows of rhabdoms. (C-E) Analysis of E1. (C) Ophthalmoscope measurements showing the object distances (in mm) for the range (represented by bars) over which the E1 photoreceptors are optimally focused for each individual (Dark N=10; Control N=10) as well as their average. (D) Measured focal lengths for dark and control individuals from isolated lenses. (E) Distance between the surface of the retina and the focal plane of the lens of E1 (in μm) based on the measured micro-ophthalmoscope measurements in combination with focal length measurements of each individual. (F-H) The same optical analysis as in C-E but on E2. (F) Object distance for E2 (N=10; Control N=10). (G) Focal length for E2. (H) The distance between the surface of the retina and the focal plane of the lens for E2 based on the focal lengths of the individual’s lenses.

Fig. 2. Phidippus audax optical analysis. (A) Schematic of the tiered jumping spider retina in principal AM eyes (modified from Stowasser et al., 2017). (B) Ophthalmoscope image of the boomerang shaped AM retina, layer 1. (C) Micro-ophthalmoscope measurements showing the object distances (in mm) including the range for which the photoreceptors of layer 2 are focused for each individual (Dark N=16; Control N=9). The average is indicated as a line. (D) Measurements of focal lengths for AM eye lenses. E) Calculation of the distance between the surface of the retina and the focal plane (and the range of focused images) of the lens for layer 2 based on the micro-ophthalmoscope and focal length measurements for each individual. (F) Object distance for layer 1 as established with the micro-ophthalmoscope, (G) Image of Phidippus audax spiderling, illustrating the prominent principal anterior median eyes. (H) The distance between the surface of the retina and the focal plane of the lens for layer 1 based on individual micro-ophthalmoscope and focal lengths measurements.

Fig. 3. Sarcophaga bullata optical analysis. (A) Ophthalmoscope post-measurement image of the surface of a S. bullata compound eye lens array illustrating bleaching of a single ommatidium (left) which was illuminated by a small square light beam (right). (B) Ophthalmoscope image showing photoreceptors of a single ommatidium. (C) Histological section of S. bullata photoreceptors; scale bar is 50 μm. (D) Ophthalmoscope measurements showing the object distances (in mm) ) for the range (represented by bars) for which the photoreceptors are focused for each individual (Dark N=10; Control daytime N=10; Control nighttime N=10). (E) Measured focal lengths for each individual calculated from the image magnification between the histological and optical images. (Dark N=7; Control daytime N=6; Control nighttime N=9). (F) The calculated distance between the surface of the retina and the focal plane of the lens, based on the micro-ophthalmoscope and lens focal measurements of each individual.