Materials and methods
One 10mg DAB tablet (Sigma D5905) was added to 15ml of Tris buffer to give a 0.66% w/v solution to which 12 µl of H2O2 was added (0.026% solution) to provide a substrate for the reaction. The reaction was halted by the addition of 70% ethanol.
Fresh specimens were added to the prepared DAB solution and left to stain at room temperature for a range of time periods. The optimal staining time was found to be: »15min for nauplii and copepodids, »6h for chalimus and »16h for preadults and adults. After these periods had elapsed the reaction was halted by carefully pipetting off the stain solution and replacing it with 70% ethanol.
Chalimus, preadult and adult stages were stained soon after collection (i.e. not exceeding 48h post-removal) to minimise any effects on gland activity that being removed from the salmon host may incur.
The same procedures were followed for nauplius, copepodid and adult specimens of the caligid copepod Caligus elongatus Nordmann, 1832. Adults of the free-living marine copepods Eurytemora affinis (Poppe, 1880)and Acartia tonsa Dana, 1849, the cladoceran Daphnia magna Straus, 1820, and larval stages of the anostracan Artemia salina (L.), and the palaemonid Macrobrachium rosenbergii (de Man) were also stained in the same way for comparison.
Small larval stages (i.e. nauplius, copepodids and chalimus I and II) were examined in a cavity slide under a cover slip. Lactic acid (87.5%) was used to aid in the clearing of the body tissues of larval stages, to enhance visualisation of gland structures without affecting the quality of staining, although this method could not be used as a permanent mountant (Huys & Boxshall, 1991).
Observations were made using an Olympus BH-2 compound microscope and photographs were taken using an Olympus C-35AD-2 camera.