The DAB stain has proven useful as a tool for highlighting a population of exocrine glands in L. salmonis and C. elongatus. Whilst the present study has not attempted to determine the identity of products giving a positive reaction to DAB, this will be addressed in future. This work has shown that DAB–positive reactants are present in both species of sea lice at all life-stages, including pre-hatch nauplii.
Several of the glands found in this study have been reported previously. Bron (1993) first described DAB-positive glands in the thoracic limbs of L. salmonis copepodids and also located glands which appear to correspond to the circum-oral glands seen in chalimus larvae (Bron, Sommerville, & Rae, 1993). The structure of the filament-producing glands in L. salmonis chalimus larvae has also been previously investigated (Bron et al. 1991; Bron, 1993) and superficially, components of the frontal gland complex found in the present study resemble some of those glands. Further analysis using histological/histochemical techniques will be required to determine the homologies of these glands. The fact that some of the filament-producing glands found by (Bron et al., 1991) stained positively with stains other than DAB, suggests a multi-component composition to the gland product and it is likely that many of the glands stained may have multiple functional components. Many exocrine secretions of copepods have a mucous component (Hicks and Grahame, 1979) and, whilst it has been demonstrated that sulphated mucopoly-saccharides may stain positively with DAB, Alcian Blue (pH 1.0 and 2.5) staining failed to demonstrate their presence in the exocrine glands of L. salmonis.
The glands in the thoracic limbs may be important with regard to the functioning of the limbs since they stain strongly only in the copepodid where swimming ability is important and in adults where swimming may similarly be important and where there are no further moults to renew the limbs and their setae. They are conspicuous by their greatly reduced appearance in the chalimus stages where limb function is relatively less important i.e. when filament-attached, and in preadult stages where there is a relatively short period between moults. As it was common to find large quantities of DAB-positive material adhering to the limb setae it may be that the secretion acts in some way to either maintain, or improve the function of those setae. This supports the suggestions of previous researchers who proposed cuticle tanning (Stevenson, 1961), anti-fouling (Boxshall, 1982; Bannister, 1993) and hydrophobic surfactants (Brunet, Cuoc, Arnaud, & Mazza, 1991) as possible actions of gland products. It has been shown (Gresty & Warren, 1993; Dumontet et al., 1996) that copepods frequently have epibiotic organisms attached to their cuticle and it is possible, therefore, that secretions from the glands may be involved in killing or preventing the establishment of such organisms on the cuticle surface, a phenomenon found in corixid beetles where gland secretions prevent bacterial growth on the hairs of the physical gill (Kovac & Maschwitz, 1991). This hypothesis does not account for why these regions appear to be unique to caligids, unless the environment in which they live is more likely to expose them to such fouling organisms.
Peroxidases have been shown to be present in red blood cells, hepatocytes, central nervous system neurons and oestrogen-sensitive tissues (Rainbow 1996) and are also known to be fundamental to the process of prostaglandin production from arachidonic acid (Bowman et al., 1996). Peroxidase enzymes and catalase have also been shown to be important neutralisers of potentially damaging free radicals, which result following tissue damage and are formed as by-products of metabolism and also from the interaction of U.V with natural organic matter. Peroxidase activity has also been show in tunicates where it is believed to be involved in iodination processes fundamental to protothyroid activity (Fredriksson et al., 1988).
The only DAB-positive glandular regions seen in free-living species are in the buccal cavity area. Whilst the function of these glands is unknown it may be suggested that peroxidases are being produced here to assist in the elimination of free radicals produced as a side-effect of feeding activity, a phenomenon witnessed in caterpillars (Felton & Duffey, 1991). If such products are not quickly neutralised they might otherwise damage the gut epithelium. Glands in the buccal cavity region of free-living marine copepods have been well documented (Boxshall, 1982; Arnaud et al., 1988ab; Vaupel Klein & Koomen, 1994) and L. salmonis and C. elongatus also have DAB-positive glands associated with their mouth-parts which may function in a similar manner, although it appears that at least one of them exits via a pore on the outside of the mouth-tube and it is therefore unclear how the secretion would interact with the food material.
It is possible that sea lice will be exposed to free-radicals, both as a result of host tissue damage, and following a host immune response which includes chemicals released at sites of tissue injury. The peroxidase compounds found in sea lice may be involved in protecting the parasite from the deleterious effects of such compounds. Prostaglandins, which are synthesized in a process involving peroxidase enzymes, have been shown to be involved in host inflammatory response modulation. Peroxidases and prostaglandins have been isolated in species of tick with unusually long host-attachment periods (Bowman et al., 1996). It seems reasonable to suppose that sea lice species may have developed methods to maintain a benevolent environment for themselves and this should be considered as a possible function of these glands.
As very few DAB-positive regions were present in free-living species, as compared to the two parasitic species examined here, the DAB stain may be picking out features which represent specific developments associated with the parasitic mode of life.
It is probable that the glands highlighted by this stain do not all have the same function. In most cases the stain highlighted secreted material and we can presume that it is not gland ultrastructural elements which are being stained, but rather the secretion itself which shows the positive reaction. This is not however, true for the DSR where no associated ducts or secreted material have been observed. The possibility that the stain here is highlighting ultrastructural or neurological features will have to be addressed using other staining techniques and TEM. The results of a preliminary SEM examination suggest that the DSR may be associated with cuticular sensillae, adding further doubt to the idea that they are exocrine glands.
In summary, the DAB-positive gland distribution of L. salmonis had a consistent pattern which could be specifically identified at each life-stage, with the exception of chalimus larvae where staining difficulties hindered interpretation. The development of the fundamental gland groups could be followed through several life-stages i.e. perianal, lateral and anterior from nauplius to chalimus, while the perianal glands (and possibly the anterior gland complexes) were retained from nauplius to the adult. The median glands could be followed from nauplius to copepodid and may possibly be represented by the circum-oral and frontal gland complexes in later stages. We can presume that these glands have a fundamental role to play in the biology of these species. Histological evidence is being sought to determine the physical characteristics of the glands and their relationship with other organs and tissues in the body.
Bron (1993) and Bron et al. (1993) have provided evidence of the existence of further glands in L. salmonis which are not DAB-positivesuggesting theDAB stain does not highlight all of the glands present in this species.