Developmental Evolution of the Visual System in the Cave- Adapted Small Carrion Beetle Ptomaphagus Hirtus

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1-1-2015 Developmental Evolution Of The iV sual System In The aC ve-Adapted Small Carrion Beetle Ptomaphagus Hirtus Jasmina Kulacic Wayne State University,

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DEVELOPMENTAL EVOLUTION OF THE VISUAL SYSTEM IN THE CAVE- ADAPTED SMALL CARRION BEETLE PTOMAPHAGUS HIRTUS

JASMINA KULA ČIĆ

THESIS

Submitted to the Graduate School

of Wayne State University,

Detroit, Michigan

in partial fulfillment of the requirements

for the degree of

MASTER OF SCIENCE

2015

MAJOR: BIOLOGICAL SCIENCES

Approved By:

______Advisor Date

JASMINA KULA ČIĆ

2015

DEDICATION

I dedicate my thesis and all my work to my husband Mladen Čepi ć, my sister

Dijana Kula čić, and my best friend Miloš Aleksi ć. None of this would be possible without

their unconditional love and support.

ACKNOWLEDGEMENTS

I would like to express my special gratitude to my advisor Dr. Markus Friedrich

for his support and guidance. I am very thankful for his patience and for directing me

during my graduate training. This thesis would not have been completed without his

advice, creativity, and profound knowledge of science. I am also grateful for his constant

optimism and humor, and that he made the lab environment very enjoyable to work in.

I would also like to thank my committee members, Dr. Aleksandar Popadi ć and

Dr. Joy Alcedo. I highly appreciate their support, creative comments, and valuable suggestions throughout the progress of this work.

I would like to thank my current and previous lab colleagues Zahabiya Husain,

Anura Shrivastava, Qing Luan, Dr. Riyue Bao (Sunny), Dr. Jason Caravas, Dr. Arun

Krishna-Appukuttan, Qing Chen and Xin Wang. They made our work environment very joyful and filled with laughter, and I will always remember our fun coffee breaks. I would like to specially thank Zahabiya Husain and Qing Luan for training me during my rotation, and for their generous help when I needed it.

I would like to thank Dr. Nansi Colley, Dr. Brian Gebelein, and Dr. Tiffany Cook for antibodies; Dr. Elke Buschbek for providing TEM images; Kurt Helf and Rick Toomey for technical support at Mammoth Cave National Park; Ajharulislam Reja Khan, Rebhi

Elder and Yumna Fatima for helping on cell counting. I must thank Mammoth Cave

National Park for the collecting permit, enabling me to perform field collection work.

iii

I also thank the staff members of the Biological Sciences department for all their assistance and help on daily basis.

I also want to thank Dr. Aleksandar Popadi ć, his wife Nela Popadi ć, and their

family for helping me settle down when I first came to the USA.

I want to express many thanks to all the friends I made during my studies in the

States. I thank all of those who stood by me, helped me, and laughed with me

throughout my graduate training. I specially want to thank my friend Dr. Kiran Koya for

helping me with the statistical analysis.

I also want to thank my family and friends from Serbia on their support.

At last, I owe my deepest gratitude that literally goes beyond words to my

husband Mladen Čepi ć, my sister Dijana Kula čić, and my best friend Miloš Aleksi ć. I

want to thank them for their love, understanding, encouragement, and endless time

spent talking to me on Skype. They unwaveringly believed in me and none of this would

be possible without their support. They are the reason I was able to go through this

graduate school journey with a constant smile on my face.

Many thanks to all of you!

TABLE OF CONTENT

DEDICATION ...... ii

ACKNOWLEDGEMENTS ...... iii

LIST OF TABLES ...... vi

LIST OF FIGURES ...... vii

CHAPTER 1: INTRODUCTION ...... 1

CHAPTER 2: MATERIAL AND METHODS ...... 5

CHAPTER 3: RESULTS ...... 8

CHAPTER 4: DISCUSSION ...... 21

REFERENCES ...... 32

ABSTRACT ...... 39

AUTOBIOGRAPHICAL STATEMENT ...... 41

LIST OF TABLES

Table 1. Life cycle of P.hirtus …………………………………………………………………..4

LIST OF FIGURES

Fig. 1. Organization of the adult P. hirtus eyelet …………………………………..…………….. 9

Fig. 2. Cellular patterning in the P. hirtus eyelets during pupal development ……………15

Fig. 3. Time course of lens deposition in the developing P. hirtus eyelets …………..……19

Fig. 4. Comparative time course of P. hirtus lens development ……………………………. 25

Supplemental Fig. 1. Optic nerve formation in P. hirtus pupae and adult ……..…………. 11

Supplemental Fig. 2. Expression of Arr2 in adult eyelet of P. hirtus …………………...….. 12

vii 1

CHAPTER 1: INTRODUCTION

The evolution of adaptive traits makes cave animals an important model system to examine developmental and evolutionary questions. Since cave species evolve from surface dwelling ancestors, the directionality of troglomorphic adaptations and evolution, such as eye reduction or loss, can be followed and understood (Protas and Jeffery,

2012). This has been demonstrated in the Mexican tetra Astyanax mexicanus (reviewed in Protas and Jeffery, 2012), the Somalian cavefish Phreatichthys andruzzii (Stemmer

et al, 2015), the Chinese cavefish Sinocyclocheilus anophthamlus (Meng et al, 2013a;

Meng et al, 2013b), as well as in amblyopsid cavefishes (Niemiller et al, 2013).

However, not much is known about the developmental genetics causing eye regression

or loss in cave species of arthropods, especially in cave insects. Two crustacean

species have been examined for better understanding of eye regression: the nearctic

cave amphipod Gammarus minus (Aspiras et al, 2012; Carlini et al, 2013) and the

palearctic isopod Asellus aquaticus (Protas et al, 2011; reviewed in Protas and Jeffery,

2012). One eyeless Australian species of Coleoptera, from the family Dytiscidae, was examined for the regressive evolution of the eye pigment genes, and the authors showed loss of pigment gene function due to frameshift mutations (Leys et al, 2005).

Tierney et al. (2015) explored the molecular basis of opsin gene regression in 3 different

Australian subterranean dytiscid beetle species and concluded that the general lack of opsin expression is an outcome of neutral evolution. Other than the above studies, the transition from surface to cave existence and the change in eye morphology in insects is

2 still a largely unresolved topic in evolutionary biology.

The cave beetle Ptomaphagus hirtus represents an emerging model for studying cave adaptation in insects at the molecular and structural level. By deep sequencing the adult head transcriptome, Friedrich et al. (2011) discovered the transcripts of all core members of the phototransduction protein machinery. Behavioral analysis showed that

P. hirtus also has a negative phototaxis response in light-dark choice tests (Friedrich et al, 2011). This suggested the functionality of the presumed rudimental lateral eyes in P. hirtus , which had been previously proposed to represent non-functional relict structures

(Packard, 1888; described in Peck, 1973). Here I present the results from investigating

the molecular basis of eye organization and development in P. hirtus .

The small carrion beetle genus Ptomaphagus is one of 14 genera in the family

Leiodidae of the coleopteran infraorder Staphyliniformia (Newton and Thayer, 2003).

The genus Ptomaphagus includes both surface-living (epigean) and obligate cave dwelling species (troglobionts). Based on the morphology of the female spermatheca,

Peck (1973) divided the nearctic representatives of the genus Ptomaphagus into three groups and named them after the oldest species in the group: the hirtus -group, the

cavernicola -group, and the consobrinus -group. The cavernicola - and consobrinus -

groups are mainly epigean, with selected species that adapted to the cave environment

(Barr, 1962; Peck, 1973; Peck, 1984). The hirtus -group is considered to be the oldest

group of the genus, and has limited geographical distribution to Nearctic regions (Peck,

1973). The hirtus -group comprises 18 troglobite representatives, which have eyes

highly reduced in size. In addition, there is one edaphophile species ( Ptomaphagus

shapardi ), which experienced eye reduction but retained regular compound ommatidial

3 organization (Peck, 1973).

Ptomaphagus hirtus is the oldest member of the group, found in Mammoth Cave in Kentucky (Barr, 1962; Peck, 1973; Peck, 1975). As a true troglobite, P. hirtus has modified morphological traits, such as the significant reduction in the size of the eyes with a lens rudiment found on the lateral portion of the head (Packard, 1888). It was believed that P. hirtus was blind and that the rudiment eye was not functional because

Packard (1888) found neither an optic nerve nor an optic ganglion (Peck, 1973). Recent molecular studies, however, produced compelling evidence of the functionality of the rudimental eye in P. hirtus (Friedrich et al, 2011).

The life cycle of the laboratory cultured P. hirtus was first described by Stewart

Peck in 1975. A span of 3 to 3.5 months is spent in the immature stages, which comprises an egg stage, 3 larval instars and the pupal stage (Table 1). After hatching from the pupal stage, adults live up to several years in the lab environment. The length of the egg and active larval stages together averages 45.7 days. After its active phase of life, the third instar larva makes a cell in the soil and spends on average 15.3 days there preparing for the pupal stage. After hatching, the pupa stays in the cell for on average 35.3 days before hatching into an adult. After eclosion, the young adult animal remains in the pupal cell for an additional average of 8.3 days before it leaves the cell and starts feeding. Then, approximately a month later, the adult animal starts mating.

My studies reveal that the P. hirtus eyelet is organized like a camera-eye. A

single compact lens covers a population of on average 130 photoreceptor cells, which

are defined by variably shaped and distributed rhabdomeres, and on average 60

accessory cells of yet uncharacterized function. The photoreceptors project axons

Table 1. Life cycle of P.hirtus . Figure adapted from Peck, 1975.

5 towards the median brain, which are organized as an optic nerve that exits the keyhole- like opening of the base of the cuticle chamber that harbors the eyelet. I further discovered that all basic components of this minimal visual organ are assembled by the beginning of pupation, except for the compact cuticle lens. The latter is formed during the first 11 weeks of the adult life span. To the best of my knowledge, P. hirtus represents the first example of adult de novo lens deposition. Here I discuss the adaptive and behavioral significance of this novel trait in this cave species.

CHAPTER 2: MATERIAL AND METHODS

2.1. Animal collection and culture

P. hirtus adults and larvae were collected in March 2013 from the White Cave entrance of Mammoth Cave (Scientific Research and Collecting Permit #MACA-2011-

SCI-0001). Beetles were cultured in the lab following previously published protocols

(Peck, 1975 and Friedrich et al, 2011), with minor additions and changes listed below.

Animals were cultured on native cave soil in larger polystyrene Petri dishes, which measure 150 mm in diameter. Animals were kept in a light-insulated temperature- controlled coldbox situated in a completely light-insulated laboratory room. During inspection and handling of cultures and individual animals, beetles were examined under red light. Animal cultures were inspected and fed with few flakes of dry baker’s yeast (Hodgson Mill, Inc) in a 2-week period.

2.2. Staging of adult animals

Newly hatched adults differ conspicuously from mature animals by the lighter color of their cuticle, and their hatching was confirmed with the presence of empty pupal cells on the plate. Young adults were transferred to new plates, and they were dissected when they reached the desired week-age of young adulthood. Newly hatched adults were taken out of the pupal cell and dissected, generating 0-1 week old samples.

Animals were also collected immediately after exiting the cell, producing adults at approximately 1.5 week-age. The rest of the adults were dissected in the increment time of two weeks, generating 1.5-3 weeks old, 3-5, 5-7, 7-9, and 9-11 weeks old samples.

Mature adults were at least 1 year old when dissected.

2.3. Immunohistochemistry

Animals were anesthetized by CO 2 gas and decapitated in ice-cold PBS under red light. Immediately after dissection, beetle heads and bodies were directly transferred to 100% ice-cold methanol for fixation. Fixed animals were stored at -20 ̊ C.

For immunohistochemistry, the following primary antibodies were used: mouse anti-α-tubulin (1:100 in PBS-Tween20 (PBT), Sigma Aldrich T6793) and rabbit anti- arrestin2 (1:100 in PBT, provided by Dr. Nansi Colley). The following secondary antibodies were used: FITC-conjugated goat anti-mouse (1:100 in PBT, Jackson lab

115-096-003), Cy3-conjugated goat anti-rabbit (1:500 in PBT, provided by Dr. Tiffany

Cook), and AlexaFlour647-labeled donkey anti-rabbit (1:500 in PBT, provided by Dr.

Tiffany Cook). Nuclei were labeled with Hoechst 33258 (2 ug/ml in PBT, Molecular

Probes H3569) or with propidium iodide (PI) (5 ug/ml in PBT, Molecular Probes P3566).

Prior to incubation with biolabels, heads were dissected in half, and washed in 1x

PBT. If PI was used for nuclei labeling, the tissues were treated with 400 ug/ml RNase for 1 hr, after which it was washed with 1xPBT. Tissues were blocked in 6 mg/ml

NGS/PBT at room temperature for 1 hr, followed by two overnight incubations at 4 ̊ C with the primary antibody combination. After this step, tissues were washed thoroughly and incubated with the appropriate secondary antibody combination overnight at room temperature. After staining, the half heads were individually mounted on slides in 70% glycerol/PBS supplemented with 2.5% DABCO.

2.4. Light microscopy

Images of cave beetle adults were taken using a Zeiss Axioskop Light

Microscope and processed using Spot Advanced software.

2.5. Confocal microscopy and image analysis

Samples were imaged using a Leica SP8 Confocal Microscope. Maximum projections and 3D images were generated using the Leica SP8 Confocal Software package. Sagittal section images were generated with 3D-image processing and adjustment. Images were additionally processed in ImageJ (Version 1.47v) (Schneider et al, 2012) for adjustment of contrast and brightness and for reduction of noise.

Measurements were conducted in ImageJ as well.

2.6. Statistical analysis

One-way ANOVA and Tukey Pairwise Comparisons in the statistical analysis of

8 lens deposition were performed in Minitab 17.

2.7. Histological Analysis and Transmission Electron Microscopy

P. hirtus eyelet samples were prepared for histological analysis and transmission electron microscopy as previously described in Mendapaka et al. (2006) and Stecher et al. (2010). Images were generated and processed as described in Stecher et al. (2010).

2.8. Image analysis and cell count

Confocal z-stacks images were imported into Microsoft Powerpoint, scaled to the same size and labeled with the correct order of z-stack numbers. Nuclei of individual cells were labeled with numbers throughout the z-stack, and total number of nuclei

(cells) was recorded. Photoreceptor cells were identified as nuclei that correlated with the cytosolic and/or patch Arrestin2 (Arr2) signal. The number of Arr2-positive photoreceptor cells was subtracted from the total number of cells, and the rest of the cells were identified as non-photoreceptor cells.

CHAPTER 3: RESULTS

3.1. Structural organization of the adult P. hirtus eyelets

Previous transcriptome and behavioral analyses (Friedrich et al, 2011) suggested the functionality of the rudimental eye in P. hirtus , but the cellular organization of the

presumed P. hirtus eyelet has not been investigated. I used immunohistochemical, laser

scanning microscopy, and ultrastructural imaging approaches to analyze the

Fig. 1. Organization of the adult P. hirtus eyelet. (A-C) Lateral view of P. hirtus head. (A’) Higher magnification of external morphology of the eyelet surface. (B) Lateral view of the eyelet showing the unfaceted eyelet surface. (C) Lateral view of the eyelet, confocal max surface projection. (D) P. hirtus camera-type eyelet, displaying a cuticle cup shape. Bracket points to the depth of the lens. Arrow points to the lack of pseudocone. (E) TEM sagittal section, low magnification. All asterisks point to randomly distributed rhabdomeres. Yellow asterisks point to two rhabdomeres that are joined together. (F) TEM sagittal section, rhabdomere in high magnification. Arrow and circle point to differently oriented microvilli. (G, H) Whole-mount immunohistochemistry for α- tubulin (green), nuclei (red), and Arr2 (magenta). (G) Horizontal section overview and (H) Sagittal section overview, showing the cellular organization. Bracket points to the depth of the lens. (I) Bar graph representing the number of Arr2-positive versus Arr2- negative cells. Scale bar in A: 100 um. Scale bars in B, C, D, E, G and H: 10 um. Scale bar in F: 2 um.

Supplemental Fig. 1. Optic nerve formation in P. hirtus pupae and adult. (A-A”) 0-2 weeks old (young) pupal eyelet. (B-B”) 3-5 weeks old (old) pupal eyelet. (C-C”) Mature adult eyelet. Whole mount immunohistochemistry for nuclei (A, B, C), α-tubulin (A’, B’, C’), and merged (A”, B”, C”). (A’-A”, B’-B”, C’-C”) Arrows point to the formation of the presumed optic nerve. Scale bar for all A, B, and C panels: 10 um.

Supplemental Fig. 2. Expression of Arr2 in adult eyelet of P. hirtus . (A-C) Whole- mount immunohistochemistry for nuclei (A), Arr2 (B), and merge (C). (B, C) Arrows point to cytosolic Arr2 signal. Asterisks point to rhabdomeric Arr2 signal. Scale bar in A, B, and C: 10 um.

13 morphology and organization of P. hirtus eyelets. Consistent with previous reports

(Peck, 1973), the lateral view of the head revealed that the relative size of P. hirtus eyelet is reduced to a very small region on the lateral-posterior side of the head (Fig.

1A). In addition, I noted the presence of several bristles at the posterior edge of the eyelet (Fig. 1B and C). The eyelet surface lacked detectable evidence of facet organization, displaying instead a continuous lens surface (Fig. 1B and C). The lack of external facet organization was confirmed by confocal imaging of surface autofluorescence (Fig. 1C). A P.hirtus eye measured 54.9 um and 71.4 um across the anterior-posterior and dorso-ventral surface, respectively.

Histological sections along the dorso-ventral axis of the eyelet confirmed presence of an approximately 10 to 12 um thick clear cuticle lens covering a defined cuticle cup that enclosed a population of unpigmented cells (Fig. 1D). This level of resolution showed the lack of pseudocones and ommatidial organisation (Fig. 1D). The bottom of the cuticle cup is medially open, forming a wide keyhole, approximately 30 um in diameter, where candidate axons project towards the median head, forming a prospective optic nerve (Sup. Fig. 1C’ and C’’). The depth of the cuticle cup, from the proximal lens surface to the keyhole opening, measured approximately 23.8 um in length.

Transmission electron microscopy sections (TEM) further confirmed the presence of a densely packed cell population in the cuticle cup and the lack of pseudocone and ommatidial subgroups (Fig. 1E). This approach also revealed irregularly shaped rhabdomere-like stacks of microvilli, which were randomly distributed within the eyelet cell population (Fig. 1E and F). Within the same rhadomere-like

14 structure, I observed that the microvilli have different directionality (Fig. 1F, arrow and circle). In some areas of the eyelet, I also observed that several rhabdomeres were joined together into a presumed rhabdom-like structure (Fig. 1E). This finding presented the first cell morphological evidence of photoreceptor cells in the P. hirtus eyelet.

To further test the presence of photoreceptor cells and to explore their organization in the eyelet, I labeled whole mount preparations of fixed eyelets with anti-

α-tubulin antibody. This approach corroborated the presence of neuronal cells that send axon-like projections through the cuticle cup proximal opening, forming a potential optic nerve (Sup. Fig. 1C”). Immunohistochemical labeling with a cross-reactive antibody against Drosophila Arrestin2 was likewise consistent with the presence of photoreceptor

cells, as defined by the low level of cytosolic Arrestin2 signal (Fig. 1G and H; Sup. Fig.

2B and C). Anti-Arrestin2 labeling was also observed as highly disorganized puncta

(Sup. Fig. 2B and C), consistent with the random distribution of the irregularly shaped

rhabdomeres visualized by TEM imaging.

Quantitative analysis revealed that the adult P. hirtus eyelet is populated by

approximately 190 cells, approximately 130 of which are photoreceptors based on

positive Arr2 labeling, whereas the absence of Arr2 label suggested the presence of

other cell fates (Fig. 1I).

Taken together, these findings reveal that P. hirtus possesses miniature camera-

like eyes, which are populated by a large number of both photoreceptor and non-

photoreceptor cells.

Fig. 2. Cellular patterning in the P. hirtus eyelets during pupal development. (A-A”’ and B-B”’) Young pupal eyelet (0-2 weeks), (A-A”’) horizontal section and (B-B”’) sagittal section representing the cellular organization. (C-C”’, D-D”’) Old pupal eyelet (3-5 weeks), (C-C”’) horizontal section and (D-D”’) sagittal section representing the cellular organization. Whole mount immunohistochemistry for nuclei (A, B, C,D), α-tubulin (A’, B’, C’, D’), Arr2 (A”, B”, C”, D”), and merge (A”’, B”’, C”’, D”’). (B”’, D”’) Arrows point to thin cuticle. Scale bar for all A, B, C, and D panels: 10 um.

3.2. Pupal development of the P. hirtus eyelets

Previous work has shown that even very strongly reduced visual systems, such as the larval eyes of holometabolous insects, can recapitulate ancestral processes of compound eye formation during their development (reviewed in Friedrich et al, 2013). I therefore probed the possibility of transient ommatidial organization of the P. hirtus eyelets during pupal development, using the same panel of cellular markers that I applied for studying the cellular organization of the adult eyelet.

In 0 to 2 weeks old pupae (defined as young pupa) as well 3 to 5 weeks old pupae (defined as old pupa), I observed the presence of randomly distributed, putative neuronal cells based on anti-α-tubulin labeling. These cells create a net-like structure

throughout the eyelet (Fig. 2A’ and C’), which terminates into a putative axon-like

projection at the base of the eyelet (Sup. Fig. 1A and B). I also detected a strong

cytosolic anti-Arrestin2 signal (Fig. 2A’’ and C’’), suggesting the presence of differentiating photoreceptor cells in the developing pupal eyelet. I observed that α-

tubulin and Arrestin2 were coexpressed, with tubulin enveloping Arrestin2 at the cell

edges (Fig. 2A”’ and C”’), which additionally confirmed the neuronal photoreceptor fates

of these cells. In young pupa, the eyelet of a single sample measured 52.5 um and 71.4

um across the anterior-posterior and dorso-ventral surface, respectively. Measurements

across the same axes in a single sample of old pupa were different: 56.5 um and 58.9

um, respectively. This discrepancy is likely due to sample damage and squeezing

during the process of tissue mounting and imaging (the experiment could not be

repeated due to low number of culturing animals). The depth of the cuticle cup in young

pupa measured approximately 21.17 um in length, and 19.74 um in old pupa. Taken

17 together, these findings indicated that most of the basic patterning steps leading to the formation of the eyelet, like cell fate specification, neural projection, and cuticle cup formation took place prior to pupation, in contrast to the temporal sequence of these processes during compound eye development in surface species (Cagan and Ready,

1989, reviewed in Charlton-Perkins and Cook, 2010 and Friedrich et al, 2013).

Moreover, the pupal stages of P. hirtus eyelet development do not appear to recapitulate aspects of compound eye development, such as ommatidial specification.

Unexpectedly, sagittal sections of both early and late pupal eyelet preparations revealed no detectable evidence of lens formation. The depth of the apical cuticle layer was similar in young and late pupae (Fig. 2B”’ and D”’) and equivalent to that of the surrounding head cuticle (Fig. 2D”’). Since this finding suggested that the P. hirtus lens was not formed during the pupal stage, as is the case in the developing compound eye of surface species (reviewed in Charlton-Perkins et al, 2011), the lens of the P. hirtus cave species is likely formed during adulthood.

3.3. Temporal dynamics of camera eye lens deposition

To explore the time window of lens deposition in adult P. hirtus , I sampled adults

in 2-week increments and performed measurements of lens thickness using the

autofluorescence signal of lens cuticle. Lens thickness was measured in the anterior-

posterior sagittal section of the eyelet at three different positions: medial (middle spot on

the lens between anterior and posterior edge of cuticular cup), anterior (spot between

medial measurement and the anterior edge), and posterior (spot between medial

measurement and the posterior edge). This approach revealed that the apical cuticle of

18 the P. hirtus eyelet progressively increases in depth during the first 11 weeks of adult development (Fig.3). I further noted that the lens was approximately the same thickness during the period when the adult was in the cell (0-1 week) and just after it exited from the cell (1.5 weeks old), measuring 1.85 (± 0.16) um for 0-1 weeks and 1.85 (± 1.01) um for 1.5 weeks at the medial position, indicating no lens secretion activity during the week of adult resting within the pupal cell (Fig. 3A, B and I). Lens deposition started after the adult exits the pupal cell (1.5-3 weeks old), and the increase in lens thickness was most pronounced until weeks 5 to 7 (Fig. 3C, D, E and I). The following measurements at the medial position in 1.5-3, 3-5, and 5-7 weeks old animals are: 3.39 um, 6.08 (± 1.57) um, and 6.87 (± 1.03) um respectively. Comparing 0-1 weeks old adults, which still rest in the pupal cell, and foraging 5-7 weeks old adults, I observed an approximate 3.5-fold increase in the posterior and medial position and 2.5-fold increase in the anterior (Fig.

3I).

By week 11 of early adult life, lens deposition appeared to plateau, measuring

7.5 (± 0.94) um and 7.47 (± 1.66) um at the medial position in 7-9 and 9-11 weeks, respectively (Fig. 3F, G and I). The adult lens thickness in 1 year old or older animals was 9.61 (± 0.85) um (medial position; Fig. 3H).

Statistical analysis showed that there was no significant difference in lens thickness when two successive stages were compared to each other. This result could be an outcome of small sample size. Alternatively, the lens increase is a continuous process so that significant differences between consecutive stages might not be detectable. However, when 0-1 week old and 1 year old adults were compared, high

Fig. 3. Time course of lens deposition in the developing P. hirtus eyelets. (A-G) Visual representation of increase in lens thickness in first 11 weeks of adult development. (A-H) Anterior is to the right, and posterior is to the left. Ocher arrows point to anterior measurement, light orange arrows to medial measurement, and dark orange arrows point to posterior measurement. (A) 0-1 week old adult. (B) 1.5 weeks old adult. (C) 1.5-3 weeks old adult. (D) 3-5 weeks old adult. (E) 5-7 weeks old adult. (F) 7-9 weeks old adult. (G) 9-11 weeks old adult. (H) Visual representation of lens thickness in ≥1 year old mature adult. (I) Bar graph representing the values of incremental increase of lens thickness in the 3 measurement spots. Scale bar in A-H: 10 um.

21 significant difference (p<0.0001) was observed in all three measurement positions

(posterior, medial, and anterior).

Furthermore, my measurements revealed that the posterior and medial thickness of the forming lens were consistently higher than in the anterior, starting from 3-5 weeks of adult lifespan (Fig.3D, E, F, G and I), implying differential lens deposition activity across the eyelet, resulting in the posteriorly and medially thickened lens that is a persistent characteristic of the eyelet in older adult animals. Consistent with this, both average posterior and medial lens depths were highly significantly different from anterior lens depths in the sample of 1 year old adult animals (p<0.0001).

CHAPTER 4: DISCUSSION

4.1. How do the structural findings of the P. hirtus visual system relate to the findings and predictions made in previous transcriptome studies?

My aim was to explore the cellular organization and development of the eye in the cave beetle P. hirtus . First insights into the nature of the cells covered by the eyelet lens have come from previous genetic studies on P. hirtus (Friedrich et al, 2011). Deep

sequencing of the transcriptome of the adult P. hirtus head revealed the absence of

transcripts of genes that encode proteins that are essential and specific for eye

pigmentation in Drosophil a, such as cinnabar , prune , scarlet , and white . However, the

same transcriptome data also revealed the expression of all core members of the

phototransduction gene cascade, such as opsin and arrestins, indicating the presence

of unpigmented photoreceptors in P. hirtus . Our ultrastructural and

22 immunohistochemical studies confirm the presence of non-pigmented photoreceptors in the P. hirtus adult eyelet. Taken together, these findings demonstrate the first identified specific cell type in the P. hirtus eyelet: photoreceptors.

Previous analyses (Friedrich et al, 2011) suggested the functionality of the P. hirtus eyelets also based on a negative phototactic response of adult P. hirtus animals in light-dark tests. My finding of axonal photoreceptor projections, which form a presumed optic nerve at the bottom of the eyelet cuticle cup and project medially towards the brain, further supports the model that the negative phototactic behavior of

P. hirtus is mediated through the response of photoreceptor cells to light stimulus.

4.2. Differences between the P. hirtus eyelet and the organization of the compound eye in surface species

The organization of the adult compound eye is well characterized in surface insect species, such as Drosophila melanogaster and Tribolium castaneum . Insect

compound eyes are structured into ommatidial units, which consist of 8 pigmented

photoreceptors surrounded by pigment cells that contain screening pigment granules

(Cagan and Ready, 1989; Friedrich et al, 1996; Tomlinson, 2012). Pigment cells are

part of the interommatidial cells (IOCs) that also include mechanosensory bristles,

which are shared among three ommatidia (Cagan and Ready, 1989). Distally from the

photoreceptor cells is a prominent layer of dioptric structure: a surface corneal

ommatidial lens and a pseudocone. Between the dioptric structure and the

photoreceptors, there are transparent cell bodies of 4 cone cells (Tomlinson, 2012).

My results show that the P. hirtus eyelet is not only externally but also internally

23 very different from this standard organization of the compound eye. A thick uniform lens covers a cuticle cup that is filled with a population of photoreceptor cells that lack pigment granules (Fig. 1D). The surface of the lens is smooth and continuous, lacking any indication of subdivisions into facets, suggesting the lack of ommatidial organization in the P. hirtus eyelet (Fig. 1). Interestingly, a layer of pseudocone is also missing, but there seems to be a specific distal-most layer of cells adjacent to the lens. I have found that out of approximately 190 cells, 60 cells do not have the photoreceptor cell fate in the P. hirtus adult eyelet (Fig. 1I). Based on the spatial location of cells adjacent to the

lens, this suggests that non-photoreceptor cells could represent a population of lens-

secreting cells that are localized under the dioptric structure, as cone cells are observed

in D. melanogaster and T. castaneum .

The rhabdomeres of surface insect photoreceptor cells are arranged in highly

organized patterns (Kumar and Ready, 1995). The rhabdomere is the site of

phototransduction (Arikawa et al, 1990), and it is a Rhodopsin-rich multi-folded

membrane (Paulsen and Schwemer, 1979; Kumar and Ready, 1995), which is

organized into stacks of parallel microvilli (Leonard et al., 1992; Ready et al, 1976;

Tomlinson and Ready, 1987). In some insects, such as Drosophila , rhabdomeres of 8 ommatidial photoreceptors face each other, and they are separated by an inter- rhabdomeral space (Tomlinson, 1985; Zelhof et al, 2006). Other insects, such as the red flour beetle Tribolium , have a closed system, in which rhabdomeres of photoreceptors are fused to each other and lack the inter-rhabdomeral space (Zelhof et al, 2006).

The arrangement of rhabdomeres in the P. hirtus eyelet, however, is very

24 different from all the known arrangements in surface species. The most dramatic difference is the highly unordered organization (Fig. 1). Individual rhabdomeres are found randomly distributed throughout the eyelet, and the ultrastructural data suggest that microvilli face several different directions within the same rhabdomere of photoreceptors. Moreover, in some areas the rhabdomeres of different photoreceptors abut to each other. However, there is no evidence of space between them; thus, the P. hirtus eyelet would be classified as a closed rhabdom system, just like the closed rhabdomere system found among its distant relatives, the beetle (Coleoptera) tribe, such as Tribolium . Interestingly, transcriptome analysis (Friedrich et al, 2011) showed

that P. hirtus has a very low level of expression of eyes shut/spacemaker (eys/spam)

(Husain et al, 2006; Zelhof et al, 2006) and no expression of prominin (prom) (Zelhof et al, 2006), which encode two extracellular proteins that guarantee the open rhabdom system (Zelhof et al, 2006). This would be consistent with the absence of the inter- rhabdomeral space in the P. hirtus adult eyelet.

The camera eyes are present in vertebrates and in cephalopods, and they have

a single corneal oval-shaped lens overlaying the pigmented retina in the optic cup

(Ogura et al, 2004; Nilsson, 2013). Morphologically, the P. hirtus lens-covered cup-

shape eyelet, which holds unpigmented retina (Fig. 1), exhibits similarity to these

animals’ camera eye.

Taken together, these findings reveal a highly modified cellular organization of

the P. hirtus eyelet. In contrast to the compound eye of surface species, P. hirtus has

camera-like eye with a reduction of signature structures characteristic for eyes of

surface insect species.

Fig. 4. Comparative time course of P. hirtus lens development. In D. melanogaster and T. castaneum , the lens is secreted during the short period of pupal development. In contrast, the P. hirtus lens is deposited during the first 11 weeks of adult development.

4.3. Differences between the development of the P. hirtus eyelet and the compound eye in surface species

Extensive studies of eye development have been completed in several different surface insect species (reviewed in Friedrich et al, 2013). These studies revealed a high level of conservation of eye development at the level of cell patterning and molecular genetic control. Since cave species originated from surface dwelling ancestors (Protas and Jeffery, 2012), I hypothesized that some aspects of compound eye development might be recapitulated during the development of the P. hirtus eyelet.

In D. melanogaster and T. castaneum , retinal differentiation starts during the last larval instar and continues through the pupal development (Cagan and Ready, 1989;

Friedrich, 2003; Friedrich and Benzer, 2000). Both in D. melanogaster and T.

castaneum , a front of differentiation moves from the posterior margin of the eye field to

the anterior edge of the prospective eye field (Friedrich, 2003; Kumar, 2011; Kumar,

2012). Thus, cell differentiation and ommatidial assembly start in the posterior portion of

the future eye (Kumar, 2011). The first cells to emerge from the precursor cells in each

ommatidium are 8 photoreceptors, followed by the differentiation of cone cells and

primary pigment cells, which together are called support cells (Bao et al, 2010). These

two cell types produce the corneal facet lenses during late pupal eye development, a

process which is completed by ~75% pupation (Cagan and Ready, 1989; Charlton-

Perkins et al, 2011).

I found little similarity between the cellular dynamics of compound eye

development and that of pupal development of the P. hirtus eyelet. Young P. hirtus

pupae (0-2 weeks old) already possess Arr2-positive cells, indicating the molecular

27 onset of terminal cell differentiation during the larval stage. I further found that there is no ommatidial organization in pupal eyelet tissue and that the photoreceptors appear already randomly distributed at this stage. This leads to the conclusion that the specification and positioning of all P. hirtus eyelet photoreceptors take place earlier, most likely during the late larval instar and is completed by the onset of pupation. This conclusion is further supported by the similarities between eyelet dimensions of pupae and those of adults. By comparing surface eyelet diameters and eyelet depths in the young pupa and the adult, I did not find significant differences in eyelet size between these two stages, indicating minimal growth of the eyelet throughout pupal development

(I excluded old pupa from comparison due to inaccurate measurements). This finding further supports the conclusion that P. hirtus eyelet assembly occurs before pupation.

In both D. melanogaster and T. castaneum , the deposition of the lens is completed during the short period of late pupal development, before hatching of the adult (Fig. 4). In contrast to this, the secretion of the enforced lens cuticle does not take place during pupal development in the P. hirtus eyelet, but instead after pupal development is completed, during the first 11 weeks of adult life (Fig. 4). Since lens secretion occurs during the early adulthood in P. hirtus , it is reasonable to assume that the support cell type that secretes the lens differentiates to full functionality by the onset of this period. This is further supported with the presence of approximately 60 Arr2- negative cells in the adult eyelet, which may represent a presumed potential of lens- secreting cells.

My findings reveal that the development of the P. hirtus eyelet is characterized by substantial modification of the ancestral patterning program that underlies the

28 development of the insect compound eye. The molecular genetic basis for these developmental changes will be interesting topics of future studies, and it will give mechanistic insight into the changes that occurred during the cellular reorganization of the P. hirtus eyelet in adaptation to the visual demands of the cave environment.

4.4. Hypotheses regarding the time frame of adult lens deposition

To the best of my knowledge, P. hirtus represents the first example among insect

species in which the lens is being added to the formed eye during the adult stage, after

pupal development has been completed (Fig. 4). Lens deposition begins after the adult

exits the pupal cell (week 1.5), which could correlate with the animal’s food intake

outside the pupal cell.

Adult lens secretion in P. hirtus could also correlate with the process of tanning.

The tanning process includes hardening and darkening of the exoskeleton through

cuticle protein deposition, and it is triggered by hormonal signals (detailed mechanism

reviewed in Andersen, 2010; also described in Arakane et al, 2005; Luan et al, 2006;

Wigglesworth, 1948). It is possible that these hormonal signals synchronize lens protein

and cuticle protein secretion. Thus, an interesting question to be addressed in future

research is whether there is a mechanistic correlation between these two processes

and whether the same hormonal signals trigger these two processes.

4.5. Hypotheses regarding the cellular mechanism and regulation of adult lens deposition

From the work in Drosophila , it is known that cone cells and primary pigment

29 cells are responsible for lens secretion during late pupal eye development (Cagan and

Ready, 1989; Charlton-Perkins et al, 2011). In the P. hirtus adult eyelet I observed approximately 130 Arr2-positive cells, representing photoreceptors, and around 60 Arr2- negative cells. I predict that the Arr2-negative cells include lens-secreting cells (cone cells and/or primary pigment cell), which are responsible for lens deposition during the first 11 weeks of adult development. I further speculate that the photoreceptors could initiate the differentiation of the lens secreting cells at the onset of pupal eclosion or during the first 1.5 weeks that the adult spends within the pupal cell, after which these cells secrete lens during the first 11 weeks of young adulthood. The onset of Arr2- negative cell differentiation and their true identity should be confirmed in future experiments.

4.6. Comparison with other cave adaptive visual systems

Several cave species have been explored for the genetic mechanisms of eye

development and regression. In many cave fish species, eyes first develop normally, but

arrest of growth and differentiation occurs followed by programmed cell death, resulting

in eye retardation and regression (Gunn 2004). The regression of the eye in A.

mexicanus involves lens cell apoptosis influenced by overexpression of Sonic

hedgehog, which in turn triggers retinal degeneration (Protas and Jeffery, 2012;

Yamamoto et al, 2004). In contrast, in the Somalian cavefish P. andruzzii , the retina degenerates first through the process of apoptosis, resulting in arrest in lens development (Stemmer et al, 2015). The scenario is similar in the Chinese cavefish

Sinocyclocheilus, where eye reduction occurs through downregulation of retinal genes,

30 leading to substantial reduction in cell proliferation, which appears to be independent of lens degeneration (Meng et al, 2013a).

Looking at invertebrates, the eye size of the cave freshwater isopod crustacean,

A. aquaticus , varies within the population, ranging from reduced-eye to completely

eyeless individuals. These phenotypes are speculated to be controlled through multiple

genetic mechanisms (Protas et al, 2011), but not much is known so far.

Compared to the species mentioned above, my data reveal that the P. hirtus

eyelet reduction is different: I neither detected the evidence of retinal apoptosis, nor the

trace of lens degeneration. In contrast, abundant lens secretion occurs in young adults,

giving evidence that there is no lens degeneration. Morphologically, P. hirtus shares the

most similarities with Typhlochoromus stolzi , the European troglobiotic ground beetle

(Bartkowiak et al. 1991). T. stolzi eyes also lack the fundamental features of compound

eye organization, but are characterized by the presence of a cuticle cup. Interestingly,

both P. hirtus and T. stolzi have a population of photoreceptors with irregularly shaped rhabdomeres that are disarranged in the eye. However, functionally, T. stolzi have been reported to be unresponsive to light (Bartkowiak et al. 1991), which is in distinct contrast to the negative phototaxis behavior of P. hirtus (Friedrich et al, 2011).

4.7. Is the adult P. hirtus lens deposition an adaptive or regressive trait?

Although showing regressive features when compared to surface compound eye structures, adaptive morphological evolution is also evident in the troglobitic P. hirtus eyelet. Microvilli assembly and maintenance are dynamic and energy-dependent processes (Alberts et al, 2007), and the presence of fully developed rhabdomeres in P.

31 hirtus adult eyelet suggests the usage of photoreceptors. In addition, P. hirtus eyelets are likely activated by light, as suggested by its negative phototaxis behavior (Friedrich et al, 2011), demonstrating the use of the eyelet structure in the discrimination of darkness from light. This could be significant for spatial and temporal orientation within a cave environment, since food source is most abundant near the cave entrance (Gunn,

2004). P. hirtus is known to feed on cave cricket guano (Peck, 1975), which is present in the disphotic zone of the cave. The P. hirtus eyelet could help the animal adapt to its disphotic environment.

The preservation of circadian gene expression (Friedrich et al, 2011) further

suggests another potential adaptation mechanism by P. hirtus to its cave disphotic

environment. However, at present, there is no firm evidence that this circadian

regulation is utilized through the P. hirtus eye structure, and that it is solely depends on

light as a zeitgeber. Taken together, the photoreceptor cells in P. hirtus eyelet seem to be under the control of stabilizing selection, since they fulfill an advantageous function, indicating adaptive evolution.

However, the fact that lens secretion occurs in young adults in P. hirtus , as opposed to pupal deposition of the lens in surface insect species, potentially represents a nonadaptive trait that occurs due to reduced purifying selection on the visual performance of young adults. This is supported by the behavior in young Tribolium adults, which avoid light exposure during the first two day after hatching from the pupal stage, until the tanning process is completed (Arbogast et al, 1973). This suggests that young P. hirtus adults are less spatially active before the completion of tanning, resulting in reduced demands on visual performance during this time window.

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ABSTRACT

DEVELOPMENTAL EVOLUTION OF THE VISUAL SYSTEM IN THE CAVE- ADAPTED SMALL CARRION BEETLE PTOMAPHAGUS HIRTUS

JASMINA KULA ČIĆ

August 2015

Advisor: Dr. Markus Friedrich

Major: Biological Sciences

Degree: Master of Science

Extensive research has been done to date on Drosophila and Tribolium eye

development; however, not much is known about the molecular basis of development of

extremely reduced and modified eyes in cave insects. Ptomaphagus hirtus represents

an emerging model system for studying the changes at the molecular level that

occurred during the evolutionary adaptation of the eye to the cave environment.

Therefore, I have started exploring the morphology and organization of the reduced P.

hirtus eyelet, using immunohistochemistry, laser scanning microscopy, and ultrastructural imaging approaches. My findings demonstrate that the adult eyelet lacks ommatidial subdivision and has a thick, clear cuticular lens that covers a cuticle cup that surrounds a population of approximately 130 randomly distributed unpigmented photoreceptor cells. I have evidence that the P. hirtus eyelet assembles before the

onset of pupation. Interestingly, in contrast to the developmental time frame of lens

40 secretion during the pupal stage in the compound eye of surface insects, lens deposition in P. hirtus occurs during the first 11 weeks of early adult development. To the best of my knowledge, this is the first example among insect species of postembryonic adult lens addition to the already formed eye. In sum, I speculate that retention of photoreceptor cells in the P. hirtus eyelet represents an adaptive evolutionary trait to the cave environment, in contrast to the non-adaptive adult lens deposition. Together my findings raise interesting questions that can be further addressed by future experiments.

AUTOBIOGRAPHICAL STATEMENT

I was born in 1988 in Belgrade, Serbia. I grew up in the capital together with my parents and my sister Dijana. After 8 years of primary school, I enrolled at the Medical High School,

Belgrade, Serbia, as a Pharmaceutical Technician major. After my High School, I decided to switch gears, and instead of continuing with getting an education in the Pharmacy field, in 2007

I enrolled in the Biological Faculty, Belgrade University, Belgrade, as a molecular biology and physiology major. During the 4 years of my undergraduate studies, I joined the student organization Biological Research Society “Josif Pančić”. There, I joined the Biospeleology group, where my love for caves and cave arthropod fauna started. I was very active in the

Society: we went for multiple field works and had multiple meetings throughout the years.

During my last year, I was vice president of the Society, as well as the leader of the

Biospeleology group.

Since I decided I was interested in further pursuing an education in science, I applied to

Wayne State University. Luckily, I got accepted into the Biological Sciences Graduate School

Program in Fall 2011. I was really excited when I discovered Dr. Friedrich’s publication on cave beetles (Friedrich et al, 2011), and I thought if I could join his lab that it would be a “dream- come-true”: I would do research on cave insects. That did happen; I officially joined Dr.

Friedrich’s lab in May 2012 and started working on my thesis project. In the meantime, one crucial event occurred: I got married in August 2012.

I had a good time working with Dr. Friedrich and his lab members, and we made satisfying progress together. I will always treasure this experience as I move forward in my career track.