Probiotic, Lactobacillus pentosus BD6 boost the growth and health status of white shrimp, Litopenaeus vannamei via oral administration.

This study aims to assess and determine the oral-administration of probiotic, Lactobacillus pentosus BD6 on growth performance, immunity and disease resistance of white shrimp, Litopenaeus vannamei. Lac. pentosus BD6 effectively inhibited the growth of aquatic pathogens, which was used in the test. Shrimp were fed with the control diet (without probiotic supplement) for 60 days and the probiotic-containing diets at 107, 108, 109, and 1010 cfu kg-1, respectively. Shrimp fed with the diet containing probiotic at the doses of 109-10 cfu kg-1 showed significant increase in growth performance as well as feed efficiency than that of the control. After a challenge test with Vibrio alginolyticus, shrimp fed with a probiotic diet at a dose of 1010 cfu kg-1 showed a significantly lower mortality as compared to the control and that of shrimp fed the diet containing probiotic at the levels up to 107-8 cfu kg-1. In addition, a therapeutic potential of Lac. pentosus BD6 was discovered because the cumulative mortalities of shrimp fed with probiotic and pathogen V. parahaemolyticus simultaneously were significantly lower when compared to control shrimp. Probiotic in diet at a dose of 109-10 cfu kg-1 significantly increased PO activity of shrimp, while shrimp receiving probiotic at the doses of 108-10 cfu kg-1 showed significant increase in lysozyme activity and phagocytic activity. Shrimp fed with the diet containing probiotic at the level of 1010 cfu kg-1 also indicated higher gene expression of prophenoloxidase (proPO) I, but not proPO II, lipopolysaccharide and ß-1,3-glucan-binding protein and penaeidin 4. Analysis of the bacterial microbiota of the shrimp intestine revealed that oral administration of probiotic increased the relative abundance of beneficial bacteria and reduced the abundance of harmful pathogenic bacteria in the gut flora of shrimp. Despite no statistically significant difference, an analysis of microbial diversity recorded higher species richness, Shannon-Weaver diversity index and evenness in the probiotic group, compared to the control group. It was concluded that Lac. pentosus BD6 has great antibacterial ability against a wide range of pathogens and has therapeutic potential to reduce the mortality of shrimp infected with V. parahaemolyticus. Additionally, dietary Lac. pentosus BD6 at the level of 1010 cfu kg-1 was recommended to improve growth performance, immunity and disease resistance of shrimp against V. alginolyticus.

Expression of Surface-associated 82 kDa proMMP-9 in Lymphatic Leukemia Blast Cells Differentially Correlates With Prognosis.

BACKGROUND/AIM: Matrix metalloproteinases (MMPs) degrade extracellular matrix and process regulatory proteins. Recently, a membrane-bound 82kDa variant of proMMP-9 identified on myeloid blasts was shown to be associated with prognosis. PATIENTS AND METHODS: To investigate the role of 82kDa proMMP-9 with acute lymphoblastic leukemia (ALL) and chronic lymphoid leukemia (CLL), we performed flow-cytometry analysis of expression on ALL blasts (n=18) and CLL lymphocytes (n=21) from blood and correlated data with clinical parameters. RESULTS: In ALL, mature B-linear blasts expressed higher levels of 82kDa proMMP-9 compared to T-linear blasts. Elevated levels of 82kDa proMMP-9 were found in elderly patients and at patients with relapse. No correlation was observed on blood cells and extramedullary disease. In CLL, the 82kDa proMMP-9 expression did not correlate with any of the clinical parameters. CONCLUSION: Our findings suggest that higher levels of 82kDa proMMP-9 expression on blast cells may correlate with a more unfavorable ALL-subtype. Further studies are required to clarify the prognostic role of the 82kDa pro-MMP-9 expression.

A trypsin-like serine protease domain of masquerade gene in crayfish Procambarus clarkii could activate prophenoloxidase and inhibit bacterial growth.

Masquerade (Mas) is a secreted trypsin-like serine protease (SPs) and involved in immune response in some arthropods. However, according to previous studies, Mas presents different functional activities. In the present study, the functional mechanisms of Mas in crayfish Procambarus clarkii immune defense were studied. A fragment cDNA sequence of PcMas was identified and characterized. From the structural analysis, it contains a trypsin-like serine protease domain. The highest expression level of PcMas was detected in hepatopancreas. The infection of A. hydrophila could induce the expression of PcMas, while the WSSV infection did not cause changes in the expression of PcMas. Through the prokaryotic expression system, the PcMas protein was expressed in E. coli. It was verified that PcMas can bind to bacteria in vitro and inhibit the growth of the bacteria. By dsRNA interference with the expression of PcMas, the decrease expression of PcMas led to a decrease in the activity of phenoloxidase in hemolymph and an increase of mortality caused by A. hydrophila infection. The injection of recombinant protein can enhance the activity of phenoloxidase and reduce mortality caused by A. hydrophila infections. Therefore, the present study confirmed that PcMas could improve the body's immune response to eliminate bacterial pathogens by binding with bacteria and activating the prophenoloxidase system. The results will enrich the molecular mechanisms of crustaceans immune defense.

The immune function of prophenoloxidase from red swamp crayfish (Procambarus clarkii) in response to bacterial infection.

Prophenoloxidase (proPO) is the zymogen form of phenoloxidase (PO), a key enzyme in melanization cascade that has been co-opted in invertebrate immune reactions. There have been reported that proPO plays many essential roles in the crustacean immune system. However, little is known about the function of proPO from red swamp crayfish (Procambarus clarkii) which is an important cultured species worldwide. Here, we cloned and expressed proPO gene from red swamp crayfish (PcproPO). Subsequently, specific antibody against PcproPO was generated. The immune function of PcproPO was further characterized in vitro and in vivo. The results showed that the expression of PcproPO mRNA could be significantly up-regulated during the challenge of Gram-positive-negative (Vibrio parahaemolyticus) and Gram-positive-positive bacterial (Staphylococcus aureus). Furthermore, the purified recombinant PcproPO protein had a strong affinity binding to both bacteria and polysaccharides. In vivo knockdown of PcproPO could significantly reduce the crayfish bacterial clearance ability, resulting in the higher mortality of the crayfish during V. parahaemolyticus infection. In addition, in vitro knockdown of PcproPO in the hemocytes significantly reduced the phenoloxidase (PO) activity and the bacterial clearance ability, indicating that PcproPO might involve in hemocyte-mediated melanization. Our results will shed a new light on the immune function of PcproPO in the crayfish.

Endocrine-disrupting chemicals impair the innate immune prophenoloxidase system in the intertidal mud crab, Macrophthalmus japonicus.

Endocrine-disrupting chemicals (EDCs), xenobiotics that interfere with endogenous hormone function, have been studied for their impacts in aquatic environments. However, there is limited information about the potentially hazardous impact of bisphenol A (BPA) and di-(2-ethylhexyl) phthalate (DEHP) on the marine environment. The aim of this study was to investigate the effects of BPA and DEHP on the immune response of the intertidal mud crab, Macrophthalmus japonicus. In order to examine immunological responses involving the prophenoloxidase (proPO) system, mRNA transcript and activity levels of six immune-related genes, including lipopolysaccharide and ß-1,3-glucan-binding protein (LGBP), proPO, phenoloxidase (PO), peroxinectin (PE), serine protease inhibitor (Serpin), and trypsin (Tryp), were assessed in M. japonicus hepatopancreas and gills exposed to BPA or DEHP. Expression of immune genes generally decreased in M. japonicus hepatopancreas and gills exposed to all concentrations of BPA by days 4 and 7. However, at day 1, expression of Serpin and Tryp genes was significantly increased in M. japonicus hepatopancreas and gills exposed to BPA. For DEHP exposure, all genes, with the exception of Serpin, were significantly downregulated in M. japonicus gills. In the hepatopancreas, gene expression of PO, proPO, and LGBP increased at day 1, and then decreased by day 7, while mRNA expression of Serpin and Tryp exhibited up-regulation over all exposure periods. In addition, PE gene expression was upregulated in hepatopancreas at day 7 in a dose-dependent manner. Taken together, these results indicated that the crab immune responses were perturbed by exposure to BPA, and, in particular, DEHP.

White Spot Syndrome Virus-Induced Shrimp miR-315 Attenuates Prophenoloxidase Activation via PPAE3 Gene Suppression.

MicroRNAs (miRNAs), the small non-coding RNAs, play a pivotal role in post-transcriptional gene regulation in various cellular processes. However, the miRNA function in shrimp antiviral response is not clearly understood. This research aims to uncover the function of pmo-miR-315, a white spot syndrome virus (WSSV)-responsive miRNAs identified from Penaeus monodon hemocytes during WSSV infection. The expression of the predicted pmo-miR-315 target mRNA, a novel PmPPAE gene called PmPPAE3, was negatively correlated with that of the pmo-miR-315. Furthermore, the luciferase assay indicated that the pmo-miR-315 directly interacted with the target site in PmPPAE3 suggesting the regulatory role of pmo-miR-315 on PmPPAE3 gene expression. Introducing the pmo-miR-315 into the WSSV-infected shrimp caused the reduction of the PmPPAE3 transcript level and, hence, the PO activity activated by the PmPPAE3 whereas the WSSV copy number in the shrimp hemocytes was increased. Taken together, our findings state a crucial role of pmo-miR-315 in attenuating proPO activation via PPAE3 gene suppression and facilitating the WSSV propagation in shrimp WSSV infection.

Two host gut-derived lactic acid bacteria activate the proPO system and increase resistance to an AHPND-causing strain of Vibrio parahaemolyticus in the shrimp Litopenaeus vannamei.

Lactic acid bacteria (LAB) are group of beneficial bacteria that have been proposed as relevant probiotics with immunomodulatory functions. In this study, we initially isolated and identified host-derived LAB from the gut of the Pacific white shrimp Litopenaeus vannamei. Analysis of the bacterial 16S rRNA gene sequence revealed two candidate LAB, the Lactobacillus plantarum strain SGLAB01 and the Lactococcus lactis strain SGLAB02, which exhibited 99% identity to the L. plantarum strain LB1-2 and the L. lactis strain R-53658, which were isolated from bee gut, respectively. The two LAB displayed antimicrobial activities against gram-positive and gram-negative bacteria, including the virulent acute hepatopancreatic necrosis disease (AHPND)-causing strain of Vibrio parahaemolyticus (VPAHPND). Viable colony count and SEM analysis showed that the two candidate LAB, administered via oral route as feed supplement, could reside and adhere in the shrimp gut. Double-stranded RNA-mediated gene silencing of LvproPO1 and LvproPO2 revealed a significant role of two LvproPOs in the proPO system as well as in the immune response against VPAHPND infection in L. vannamei shrimp. The effect of LAB supplementation on modulation of the shrimp proPO system was investigated in vivo, and the results showed that administration of the two candidate LAB significantly increased hemolymph PO activity, the relative mRNA expression of LvproPO1 and LvproPO2, and resistance to VPAHPND infection. These findings suggest that administration of L. plantarum and L. lactis could modulate the immune system and increase shrimp resistance to VPAHPND infection presumably via upregulation of the two LvproPO transcripts.

Clip domain prophenoloxidase activating protease is required for Ostrinia furnacalis Guenée to defend against bacterial infection.

The prophenoloxidase (PPO) activating system in insects plays an important role in defense against microbial invasion. In this paper, we identified a PPO activating protease (designated OfPAP) containing a 1203 bp open reading frame encoding a 400-residue protein composed of two clip domains and a C-terminal serine protease domain from Ostrinia furnacalis. SignalP analysis revealed a putative signal peptide of 18 residues. The mature OfPAP was predicted to be 382 residues long with a calculated Mr of 44.8 kDa and pI of 6.66. Multiple sequence alignment and phylogenetic analysis indicated that OfPAP was orthologous to the PAPs in the other lepidopterans. A large increase of the transcript levels was observed in hemocytes at 4 h post injection (hpi) of killed Bacillus subtilis, whereas its level in integument increased continuously from 4 to 12 hpi in the challenged larvae and began to decline at 24 hpi. After OfPAP expression had been silenced, the median lethal time (LT50) of Escherichia coli-infected larvae (1.0 day) became significantly lower than that of E. coli-infected wild-type (3.0 days, p < 0.01). A 3.5-fold increase in E. coli colony forming units occurred in larval hemolymph of the OfPAP knockdown larvae, as compared with that of the control larvae not injected with dsRNA. There were notable decreases in PO and IEARase activities in hemolymph of the OfPAP knockdown larvae. In summary, we have demonstrated that OfPAP is a component of the PPO activation system, likely by functioning as a PPO activating protease in O. furnacalis larvae.

A novel peptidoglycan recognition protein involved in the prophenoloxidase activation system and antimicrobial peptide production in Antheraea pernyi.

Pattern recognition receptors (PRRs) are employed in insects to defend against infectious pathogens by triggering various immune responses. Peptidoglycan recognition proteins (PGRPs), a vital family of PRRs, are widely distributed and highly conserved from vertebrates to invertebrates. To date, five PGRP genes have been identified in Antheraea pernyi, but their biochemical roles still remain unknown. In this study, we focused on the immune functions of PGRP-SA in A. pernyi (ApPGRP-SA), which was confirmed to be immune-related according to its significantly up-regulated expression level post microbial injection. In addition, the binding properties of ApPGRP-SA were investigated using a recombinant protein produced in a prokaryotic expression system, revealing that rApPGRP-SA displayed a multi-binding ability to various microbes, including the Gram-positive bacteria Staphylococcus aureus and Micrococcus luteus, Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, and fungus Candida albicans, together with their surface pathogen associated molecular patterns (PAMPs). Further studies showed that after recognition, the mixture of rApPGRP-SA/PAMP remarkably stimulated prophenoloxidase (PPO) activation in the hemolymph of A. pernyi in vitro, while the ds-PGRP-SA-treated hemolymph exhibited a lower sensitivity to PAMPs in comparison to the native sample. Moreover, the transcriptional level of the three antimicrobial peptides was also decreased in PGRP-SA knock-down larvae in response to immune-challenge. In summary, we conclude that ApPGRP-SA is a novel identified PGRP in A. pernyi that might act as a broad-spectrum pattern recognition receptor and is involved in the PPO activation system as well as antimicrobial peptide production.

Dual role of the serine protease homolog BmSPH-1 in the development and immunity of the silkworm Bombyx mori.

Serine proteases and serine protease homologs are involved in the prophenoloxidase (proPO)-activating system leading to melanization. The Bombyx mori serine protease homolog BmSPH-1 regulates nodule melanization. Here, we show the dual role of BmSPH-1 in the development and immunity of B. mori. BmSPH-1 was expressed in hemocytes after molting and during the larval-pupal transformation in normal development. In contrast, following infection, BmSPH-1 was expressed in hemocytes and cleaved in the hemolymph, which resulted in the induction of PO activity. Moreover, BmSPH-1 was cleaved in the cuticle during the larval-pupal transformation and early pupal stages. In BmSPH-1 RNAi-treated silkworms, the reduced BmSPH-1 mRNA levels during the spinning stage or the prepupal stage resulted in the arrest of pupation or pupal cuticular melanization, respectively. The binding assays revealed that BmSPH-1 interacts with B. mori immulectin, proPO, and proPO-activating enzyme. Our findings demonstrate that BmSPH-1 paticipates larval-pupal transformation, pupal cuticular melanization and innate immunity of silkworms, illustrating the dual role of BmSPH-1 in development and immunity.

Identification, characterization and immune response of prophenoloxidase from the blue swimmer crab Portunus pelagicus and its antibiofilm activity.

Prophenoloxidase is a conserved Cu-containing enzyme acting as a major defense molecule in the immune response of crustaceans. In the present research, we purified prophenoloxidase from the haemolymph of Portunus pelagicus (Pp-proPO) by Blue Sepharose CL-6B chromatography. Pp-proPO exhibited only one band with molecular weight of 75kDa on SDS-PAGE. The purified Pp-proPO was characterized through X-ray diffraction (XRD) and high-performance liquid chromatography (HPLC). Pp-proPO showed phagocytic activity on the yeast Saccharomyces cerevisiae as well as encapsulation on sepharose CL-6B beads associated with CM sepharose and beads of sodium alginate. Pp-proPO also led to strong agglutination on human erythrocytes. Furthermore, Pp-proPO showed magnified PO activity when altered with activated particles acting as pathogen combined molecular patterns (PAMPs), metal ions or other chemicals. Pp-proPO showed relevant antibiofilm activity on Gram negative bacteria Pseudomonas aeruginosa and Escherichia coli. Overall, the above results allowed us to claim that Pp-proPO play a key role in immune defense mechanisms of P. pelagicus crabs, in particular towards microbial pathogens; notably we added basic information to the functional characterization of Pp-proPO, as well as to understand its immunological role in crustaceans defense systems.

An immunoproteomic approach revealed antigenic proteins enhancing serodiagnosis performance of bird fancier's lung.

BACKGROUND: Bird fancier's lung (BFL) caused by repeated inhalation of avian proteins is the most common form of hypersensitivity pneumonitis. However, the exact identification of proteins involved is unknown, and serological test use for diagnosis need to be standardized. The objectives of this study were (i) to identify antigenic proteins from pigeon droppings (ii) to provide information about their location in avian matrices and (iii) to produce them in recombinant proteins to evaluate their diagnostic performances. METHOD: Antigenic proteins of pigeon dropping extracts were investigated using 2-dimensional immunoblotting with sera from patients with BFL, asymptomatic exposed controls and healthy volunteers. We investigated the origin of these antigenic proteins by analyzing droppings, blooms and sera using a shotgun proteomic analysis. BFL-associated proteins were produced as recombinant antigens in E. coli and were assessed in ELISA with sera from patients (n=25) and subject exposed controls (n=30). These diagnostic performances were compared with those obtained by precipitin techniques (agar gel double diffusion, immunoelectrophoresis). RESULTS: We identified 14 antigenic proteins mainly located in droppings and blooms. These proteins were involved in either the digestive or immune systems of pigeons. Using the recombinant BFL-associated proteins: Immunoglobulin lambda-like polypeptide-1 (IGLL1: sensitivity: 76%; specificity: 100%; AUC: 0.93) and Proproteinase E (ProE: sensitivity: 84%; specificity: 80%; AUC: 0.85), the ELISA test showed better performance than precipitin assays with pigeon dropping extracts (sensitivity: 60%; specificity: 93.3%; AUC: 0.76). CONCLUSION: IGLL1 and ProE were identified as the biomarkers of the disease. The use of these highly standardized antigens discriminates BFL cases from exposed subjects in serological assays. The results of this study offer new possibilities for the serological diagnosis of the disease. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov: Identifier NCT03056404.

A probiotic Bacillus strain containing amorphous poly-beta-hydroxybutyrate (PHB) stimulates the innate immune response of Penaeus monodon postlarvae.

In this study, the PHB-accumulating Bacillus sp. JL47 strain (capable of accumulating 55% PHB on cell dry weight) was investigated for its effects on the immune response of giant tiger shrimp (Penaeus monodon) postlarvae (PL) before and after the Vibrio campbellii challenge. Briefly, shrimp PL were cultured and fed with Artemia nauplii enriched with Bacillus sp. JL47. Shrimp receiving the Artemia nauplii without JL47 enrichment were used as control. After 15 days of feeding, the shrimp were challenged with pathogenic V. campbellii LMG 21363 at 106 cells mL-1 by immersion. Relative expression of the immune related genes encoding for prophenoloxidase (proPO), transglutaminase (TGase) and heat shock protein 70 (Hsp70) in the shrimp were measured before (0 h) and after (3, 6, 9, 12, 24 h) the Vibrio challenge by quantitative real-time PCR using ß-actin as the reference gene. The expressions of TGase and proPO were significantly up-regulated (p < 0.05) within 9 h and 12 h, respectively after challenge in shrimp receiving the Bacillus sp. JL47 as compared to the challenged and non-challenged controls. Hsp70 expression was significantly increased (p < 0.05) at 3 h post-challenge in all challenged shrimp. Interestingly, proPO and TGase genes were significantly up-regulated (p < 0.05) in Bacillus sp. JL47 treated shrimp even before the Vibrio challenge was applied. No up-regulation in the Hsp70 gene, however, was observed under these conditions. The data suggest that the protective effect of the PHB-accumulating Bacillus sp. JL47 in shrimp was due to its capacity to stimulate the innate immune related genes of the shrimp, specifically the proPO and TGase genes. The application of probiotic Bacillus species, capable of accumulating a significant amount of PHB, is suggested as potential immunostimulatory strategy for aquaculture.

A chymotrypsin-like serine protease from Portunus trituberculatus involved in pathogen recognition and AMP synthesis but not required for prophenoloxidase activation.

Clip domain serine proteases (clip-SPs) play critical roles in various immune responses in arthropods, such as hemolymph coagulation, antimicrobial peptide (AMP) synthesis, cell adhesion and melanization. In the present study, we report the molecular and functional characterization of a clip domain serine protease (PtcSP2) from the swimming crab Portunus trituberculatus. The N-terminal clip domain and the C-terminal SP-like domain of PtcSP2 were expressed in Escherichia coli system, and assayed for their activities. Sequence similarity and phylogenetic analysis revealed that PtcSP2 may belong to the chymotrypsin family, which was confirmed by protease activity assay of the recombinant SP-like domain. The clip domain of PtcSP2 exhibited strong antibacterial activity and microbial-binding activity, suggesting the potential role in immune defense and recognition. Knockdown of PtcSP2 by RNA interference could significantly reduce PtcSP2 transcript levels, but neither decrease the total phenoloxidase (PO) activity in crab nor significantly alter the expression levels of serine protease inhibitors PtPLC and PtSerpin. These results indicate that PtcSP2 is not involved in the proPO system. However, suppression of PtcSP2 led to a significant change in the expression of AMP genes PtALFs and PtCrustin but not PtALF5. All these findings suggest that PtcSP2 is a multifunctional chymotrypsin-like serine protease and may participate in crab innate immunity by its antibacterial activity, immune recognition or regulation of AMP expression.

Immune mediators associated to male infertility in a mouse model of DNA immunization with the sperm protease proacrosin.

The immune response has relevant physiological functions both in the male and female reproductive system, and must be tightly controlled to achieve a successful pregnancy. Several immune factors have been related to infertility, among them humoral and cellular immune responses triggered by sperm antigens. The present study was aimed at evaluating the immune profile induced by DNA immunization against the sperm protease proacrosin in CF1 male mice and its effect upon fertility. Immunized animals exhibited higher anti-proacrosin antibodies levels than controls (indirect ELISA), both in serum (p<0.01) and in seminal vesicle fluid (SVF; p<0.05). IgG2a levels were higher than IgG1 in serum (p<0.01) and similar in SVF. IL-10 and TGF-ß1 mRNA levels were lower in testis (p<0.05), whereas TNF-α and IFN-γ transcript levels were increased in SV tissue (p<0.05). Immunized mice showed a trend toward higher IFN-γ concentration in serum and SVF than controls. Male fertility rate was diminished in immunized mice (p<0.01) and inversely correlated with serum and SVF anti-proacrosin IgG levels (p<0.001). Immunized animals also had fewer pups born than controls (p<0.01). To our knowledge, this is the first report on DNA immunization done in CF1 mice. Injection of proacrosin DNA induces an immune response in the male reproductive tract characterized by high levels of specific antibodies and cytokine changes. These factors may alter the crucial balance of the genital tract microenvironment required for adequate fertilization and pregnancy.

Structural and Functional Characterization of the Major Allergen Amb a 11 from Short Ragweed Pollen.

Allergy to the short ragweed (Ambrosia artemisiifolia) pollen is a major health problem. The ragweed allergen repertoire has been recently expanded with the identification of Amb a 11, a new major allergen belonging to the cysteine protease family. To better characterize Amb a 11, a recombinant proform of the molecule with a preserved active site was produced in Escherichia coli, refolded, and processed in vitro into a mature enzyme. The enzymatic activity is revealed by maturation following an autocatalytic processing resulting in the cleavage of both N- and C-terminal propeptides. The 2.05-Å resolution crystal structure of pro-Amb a 11 shows an overall typical C1A cysteine protease fold with a network of molecular interactions between the N-terminal propeptide and the catalytic triad of the enzyme. The allergenicity of Amb a 11 was confirmed in a murine sensitization model, resulting in airway inflammation, production of serum IgEs, and induction of Th2 immune responses. Of note, inflammatory responses were higher with the mature form, demonstrating that the cysteine protease activity critically contributes to the allergenicity of the molecule. Collectively, our results clearly demonstrate that Amb a 11 is a bona fide cysteine protease exhibiting a strong allergenicity. As such, it should be considered as an important molecule for diagnosis and immunotherapy of ragweed pollen allergy.

A shrimp pacifastin light chain-like inhibitor: molecular identification and role in the control of the prophenoloxidase system.

Pacifastin is a recently classified family of serine proteinase inhibitors that play essential roles in various biological processes, including in the regulation of the melanization cascade. Here, a novel pacifastin-related gene, termed PmPacifastin-like, was identified from a reverse suppression subtractive hybridization (SSH) cDNA library created from hemocytes of the prophenoloxidase PmproPO1/2 co-silenced black tiger shrimp Penaeus monodon. The full-length sequences of PmPacifastin-like and its homologue LvPacifastin-like from the Pacific white shrimp Litopenaeus vannamei were determined. Sequence analysis revealed that both sequences contained thirteen conserved pacifastin light chain domains (PLDs), followed by two putative kunitz domains. Expression analysis demonstrated that the PmPacifastin-like transcript was expressed in all tested shrimp tissues and larval developmental stages, and its expression responded to Vibrio harveyi challenge. To gain insight into the functional roles of PmPacifastin-like protein, the in vivo RNA interference experiment was employed; the results showed that PmPacifastin-like depletion strongly increased PO activity. Interestingly, suppression of PmPacifastin-like also down-regulated the expression of the proPO-activating enzyme PmPPAE2 transcript; the PmPacifastin-like transcript was down-regulated after the PmproPO1/2 transcripts were silenced. Taken together, these results suggest that PmPacifastin-like is important in the shrimp proPO system and may play an essential role in shrimp immune defense against bacterial infection. These results also expand the knowledge of how pacifastin-related protein participates in the negative regulation of the proPO system in shrimp.

Ant sperm storage organs do not have phenoloxidase constitutive immune activity.

The prophenoloxidase system (proPO-AS) is a primordial constituent of insect innate immunity. Its broad action spectrum, rapid response time, and cytotoxic by-products induced by phenoloxidase (PO) production contribute to the effective clearing of invading pathogens. However, such immune reactions may not be optimal for insect organs that evolved to have mutualistic interactions with non-self-cells. Ant queens are long-lived, but only mate early in adult life and store the sperm in a specialized organ, the spermatheca. They never re-mate so their life-time reproductive success is ultimately sperm-limited, which maintains strong selection for high sperm viability before and after storage. The proPO-AS may therefore be inappropriate for the selective clearing of sexually transmitted infections, as it might also target sperm cells that cannot be replaced. We measured PO enzymatic activity in the sperm storage organs of three ant species before and after mating. Our data show that no PO is produced in the sperm storage organs, relative to other somatic tissues as controls, and that these negative results are not due to non-detection in small volumes as non-immune-relevant catalase activity in single spermatheca fluid samples of both virgin and mated queens was significant. The lack of PO activity in sperm storage organs across three different ant species may represent an evolutionarily conserved adaptation to life-long sperm storage by ant queens. We expect that PO activity will be similarly suppressed in queen spermathecae of other eusocial Hymenoptera (bees and wasps) and, more generally, of insect females that store sperm for long periods.

[Cellular immune system of surgical maggots Lucilia sericata (Diptera, Calliphoridae)].

In the hemolymph of surgical maggots Lucilia sericata seven types of hemocytes were revealed. These are prohemocytes, stable and unstable hyaline cells, thrombocytoids, spindle cells, larval plasmatocytes and plasmatocytes I-IV, which represent sequential stages of one cell line differentiation. In contrast to Calliphora hyaline cells, this type of hemocytes in cropemptying larvae of Lucilia is elongated or vermiform in shape. Hyaline cells may be transformed to both prothrombocytoids and unstable prophenoloxydase-producing cells. Appearance and differentiation of each hemocyte type is rigidly linked with a definite stage of development. In cellular defense the main role play juvenile plasmatocytes, plasmatocytes II and III and trombocytoides. Juvenile plasmatocytes are the most active ones. After charcoal particles injection they were instantly surrounded by the thick envelope of adhered alien particles and form uniform morules aggregations or conglomerates together with thrombocytoidal agglutinates. Plasmatocytes II and III during the early stages of differentiation may be involved in adhesion and phagocytosis of alien particles and during the last stages in the engulfing of apoptose desintegrated tissues. Thus the cellular defense reaction is assisted by 4 hemocyte types--prophenoloxydase-unstable hyaline cells, thrombocytoids, juvenile plasmatocytes and plasmatocytes I-IV.

[Cellular defense system of some synanthropic dipterans inhabitant of bacterially aggressive environment].

The hemocytic count and defense reaction within 4 families of higher Diptera: Tabanidae, Syrphidae, Muscidae and Sarcophagidae, whose larvae inhabit bacterially aggressive environment, were investigated. The least hemocytes types (3) were revealed in Tabanidae and Syrphidae larvae--prohemocytes, plasmatocytes and prophenoloxydase-containing unstable hyaline cells (oenocytoids). In Sarcophaga crassipalpis and Musca domestica stable hyaline cells and thrombocytoids or podocytoid-like cells can be added to this set. At the time of pupariation in Sarcophaga, new generation of prohemocytes is segregated into the hemolymph, which form small round or spindle-shaped hyaline cells. So, the number of plasmatocyte types in Sarcophaga increase to six. Typical to Calliphoridae juvenile plasmatocytes in the members of investigated families are absent. Among the one hemocyte type morphology also can vary, especially in unstable prophenoloxydase hyaline cells. In Drosophila there are crystal cells containing in the cytoplasm paracrystalloidal inclusions. In Calliphoridae there are big hyaline cells with homogenous cytoplasm producing circumferential bubbles. Both in Sarcophaga and Tabanidae they contain in their cytoplasm big globules. However in Sarcophaga they rapidly disintegrate, while in Tabanidae are maintained unchanged during hours. In Muscidae and Syrphidae prophenoloxydase extrusion occurs very early and these cells obtain pycnotic nuclei and very liquid cytoplasm with strings of granules. Thrombocytoids in Musca larvae are represented by big flattened anucleated irregular cytoplasm and "naked" nuclei and cytoplasmic fragments often with fan-like projections. Plasmatocytes in all species studied are the cells with pronounced phylopodies. In larvae they contain cytoplasmic catabolic inclusions and in pupa--ragments of apoptotic tissues. Clearance of hemolymph from alien particles in Sarcophagidae and Muscidae occur by thrombocytoides, while in Tabanidae by plasmatocyte nodulation. A differing case is Syrphidae whe-e charcoal injection produce depletion of hemolymph both from particles and all types of hemocytes. So the specimen of different higher Diptera families can use different schemes of cellular defense reaction.