Diagnostic and clinical techniques for invertebrates (Proceedings)

August 1, 2011

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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)

Diagnostic and clinical techniques for invertebrates (Proceedings)

Porifera (Sponges)

Further reading

Coelenterates

Further reading

Gastropod Mollusks

Further reading

Cephalopod Mollusks

Further reading

Bivalve mollusks

Further reading

Annelids

Further reading

Crustaceans

Further reading

Spiders

Further reading

Limulus

Further reading

Insects

Further reading

Echinoderms

Further reading

Clinical techniques

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

Imaging

Chemotherapeutics

Surgery

General invertebrate zoology and medicine references

Webliography