Olfactory Organs of Mesopelagic Cephalopods: Comparative Morphology and Ecological Function

Home , Galiteuthis phyllura, Histioteuthis heteropsis, Japetella

OLFACTORY ORGANS OF MESOPELAGIC CEPHALOPODS: COMPARATIVE MORPHOLOGY AND ECOLOGICAL FUNCTION

A thesis submitted to the faculty of San Francisco State University and Moss Landing Marine Laboratories in partial fulfillment of the requirements for the degree

Master of Science m Marine Science .

Caren Eliza beth B raby

San Francisco, California

December 1998 Copyright by Caren Elizabeth Braby 1998 OLFACTORY ORGANS OF MESOPELAGIC CEPHALOPODS: COMPARATIVE MORPHOLOGY AND ECOLOGICAL FUNCTION

Caren Elizabeth Braby San Francisco State University 1998

To better understand the sensory capabilities of coleoid cephalopods, this study examined olfactory organ comparative morphology and whole animal behavioral response to chemoreception experiments. Six species were used, all relatively abundant members of the mesopelagic cephalopod community in Monterey Bay, California:

Histioteuthis heteropsis, Gonatus onyx, Chiroteuthis calyx, Galiteuthis phyllura,

Vampyroteuthis infemalis and Japetella lzeathi. The length of the olfactory organ stalk is variable, with C. calyx having the most elongated, G. onyx having the most truncated and the other species having intermediate lengths. The olfactory organ microstructure revealed a large morphological diversity and unique ciliary and microvillar structures in each species. Ciliary and microvillar cones found in G. onyx and J. heathi suggest a possible mechanoreception function, while the ultrastructure is consistent with previous research, suggesting a chemoreception function as well.

Behavioral experiments targeted three known loci of sensory epithelia: the arms/mouth region, the olfactory organs and the sensory filaments of V. infemalis. The experiments, the first chemoreception experiments on mesopelagic cephalopods, showed that, like epipelagic and benthic cephalopods, mesopelagic cephalopods use their arms for chemoreception of prey items. The experiments also showed that there is a mechanoreception response in the olfactory organ region of C. calyx and the sensory filaments of V infenzalis. The combination of morphological and behavioral data suggests that there are multiple functions of the olfactory organs, including both mechano- and chemoreception, and that these sensory pathways may be important complemems to vision for ecological interaction in mesopelagic cephalopods. ACKNOWLEDGMENTS

There are many individuals and institutions who made this study financially, intellectually, logistically and emotionally feasible.

Generous financial support carne from many sources, without which this research truly would not have been possible. I have received grants from the Earl and Ethel Myers

Marine Biological and Oceanography Trust and the Packard Foundation. I have received scholarships from the John H. Martin Memorial Scholarship Fund and the Kim Peppard

Memorial Scholarship Fund. The Monterey Bay Aquarium Research Institute (MBARI) and particularly Bruce Robison and the Midwater Ecology Lab, have provided not only funding but also an incredible place in which I conducted this research, including unlimited use of lab space, tools, supplies and other facilities.

For intellectual and logistical support, as well as continual encouragement, thank you to (people listed in time order, relative to when they became involved in my thesis):

Eric Hochberg (SBMNH) for encouraging my interest in cephalopods and providing important discussion; Jim Nybakken (MLML) for giving me the opportunity to study cephalopods and encouraging the progression of my career; Alissa Arp (SFSU) for serving as my committee member and being a great role model; Joan Parker and Gail

Johnston (MLML) for so many things I can not list them all; Brad Seibel and Jim

Childress (UCSB) for allowing me to participate in their midwater collection cruises;

VI Bruce Robison (MBARI) for exposure to cephalopod behavioral ecology as viewed through the eyes ofMBARI's ROV Ventana, for providing me with invaluable specimens, and for having faith in my abilities; George Matsumoto (MBARI) for being a great mentor; Rob Sherlock (MBARI) for answering questions about everything, especially statistics; W.F. Gilly (Hopkins Marine Station [HMS]) for help with neurophysiology of olfactory organ cells; Heike Neumeister (HMS) for help with specimen preparation; Kurt Buck (MBARI) for discussions about, training for and interpretation of electron microscopy; Kevin Raskoff (MBARI & UCLA), the computer wizard, for always dropping whatever he was doing to help me; Sue Service (UCSF) for discussions about statistics; Mario Tamburri (MBARI) for discussions about chemical ecology; John Krupp (UCSC) for allowing me access to and showing me how to best utilize the electron microscopy lab at UCSC; Don Pardoe and Chris Morgan (CSU

Hayward) for introducing me to the world ofremote scanning electron microscopy; and

Michele Jacobi, Chip Rerig and Bruce Robison for reading drafts of this manuscript.

Emotional support has come from many places. Thank you to: my parents; my house mates Brian, Kyra, Brynie and Gypsy; the family; and again to Kyra (for company during all those weekends spent at MBARI). Lastly and most of all, thank you to Chip and Jacaranda, without whom the sun shines less brightly.

Vll TABLE OF CONTENTS

List of Tables . ix

List of Figures X

List of Appendices Xll

Introduction 1

Methods 8 Animal Collection 8 Scanning Electron Microscopy 8 Transmission Electron Microscopy . 9 Behavioral Experiments 9

Results . 12 Scanning Electron Microscopy 12 Transmission Electron Microscopy 15 Behavioral Experiments 16

Discussion 18

References 27

Appendices. 76

viii LIST OF TABLES

Table Page

l. Study species: natural history and distribution . 32

2. Specimens used in this study 33

3. Results from behavioral experiments . 34

4. Results from behavioral experiments: arms . 35

5. Results from behavioral experiments: olfactory organs and sensory filan1ents . 36

lX LIST OF FIGURES

Figures Page

I. Sites of sensory epithelia. 37

2. Diagram of the brain 39

3. Images of study species and olfactory organ location 41

4. Olfactory organs: scanning electron micrographs (SEMs) of study species. 43

5. Histioteuthis heteropsis: SEMs of olfactory organ. 45

6. Gonatus onyx: SEMs of olfactory organ . 47

7. Chiroteuthis calyx: SEMs of olfactory organ 50

8. Galiteuthis phyllura: SEMs of olfactory organ. 52

9. Vampyroteuthis infernal is: SEMs of olfactory organ. 54

10. Japetella heathi: SEMs of olfactory organ 56

II. Diagrams of structures in SEMs 58

12. Histioteuthis heteropsis: transmission electron micrographs (TEMs) of olfactory organ tissue . 60

13. Gonatus onyx: TEMs of olfactory organ tissue. 62

14. Chiroteuthis calyx: TEMs of olfactory organ tissue 64

15. Vampyroteuthis infernal is: TEMs of olfactory organ tissue 66

X Figures Page

16. Japetella heathi: TEMs of olfactory organ tissue . 68

17. Results from behavioral experiments: arms as target site 70

18. Results from behavioral experiments: olfactory organs and sensory filament as target sites. 72

19. Evolutionary model of flattened olfactory organ development 74

XI LIST OF APPENDICES

Appendix Page

1. Primary scientific literature on olfactory organs 76

XII INTRODUCTION

The complex behavior of cephalopods is largely attributed to their notable visual acuity (Williamson, 1995). Along with fishes, cephalopods are the dominant visual predators in sunlit marine habitats and it is assumed that they also play a dominant role in mid water habitats. Because solar irradiatiml penetrating the water column attenuates to less than 0.01% at 200 meters in clear oceanic waters (Jerlov, 1968), mesopelagic cephalopods live in a habitat where they must rely on non-visual sensory input (Hanlon &

Budelmann, 1987) or on light sources other than solar irradiation. Although cephalopods are able to utilize dinoflagellate bioluminescence as a motion detector to facilitate prey capture (Fleisher & Case, 1995), bioluminescence is probably not the only source of environmental information used in hunting. Bioluminescence also is not likely to be a mechanism for other complex ecological interactions, such as predator avoidance and location of mates. Chemical signaling is another possible source of sensory input, which may work alone or in combination with visual signals to inform cephalopods of important ecological factors, especially those species that live in light-limited habitats. There are few studies on the behavior resulting from chemical signals, or direct evidence of the effects of these signals on the ecology of cephalopods (Packard, 1972; Williamson,

1995).

There are three known loci of sensory epithelia in coleoid cephalopods: buccal lips (Emery, 1975a), arm suckers (Graziadei & Gagne, 1976), and olfactory organs

(Zernoff, 1869) (Figs. 1A and lB). The olfactory organs are paired structures which are

1 located on the head, both posterior and ventro-lateral to the eye. There is also a presumed fourth locus in the Vampyromorpha, the poorly understood paired sensory filaments (Fig.

I C). The function of sensory epithelia in the buccal lips and arm suckers has been well studied and shows capabilities for both tactile and distance chemoreception. Stimulation of the buccal lips of squids with prey items Ras been shown to be necessary to initiate feeding behavior (Emery, 1975b). In octopuses, the arm suckers are able to discriminate between changes in the chemical and physical nature of items being held by the arms

(Wells 1963-1964; Wells eta!., 1965) and are thought to play a role in chemotaxis as well

(Chase & Wells, 1986).

Despite the knowledge of the chemoreceptive function of buccal lips and arm suckers, there is little known about the ultrastructurally similar olfactory organs

(Wilden burg, 1997). The olfactory organs are densely innervated with efferent nerve fibers leading to the olfactory and dorsal basal lobes of the brain in both octopus and squid (Messenger, 1967; Messenger, 1979). These organs are associated intimately with the optic lobe, the photosensitive vesicles, the optic gland, and the major motor centers

(Fig. 2). Because of these associations, one may expect that sensory signals received by the olfactory organ could be integrated with visual and light signals, could affect sexual maturity cycles, and could induce visible motor responses. Thus it is these physiological outputs that may indicate when the olfactory organ has been stimulated.

Olfactory organs were first described by Kolliker (1844) and Zernoff (1869) later named them "Geruchsorgan," or smelling organs, because of their location at the inhalant

2 entrance to the mantle cavity (in Wildenburg & Fioroni, 1989). Originally it was thought that olfactory organs were used to monitor the incoming water quality to protect the delicate tissues lining the mantle cavity (Woodhams & Messenger, 1974). Watkinson

(1909) continued Zernoff' s work with a seminal study on comparative morphology of olfactory organs in 23 species. Based on he~ work, she suggested that olfactory organs were present in all coleoid cephalopods and that they were analogous to nautiloid rhinophores. This paradigm persists today, although Watkinson's suggestion that olfactory organs were not homologous to gastropod osphradia has since been superceded by the idea that they are indeed homologous (Emery, 1975b, 1976; Wildenburg &

Fioroni, 1989; Wildenburg, 1997). Watkinson described a gradient in the gross morphology of the organs, ranging from a pit of sensory cells in Octopus, to a flattened pad of cells in Sepia, to an elongate papilla in Chiroteuthis. Subsequent to Watkinson's work, there have been a number of studies on olfactory organ morphology, many using light microscopy and several using electron microscopy (Appendix 1).

Our understanding of olfactory organ ultrastructure is derived from studies on

Lolliguncula adults (Emery, 1975b), Loligo adults (Gilly & Lucero, 1992), Sepia juveniles (Wildenburg & Fioroni, 1989; Wildenburg, 1991) and Octopus juveniles

(Wilden burg, 1997). There are several morphological characteristics that allow olfactory sensory cells to be identified. Olfactory organ tissue is composed of epithelial and sensory cells, typically with a complex surface microstructure of cilia and microvilli (Fig.

5). The sensory cells are primary sensory neurons (Messenger, 1979), characterized by

3 having ciliated vacuoles (Emery, 1975b) (Fig. 12). Vacuoles are found in a morphological continuum from exposed ciliated pads flush with the epithelial surface to completely internalized vacuoles (Emery, 1975b; Wildenburg & Fioroni, 1989).

Vacuoles often, but not always, have pores leading to the external environment (Emery,

l975b; Graziadei & Gagne, 1976). The cilia typically have a standard 9+2 microtubule arrangement (Bubel, 1989). Electron dense membrane thickenings often are noted at the tissue surface between the epithelial and sensory cells (Emery, 1976). Because of the wide range of morphologies observed, it has been suggested that there may be an ontogenetic maturation of the cells (Emery, 1975b; Wilden burg, 1997) or that there is a specialization in cell function among the many sensory cells (Graziadei, 1965; Boyle,

1986). Cells that lack a pore leading to the external environment have been labeled as either immature chemoreceptors or as mechanoreceptors (Wilden burg & Fioroni, 1989;

Wildenburg, 1997). Because of the similarity between cells of the olfactory organ and sensory cells in other cephalopod tissues, the olfactory organ is considered by most workers to be sensory. However, further comparative morphology and experimentation is needed to demonstrate its specific function.

Ecologically, there are only a few critical functions that require sensory input.

Among these are prey capture, predator avoidance, and mate location/selection. All of these functions can, theoretically, be mediated by chemical cues. Although there has been experimentation on chemoreception in cephalopods it has been done without targeting a particular locus (Boyle, 1983; Chase & Wells, 1986). In the past, the suckers,

4 and not the olfactory organs, were considered to represent the most significant site of chemoreception. Gilly and Lucero (1992) described the first behavioral evidence of olfactory organ stimulation and thus confirmed that it is indeed chemoreceptive. In their study, they targeted Loligo opalescens Berry ( 1911) olfactory organs with many substances including K+ channel blocking agents, squid ink and its precursors, and prey mimics (homogenates and amino acids). Although several compounds produced positive responses, the results from squid ink were the most ecologically interesting. Both squid ink and its precursors, melanin and L-dopamine, consistently elicited escape behavior in

Loligo. In this schooling species, inking probably functions as an intraspecific alarm mechanism against predators. Mesopelagic cephalopod alarm signaling is not a logical ecological function because most species are solitary animals, as observed in their habitat by submarines and as inferred by their rare appearance in mid water traw Is (regardless of species). Therefore, although significant progress has been made in this area, the function of the olfactory organ is still largely unknown and yet the potential for its contribution to the sensory input of cephalopods is great.

Because the visual system of cephalopods is so highly developed, the hypothesis that chemical signals may be more important ecological cues than visual signals requires an assumption that visual cues are either no longer available or are not sufficient for the sensory needs. While adults of many mesopelagic species migrate to shallow waters at night, only paralarvae/juveniles reach shallow depths during sunlight hours. Because less of their lifetime is spent in sunlit waters and because activity levels are so low (Seibel et

5 a!., 1997), vision most likely is not as important to mesopelagic species as it is to epipelagic ones. There is a positive correlation between minimum depth of occurrence

(the depth below which 95% of all animals were captured) and metabolism, which suggests that light plays an important part in structuring the distribution of mid water species in the water column, as well as defining physiological attributes (Childress,

1995). As the minimum depth of occurrence for cephalopod species deepens, the less light is available in the habitat and the less likely it is that that species depends on vision for predation. This leads to the assumption that the inadequacy of available light of mid water habitats may produce a shift in the importance of sensory signals from visual to non-visual signals, and particularly chemical ones, is plausible.

The literature has established that: 1) little or no light is present in the mesopelagic depth range and therefore organisms need to compensate for low light conditions; 2) morphological information supports a chemoreceptive function for olfactory organs in epipelagic and benthic cephalopods but the function is not known for mesopelagic species; and 3) experimental evidence demonstrates that the olfactory organ is effective as a chemoreceptor in schooling epipelagic species (i.e. Loligo opalescens), but the function described does not make ecological sense for solitary mesopelagic species. Based on this knowledge, this study focuses on the comparative morphology and functional aspects of olfactory organs in mesopelagic cephalopods, a group that has significantly different sensory requirements than either their benthic or epipelagic relatives. The intention of this work is to lay the foundation for further study on olfactory

6 organ function through presenting microstructure and ultrastructure on a comparative basis, and relying on the rich literature for ultrastructural analysis. It is also an intention to spark ideas about the possible utilization of multiple sensory sources in this light limited group of animals and suggest that there may be alternatives to the paradigm that cephalopods are only interpreting their environment through visual signals. In this paper, the morphological characters of midwater species are described in comparison to previous work on epipelagic and benthic species using scanning and transmission electron microscopy. Then, preliminary chemoreception experiments suggest directions for future work in this area. This research will help to interpret the sensory ecology of mesopelagic cephalopods, an important component of the largest ecosystem on earth, the mesopelagic habitat.

7 METHODS

Animal Collection

Cephalopods were captured during several cruises on the RJV Point Sur using opening/closing mid water trawls and by the RJV Point Lobos and its submersible, the

ROV Ventana (remotely operated vehicle, or ROY). Because mesopelagic cephalopods are extremely fragile animals, great care was taken to c9llect animals with the least amount of physical trauma. The midwater trawl used in this study was designed by J.J.

Childress for collection of delicate mid water organisms with the intention of using the animals for behavioral and physiological studies. In contrast with traditional midwater trawls, the net is more elongated and the cod end is both optically and thermally protected

(Childress et al., 1978). After capture, animals were transferred immediately to chilled seawater (5-7°C). Two sources were used to identify the animals used in this study,

Young (1972) and Nesis ( 1987). Specimens that were not destroyed in the course of the research are archived at the Santa Barbara Museum of Natural History.

Scanning Electron Microscopy

Scanning electron microscopy (SEM) was employed to examine the external anatomy of the olfactory organ sensory epithelium. Immediately following excision from anaesthetized animals, olfactory organs were fixed in 2-3% glutaraldehyde in saltwater.

After varying lengths of time (ranging from several days to several weeks; for Galiteuthis phyllura it was 5 years), the tissues were secondarily fixed in 1% osmium tetroxide and put through an 8-step graded dehydration series using either acetone or ethanol. They

8 were then critical point dried and sputter coated with gold-palladium. Images of

Chiroteuthis calyx were recorded using traditional photographic methods but all other

samples were captured digitally using a remotely operated scanning electron microscope

(Morgan eta!., 1998).

Transmission Electron Microscopy

Transmission electron microscopy (TEM) was employed to examine the external

anatomy of the olfactory organ sensory epithelium and to confirm the ultrastructure of

surface topologies and structures observed with the SEM. The overall structure and innervation of the sensory cells has been described by other authors, as cited above.

Tissues were available for all species, except Galiteuthis phyllura. Immediately

following excision from anaesthetized animals, olfactory organs were fixed in 2-3%

glutaraldehyde in seawater. After varying periods of time (ranging from a few weeks to a

few months) the tissues were processed by microwave fixation and embedding (Giberson et al., 1997). The microwave technique is not only more rapid than traditional

techniques, it also provides superior fixation results. The reagents used were osmium

tetroxide, acetone for dehydration, 2% uranyl acetate stain and Reynolds' lead citrate counterstain. Sections were examined using a JEOL 1OOB transmission electron microscope at 80 kV accelerating voltage.

Behavioral Experiments

Laboratory experiments were conducted to determine the relative behavioral

response of whole animals to food stimuli, targeting each sensory epithelium. Only

9 animals in the best physical condition were chosen for behavioral experimentation.

Physical condition was determined by evaluating the steadiness of inhalation rate and fin movement, the ability to maintain position in the water column, and the appearance of the skin. Animals selected for study were allowed to acclimate to laboratory conditions for several hours before experimentation. Stud)/ animals were not restrained and could move freely within a 40 liter tank with a flow-through temperature-controlled recirculating sea water system (1 ,500 liter capacity). Experiments were conducted under lighted conditions. Because these midwater species are relatively large and sedentary (Table 1), a baseline was established by observing hovering behavior in each animal for a short period of time prior to each experiment.

After baseline behavior was established through observation, animals were subjected to a series of stimulus trials. Each trial consisted of hand-pipetting a stimulus or sea water control onto a target site and observing for 20 seconds. Two stimuli were used to mimic aqueous masticated food sources: 1) homogenized krill, and 2) amino acid mixture (cell culture medium). Seawater was used as a control for both mechanical stimulation and experimental effects. The arms and lips region (here, considered as one site) and the olfactory organs were targeted for each of the 6 species, and the sensory filament was targeted for Vampyroteuthis irifemalis. This design resulted in a total of 26 trial types (6 species X 2 stimuli X 2 target sites, plus 2 stimuli X sensory filaments).

Data were collected using a Hi-8 video camera. A positive response was defined as any change in the animal's baseline behavior within a 20 second period foliowing

10 stimulation, with the underlying assumption being that there would be a visible motor response accompanying a significant chemical reception event. Because each animal underwent repeated experiments, the assumption of independent data was violated and, hence, no statistical tests were applied to the resulting data.

11 RESULTS

The species used in the study and some aspects of their natural history are listed in Table 1, along with data for the epipelagic Loligo opalescens Berry, 1911 for comparison. A total of 16 specimens at various stages of development, representing 6 mesopelagic species, and all 3 orders of colt;oid cephalopods, were collected from

Monterey Bay, California (Table 2). Histioteuthis heteropsis Berry (1913), Chiroteuthis calyx Young (1972), Galiteuthis phyllura Berry (1911), Vampyroteuthis infemalis Chun

(1903), and Jape tel/a heathi Berry (1911) are all very sedentary organisms, while

Gonatus onyx Young (1972) is more active. All 6 species live at depths well below that to which ambient light reaches.

The locations of the olfactory organs vary somewhat with each species. In all species except Galiteuthis phyllura, the olfactory organs are located on the lateral face of the head, posterior and ventral to the eye, close to or just under the mantle edge. In both juvenile and adult G. phyllura, the olfactory organs are on the eye stalks rather than on the head itself. Figure 3 shows the location of the olfactory organ on each species.

Scanning Electron Microscopy

In all 6 species the olfactory organs are either stalked papilla or raised above the epithelial surface. Figure 4 shows the olfactory organ projecting out from the ventro lateral surface of the head. There is a visible distinction between the sensory epithelium at the tip of the olfactory organ and the regular epithelium on the stalk. Although the general structure of the olfactory organ in all species is similar, there are characteristic

12 species-specific differences in gross morphology. The most obvious of these differences is length of the olfactory organ stalk, which is quite variable among the species studied.

In Chiroteuthis calyx, the stalk is very elongate and slender, while in Histioteuthis heteropsis, Galiteuthis phyllura, V. infemalis, and Japetella heathi it is short and stout, and in Gonatus onyx there is an olfactory ridge without a stalk.

At a finer scale these species show dramatic morphological differences in terms of the arrangement of cilia and microvilli (Figs. 5-10). Cilia emerge directly from the epithelial surface in all species, except Gonatus onyx and Japetella heathi. In the cases of G. onyx and J. heathi, a reticulate network of microvilli covers the epithelium and discrete patches or bundles of cilia periodically erupt from that network (Figs. 6C.1-C.5

& 10B.1-B.3). Cilia structure appears to be similar among all species studied except in

G. onyx, which also appears to have paddle-shaped, chemosensory disco-cilia (Figs.

6C.2-C.3) CLaverack, 1988), although they do not appear in cross sections.

Unique ciliary and microvillar structures are evident in several species and are diagramatically depicted in Figure 11. These structures may be categorized as either unoriented or oriented. Unoriented ciliary plumes, which occur in high density across the sensory epithelium surface, are present in Histioteuthis heteropsis (Figs. 5 B.1-B.3). The ciliary plumes are approximately 7!J.m in diameter and occur 1/1 OO!J.m2 at the highest density (Fig. 5B.1). Oriented structures include the microvillar cones in Gonatus onyx and Japetella heathi and ciliary bundles in J. heathi. There are two morphological types of cones in G. onyx: pointed cones with approximately 35-40 microvilli around the

13 perimeter (Fig. 6C.4) and truncated cones with approximately 50 microvilli around the perimeter (Figs. 6C.3 & 6C.5). The microvillar cones of 1. heathi are much less apparent than those of G. onyx, perhaps because of the thickness of the reticulate network covering the epithelium. They appear as small projections just emerging from the microvillar network (Fig. 10B.2). In 1. heathi, the cilia appear to be fused together in elongate bundles, with approximately 25-30 cilia/bundle (Figs. 10B.l-B.3). The bundles also appear to be covered by a thick coat of mucus. Chiroteuthis calyx appears to have ridges of cilia (Fig. 7 A.3), although it is unclear whether these are due to artifacts of preservation. Galiteuthis phyllura and Vampyroteuthis infemalis are not particularly distinctive, having rather undifferentiated fields of cilia and microvilli (Figs. 8A.3 &

9B.3, respectively) .

. On the surface of the olfactory organ, cilia in most of the study species appear to be directional in distribution. In some, the cilia themselves are localized on the epithelial surface and in others the specialized ciliary structures (discussed above) are localized. In

Chiroteuthis calyx (Fig. 7 A.!) and Vampyroteuthis infemalis (Fig. 9), cilia are localized in one portion of the epithelial surface. In Gonatus onyx, cilia occur on the lateral side of the olfactory surface but not in the central and most distal portion of the epithelial surface

(Fig. 6). In Histioteuthis heteropsis (Fig. 5, regions B & C) and 1apetella heathi (Fig. 10, regions B & C), the ciliary plumes and bundles, respectively, are localized in one region of the olfactory epithelium.

14 Transmission Electron Microscopy

Similarities among the tissues of the 5 species studied and those previously described in the literature are shown in transmission electron micrographs (TEMs) (Figs.

12-16). In most of the TEMs and in all species, cilia and microvilli at the epithelial surface occur both singly and in clusters, cre~ting a carpet of microstructure above the epithelial surface. Another character common to all species is the presence of many ciliated vacuoles within the epithelial layer, some exposed to the external environment and some apparently completely internal. Sensory cells are interspersed with epithelial cells. Electron dense cell junctions are evident at the epithelium surface between the sensory cells and the epithelial cells (Figs. 12C, 13A, 14A, 15C & 16B). The cilia structure is the standard 9+2 microtubule arrangement (Bubel, 1989).

The cross-sectional perspective of the TEMs confirms the ultrastructure ofthe specialized structures visible in SEMs. Ciliary bundles, apparent in SEMs of Japetella heathi, are clearly visible in cross-section (Fig. 16C), as are the ciliary plumes of

Histioteuthis heteropsis (Fig. 12C). Cones of Gonatus onyx and J. heathi appear to have similar structural elements: a few cilia interspersed among densely stained structures which may be either microvilli or stereocilia (Wildenburg, 1997; Westfall et al., 1998)

(Figs. !3D & 16D). Functionally, stereocilia surround anywhere from one to a few kinocilia and amplify any movement in the kinocilia from vibrational or mechanical sensory input. Stereocilia are microvilli projecting from the cells bordering the kinocilia's cell. Structurally, the stereocilia have a filamentous actin core, whereas in

15 true cilia the core is polymerized tubulin. Core composition can only be identified through immunohistochemistry, not microscopy (Westfall et a!., 1998). Because these structures are more darkly stained than the cytoplasm of the cell, it suggests that there may be a difference in cytoplasmic composition, which could be explained by a high concentration of actin. Given this interpretation of the TEMs, each microvillar cone of

G. onyx and J. heathi may be composed of multiple cells. However, the ciliary plume of

H. heteropsis and ciliary bundle of J. heathi is likely traceable to a single vase-shaped sensory cell.

Cross-sections also show differences below the surface of the epithelium and details of structural elements that were not apparent in SEMs. Histioteuthis heteropsis,

Gonatus onyx and Chiroteuthis calyx show high densities of ciliated vacuoles in the epithelium (Figs. 12A, 13A & 14A), while Vampyroteuthis infernalis and Japetella heathi have a sparse distribution of these cell types (Figs. !SA & 16A).

Behavioral Experiments

Twelve animals were tested for response to chemical and mechanical stimulation.

There were a total of 55 trial types, each trial type being a combination of specimen number, stimulus used, and receptor site targeted. Because of the difficulties encountered in working with mid water cephalopods, the number of trials for each trial type varied considerably. Table 3 details trial types, as well as the number of trials for each type, and the behavioral responses. To keep analysis of these data as rigorous as possible, all trial types that had less than 3 trials were not used and the resulting data sets are presented by

16 target site in Tables 4· and 5, and graphed in Figures 17 and 18. Table 4lists all data from trials that targeted the ann/lip region and Table 5 lists data from trials that targeted either the olfactory organs or the sensory filaments.

When arms were used as the target site, there was clearly a stronger response to the food stimulus thim to the seawater controls. In all 7 animals, the individual was at least 60% more likely to have a positive response to the food stimulus. As mentioned in the methods, because of the lack of independence of the trials, there is no analysis of the variability associated with those responses.

When olfactory organs and sensory filaments were used as target sites, there was little or no difference in response to seawater controls or food stimulus. However, there were positive responses for both stimuli in Chiroteuthis calyx olfactory organs and

Vampyroteuthis infemalis sensory filaments. This suggests that the mechanical portion of the stimulus treatment, not the chemical stimuli themselves, which induced behavioral responses. Because seawater controls did not induce relatively strong behavioral responses when arms were targeted, the experimental approach (delivering the pipette, experimenter movements, etc.) was not inducing behavior. Instead, mechanical stimulation of these tissues by the water stream was inducing the observed behavioral response.·

17 DISCUSSION

The cephalopod olfactory organ has been the subject of study for over 150 years and we are little closer to understanding its function now than we were when Ktilliker first described these structures as acoustic organs. Both comparative morphology and behavioral data presented in this study provide insight into how mesopelagic cephalopods may be using their sensory adaptations- including the olfactory organs- to interpret their environment. Behavioral data support previous work done on ecologically diverse groups of cephalopods. These are the first studies on chemosensory behavior in deep-sea cephalopods, one of the most difficult groups of marine animals with which to work.

Mesopelagic cephalopods have not been observed in their natural habitat for a long enough period of time to have collected sufficient data on foraging, mating and predator avoidance. Hence, we must rely on indirect data and make inferences to begin to understand the biology of these animals.

Olfactory Organ Stimulation

The neurological structure of the cephalopod brain suggests that olfactory organ stimulation may result in several responses. These responses include motor responses, such as escape behavior or chromatophore displays, and physiological responses, such as sexual maturation (Messenger, 1979). What types of ecological signals might there be that could elicit these responses and what are the functions of each? There are three classifications of ecological functions that are likely to be of primary importance to mid water cephalopods: predator avoidance, foraging and mate selection. Taking each of

18 these possible signals in turn, an intraspecific alarm signal for predator avoidance, as seen in Loligo opalescens (Gilly & Lucero, 1992), does not make ecological sense for the study species because the midwater cephalopods in this .study do not school (with the possible exception of gonatids; Hunt, 1996). Foraging signals could come from sensing masticated prey being eaten by another predator in the nearby environment (for distance chemoreception in gastropod osphradia, see Kahn, 1961 ), or from the prey item itself emitting a chemical signal itself. Individuals may emit a hormone for mate location and selection, or possibly for sexual maturation. If a hormone were used for mate location, one would expect to see sexual dimorphism in hormone production. Sexual maturation in mesopelagic cephalopod females is a significant issue. Females of some mesopelagic species are known to retain intact spermatophores embedded within the mantle epithelium or carried within the mantle cavity for an indeterminate amount of time after mating (Mangold, 1987; Norman & Lu, 1997). In this case, the mating act could potentially stimulate maturation of the female reproductive organs and the female is able to hold onto the sperm until she is sexually mature. There clearly is potential for chemical signals to make a significant contribution to the ecology of cephalopods.

Olfactory Organ Function

The morphological diversity seen in the olfactory organs of the study species, using electron microscopy, is quite striking. The origin and ontogeny of the sensory cells may suggest the olfactory organ function. Emery (!976) has suggested a hypothesis that the morphological continuum of cell types seen in the olfactory epithelium could possibly

19 have evolved from a simple sensory cell, as seen in octopod suckers. These cells have a shallow cup-shaped pad of cilia at the epithelial surface with no internal ciliated vacuoles.

This suggests that sensory cells on the olfactory epithelium were originally chemoreceptors and that, on an evolutionary time-scale, the sensory cells may have differentiated into more complex forms, with the sensory surfaces becoming internalized vacuoles. In this way, cells with the original function of being chemoreceptors may have evolved into more complex cells with a derived function of being mechanoreceptors. To date, there has been no information on the function of the olfactory organs in mesopelagic cephalopods. This study suggests that the olfactory organs serve as mechanoreceptors, although more sampling is needed to get definitive answers.

Specific morphological characteristics that suggest a mechanoreception function are the microvillar cones seen in Gonatus onyx and Japetella heathi. These cones are reminiscent of the well-studied mechanoreceptors of the cephalopod statocyst, in which the highly oriented structure becomes displaced by pressure waves and the stereocilia of the supporting cells amplify the displacement. Similar microvillar cones have been seen in SEMs of the mantle tissue of adult Loligo plei Blain ville (1823) (Hanlon &

Budelmann, 1987). The stereocilia/kinocilia arrangement seen in TEMs also have been seen in Loligo sp. (Hulet, 1982) and Octopus vulgaris Lamarck (1798) (Wildenburg,

1997). Because the structure of mechanical and chemical sensory epithelia are similar, structure alone does not indicate function. However, there is a distinct similarity between the microvillar cones in G. onyx and J. heathi reported here and identified

20 mechanoreceptors reported in the literature. This similarity suggests that the olfactory organ epithelium has a mechanoreception function. In fact, if it turns out to have a function other than mechanoreception, the entire mechanoreceptor literature would need to be re-evaluated. A contradictory problem relates to the placement of the olfactory organs and a putative mechanoreceptor function of the microvillar cones. On the two study species that bear microvillar cones, G. onyx and J. heathi, the olfactory organ is just underneath the mantle edge so that they are directly affected by the inhalant water stream, which flushes the mantle cavity. How such a placement would allow for the mechanoreception of environmental signals is unclear.

Cilia play a major role in chemoreception, as well, and the species in this study have widely distributed unoriented cilia on the sensory epithelium. Subsequent to the first behavioral evidence for chemoreception in the olfactory organs of Loligo opalescens

(Gilly & Lucero, 1992), additional research has been focused on the electrophysiology of the primary neurons (Lucero & Chen, 1997). However, no further stimuli have been found that have ecological relevance. The disco-cilia found in Gonatus onyx have been found in many animal groups including other molluscs (Tarnarin et al., 1975; Arnold &

Williams-Arnold, 1980; Matera & Davis, 1982). The cells bearing these cilia have been shown to be primary neuron receptors (Matera & Davis, 1982), like the sensory cells of the olfactory organs (Messenger, 1979), and are associated with experimentally demonstrated chemosensory tissues (Matera & Davis, 1982).

The location of the olfactory organs has led researchers to conclude that this

21 function is chemosensory ever since the 1800's. Unlike mechanoreception, which would be confounded by the movement of the inhalant water stream, chemoreception would be enhanced by such a location. When comparing the relative reaction of the three target sites used in the behavioral experiments, namely, the arms, the olfactory organs and the sensory filaments, the olfactory organs are npt sensitive to food stimuli. Experimental evidence indicates that the lips and arm suckers of Octopus vulgaris (Wells, 1963;

Emery, 1975a) play a role in food detection. The behavioral results reported here support this paradigm that the arms and suckers are used for food detection and foraging and that this is mediated by a chemical signal. In all study animals, the arms were clearly more sensitive to food stimuli than to the sea water control. This is in contrast to the olfactory organs, which were equally sensitive to both food and control stimuli. Although statistical analysis was inappropriate for these data, a pattern is evident even in the absence of statistical analysis. There is a difference in the effectiveness of the stimuli for the various tissues tested.

There is evidence from other groups of mesopelagic organisms that chemoreception may be more important in mesopelagic habitats than in epipelagic or benthic ones. Previous chemoreception experiments have been done on either epipelagic or benthic cephalopods. However, some research on mid water crustaceans may be applicable. Chemical cues have been shown to be important in the case of the mesopelagic mysid Gnathophausia ingens (Fuzessery & Childress, 1976).

Electrophysiology on the dactyl receptors of this mysid demonstrates that it has detection

22 limits 2 orders of magnitude lower than those of littoral crustacean species tested. This sensitivity suggests that the specialized distance chemoreception of mid water species may be a generalized adaptation to the mesopelagic habitat. This evidence is encouraging that chemoreception may be important for cephalopods, too, and that the lack of experimental data stems from not ha>.ring found the right chemical signals.

Correlations of Morphology and Ecology

The purpose of using a comparative morphology approach is to look for similarities among these mesopelagic species, which then might lead to hypotheses about ecological function, perhaps even a function unique to the mesopelagic group. However, morphological data emphasize the variability of olfactory organs at the microstructural level. The characters available for correlative comparative use are the relative lengths of the olfactory organ stalk and the presence or absence of specialized ciliary or microvillar structures, since all other characters seen are species-specific.

The length of the olfactory organ varies greatly with species. Differences in this character may be correlated to physiological or ecological factors such as, I) metabolic activity, 2) migratory patterns, 3) minimum depth of occurrence (MDO), or 4) habitat type. When analyzing the importance of these factors, it must be done under the assumption that the ecomorphological paradigm- morphology is directly related to ecology (Wainwright, 1991; Motta & Kotrschal, 1992)- is correct. With this assumption in mind, one may begin to look for correlations between morphology and environmental or physiological factors.

23 Metabolic activity indirectly affects the flow rate of water past the olfactory

organ. The amount of turbulent mixing _is directly proportional to the distance from a

given surface. This suggests that with a lower metabolism, and assuming the olfactory

organ is a chemical sensor, effective chemoreception would require highly elongated

olfactory organs for sedentary animals. How!?ver, within the range of the metabolic

activities of study species (Table 1), there is little to suggest such a pattern.

Vampyroteuthis infemalis and Japetella heathi have the lowest metabolism but an

intermediate olfactory organ length; Chiroteuthis calyx has the most elongate olfactory

organ but an intermediate metabolism. Go1wtus onyx does fit this model because it has

the shortest olfactory organ and the highest metabolism, almost an order of magnitude

above the others. Gonatus onyx also has the most shallow MDO of all the study species.

For comparison, Loligo opalescens also has a very flattened olfactory organ and a high

metabolism, over twice that of G. onyx at resting levels. Loligo opalescens is truly an

epipelagic species, and may be found near the surface during both the day and night

(Hunt, 1996). Gonatus onyx and L. opalescens may therefore have an olfactory organ morphology that synergistically correlates with vertical distribution and metabolism.

Childress (1995) suggests that light is the determining feature of metabolic activity in

mesopelagic organisms. So, it is not unlikely that in addition to affecting metabolism, the presence of light may have a profound effect on morphology as well. Because olfactory

organ length separates the study species into two distinct groups, shallowly distributed G.

onyx and all the other deeper-dwelling species, this hypothesis may have some bearing on

24 the observations presented here.

Habitat characteristics also may be correlated with olfactory organ length.

Japetella has a papilla-form olfactory organ, as does Bolitaena, another meso pelagic

octopod (Watkinson, 1909). However, its benthic relative Octopus (Watkinson, 1909)

has an olfactory organ which is either pit-shaped or flush with the epithelial surface

depending on the stage of development. Other demersal cephalopods, namely Sepia

(Watkinson, 1909) and Euprymna (Novicki, 1984), have flattened olfactory organs as

well. This not only suggests habitat as an influencing factor but also suggests that if there

are effects of phylogeny on this characteristic, it is seen not at the ordinal or subordinal

level, but at the family level. In recent phylogenetic analyses, the bolitaenids are tightly

clustered with Octopus, one using morphological characters (Young & Vecchione, 1996)

and the other using biochemical and molecular characters (Boucher-Rodoni & Bonnaud,

1996). Both the longest and shortest olfactory organs in this study (Chiroteuthis calyx

and Gonatus onyx, respectively) occur in the Teuthoida, with the other teuthoids, the

vampyromorph and the octopod, J. heathi, being intermediate.

Ancestral vs. Derived State of Olfactory Organ Length

From an evolutionary perspective, it is interesting to consider how the papilla

form evolved. The nautiloid rhinophore, thought to be homologous to the coleoid

olfactory organ (Emery, 1975b & 1976; Wildenburg & Fioroni, 1989; Wildenburg,

1997), is distinctly papilla-form. It is possible the papilla is the ancestral form and the

flattened olfactory organs of benthic or epipelagic, high-metabolism teuthoid families

25 (which include Loligo, Gonatus and Sepia) are convergent with the flattened olfactory organs of benthic or demersal octopod families (which includes Octopus). If this were the case, all study species except G. onyx would be considered closer to the papilla-form ancestor. The evolutionary model for transition from a papilla-form ancestor to a derived flattened olfactory organ is depicted in Figur_t: 18, using olfactory organ length and habitat as comparative characters. This model suggests that both ancestral and divergent characters are present in extant cephalopods.

Conclusions

Olfactory organs clearly are important to the biology of coleoid cephalopods because of the complexity and diversity seen in the microstructure of the sensory epithelium. Although evidence presented herein suggests mechanoreception as a function, there is no evidence that this is the olfactory organ's primary function. In fact, it may be that the olfactory organ epithelium receives signals for both mechano and chemoreception and simply uses different cells for each type of signal. Difference in stalk length among species may be a key to understanding how the organ performs and should be considered as an important characteristic for phylogenetic analyses. Future work in electrophysiology and behavioral experimentation may help to clarify the importance of chemoreception in mesopelagic cephalopods. The sensory adaptations of mesopelagic cephalopods are important not only for understanding more about the biology and ecology of the cephalopods themselves, but also for understanding more about survival in the mesopelagic habitat.

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31 Table 1. Natural history and other relevant data from published works on study species and Loligo opalescens as a reference. Distribution data are from the following sources: (1) Roper & Young, 1975; (2) Hunt, 1996; (3) Seibel, eta!., 1997.

Vertical Migration (1) Max Depth (2) Min Depth Activity (3) Daily Ontogenetic ROV ROY Trawling (M02) Day/Night Day/Night (2) Day/Night (3) SubClass Coleoida Order Teuthoida Loligo opalescens yes unknown 600/300 na na 14.00 w Histioteuthis heteropsis yes unknown na 300/na ~00/150 0.73 N Gonatus onyx yes yes na 200/50 400/100 6.55 Chiroteuthis calyx yes yes na 200/na 500/300 0.67 Galiteuthis phyllura yes yes na 300/na 500/300 0.54 Order Vampyromorpha Vampyroteuthis infemalis no yes na 600/na 600/600 0.16 Order Octopoda ]apetella heathi no yes na na 600/600 0.09 Table 2. Specimens used for both morphological and behavioral aspects of the study. Specimen numbers are in the form: ship-year-month/date-animal# (PL =Pt. Lobos/ROV Ventana; PS =Pt. Sur /Trawling). Specimens caught with the Pt. Lobos are archived with the Monterey Bay Aquarium Research Institute and those caught with the Pt. Sur are archived with the SantaBarbara Museum of Natural History.

life stage ML (mm) SEM TEM Behavior Histioteuthis lzeteropsis 1. PS-97-Dec4 adult 40 yes yes yes 2. PL-98-May29 adult 40 yes

Gonatus onyx 1. PS-98-Aprl4 juv 30 yes yes

2. PS-97-0ct21 JUV 30 yes

Chiroteuthis calyx 1. PL-97-June26-l juvenile 40 yes 2. PL-97-June27-3 adult 70 yes yes 3. PS-97-Dec4 adult 70 yes 4. PL-98-June9 adult 70 yes

Galiteuthis phyllura 1. PL-93-FebS adult 150 yes 2. PL-97-0ct30 juvenile 40 yes 3. PS-97-Dec2-3 juvenile 40 yes

Vampyroteuthis infemalis 1. PS-97 -Dec2-4 adult 130 yes yes 2. PS-97 -Dec2-5 adult 50 yes yes

Japetella lzeathi 1. PS-97-Dec2 adult 50 yes 2. PS-97-Dec2-l adult 40 yes 3. PS-97-Dec2-4 adult 40 yes yes yes

33 Table 3. Results from behavioral experiments. Data are displayed as a ratio(%) of positive responses/total number of trials.

Target Site: Olfactory Organ Arms Filament

Stimulus: Control Krill AA Control Krill AA Control Krill Histioteuthis 1 0 100 0 100 (1) (3) . (3) (3) Histioteuthis 2 10 0 30 100 (9) (2) (11) (6) Gonatus 2 20 0 30 100 (5) (1) (4) (3) Chiroteuthis 3 40 40 100 100 (5) (5) (2) (5) Chiroteuthis 4 10 20 40 100 (7) (6) (20) (9) Galiteuthis 2 0 0 0 0 100 100 (3) (1) (3) (1) (2) (4) Galiteuthis 3 0 0 0 70 50 (1) (1) (4) (3) (2) Vampyroteuthis 1 0 0 0 0 30 0 (1) (2) (I) (1) (4) (1) Vampyroteuthis 2 0 0 0 0 70 100 50 70 (6) (2) (1) (4) (7) (1) Japetella 1 0 100 (1) (2) Japetella 2 0 0 0 50 (1) (2) (I) (2) Japetella 3 0 0 100 100 (1) (3) (1) (5)

(n) =number of trials = n/a AA = amino acid mixture - = not tested

34 Table 4. Results from behavioral experiments using the arms as the target site. Data are the ratio of the positive responses/total number of trials expressed in percentage (%). Only includes data from specimens where n-trials>2 and where a comparison was possible.

Animal Control Food Histioteuthis I 0 100 (3) (3) Histioteuthis 2 30 100 (11) (6) Gonatus 2 30 100 (4) (3) Chiroteuthis 4 40 100 (20) (9) Galiteuthis 3 0 70 (4) (3) Vampyroteuthis 2 0 70 (4) (7) Japetella 3 0 100 (3) (5) food =krill+ amino acid data (n) = n trials

35 Table 5. Results from behavioral experiments using the olfactory organs (oo) or sensory filaments (fil) as the target site. Data are the ratio of the positive responses/total number of trials expressed in percentage (% ). Only includes data from specimens where n trials> 2 and where a comparison was possible.

Animal- site Control Food Chiroteuthis 3 - oo 40 40 (5) (5) Chiroteuthis 4 - oo 10 20 (7) (6) Galiteuthis 2 - oo 0 0 (3) (3) Vampyroteuthis 2- fil 50 70 (8) (9)

food = krill + amino acid data (n) = n trials

36 Figure 1. Sensory epithelia of cephalopods (shaded regions), grouped as in this study: A) the arms suckers (as) and buccal lips (bl), frontal view; B) the lateral view of one of the paired olfactory organs (oo); and C) a dorsal view of the paired sensory filaments (fil), present only in Vampyroteuthis inferno/is.

37 A

00 c

38 Figure 2. The region of the cephalopod brain involved in olfactory and light signal transduction. Of particular interest is the inter-connectedness of the lobes: the dorsal basal lobe integrates information from the optic, peduncle and olfactory lobes and is one of the major loci for higher motor control. Also, the optic gland is downstream from the olfactory lobe, suggesting possible function and response mechanisms. Redrawn from Messenger, 1979.

39 olfactmy photosensitive organ vesicles . eyes

- .. - .. ··-··-··-··-··-·· -"- .. - .. - .. - .. - ··- .. - .. - .. - .. - .. - .. - .. - .. "

I BRAIN '( I I r ' I I ' optic I lobe I peduncle . I lobe

I I ..."" olfactory lobe '( ., ,. :~ '( ' I " '= ' ' !lI I dorso-lateral dorsal basal I. : ..,. I lobe lobe ., ,. I I I

I ; I I optic ' subpedunculate . I ~ ..t/1..... I gland lobe I

I BRAIN I ··-··-··-··-··-··-··-··-··-··-··-··-··-··-··-··-··-··-··-··-··-··-··

40 Figure 3. In situ photographs of each of the six species in this study, taken from the ROV Vent ana. Arrows indicate olfactory organ location and sensory filament location (in Vampyroteuthis infernalis only). A) Histioteuthis heteropsis; B) Gonatus onyx; C) Chiroteuthis calyx; D) Galiteuthis phyllura; E) Vampyroteuthis infernalis; F) Japetella heathi. Photographs courtesy of MBARJ, copyright 1998.

42 Figure 4. Gross comparative morphology of olfactory organs using scanning electron microscopy. A) Histioteuthis heteropsis (scale bar= 80.6 J.lm); B) Gonatus onyx (scale bar= 50.8 J.lm); C) Chiroteuthis calyx (scale bar= 215 J.lm); D) Galiteuthis phyllura (scale bar= 42.4 J.lm); E) Vampyroteuthis infernalis (scale bar= 121 J.lm); F) Japetella heathi (scale bar= 50.0 J.lm).

43 44 Figure 5. Histioteuthis heteropsis: scanning electron micrographs of the olfactory organ with Fig. 4A in the upper left comer for reference (scale bar= 80.6 Jlm). Close-ups of three regions: A, epithelium adjacent to the sensory tissue; B, differentiated sensory epithelium; and C, undifferentiated sensory epithelium. A) no cilia are present on the epithelium adjacent to the sensory epithelium but it appears to be highly secretory (scale bar= 13.6 Jlm); B.l) dense patch of ciliary plumes (scale bar= 5.0 Jlm); B.2) a single ciliary plume (scale bar= 2.5 Jlm); B.3) a close-up of the ciliary plume seen in B.2 (scale bar= 1.3 Jlm); C) ciliary and microvillar carpet of undifferentiated region (scale bar= 10 Jlffi).

45 46 Figure 6. Gonatus onyx: scanning electron micrographs of the olfactory organ with Fig. 4B in the upper left comer for reference (scale bar= 50.8 j.!m). Close-ups of three regions: A, epithelium adjacent to the sensory tissue; B, undifferentiated sensory epithelium; and C, differentiated sensory epithelium. Plate I: A) no cilia are present on the epithelium adjacent to sensory epithelium (scale bar = 4. 7 1-1m); B) a few cilia are present in the middle region of the olfactory organ (scale bar = 7.5 j.!m); C.l) the lateral region of the organ has dense patches of cilia interspersed with empty spaces (scale bar= 7.5 j.!m). Plate II: C.2) a pointed cone in a patch without long cilia and a truncated cone within a patch of long cilia, some of which are disco-cilia (scale bar= 2.8 j.!m); C.3) a close-up of the truncated cone seen in C.2 (scale bar = 1.2 j.!m); C.4) a close-up of the pointed cone seen in C.2 (scale bar= 0.8 j.!m); C.5) a truncated cone from a different cilia patch (scale bar= 1.0 1-1m).

47 48 49 Figure 7. Chiroteuthis calyx: scanning electron micrographs of the olfactory organ with Fig. 4C in the upper left comer for reference (scale bar= 215 J-tm). Close-ups of two regions: A, differentiated sensory epithelium; and B, epithelium on stalk, adjacent to sensory tissue. A. I) cilia tufts are visible on left side of the sensory epithelium only (scale bar= 61.0 J-tm); A.2) dense ciliary mat (scale bar= 11.0 J-tm); A.3) ridges may be indicative of sensory function (scale bar= 4.35 J-lm); B) no cilia are present on the stalk (scale bar= 59.7 11m).

50 51 Figure 8. Galiteuthis phyllura: scanning electron micrographs of the olfactory organ with Fig. 4D in the upper left corner for reference (scale bar= 42.4 J.lm). Close-ups of region A, undifferentiated sensory epithelium. A.l) dense ciliary mat (scale bar= 7.5 J.lm); A.2) another region of the ciliary mat (scale bar= 7.5 J.lm); A.3) close-up of ciliary mat (scale bar= 3.8 J.lm).

52 53 Figure 9. Vampyroteuthis infernalis: scanning electron micrographs of the olfactory organ of with Fig. 4E in the upper left corner for reference (scale bar= 50.8 Jlm). Close ups of two regions: A, epithelium adjacent to the sensory tissue; and B, undifferentiated sensory epithelium. In Fig. 4E, the portion of the sensory epithelium in the lower part of the micrograph (opposite from region B) has little or no cilia. A) no cilia are present on the tissue adjacent to the sensory epithelium (scale bar= 2.8 Jlm); B.!) ciliated areas are undifferentiated and appear to be highly secretory (scale bar= 6.0 Jlm); B.2) another patch of cilia (scale bar= 3.8 Jlm); B.3) close-up ofB.2 (scale bar= 1.9 Jlm).

54 55 Figure 10. Japetella heathi: scanning electron micrographs of the olfactory organ with Fig. 4F in the upper left corner for reference (scale bar= 50.0 11m). Close-ups of two regions: A, epithelium adjacent to the sensory tissue; and B, differentiated sensory epithelium. A) no cilia are present on tissue adjacent to the sensory epithelium (scale bar = 1.6 11m); B.l) at the distal tip of the organ, ciliary bundles errupt from the surface (scale bar= 18.8 11m); B.2) close-up of B.l showing ciliary bundles and microvillar cones (scale bar= 9.4 11m); B.3) close-up ofB.2 showing ciliary bundle (scale bar= 2.3 11m).

56 57 Figure 11. Diagrams of olfactory organ stalk types and ciliary and microvillar structures. A) stalk types: elongate stalked organ of Chiroteuthis calyx; intermediate stalked organ of Histioteuthis heteropsis; and the unstalked ridged organ of Gonatus onyx. B) ciliary and microvillar structures: unoriented ciliary plume found in H heteropsis; oriented ciliary bundle found in Japete/la heathi; truncated and pointed microvillar cones found G. onyx, and J. heathi.

58 A

Chiroteuthis Histioteuthis Gonatus

ciHary plume ciliary bundle

truncated cone pointed cone

59 Figure 12. Histioteuthis heteropsis: transmission electron micrographs of the olfactory organ. A) shows densely packed ciliated surface of the olfactory organ and 2 ciliated vacuoles of sensory epithelial cells (scale bar= 3.3 ~m); B) cilia are sometimes enveloped in small pockets and there are often dense cell junctions at the epithelium surface (scale bar= 1.4 ~m); C) a cluster of cilia, presumably a cross-section through the ciliary plumes visible in SEMs (scale bar= 1.3 ~m). Symbols used: bb =basal body, c = cilium,j =dense cell junction, m =mitochondrion, mv =microvilli, p = ciliary plume, v = ciliated vacuole.

60 61 Figure 13. Gonatus onyx: transmission electron micrographs of the olfactory organ. A) dense packing of cilia and microvilli at epithelial surface and many densely packed ciliated vacuoles below the epithelial surface (scale bar= 3.0 [lm); B) close-up of microvilli at epithelial surface and of a dense cell junction (scale par= 3.8 [lm); C) close up of two ciliated vacuoles (scale bar = 1.2 [lm); D) close-up of a cluster of darkly stained microvilli surrounding four cilia, presumably a microvillar cone as seen in SEMs (scale bar= 1.2 [lm). Symbols used: bb =basal body, c = cilium, co = microvillar cone, j = dense cell junction, mv = microvilli, v = ciliated vacuole.

62 63 Figure 14. Chiroteuthis calyx: transmission electron micrographs of the olfactory organ. A) dense packing of cilia and microvilli at epithelial surface and many densely packed ciliated vacuoles below the epithelial surface (scale bar= 6.5 j.!m); B) close-up of a single ciliated vacuole and rootlets of cilia penetrating down into the sensory cell (scale bar= 2.2 llm); C) close-up of a single ciliated vacuole with· an opening leading to the external environment (scale bar= 2.4 j.!m); D) close-up of another single ciliated vacuole with an opening leading to the external environment (scale bar= 1.2 j.!m). Symbols used: bb = basal body, c = cilium,j =dense cell junction, mv =microvilli, r =rootlet, v = ciliated vacuole.

64 65 Figure 15. Vampyroteuthis infemalis: transmission electron micro graphs of the olfactory organ. A) the sensory epithelium has few cilia compared to other species and ciliary bundles, not apparent in the SEMs, are visible here (scale bar= 4.3 [lm); B) close-up of a single ciliated vacuole (scale bar= 2.4 [liD); C) close-up of a single ciliated vacuole with an opening leading to the external environment (scale bar= 1.8 [liD). Symbols used: b = ciliary bundle, j =dense cell junction, mv =microvilli, v =ciliated vacuole.

66 67 Figure 16. Japetella heathi: transmission electron micrographs of the olfactory organ. A) the transition between the non-sensory epithelium to the left and the sensory epithelium to the right; a ciliated vacuole with an opening leading to the external environment (scale bar= 2.4 [lm); B) close-up of a single ciliated vacuole and of dense cell junctions at the epithelial surface (scale bar = 1.6 [liD); C) close-up of a ciliary bundle surrounded by a dense packing of microvilli (scale bar= 1.8 [lm); D) close-up of two microvillar cones (scale bar= 1.8 [liD), the one in the lower portion of the micrograph shows the cilia within a dense cluster of microvilli, whereas the one in the upper portion of the micrograph has basal bodies, suggesting that cilia are present either before or after the section shown. Symbols used: bb =basal body, c =cilium, j =dense cell junction, m = mitochondrion, mv = microvilli, p = ciliary plume, v = ciliated vacuole.

68 A

69 Figure 17. Results from behavioral experiments using the arms as the target site. Only includes data from specimens where n trials>2 and where a comparison was possible (data listed in Table 4). NR =no response.

70 Target Site: Arms

100 ,...... D Histioteuthis I of [;:) Histioteuthis 2 '-' til 80 DGonatus 2 o::l - IIIIChiroteuthis 4 ·~l:l [] Galiteuthis 3 60 -z l:l Vampyroteuthis 2 til 3 =0 ~ til 40 a.> ...'"'a.> ·~.... 20 ·~til 0 i::l-< NR NR NR NR 0 Control Food

71 Figure 18. Results from behavioral experiments using either the olfactory organ (oo) or the sensory filament (fil) as the target site. Only includes data from specimens where n trials>2 and where a comparison was possible (data listed in Table 5). NR =no response.

72 Target Sites: Olfactory Organs (oo) and Sensory Filaments (fil)

100~------~====~ ,....._, ~ !lilliControl '--' CJ.l 80 li!il Food .E ·.o...... 20 CJ.l 0 0... NR NR 0

73 Figure 19. An evolutionary model of how the flattened olfactory organ form may be convergent in the several lines of cephalopod taxonomy using habitat type and olfactory organ shape as the characters. The papilla seems to be ancestral because the species with a flattened olfactory organ are highly specialized, high metabolism organisms, vs. the ancestral nautiloid-type cephalopod.

74 flat flat flat midwater benthic benthic

F. Gonatidae F. Sepiidae . F. Octopodidae / papilla papill a papilla midwater midwater midwater

F. Chiroteuthidae F. Vampyro morpha F. Bolitaenidae

0. Teuthoidea 0. Vampyromorpha 0. Octopoda

papilla demersal

F. Nautilidae

0. Nautiloidea

75 Appendix I. Primary literature on olfactory organ morphology and function.

Year Author(s) Species Studied Findings

1844 Kii lliker squids & octopuses paired pits on head; thought they might be acoustic

1852 Hancock Ommastrephes todarus noted presence; thought they might be (= Todarodes sagitiatus) olfactory

1866 Cheron many noted presence; thought they might be olfactory

1869 Zemoff Sepia officina/is 1. termed them "Geruchsorgan," or Eledone moschata olfactory organs Lo/igo sp. 2. identified two cell types: sensory cells & ciliated cells 3. showed in Sepia that cells appear early in development

1909 Watkinson Sepia officianalis 1. comparative morphology of many S. elegans taxa from preserved specimens S. orbignyana 2. olfactory organs in all co leo ids; Sepia/a rondeletii chemoreceptors due to position & Rossia macrosoma structure, to monitor inhalant water Loligo vulgaris stream L. mannorae 3. suggested homologous to nautiloid (= Alloteuthis media) rhinophores, but not to gastropod L.forbesi osphradia Illex coindetii Todaropsis veranyi (= T. eblanae) Abraliopsis morisii (=A. pfefferi) Chiroteuthis veranyi Liocranchia valdiviae Octopus vulgaris 0. macropus 0. defilippi

76 Appendix I. Primary literature on olfactory organ morphology and function (cont.)

Year Author(s) Species Studied Findings

1909 Watkinson Eledone moschata (cont.) E. aldrovandi (= E. cirrhosa) Scaeurgus unicirrus. Ocythoe tuberculata Argonauta argo Tremoctopus violaceus Bolitaena steenstrup 1913 Polimanti Sepia officianalis olfactory organs chemosensory 1926 Giersburg Octopus vulgaris olfactory organs not chemosensory based on excision & resulting behavioral experiments

1963 Wells Octopus vulgaris olfactory organs not chemosensory based on excision & resulting behavioral experiments; tried to repeat Polimanti's 1913 experiment

1967 Messenger Sepia officina/is olfactory organs not chemosensory based on excision & resulting behavioral experiments

1974 Woodhams & Octopus vulgaris 1. ultrastructural study of olfactory Messenger organ tissue; suggested that ciliated vacuoles created by more than one cell per vacuole 2. found bundles of cilia 3. because of lack of organization of cilia, suggested not for spatial resolution (mechanoreception) 4. hypothesis: may be for reception of sexual signaling compound

77 Appendix I. Primary literature on olfactory organ morphology and function (cont.)

Year Author(s) Species Studied Findings

1975b Emery Lolliguncula brevis I. ultrastructure of olfactory organs; morphological continuum of sensory cell types; did not find vacuoles associated with more than one cell per vacuole (per Woodhams & Messenger) 2. suggested may be for hormone reception 3. describes connection of olfactory lobe to optic gland 4. olfactory organs analogous to osphradia 1976 Emery Octopus joubini adult vs. juvenile morphology of (= Paroctopus joubini) olfactory organs 1983 Boyle Octopus vulgaris crab extracts in water produced increase in ventilation rate; have distance chemoreception (smell)

1984 Novicki Euprymna scolopes ultrastructure of olfactory organ tissue

1989 Wildenburg & Loligo vulgaris ultrastructure of embryonic & Fioroni hatchling olfactory organs; cells function as mechanoreceptors, stimulated by pressure waves on epithelial surface

1990 Otis & Gilly Loligo opalescens preliminary work on olfactory organ stimulation & behavioral response

1992 Gilly & Lucero Loligo opalescens escape response when olfactory organs stimulated by own ink, potassium channel blockers, various stimuli; negative response to food stimuli

78 Appendix I. Primary literature on olfactory organ morphology and function (cont.)

Year Author(s) Species Studied Findings

1992 Lucero, Loligo opalescens electrophysiology of extracted Horrigan & olfactory organ neurons to various Gilly stimuli tested by Gilly & Lucero (1992)

1997 Lucero & Chen Lolliguncula brevis electrophysiology of extracted olfactory organ neurons

1997 Wildenburg Octopus vulgaris ultrastructure of embryonic olfactory Eledone moschata organ