Schistosoma mansoni: passive transfer of resistance by serum in the vector snail, Biomphalaria glabrata

Passive transfer of natural resistance to Schistosoma mansoni (PR-1 strain) has been successfully accomplished in the snail intermediate host, Biomphalaria glabrata (PR albino, M-line strain). Injection of serum (cell-free hemolymph) from a naturally schistosome-resistant strain of B. glabrata (10-R2) into PR albino snails induced a complete protection from a primary infection with the parasite in 29 of 48 snails (60.4%). In comparison, inoculation of homologous PR albino serum or heterologous proteins (fetal calf serum) had no effect. Moreover, this protection could be induced 24 hr prior to, or 24 hr after, exposure to the parasite, although heating of 10-R2 serum to 70 C for 30 min destroyed its protective ability. When in vitro transformed sporocysts were preincubated in 10-R2 or PR albino serum and then were injected into susceptible snails, a high level of infection (88.5 and 83.3%, respectively) was produced in both groups. Thus, the 10-R2 serum factor does not appear to be mediating specific parasite recognition by host hemocytes. Alternatively, our results suggest that 10-R2 serum possesses a heat-labile factor which specifically activate B. glabrata hemocytes to encapsulate and destroy sporocysts whereas PR albino serum lacks this factor.     

Phenoloxidase Activity in Hemolymph of Plasmodium-Melanizing and Nonmelanizing Strains of Anopheles gambiae

ABSTRACT Phenoloxidase (PO) activity was compared in two strains of the mosquito, Anopheles gambiae , one of which melanotically encapsulates and kills malaria parasites ( Plasmodium spp.) and a second which permits normal development of these parasites. The enzyme assay, based on the formation of dopachrome from L-dopa, was linear with respect to enzyme concentration and time and was as inhibitable by phenylthiourea. There were no significant differences in the K m or V max of hemolymph samples taken from 3 days old naive mosquitoes of each strain. An age course demonstrated that PO activity was slightly higher in the refractory strain on Days 1 and 2 after emergence but decreased to similar stable levels in both strains by Day 3. PO activity differed in the two strains following an uninfected blood meal. While PO activity significantly increased by 48 h post blood feeding in Plasmodium -susceptible strain, activity remained level or slightly decreased in the Plasmodium -refractory strain. Finally, protease inhibitors and proteases were tested for effects on PO activity. Leupeptin, TLCK, and TPCK all inhibited PO activity in both strains. PO in both strains was activated by bovine trypsin but not by bovine chymotrypsin.     

In search of a function of Manduca sexta hemolymph protease-1 in the innate immune system

Extracellular serine protease cascades mediate immune signaling and responses in insects. In the tobacco hornworm Manduca sexta, nearly 30 serine proteases (SPs) and their homologs (SPHs) are cloned from hemocytes and fat body. Some of them participate in prophenoloxidase (proPO) activation and proSpätzle processing. Here we report the cDNA cloning of hemolymph protease-1b (HP1b), which is 90% identical and 95% similar to HP1a (formerly HP1). The HP1a and HP1b mRNA levels in hemocytes was down- and up-regulated after an immune challenge, respectively. Quantitative real-time polymerase chain reactions revealed their tissue-specific and development-dependent expression, mostly in hemocytes of the feeding larvae. We isolated HP1 precursor (proHP1) from larval hemolymph and observed micro-heterogeneity caused by N-linked glycosylation. Supplementation of the purified proHP1 to plasma samples from naïve larvae or induced ones injected with bacteria caused a small PO activity increase, much lower than those elicited by recombinant proHP1a/b, but no proteolytic cleavage was detected in the zymogens. Incubation of proHP1a/b or their catalytic domains with a cationic detergent, cetylpyridinium chloride, induced an amidase activity that hydrolyzed LDLH-p-nitroanilide. Since LDLH corresponds to the P4–P1 region before the proteolytic activation site of proHP6, we propose that the active but uncleaved proHP1 may cut proHP6 to generate HP6 that in turn activates proPAP1 and proHP8. The catalytic domain of HP1a/b, which by itself does not activate purified proHP6 or hydrolyze LDLH-p-nitroanilide, somehow generated active HP6, HP8, PAP1 and PO in plasma. Together, these results indicate that proHP1 participates in the proPO activation system, although detailed mechanism needs further exploration.     

Purification and characterization of a lectin from the banana shrimp Fenneropenaeus merguiensis hemolymph

A lectin from the hemolymph of the banana shrimp Fenneropenaeus merguiensis was purified by affinity chromatography on a fetuin-agarose column following by gel filtration on a Superose-12 column. The native molecular mass of purified F. merguiensis lectin (FmL) determined by gel filtration was 316.2 kDa and its carbohydrate content was estimated to be 4.4%. By SDS-PAGE analysis, purified FmL consisted of 32.3 kDa and 30.9 kDa subunits. These data suggest that this lectin is an oligomer. Two-dimensional electrophoresis showed that it had a pI value of 6.0 and was mainly composed of glycine, serine, histidine, glutamic acids and glutamine, with relatively lower amounts of methionine and tyrosine. Purified FmL expressed higher agglutination activity against rabbit and rat erythrocytes than with those from human, and its activity was Ca²⁺-dependent. The hemagglutinating activity of FmL was stable up to 55 °C and at pH 7.5–8. N-acetylated sugars, such as ManNAc, GlcNAc, GalNAc, and NeuNAc were strong inhibitors of the FmL induced hemagglutinating activity with NeuNAc being most effective. Porcine stomach mucin and fetuin were the most potent inhibitors of FmL. Purified FmL caused selective agglutination of Vibrio harveyi, and Vibrio parahemolyticus both pathogens of this Penaeus species and to a lesser extent Vibrio vulnificus but had no effect on the non-pathogenic strains; Vibrio cholerae, Salmonella typhi and Escherichia coli. Its bacterial agglutination was also completely inhibited by NeuNAc, mucin, fetuin and also anti-FmL antibody. This observation indicates that FmL may contribute to the defense response of this species of penaeid shrimps to potentially pathogenic bacteria.     

A serine protease inhibitor from hemolymph of green mussel, Perna viridis

Bioactivity guided fractions of cell-free hemolymph of bacterially challenged marine mussel, Perna viridis led to the isolation of a novel quaternary alkaloid 1, which was identified by its spectral data. The isolated molecule 1 has been found to be a potent serine protease inhibitor (SPI) showing IC₅₀ and K_i values of 102.5 and 97.1–104.68 μM, respectively. The E_t/K_i value of SPI is 6.3, whereas E_t/K_m value is 1.04. The Van’t Hoff analysis showed that the value of K_i decreases with increase in temperature, and the binding of the inhibitor is entropically driven.     

Pathogenesis of Helicosporidium sp. (Chlorophyta: Trebouxiophyceae) in susceptible noctuid larvae

Helicosporidium sp. is a unique, achlorophyllous green alga that has been reported to infect various insect orders, including Lepidoptera, Diptera, and Coleoptera. The infectious cyst stage is ingested by the host, ruptures in the midgut lumen, and releases a filamentous cell. Histopathological examinations using larvae of a susceptible noctuid host, Spodoptera exigua, showed both cysts and filamentous cells affiliated with the microvillar lining of the midgut epithelium. A considerable proportion of the ingested cysts (22-39%) were recovered in feces collected 24 h after ingestion. A small number of filamentous cells passed the midgut epithelium and entered the hemocoel within 4-24 h after cyst ingestion. After 48 h, vegetative cell stages were detected in the hemolymph, followed by a 4- to 5-day period of increasing multiplication. Cyst differentiation in the colonized hemolymph began 6-7 days after the treatment.     

Hyper-osmoregulatory capacity of the Chinese mitten crab (Eriocheir sinensis) exposed to cadmium; acclimation during chronic exposure

The aim of this study was to evaluate the effects of waterborne cadmium on hyper-osmoregulatory capacity of the Chinese mitten crab Eriocheir sinensis acclimated to freshwater. For this purpose, crabs were submitted to acute (0.5 mg Cd L(-1) for 1, 2 or 3 days), chronic (10 or 50 microg Cd L(-1) for 30 days) or chronic, immediately followed by acute, exposure. While no effect was observed after 1 or 2 days, hemolymph osmolality, Na(+) and Cl(-) concentrations were significantly reduced after 3 days of acute exposure. Under this latter condition, the respiratory anterior gill ultrastructure, Na(+)/K(+)-ATPase and cytochrome c oxidase activities were significantly impaired. In contrast, the osmoregulatory posterior gill was unaffected for all treatments. As a consequence, we suggest that the observed hyper-osmoregulatory capacity impairment is the result of increased dissipative flow of ions and/or water through anterior gills. In contrast to acute exposure, chronic exposure did not induce any observable effect. However, crabs submitted to a known deleterious acute condition (0.5 mg Cd L(-1) for 3 days) directly after chronic exposure to 50 microg Cd L(-1) for 30 days showed normal hyper-osmoregulatory capacity with no change in gill Na(+)/K(+)-ATPase activity, and only little disturbance of anterior gill ultrastructure. These results demonstrate that a chronic cadmium exposure can induce acclimation mechanisms related to osmoregulation in this euryhaline decapod crustacean.     

Structure and mechanical strength of larval cuticle of sawflies capable of "easy bleeding" a defence strategy against predators evolved in Tenthredinidae (Hymenoptera)

The cuticle of Tenthredinidae (Hymenoptera) larvae is ruptured after a very mild mechanical stress and hemolymph is released. This phenomenon, called "easy bleeding" is considered as a defence strategy against predators. We investigated the cuticle structure of some selected species capable of "easy bleeding" by light-, transmission- and scanning microscopy and compared it with phylogenetically-related sawfly species without this defense strategy (control). Cuticle thickness has no major influence on mechanical stability, but pronounced local differences in thickness seem to be inversely correlated with mechanical strength. The density of chitin fibrils is considerably reduced in animals capable of easy bleeding and the fibres form a loose 3D network in contrast to the densely packed layers seen in related "non easy bleeding" species like Strongylogaster multifasciata, where a pressure of 2584 kPa is needed to pierce the integument, and which served as a control. Efficient bleeders like Phymatocera aterrima (203 kPa) and Rhadinoceraea nodicornis (219 kPa) are devoid of any defined layers or other structural elements. In Athalia rosae (Allantinae) (539 kPa), and Aglaostigma discolor (Tenthredininae) (1494 kPa)--bleeders with medium efficiency--a reduced number of layers are observed by LM and TEM and a loose network of chitin fibres is visible after treatment with KOH.     

A novel protein produced by the vitellogenic fat body and accumulated in mosquito oocytes

Summary The fat body of vitellogenic mosquitoes was found to synthesize and secrete another protein in addition to vitellogenin, that accumulated in developing oocytes. In the tissues, this protein has M_r = 53000 on SDS-PAGE under reducing or non-reducing conditions. This protein is glycosylated as shown by [³H]mannose incorporation and experiments with tunicamycin. Polyclonal antibodies were produced using the ovarian 53-kDa peptide. Immunoblot analysis demonstrated the immunological identity of 53 kDa peptides from the fat body and the ovary. Furthermore, the 53-kDa protein (53KP) is synthesized and secreted exclusively by the vitellogenic fat body. Radioimmunoassay showed that 53KP is produced by the female fat body as early as 4 h and reaches its peak near 24 h after the initiation of vitellogenesis. Synthesis then drops to low levels by 36 h and declines to background levels by 48 h. In vitro experiments conducted on fat bodies of previtellogenic females demonstrated that the synthesis and secretion of 53KP can be stimulated by a physiological dose of 20-hydroxyecdysone (10^–6 M). Immunocytochemical studies of the ovary demonstrate that 53KP is present in channels between follicle cells, in the perioocytic space and in yolk granules of the developing oocytes. This suggests that 53KP is accumulated in the oocytes by a pathway similar to that of vitellogenin.     

Expression and characterization of recombinant Manduca sexta serpin- 1B and site-directed mutants that change its inhibitory selectivity

Hemolymph of Manduca sexta contains a number of serine proteinase inhibitors from the serpin superfamily. During formation of a stable complex between a serpin and a serine proteinase, the enzyme cleaves a specific peptide bond in an exposed loop (the reactive-site region) at the surface of the serpin. The amino acid residue on the amino-terminal side of this scissile bond, the P₁ residue, is important in defining the selectivity of a serpin for inhibiting different types of serine proteinases. M. sexta serpin-1B, with alanine at the position predicted from sequence alignments to be the P₁ residue, was previously named alaserpin. This alanyl residue was changed by site-directed mutagenesis to lysine (A343K) and phenylalanine (A343F). The serpin-1B cDNA and its mutants were inserted into an expression vector, H6pQE-60, and the serpin proteins were expressed in Escherichia coli. Affinity-purified recombinant serpins selectively inhibited mammalian serine proteinases: serpin-1B inhibited elastase; serpin-1B(A343K) inhibited trypsin, plasmin, and thrombin; serpin-1B(A343F) inhibited chymotrypsin as well as trypsin. All three serpins inhibited human cathepsin G. This insect serpin and its site-directed mutants associated with mammalian serine proteinases at rates similar to those reported for mammalian serpins. Serpin-1B and its mutants formed SDS-stable complexes with the enzymes they inhibited. The scissile bond was determined to be between residues 343 and 344 in wild-type serpin-1B and in serpin-1B with mutations at residue 343. These results demonstrate that the P₁ alanine residue defines the primary selectivity of serpin-1B for elastase-like enzymes, and that this selectivity can be altered by mutations at this position.