HOW TO ASSESS THE HEALTH OF YOUR CRUSTACEANS

History

The history of each group of crustaceans being examined should be closely followed, particularly that information related to water source, pond, cage or system number, species, age, stocking rate, growth performance, survival rates, feeding activity, food conversion rate, grading history, parasite or pathogen burdens and treatment history. Any abnormalities in any of these areas should be noted when they are observed so that these data are available for future reference if required.

Water quality

Basic water quality parameters such as temperature and dissolved oxygen should be regularly monitored. Other variables such as salinity, and presence or absence of toxic phytoplankton and jellyfish should be recorded for crustaceans held in seacages, while on land and particularly in recirculation systems monitoring of additional parameters such as ammonia, nitrite and nitrate, water hardness and pH should be implemented. Ideally these records should be available for the entire history of each group of crustaceans being examined.

Gross examination

Detailed investigations should be restricted only to live or moribund crustaceans. Dead crustaceans are generally of very limited usefulness when it comes to attempting to determine their disease status or cause of death. The carapace length (or width for crabs) should be measured to the nearest mm with vernier calipers Figure 1 and the animal should be sexed. Each animal should then be examined for external lesions, damage, melanisation (blackened areas on the carapace) and abnormal behaviours, such as lethargy, or other visible signs of stress. These signs should be noted on a data sheet prior to capturing and handling the fish. A web site which outlines the anatomy of lobsters can be found Here or for prawns and crabs, Here

Collection of haemolymph

Haemolymph is collected from the arthrodial membrane covering the articulated base of the 5th walking leg Figure 2. The area is swabbed or flushed with 70% ethanol (see Recipes page) to sterilise the surface and a 25 gauge sterile hypodermic needle and syringe is used to extract around 1 ml of haemolymph. The haemolymph can be placed directly onto a handheld serum refractometer to determine its refractive index (useful for determining nutritional status and moult stage in lobsters) Figure 3. Drops of haemolymph can also be inoculated into microbiological media to examine for systemic bacterial infections. Alternatively haemolymph can be drawn into a syringe containing a fixative (e.g. 2.5% gluteraldehyde or 10% seawater formalin (see Recipes page)) and the fixed cells can be dropped directly onto a microscope slide, air dried and stained with Diffquick (see Recipes page) to examine for systemic infections by bacteria or protozoans. Differential cell counts and other more applied tests on the haemolymph should be done using an anticoagulant (citrate/EDTA anticoagulant - 0.45 M NaCl, 0.1 M glucose, 30 mM sodium citrate, 26 mM citric acid, 10 mM EDTA; pH 5.4) which is placed into the syringe prior to taking haemolymph, so that the haemolymph is drawn directly into the anticoagulant. If more applied tests are required, contact DigsFish Services for more details.

Histopathology

Larvae

Larval stages of all crustaceans can be fixed by simply placing them directly into 10% seawater formalin or Davidsons fixative (see Recipes page) for at least 24 hours (ensure that there is at least 5 times the volume of fixative to the volume of the larvae). Once this is done they are generally ready for histological onprocessing. Since most larvae are small and delicate their onprocessing for histopathology requires a few special techniques. The onprocessing is probably best left to DigsFish Services personnel.

Prawns/shrimp/lobster puerulus

Small crustaceans such as prawns, shrimp and lobster puerulus can be typically processed for histopathology as follows:

1. Prepare the fixative to be used prior to sampling - ensure that the volume of fixative available is at least 5 times that of the tissues being fixed. Davidsons fixative (see Recipes page) has been standardised for use with prawns and shrimp, while lobsters fix well using 10% seawater formalin (see Recipes page).

2. Draw the fixative into an appropriate sized needle and syringe (0.1 ml to 5 ml volume required, tailor needle gauge and syringe size to crustacean size).

3. Record sex Figure 4 and moult stage if appropriate (A-E, and postmolt, intermolt, premolt, Figure 4. Also note any grossly visible lesions Figure 5 on the data sheet.

4. Inject the fixative laterally into the hepatopancreas first Figure 6 (into at least 3 separate areas with larger crustaceans), then into the anterior and posterior abdominal regions Figure 7, Figure 8. Continue injecting fixative until the entire body of the crustacean changes to an opaque colour. Then slit the cuticle with a pair of small sharp scissors along the midline Figure 9 to ensure complete entry of the fixative and place the specimen in a container with at least 5 volumes of the fixative for a minimum of 24 hours.

5. Transfer to 50-70% ethanol (see Recipes page) for storage or transport to DigsFish Services.

Larger crustaceans (Crabs/lobsters)

1. Place the crab/lobster in freezer until it is suitably anaesthetised to allow easy handling.

2. Record sex and moult stage (A-E, and postmolt, intermolt, premolt), or snip off the tip of a pleopod Figure 4 and fix in 10% seawater formalin for later moult staging. Also note any grossly visible lesions, such as shell disease Figure 5 on the data sheet.

3. Cut open the carapace with a strong pair of scissors to expose the internal organs Figure 10. Excise samples of hepatopancreas, antennal gland, gill, heart, midgut, and hindgut and place them into individual histology cassettes (Simport M492). Any grossly visible lesions on the internal organs should also be fixed after taking a digital photograph. The volume of each tissue sample should be around 0.5 to 1 cm3. Immediately place the samples into 10% seawater formalin (see Recipes page) for at least 24 hours.

4. Transfer to 50-70% ethanol (see Recipes page) for storage or transport to DigsFish Services.

Bacteria and Virus testing

Bacteriology and virology testing use specialised techniques which should be conducted using methods recommended by the diagnostic laboratory which will undertake the sample screening. Contact DigsFish Services or the laboratory responsible for processing your samples for more information on their desired methods of sample collection.