Brain and Sense Organ Anatomy and Histology in Hemoglobinless Antarctic Icefishes (Perciformes: Notothenioidei: Channichthyidae)

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1 JOURNAL OF MORPHOLOGY 260: (2004) Brain and Sense Organ Anatomy and Histology in Hemoglobinless Antarctic Icefishes (Perciformes: Notothenioidei: Channichthyidae) Joseph T. Eastman 1 * and Michael J. Lannoo 2 1 Department of Biomedical Sciences, College of Osteopathic Medicine, Ohio University, Athens, Ohio Muncie Center for Medical Education, Indiana University School of Medicine, Ball State University, Muncie, Indiana ABSTRACT The Channichthyidae, one of five Antarctic notothenioid families, includes 16 species and 11 genera. Most live at depths of m and are a major component of fish biomass in many shelf areas. Channichthyids are unique among adult fishes in possessing pale white blood containing a few vestigal erythrocytes and no hemoglobin. Here we describe the brains of seven species and special sense organs of eight species of channichthyids. We emphasize Chionodraco hamatus and C. myersi, compare these species to other channichthyids, and relate our findings to what is known about brains and sense organs of red-blooded notothenioids living sympatrically on the Antarctic shelf. Brains of channichthyids generally resemble those of their bathydraconid sister group. Among channichthyids the telencephalon is slightly regressed, resulting in a stalked appearance, but the tectum, corpus cerebellum, and mechanoreceptive areas are well developed. Interspecific variation is present but slight. The most interesting features of channichthyid brains are not in the nervous tissue but in support structures: the vasculature and the subependymal expansions show considerable elaboration. Channichthyids have large accessory nasal sacs and olfactory lamellae are more numerous than in other notothenioids. The eyes are relatively large and laterally oriented with similar duplex (cone and rod) retinae in all eight species. Twin cones are the qualitatively dominant photoreceptor in histological sections and, unlike bathydraconids, there are no species with roddominated retinae. Eyes possess the most extensive system of hyaloid arteries known in teleosts. Unlike the radial pattern seen in red-blooded notothenioids and most other teleosts, channichthyid hyaloid arteries arise from four or five main branches and form a closely spaced anastomosing series of parallel channels. Cephalic lateral line canals are membranous and some exhibit extensions (canaliculi), but canals are more ossified than those of deeper-living bathydraconids. We conclude that, with respect to the anatomy and histology of the neural structures, the brain and sensory systems show little that is remarkable compared to other fishes, and exhibit little diversification within the family. Thus, the unusual habitat and a potentially deleterious mutation resulting in a hemoglobinless phenotype are reflected primarily in expansion of the vasculature in the brain and eye partially compensating for the absence of respiratory pigments. Neural morphology gives the impression that channichthyids are a homogeneous and little diversified group. J. Morphol. 260: , Wiley-Liss, Inc. KEY WORDS: brain histology; retinal histology; cephalic lateral line system; olfactory system; brain and ocular vasculature; hyaloid arteries Approximately 25 million years of isolation have endowed Antarctica with the world s most distinctive marine biota (Briggs, 2003). With respect to fishes, perciforms of the suborder Notothenioidei dominate the diversity, abundance, and biomass in the subzero waters of the continental shelf. Here, the nearly 100 Antarctic species form an adaptive radiation (Eastman, 1993, 2000; Clarke and Johnston, 1996) and possibly a species flock (Eastman and Clarke, 1998; Eastman and McCune, 2000). A species flock is an assemblage of a disproportionately high number of closely related species that have evolved rapidly within a circumscribed area where most species are endemic (Ribbink, 1984). With little competition from a sparse nonnotothenioid fauna, notothenioids underwent phyletic as well as morphological and ecological diversification. The notothenioid group most widely known to biologists is the icefishes of the family Channichthyidae unique among adult fishes in possessing pale white blood with only a few vestigal erythrocytes and no hemoglobin. Although a number of channichthyid species were discovered and described during the earliest period of Antarctic exploration (Richardson, ; Günther, 1861; Dollo, 1900; Lönnberg, 1905), a century ensued before the Contract grant sponsor: National Science Foundation; Contract grant number: OPP (to J.T.E.); Contract grant sponsor: National Institutes of Health; Contract grant number: NS (to M.J.L.). *Correspondence to: J.T. Eastman, Department of Biomedical Sciences, College of Osteopathic Medicine, Ohio University, Athens, OH eastman@ohiou.edu DOI: /jmor WILEY-LISS, INC.

2 118 J.T. EASTMAN AND M.J. LANNOO Fig. 1. The channichthyid Chionodraco hamatus (Lönnberg, 1905) showing general morphology, eye size, and location of the single nasal aperture anterior to the eye. From Regan (1914). Approximately 0.5. significance of the hemoglobinless condition was widely recognized and brought to the attention of scientists (Ruud, 1954). The Channichthyidae, one of the five Antarctic notothenioid families, includes 16 species and 11 genera (Eastman and Eakin, 2000; La Mesa et al., 2002). Channichthyids (Fig. 1) are fusiform pike-like fishes with large heads and depressed, elongated snouts. As a family they are the largest notothenioids, with adults ranging in size from cm total length (TL) (Iwami and Kock, 1990). Channichthyids are confined to the Antarctic Region, with the exception of Champsocephalus esox from southern South America and the Falkland Islands. Most live at depths of less than 800 m, although Chionobathyscus dewitti is found as deep as 2,000 m (Iwami and Kock, 1990). Channichthyids are a major component of fish biomass in many shelf areas (Ekau, 1990; Eastman and Hubold, 1999). Channichthyids show remarkable similarities in morphology, ecology, and behavior (Iwami, 1994). While some species are suspected to be primarily benthic and others primarily pelagic, with few exceptions differences in measured buoyancies between the two groups are minimal and probably reflect the combined pelagic-benthic lifestyles of many species (Eastman and Sidell, 2002). Most channichthyids exhibit an active vertical migration to feed on pelagic prey (Iwami and Kock, 1990), especially fish and krill (Pakhomov, 1997). Since there are no obligatory benthivores in this family (Voronina and Neelov, 2001), channichthyids are less dependent on the substrate for food than are most other notothenioids. Skeletal underdevelopment with the persistence of cartilage facilitates the semipelagic lifestyle of many channichthyids (Voskoboinikova, 2001). Among notothenioids, delayed ossification of bones is most pronounced among channichthyids (Voskoboinikova, 1997) and is a mechanism of pedomorphic evolution (Voskoboinikova, 1994). Heterochrony, and especially pedomorphy, has been important in the evolution of notothenioids in general (Balushkin, 1984) and channichthyids in particular (Iwami, 1994). Channichthyids are phyletically derived within the Notothenioidei (Iwami, 1985; Balushkin, 2000; Bargelloni et al., 2000). There are several fully resolved hypotheses of relationships within the Channichthyidae, including those based on morphology (Iwami, 1985; Balushkin, 2000; Voskoboinikova, 2000) and on partial mitochondrial DNA gene se-

3 BRAIN AND SENSE ORGANS OF CHANNICHTHYIDS 119 Fig. 2. Cladogram of relationships for the 16 commonly recognized species of the family Channichthyidae. The tree is the result of a maximum parsimony analysis of combined molecular and morphological datasets. From Near et al. (2003). quences (Chen et al., 1998). Near et al. (2003) employed complete sequences from two mitochondrial genes as well as all morphological data and found that the greatest phylogenetic resolution is obtained when these data are combined in a single maximum parsimony analysis (Fig. 2). Nevertheless, they also found that channichthyids are monophyletic when these datasets are analyzed either separately or together. The loss and reduction of structures is prominent in the evolution of Antarctic notothenioid fishes (Iwami, 1985); however, the lack of erythrocytes and hemoglobin and the variable patterns of myoglobin expression in muscle tissues of channichthyids are especially novel. These features have ensured an intensive study of channichthyids from morphological, physiological, genetic, and evolutionary perspectives (Iwami, 1985; Cocca et al., 1995; Sidell et al., 1997; Tota et al., 1997; Chen et al., 1998; Zhao et al., 1998; Detrich, 2000; Moylan and Sidell, 2000; O Brien and Sidell, 2000; di Prisco et al., 2002; Near et al., 2003; O Brien et al., 2003; Patarnello et al., 2003; Small et al., 2003). The absence of respiratory pigments is a highly specialized condition that is compatible with life under Antarctic conditions because waters are always cold and well oxygenated. However, there are functional consequences associated with the lack of respiratory pigments, and considerable morphological and physiological compensation in the cardiovascular system is necessary to support the hemoglobinless state. It may be that this condition is nonadaptive (Wells, 1990), or a disaptation that has been followed by an adaptive recovery (Montgomery and Clements, 2000) facilitated by the lack of competition from the sparse non-notothenioid fauna. We have been investigating the diversity of sensory and nervous systems among Southern Ocean fishes and the sensory-neural response to conditions found on the Antarctic continental shelf. Our previous studies of notothenioids (Eastman and Lannoo, 1995, 2003a,b; Lannoo and Eastman, 1995, 2000)

4 120 J.T. EASTMAN AND M.J. LANNOO TABLE 1. Cell counts 1 in central area of the retina of channichthyids, with species arranged by increasing depth based on shallowest reported depth of occurrence Species Habitat depth (m) 2 Cones Rods Cones rods Ratio cones:rods 3 Cells in internal nuclear layer Ganglion cells Convergence ratio (cones rods: ganglion cells) 4 Champsocephalus gunnari : :1 Chionodraco hamatus : :1 Pagetopsis macropterus : :1 Chaenocephalus aceratus : :1 Pagetopsis maculatus : :1 Chaenodraco wilsoni : :1 Chionodraco myersi : :1 Chionodraco rastrospinosus ;1, : :1 1 Counts are mean number of nuclei for three replicates in an area of 100 m along the various layers of one Bodian-stained histological section, viewed at 1, Iwami and Kock (1990). 3 Values for cone:rod ratios in notothenioids (modified from Eastman, 1988): high (1:2 4), moderate (1:5 11), and low (1:14 57). 4 Values for convergence ratios in notothenioids (Eastman, 1988): high (58:1), moderate (30 12:1), and low (10 5:1). and non-notothenioids (Eastman and Lannoo, 1998, 2001) serve as a basis for the phyletic and ecological comparisons presented here. In this article we describe the brain and special sense organs of channichthyids in the context of these previous findings. We provide a detailed drawing of the brain and cranial nerves of Chionodraco hamatus and document the anatomy and histology of the brain, olfactory apparatus, retina and ocular vasculature, and cephalic lateral line, with an emphasis on C. hamatus and C. myersi. We compare these species to other channichthyids and relate our findings to what is known about these organs in red-blooded notothenioids living sympatrically on the Antarctic shelf. We also address the following questions: Are the brains and sense organs of channichthyids different from those of other notothenioids and other teleosts? Is the neural morphology of channichthyids influenced by the hemoglobinless condition? How much neural and sense organ divergence is seen in this radiation? MATERIALS AND METHODS Specimens and Nomenclature We collected channichthyids in the southwestern Ross Sea during bottom-trawling on cruises 96-6 (11 December 1996 to 8 January 1997) and 97-9 (20 December 1997 to 10 January 1998) of the RV Nathaniel B. Palmer (Eastman and Hubold, 1999). Chionodraco hamatus, C. myersi, Pagetopsis macropterus, and P. maculatus were the major channichthyid components of the catch and the primary subjects for our current work. Bottom depth at the stations where these species were collected was m. We used a 9.1-m long, 7.6-m effective width Marinovich Gulf Coast style flat trawl, a type of otter trawl. We trawled at a speed of knots for h. Bottom temperature varied from 1.5 to 1.9 C at our sampling stations. We also collected channichthyids during cruise of the ARSV Laurence M. Gould (11 June to 16 July 2001). Our primary trawling site was in Dallmann Bay, Brabant Island ( S, W) in the South Shetland Islands off the Antarctic Peninsula. Here we obtained Chionodraco rastrospinosus, Chaenocephalus aceratus, Champsocephalus gunnari, and Chaenodraco wilsoni. Water depth at this location was m and water temperature was 0.8 to 1.1 C. Nomenclature for channichthyids is that of Iwami and Kock (1990). There are 16 known species and 11 genera of channichthyids; our study includes eight species and five genera (Table 1) representing good phylogenetic coverage (Fig. 2). We utilize the channichthyid phylogeny (Fig. 2) of Near et al. (2003) when evaluating hypotheses of neural diversification. Terminology used for the cephalic lateral line canals is that of Iwami et al. (1999). In describing brain nuclei, we follow the nomenclature of authors included in Northcutt and Davis (1983) and Davis and Northcutt (1983), with the exception of the lateral line nerves. What have been traditionally termed the anterior and posterior lateral line nerves of fishes are actually complexes consisting of several distinct cranial nerves (Northcutt, 1989; Northcutt and Bemis, 1993). We follow Northcutt (1989) in distinguishing the anterodorsal and anteroventral lateral line nerves, although the latter were not obvious in proximal dissections of the cranial nerves in all channichthyids. Histology Specimens, including most used for histological analyses, were fixed by transcardial perfusion of Bouin s fixative onboard ship, according to procedures described previously (Eastman and Lannoo, 1995) and summarized briefly here. After anesthetization in a 150-mg L 1 solution of 3-aminobenzoic acid ethyl ester (MS-222, Sigma, St. Louis), the heart and bulbus arteriosus were exposed. Notothenioid saline solution (O Grady et al., 1982) was prepared, adjusted with NaCl to a concentration of 600 mosm L 1, maintained at ambient seawater temperature ( 1.5 C), and perfused through the heart. Saline was followed by Bouin s fixative. During this perfusion the gills were periodically irrigated with subzero water. Other specimens were preserved by immersion in 10% formalin. We removed brains and proximal spinal cords from four perfused specimens of Chionodraco hamatus and two of C. myersi. Brains were dehydrated in alcohol, cleared in Hemo-De (Fisher Scientific, Pittsburgh, PA) and embedded in paraffin according to standard procedures (Kiernan, 1990). Embedded brains and spinal chords were cut in transverse and longitudinal planes on a rotary microtome to produce sections m thick. Sections were mounted on slides, dried, deparaffinized, stained with either hematoxylin and eosin or Bodian s Protargol for 24 h at 50 C (Clark, 1981), dehydrated, and coverslipped using Cytoseal 60 as the mounting medium. We performed a gross examination on the unperfused brains of five other channichthyid species in the gen-

5 era Chionodraco (one additional species), Chaenocephalus (one species), Chaenodraco (one species), Champsocephalus (one species), and Pagetopsis (one species). Thus, our study includes brains of seven species, although only Chionodraco hamatus and C. myersi were subjected to detailed histological examination. We cut histological sections of the eyes of one to three specimens from eight species with emphasis on Chionodraco hamatus and C. myersi. In examining the eyes, we took dorsoventral strips from the central retina immediately temporal (lateral) to the optic disk. We employed the histological protocol outlined above except that sections were cut at 7 m. Sections were stained with hematoxylin and phloxine B, periodic acid-schiff (PAS) procedure including Harris hematoxylin with glacial acetic acid, Gomori s one step trichrome, PAS-Alcian blue at ph 2.5, or Bodian s Protargol for 24 h at 50 C. We counted olfactory lamellae in two to nine adult specimens from each of eight species. We included the most rostrally located rudimentary lamellae in the count. In some species we also examined histological sections of the olfactory apparatus and transverse and longitudinal sections of the head for the cephalic lateral line system. We used the same histological techniques outlined above. Because of epidermal damage produced by long periods of trawling, we were unable to study free neuromasts and taste buds, and canal neuromasts were damaged by debris forced into the canals. Other Morphological Procedures BRAIN AND SENSE ORGANS OF CHANNICHTHYIDS We examined the bony cephalic lateral line canal system in seven species to determine whether or not channichthyid possess enlarged membranous canals. For this we used specimens that had been cleared and stained with alizarin red S (Taylor, 1967) dissolved in 75% ethyl alcohol (Springer and Johnson, 2000). The head skin was left intact to preserve the canals. We used Microfil (Flow Tech, Carver, MA), a liquid silicon rubber injection compound, to demonstrate ocular blood vessels. After anesthetization, we placed specimens of Chionodraco hamatus and C. myersi ventral side up on an iced surgical board. We cut the bulbus-ventricle junction and cannulated the ventral aorta with a 34-cm length of PE-160 tubing (1.57 mm OD). We blunted the free end on a hot plate and secured the cannula with two sutures. The cannula was in turn connected to an 18-G needle, an 84-cm extension tube, and a 20-mL syringe. The entire apparatus was packed in ice to maintain body temperature. We placed the syringe in a Sage model 341B syringe pump and began the perfusion with heparinized (10 mg ml 1 ) notothenioid saline followed by Microfil. Flow rate was ml min 1. We allowed the Microfil to polymerize while maintaining the specimen on ice for about 1 h, and then preserved the specimen in 10% formalin with subsequent storage in 70% ethanol. We used low viscosity Reprosil (Dentsply International, York, PA), a vinyl polysiloxane dental impression material, to make casts of the nasal sac system of ethanol-stored specimens. In order to enhance the color of the pale orange Reprosil and to reduce the viscosity, we dissolved sufficient oil red O in acetone to produce a dark red solution. We diluted one part of Reprosil base with two parts of the red acetone and mixed thoroughly. We then added one part catalyst with additional mixing. We drew the final mixture into a syringe, attached a cannula of PE-160 tubing, and injected the nasal sac. The specimen was returned to 70% ethanol for h while the cast polymerized. 121 RESULTS Gross Brain Morphology The brain morphology of Chionodraco hamatus is a mosaic of reduced structures when considered from an axial perspective, and expanded structures when considered from the perspective of lobation (Fig. 3). For example, in lateral view the telencephalon is reduced, the inferior lobes are small, and in dorsal view the telencephalon is stalked. A stalked appearance is produced when telencephalic and/or tectal lobes are reduced in size without a concomitant reduction in the length of the neural axis (see Eastman and Lannoo, 1998, 2003b). Within the telencephalon, the rostral portions of the dorsodorsal, dorsomedial, and dorsolateral lobes are small when compared to their caudal components and the dorsoposterior lobe is reduced. In contrast, several structures of the Chionodraco hamatus brain are prominent, including the olfactory bulbs, the caudal lobes of the telencephalic dorsomedial and dorsolateral nuclei, the tectal lobes, the corpus cerebellum (which is dorsally expanded), and the eminentia granulares and crista cerebellares portions of the lateral line system, which form a continuous structure (i.e., meet with each other and the caudal corpus cerebellum without forming a sulcus). Brain Histology Nervous tissue. In the telencephalon, the proportionally larger sizes of the dorsomedial and dorsolateral lobes, and the smaller size of the dorsoposterior lobe, confirm the observations based on gross morphology. At the telencephalic diencephalic junction, the preoptic region is extensive and consists of parvocellular and magnocellular neurons, but no gigantocellular neurons. The diencephalic habenulae are well developed and have a dorsally positioned commissure 80 m (rostrocaudal extent) by 100 m (dorsoventral extent) connecting them. The thalamic nuclei can be divided into dorsal (anterior, dorsal posterior, and central posterior nuclei) and ventral (intermediate, ventrolateral, and ventromedial nuclei) regions. The thalamic region appears normal, with the exception of subependymal expansions (see below) and paired, large-diameter arteries positioned immediately below the central posterior nucleus coursing from the nervous tissue caudally into the subependymal expansion. The glomerular complex, including the preglomerular and glomerular nuclei, is large. The hypothalamic tuberal (anterior, lateral, and posterior) nuclei and the mammillary bodies are exceptionally well developed. The inferior lobes are also large and the recessus lateralis opens subpially (see Wullimann, 1987). The paraventricular organ is a large midline structure and is associated with its own subependymal expansion. In the mesencephalon, the tectal lobes, which appear large from the perspective of gross morphology, exhibit thin laminae, and a narrow overall appearance. The caudalmost portions of the tectum show dense cells characteristic of growth zones in imma-

6 122 J.T. EASTMAN AND M.J. LANNOO Fig. 3. Brain and cranial nerves of Chionodraco hamatus (SL 378 mm) in left lateral and dorsal views ADLL, anterodorsal lateral line nerve; AVLL, anteroventral lateral line nerve; CC, crista cerebellaris of the rhombencephalon; CCb, corpus division of the cerebellum; Dd, dorsal dorsal subdivision of the telencephalon; Dl, dorsal lateral subdivision of the telencephalon; Dm, dorsal medial subdivision of the telencephalon; Dp, dorsal posterior subdivision of the telencephalon; EG, eminentia granularis division of the cerebellum; Ha, habenula; IL, inferior lobe of diencephalon; OB, olfactory bulb; Pit, pituitary gland; PLL, posterior lateral line nerve; SN1, first spinal nerve; SN2, second spinal nerve; SN3, third spinal nerve; SV, saccus vasculosus; Tec, tectum of the mesencephalon; Tel, telencephalon; I, olfactory nerve; II, optic nerve; III, oculomotor nerve; IV, trochlear nerve; V, trigeminal nerve; VII, facial nerve; VIII, auditory/vestibular nerve; IX, glossopharyngeal nerve; X, vagus nerve. ture individuals. The torus longitudinalis is large. There are two midbrain commissures, one dorsally positioned and one ventrally positioned. Both of these split a large, irregularly shaped blood vessel, which is likely venous (see below). The valvula of the cerebellum (VCb) is averagesized and consists of four stacked lobes, although the dorsalmost lobe is incomplete, with only lateral portions developed. The molecular portions of adposed layers do not meet, as is typical of other teleosts (Finger, 1983; Meek and Nieuwenhuys, 1998), but instead holds a ventricular out-pocketing (see below). The corpus of the cerebellum (CCb) consists of a medium- to large-sized lobe that projects caudally. The eminentia granulares (EG) and crista cerebellares (CC) are robust, with a narrow decussation about halfway along their rostrocaudal extent. As was seen in gross appearance, the rostral portions of the crista cerebellares are large and continuous with the ventral portions of the molecular layer of the corpus cerebellum. There are no unusual features associated with the brainstem. For example, the motor neuron pools of cranial nerves (CN) V, VII, IX, and X are typical both in cell size and cell number. Similarly, the sensory areas of the auditory and vestibular systems, the lateral line system, the vagus nerve, and the somatosensory system all appear normally proportioned, although there is a strong decussation of the sensory component of CN V. A second decussation, the cerebellar commissure, is present farther caudally. Mauthner cells are absent; the absence of Mauthner cells has been a characteristic of all notothenioids examined to date (Eastman and Lannoo,

7 BRAIN AND SENSE ORGANS OF CHANNICHTHYIDS 1995, 2003a,b; Lannoo and Eastman, 2000), as well as Southern Ocean scorpaeniform liparids (Eastman and Lannoo, 1998), gadiform muraenolepidids (Eastman and Lannoo, 2001), and perciform zoarcids (unpubl. data). While these features of nervous tissue are interesting, and distinctive for channichthyids, perhaps the most interesting features of channichthyid brains are in what can be considered support structures: the vasculature and the subependymal expansions. Vasculature. Teleost brains are supplied by paired internal carotid arteries that branch to form a series of arteries that anastomose, forming a circle at the base of the brain (Pollak, 1960; Petukat, 1965; Harder, 1975). Branches from these vessels then penetrate the brain from the ventral surface in a roughly bilaterally symmetrical pattern. The primary arteries to the brain, and their major branches, are extraordinarily numerous and large in Chionodraco hamatus. In the telencephalon, arteries penetrate at or near the ventromedial edge of the dorsoposterior lobe (Fig. 4A). Two sets of large, paired arteries are present. The largest (lumen diameter 100 m) set penetrates the ventral telencephalon rostral to the anterior commissure (Fig. 4A); the smaller (80 m) set penetrates in the lateral sulcus between the telencephalic lobe and the preoptic region at the level of the anterior commissure. At least a dozen small- (10 25 m) to medium- (25 50 m) sized penetrating arteries are also observed. In the telencephalon, penetrating arteries form an extensive network of large-diameter vessels (Fig. 4B). From this network, at least one branch runs medially towards the midline (arrows; Fig. 4C,D), penetrates the ipsilateral subependymal expansion (Fig. 4D), and courses caudally within the expansion (arrows; Fig. 4E,F). In the lateral diencephalon, penetrating arteries follow sulci between nuclei and brain divisions except for the inferior lobes, where large, paired arteries penetrate the lateral surfaces of the nucleus diffusus, course caudally, divide into smaller branches, and run in a position immediately lateral to the lateral recess and ventral to the nucleus glomerulosus. These arteries continue in association with the lateral recesses throughout their caudal extent. Medial diencephalic structures receive their blood supply from arteries that course along the pial surface, within the subependymal expansions (see below) associated with the third ventricle. Mesencephalic structures are supplied by arteries that course along the pial surface, within the subependymal expansions (see below) associated with the cerebral aqueduct. These arteries also travel in the tectal ventricles, where they supply the tectum. Medium and long circumferential arteries may also supply the tectum. The torus longitudinales have a 123 single artery, coursing just off the midline, that supplies these paired structures. In the tegmentum, numerous small- to medium-diameter (to 50 m) blood vessels are observed penetrating the ventral and ventrolateral surfaces. A large, triangular-shaped venous sinus drains the diencephalon and mesencephalon (Fig. 5C). This sinus is fed from rostrally positioned veins that are split by decussations as they course dorsally within subependymal expansions (Fig. 5A,B). This sinus is in turn divided (Fig. 5D) and drains through paired, midline veins located at the base of the tegmentum (Fig. 5E,F). This pattern of venous drainage is repeated twice farther caudally in the mesencephalon (see large vein along the dorsal medial surface of the tegmentum on the left side of Fig. 5F). These venous sinuses are substantially larger than those seen in the Bathydraconidae (Fig. 6). Cerebellar regions also exhibit numerous largediameter blood arteries. In the valvula, vessels are conspicuous in the lateral granule cell layer regions, especially where the valvula contacts the midbrain, in the region of the lateral valvular nucleus. In the corpus cerebellum, large ( m) arteries penetrate the nervous tissue in a dorsolateral position, between the molecular and granule cells layers. These arteries then course caudally and in a ventromedial direction in the dorsolateral granule cell layer as they divide. The dorsalmost of these arteries bear caudally within the granule cell layer as large-diameter vessels and continue into the lobe of the corpus as bilaterally paired vessels which eventually branch. The blood supply to the rhombencephalon resembles the pattern seen in the mesencephalic tegmentum. In particular, many small- to medium-diameter (to 50 m) arteries penetrate the ventral and ventrolateral surfaces, which are drained by a network of smaller veins positioned inside the subependymal expansions. A large ventral midline venous sinus receives these veins. This pattern of vascular organization continues into the spinal cord. Subependymal expansions. Subependymal expansions are extensive, occurring in each of the major brain divisions. These expansions are better developed than in the Bathydraconidae (Fig. 7). Expansions consist of either nervous tissue that is less dense than the adjacent tissue or saccular structures that contain cerebrospinal or extracellular fluid. Within each brain division, expansions containing nervous tissue tend to be more extensive than saccular expansions. The cerebellum has only saccular expansions, associated with the valvula, corpus, and decussation of the crista cerebellares. Within the major brain divisions, there is a tendency for saccular expansions to be associated with sulci. Based on the classification scheme of diencephalic subependymal expansions proposed by Lannoo and Eastman (1995), Chionodraco

8 124 J.T. EASTMAN AND M.J. LANNOO Fig. 4. Blood supply to the telencephalic lobes of Chionodraco hamatus. The lumina of vessels show as empty spaces in these perfused specimens. Arteries penetrate the telencephalic lobe along the basal surface (A) to form an extensive network of largediameter vessels within the nervous tissue (B). From this network, at least one branch courses towards the midline (C, arrow), enters the subependymal expansion (D), and courses caudally with the expansion (E F). Hematoxylin and eosin. 20. AC, anterior commissure; Dl, dorsal lateral subdivision of the telencephalon; Dm, dorsal medial subdivision of the telencephalon; IL, inferior lobe of diencephalon; PreO, preoptic area; Tec, tectum of the mesencephalon; Ven, ventral region of the telencephalon. hamatus would rate Type 4, with ependymal cells from each side fusing to form a septum along the midline (Fig. 7E). Atrial natriuretic peptide-like substances, hormones that regulate activity of neurons and monitor homeostasis of cerebrospinal fluid, are present in tanycytes in the subependy-

9 BRAIN AND SENSE ORGANS OF CHANNICHTHYIDS 125 Fig. 5. Venous drainage in the mesencephalon of Chionodraco hamatus. Small, dorsally positioned midline veins (A) course caudally and ventrally within the subependymal expansions and merge (B) to form a large venous sinus (C). This sinus is in turn divided (D) and drained caudally and ventrally by paired veins positioned along the base of the tegmentum (E,F). This pattern is repeated farther caudally (see dorsally positioned feeder veins in E). Hematoxylin and eosin IL, inferior lobe of diencephalon; TL, torus longitudinalis of the mesencephalon; TS, torus semicircularis of the mesencephalon; VCb, valvula division of the cerebellum. mal expansions of C. hamatus (Pestarino et al., 2000). As subependymal expansions and their associated tissues are quasi-vascular structures, their large size in channichthyids may reflect the extensive vasculature and large fluid volumes associated with the hemoglobinless state. Comparative Anatomy: Descriptions of the Brains of Other Channichthyid Species Chionodraco rastrospinosus. This brain is stalked. Olfactory bulbs are smooth-sided. The telencephalon is less lobated than in Chionodraco

10 126 J.T. EASTMAN AND M.J. LANNOO Fig. 6. Vasculature in the brains of channichthyids is more extensive than in their red-blooded sister group the bathydraconids. A comparison of the mesencephalic venous sinus in (A) the channichthyid Chionodraco hamatus and (B) the bathydraconid Gymnodraco acuticeps. Hematoxylin and eosin and 31. Teg, tegmentum of the mesencephalon. hamatus. The telencephalic dorsoposterior nucleus is small. A habenular commissure is present. The tectal lobes do not meet in midline and are small. The torus longitudinales are visible in dorsal view. The corpus cerebellum is small, has no caudal lobe (a sulcus is absent), and projects more dorsally than C. hamatus. The eminentia granulares and crista cerebellares are large and morphologically similar to Chionodraco hamatus. Chionodraco myersi. This brain is stalked. The telencephalic dorsoposterior nucleus is small and the entire telencephalon is reduced compared to the other two species of Chionodraco. A habenular commissure is present. The tectal lobes are relatively large and meet in midline along their caudal extent. The torus longitudinales are visible in dorsal view. The corpus cerebellum is lobed; this species has a large corpus, like C. hamatus. The eminentia granulares and crista cerebellares are large and morphologically similar to C. hamatus and C. rastrospinosus. Chaenocephalus aceratus. This brain is more stalked and larger in dorsoventral extent than in the three Chionodraco species. The olfactory lobes are smooth-sided. The diamond-shaped midline fissure formed by the caudal olfactory bulbs and the rostral telencephalic lobes is not as prominent as in Chionodraco. The telencephalic dorsolateral lobe is reduced caudally; the dorsoposterior nucleus is small. A habenular commissure is present. The tectal lobes are large. The corpus cerebellum is large and lobated. The eminentia granularis and crista cerebellaris are large and morphologically similar to Chionodraco hamatus. Chaenodraco wilsoni. The brain of this species is stalked; it is also rostrocaudally compressed and dorsoventrally expanded. The surface of the olfactory lobes is irregular. The diamond-shaped midline fissure between the olfactory bulbs and the medial surface of the telencephalic lobes is not as prominent as in Chionodraco, but is morphologically similar to Chaenocephalus aceratus. The telencephalic dorsomedial lobe and the dorsolateral lobe are large; the dorsoposterior lobe is small. A habenular commissure is present. The tectal lobes are medium- to large-sized. While the corpus cerebellum is large and more dorsally oriented than in Chionodraco, there is some expansion dorsocaudally and it does not exhibit a lateral sulcus. Champsocephalus gunnari. This brain is slightly stalked. Large olfactory bulbs and olfactory nerve expansions are present. The diamond-shaped midline fissure formed by the medial surface of the olfactory bulbs and the medial surface of the telencephalic lobes is not as prominent as in Chionodraco, but instead tends to be more similar to Chaenocephalus aceratus and Chaenodraco wilsoni. The telencephalon appears scalloped in dorsal view; the dorsomedial lobe and the dorsolateral lobe are large and the dorsoposterior lobe is small. The habenulae are hidden by the rostral tectum. Tectal lobes are medium- to large-sized and meet along the midline. The corpus cerebellum is large and dorsally oriented, expanded dorsocaudally, but does not exhibit a lateral sulcus. The eminentia granulares and crista cerebellares are large; the decussation of the cristae is broad. Pagetopsis macropterus. This brain is not stalked and, unlike all other channichthyids examined, is depressed in dorsoventral extent. While in other channichthyids the tectum extends farther dorsally than the telencephalon, and the corpus cerebellum extends farther dorsally than the tectum, in Pagetopsis macropterus the dorsal plane of these three structures is about level. The surface of the olfactory lobes is irregular. The midline fissure along the olfactory bulbs and the rostral telencephalon does not form a simple pat-

11 BRAIN AND SENSE ORGANS OF CHANNICHTHYIDS 127 Fig. 7. Subependymal expansions and brain vasculature are more extensive in channichthyids than in bathydraconids. The left column shows the brain of Chionodraco hamatus, the right the brain of the bathydraconid Gymnodraco acuticeps. A,B: At the level of the telencephalic anterior commissure. 18 and 28. C,D: At the level of the diencephalic preoptic area and E,F: At the level of the diencephalic posterior commissure G,H: At the level of the rhombencephalic decussation of the crista cerebellaris. 13 and Hematoxylin and eosin.

12 128 J.T. EASTMAN AND M.J. LANNOO tern. The telencephalic dorsomedial lobe and the dorsolateral lobe are large; there is a large sulcus between the two. The dorsoposterior lobe is small and appears oriented towards the midline. A habenular commissure is present. The tectal lobes are medium- to large-sized and meet along the midline. The corpus cerebellum is lobated. The eminentia granularis and crista cerebellaris are large but do not exhibit the distinctive morphology of Chionodraco. Sensory Systems Olfactory apparatus. Nasal and Accessory Nasal Sacs. The nasal sac of channichthyids has a single opening at the end of a short tube (Figs. 1, 8C). This aperture is located over the middle of the oval-shaped olfactory rosette containing closely set lamellae arranged perpendicular to a central raphe. In the classification of lamellar arrangements by Yamamoto (1982), this is a Type G pattern that is widely distributed among various teleostean orders. The olfactory rosette occupies a soft tissue shelf on the floor of the nasal (or sensory) sac. Lateral to the rosette the nasal sac communicates via a long cleft with a well-developed accessory nasal sac (Jakubowski, 1975; Iwami, 1986; Eastman, 1993) located ventral to the nasal sac on each side of the head (Fig. 8A C). The sacs are lined by a thin, stratified squamous epithelium. Jakubowski (1975) describes a system of two small dorsal and two large ventral accessory sacs, designated lacrimal and palatal, in the bathydraconid Gymnodraco acuticeps, and indicates that the arrangement is similar in channichthyids. Our casts of the sacs of Chionodraco hamatus reveal that the dorsomedial and dorsolateral sacs are slight anterior extensions of the main (or sensory) nasal sac. The ventral palatal sac, also visible through the roof of the oral cavity (Fig. 8A), is large, occupying most of the volume of the cast. The lacrimal sac, small and vertically oriented, is the cleft-like connection from the main to the palatal portion of the accessory sac. Dye injections and casts reveal that the accessory sac complex is shaped like an elongated dorsoventrally flattened cone, with the apex directed anteriorly (Fig. 8A C). Ventrally, the palatal portion of the sac is in contact with the skin of the roof of the oral cavity (Fig. 8A). In mm SL specimens of Chionodraco hamatus, weighing 400 g, one accessory sac complex occupies a volume of ml and, assuming weight of the fish is approximately equal to body volume in ml, both sacs therefore constitute % of total body volume. Olfactory Lamellae. Channichthyids have more lamellae than most other notothenioids (Eastman, 1993) with inter- and intraspecific variation for adults as follows (arranged phyletically as in Fig. 2): Champsocephalus gunnari (33 39), Pagetopsis macropterus (29 31), P. maculatus (30 31), Chaenodraco wilsoni (38 47), Chaenocephalus aceratus (45 52), Chionodraco myersi (40 44), C. hamatus (41 46), and C. rastrospinosus (42 46). There is a weak phyletic trend toward increasing numbers of lamellae; phyletically derived species have about 1.3-fold more lamellae than basal species. There is also an ontogenetic increase in the number of lamellae with new lamellae added to the anterior aspect of the rosette. For example, subadult (SL mm) C. myersi have lamellae and subadult ( mm) P. macropterus and P. maculatus have 24 lamellae. The olfactory lamellae of Chionodraco hamatus lack secondary folds and are covered by pseudostratified columnar epithelium that is m thick (Fig. 9A,B). Near the tips of the lamellae the epithelium becomes stratified squamous and thins to m. There are relatively few mucous cells in the epithelium. Other recognizable cell types include basal cells, sustentacular cells, and primary olfactory neurons. Silver staining demonstrates the Fig. 8. Olfactory apparatus of channichthyids and comparison of ocular vasculature of channichthyids and red-blooded notothenioids. A: Palatal portions of accessory nasal sacs of Chionodraco myersi (SL 249 mm) viewed ventrally through the roof of the oral cavity. Sacs filled with 0.1% cresyl violet acetate B,C: Reprosil casts of nasal (sensory) and accessory nasal sacs of two different specimens of Chionodraco hamatus. Anterior is to the left. Dorsal view (B) of the left sac system of a 350-mm SL specimen showing small nasal (sensory) sac with dorsomedial and dorsolateral extensions located dorsally and large, ventrally located accessory sac. The lacrimal portion of the accessory sac unites the nasal sac with the more ventrally located palatal portion of the accessory sac Ventromedial view (C) of the right sac system of a 355-mm SL specimen showing single nasal aperture and impression of lamellae of olfactory rosette on underside of cast of nasal sac. Most of the cast consists of the palatal portion of the accessory sac D G: Left eyes of four notothenioids showing yellow or orange Microfil in hyaloid arteries at the vitreoretinal interface. These species lack a choroid rete. Cornea, iris, lens, and vitreous body have been removed; the retractor lentis muscle is situated midventrally in all specimens. D: Red-blooded nototheniid Pagothenia borchgrevinki (SL 193 mm) with a widely spaced radial branching pattern emanating from optic disk E: Red-blooded bathydraconid Gymnodraco acuticeps (SL 254 mm) with a more dense radial pattern. Temporal avascular area is artifact of incomplete filling F: Hemoglobinless channichthyid Chionodraco hamatus (SL 390 mm) showing five main branches of hyaloid artery that repeatedly give off other branches forming a dense network of vessels. Smaller branches near plus ( ) are m in diameter; larger vessels near asterisk (*) and elsewhere are m. Areas not filled are artifacts where vessels attached to vitreous body were removed G: Hemoglobinless channichthyid Chionodraco myersi (SL 366 mm) with five main branches of hyaloid artery, dense branching pattern, and prominent annular vessel D, dorsal; N, nasal; T, temporal; V, ventral; av, annular vein; dl, dorsolateral extension of nasal sac; dm, dorsomedial extension of nasal sac; lac, lacrimal portion of accessory nasal sac; mv, maxillary valve; na, nasal aperture; ns, nasal sac; olam, olfactory lamellae; pal, palatal portion of accessory nasal sac; rl, retractor lentis muscle.

13 BRAIN AND SENSE ORGANS OF CHANNICHTHYIDS C O L O R Figure

14 130 J.T. EASTMAN AND M.J. LANNOO C O L O R Figure 9

15 BRAIN AND SENSE ORGANS OF CHANNICHTHYIDS nuclei of all cell types but does not clearly identify features of the primary olfactory neurons (Fig. 9B). However, when stained with Gomori s trichrome, olfactory neurons have red fusiform nuclei in the mid-epidermis and slightly acidophilic dendrites that reach the free surface of the epithelium (Fig. 9A), characteristics of primary olfactory neurons (Grizzle and Rogers, 1976; Groman, 1982). Axons are thin and not usually identifiable in the basal epithelium. The olfactory neurons are scattered throughout the length of the lamellae with the exception of the tip. Detail of the shape of the receptor endings at the free surface was not preserved in the epithelium of our trawled specimens. Visual system. Eye Size. The eyes are relatively large and laterally oriented in channichthyids (Fig. 1). When expressed as a percentage of head length, the modal range for eye diameter is 19 22% in many species including Chionodraco (Iwami and Kock, 1990). Ocular Vasculature. As background information we note that the eyes of most teleosts have a dual set of arteries supplying blood to the sclerad and vitread surfaces of the retina (Nicol, 1989). With the exception of anguilliforms (eels) the retina is avascular, lacking vessels within its substance (Nicol, 1989). In phyletically basal notothenioid species with a choroid rete mirabile, eyes also have a dual blood supply (Eastman, 1988, 1993). The ophthalmic artery, a continuation of the efferent pseudobranchial artery, enters the sclera dorsal to the optic nerve and supplies the choroid rete and the choriocapillaris adjacent to the sclerad aspect of the retina. The choriocapillaris (Fig. 9C,D) is a system of interconnected capillaries located between the choroidal pigment layer and the sclera. Eyes also receive blood from the optic artery, an anterior branch of the arterial circle, formed in part by the internal carotids, at the base of the brain. The optic artery penetrates the sclera ventral to the optic nerve, enters the optic nerve, Fig. 9. Olfactory and retinal histology of channichthyids. A,B: Epithelium on one side of an olfactory lamellus of Chionodraco hamatus. Staining with Gomori s trichrome (A) shows red fusiform nuclei and slightly acidophilic dendrites of primary olfactory neurons. Staining with Bodian s Protargol (B) shows darkly stained nuclei of all cell types, especially those of basal and sustentacular cells C F: Retina of three channichthyid species showing layering and distribution of rods and cones. All panels are the same magnification and aligned horizontally along the external limiting membrane. Müller cells, a type of glial cell, are seen extending radially through the retina. There is artifactual separation of layers in C. C E: stained with Gomori s trichrome. F: stained with Bodian s Protargol to demonstrate nuclei in various layers. C: Chionodraco hamatus. D: Chionodraco myersi. E F: Pagetopsis maculatus , retinal pigment epithelium; 2, outer segments of photoreceptors; 3, inner segments of photoreceptors; 4, external limiting membrane; 5, external nuclear layer; 6, internal nuclear layer (primarily bipolar cells); 7, ganglion cell layer; 8, optic nerve fibers; c, cone ellipsoids; cc, choriocapillaris; ha, hyaloid arteries; mu, process of Müller cell; r, rod photoreceptors. 131 and, at the optic disk, ramifies into a series of hyaloid arteries at the vitreoretinal interface. All notothenioids lack additional vascular specializations such as a falciform process and lentiform body. We filled the ocular vasculature with Microfil in Chionodraco hamatus (Fig. 8F), C. myersi (Fig. 8G), and Chaenocephalus aceratus. Unlike the phyletically basal notothenioids, channichthyids lack a choroid rete. Our perfusions did not display vessels identifiable as efferent pseudobranchial or ophthalmic arteries, and these may not be present in channichthyids. A large optic artery is the primary blood supply to the channichthyid eye. This vessel parallels the optic nerve and enters the sclera ventral to the nerve. It enters the optic nerve and at the optic disk it divides into four or five main branches (diameter m) aligned along dorsoventral and nasotemporal axes (Fig. 8F,G). The dorsal branch is paired and the ventral branch supplies the retractor lentis muscle. These main branches ramify into the exceptionally well-developed series of hyaloid arteries shown in Figure 8F,G. They are variable in size the smaller arteries measure m, with a typical diameter of 60 m in the three species, but larger arteries ( m) are also present (Fig. 8F,G). Smaller hyaloid arteries in red-blooded nototheniids measure 30 m (Fig. 8D,E). The branching architecture and spacing of hyaloid arteries in channichthyids is different than in redblooded notothenioids that also lack retia (Fig. 8D,E) and, we believe, in other teleosts studied to date. Channichthyid hyaloid arteries arise from four or five main branches and form an extensively anastomosing series of parallel channels (Fig. 8F,G) rather than the radially arranged system of discrete, partially dichotomous branches seen in red-blooded notothenioids (Fig. 8D,E). This dense pattern of closely aligned vessels is remarkably homogeneous over most of the retinal surface in channichthyids, although it becomes less dense at the extreme periphery as vessels coalesce before joining the annular vein. The hyaloid arteries are more densely arranged in the ventral hemiretina, although this is not conspicuous. Generally, the pattern in the nasal and temporal hemiretinae is of similar density, but in Chionodraco myersi the pattern is slightly more dense nasally than temporally (Fig. 8G). The hyaloid arteries drain to a single circumferential annular vein ( m) located near the ora serrata (Fig. 8E G), and this exits the interior of the eye midventrally as the ventral choroidal vein. Traveling to the back of the eye, the ventral choroidal vein widens into a large triangular-shaped sinus that probably represents part of the choriocapillaris. Histology indicates that the choriocapillaris in this area shows little subdivision into discrete vessels and appears as a continuous channel (Fig. 9C,D). The sinus drains dorsally to an optic vein that parallels the optic artery.

16 132 J.T. EASTMAN AND M.J. LANNOO In the apparent absence of the ophthalmic artery in channichthyids, other arteries may contribute to the choriocapillaris, although their input is not extensive. For example, our casts filled branches of the orbitonasal artery that enter the dorsal aspect of the sclera and choroid, and arteries supplying the extrinsic eye muscles also continue into the sclera and choroid. In summary, the vasculature on the vitread side of the channichthyid retina is extensive and homogeneously distributed, and the vessels are larger in diameter and more closely spaced in channichthyids than in Pagothenia borchgrevinki and Gymnodraco acuticeps, two red-blooded species used for comparison. For example, in channichthyids the smaller hyaloid arteries are 2-fold larger in diameter, the main stem hyaloid arteries are 2 4-fold larger, and the annular vein is 3.5-fold larger. Retinal Histology. The central retina is 200 m thick, excluding the optic nerve fiber layer. Layering of the retina is similar to that in other teleosts and other notothenioids and, with minor exceptions, the retinae are histologically similar in the eight species we examined. All possess a duplex retina with both cone and rod photoreceptors. Although rods are dominant by number in all species (Table 1), twin cones are the qualitatively dominant and most conspicuous photoreceptor in cross sections of the central retina (Fig. 9C F). Most species also have single cones, especially in the peripheral retina. Rods are arranged in a single bank and in some species occupy gaps between cones; gaps are most prominent in Chionodraco myersi (Fig. 9D), C. hamatus (Fig. 9C), C. rastrospinosus, and Chaenocephalus aceratus. Among the eight species we examined there is a 1.3-fold difference in the number of cones, a 1.8-fold difference in the number of rods, a 2.2-fold difference in the ratio of cones:rods, and a 2.0-fold difference in the number of cells in the internal nuclear layer (Table 1). The magnitude of these differences is relatively modest compared to the fold differences seen among members of the closely related Bathydraconidae (Eastman and Lannoo, 2003b). For channichthyids in general, cone:rod ratios are moderate, with Chionodraco myersi tending to be lower. Convergence ratios are also moderate, with C. rastrospinosus having a higher value. Although species in Table 1 are arranged according to increasing depth of occurrence, most species are eurybathic and the expected decrease in the number of cones and increase in the number of rods with depth is not obvious. This is illustrated by Champsocephalus gunnari the large number of rods suggests that they normally inhabit depths greater than the shallow extreme of their reported depth range. Furthermore, the small number of rods in Pagetopsis maculatus and Chaenodraco wilsoni may indicate that these species normally live within the shallower portions of their depth ranges. Although the internal nuclear layer (Fig. 9C F), composed of horizontal, bipolar, and amacrine cells, generally has more cells in species from shallow water, this tendency is also not obvious in channichthyids. Finally, counts for Chionodraco hamatus and C. myersi indicate that the proportion of photoreceptors is phyletically plastic, as these two closely related species (Fig. 2) are at the opposite extremes for numbers of rods in our sample of eight species. Cephalic lateral line canals. Figure 10A shows the pattern of the dorsally located cephalic lateral line canals as well as the degree of ossification and pore size in Chionodraco myersi. Additional description is unnecessary since complete details of the canal and pore system are available for 15 of the 16 channichthyid species (Iwami, 1985; Iwami et al., 1999). Furthermore, interspecific variation in the canal and pore pattern in this family is less marked than in other notothenioid groups (Iwami et al., 1999). It has been suggested, but not confirmed anatomically, that the canals in Pagetopsis macropterus and Chionodraco hamatus are membranous (Montgomery et al., 1994). Our purpose here is to document the size and the extent of the ossification of the canals or, from another perspective, to determine how membranous they are. The infraorbitals of the four species shown Figure 10B E encompass the range we encountered in specimens of seven alizarin-stained species. Among these four species of the same size ( mm SL), the third infraorbital ranges from mm at the widest point and from mm at the narrowest point. The infraorbitals of Chionodraco hamatus are more bony (or less membranous, Fig. 10B) than those of C. myersi (Fig. 10C), Chaenocephalus aceratus (Fig. 10D), and Champsocephalus gunnari (Fig. 10E). The mean percentage of the infraorbital canal roof that is ossified for the series in Figure 10B E is 40%, 26%, 20%, and 25%, respectively. Therefore, with Fig. 10. Cephalic lateral line system of channichthyids. Dorsal view (A) of alizarin-stained and cleared specimen of Chionodraco myersi (SL 330 mm) showing arrangement, size, and degree of ossification of the dorsal portion of the canal and pore system. Removal of lower jaw and many ventral skull bones has produced the dorsoventral flattening necessary to demonstrate the infraorbital series B E: Lateral view of alizarinstained right infraorbital (io) canal bones of channichthyids of the same size demonstrating interspecific differences in size and degree of ossification of these membranous canals. More anterior infraorbitals are to the right. B: Chionodraco hamatus (SL 340 mm), io 2 5. C: Chionodraco myersi (SL 330 mm), io 2 5. D: Chaenocephalus aceratus (SL 345 mm), io 2 6. E: Champsocephalus gunnari (SL 345 mm), io F: Cross section of membranous infraorbital canal of Chionodraco hamatus. A neuromast is not present in this area. Epidermis has been abraded away in this trawled specimen. Gomori s trichrome. 41. b, bone of canal; ci, canaliculi; d, dermis of head skin; hy, hypodermis; io, infraorbital canals; lum, lumen of canal; m, mucosa of canal; mus, muscle; n, nerve; so, supraorbital canals; soc, supraorbital commissure; st, supratemporal canals; t, temporal canals; ull, upper lateral line canals; v, blood vessels.

17 BRAIN AND SENSE ORGANS OF CHANNICHTHYIDS C O L O R Figure