Phenoloxidase (PO) is a significant biomolecule involved in humoral defence mechanism of invertebrates. Spontaneous melanization of insect haemolymph is the major hinderance for studying PO activity, as haemolymph was collected devoid of phenylthiourea. In the study, no visible melanization was observed in crude serum from the grub of Oryctes rhinoceros up to 30 min of incubation amongst crude haemolymph, diluted haemolymph, crude serum and diluted serum that were subjected to visual observation for spontaneous melanization reaction. Accordingly, crude serum was taken for evaluating PO activity. At the same time, as PO substrates tend to auto-oxidize and provide false optical density value, tris-buffered saline devoid of any substrates were used as blank for PO assays. The ideal wavelength at which maximum PO activity occurred for each substrate, namely, tyrosine, tyramine, dopamine, L-dopa, DL-dopa, catechol, protocatechuic acid and pyrogallol was determined as 407, 410, 429, 465, 403, 466, 428 and 400 nm, respectively. Additionally, time course of oxidation for each phenolic substrate by the serum PO were examined and DL-dopa was identified as the specific substrate for serum PO in the grub of O. rhinoceros. Furthermore, maximum PO activity was observed at 5 min of incubation for 10 mM of DL-dopa that was considered as optimum concentration. The ideal pH and temperature for serum PO activity was observed as 7.5 and 20°C, respectively. These results suggested that standardizing a suitable substrate is an essential prerequisite to evaluate the real PO activity of serum which might significantly fluctuate in each insect model.

This study constitutes a first attempt to investigate sex differences in osmoregulatory capacity and metabolic responses in relation to hyper- and hypo-osmoregulation in the intertidal euryhaline crab Uca tangeri. Adult male and female specimens from Cadiz Bay, Spain (36°23′–37′N 6°8′–15′W), were acclimated to three different environmental salinities (12, 33 and 55 psu) during 7 days, and several parameters were assessed in haemolymph (osmolality, glucose, amino acids, triglycerides and lactate) as well as in metabolic key organs (hepatopancreas, anterior and posterior gills: glycogen, free glucose, amino acids and triglycerides). Specimens from both sex exhibited high and similar hyper- and hypo-osmoregulatory capacities. However, metabolite levels were differentially affected upon acclimation to low and high salinity in several metabolic organs and haemolymph of male and females: (i) glycogen in gills, (ii) free glucose in gills and hepatopancreas, (iii) amino acids in hepatopancreas, (iv) triglycerides in haemolymph, hepatopancreas and posterior gills, and (v) lactate in haemolymph. The results suggest the occurrence of differential metabolic adjustments upon hyper- and hypo-osmoregulation related to sex in the intertidal euryhaline crab U. tangeri.

Sarcocystis neurona is a terrestrial parasite that can cause fatal encephalitis in the endangered Southern sea otter (Enhydra lutris nereis). To date, neither risk factors associated with marine contamination nor the route of S. neurona infection to marine mammals has been described. This study evaluated coastal S. neurona contamination using California mussels (Mytilus californianus) as sentinels for pathogen pollution. A field investigation was designed to test the hypotheses that (1) mussels can serve as sentinels for S. neurona contamination, and (2) S. neurona contamination in mussels would be highest during the rainy season and in mussels collected near freshwater. Initial validation of molecular assays through sporocyst spiking experiments revealed the ITS-1500 assay to be most sensitive for detection of S. neurona, consistently yielding parasite amplification at concentrations ⩾5 sporocysts/1 mL mussel haemolymph. Assays were then applied on 959 wild-caught mussels, with detection of S. neurona confirmed using sequence analysis in three mussels. Validated molecular assays for S. neurona detection in mussels provide a novel toolset for investigating marine contamination with this parasite, while confirmation of S. neurona in wild mussels suggests that uptake by invertebrates may serve as a route of transmission to susceptible marine animals.

The tick Rhipicephalus (Boophilus) microplus is an important parasite of cattle in many areas of the tropics. Characterization of molecules involved in mechanisms such as vitellogenesis and embryo development may contribute to a better understanding of this parasite's physiology. The vitellin-degrading cysteine endopeptidase (VTDCE) is the most active enzyme involved in vitellin hydrolysis in R. microplus eggs. Here we show an association between VTDCE and vitellin in an additional site, apart from the active site. Our data also demonstrate cysteine endopeptidase activity in different tissues such as ovary, gut, fat body, salivary gland and female haemolymph, where it is controlled by a physiological inhibitor. In R. microplus female gut, VTDCE is localized in areas of protein synthesis and trafficking with the underlying haemolymph. VTDCE is also localized in the ovary basal region, in vesicle membranes of ovary pedicel cells and in oocyte cytosol. These results suggest that VTDCE plays a role in vitellin digestion during tick development.

Activity of the following glycosidases was detected in the plasma of the freshwater snail Biomphalaria glabrata: β-D- fucosidase, β-D-glucosidase, β-D-galactosidase, β-D-mannosidase, β-D-glucuronidase, N-acetyl-β-D-galactosaminidase, N-acetyl-β-D-glucosaminidase, and lysozyme. At the physiological pH (7·2–7·4) of snail haemolymph, enzymatic activity was about 10–50% of the maximum activity at each enzyme's respective acid pH-optimum. Schistosome-susceptible B. glabrata showed lower plasma protein concentration and significantly lower enzymatic activities (U/mg protein) than schistosome-resistant snails. Changes in glycosidase activity levels correlate with the progress of infection. After successful schistosome invasion, activities of plasma glycosidases but not the concentration of total plasma proteins increased significantly during the first 2 days in both snail strains. Thus, most tegumental glycoproteins of schistosome larvae can be altered by humoral host glycosidases. The detection of only very low activities of hexosaminidases leads to the hypothesis that GalNAc/GlcNAc may be involved in the process of non-self recognition. At 4 days post-infection, glycosidase activities were identical or slightly below the levels found in naive snails. At this time of infection the parasite is encapsulated and destroyed by haemocytes of resistant snails. In susceptible snails, however, the schistosomes have transformed into sporocysts and will complete their life-cycle without eliciting effective defence reactions. After >30 days post-infection, when cercariae are fully developed in susceptible snails, plasma protein concentration decreased significantly, whereas glycosidase activities were elevated.

An in vitro bioassay was used to examine [14C]glucose incorporation into polysaccharides in albumen glands (AGs) of susceptible M-line Biomphalaria glabrata infected with the NMRI strain of Schistosoma mansoni. Polysaccharide and galactogen synthesis were unaffected by larval trematode infection in AGs of snails at days 14, 21, and 28 post-infection (p.i.) when compared to uninfected controls. Further experiments were conducted to determine if daughter sporocysts, hypothesized to be primary mediators of parasitic castration in this system, were able to exert direct effects on synthetic activity of uninfected AGs via haemolymph-borne molecules or in vitro culture-generated larval excretory–secretory (ES) products. When AGs were incubated in the presence of infected snail haemolymph, significant differences in quantities of polysaccharides and galactogen were detected only in test organs incubated in day 28 p.i. haemolymph. Daughter sporocyst ES products generated during the first 48 h of culture caused a significant reduction in polysaccharide and galactogen synthesis in test organs. When ES products from days 3 to 6 of in vitro culture were tested similarly, no significant differences in either polysaccharide or galactogen synthesis were observed between control and test organs. These data demonstrate that daughter sporocysts are able to modulate a specific aspect of the reproductive activity of the snail host through haemolymph-borne molecules of host or parasite origin, or directly through in vitro culture-generated ES products.

Trials were carried out to study the humoral immune response of blackflies to filariae following infection using the intrathoracic injection technique. An induced 66 kDa protein was abundant in the haemolymph of the European species Simulium ornatum following infection with bovine Onchocerca lienalis. This protein was apparently at higher concentrations in the haemolymph of sham-inoculated flies, i.e. flies that received sterile medium without the parasites. A molecule of the same size was also observed in the haemolymph of infected S. damnosum s.l. following infection with human O. volvulus or bovine O. ochengi. However, the level of this protein was lower in blackflies injected with microfilariae of bovine O. dukei. Unlike O. volvulus and O. ochengi this species is not transmitted by S. damnosum s.l. under natural conditions. No such reaction was observed if the African blackflies had received a sham inoculation. Feeding experiments with wild-caught nulliparous S. damnosum sl. on Onchocerca-infected cattle supported the results of the injection trials. The 66 kDa protein could only be found in the haemolymph of specimens infected via a blood meal. This 66 kDa molecule was identified as phenoloxidase. It appeared in the haemolymph due to the activation of the prophenoloxidase system following the filarial infection and we hypothesize that it may be sequestered by the parasites, as part of a non-self recognition system.

Comparisons were made between the protein concentrations and protein profiles of uninfected Gammarus pulex and those infected with cystacanths of Pomphorhynchus laevis. Comparisons of the extractable haemolymph volumes from uninfected and infected G. pulex, when related to host tissue weight, showed that there was no difference in the host tissue/haemolymph volume ratio between infected and uninfected individuals. SDS-PAGE of haemolymph samples resolved 36 bands of protein, the major components being band 6(19 kDa) and 21(33 kDa). Densitometric analysis of haemolymph samples showed that infected gammarids had total protein concentrations elevated by 84%. The (88 kDa) subunit was identified as the monomer of the respiratory protein haemocyanin. This band was significantly elevated in infected individuals (one-way ANOVA P < 0.001); increasing from 30 to 45% of the total haemolymph proteins.

Molecular interplay during snail-schistosome interactions is poorly understood and there is much to discover concerning the effect of snail host molecules on molecular processes in schistosomes. Using the Biomphalaria glabrata – Schistosoma mansoni host-parasite system, the effects of exposure to haemolymph, derived from schistosome-resistant and susceptible snail strains, on protein tyrosine phosphorylation in miracidia have been investigated. Western blotting revealed several tyrosine phosphorylated proteins in this larval stage. Exposure of miracidia to haemolymph from susceptible snails for 60 min resulted in a striking, 5-fold, increase in the tyrosine phosphorylation of a 56 kDa (p56) S. mansoni protein. In contrast, haemolymph from resistant snails had little effect on protein tyrosine phosphorylation levels in miracidia. Confocal microscopy revealed that tyrosine phosphorylation was predominantly associated with proteins present in the tegument. Finally, treatment of miracidia with the tyrosine kinase inhibitor genistein significantly impaired their development into primary sporocysts. The results open avenues for research that focus on the potential importance of phospho-p56 to the outcome of schistosome infection in snails, and the significance of protein tyrosine kinase-mediated signalling events to the transformation of S. mansoni larvae.

Sex pheromonal components of the tephritid fruit fly Bactrocera dorsalis (Hendel), 2-allyl-4,5-dimethoxyphenol and (E)-coniferyl alcohol, are biosynthesized from a highly potent male attractant, methyl eugenol, then sequestered and stored in the rectal gland prior to their release during courtship at dusk. These sex pheromonal components have been detected in the haemolymph and crop organ. Hence, attempts were made to separate and identify the haemolymph fractions which contained the sex pheromonal components. Identification of these bioactive fractions in methyl eugenol-fed male flies using gel filtration column chromatography and biodetection using live male flies showed two fractions as highly attractive to conspecific males. These fractions show a significant increase in protein absorbance in the elution profile of haemolymph from methyl eugenol-fed males compared with that from methyl eugenol-deprived males. The molecular mass of these bioactive fractions as determined by using gel filtration was in the peptide range of 3.3 to 5.5 kDa. Subsequent gas chromatography–mass spectrometry analyses further confirmed the presence of the pheromonal components in the bioactive fractions. The presence of these methyl eugenol-derived sex pheromonal components in specific haemolymph fractions suggests the involvement of a sex pheromone binding complex.

A number of cephalopod species present substantial ecological and economical importance; however, data on the physiology of stress and on regulatory processes linking stress to immune defence against pathogens remain extremely scarce in these organisms. The present study examined the influence of a 5 min air exposure, a common perturbation associated with handling in aquaculture settings and fisheries, on neuroendocrine and immune parameters in the octopus Eledone cirrhosa. Measurements of circulating concentrations of noradrenaline and dopamine, two hormones that are released in the haemolymph during stress in bivalves and gastropods, showed that the 5 min air exposure represents a real stress to octopus. Indeed, blood levels of both hormones increased by about 2–2.5-fold in stressed animals. Concomitantly, a significant decrease in the number of circulating haemocytes was observed, whereas haemocyte phagocytotic activity and superoxide anion production increased transiently between 5 and 60 min after the beginning of the stress. These results provide a first insight into the effects of stress on catecholamine levels and immune functions in cephalopods and suggest that stress and immunity may be associated in these organisms.

Changes in the pattern of haemolymph polypeptides were studied to map the innate immune response of the malaria vector Anopheles stephensi (Diptera: Culicidae) to infection by the parasite Plasmodium yoelii yoelii. In addition, changes in the ontogenetic pattern due to glucose feeding, age, sex and gonotrophic cycle-related proteins were analysed. Six polypeptides were induced and 22 polypeptides disappeared due to glucose feeding during various stages of the mosquito's adult life. Five polypeptide patterns (91, 100, 108, 133 & 145 KDa) were found exclusively in female haemolymph. The changes in these polypeptide patterns have been correlated with sexual dimorphism in their feeding behaviour.

In total, 18 polypeptides were induced in the haemolymph of parasitised mosquitoes during different stages of development. Most of the polypeptides were induced in the early stages of infection, i.e. immediately after a bloodmeal. One polypeptide (25 KDa) was induced in all the stages. The expression of two polypeptides (32.5 and 70 KDa) on day 9 and one polypeptide (42 KDa) on day 15 was also enhanced following parasitism. The different induced proteins may help mosquitoes of different ages cope with parasite infection. The parasite evidently elicits immune responses in multiple tissues of the mosquito, including two epithelia which the parasite must traverse to complete its development. The mechanism of these responses and their significance in malaria transmission are discussed.

The effect of photoperiod on the haemolymph protein profile in the silkworm, Bombyx mori L. larvae was investigated at photoperiod treatments of light to dark (L:D) ratios of 13:11 (control), 24:0, 18:6, 12:12, 6:18 and 0:24 hours using fifth instar larvae. The intensity of protein bands in haemolymph extracts from larvae was measured with a densitometer. The intensity was highest in the haemolymph from larvae in the 24:0 (L:D) h photoperiod. Strongly reactive antigens of SP-1 and SP-2 and weakly reactive bands of storage proteins for both male and female larvae were observed in the 24:0 (L:D) h photoperiod.

Eucalyptus oil vapour treatment provided to Dysdercus koenigii female individuals as virgin adults or during their fifth instar nymphal life led to a fall in the total protein content accompanied by a rise in the free amino acid titre of haemolymph and fat body. However, the level of total lipids in these two tissues under such condition varied in that a decrease and an increase were recorded in haemolymph and fat body respectively. In the ovary of this adult female, the amounts of total proteins and lipids were low, while the value for total free amino acids was high. The implications of all these findings are discussed chiefly in relation to transport, synthesis and degradation of biochemical constituents in the three tested tissues.

Polyacrylamide disc gel electrophoresis (PAGE) was performed on soluble proteins from the fat body, haemolymph (male and female), and ovary of the adult fresh water-bug, Laccotrephes rubra, in order to determine their protein patterns. The electrophoresis resulted in the separation of 16 and 17 protein components in male and female haemolymph respectively. In female fat body extracts 15 protein bands could be detected; whereas ovary extracts resolved into 11 protein bands only. Comparison of the electropherograms of protein pattern of fat body (FBPP), haemolymph (HPP) and ovary (OPP) revealed that the extra band present in the female haemolymph is common to both female fat body and ovary, which is suggestive of it being the female specific protein, i.e. vitellogenin. A careful examination of the various electropherograms further revealed that the majority of the protein bands of haemolymph and ovary are common to its corresponding fat body protein bands. This might suggest that the ovary derives most of its protein components from the surrounding haemolymph, which have presumably been synthesized and secreted by the fat body.

The histochemical analyses demonstrated that the female-specific protein band is a glycolipoprotein; and most of the haemolymph protein zones stain as glycoprotein, lipoprotein or ribonucleoprotein. ‘Stains-all’ technique however, revealed the presence of four possible RNA bands in fat body extracts.