‘O’ group mullet, Chelon labrosus. were experimentally infected with Cryptocotyle lingua (Heterophyidae) by tail dip in a suspension of cercariae. Metacercariae were excised after 1 and 24 hours and prepared for TEM and post-embedding immunogold labelling. Antisera to cercariae of C. lingua were raised in adult mullet by natural infection via the skin and by intra-peritoneal injection of sonicate. The membrane-bound vesicles within the syncytial lining of the metacercarial excretory vesicle were found to be intensely antigenic with both antisera: the epidermal secretory bodies type 5 within the cystogenousglands gave a positive response. Penetration gland contents were not found to be antigenic with either antiserum. Discharge of the membrane-bound vesicles coinciding with both the reorganization of the lining of the metacercarial excretory vesicle and with cyst wall formation appears to be of significance in the initiation of the host immune response. That the term ‘excretory vesicle’ in Digenea may be a misnomer is discussed in the light of current information regarding the wide range of functions attributed to this structure.

A series of monoclonal antibodies was prepared against tegumental and internal antigens of Fasciola hepatica by immunizing mice with whole adult-fluke homogenates prior to harvesting the splenic lymphocytes for fusion. Preliminary screening by the Indirect Fluorescent Antibody technique indicated the occurrence of discrete groups of monoclonals differing from one another in tissue-specificity but within which IFA labelling patterns were fairly consistent. Representative hybridomas for 5 of these groups were stabilized and used to produce ascites fluid in mice. By application of an immunogold labelling technique it was possible to map the distribution of antigens for which each monoclonal antibody had affinity throughout the tissues of 4-week and 12-week flukes. Several monoclonals specifically labelled antigenic determinants on the important tegumental antigen T1. However the distribution of gold colloid labelling suggested that epitopes other than that normally exposed to the infected host were recognized; and several monoclonals specifically attached to T1 antigen in the tegument of juvenile worms only. The glycocalyx of the gut and excretory system of flukes shared T1 antigenicity with the tegument. Monoclonal antibodies were produced against an internal immunogen associated with ribosomes and heterochromatin in active protein-producing cells, and against interstitial material of adult flukes. Monoclonals against antigens in parenchymal cell cytoplasm and in mature vitelline cells were recognized but the corresponding hybridomas were not stabilized.

An electron microscopical examination has been made of the fine structure and disposition of pancreatic polypeptide immunoreactive cells associated with the egg-forming apparatus in Diclidophora merlangi. The cell bodies are positioned in the parenchyma surrounding the ootype and taper to axon-like processes that extend to the ootype wall. The terminal regions of these processes branch and anastomose and, in places, the swollen endings or varicosities form synaptic appositions with the muscle fibres in the ootype wall. The cells are characterized by an extensive GER-Golgi system that is involved in the assembly and packaging of dense-cored vesicles. The vesicles accumulate in the axons and terminal varicosities, and their contents were found to be immunoreactive with antisera raised to the C-terminal hexapeptide amide of pancreatic polypeptide. It is concluded that the cells are neurosecretory in appearance and that, functionally, their secretions may serve to regulate ootype motility and thereby help co-ordinate egg production in the worm.

In the present study ultrastructural immunocytochemical techniques have been used to localize the cells producing two soluble egg antigens present in the hatching fluid of Schistosoma mansoni, using two recently described monoclonal antibodies (MAbs). In the miracidium of S. mansoni, the one apical and the two lateral unicellular glands showed immunoreactivity for the egg antigens, using a post-embedding immunogold technique. Immunoreactivity was associated with the contents of vesicles for both the MAbs, although the intravesicular distribution was not identical. This difference in ultrastructural staining pattern provides additional evidence that the two MAbs are binding with different epitopes of the same antigen or with different antigens altogether. The finding of free unbound label around the embedded miracidium is consistent with secretion having been released from the glands.

We have used immunoelectron microscopy to investigate the subcellular location of Pfs16 in Plasmodium falciparum. It was detected in the outer membrane region of gametocytes and more specifically on the parasitophorous vacuole membrane (pvm), since, during gametogenesis when the pvm disintegrates, the majority of the antigen was detected on the remains of this membrane in multilaminated whorls and not on the gamete plasma membrane. The antigen was also present on other gametocyte cellular structures, including those which we believe to be Garnham bodies, present in the host cell cytoplasm of some gametocytes. The antigen was present too on the membrane surrounding cytostomes and the resulting food vacuoles in the parasite cytoplasm.

A post-embedding immunogold technique has been used to examine the subcellular distribution of immunoreactivities to vertebrate pancreatic polypeptide (PP) and to the invertebrate peptide, FMRFamide within the central nervous system (CNS) of the nematode, Ascaris suum. Gold labelling of peptide was localized exclusively over dense-cored vesicles within nerve cell bodies, nerve axons and nerve terminals of the main ganglia and nerve cords in the CNS. Double-labelling of peptides demonstrated an apparent co-localization of PP and FMRFamide immunoreactivities in the same dense-cored vesicles, although populations of dense-cored vesicles that labelled solely for FMRFamide were also evident. Antigen preabsorption studies indicated little or no cross-reactivity between the two antisera.

Immunocytochemical techniques used in conjunction with confocal scanning laser microscopy (CSLM) and electron microscopy have been used to demonstrate, for the first time, the distribution of the parasitic platyhelminth neuropeptide, neuropeptide F (NPF) in the cestode, Moniezia expansa. Antisera were raised to intact NPF(1–39) and to the C-terminal decapeptide of NPF(30–39). These antisera were characterized and validated for use in both immunocytochemistry and radioimmunoassay (RIA). NPF immunoreactivity (IR) was detected using both antisera throughout all of the major components of the central and peripheral nervous systems of the worm. The pattern of NPF-IR was found to mirror the IR obtained using a C-terminally directed pancreatic polypeptide (PP) antiserum and FMRFamide antisera; blocking studies using these antisera revealed that FMRFamide and PP antisera cross-react with NPF(M. expansa). RIA of acid-alcohol extracts of the worm measured 114 ng/g using the C-terminal NPF antiserum and 56 ng/g using the whole-molecule-directed antiserum. While the C-terminally-directed NPF antiserum cross-reacts with NPF-related peptides from other invertebrates, the whole-molecule-directed NPF antiserum is specific for NPF(M. expansa). The C-terminal NPF antiserum has potential for use in the identification and purification of NPF analogues from other platyhelminth parasites.

The localization and distribution of SALMFamide immunoreactivity (IR), SI(GFNSALMFamide), in the nervous system of both the adult and larval stages of the trematode Schistosoma mansoni has been determined by an indirect immunofluorescent technique in conjunction with confocal scanning laser microscopy (CSLM). Immunostaining was widespread in the nervous system of adult male and female S. mansoni. In the central nervous system (CNS), IR was evident in nerve cells and fibres in the anterior ganglia, cerebral commissure and dorsal and ventral nerve cords. In the peripheral nervous system (PNS), IR was apparent in nerve plexuses associated with the subtegmental musculature, oral and ventral suckers, the lining of the gynaecophoric canal, and in fine nerve fibres innervating the dorsal tubercles of the male worm. In the reproductive system of male and female worms, Sl-IR was only observed around the ootype/Mehlis' gland complex in the female. Immunostaining was also evident in the nervous system of both miracidium and cercarial larval stages. A post-embedding, IgG-conjugated colloidal gold immunostaining technique was employed to examine the subcellular distribution of SALMFamide-IR in the CNS of S. mansoni. Gold labelling of peptide was localized over dense-cored vesicles within nerve cell bodies and fibres constituting the neuropile of the anterior ganglia, cerebral commissure and nerve cords of the CNS. Antigen pre-absorption studies indicated that the results obtained do suggest S1-like immunostaining and not cross-reactivity with other peptides, in particular FMRFamide.

Immunochemical techniques were used to determine the distribution, chemical characteristics and relative abundance of immunoreactivity (IR) to two native platyhelminth neuropeptides, neuropeptide F (NPF) (Moniezia expansa) and the FMRFamide-related peptide (FaRP), GNFFRFamide, in the trematodes, Fasciola hepatica and Schistosoma mansoni; the larger S. margrebowiei was used in the chemical analysis. Extensive immunostaining for the two peptides was demonstrated throughout the nervous systems of both F. hepatica and S. mansoni, with strong IR also in the innervation of muscular structures, including those associated with the egg-forming apparatus. The patterns of immunostaining were similar to those previously described for the vertebrate neuropeptide Y superfamily of peptides and for FMRFamide. Ultra-structurally, gold labelling of NPF- and GNFFRFamide-IRs was localized exclusively to the contents of secretory vesicles in the axons and somatic cytoplasm of neurones. Double-labelling experiments showed an apparent homogeneity of antigenic sites, in all probability due to the demonstrated cross-reactivity of the FaRP antiserum with NPF. Radio-immunoassay of acid-ethanol extracts of the worms detected 8·3 pmol/g and 4·7 pmol/g equivalents of NPF- and FMRFamide-IRs, respectively, for F. hepatica, and corresponding values of 4·9 pmol/g and 4·3 pmol/g equivalents for S. margrebowiei. Gel-permeation chromatography resolved IR to both peptides in discrete peaks and these eluted in similar positions to synthetic NPF (M. expansa) and GNFFRFamide, respectively.

An immunogold ultrastructural study was made of Cryptosporidium parvum-infected intestinal cells from SCID mice to locate parasite antigens recognized by monoclonal antibodies raised against sporozoite or oocyst wall antigens. The results suggested that these antigens were present in more than one life-cycle stage and demonstrated that the intracellular parasite modified the parasitophorous vacuole membrane and villous membrane surrounding the parasite. In an immuno-fluorescence antibody test monoclonal antibody (MAb) IB5 reacted with the oocyst wall, MAb 2C3 with the whole sporozoite and MAb 2B2 with the sporozoite surface. Western and dot-blot studies demonstrated that different carbohydrate epitopes were recognized by the respective sporozoite-reactive antibodies. In the ultrastructural examination MAb 1B5 reacted with macro- and microgametocytes as well as the oocyst wall. In the macrogametocyte MAb 1B5 recognized the large electron-dense bodies characteristic of this stage and, in some parasites, the parasitophorous vacuole and the parasite pellicle. The sporozoite-reactive MAbs were able to bind to all developmental stages. These antibodies recognized the parasite cytoplasm and, additionally, MAb 2B2 produced substantial labelling of the parasite membrane. Significantly, both these antibodies also detected antigen in the parasitophorous vacuole membrane and, to a lesser extent, the villous membrane surrounding the parasite.

The transformation from larval caterpillar to non-feeding pupa and adult moth involves a complete remodelling and restructuring of an insect and its organs. In the groundnut pest Amsacta albistriga Walker, the female sex-specific storage protein 1 (SP1) was monitored from last larval instars, during pupal development to the adult stage. Staining intensity of SP1 (which resolved at 82 kDa) in the peripheral fat body (PF) tissues subjected to 10% SDS-PAGE was maximum at mid-stage last larval instars and declined subsequently. However, in perivisceral fat body (PVF) tissues, the staining intensity of SP1 increased significantly from mid-stage final instar. The presence of SP1 during pupal ovary and adult ovariole development and its availability during egg development was confirmed by immunoblot analysis. Electron microscopy data revealed that the decline of biosynthesis of SP1 in PF tissues and its disintegration were associated with the appearance of irregular nucleus and autophagic vacuoles during transformation of larva to pupa. In vitro and in vivo studies using [35S]-methionine-labelled storage proteins showed that, unlike PF tissues, PVF tissue sequestered a significant amount of labelled SP1. During this transformation, SP1 was mainly sequestered as storage protein granules until they served as sources of amino acids for the production of egg yolk proteins. Localization of clathrin-coated pits and crystalline storage proteins was confirmed by immunogold tracer techniques.

The abundance and cellular location of Fe-containing superoxide dismutase (Fe-SOD) in trichomes of Nodularia, Aphanizomenon and Anabaena collected from various depths in the Baltic Sea, and in trichomes of a cultured Nodularia strain, BC Nod-9427, isolated from the Baltic Sea, was examined by immunogold labelling. For trichomes collected from natural populations the areal concentration of Fe-SOD labelling decreased with depth: trichomes collected from surface accumulations had between 8 and 11 gold particles μm−2 whereas trichomes collected from a depth of 18 m were unlabelled. When trichomes collected from a depth of 10 m (mean areal labelling density 0·5 gold particles μm−2) were exposed to the higher irradiances present at 1 m, the areal concentration of Fe-SOD increased to 3·5–4 gold particles μm−2 within 4 h. When cultures of Nodularia strain BC Nod-9427, adapted to low light (10 μmol m−2 s−1), were transferred to an incident irradiance of 1350 μmol m−2 s−1, a doubling of the areal concentration of Fe-SOD gold label was observed within 1 h. Addition of 3-(3,4-dichlorophenyl)-1,1′-dimethylurea (DCMU) to cultures immediately before their transfer to increased illumination resulted in a decrease in areal Fe-SOD concentrations whereas addition of CdCl2 caused an increase over and above that induced by the elevated irradiance. These results suggest that Baltic Sea cyanobacteria are able to modulate their Fe-SOD content but that this might be in response to oxidative stress rather than to light per se.