COMPARATIVE ORGANIZATION OF OPTIC LOBES IN CRUSTACEANS: HEMIGRAPSUS NUDUS

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Authors Chung, Tzu-Han

Citation Chung, Tzu-Han. (2020). COMPARATIVE ORGANIZATION OF OPTIC LOBES IN CRUSTACEANS: HEMIGRAPSUS NUDUS (Bachelor's thesis, University of Arizona, Tucson, USA).

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Link to Item http://hdl.handle.net/10150/650937 COMPARATIVE ORGANIZATION OF OPTIC LOBES IN CRUSTACEANS:

HEMIGRAPSUS NUDUS

By

TZU-HAN CHUNG

______

A Thesis Submitted to The Honors College

In Partial Fulfillment of the Bachelor’s degree With Honors in

Ecology and Evolutionary Biology

THE UNIVERSITY OF ARIZONA

M A Y 2 0 2 0

Approved by:

Dr. Nicholas J. Strausfeld Department of Neuroscience Abstract The common structure of optic neuropils connected by chiasmata has been used to propose that crustaceans and insects share a common ancestor. It was expected that comparisons of common organizations across taxa would reflect a sister group relationship between crustaceans and insects. However, recent studies revealed profound differences between the four synaptic regions across taxa, suggesting that the organization of neurons in these systems do not immediately reflect the common ancestry background. Here we describe the neuroanatomy of Hemigrapsus nudus (Malacostraca, Crustacea) focusing on optic lobe organization characterizing the medulla and lobula, the nested second and third synaptic visual centers. It is shown that unlike the equivalent region of insects, the malacostracan lobula is densely packed with columns that are distributed similarly to those in the medulla. Towards the medial side of the lobula, the dendrites converge into a thick bundle of axons that can be traced towards the midbrain. While similarities in neuronal organizations have been demonstrated to argue a monophyletic relationship between crustaceans and insects, differences in these systems could still indicate independent origins.

Introduction The same denomination is used in both insects and crustaceans when referring to neuropils in the visual system. From proximal to distal, the neuropils are in the lamina, medulla, and the lobula complex which contains the lobula and the lobula plate. In both groups, the chiasmata connects the lamina to the medulla and the medulla to the lobula. Early studies of the morphological features of the brain proposed that the evolutionary relationship between arthropods to be monophyletic (Hanström, 1926), and that malacostracan crustaceans and insects should be viewed as sister groups due to common organizations of their nervous system. Osorio (1991) proposed that the lamina and medulla in malacostracans and insects are unlikely to have originated independently or have evolved convergently since they have such similar organization and cell types. This hypothesis was reiterated by Nilsson and Osorio (1997). Harzsch (2002) proposed that the lamina, medulla, lobula, and their chiasmata are synapomorphic and that the presence of three optic neuropils linked by two chiasmata in malacostracans and insects strongly support that these two groups share a common origin.

While recent studies based on molecular biology and anatomical development with respect to phylogeny suggested that insects and crustaceans share a common ancestor (Oakley et al., 2013; Schwentner et al., 2019), whether insects have evolved from crustaceans or whether crustaceans, as an entire subphylum, are the sister group of insects remains undetermined. If arthropods are monophyletic, we would expect insects and crustaceans to possess the same number of neuropils and similar anatomical characteristics of the optic lobe. However, previous studies suggested otherwise.

Diverged from the general structure, the optic lobes of brachiopods, a basal crustacean taxon, consist of only two retinotopic neuropils linked by uncrossed axons, the second neuropil also characterized by large tangential neurons the dendrites of which receive an entire representation of the retina. In dipteran insects, the enlarged lobula plate, part of the third neuropil, the lobula complex, is connected by uncrossed axons to the medulla and lobula. It also typically contains wide networks of tangential neurons. In flies, these are known to respond to the direction of motion across the retina (Hausen and Egelhaaf, 1989). While crustacean optic lobes were thought to contain only three neuropils in early studies, the presence of an additional structure, shaped like the lobula plate of insects, has been discovered in several malacostracans. The neuropil has been identified in the anomuran hermit crab Coenobita clypeatus (Harzsch and Hansson, 2008) and in the brachyuran grapsid crabs Neohelice granulata and Hemigrapsus sanguineus (Sztarker, Strausfeld, and Tomsic, 2005), and its general structure has been described in detail (Bengochea et al., 2017). The shared organizations and differences between optic lobes of crustaceans and insects led to the question of whether they have evolved divergently from a common ancestor or whether they have evolved convergently.

Compared to insects, there are few studies regarding the neuroanatomy and structural development of crustacean optic lobes. Specifically, previous research focused on entomostracan and malacostracan crustaceans. Neuroanatomical studies have been heavily used to support recent debate regarding the evolutionary relationship between insects and crustaceans. To explore this question, we have used specimens of Golgi impregnations, optical microscopy, and digital neural reconstruction to reveal organizations of the visual system in Hemigrapsus nudus, also known as the Eastern Pacific shore crab. As the name suggests, the crab can be found in intertidal areas along the west coast of North America, whose habitat ranges from Alaska to Baja California in Mexico. H. nudus possesses carapaces that are dark purple in general, although some are olive green or red, with white or cream markings. The color of their legs matches the color of the carapace, except the white-tipped chelipeds with purple or red spots. A small crab, adult H. nudus reaches approximately 40–56mm in size and are opportunistic omnivores. Here, we describe the neuroanatomy of the crustacean medulla and lobula followed by a comparison with that of insects. We also identified the general structure of the reniform body in H. nuduss. By identifying fundamental structures shared by modern representatives from both groups, it should be possible to suggest either a malacostracan/insect-clade relationship or divergent evolution. Material and Methods Golgi impregnation The Golgi method involves sequential immersion of neural tissue in solutions of potassium dichromate and silver nitrate, followed by sectioning for light microscopy. Through a mechanism that is yet unknown, a fraction of neurons are filled with brown precipitate of silver chromate, which contrast against a yellow background. This allows the staining of structural details such as dendritic spines and blood vessels. While random staining fails to reveal the complete axonal network and can be a limiting characteristic, the technique remains a key method to study neuronal morphology since individual neurons can be identified more easily.

The presented results are based on Golgi-impregnated optic lobes of H. nudus, a malacostracan also known as the Eastern Pacific shore crab. After the thorax was cooled to immobility, it was fixed in a standard mixture of 2.5% potassium dichromate with 25% glutaraldehyde containing 1.2 g sucrose/100 ml. The eyestalks were then dissected, separating the optic neuropils from the cuticle and sheaths of connective tissue. Next, the optic lobes were incubated in fresh standard solution in the dark for 48 hours at room temperature. The fragments of tissue were then dipped in a series of fresh solutions of 0.75% silver nitrate with wooden sticks until no more precipitation occurred. The neuropils were kept in 0.75% silver nitrate in the dark for 24 hours at room temperature. After two washes in distilled water, the tissue was placed in standard dichromate-osmium solution (99ml 2.5% dichromate, 1ml 1% osmium tetroxide) and left for 24 hours in the dark at room temperature. For second rounds of staining, the tissue was once again taken through the steps that involved washes of silver nitrate and immersion in dichromate- osmium solution. After a distilled-water wash, the tissue fragments were transferred through increased concentrations of alcohol solutions, then immersed in propylene oxide for 10 minutes before being transferred to a mixture of propylene oxide and Durcupan plastic (1:1). The mixture was left in the fume hood under room temperature for 24 hours, allowing the propylene oxide to evaporate. The optic lobes were then placed in fresh Durcupan on a shaker for another 24 hours, after which they were transferred to a small polypropylene container and orientated such that both optic lobes were leveled horizontally and facing the same direction. The specimens were polymerized at 60°C for 12 hours. After cooling, the polypropylene container was removed from the fresh cube of plastic, which was trimmed and serial sectioned at 15-20 μ mon a sliding microtome. Sections were mounted under coverslips in Permount.

Reconstruction Images of an entire section of an optic lobe were displayed as montages of 30-50 optical sections, at x100 oil immersion, of several overlapping areas of the observed neuropil. First, a section was observed with light microscopy to identify neurons of interest. Specifically, individual neurons in the medulla and the lobula. Fragments of the optic lobe were imaged with a video camera that was connected to a bright-field microscope. For each final image, between 20 to 30 optical images were captured at different focal planes in a section and combined digitally with Helicon Focus, a program that focus-stacked multiple sources and produced one image that was focused in all areas. The processed fragments were then merged with Adobe Photoshop, resulting in one extended depth-of-field image of the entire section. Different sections of the same specimen were further compared and combined based on the structure of stained neuronal processes on the same software. Layers are made translucent by using the darkening function. Shadows were removed, and the images were then flattened. In some cases, contrast was enhanced and the dynamic range extended by using the image adjust tools from the same software. The end result of this process was a hierarchical skeletal representation of the branching organization of neurites. This reconstruction method often resulted in some additional information such as markers for synaptic boutons and dendritic spines and soma profiles.

Results In this section, I describe experimental data of the second and third optic neuropils of the crab Hemigrapsus and compare the results with data on other arthropods to analyze the structural pattern with respect to the visual system across taxa.

The Medulla Specific differences exist in the visual organization of neurons between crustaceans and insects. As described previously, the retinotopic structure of malacostracans and pterygotes possess discrete ensembles of neurons in the lamina and medulla, presented respectively by units of optic cartridges and columnar processes (Strausfeld and Nässel, 1980). This observation holds true even in nocturnal insects. While the lamina of Sphinx ligustri lacks clearly defined cartridges (Strausfeld and Blest, 1970), certain photoreceptor axons from the retina and monopolar-cell axons from the lamina project point for point into the medulla. This results in an equivalent number of columns in the medulla as there are ommatidia in the retina.

The second neuropil of H. nudus, the medulla, is dome shaped and slightly elongated in the latero–medial axis (Sztarker et al., 2005). The majority of cell bodies that are associated with the medulla is located above the neuropil, which consists mainly of transmedullary neurons. In contrast to that of insects (Sinakevitch et al., 2003), different sectional layers of preparations show that the crab medulla has a relatively homogenous structure across its depth. No evident separation such as a serpentine layer, which distinguished a proximal and distal region. Instead, tangential neurons are present across strata (Sztarker et al., 2005).

Retinotopic information is sent to the medulla via the first optic chiasma from R8 photoreceptor cells in the retina and from T-cells and monopolar cells from the lamina (Sztarker et al., 2009). The arborizations of these input terminals in the medulla of H. nudus are displayed in Figure 1. Identification of these processes and the other neuronal elements in different areas of the optic lobe was achieved by comparing both the organizational pattern and the overall shape of the neurons. The long photoreceptor R8 terminal is displayed in Figure 1a. Such processes with shallow endings have been observed in other crustaceans (Strausfeld and Nässel, 1980). The next two terminals are identified as T-cell type 1 (Figure 1b) and T-cell type 2 (Figure 1c), which were previously described as efferent neurons from the lamina that has a similar role to that of monopolar cells (Wang-Bennett and Glantz, 1987; Sztarker et al., 2009).

Figure 1. Golgi impregnations demonstrating medulla inputs from the lamina into the medulla. (a) Terminals of photoreceptor R8 with shallow processes. (b) Terminals of T-cell type 1 projecting into the medulla with processes that are slightly longer and more densely packed than those of photoreceptor R8. (c) Terminals of T-cell type 2 that are slightly longer and less densely-packed than those of T-cell-type 1

Cell bodies above the medulla’s distal area derived into transmedullary neurons (Tm), which are commonly described as the columnar pattern in the medulla (Figure 2). Depending on the cell type, these neurons possess processes that are similar to dendrites at certain levels of the medulla. A wide variety of transmedullary neurons have been described in insects. As described by Fischbach and Dittrich (1989), 26 subtypes of such neurons have been observed in fruit flies (Drosophila). The arrow in Figure 2b indicates a type of Tm that lacks processes in the medulla. Such cell type is similar to Tm23 and Tm24 of Drosophila (Fischbach and Dittrich, 1989). On the other hand, the distribution of transmedullary neurons that possess arborizations in the distal medullary area (Figure 2c) indicates that they are postsynaptic to specific inputs from the lamina. This further suggests a parallel organization of information processing pathways. Based on the narrow branching patterns presented by some transmedullary neurons, another characteristic of parallel channeling may be that information is collected mainly from few input processes. In addition to these cell types displayed by Golgi impregnations, Lucifer yellow injections show two types of wide-field tangential neurons (Kirk et al., 1982).

Figure 2. The columnar distribution and unique structures of certain neurons in the medulla. (a) A montage of four consecutive sections of the optic lobe demonstrates the columnar distribution of neurons in the medulla and the lobula. Spatial distribution of columnar neurons in the medulla is approximately the same as that across the lobula. (b) A type of transmedullary neuron that lacks processes in the medulla, similar cell types in insects were proposed by Fischbach and Dittrich (1989) (c) Transmedullary neurons with arborizations in the distal medullary area suggests specific postsynaptic relationship with lamina inputs

The Lobula Similar to the medulla, the lobula of H. nudus is dome-shaped and slightly elongated on the lateromedial axis. Retinotopic information is transferred to the lobula via the second optic chiasma from the medulla. Previous studies based on reduced silver preparations show that the spatial distribution of columnar neurons in the medulla is approximately the same as that across the lobula (Figure 2a), matching a bundle of axons from each medullary column to another one in the lobula with respect to the retinotopic mosaic. In contrast to that of terrestrial insects (Strausfeld, 1998), retinotopic organizations in the lobula of the crab H. nudus are not coarsened. As described by Strausfeld and Nässel (1980), the lobular organization of neurons in many arthropod species is characterized by columnar arrays and inverted arborizations (Figure 3c). However, spacing of the lobular neurons can differ significantly between insects and crustaceans (Strausfeld and Nässel, 1980). While Golgi impregnations display narrow spacing between the columns and overlapping of the dendrites of neighboring neurons in the crustacean lobula (Figure 3b), visual organization in the lobula of the fly Musca domestica exhibit wide dendritic fields that each represent multiple retinotopic columns (Strausfeld and Hausen, 1977). Between layers of strata exists an evident distribution of columnar components that run parallel to each other on the lateromedial axis of the neuropil. Towards the medial side of the lobula, the dendrites converge into a thick bundle of axons that can be traced towards the midbrain (Figure 3a).

Figure 3. Connection between the lobula and the midbrain and organizational structures in the lobula. (a) A montage of four consecutive sections of the optic lobe shows dendrites converging into one bundle of axons towards the midbrain. (b) Narrow spacing between two columns that contains overlapping dendrites of neighboring neurons (c) Neurons with inverted arborizations in the lobula

The Reniform Body As revealed by Wolff and her colleagues (2017), the reniform body is not unique to stomatopods but also exists in Brachyura, which is one of the most recent branches of decapod crustaceans (Wolfe et al., 2019). Here we demonstrate the reniform body found in the decapod H. sanguineus, the structure of which appears to be strikingly similar to that of stomatopods. The reniform body lies proximal to the optic lobe’s medulla and lobula. Golgi impregnations show two bundles (Figure 4a) of columnar pedestal that give rise to four areas consisting of densely branched collaterals. In stomatopods, these regions are named the lateral, distal, proximal, and medial zones (lz, dz, pz, and mz) (Thoen et al., 2019) and the initial zone (Figure 4b), where the columnar pedestal originated. Large bundles of significantly thin axons projecting into the reniform body indicate the participation of massive amounts of neurons originating from the lateral protocerebral neuropils.

Figure 4. Reniform body of the shore crab, Hemigrapsus nudus. (a) General structure of the reniform body showing dendritic and terminal zones. Axons of the pedestal (pds) gave rise to the dense arborizations of the initial, lateral, distal, and proximal zones (iz, lz, dz, and pz). (b) Detail of the initial zone (iz) of the reniform body showing its arborizations.

The four regions of the reniform body was demonstrated to be discrete by Theon and her colleagues (2019) in a study featuring immunostaining with anti-α-tubulin that showed extensive processes branching into the reniform body neuropil from tracts, from which numerous tributaries projected into the various domains. Immunoreactivity demonstrated that each zone was defined by their specific affinities to 5HT antibodies and to antibodies that raised against GAD. Reniform bodies have not been identified in insects and may be unique to crustaceans. The shrub-like terminals in the reniform body that are associated with elaborate multineuronal subunits of the olfactory lobes was proposed to be homologous to the lateral horn in insect brains, which is positioned between the mushroom bodies and the optic lobes and plays an important role in comprehensive understanding of olfactory information (Schultzhaus et al., 2017). Discussion Differences between the Insect and Crustacean Medulla The present study illustrates the fundamental organization of neurons in the crustacean medulla. Golgi impregnations show that in H. nudus, tangential neurons enter or leave the medulla, extending their axons over the lobula into the midbrain. In contrast, the insect medulla is separated into two parts by axons of a variety of tangential neurons contained in the Cuccati bundle, which projects a large number of neuron processes into the serpentine layer. The detailed structure of the insect medulla was described by Singh and Strausfeld (2013). Reduced silver staining showed that in brachycerans, a stratum of large tangential axons which comprise the serpentine layer separates the outer two-thirds of the medulla from the inner third. In each dipteran species, transmedullary neurons that cluster into columns are traversed by synaptic layers derived from the axon collaterals and dendrites of transmedullary neurons, and wide-field tangential neurons (Strausfeld, 1970). Specifically, the serpentine layer connects wide-field tangential neurons to axons that enter or leave the medulla. As the junction between the inner and outer medulla, the serpentine layer was proposed to be an active synaptic layer that mediates the different types of signal transfer and modulation that occur in a densely-packed stratum.

While the medulla of H. nudus provides and receives tangential neurons to and from the protocerebrum, it is not evident that axons of these neurons cluster into a discrete stratum. Instead, these neuron processes distribute across the medulla and project into the lobula, creating a pattern in which columnar neurons run parallel to each other in both the second and third optic neuropil before dendrites converge into a thick bundle of axons that can be traced towards the midbrain. Another difference between tangential neurons in the medulla of insects and crustaceans is their immunoreactivity to gamma-aminobutyric acid (GABA) (Sinakevitch et al,. 2013). In contrast to the few medullary neurons in crustaceans that react to GABA, clustered axon bundles of tangential neurons are revealed in insects. The presence of a discrete inner medulla in insects, which is not evident in crustaceans, suggests a major distinction between these two groups of arthropods.

These differences indicate that the insect medulla may have developed from two origins while the crustacean medulla from one, an argument that was proposed and supported by previous studies. In insects, the neuroblasts in the outermost of two Anlagen give rise to the lamina and outer medulla while the inner Anlagen derives into the inner medulla and the lobula complex (Meinertzhagen and Hanson, 1993; Hofbauer and Campos- Ortega, 1990). The independent development of the outer and inner medulla of insects was illustrated by a study on Drosophila mutants that lack certain neuropils of the compound eyes. It was demonstrated that, despite the absence of the lamina and outer medulla, development of the inner medulla and the lobula complex can still occur (Fischbach, 1982).

Color and Motion Detection in Insects and Stomatopods The present study identified terminals of monopolar-cell axons from the lamina that project into the medulla. Golgi impregnations suggest that the first neuropil of the crab provides two types of T cell efferents: type 1 T-cells (narrow field) and type 2 T-cells (wide-field). With both types of T-cells being postsynaptic in the lamina, this organization provides additional efferent channels, possibly twice the number of output pathways from an optic cartridge of an insect. This result is expected since insects that have restricted color perception detect linearly polarized light for navigation. This behaviour was also suggested by the organization of the first neuropil in the crab Chasmagnathus granulatus, although there is yet no known specialized dorsal zone in the retina that mediates this function (Sztarker et al., 2009).

Insects detect color and form from their visual scene and integrate information about panoramic visual motion for flight control. In contrast to arthropods that employ different visual systems for different tasks, such as spiders, each optic unit of the insect compound eye possesses both color- sensitive and achromatic photoreceptors (Rister & Desplan, 2011). Achromatic inputs terminate in the lamina whereas color-sensitive inputs terminate in the medulla. Channels that are associated with these processes divide and transfer color-related information into the lobula. On the other hand, information regarding visual motion across the retina is signaled into the lobula plate. Ultimately, information associated with visual balance and optokinetic movement is delivered from the lobula and lobula plate to corresponding motor pathways through further segregation of outputs from these neuropils (Strausfeld & Lee, 1991).

Among crustaceans, only one group is known to possess multichannel color vision: the stomatopods. the retinal organization of which contains a unique horizontal band of photoreceptors sensitive to 12 different color channels as well as both linearly and circularly polarized light (Chiou et al., 2008; Marshall, Cronin, & Kleinlogel, 2007). However, how deeper neuropils integrate color and achromatic information is yet unclear due to differences in the representative zones of the channels and retinotopic arrangements of output neurons from the lobula (Thoen et al., 2017, 2018) from those of insects. Regardless, it is certain that color plays a crucial role in stomatopod behavior (Marshall & Oberwinkler, 1999). As one of the most important predators in many shallow marine habitats, one potential function of these color channels is color cancellation in brightly-lit water, where spectral and lenticular movements of the water surface create a variety of chromatic noises that may interfere with motion detection.

Observations in the present study also suggest that the lobula plate of the crab H. nudus is relatively small compared to its lobula and the lobula plate of insects. As mentioned, the lobula plate in dipteran insects is typically enlarged and contains wide-field tangential neurons that respond selectively to motion direction and orientation (Buchner et al., 1984) and further relays them to premotor neurons that control head movements and flight direction. Comparative studies focused on motion-detecting circuits across the dipteran taxa demonstrated homologues of small- field retinotopic neurons involved in motion computation pathways that link the retina to the lobula plate (Buschbeck and Strausfeld, 1996). Conserved cell types and arrangements in basal groups suggested that fundamental motion-sensitive circuits have evolved before the appearance in the Jurassic of flies with short antennae (Kovalev, 1981). In addition, many types of insects possess analogous motion-detecting circuits since they show general similarities in responses to optomotor stimuli by premotor descending neurons (Odonata, Olberg, 1986; Lepidoptera, Rind, 1983). Specifically, both the moth Sphinx ligustri (Lepidoptera) and the honeybee Apis mellifera (Hymenoptera) possess transmedullary neurons that are similar to those found in flies. In bees, these processes terminate in the undivided lobula (Strausfeld, 1976), whereas in Lepidoptera, they project into the lobula plate and are associated with directional motion-sensitive tangentials (Wicklein and Varjú, 1999). In contrast, no evidence supports a structural equivalent in the crustacean lobula plate with respect to motion detection and computation. While the lobula plate was described in certain isopod (Strausfeld, 1998) and decapod crustaceans (Sztarker et al. 2009), there was no evident segregation of motion-sensitive channels from other neuropils.

The Lateral Horn as a Potential Equivalent of the Reniform Body Lastly, the present study identified the presence of the reniform body and its general structure in the H. nudus. While this center and the lateral horn in insects was proposed to be homologous, recent studies suggest otherwise. Studies on the evolution of arthropod brains show that olfactory lobes project tributaries into the neuropils lateral to the mushroom body and their calyces in both eumalacostracans and insects (Strausfeld, 2018). In insects, these tributaries are provided to the lateral horn either directly from the olfactory lobes or via the calyces (Galizia & Rössler, 2010). In eumalacostracans, such processes reach the hemiellipsoid bodies, which are proposed to be equivalent to the mushroom bodies in insects, and the neuropils ventral and lateral to it (Sullivan & Beltz, 2005). Such comparison suggests that the reniform body may have no known homologue in the hexapod brain (Wolff et al., 2017).

Due to the arrangement of parallel axons that comprise its pedestal, the reniform body in the crab Neohelice granulata was mistakenly identified by Maza and his colleagues (Maza et al., 2016) as a mushroom body that participates in context-dependent visual memory. As demonstrated by Thoen et al. (2019), this center is not a homologue of the insect mushroom body or the hemiellipsoid body but is present with either of those centers. Nevertheless, the two studies show compelling evidence that aligns the center of N. granulata with the reniform body of stomatopods, which is directly linked to the lobula and to the mushroom body calyces, demonstrating its association with visual learning and memory. While the functional relevance of the reniform body has yet to be discovered, possibly through behavioral studies and ecological constraints of these species, nothing equivalent has been identified in the lateral protocerebrum of insects (Wolff et al., 2017). Thus far at last, the reniform body is an intriguing visual association center that may be unique to crustaceans.

Taken together, differences in the four neuropils of the optic lobes between insects and crustaceans suggest convergence evolution. Morphological similarities can be deceptive, therefore, the presence of common structures in related species does not directly imply a monophyletic relationship. One overarching objective of future research is to consider the mushroom body, another brain region proposed to evolve very early in pancrustacean or even in panarthropod evolution (Wolff and Strausfeld, 2016). The fact that these centers with such complexity, along with the other fascinating features of the visual system, may have evolved convergently in crustaceans and insects should lead to further understanding of their significance in the behavior and evolution of arthropods. References

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