Here we focused on the dynamic processes in the snail at different time after short-term hypergravity load (STHL) by monitoring the state of neuroendocrine and immune systems, the nucleic acid synthesis levels in the atrial cells, and the behaviour of the atrial granular cells (GCs). We observed that immediately after centrifugation (14 g for 15 min) in the snail haemolymph concentration of dopamine and noradrenaline (measured by high-performance liquid chromatography) and the number of circulating haemocytes and their proliferative activity (estimated by the direct cell counting and [3H]thymidine incorporation, respectively) increased significantly, whereas the concentration of adrenaline decreased. Twenty-four hours after STHL, the levels of catecholamines and haemocytes returned to their control values. In the atrial epicardial and endothelial cells, a notable drop of transcription activity (evaluated by [3H]uridine autoradiography) from the baseline in the immediate post-STHL period was followed by its gradual increase reaching a maximum at the day 5 and subsequent decrease to control value by the day 10. In endothelial cells, DNA-synthesizing activity (evaluated by [3H]thymidine autoradiography) equal to zero before and just after STHL, increased significantly at the day 5, and decreased by the day 10. The atrial GCs underwent total degranulation. Formed as a result small ungranulated cells exhibited DNA synthesis. Afterwards, most probably, the GCs divided and regranulated. One month after STHL the GC population had been restored. Overall, STHL has triggered an immediate reaction of the neuroendocrine and immune systems and initiated long-lasting processes at a cellular level, which included alterations in activity of nucleic acid syntheses in the epicardial and endothelial cells and remodelling of the GC population in the atrium.

The larval haemolymph of three cyclorrhaphous dipteran flies, Sarcophaga ruficornis Fab., Musca domestica L. and Chrysomya megacephala Fab., possesses five different types of haemocytes: prohaemocytes; plasmatocytes; granulocytes; spherulocytes; oenocytoids. In addition, vermicytes are found in the haemolymph of S.ruficornis and adipohaemocytes in the haemolymph of M. domestica. The total haemocyte counts in the three dipteran flies show an increasing trend throughout the larval stage, attaining a peak value in the freshly formed white puparium stage and declining thereafter. The differential haemocyte count shows a decline in the number of prohaemocytes with an increase in the number of plasmatocytes, granulocytes and spherulocytes.

The nucleus diameter/cell diameter (N/C) ratio and morphological characteristics of the haemocytes of the snail Babylonia areolata were studied using microscopy. Our results revealed two major types of haemocytes, namely granulocytes and hyalinocytes. In granulocytes, the cytoplasm was purplish red with Wright's staining, but it was blue in hyalinocytes. Hyalinocytes were smaller than granulocytes and had a higher N/C ratio. The granulocytes were sub-categorized into type I granulocytes and type II granulocytes based on the shape and the number of granules. Hyalinocytes were sub-categorized into large and small hyalinocytes based on the diameter and N/C ratio. Snails with a shell length from 2.7 to 3.3 cm showed no differences in the abundance of haemocytes.

The presence of ecdysteroids during the larval life cycle and in the abdomens and ovaries of pregnant tsetse flies was established using radioimmunoassay. The hormone titre rises on day 9 of the larval life cycle 2–3 days after the hardening of the polypneustic lobes, mouthparts, phragma and anus. How initiation of tanning of the cuticle of the polypneustic lobes and later the puparium of tsetse fly is regulated is discussed as these events are separated in time by several days. It would seem to be unlikely that the pyrokinin neuropeptide, which is responsible for initiating the events that lead up to pupariation, is also involved 2–3 days earlier in the life cycle. The objective was to ascertain whether 14C-l-p-tyrosine enters the lobe cuticle in vivo and in vitro much faster than into integument cuticle on the 7th day of the pregnancy cycle. The result showed that the uptake of tyrosine into the lobes was 11–12 times faster than into the rest of the cuticle. Some evidence is presented that haemocytes underlying the epidermis are involved in overcoming the barrier to uptake of the phenolic compounds required for sclerotization of the lobes. Pupariation could be retarded by physical means such as ligaturing of the larvae. To try to break this inhibition (retardation) of pupariation in ligatured larvae, 14C-l-p-tyrosine metabolites were injected, with and without ecdysteroids, into the anterior of ligated larvae. The result of this experiment showed that the steroid hormone did not overcome the inhibition but neither does injecting tyrosine, dihydroxyphenylalanine, dopamine or tyramine. Evidence obtained from chromatography and electrophoresis indicated that N-acetylation of dopamine did not occur in the haemolymph of larvae that were artificially retarded. This impact raises the possibility that N-acetyltransferase and acetyl-coenzyme A could be released in pre-pupae after the action of a neuropeptide breaks the retardation of tanning.

The secretions of the two colleterial glands give rise to the walls of the ootheca which, when hardened, serve to protect fertilised eggs in the cockroach P. americana. The larger left gland (LCG) secretes a β-D-glucoside of 3,4-dihydroxybenzoate, several proteins (oothecins), calcium oxalate crystals and a latent phenoloxidase enzyme. The smaller right gland (RCG) secretes a β-glucosidase. When the two secretions mix in the genital vestibulum, the glucoside is hydrolyzed to glucose and free dihydroxybenzoate, which is then oxidized by the phenoloxidase to the o-benzoquinone, which cross-links the oothecins Scanning and thin section electron microscopy (EM) showed haemocytes adhering to the LCG. The haemocytes were obtained by washing the gland with insect saline; and, when they were incubated with labelled tyrosine, they showed an enhanced ability to decarboxylate L-p-tyrosine to tyramine and then deaminate and oxidize tyramine to give p-hydroxyphenylacetate. After removal of adhering haemocytes, the LCG was no longer able to decarboxylate tyrosine. Injection of α-ecdysone into the abdomens of recently emerged adult females inhibited synthesis of a phenolic glucoside in the developing LCG but not of β-glucosidase produced by RCG. Furthermore, injecting inhibitors of the decarboxylase and monoamineoxidase enzymes partly closed down synthesis in vivo of the phenolic glucoside by LCG. Therefore, in the adult female cockroach, tyramine was converted to p-hydroxyphenylacetate in the haemocytes and then transferred to the gland where it was hydroxylated to 3,4-dihydroxyphenylacetate, which gave rise to a dihydroxybenzoate. Evidence suggested that biosynthesis of the oothecal sclerotizing agent could be controlled by juvenile hormone (JH) acting on the LCG or on haemocytes adhering to the gland.

Biological invasions expose parasites to new invasive hosts in addition to their local hosts. However, local parasites are often less successful in infecting and exploiting their new hosts. This may have major consequences for the competitive ability of hosts, and finally on the fate of the parasite-host community. In Burgundy (Eastern France), the acanthocephalan parasite, Pomphorhynchus laevis, infects 2 amphipod species living in sympatry: the native Gammarus pulex and the invasive Gammarus roeseli. While P. laevis affects the behaviour and the immunity of G. pulex, G. roeseli seems unaffected by the infection. In this study, we examined in detail the ability of the parasite to affect the immune system and resource storage of both gammarid species. We found that the infection was associated with a general decrease of the prophenoloxidase activity, haemocyte density, resistance to an artificial bacterial infection and level of sugar reserves in G. pulex, but not in G. roeseli. These results demonstrate a differential ability of P. laevis to exploit its local and its invasive gammarid hosts. Potential mechanisms of these differential physiological alterations and their potential consequences on the coexistence of both gammarid species in sympatry are discussed.

Eggs of an immune suppressive strain ( = virulent) of the parasitoid Leptopilina boulardi are encapsulated neither in resistant nor in susceptible strains of Drosophila melanogaster but are encapsulated in Drosophila yakuba. Eggs of a non-immune suppressive strain ( = avirulent) of the same parasitoid are encapsulated in a resistant strain of D. melanogaster and in D. yakuba but are not encapsulated in a susceptible strain of D. melanogaster. Egg chorion in the 2 parasitoid strains showed the same morphology and the same modifications after egg laying whatever the host strain. When a capsule is built, a small dotted dense layer was first spread on the chorion, followed by accumulation layers of cells (plasmatocytes and lamellocytes) and lastly necrosis of the inner haemocytes. The encapsulated eggs darken only at the time of necrosis of haemocytes. In susceptible hosts, neither the tiny dense layer nor haemocyte accumulation occured. We concluded that (1) this tiny dense layer was present before the deposition of the first haemocytes, (2) inhibition of deposition of this dense layer was the initial event of the induced immunosupression, (3) haemocytes other than lamellocytes were engaged in caspsule formation, (4) the immunosupressive factors did not target only the lamellocytes but also the plasmatocytes, (5) darkening of the encapsulated eggs was due to cell necrosis rather than to extracellular melanin deposition.

The ability of haemocytes, from the haemolymph of the gastropod mollusc Lymnaea stagnalis, to recognize and eliminate the bacterium Aeromonas salmonicida was shown using an in vitro bacterial clearance assay. The assay employs a dye which is reduced by A. salmonicida in direct proportion to the number of viable bacteria resulting in a colour change which can be determined spectrophotometrically. Addition of cytochalasin B resulted in a marked decrease in bacterial clearance, implicating both intracellular and extracellular cytotoxicity of haemocytes. A comparison of haemocytes from uninfected snails and snails infected with the avian schistosome parasite Trichobilharzia ocellata showed that both juveniles and adults of L. stagnalis were susceptible to infection with T. ocellata. After exposure to the trematode for 1·5 h the haemocytes from these infected snails had an enhanced clearance capacity, whilst cells obtained from snails with 24–96 h infections showed decreased clearance of the bacteria, indicating suppression by the parasite. Haemocytes, as well as plasma, which was tested on haemocytes from uninfected snails, were used and hence a distinction was made between cell and humoralassociated effects. The results show that both cellular and humoral components of immunity were activated, then suppressed, following exposure to the parasite. Infection with T. ocellata seems to have a modulating effect on the bactericidal activity of the internal defence system of the snail host, L. stagnalis.

In this in vitro study we investigated whether previously described in vivo plasma-associated effects, that occurred in the period shortly after penetration of Trichobilharzia ocellata into the snail host Lymnaea stagnalis (1.5–72 h post-exposure; p.e.) were direct and/or indirect effects of parasite-derived factor(s). It was investigated whether the effect is mediated by the central nervous system (CNS) of the host. Phagocytic activity of the haemocytes was taken as a parameter for the activity of internal defence of the host. A number of preliminary experiments were performed. When the supernatant of in vitro cultured parasites (33 h; corresponding with their developmental stage in vivo when plasma-associated activation was found) was applied directly to monolayers of haemocytes, it appeared to enhance their phagocytic activity. No direct effect, however, was found with a supernatant of parasites cultured for a longer period of time (72 h; when, in vivo, a plasma-associated suppression was found). In this case, indirect suppression was detected: the parasites appeared to have released a factor that induced the CNS of the host to release material suppressing the activity of the internal defence system of the host. To date the nature of this factor is unknown.

Effects of infection with the avian schistosome Trichobilharzia ocellata on the activity of the internal defence system of the intermediate snail host Lymnaea stagnalis were studied, utilizing an in vitro phagocytosis assay for determining haemocyte activity. A distinction was made between plasma- and cell-associated effects. The period immediately after penetration of the parasite into the snail host (1.5–72 h post-exposure (p.e.)) was extensively studied. In addition, several time-points coinciding with the later–successive–stages of parasite development (2, 4, 6, 8 and 10 weeks p.e.) were investigated. Plasma-associated enhancement of defence activity was found between 1.5 and 6 h p.e., followed by plasma-associated suppression between 12 and 72 h p.e. A cell-associated activation was found between 1.5 and 6 h p.e. and also at 8 and 10 weeks p.e. How these effects on the defence system may be related to phenomena observed in infected snails at these time-points is discussed.

Injection trials with compatible and non-compatible Onchocerca species into S. damnosum s.l., the vector of human and bovine onchocerciasis, demonstrated that the rapid killing of microfilariae within the blackfly's haemocoel is species specific. In the presence of the peptide RGDS as a blocking agent for integrin-like receptors of haemocytes, the survival of O. ochengi microfilariae in its natural intermediate host was significantly increased. This increased survival 24 h p.i. correlated with a significant decrease of apoptosis levels in the microfilariae following a 2 h exposure to the haemolymph in vivo. These findings suggest that haemocytes are directly involved in the killing of Onchocerca microfilariae in the blackfly.

In contrast to the growing knowledge accumulated on plant resistance to pathogens, mechanisms of parasite resistance largely remain to be elucidated in animal species. In the present study we investigated mechanisms underlying resistance/susceptibility in the snail–trematode system Biomphalaria glabrata–Echinostoma caproni. In particular, we compared the effect of the parasite excretory–secretory (E–S) products on the defence functions of haemocytes from 2 susceptible and 2 resistant snail strains. In vitro experiments showed that E. caproni E–S products inhibit adhesion and phagocytosis of haemocytes from susceptible snails. A partial biochemical characterization also suggested that the interfering factor(s) is (are) heat-labile glycosylated polypeptides of molecular mass between 10 and 30 kDa. Interestingly, haemocytes from resistant snails remained unaffected by the parasite E–S products, suggesting that a constitutive difference results in their refractoriness to the parasite's immunosuppressive factor(s).

In order to demonstrate the involvement of nitric oxide in the defence systems of marine bivalves, we investigated the production of superoxide and nitrite, following in vitro phorbol myristate acetate stimulation of Mytilus galloprovincialis, Crassostrea gigas and Ruditapes decussatus haemocytes. Whereas M. galloprovincialis and C. gigas haemocytes were found to produce superoxide and nitrite, R. decussatus haemocytes were found to be unable to generate either of these mediators. Nitrite is a stable end product of nitric oxide and peroxynitrite as well; it appeared therefore that some marine bivalves, to kill microbial pathogens, use NADPH-oxidase and nitric oxide-synthase pathways. This was confirmed at an experimental level where inhibitors of both enzymatic pathways blocked the production of nitrite. Moreover, this notion was strengthened by the inability of the haemocytes from R. decussatus, which cannot produce superoxide, to release nitrite when stimulated.

Factors which may determine trematode–snail interactions were assessed in the present study. Compatibility was examined using a bacterial clearance assay to detect the modulatory effects of both compatible and incompatible trematode infections on the activity of haemocytes from Lymnaea stagnalis, during the early stages of infection. Exposure to and injection with Trichobilharzia ocellata, a compatible trematode, or the incompatible Schistosoma mansoni, resulted in modulation of haemocyte activity. However, T. ocellata activated haemocytes 1·5 h post-infection (p.i.) and then suppressed activity 24–72 h p.i. whereas with S. mansoni no suppression, only activation of haemocytes was observed throughout the test period (1·5–72 h p.i.). In previous studies, modulation of the haemocyte clearance activity by T. ocellata was found to be mediated by 2 E–S fractions, an activating fraction and a suppressing one. Investigations to assess whether the lack of suppression of haemocyte activity, observed in the S. mansoni–L. stagnalis incompatible trematode–snail interaction studied, was due to either the absence or ineffectiveness of the suppressing E–S fraction, were performed on a second incompatible combination, T. ocellata–Planorbis corneus. Using this combination it was revealed that only the activating E–S fraction had modulatory effects on P. corneus haemocytes, indicating that the suppressing E–S fraction, which actively interferes with the clearance activity of haemocytes from L. stagnalis, appears to act in a host-specific manner. In conclusion, the suppressing E–S fraction determines, at least in part, compatibility in the trematode–snail association studied. This is also probably likely in other trematode–snail combinations.

Excretory–secretory (E–S) products, released by Trichobilharzia ocellata cultured in vitro, were assessed for their modulatory effects on the internal defence system of the freshwater snail, Lymnaea stagnalis. E–S products were concentrated and fractionated using high-pressure liquid chromatography (HPLC) and bacterial clearance activity of haemocytes was taken as a parameter for the internal defence system. Two main fractions of E–S products were produced by T. ocellata, a low molecular fraction (low MW) and a high molecular fraction (high MW), weighing approximately 2 kDa and 40 kDa, respectively. Stage-dependent release of E–S products by transforming miracidia and mother sporocysts was indicated by the difference in proportions of these two fractions, which varied over time. After a culture time of 0–33 h, the smaller fraction was prominent whilst E–S products released between 33 and 72 h consisted of a higher proportion of the high MW fraction. After a culture of 72–96 h, lower and comparable quantities of both fractions were produced by the same batch of cultured parasites. Assaying the 2 fractions released during 0–33 h revealed that the high MW fraction had a suppressing effect on the bacterial clearance activity of haemocytes from juvenile snails and the low MW fraction activated haemocyte activity. Combined fractions obtained during 0–33 h had an activating effect on haemocyte activity but fractions separated from media collected at 33–72 h culture had a suppressing effect on the bacterial clearance activity of the haemocytes. The combined E–S fractions from the 72–96 h incubation had no effect on haemocyte activity. The present study shows that T. ocellata modulates the internal defence system of L. stagnalis by actively interfering with haemocyte function. MALDI-MS analysis of the low MW fraction revealed several components ranging from 450 to 1600 Da. Although it is as yet not clear which factor is responsible for the activating effect it is supposedly a peptide as it is pronase-sensitive and resistant to TFA and acetonitrile.

The phytochemical ptumbagin, isolated from Plumbago zeylanica L., was applied in 2 and 5 μl doses of 0.1% concentration on the surface of Dysdercus koenigii F. adults and its effect on the haemocytes of the bug, studied by transmission electron microscopy. All the five haemocyte types viz. the prohaemocyte, plasmatocyte, granular haemocyte, oenocytoid, and the adipohaemocyte are affected within a period of 24–48 hr. The effect begins with an acute state of vacuolization of the cells and a gradual destruction of the organelles, followed by dissolution of the plasma membrane and passing out of the internal organelles, like the mitochondria and the endoplasmic reticulum. This phenomenon is responsible for a consistent elimination of the haemocytes from the blood, leading to lowering of the resistance and mortality of the insects.

Five types of haemocytes were identified in Poecilocerus pictus (Fab.) and their ultrastructural details and differential counts were studied. The types identified are the prohaemocyte, plasmatocyte, granular haemocyte, oenocytoid and coagulocyte. In response to the injection of yeast cells into the haemocoel, changes in haemocyte morphology and differential counts occurred. The predominant cell involved in phagocytosis is the plasmatocyte, followed by granular haemocyte.

Both free and sessile haemocyte counts in Alphitobius piceus showed wide variations between 06.00 and 22.00 hr. Variations in the haemogram appeared to be related to sex related cycles of activity, presumably with special reference to feeding patterns. Males showed higher total haemocyte counts during the day in comparison to females. In both sexes, the percentage of prohaemocytes and spherule cells and only in males, plasmatocytes increased during the early hours of night. In females, the percentage of granular haemocytes decreased notably between 18.00 and 20.00 hr. Number of adipohaemocytes and oenocytoids did not vary significantly. Heat treatment caused release of sessile haemocytes into the circulation resulting in a notable increase in total haemocyte counts during the day. This increase was comparatively high in females in comparison with males. Altered photoperiod had little effect on these haemocyte variations. Blood volume was slightly higher in the night than in the day in both sexes. The variation of blood volume was insignificant under both heat treatment and altered photoperiod conditions.

Three morphological classes of haemocytes (prohaemocytes, plasmatocytes and granulocytes) were observed in the haemolymph of Glossina morsitans morsitans and G. pallidipes. In addition to the three types, a category of spindle cells were also observed in haemolymph especially in the newly emerged Glossina. Based on their unusual morphology, as well as on their inverse relationship with the number of thrombocytoids (a filamentous plasmatocyte), it is suggested that these spindle cells might be precursors of thrombocytoids observed mostly in older Glossina. The number of thrombocytoids increased progressively with the increasing age of the flies. Total haemocyte counts (THCs) dropped significantly during the first 48 hr following emergence, after which the values levelled off with the exception of minor fluctuations. This sudden drop in THCs resulted primarily from a remarkable decrease in the number of the spindle cells in the haemolymph. The number of round plasmatocytes decreased gradually and progressively with age of the flies, while the number of the granulocytes increased, suggesting a possible transformation of plasmatocytes into granulocytes. Newly emerged female G. morsitans morsitans were observed to have significantly higher THCs (P < 0.01) than males of the same age. Although no difference was observed between THCs of 5-week-old pregnant and non-pregnant female G. morsitans morsitans, the differential haemocyte counts (DHCs) showed that the concentration of thrombocytoid fragments was significantly lower (P < 0.01) in the pregnant than in the non-pregnant females.