Diagnostic and clinical techniques for invertebrates (Proceedings)

Article

Invertebrate animals comprise 95% of the animal kingdom's species, yet non-parasitic invertebrates are vastly underrepresented in the typical veterinary school curriculum. These notes and the accompanying lecture provide a brief introduction to some of the more prominent invertebrate groups (coelenterates, mollusks, crustaceans, echinoderms, insects, the horseshoe crab, and spiders) and review the state of the science with regards to clinical techniques.

*These notes have been modified and significantly revised from a 2004 Special Species Symposium handout and subsequently the 2006 International Conference on Exotics Proceedings, the 2007 & 2008 AVMA Conventions, and the 2010 Central Veterinary Conference Proceedings. Portions of these notes are adapted from Lewbart GA (ed). 2006. Invertebrate Medicine, Blackwell Publishing, Ames, IA., 327 pp. and Lewbart GA (ed). 2012. Invertebrate Medicine, 2nd Ed., Wiley-Blackwell Publishing, Ames, IA., In Press. Where indicated, these notes contain excerpts from the following book chapters (specific references not included)

**These notes are applicable for all four Lewbart invertebrate lectures at the 2011 AVMA Conference.

Gunkel C and Lewbart GA. Anesthesia and Analgesia of Invertebrates. In Anesthesia and Analgesia In Laboratory Animals, 2nd ed. (R. Fish, P. Danneman, M. Brown, and A. Karas editors). Elsevier Publishing, 2008.

Gunkel C and Lewbart GA. Invertebrates. In Zoo Animal & Wildlife Immobilization and Anesthesia (G West, D Heard, N Caulkett eds.). Blackwell Publishing, Ames, IA, 2007;147-158.

Van Wettere A, Lewbart GA. Cytologic Diagnosis of Diseases of Invertebrates. Veterinary Clinics of North America Exotic Animal Practice (M. Garner ed.), Elsevier, 2007;10235-254.

Invertebrate animals comprise 95% of the animal kingdom's species, yet non-parasitic invertebrates are vastly underrepresented in the typical veterinary school curriculum. These notes and the accompanying lecture provide a brief introduction to some of the more prominent invertebrate groups (coelenterates, mollusks, crustaceans, echinoderms, insects, the horseshoe crab, and spiders) and review the state of the science with regards to clinical techniques. These notes are by no means comprehensive, and are primarily meant to inform the interested clinician on the clinical possibilities related to working with invertebrates.

Porifera (Sponges)

The phylum Porifera is a diverse group of primitive animals commonly referred to as the sponges. Until the early 1800's sponges were actually classified as plants. Sponges occur in the fossil record back to the Precambrian (over 600 million years ago) and were the most important contributors to reefs during the Palaeozoic and Mesozoic Eras (Hooper and Van Soest, 2002). All members lack defined organs; differentiated cells within connective tissue perform necessary biological functions. A unique system of water canals facilitate transport of food, waste products, and gametes. Nearly all are sessile and most species are marine. Most of the 15,000 species are marine but about 3% of sponges live in freshwater environments. Sponges are normally found on firm substrates in shallow water, although some occur on soft bottoms.

Key points

     1. Sponges maintain a close association with a variety of bacterial genera, some of which can be pathogenic.

     2. Virtually nothing is known about analgesia, anesthesia, and therapeutics of sponges.

     3. Sponges seem to tolerate surgical manipulation in the form of cutting and auto-grafting.

     4. Sponges are an integral part of coral reef and other aquatic communities.

     5. Natural products produced by sponges are important to biomedical science.

Further reading

Hooper JNA and RWM Van Soest. Systema Porifera A Guide to the Classification of sponges (Vols. 1 and 2). Kluwer Academic/Plenum Publishers, New York. 2002.

Kuhns WJ, Ho M, Burger MM, and R Smolowitz. Apoptosis and tissue regression in the marine sponge Microciona prolifera. Biol. Bull. 1997; 193239-241.

Lewbart GL. Porifera. In Invertebrate Medicine, Lewbart GA (ed.). Blackwell Publishing, Ames, IA, 2006;7-17.

Lauckner G. Diseases of Porifera. In Diseases of Marine Animals (vol. 1), O. Kinne ed. John Wiley & Sons, 1980;139-165.

Rützler K (Ed.). New perspectives in Sponge Biology. Smithsonian Institution Press, Washington, DC, 1990;188 pp.

Coelenterates

This large phylum includes the comb jellies (Ctenophores), Hydrozoans (hydras, fire coral, Portuguese Man-O-War), Scyphozoans (jellyfishes), and Anthozoans (stony corals, soft corals, sea anemones). This is an economically important group for research, environmental monitoring, public and private display, and tourism. Coral reefs collectively are one of the most beautiful, diverse, and fragile ecosystems on the planet. Jellyfish exhibits are now some of the most popular displays in public aquariums and upscale restaurants throughout the world.

Key points

     1. Some diseases of hard and soft corals have been well documented. The nomenclature for many of the infectious diseases is in the process of standardization.

     2. Many coelenterates are important indicators of ecosystem health.

     3. Trauma and "inversion syndrome" are major concerns when keeping captive jellyfish.

     4. A variety of therapeutic compounds have been used in this phylum, almost exclusively on an empirical basis.

     5. Our overall knowledge of coelenterate medicine and surgery is minimal but growing steadily. Fragging is a term used to describe "surgical propagation" of hard and soft corals utilizing a variety of instruments, adhesives, and substrates.

Further reading

Dorton D. The "cure" for red acro bugs. Reefs.Org, 2004; http//www.reefs.org/forums/viewtopic.php?p=439155. Accessed Jan 18, 2011.

Freeman KS, Lewbart GA, Robarge WP, Law M, Harms, CA, Stoskopf MK. Characterizing eversion syndrome in captive scyphomedusa jellyfish. American Journal of Veterinary Research 2009; 70(9)1087-1093.

Sprung, J. and J.C. Delbeek. The Reef Aquarium, A Comprehensive Guide to the Identification and Care of Tropical marine Invertebrates, Volume One. Ricordea Publishing, Inc, Coconut Grove, Florida,1994.

Sprung, J. and J.C. Delbeek. The Reef Aquarium, A Comprehensive Guide to the Identification and Care of Tropical marine Invertebrates, Volume Two. Ricordea Publishing, Inc, Coconut Grove, Florida, 1997.

Stoskopf MK. Coelenterates. In Invertebrate Medicine, Lewbart GA (ed.). Blackwell Publishing, Ames, IA, 2006;19-51.

Gastropod Mollusks

(Adapted from R. Smolowitz. 2006. Gastropods, Invertebrate Medicine, Blackwell Publishing, Ames, IA)

The gastropods belong in the phylum Mollusca and include over 80,000 marine, fresh water and terrestrial species. All gastropods have a ventrally flattened foot that provides locomotion along the various surfaces of their habitats. The group includes snails, slugs, sea hares, nudibranchs, slipper shells, conchs, whelks, and abalone, among many others. The use of gastropods as laboratory animals and in aquaculture is limited but does occur. They are, however, important display and food animals. Investigators working on the sea hare, Aplysia, were awarded a Nobel Prize for medicine or physiology in 2000.

Key points

     1. Some species can be quite large and these animals are relatively easy to work with.

     2. A number of infectious diseases have been well described in this group.

     3. Fractured shells can be repaired with external fixation methods.

     4. A number of therapeutic and anesthetic techniques have been described.

     5. Some species can be long-lived and may be quite valuable.

Further reading

Anderson ET, Davis AS, Law JM, Lewbart GA, Christian LS, and Harms CA. Gross and histological evaluation of five suture materials in the skin and subcutaneous tissue of the California sea hare (Aplysia californica). Journal of the American Association for Laboratory Animal Science, 2010;49(1)1-5.

Araujo R, Remon JM, Moreno D, Ramos, MA. Relaxing techniques for freshwater molluscs trials for evaluation of different methods. Malacologia, 1995;3629-41.

Friedman CS, Trevelyan G, Robbins TT, Mulder EP, Fields R. Development of an oral administration of oxytetracycline to control losses due to withering syndrome in cultured red abalone Haliotis refescens. Aquaculture, 2003;2241-23.

Lauckner, G. Diseases of Mollusca Gastropoda. In diseases of Marine Animals (ed. O. Kinne) pp. 311-424. John Wiley and Sons, New York. 1980.

Leibovitz L and Capo TR. Diseases of a mass cultured marine laboratory animal, the sea hare, Aplysia californica. In 3rd. Internat. Colloq. Pathol. Marine Aquacul. 2-6 Oct. 1988, Gloucester Point, VA.

Nicolas JL, Basuyaux O, Mazurie J, Thebault A. Vibrio carchariae, a pathogen of the abalone Haliotis tuberculata. Dis. Aquat. Org. 2002;5035-43.

Smolowitz R. Gastropods. In Invertebrate Medicine, Lewbart GA (ed.). Blackwell Publishing, Ames, IA, 2006;65-78.

White HI, Hecht T, Potgieter B. The effect of four anaesthetics on Haliotis midae and their suitability for application in commercial abalone culture. Aquaculture, 1996;140145-151.

Cephalopod Mollusks

There are about 650 species of cephalopods, a group that includes the octopuses, squids, cuttlefish, and the chambered nautilus. This is an important economic group in that they serve as a food source for humans and other animals, are popular display animals, and have been frequently employed in a variety of research projects. Their acute vision, manual dexterity, and intelligence make them fascinating animals to observe and study. Unfortunately, most species are short-lived in the wild and captivity.

Key points

     1. These are highly visual, intelligent animals that can make good clinical patients.

     2. Common problems in captivity include trauma, anorexia, microbial infections, and water quality problems. Recent clinical work includes publications on pharmacokinetics (Gore et al., 2005) and surgery (Harms et al., 2006).

     3. Anesthetic and surgical protocols have been established for some species.

     4. In Great Britain an IACUC (Institutional Animal Care and Use Committee) application is required to perform research on cephalopods.

     5. With their closed circulatory system, these animals make good subjects for pharmacokinetics studies.

Further reading

Forsythe JW, DeRusha RH, and Hanlon RT. Growth, reproduction and life span of Sepia officinalis (Cephalopoda Mollusca) cultured through seven consecutive generations. J. Zool. Lond. 1994;233175-192.

Gore SR, Harms CA, Kukanich B, Forsythe J, Lewbart GA, Papich MG. Enrofloxacin pharmacokinetics in the European cuttlefish, Sepia officinalis, after a single i.v. injection and bath administration. Journal of Veterinary Pharmacology and Therapeutics 2005;28433-439.

Hanlon RT and Forsythe JW. 1. Diseases of Mollusca Cephalopoda 1.1 Diseases caused by microorganisms. Kinne O (ed.). Diseases of Marine Animals, Vol. 3. pp. 23-46. Biologische Anstalt Helgoland, Hamburg, Germany. 1990.

Harms CA, Lewbart GA, McAlarney R, Christian CS, Geissler K, and C Lemons. Surgical excision of mycotic (Cladosporium sp.) granulomas from the mantle of a cuttlefish (Sepia officinalis). Journal of Zoo and Wildlife Medicine 2006;37(4)524–530.

Mather JA, Anderson RC. Ethics and invertebrates a cephalopod perspective. Diseases of Aquatic Organisms 2007;75 119-129.

Messenger JB, Nixon M, and Ryan KP. Magnesium chloride as an anaesthetic for cephalopods. Comp. Biochem. Physiol. 1985;82(C1)203-205.

Oestmann DJ, Scimeca JM, Forsythe JW, Hanlon RT, and Lee PG. Special considerations for keeping cephalopods in laboratory facilities. Contemp. Top. Assoc. Lab. Anim. Sci. 1997;36 89-93.

Reimschuessel RM, Stoskopf MK, and Bennett RO. Myocarditis in the common cuttlefish (Sepia officinalis). J.Comp. Pathol. 1990;102291-298.

Scimeca JM and Oestmann DJ. Selected diseases of captive and laboratory reared cephalopods. Proc. Int. Assoc. Aquat. Anima. Med. 1995;2788.

Scimeca J. Cephalopods. In Invertebrate Medicine, Lewbart GA (ed.). Blackwell Publishing, Ames, IA, 2006;79-89.

Sherrill J, Spelman LH, Reidel CL, and Montali RJ. Common cuttlefish (Sepia officianalis) mortality at the National Zoological Park Implications for clinical management. Journal of Zoo and Wildlife Medicine 2000;31(4)523-531.

Bivalve mollusks

(Adapted from Levine J, Law M, and Corsin F, Bivalve chapter, Invertebrate Medicine, 2006)

This class of mollusks contains many common animals including the clams, mussels, oysters, and scallops. This is an extremely economically important group, especially as a food source for humans. Many species are both captured and cultured for food worldwide. There are more than 10,000 recognized species, found in freshwater, estuarine and marine surface waters. Bivalves fill a critical niche within aquatic ecosystems, the majority functioning as living filters. They comprise a large portion of the shell fauna collected by amateur or professional conchologists on our beaches and freshwater stream banks, and historically have played a significant role in the apparel industry as a source of buttons, or pearls, and as a frequent item on the shelves of novelty shops. Bivalves are popular in display aquariums (private and public aquaria) and as research animals.

Key points

     1. A number of major bacterial, viral, and protozoal diseases of bivalves have been described.

     2. Despite the above statement, knowledge of appropriate chemotherapeutics is minimal.

     3. Diagnostic techniques have been described, including antemortem hemolymph collection.

     4. Many bivalves can live for decades and as such can be quite valuable.

     5. Some species are critically endangered, including a number of freshwater mussels. Veterinarians have already contributed to health assessment efforts on behalf of endangered freshwater bivalves.

Further reading

Allam, B., C. Paillard, A. Howard, and M. Le Pennec. Isolation of the pathogen Vibrio tapetis and defense parameters in brown ring diseased Manila clams Ruditapes philippinarum cultivated in England. Diseases of Aquatic Organisms, 2000;41105-113.

Burkhard M.J., Leavell S., Weiss R.B., Kuehn K., Valentine H., Watters G.T., Wolfe B.A. Analysis and cytologic characterization of hemocytes from freshwater mussels (Quadrula sp.). Veterinary Clinical Pathology 2009;38(4)426-436.

Bower, S.M., and S.E. McGladdery SE. Synopsis of infectious diseases and parasites of commercially exploited shellfish. 2003. URL http//www-sci.pac.dfo-mpo.gc.ca/shelldis/title_e.htm.

Levine J, Law M, and Corsin F. Bivalves. In Invertebrate Medicine, Lewbart GA (ed.). Blackwell Publishing, Ames, IA, 2006;91-113.

Nakayama, K., A.M. Nomoto, M. NishijimaT. Maruyama T. Morphological and Functional characterization of Hemocytes in the Giant Clam Tridacna crocea. Journal of Invertebrate Pathology 1997;69105-11.

Nakayama, K., M. Nishijima, and T. Maruyama T. Parasitism by a protozoan in the hemolymph of the giant clam, Tridacna crocea. Journal of Invertebrate Pathology 1998;71193-198.

Smolowitz, R., D. Leavitt, and F. Perkins. Observations of a protistan disease similar to QPX in Mercenaria mercenaria (hard clams) from the Coast of Massachusetts. Journal of Invertebrate Pathology 1998;719-25.

Annelids

The Annelids are a large and diverse group of segmented vermiform animals that are divided into three main classes the Polychaetes, Oligochaetes, and Hirudineans. All are characterized by regular segmentation of the trunk. It is believed this segmentation evolved as a means of burrowing via peristaltic contractions (Ruppert and Barnes, 1994). Annelids possess a coelomic cavity that is divided into segments by regular septa. The circulatory, excretory, and nervous systems are also segmented. A cuticle covers the animal and segmented setae occur in nearly all members of the phylum. The mouth is located anteriorly and the anus posteriorly with a straight gut between the two openings (Ruppert and Barnes, 1994).

Key points

     1. Some polychaetes and oligochaetes have the capacity to regenerate portions of their bodies.

     2. Much of the early research on tissue grafting and rejection was performed on earthworms (terrestrial oligochaetes).

     3. Virtually nothing is known about the chemotherapeutic treatment of these animals.

     4. Very few infectious diseases have been described from this group (except where these animals are the intermediate host for diseases of vertebrates).

     5. Some species (tropical marine polychaetes like feather duster and Christmas tree worms) are important and valuable display animals.

Further reading

Benkendorff K. Chemical defense in egg masses of benthic invertebrates An assessment of antibacterial activity in 39 mollusks and 4 polychaetes. Journal of Invertebrate Pathology 2001;78(2) 109-118.

Bilej M, de Baetselier P and A Beschin. Antimicrobial defense of the earthworm. Fol. Microbiol. 2000;45283-300.

Cooper JE, Mahaffey P, and K Applebee. Anaesthesia of the medicinal leech (Hirudo medicinalis). Veterinary Record 1986;118589-590.

Cooper JE. A veterinary approach to leeches. Veterinary Record 1990;127226-228.

Davila VJ, Hoppe IC, Landi R and FS Ciminello. The effect of anchoring sutures on medicinal leech mortality. Open Access J of Plastic Surgery 2009;278-281.

Federov A and Federova L. Where is the difference between the genomes of humans and annelids? Genome Biology 2006;7(1)Article 203.

Heimpel AM. A crystalliferous bacterium associated with a "blister disease" in the earthworm, Eisenia foetida (Savigny). Journal of Invertebrate Pathology 1966;8295-298.

Lewbart GL. Annelids. In Invertebrate Medicine, Lewbart GA (ed.). Blackwell Publishing, Ames, IA, 2006;115-131.

Papavramidou N and H Christopoulou-Aletra. Medicinal use of leeches in the texts of ancient Greek, Roman and early Byzantine writers. Internal Medicine J 2009;39(624-627).

Lowers JM and JL Bartholomew. Detection of myxozoan parasites in oligochaetes imported as food for ornamental fish. Journal of Parasitology, 2003;89(1)84-91.

Snower DP, Ruef C, Kuritza AP, and SC Edberg. Aeromonas hydrophila infection associated with the use of medicinal leeches. Journal of Clinical Microbiology 1989;27(6)1421-1422.

Terzioglu A and D Tuncali. The re-usable medicinal leech. Plastic and Reconstructive Surgery 2003;111(3)1358-1359.

Crustaceans

The crustaceans are a highly successful class of the Phylum Arthropoda. This group includes the well-known lobsters, crabs, crayfish, shrimp, barnacles, and hermit crabs. Numerous other taxa belong to this class isopods, amphipods, and brine shrimp. Economically, this is one of the most important groups of invertebrates. Its members are important for food, research, and as display animals.

Key points

     1. The infectious diseases of the penaeid shrimp (a family of shrimp with economic importance as food animals) have been described in great detail, especially some of the viral and bacterial problems.

     2. There is data for some antimicrobial treatment in the literature for economically important groups like panaeid shrimp.

     3. There are published protocols for anesthesia and euthanasia for some crustaceans.

     4. Funds are available to study the disease of economically important groups like crabs, shrimp, and lobsters.

     5. Hemolymph is relatively easy to extract and analyze from animals like lobsters, crabs, and hermit crabs.

Further reading

Brock JA and Lightner DV. Diseases of crustaceans Diseases caused by microorganisms. In Diseases of Marine Animals. Vol. 3 Cephalopoda, Annelida, Crustacea, Chaetognatha, Echinodermata, Urochordata. (ed. by O Kinne), Biologische Anstalt Helgoland, Hamburg,1990;245-349.

Brock JA and Main KL. A Guide to the Common Problems and Diseases of Cultured Penaeus vannamei. The Oceanic Institute, Honolulu, HI. 1994.

Edgerton, BF, Evans LH, Stephens FJ and Overstreet RM. Synopsis of freshwater crayfish diseases and commensal organisms. Aquaculture 2002;20657-135.

Elwood RW, Appel M. Pain experience in hermit crabs? Animal Behavior 2009;77 1243-1246.

Fotedar S and Evans L. Health management during handling and live transport of crustaceans A review. Journal of Invertebrate Pathology 2011;106143-152.

Gardner C. Options for humanely immobilizing and killing crabs. Journal of Shellfish Research 199;1619-224.

Ingle RW. The UFAW Handbook on the Care and Management of Decapod Crustaceans in Captivity. Universities Federation for Animal Welfare, Potters Bar, Hertfordshire, England. 1995.

Lightner DV (ed.). A Handbook of Shrimp Pathology and Diagnostic Procedures for Diseases of Cultured Penaeid Shrimp. World Aquaculture Society, Baton Rouge, LA (looseleaf). 1996.

Lightner DV and Redman RM. Shrimp diseases and current diagnostic methods. Aquaculture 1998;164201-220.

Meyers TR. Diseases of crustaceans Diseases caused by protistans and metazoans. In Diseases of Marine Animals. Vol. 3 Cephalopoda, Annelida, Crustacea, Chaetognatha, Echinodermata, Urochordata. (ed. by O Kinne), pp. 350-389. Biologische Anstalt Helgoland, Hamburg. 1990.

Noga EJ, Sawyer TK and Rodon-Naveira M. Disease processes and health assessment in blue crab fishery management 1998;J Shellfish Res 17567-577.

Noga EJ, Smolowitz R and Khoo L. Pathology of shell disease in the blue crab, Callinectes sapidus. J Fish Dis 2000;23389-399.

-Noga EJ, Hancock AL, and RA Bullis. Crustaceans. In Invertebrate Medicine, Lewbart GA (Ed.). Blackwell Publishing, Ames, IA, 2006;179-193.

Spiders

This is a huge group of animals (over 30,000 species) that belong to the class Arachnida. Less conspicuous arachnids include the mites, ticks, and scorpions. Numerous texts describe the biology, natural history, and husbandry of these fascinating creatures. Tarantulas (not true spiders) represent an important group of commonly kept arachnids that commonly require medical care.

Key points

     1. By far the most popular group of "spiders" kept at pets in the home are the tarantulas.

     2. Many clinical techniques, including hemolymph collection and anesthesia have been described for tarantulas.

     3. Female spiders can be long-lived (several decades) and may be quite valuable.

     4. Common clinical problems include trauma, limb autotomy, and dysecdysis.

     5. Surgical repair of the fractured exoskeleton is commonly accomplished with surgical adhesives.

Further reading

Breene RG and O'Brien MR. Concise care guide for the 80 plus most common tarantulas. Am. Tarantula Soc. Carlsbad, NM 90 pp. 1998.

Breene RG. The ATS arthropod medical manual Diagnoses & treatment. Am. Tarantula Soc. Carlsbad, NM 32 pp. 1998.

Cooper JE. A veterinary approach to spiders. Journal of small animal practice, 28, 229-239. 1987.

Frye FL. Arachnids. In Captive invertebrates; a guide to their biology and husbandry, Krieger. 1992.

Pizzi R. Spiders. In Invertebrate Medicine, Lewbart GA (ed). Blackwell Publishing, Ames, IA., 2006. 143-168.

Zachariah TT, Mitchell MA, Guichard CM, and Singh RS. Hemolymph biochemistry reference ranges for wild-caught goliath birdeater spiders (Theraphosa blondi) and Chilean rose spiders (Grammostola rosea). Jou Zoo Wild Med 2007;38(2)345-251.

Limulus

Limulus polyphemus, the American horseshoe crab, is actually not a crab at all but a member of the Class Merostomata in the Phylum Chelicerata. Horseshoe crabs are more closely related to arachnids than crustaceans. This is the only species that occurs on our coast (Western Atlantic) but there are other species of horseshoe crabs that occur in Asia. Limulus is a very important animal for biomedical research and is used as bait and fertilizer (controversial) as well as being an important display and "touch tank" animal in public aquaria. Investigators examining vision and the Limulus lateral eye were awarded the Nobel Prize for medicine or physiology in 1967.

Key points

     1. The anatomy and physiology of this animal has been thoroughly researched.

     2. These animals are easy to handle and work with.

     3. Trauma cases can be surgically repaired with external fixators like epoxy.

     4. Very little work has been done with regards to chemotherapeutic treatment.

     5. These animals make good subjects for pharmacokinetics studies and some papers on pharmacokinetics are now appearing in the literature.

Further reading

Allender MC, Schumacher J, Milam J, George R, Cox S, and Martin-Jimenez T. Pharmacokinetics of intravascular itraconazole in the American horseshoe crab (Limulus polyphemus). J. Vet. Pharmacol. Therap 2007;3183-86.

Bullis, R.A. Care and maintenance of horseshoe crabs for use in biomedical research. In Techniques in Fish Immunology, Volume 3 (J.S. Stolen, T.C. Fletcher, A.F. Rowley, J.T. Zelikoff, S.L. Kaattari and S.A. Smith, eds.). SOS Publications, Fair Haven, NJ. 1994;A9-A10.

Groff, J.F. and L. Leibovitz. A gill disease of Limulus polyphemus associated with triclad turbellarid worm infections. Biological Bulletin 1982;163392.

Leibovitz, L. Cyanobacterial diseases of the horseshoe crab (Limulus polyphemus). Biological Bulletin 1986;171482.

Leibovitz, L. and G.A. Lewbart. A green algal (chlorophycophytal) infection of the dorsal surface of the exoskeleton and associated organ structures in the horseshoe crab (Limulus polyphemus). Biological Bulletin 1987;173430.

Leibovitz, L. and G.A. Lewbart. Diseases and Symbionts Vulnerability Despite Tough Shells. In The American Horseshoe Crab (C.N. Shuster, Barlow RB and HJ Brockmann, eds.). Harvard University Press, Cambridge, Mass. 2004;245-275.

Rudloe, J. The effect of heavy bleeding on mortality of the horseshoe crab, Limulus polyphemus, in the natural environment. Journal of Invertebrate Pathology 1983;42167-176.

Smith, S.A., J.M. Berkson and R.A. Barratt. Horseshoe crab (Limulus polyphemus) hemolymph, biochemical and immunological parameters. Proceeding of the International Association for Aquatic Animal Medicine 2002;33101-102.

Smith, S.A. and J.M. Berkson. Laboratory culture and maintenance of the horseshoe crab (Limulus polyphemus). Lab Animal 2005;34(7)27-34.

Spotswood T. and SA Smith. Cardiovascular and gastrointestinal radiographic contrast studies in the horseshoe crab (Limulus polyphemus). Veterinary Radiology and Ultrasound 2007;48(1)14-20.

Insects

This is by far the largest group of invertebrates and possibly the most economically important. Insects are loved and despised worldwide and occupy nearly all niches except the marine environment. They are important as a human food source in parts of the world and both sustain and destroy agricultural crops, depending on the species of insect and plant.

Key points

     1. Important research and display animals include beetles, butterflies, grasshoppers, walking sticks, and ants.

     2. Much research has focused on the diseases of insects; in some cases to help the insects and in some cases to harm them.

     3. Butterfly houses and "arthropod zoos" are very popular at zoological gardens and natural history museums.

     4. A fair amount of research has been published on managing infectious diseases of honeybees (which are not native the North America).

     5. These animals can be successfully anesthetized prior to a variety of diagnostic procedures.

Further reading

Boucias DG and Pendland JC. Principles of Insect Pathology. Kluwer, The Netherlands. 1999.

Cooper JE and Cunningham AA. (Pathological investigation of captive invertebrates. International Zoo Yearbook 1991;30137-143.

Cooper JE. Insects. In Invertebrate Medicine, Lewbart GA (ed.). Blackwell Publishing, Ames, IA, 2006;205-219.

Cox-Foster et al. A metagenomic survey of microbes in honey bee colony collapse disorder. Science 2007;318283-287.

Echinoderms

This interesting and diverse group of animals includes the sea stars, brittle stars, sea cucumbers, sea urchins, sea biscuits, and crinoids. Many are commonly displayed in aquaria and used in research. Humans do not consume most species but the gonads of sea urchins are a popular food item in some sushi restaurants.

Key points

     1. Very little is published with regards to the medicine and surgery of this group.

     2. Some species are easy to maintain in captivity, making them popular as "pets" and for research.

     3. Some species have regenerative capabilities and generally heal well and quickly.

     4. Information on anesthesia and sedation can be found in the basic scientific literature.

     5. In some groups the external "test," or skeleton, can make clinical evaluation difficult.

Further reading

Harms CA. Echinoderms. In Invertebrate Medicine, Lewbart GA (ed.). Blackwell Publishing, Ames, IA, 2006;245-256.

Jangoux M. Diseases of Echinodermata. In Diseases of Marine Animals, Vol. 3 (ed. O Kinne) pp. 439-567. Biologische Anstalt Helgoland, Hamburg. 1990.

Jellett JF, Wardlaw AC, and Scheibling RE. Experimental infection of the echinoid Strongylocentrotus droebachiensis with Paramoeba invadens quantitative changes in the coelomic fluid. Dis Aquat Org 1988;4 149-157.

Jones GM, Hebda AJ, Scheibling RE, Miller RJ. Histopathology of the disease causing mass mortality of sea urchins (Strongylocentrotus droebachiensis) in Nova Scotia. J Invertebr Pathol 1985;45(3)260-71.

Lessios HA. Mass mortality of Diadema antillarum in the Caribbean What have we learned? Ann Rev Ecol Syst 1988;19 371-393.

Clinical techniques

Anesthesia & analgesia (from gunkel and lewbart, 2007 & 2008)

Invertebrate anesthesia is still in its infancy and relatively little research has been done to improve the understanding of the various anesthetic agents used for invertebrates. Still, there is a body of work that supports and defines this topic, and key points and references have been included where appropriate. Until more information is available regarding pain perception by invertebrates, an analgesic should be give to any animal that is subjected to a painful procedure, and the amount of stress and pain induced should be reduced by decreasing awareness via appropriate choices of anesthetic agents.

The use of analgesic agents in invertebrate species has its own limitations since very few reports can be found documenting the administration and dosing of analgesics agents, especially in the very small species. Assessment of pain or discomfort in invertebrates is very difficult, despite the fact that some avoidance behavior has been described. The effect of analgesia is even harder to evaluate.

More research is required in this area, but in the meantime, if possible, drugs with analgesic properties should be used to anesthetize invertebrates when invasive procedures are performed to decrease the nociceptive pathway. Hypothermia and CO2 do not possess analgesic properties and may even show hyperalgesic characteristics. Inhalant agents are preferred over the latter methods. Although inhalant agents do not possess true analgesic properties, they do render mammalian patients insensible to painful stimuli when administered at sufficient doses. Unfortunately, the insensibility to painful stimuli only lasts as long as the animal is anesthetized, and administration of an analgesic would be advisable if post-operative pain is anticipated.

Sample collection, preparation, and evaluation (from vanwettere and lewbart, 2007)-

Cytologic samples can be collected, as with vertebrates, by swabbing, scrapping, imprints or fine needle biopsy. One major difference between vertebrates and invertebrates is hemolymph collection. Hemolymph of invertebrates is comparable to the blood of vertebrates. Many invertebrates have an open circulatory system in which hemolymph flows to the organs through arteries, passes into a hemocoelomic cavity, and drains to the heart and respiratory organs through progressively larger venous channels. In most cases, hemolymph is composed of circulating hemocytes, soluble defense molecules, and a respiratory pigment (usually the copper-containing hemocyanin). Hemocytes are considered the equivalent of the vertebrate white blood cells with the additional function of coagulation. Hemolymph is the most readily accessible and often relatively easily antemortem collected tissue and can even be obtained from very small invertebrates such as flies and mosquitoes.

Generally, it is recommended to clean and disinfect the shell and/or integument at the collection site. In species with a hard cuticle, if the bleeding does not stop rapidly after sample collection, closure of the puncture site can be made using cyanocrylate (tissue glue) or a similar adherent material. Anesthesia may be required for sample collection in some species. There is wide variation in the anatomy between different species and description of hemolymph collection sites and details will be presented during the lecture and are accessible via the references.

Imaging

There are examples of diagnostic imaging being used on invertebrate species and a recent paper on Limulus (see section X) will be reviewed and discussed. Davis et al., (2008) have published a very informative work on imaging a number of terrestrial species with standard and contrast radiography.

Chemotherapeutics

There have been some recent papers dealing with chemotherapeutics and pharmacokinetics pertaining to invertebrates. Appropriate examples appear in this handout.

Surgery

The literature contains a slowly expanding database of surgical procedures being performed on invertebrates. Some cases with the applicable references have been included and will be reviewed.

General invertebrate zoology and medicine references

Barnes RD. Invertebrate Zoology (5th ed.). Saunders College Publishing, Philadelphia, 1987;71-91. Earlier editions were published in 1963, 1968, 1974, and 1980.

Breene RG. The ATS arthropod medical manual Diagnoses & treatment. Am. Tarantula Soc. Carlsbad, NM 1998;32 pp.

Cooper JE. Emergency care of invertebrates. In Veterinary Clinics of North America, Exotic Animal Practice 1998;1251-264.

Cooper JE. Invertebrate anesthesia. In "Analgesia and Anesthesia." Veterinary Clinics of North America Exotic Animal Practice 2001;4(1)57–67.

Davis MR, Gamble KC & Matheson JS. Diagnostic Imaging in terrestrial Invertebrates Madagascan Hissing Cockroach (Gromphadorina pertentosa), Desert Millipede (Orthoporus sp), Emperor Scorpion (Pandinus imperator), Chilean Rosehair Tarantula (Grammostola spatulata), Mexican Fireleg Tarantula (Brachypelma boehmi ) and Mexican Redknee Tarantula (Brachypelma smithi). Zoo Biology 2008;27 109-125.

Dombrowski D, De Voe R. Emergency care of invertebrates. Vet Clin North Am Exotic Anim Pract 2007;10621-645.

Frye FL. Care and feeding of invertebrates as pets or study animals. In "Zoo and Wild Animal Medicine" (Ed. Fowler ME) 2nd ed. WB Saunders, Philadelphia, USA. 1986.

Frye FL. Captive Invertebrates A Guide to their Biology and Husbandry. Krieger, Malabar, Florida. 1992.

Howard DW, Lewis EJ, Keller BJ, and Smith CS (eds.). Histological Techniques for Marine Bivalve Mollusks and Crustaceans. NOAA Tech. Memo. NOS NCCOS 2004;5, 218 pp.

Hyman LB. The invertebrates. Vol. I-VI (multiple years). McGraw-Hill Book Co., New York.

Kinne O. (Ed). Diseases of Marine Animals (Vols. 1-3), John Wiley & Sons. 1980-1990. Available online http//www.int-res.com/book-series/diseases-of-marine-animals-books/

Lewbart G.A. (Ed.). Invertebrate Medicine, Blackwell Publishing, Oxford, UK, 2006;327 pp.

Lewbart G.A. Invertebrates. In Exotic Animal Formulary, 4th Ed., J. Carpenter editor, Elsevier Publishing, 2012; In Press.

Lewbart G.A. (Ed.). Invertebrate Medicine, 2nd Ed., Blackwell Publishing, Oxford, UK, 2012; In Press.

Meglitsch PA and Schram FR. Invertebrate zoology. Oxford University Press, New York, 1991;623 pp.

New TR. An Introduction to Invertebrate Conservation Biology. Oxford Science Publications, Oxford University Press, New York, NY. 1995;194 pp.

Pearse V, Pearse J, Buchsbaum M, Buchsbaum R. Living invertebrates. Blackwell Scientific Publication, Palo Alto, California, 1987;848 pp.

Ruppert EE, Fox RS and Barnes RD. Invertebrate Zoology A Functional Evolutionary Approach. 7th Ed., Thompson-Brooks/Cole, Belmont, California, 2004;963 pp.

Stolen J.S., Fletcher T.C., Smith, S.A., Zelikoff J.T., Kaattari S.L., Anderson R.S., Soderhall K., and B.A. Weeks-Perkins. Techniques in Fish Immunology, Fish Immunlogy Communications 4 (FITC 4), SOS Publications, Fair Haven, NJ 07760, 1995; 258 pp. plus appendices.

Williams DL. Sample taking in invertebrate veterinary medicine. In Veterinary Clinics of North America, Exotic Animal Practice 1999;2, 777-801.

Williams DL. Invertebrates. In Manual of Exotic Pets (eds. Meredith A and Redrobe S) British Small Animal Veterinary Association, Gloucester, UK. 2002.

Webliography

http://www.apivet.eu/ -This site is written/supervised by a French veterinarian familiar with honeybees. (Accessed June 4, 2011)

http://www.oie.int/eng/en_index.htm -This is the general site/home page for the OIE (World Health Organization for Animals). (Accessed June 4, 2011)

http://www.oie.int/eng/maladies/en_classification2010.htm?e1d7 -This is the specific OIE page for reportable/notifiable diseases. (Accessed June 4, 2011)

http://www.elsevier.com/wps/find/journaldescription.cws_home/622883/description#description -This is the link to the Journal of Invertebrate Pathology, an important primary literature resource. (Accessed June 4, 2011)

http://www.xerces.org/ -This site provides information on invertebrate conservation and members receive a hard copy periodical (Wings Magazine) twice yearly. (Accessed June 4, 2011)

http://awic.nal.usda.gov/nal_display/index.php?info_center=3&tax_level=3&tax_subject=180&topic_id=1480&level3_id=5398&level4_id=0&level5_id=0&placement_default=0 -The general USDA site for zoological species including invertebrates. (Accessed June 4, 2011)

http://www.thecephalopodpage.org/ -A specific site for cephalopod mollusks that is linked to the above USDA web page. (Accessed June 4, 2011)

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