Prepared by Judith Handlinger

Systematic Fish Pathology Part 1. “Consider the Fish”An evolutionary perspective on comparative anatomy and physiology Prepared by Judith Handlinger Fish Health Unit, Animal Health Laboratory, Department of Primary Industries & Water, PO Box 46, KINGS MEADOWS, Tasmania 7249 for The Australian Animal Pathology Standards (AAPSP) program

Before Entering Training Program READ ME Welcome to this series of training modules, prepared for The Australian Animal Pathology Standards (AAPSP) Program as part of its Veterinary Pathology training and further education programs. This has been undertaken with financial and in-kind support from Animal Health Australia and from the Tasmanian Department of Primary Industries and Water. It is based on a teaching slide set used for short courses on fish histopathology, aimed at students with a range of academic backgrounds, and varying prior knowledge of pathology or of fish. It will therefore provide opportunities for revision of basic pathology principles, as well as fish-specific pathology. Slides are representative of the pathology found in Australian fish in this (Tasmanian) fish laboratory, rather than a comprehensive record of all fish diseases, or diseases of all fish. There are some examples from other laboratories, including exotic diseases.

Acknowledgments Most material used was generated in the Fish Health Unit within the Tasmanian Department of Primary Industries & Water, and includes cases and photographs from many other contributors including Jeremy Carson, David Taylor, Stephen Pyecroft, Richmond Loh, Kevin Ellard, Paul Hardy-Smith and Barry Munday. Contributors of cases from other laboratories have been acknowledged wherever possible, and specific material and photographs used with permission. Any inadvertent omissions in this regard are unintended. Material exotic to Australia includes slides distributed for general teaching purposes, and slides contributed specifically for the DPIW Fish Teaching set, and are acknowledged as such. In this presentation, photographs of animals have also been included from other sources such as web-sites, when other photographs were not available, and these are acknowledged whenever possible.

Aim of the course The major aim of this course is to convey an approach to diagnosis, not to cover all fish diseases. The systematic approach is relevant, regardless of species (this is just applying mammalian pathology training to fish). Nevertheless, the pathology of any species can only be interpreted by comparison with the known normal. As there are more fish species (>30,000) than all the other vertebrates together, we must appreciate the diversity of fish species, and resist the temptation to consider fish just a new “species” for study. This course does not cover all Australian fish. It uses available case material to impart knowledge of general fish pathology, including general patterns of fish diseases, and to introduce the types of pathogen common in fish.

Course Outline A. Systematic Fish Pathology 1. Consider the Fish": An evolutionary perspective on comparative anatomy and physiology (this presentation) 2. Pathology of the kidney I – interstitial tissue Part A 3. Pathology of the kidney II – interstitial tissue Part B 4. Pathology of the kidney III – the nephron 5. Pathophysiology of the spleen 6. Fish haematology 7. Fish immunology – evolutionary & practical aspects 8. Pathology of the digestive system I – the oesophagus, stomach, and intestines. 9. Pathology of the digestive system II – the liver and pancreas. 10. Pathology of fish skin 11. Pathology and diseases of circulatory / respiratory system – heart, gills and vessels 12. Pathology of the musculoskeletal system and nervous systems 13. Pathology of gonads and fry (Course B: Presentations 14-16 - mollusc pathology)

Principles covered To apply a knowledge of mammalian pathology to fish you need: Comparative pathology (studying fish is therefore also a route to greater understanding of mammalian responses) Basic knowledge of fish anatomy, histology and responses, and how these differ from mammals Some knowledge of pathogens likely to be encountered in fish. This presentation concentrates on comparative anatomy.

So what’s different about fish? So what’s different about terrestrial animals? Fish were here first. They’ve been highly successful, & are our ancestors! Fish (and invertebrates) should not be considered as “primitive” (having survived on earth for longer than any land vertebrates), & we need to know a little about our evolution.

What’s a fish? When we discuss fish pathology we are mainly talking about the ray-finned fish, which make up the major group of bony fish. The other bony fish group is the less common lobe-fin fish. There are other groups of fish that may be encountered, particularly the sharks and raysthat together make up the cartilaginousfish, while lampreys & hagfish, the extant (i.e current) jawless fish, are rarely presented. The ray-finned fish share many characteristics, but are a very diverse group, comprising more species than all other land vertebrates together. The relationships of these groups is shown below:

Where did they come from? Reminder – vertebrate evolution (just to get the terms right): Vertebrates belong to the phylum chordata, which has three subphyla: Urochordata (tunicates), Cephalochordata (lancelets or amphioxus), and Vertebrata (vertebrates). All chordates, at some time in their lives, have four distinctive features: A notochord which is a long rod of stiffened tissue that supports the body. Later in development, it changes to bony units in vertebrates. A dorsal, tubular nerve cord lying above the notochord and gut A muscular pharynx with gill slits at the entrance to the digestive tract (at least in the embryo). A tail near the anus (at least in the embryo; may be rudimentary).

Vertebrate ancestors - early chordates Urochordates (tunicates – “sea squirts”) only have a notochord and look like tadpoles at the larval stage: Probably we evolved by just keeping the larval stage. Photos fromhttp://trc.ucdavis.edu/biosci10v/bis10v/week9/08tunicates.html Lancelets (Amphioxus) are the first fish-like animals, with tapered bodies, segmental fish-like muscles, a closed circulation (but no red cells), a dorsal nerve cord but no brain, no jaws, no vertebrae.

Jawless fish – Hagfish & Lampreys Jawless fish (the cyclostomes or agnatha), are the most primitive of true vertebrates (though the vertebrae are rudimentary ) . They look eel-like but have cartilaginous skeletons with no jaws derived from gill arches, no ribs, no shoulder or pelvic girdles and no paired appendages. The gill passages are expanded into pouches to give internal gills connected through pores (rather than slits) with the exterior; with more than five external openings of the gills. Modern species are secondarily adapted for parasitic life. Hagfish have no externally visible eyes, though lampreys do (with a lens) Lampreys show other vertebrate features not seen in hagfish (probably lost). They show fish-like haematopoietic tissue in the kidney and along the gut. These are poorly developed in hagfish. In the lamprey they are better developed in the long larval stage (including a protospleen), than in the adult. They produce lymphocyte-like cells (but no antibody), erythrocytes and granulocytes, but no cells differentiated as monocytes or thrombocytes. This includes lymphoid accumulations in the pharynx region (not yet a thymus). Following photos from: http://universe-review.ca/option2.htm#L www.english-nature.org.uk/.../lamprey.html http://www.fhwa.dot.gov/environment/wildlifeprotection/index.cfm?fuseaction=home.viewPhotos&articleID=76 Hagfish mouth – multiple sites, source unknown.

Hagfish mouth Lamprey http://universe-review.ca/option2.htm#L Lamprey mouth Pacific lamprey Lampreys on Great Lakes fish

Larval lampreys, showing gill arches but no jaws. Notochord Spinal chord

Cartilaginous fish (sharks & rays) Sharks and their relatives are the most primitive extant group of jawed fish. They have a vertebrate-type immune system, including a thymus and antibody production, that apparently developed along with jaws(see presentation 7), although there are some differences from both the teleost bony fish and land vertebrates. They retain multiple gill slits (one for each gill arch). The thymusis seen as paired lobed masses dorsal to the first 2 gill arches of young juveniles,with a clear cortex and medulla structure that is often (but not always) lost in higher fish, and typically involutes after a few weeks. Internal organs are similar to bony fish, and include a well-defined spleen (with red and white pulp), and some lymphoid organs unique to this group (the Leydig and epigonal organs located in oesophagus and gonads, & smaller accumulations in other organs including heart).

The jaws are obvious. Note the 5 gill slits

and also for the rays

So who is our ancestor? The diagram on the next slide shows that land vertebrates evolved from an early branch of teleost or bony fish. This was thelobe-fin group, which includes lung-fish & the coelacanth: we inherited and then modified the immune and anatomical heritage of one early fish group. The other main groups, the ray-finteleosts, later diversified into multiple fish classes and evolved some features not present in our ancestors. The cartilaginous fish (sharks and rays), and the jawlesslampreys and hagfish are progressively further away from the evolutionary point of the fish: terrestrial divergence. Lobe-finned Coelacanth uwinnipeg.ca

From Pilstrom and Bengten, 1996. Immunoglobulins in fish – genes, expression and structure. Fish & Shellfish Immunology 6: 243-262. (Boxed at right = species with immunoglobulin data available) Note terrestrial animals arose from a relatively primitive branch of bony fish, while other teleosts diversified.

Back to fish v mammals:So what is different? The question, then, is not “what are fish adaptations to an aquatic environment”? Rather, what anatomical and physiological changes were necessary to colonise the land, and how have these defined the terrestrial animal that you are likely to be using as a template to interpret pathology of fish?

Basic Anatomic Differences Compare the sleek lines of the fish….

…with these angular shapes

fish Shape mammal Sleek streamlined shapes important to reduce drag - except for sedentary fish where camouflage or display is more important. Many terrestrial animals are angular - except for marine mammals & flying birds: aerodynamics important only for high speed. Sedentary sea-dragon

fish Mechanics of locomotion mammal Limbs (fins) small, with minimal joints, largely for stability & manoeuvrability. Large jointed limbs needed to support body above the ground. Result: More energy is expended by all land animals for supporting the body above the ground. (Reptiles may minimise this by spending most time resting on the surface, with elevation exertion only when required). Minimal energy is used for vertical support: this is maintained by the swim bladder, which is pressurised to float at a desired level, allowing short bursts of vertical propulsion.

fish Propulsion terrestrials • The major propulsion force (and muscle mass) is usually trunk muscles. • These are attached to the spine as parallel myomere segments rather than individual muscles with specific directional pull. • The myomere shape is accentuated by the muscle haemorrhage in the photo below Propulsion is accomplished through muscles anchored to limb bones. Limbs are the major propulsion force & bear a much greater % of muscle mass. Movement of land animals is in general less efficient. The combination of extra energy needs & vulnerability to fluctuating air temperatures was a driver for the evolution of homeothermy. Homeothermy is also energy-expensive Result: fish have lower basal energy needs, thus much greater food conversion efficiency (a major attraction of aquaculture as a food industry). This arrangement is very efficient biodynamically.

fish Skeleton terrestrials Typical bones arose in early jawed fish as calcified collagen/cartilage To maintain the limb strength needed to support large mammals in air & provide muscle anchor points, the bones have become thick and heavily calcified. So the cartilaginous bones of sharks and rays represent secondary loss (not an earlier stage of evolution) To provide strength with minimal weight, the centre is not filled with heavy bone, creating a medulla. Fish limbs bones are small, with minimal joints, with no hollow medulla (haematopoietic tissue in located in the kidney and spleen) • Results: • Mammal bones need additional blood supply (development of Haversian system) and to reduce weight, the centre has become hollow. Osteogenesis of bone extremities was accomplished by ending the bone with a simple plug of cartilage 2. The resulting bone medulla is a handy place to park haematopoietic tissue. 3. Cartilage at end of long bones is too flexible for weight bearing, so terrestrial animals need growth-plates and well-lubricated synovial joints Note: There is speculation that moving the HP tissue to bone medulla provides protection from the additionalradiation encountered by terrestrial animals.

Joint Simple cartilage joint

Strong limb joints with growth plates and well-developed synovial surfaces evolved with the need for weight bearing (exemplified by the frog’s equipment for long hops) Fish skeleton From http://www.zoology.ubc.ca/courses/bio204/lab7_photos.htm Frog skeleton From Myspace – origin uncertain

Bones of our ancestors From mbscientific.com/wiki Terrestrial limbs adopted the bones of our lobe-finned ancestor Above; the fin bone structure of: 1) A typical ray-finned fish (perch), showing fins that are webs of skin supported by bony spines attached directly to a single bone linking them to the main skeleton. 2) Jointed bones of the fleshy, lobed fins of a modern lungfish 3) The jointed fins of our ancient lobe-finned rhipidistian ancestor.

Smooth mucus-covered epithelial surface gives maximum hydrodynamics & protection. Actual skin epithelium is thin and fragile, so scales (small bones) are embedded within the dermis. Surface epithelium has become thick & cornified to prevent moisture loss in air. Hair/feathers for protection and insulation (and for flight) fish Surface terrestrials

Fish skin Fish skin, showing scales, covered by epithelium Histological appearance of fish skin, with embedded scales (small bones, arrows)

Actual skin epithelium is thin, fragile, hence scales. Eyes remain bathed in fluid & mucus - no need for eyelids in typical fish Internalised auditory canals readily transmit sound through water. These are specialised parts of the lateral line sensory system that also detects water movement & orientation. Because of the need to maintain homeothermy, hair & feathers have developed for insulation. Eyelids & tear glands developed to maintain corneal moisture External ears of various sizes developed to capture airborne sound waves. fish Surface Appendagesterrestrials

fish Reproduction terrestrials Relatively large numbers of eggs are usually produced, with fertilization in water outside the body. Water environment allows hatch at early stage of development: typically small amounts of yolk. A few species show parental care, some are live bearers (internal fertilization) Long foetal stage (need well developed skin and lungs to cope with air environment). Longer, if immediate mobility is required. Larger investment in each individual egg (with shells or placenta). Fewer offspring: increased parental care.

Variation in fish reproductive strategies Eggs laid in stony hollow, fertilized externally and left (eg salmonids – but extraordinary efforts to return to a suitable spawning environment). One to several spawnings / fish, moderate numbers of ova, relatively large amount of yolk Marine fish typically have many small eggs and a prolonged larval feeding stage. Salmon fry Small fish (eg aquarium species) often show more parental care, smaller but more frequent batches, eg: • discus lay eggs on surfaces in nest area, clean the developing eggs, & produce a skin “milk” for early nutrition. • mouth brooding fish Pregnant Guppy • pouch brooding fish (seahorse) - the male receives the eggs from the female, fertilizes them within the pouch (and produces very few sperm) • internal larval development eg “live bearers” such as the guppy (internal fertilization) Thus there is varying but increasing parental care and investment per egg Larval Guppy

Fish Internal Anatomy

Brown trout (above) showing the operculum or gill cover over the single gill opening typical of bony fish. Operculum reflected (below) to show multiple gill arches (typically 5).

Shark anatomy, including internal gill structure, is similar except for a very pale fatty liver (this species).

fish Respirationmammal Mammals are homeotherms with high energy need – require high levels of oxygen absorption. Very large surface area of lungs but less effective ventilation (not flow-through) Still need moist surfaces for exchange Water is denser than air, holds a relatively small amount of dissolved oxygen, and absorbs light more than does air Result: increased vascular resistance, so need more heart chambers & thicker vessels. Oxygen exchange in fish is via the gills – a thin epithelium over a vascular network through extensive secondary lamellae. Relatively high surface area. Note: Mammals discarded the erythrocyte nucleus, apparently to increase hemoglobin content per cell (and to ensure only non-viable cells in circulation?) - but most vertebrates retain nucleated erythrocytes. Counter-current flow (blood flows in reverse direction to water flow) aids oxygen exchange Oxygen is carried by nucleated elliptical erythrocytes rich in haemoglobin (not seen in early chordates, though does occur in some invertebrate worms)

fish Circulation mammal Fish hearts • Mammals require a dual pump heart to overcome: • the resistance of the larger pulmonary bed • the greater vertical pump pressures needed in erect mammals A 2-chambered, spongy heart effectively maintains blood flow through the gills and the tissues of fish. Results in a short sharp pulse – vessel walls need to be thicker. Ventricles empty into an elastic bulbus that helps to maintain a pulse of low pressure but prolonged amplitude, to maintain an even slow blood flow.

Evolution of the extra heart chambers apparently started with auxiliary circulation through the air sacs of air-breathing lobe-finned fish Images from mbscientific.com/wiki

fish Haematopoiesis mammal Major sites of haematopoiesis in teleosts are the head kidney (pronephros that has no nephrons); its extension as the interstitium of the tail kidney (mesonephros) of most fish; and the spleen. The major site of mammal haematopoiesis is the bone marrow Specialized lymphoid organs include the thymus, spleen, lymph notes. Lymphoid organs include the thymus, spleen, kidney and GALT (gut associated lymphoid tissue). Smaller lymphoid aggregates occur as the GALT, more broadly expressed as part of the MALT (mucusa-associated lymphoid tissue). Kidney Of these, Peyer’s patches are large enough to be visible grossly Spleen

Fish haematopoietic tissueanatomy Kidneys are paired, located against the dorsal margin of the peritoneal cavity close to the midline, sometimes coalescing. Though grossly they may appear as a single organ running the length of the abdominal cavity, there are 2 distinct zones. In some fish the transition is invisible grossly, or they can be lobed (anterior & posterior) Anterior (head) kidney Only the posterior kidney has the renal (nephron) elements. Haematopoietic tissue is present in both lobes (most fish), giving the dark colour. Posterior (tail) kidney Haematopoiesis also occurs in the spleen. Salmon kidney: head - tail junction

fish Immune systemmammal(simple version: you can’t see it, but you need to understand it) Fish have an adaptive antibody-based immune system, with B- and T-cells, athymus, and immune memory. This combination arose relatively suddenly (in evolutionary terms) in jawed fish. Higher vertebrates have an adaptive antibody-based immune system using more “highly evolved”, varied and generally smaller immunoglobulins, though IgM is still retained as the first antibody produced. This followed the evolution of recognisable lymphoid organsin primitive fish groups. Ig range includes specialized surface immunoglobulin (IgA), and an allergic form (IgE). Innate immunity is also present (but its complexity is now only becoming fully appreciated). Fish immunoglobulin is of IgM type All fish groups also have innate immunity, similar to invertebrates. Invertebrates show neither an adaptive response nor immune memory, and were regarded as having no immune system – rather surprising, considering their long & successful history.

The thymus was first seen in jawed fish, located in the roof of the gill chamber. Note the close proximity to the head kidney. Anterior Sagittal section of salmonid. Gonad Liver Gill

Thymus Gills

Note the thin covering epithelium Thymus

Another thymus located next to gill cavity (salmon fry).

Fish thymus histology: Note lack of well-defined cortical and medullary regions. Hassall’s corpuscles are poorly defined (relative to mammals), though similar cells are recognisable.

Details of thymus. Note thin epithelial surface separating thymus cells from direct interaction with the aquatic environment

Adult amphioxus of the species Branchiostoma lanceolatum with mature gonads (yellow). From http://www.biolsci.org/v02p0030.htm So typical fish have evolved to have a similar immune system and generally similar organs to land vertebrates, and we are ready to move to Presentation 2, to start an overview of the histological appearance of fish pathology and reactions, using the kidney to show these. Before you do, just to finish this evolutionary ramble, a tribute to our ancestral relative, amphioxus: “the amphioxus looks like nothing more than a pallidly animated anchovy fillet” (Henry Gee) In the early XX century, amphioxus became a favorite of the summer students at the Biological Laboratories of the Cold Spring Harbor Laboratories on Long Island, New York, who invented the chorus of a song that became the “It’s a long way from amphioxus” song, authored by the marine biologist Phylip Pope in 1921 and popularised by the folk singer Sam Hilton in 1961. This is a catchy little summary of our shared evolution.

IT'S A LONG WAY FROM AMPHIOXUS A fishlike thing appeared among the annelids one dayIt hadn't any terrapods or cetae to displayIt hadn't any eyes or jaws or ventral nervous chordBut it had a lot of gillslits and it had a notochord.Chorus.It's a long way from amphioxusIt's a long way to us.It's a long way from amphioxusTo the meanest human cuss.So goodbye to fins and gillslitsHello lungs and hair,It's a long, long way from amphioxusBut we all came from there.Well, it wasn't much to look at and it scarce knew how to swimAsterius was very sure it hadn't come from him.The mollusks wouldn't own it and the arthropods got soreSo the poor thing had to burrow in the sand along the shore.Chorus. It burrowed in the sand before it grabbed in with its tailAnd said gillslits and myotomes are all to no avail.I've grown some metapleural folds and sport an oral hoodBut all these fine new characters don't do me any good.Chorus.He soaked a while down in the sand without a bit of pepThen he stiffened up his notochord and said: "I'll beat 'em yet."They laugh and show their ignorance, but I don't mind their jeersJust wait until they see me in a hundred million years.Chorus.My notochord will stiffen to a chain of vertebraeAs fins, my metapleural folds will agitate the seaMy tiny dorsal nervous chord will be a mighty brainAnd vertebrates will dominate the animal domain.Chorus

Prepared by Judith Handlinger