Granular Hemocytes Research Articles

Morphology and ultrastructure of the somatic cells in Astacus leptodactylus ovary

We defined the somatic environment in which female germinal cells develop, and performed ultrastructural analyses of various somatic cell types, with particular reference to muscle cells and follicle cells, that reside within the ovary at different stages of oogenesis. Our findings show that ovarian wall of the crayfish is composed of long muscle cells, blood cells, blood vessels and hemal sinuses. The follicle and germinal cells lie within a common compartment of ovarian follicles that is defined by a continuous basal matrix. The follicle cells form branching cords and migrate to surround the developing oocytes. A thick basal matrix separates the ovarian interstitium from ovarian follicles compartment. Transmission electron microscopy shows that inner layer of basal matrix invaginates deeply into the ovarian compartment. Our results suggest that before being surrounded by follicle cells to form follicles, oogonia and early previtellogenic oocytes reside within a niche surrounded by a basal matrix that separates them from ovarian interstitium. We found coated pits and coated vesicles in the cortical cytoplasm of previtellogenic and vitellogenic oocytes, suggesting the receptor mediated endocytosis for transfer of material from the outside of the oocytes, via follicle cells. The interstitial compartment between the inner muscular layer of the ovarian wall and the basal matrix of the ovarian follicle compartment contains muscle cells, hemal sinuses, blood vessels and blood cells. Granular hemocytes, within and outside the vessels, were the most abundant cell population in the ovarian interstitium of crayfish after spawning and in the immature ovary.

Antioxidant enzyme activities and immunological system analysis of Litopenaeus vannamei reared in biofloc technology (BFT) at different water temperatures

The purpose of this study was to evaluate the effect of water temperature on the antioxidant (gills, hemolymph and hepatopancreas) and immunological systems (hemolymph) of Litopenaues vannamei reared with no water exchange. A 60-day trial was conducted at different temperatures (15, 21, 27—control and 33°C), with 3 replicate tanks for each treatment. The immunologic parameters analyzed were as follows: hyaline and granular hemocyte count, total protein and apoptosis. The enzymatic and lipid peroxidation assays consisted of glutamate cysteine ligase (GCL), reduced glutathione (GSH) and thiobarbituric acid-reactive substances (TBARS). There were no significant differences (p>0.05) in immunological parameters among treatments throughout the experimental period. Shrimp exposed to 15°C at 30days exhibited significantly higher GCL activity in gills compared to 21°C (1166.28%), 27°C (422.96%) and 33°C (809.37%). L. vannamei reared at 15°C had the highest GSH concentration at 30days. The GSH increase in gills was 390.29, 376.94 and 361.75% at 21, 27 and 33°C, respectively. On day 30, shrimp subjected to 33°C had higher TBARS levels (6157.14; 8620.00 and 14,336.66% at 15, 21 and 27°C, respectively). Shrimp reared at 15°C exhibited higher activity of the rate-limiting compound for GSH production (GCL) and higher concentrations of this non-enzymatic antioxidant. This increased activity may counteract the overproduction of reactive oxygen species generated by temperature stress. We further noted that at 33°C, the animals showed higher lipid peroxidation. The lowest stress response was observed in shrimp reared at 27°C (the control group). Therefore, the extreme temperatures should be avoided. Statement/relevanceThis study present new results about handling and welfare.

Yellow head virus infection in black tiger shrimp reveals specific interaction with granule-containing hemocytes and crustinPm1 as a responsive protein

Yellow head virus (YHV) causes acute infections and mass mortality in black tiger shrimp culture. Our study aims to investigate molecular interaction between YHV and circulating hemocytes of Penaeus monodon at early infection. Total shrimp hemocytes were isolated by Percoll gradient centrifugation and identified by flow cytometric analysis. At least three types of hemocyte cells were identified as hyaline, semi-granular, and granular hemocytes. Experimental infection of YHV in shrimp culture demonstrated drastic changes in total and each hemocyte cell counts. Immunohistochemistry analysis demonstrated interaction and replication of YHV mainly with the granule-containing hemocytes and little to none in hyaline cell. These granule-containing hemocytes are proposed to be YHV targets providing the first line of defense to viral infection. Protein expression profiling of granule-containing hemocytes revealed several immune-responsive proteins including antimicrobial protein crustins (crustinPm1 and crustinPm4), alpha-2-macroglobulin, and kazal-type serine proteinase inhibitor. During an early phase of YHV infection at 6 hpi crustinPm1 illustrated a significant increase of mRNA and protein expression level in plasma. The results suggest that an antimicrobial crustinPm1 may participate in shrimp defense mechanism against YHV, especially on the granule-containing hemocytes.

Purification and Characterization of a Cysteine-Rich 14-kDa Antibacterial Peptide from the Granular Hemocytes of Mangrove Crab Episesarma tetragonum and Its Antibiofilm Activity.

Antimicrobial peptide (AMP) crustin is a type of immune molecule present in the immune system of crustaceans and response against microbial invasion. In the present study, we have identified and characterized the cationic, amphipathic structure consisting of AMP crustin from a mangrove crab Episesarma tetragonum using CM Sepharose-based cation exchange column chromatography. E. tetragonum crustin showed a single band of 14kDa on SDS-PAGE and the homogeneity showed retention time of 8.4min in RP-HPLC. Functional studies of E. tetragonum crustin exhibits the antibacterial activity (2-4μg/ml) and biofilm inhibition (20μg/ml) against the Gram-positive bacteria Staphylococcus aureus and Enterococcus faecalis. Hydrophobicity and extrapolysaccharide production of Gram-positive bacteria were inhibited through the bactericidal inhibitory concentration. In situ visualization analysis of biofilm inhibition was observed through light and confocal laser scanning microscopy. Surface morphology and the bacterial biofilm inhibition were viewed by scanning electron and atomic force microscopy. This study emphasizes the potential activity of E. tetragonum crustin, an interesting candidate for the development of novel broad-spectrum antimicrobial agent against bacterial pathogens. Graphical Abstract Antimicrobial peptide synthesis and host-pathogen interaction lead to production of immune molecules directed to destruction of pathogens.

Prolonged pre-incubation increases the susceptibility of Galleria mellonella larvae to bacterial and fungal infection

Galleria mellonella larvae are widely used for assessing the virulence of microbial pathogens and for measuring the in vivo activity of antimicrobial agents and produce results comparable to those that can be obtained using mammals. The aim of the work described here was to ascertain the effect of pre-incubation at 15°C for 1, 3, 6 or 10 weeks on the susceptibility of larvae to infection with Candida albicans and Staphylococcus aureus. Larvae infected with C. albicans after 1 week pre-incubation at 15°C showed 73.3 ± 3.3% survival at 24 hours post-infection while those infected after 10 weeks pre-incubation showed 30 ± 3.3% survival (P < 0.01). Larvae infected with S. aureus after 1 week pre-incubation showed 65.5 ± 3.3% survival after 24 hours while those infected after 10 weeks pre-incubation showed 13.3 ± 3.3% (P < 0.001). Analysis of the haemocyte density in larvae pre-incubated for 3–10 weeks showed a reduction in haemocytes over time but a proportionate increase in the density of granular haemocytes in the population as determined by FACS analysis. Proteomic analysis revealed decreased abundance of proteins associated with metabolic pathways (e.g. malate dehydrogenase, fructose-1,6-bisphosphatase, glyceraldehyde-3-phosphate dehydrogenase) and prophenoloxidase. G. mellonella larvae are a useful in vivo model system but the duration of the pre-incubation stage significantly affects their susceptibility to microbial pathogens possibly as a result of altered metabolism.

A recombinant horseshoe crab plasma lectin recognizes specific pathogen-associated molecular patterns of bacteria through rhamnose.

Horseshoe crab is an ancient marine arthropod that, in the absence of a vertebrate-like immune system, relies solely on innate immune responses by defense molecules found in hemolymph plasma and granular hemocytes for host defense. A plasma lectin isolated from the hemolymph of Taiwanese Tachypleus tridentatus recognizes bacteria and lipopolysaccharides (LPSs), yet its structure and mechanism of action remain unclear, largely because of limited availability of horseshoe crabs and the lack of a heterogeneous expression system. In this study, we have successfully expressed and purified a soluble and functional recombinant horseshoe crab plasma lectin (rHPL) in an Escherichia coli system. Interestingly, rHPL bound not only to bacteria and LPSs like the native HPL but also to selective medically important pathogens isolated from clinical specimens, such as Gram-negative Pseudomonas aeruginosa and Klebsiella pneumoniae and Gram-positive Streptococcus pneumoniae serotypes. The binding was demonstrated to occur through a specific molecular interaction with rhamnose in pathogen-associated molecular patterns (PAMPs) on the bacterial surface. Additionally, rHPL inhibited the growth of P. aeruginosa PAO1 in a concentration-dependent manner. The results suggest that a specific protein-glycan interaction between rHPL and rhamnosyl residue may further facilitate development of novel diagnostic and therapeutic strategies for microbial pathogens.

The phylogenetic profile of mast cells.

Mast cells (MCs) are tissue-based immune cells that participate to both innate and adaptive immunities as well as to tissue-remodelling processes. Their evolutionary history appears as a fascinating process, whose outline we can only partly reconstruct according to current remnant evidence. MCs have been identified in all vertebrate classes, and a cell population with the overall characteristics of higher vertebrate MCs is identifiable even in the most evolutionarily advanced fish species. In invertebrates, cells related to vertebrate MCs have been recognized in ascidians, a class of urochordates which appeared approximately 500 million years ago. These comprise the granular hemocyte with intermediate characteristics of basophils and MCs and the "test cell" (see below). Both types of cells contain histamine and heparin, and provide defensive functions. The test cell releases tryptase after stimulation with compound 48/80. A leukocyte ancestor operating in the context of a primitive local innate immunity probably represents the MC phylogenetic progenitor. This cell was likely involved in phagocytic and killing activity against pathogens and operated as a general inducer of inflammation. This early type of defensive cell possibly expressed concomitant tissue-reparative functions. With the advent of recombinase activating gene (RAG)-mediated adaptive immunity in the Cambrian era, some 550 million years ago, and the emergence of early vertebrates, MC progenitors differentiated towards a more complex cellular entity. Early MCs probably appeared in the last common ancestor we shared with hagfish, lamprey, and sharks about 450-500 million years ago.

Yellow head virus binding to cell surface proteins from Penaeus monodon hemocytes

Our previous data revealed that viral particles of yellow head virus (YHV) specifically interacted with granule-containing hemocytes. After isolation of targeted hemocytes, biotinylation was performed using Biotin–NSH–LC. Biotinylated protein was extracted and separated by 2-D PAGE. Electro-transferred proteins on a nitrocellulose membrane were probed with streptavidin-HRP complex to detect biotinylated proteins. The data from 2-D PAGE combined with affinity pull down purification revealed 8 and 6 biotinylated proteins specific to hyaline and granule containing hemocytes, respectively. Four proteins were found in common for both two hemocytes. The majority of proteins detected in granular hemocytes are membrane-associated proteins and immune-related proteins such as alpha-2-macroglobulin (A2M), kazal-type serine protease inhibitor (SPI) and crustin. CrustinPm1 was found to bind to YHV as shown with biotinylation pull-down assay and confirmed with two-dimensional virus overlay protein binding assay (2-D VOPBA). The expression of crustinPm1 was observed in semigranular and granular hemocytes whereas very low or no expression occurred in hyaline hemocytes. CrustinPm1 appears to either be directly involved in cellular binding or mediating virus internalization into permissive hemocytes.

Characterization of a lectin from the craysfish Cherax quadricarinatus hemolymph and its effect on hemocytes

Lectins participate in the immune mechanisms of crustaceans. They have been considered as humoral receptors for pathogen-associated molecular patterns; however, some reports suggest that lectins could regulate crustacean cellular functions. In the present study, we purified and characterized a serum lectin (CqL) from the hemolymph of Cherax quadricarinatus by affinity chromatography and determined its participation in the regulation of hemocytes' oxidative burst. CqL is a 290-kDa lectin in native form, constituted by 108, 80, and 29-kDa subunits. It is mainly composed of glycine, alanine, and a minor proportion of methionine and histidine. It showed no carbohydrates in its structure. CqL is composed of several isoforms, as determined by 2D-electrophoresis, and shows no homology with any crustacean protein as determined by Lc/Ms mass spectrometry. CqL agglutinated mainly rat and rabbit erythrocytes and showed a broad specificity for monosaccharides such as galactose, glucose, and sialic acid, as well as for glycoproteins, such as porcine stomach and bovine submaxillary mucin and fetuin. It is a Mn2+-dependent lectin. CqL recognized 8% of crayfish granular hemocytes and increased 4.2-fold the production of hemocytes' superoxide anion in vitro assays when compared with non-treated hemocytes. This effect showed the same specificity for carbohydrates as hemagglutination; moreover, superoxide dismutase and diphenyleneiodonium chloride were effective inhibitors of CqL oxidative-activation. The CqL homoreceptor is a 120-kDa glycoprotein identified in the hemocytes lysate. Our results suggest that CqL participates actively in the regulation of the generation of superoxide anions in hemocytes using NADPH-dependent mechanisms.

Granular hemocytes are phagocytic, but agranular hemocytes are not, in the Eastern Oyster Crassostrea virginica

Granular hemocytes are phagocytic, but agranular hemocytes are not, in the Eastern Oyster Crassostrea virginica

Characterization of the circulating hemocytes in mud crab (Scylla olivacea) revealed phenoloxidase activity

This study focused on an isolation and characterization of the circulating hemocytes in mud crab, Scylla olivacea. Isolation of specific cell types of hemocytes from crab hemolymph was accomplished by using 60% Percoll density gradient centrifugation. Four separated bands of the hemocytes were successfully obtained. Characterization of these isolated hemocytes by light microscope using trypan blue-rose bengal staining, rose bengal–hematoxilin staining, and phase contrast revealed four distinct types of hemocyte cells. Using their specific morphology and granularity, they were identified as hyaline cell (HC), small granular cell (SGC), large granular cell (LGC) and mixed granular cell (MGC). Transmission electron microscopy (TEM) revealed more details on specific cell size, size of cytoplasmic granule, and nuclear to cytoplasmic ratio, and confirmed the classification. Relative abundance of these cells types in the hemolymph of an adult crab were 15.50±8.22% for HC, 55.50±7.15% for SGC, 13.50±5.28% for LGC, and 15.50±3.50% for MGC. Proteomic analysis of protein expression for each specific cell types by two-dimensional electrophoresis identified two highly abundant proteins, prophenoloxidase (ProPO) and peroxinectin in LGC. Determination of phenoloxidase (PO) activity in each isolated cell types using in vitro and in situ chemical assays confirmed the presence of PO activity only in LGC. Based on an increased PO activity of crab hemolymph during the course of White Spot Syndrome Virus (WSSV) infection, these results suggest that prophenoloxidase pathway was employed for host defense mechanism against WSSV and it may link to the role of large granular hemocyte.

Interaction between Kazal serine proteinase inhibitor SPIPm2 and viral protein WSV477 reduces the replication of white spot syndrome virus

White spot syndrome (WSS) is a viral disease caused by white spot syndrome virus (WSSV) which leads to severe mortality in cultured penaeid shrimp. In response to WSSV infection in Penaeus monodon, a Kazal serine proteinase inhibitor SPIPm2, normally stored in the granules of granular and semi-granular hemocytes is up-regulated and found to deter the viral replication. By using yeast two-hybrid screening, we have identified a viral target protein, namely WSV477. Instead of being a proteinase, the WSV477 was reported to be a Cys2/Cys2-type zinc finger regulatory protein having ATP/GTP-binding activity. In vitro pull down assay confirmed the protein–protein interaction between rSPIPm2 and rWSV477. Confocal laser scanning microscopy demonstrated that the SPIPm2 and WSV477 were co-localized in the cytoplasm of shrimp hemocytes. Using RNA interference, the silencing of WSV477 resulted in down-regulated of viral late gene VP28, the same result obtained with SPIPm2. In this instance, the SPIPm2 does not function as proteinase inhibitor but inhibit the regulatory function of WSV477.

Effects of the probioticBacillus subtilis(BP11 and BS11) on the growth and survival of Pacific white shrimp,Litopenaeus vannamei

The effect of Bacillus S11 (BS11)- and/or Bacillus P11 (BP11)-supplemented feeds on the growth performance, survival, immunoenhancement and disease resistance of cultured Pacific white shrimp, Litopenaeus vannamei, was evaluated. Four feeding treatments of (i) regular feed (control), (ii) BS11-supplemented feed, (iii) BP11-supplemented feed and (iv) BS11-and BP11-supplemented feed were prepared and used for shrimp cultivation in closed recirculating cement tanks (~400 L) in two trials, one for juvenile and PL-30 shrimp at 60 and 90 days, respectively. The results showed that BS11 gave a higher probiotic potential than BP11 for both age groups of L. vannamei in cultivation, because the average weight and survival of shrimp fed BS11-supplemented feed were significantly higher (P < 0.05) than those of the control and the other two groups. The survival of shrimp fed either BS11-or both BS11-and BP11-supplemented feed was significantly higher (P < 0.05) than that of the control group. In addition, the highest total haemocyte and granular haemocyte counts and phenoloxidase activity were found in shrimp fed with the BS11-supplemented feed. After challenge with Vibrio harveyi 639 (~107 CFU mL−1) by immersion, the lowest cumulative death (%) and disease resistance were clearly found in shrimp fed with the BS11-supplemented feed.

Comparative analysis of circulating hemocytes of the freshwater snail Pomacea canaliculata

Molluscs are invertebrates of great relevance for economy, environment and public health. The numerous studies on molluscan immunity and physiology registered an impressive variability of circulating hemocytes. This study is focused on the first characterization of the circulating hemocytes of the freshwater gastropod Pomacea canaliculata, a model for several eco-toxicological and parasitological researches.Flow cytometry analysis identified two populations of hemocytes on the basis of differences in size and internal organization. The first population contains small and agranular cells. The second one displays major size and a more articulated internal organization. Light microscopy evidenced two principal morphologies, categorized as Group I (small) and II (large) hemocytes. Group I hemocytes present the characteristics of blast-like cells, with an agranular and basophilic cytoplasm. Group I hemocytes can adhere onto a glass surface but seem unable to phagocytize heat-inactivated Escherichia coli. The majority of Group II hemocytes displays an agranular cytoplasm, while a minority presents numerous granules. Agranular cytoplasm may be basophilic or acidophilic. Granules are positive to neutral red staining and therefore acidic. Independently from their morphology, Group II hemocytes are able to adhere and to engulf heat-inactivated E. coli. Transmission electron microscopy analysis clearly distinguished between agranular and granular hemocytes and highlighted the electron dense content of the granules. After hemolymph collection, time-course analysis indicated that the Group II hemocytes are subjected to an evident dynamism with changes in the percentage of agranular and granular hemocytes. The ability of circulating hemocytes to quickly modify their morphology and stainability suggests that P. canaliculata is endowed with highly dynamic hemocyte populations able to cope with rapid environmental changes as well as fast growing pathogens.

Microscopic and cytochemical characterisation of haemocytes of the mud crab Scylla serrata (Forskål, 1775) (Decapoda, Portunidae)

Haemocytes of the mud crab Scylla serrata (Forskål, 1775) were characterised based on morphological features using light and electron microscopy, and cytochemistry. The cells were identified as hyaline, semigranular and granular haemocytes. Hyaline cells were the smallest haemocytes among the three types identified, having the highest nucleo-cytoplasmic ratio. The cells showed a number of cytoplasmic organelles and also contained a few small as well as large-sized granules. Semigranular haemocytes possessed moderate numbers of large-sized granules or numerous small-sized granules and comparatively less numbers of organelles. Granular haemocytes were the largest haemocytes with the lowest nucleo-cytoplasmic ratio and contained many large-sized granules. Cytoplasmic organelles were least observed in the granular haemocytes. These three haemocyte morphotypes constituted 60, 21 and 19%, respectively, of the total haemocyte population, while the total haemocyte count was 7.31 × 106 to 7.18 × 107 with a mean of 2.86 × 107 cells ml−1. In cytochemical studies performed to localize carbohydrates, lipids and prophenol oxidase, all the haemocyte types were positive for PAS and toluidine blue, indicating the presence of mucopolysaccharides, whereas semigranular and granular haemocytes were rich in carbohydrates and lipid moieties. Besides, prophenol oxidase was localised within the granules of semigranular and granular haemocytes. Hyaline haemocytes showed an abundance of well differentiated cytoplasmic organelles and granules, and there was a distinct differentiation between semigranular and granular haemocytes in terms of granules and organelles. This is the first report of the characterisation of haemocytes of the mud crab.

Evidence for cell apoptosis suppressing white spot syndrome virus replication in Procambarus clarkii at high temperature

In shrimp, higher water temperatures (~32°C) can suppress the ability of white spot syndrome virus (WSSV) to replicate and cause mortality, but the mechanisms remain unclear. To investigate whether cell apoptosis might be involved, a Tdt-mediated dUTP nick-end label (TUNEL) method was used to assess levels of chromosomal DNA fragmentation in hepatopancreas and gill cells of Procambarus clarkii crayfish infected with WSSV and maintained at either 32 ± 1°C or 24 ± 1°C. Based on relative cell numbers with yellow-green colored TUNEL-positive nuclei, the apoptotic index was elevated in WSSV-infected crayfish maintained at 32°C. In gill tissue sections examined by transmission electron microscope, cells with nuclei displaying apoptotic bodies or marginated, condensed and fragmented chromatin without concurrent cell cytoplasm damage were also more prevalent. Flow cytometry sorting of annexin-stained cells showed apoptosis to be most prevalent in granular haemocytes, and assays for caspase-3 activity showed it to be most elevated in hepatopancreas tissue. Despite these indicators of cell apoptosis but consistent with WSSV replication being restricted at elevated temperatures, no increases in transcription of the viral anti-apoptosis genes ORF390 and ORF222 were detected by RT-PCR in shrimp maintained at 32°C, possibly due to the elevated levels of cellular apoptosis.

Production and characterization of monoclonal antibodies to the hemocytes of mud crab, Scylla serrata

Monoclonal antibodies (MAbs) to hemocytes of mud crab, Scylla serrata, were produced by immunizing mice with formalin-fixed hemocytes. Of the six MAbs produced, two (MAb 1D11 and MAb 1A2) reacted specifically with hemocyte proteins in western blot. MAb 1A2 showed strong immunofluorescent reaction with granular hemocytes and a weak reaction with semigranular cells. However, hyaline cells did not show any reaction. The MAbs also showed strong cross-reactivity with the hemocytes of different crab species but not with other crustaceans. The MAbs produced can be used as a marker for granular hemocytes of crabs.

Gene expression and characterization of a serine proteinase inhibitor PmSERPIN8 from the black tiger shrimp Penaeus monodon

The ubiquitous SERPINs or serine proteinase inhibitors are essential for controlling proteinases in several biological processes in various organisms. A PmSERPIN8, one of eight SERPINs identified from the Penaeus monodon database, is studied and reported herein. The open reading frame of PmSERPIN8 gene derived from a genomic gene contains 5 exons of 320, 139, 244, 239 and 312 bp separated by 4 introns of 447, 657, 326 and 479 bp. The PmSERPIN8 gene is highly expressed at nauplius stage and gradually subsided as the shrimp grow through zoea, mysis and postlarva stages. At sub-adult stage, the PmSERPIN8 gene is expressed mainly in the hemocyte and epipodite. The expression in response to Vibrio harveyi and YHV injection is up-regulated, respectively, at 24 and 48 h post-injection. The number of PmSERPIN8-producing hemocytes, however, is observed highest at 48 h post V. harveyi injection. All three hemocyte cell types: hyaline, semigranular and granular hemocytes are able to produce PmSERPIN8. The recombinant mature PmSERPIN8 (rPmSERPIN8) with a predicted size of 45.5 kDa was over-produced in an Escherichia coli system, solubilized from the inclusion bodies, purified and tested for its activity. We have found that the rPmSERPIN8 is able to inhibit the growth of Gram-positive bacterium, Bacillus subtilis, but not Gram-negative bacterium, V. harveyi 639, and inhibit the shrimp prophenoloxidase system. The PmSERPIN8 is, thus, involved in the shrimp innate immunity.

Immune Response of Harmonia axyridis (Coleoptera: Coccinellidae) Supports the Enemy Release Hypothesis in North America

Abstract The multicolored Asian lady beetle, Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae), is an invasive species in North America that has a low susceptibility to indigenous natural enemies. We investigated the role of the immune system of H. axyridis in its resistance to North American population of the hymenopteran parasitoid Dinocampus coccinellae Schrank. An ultrastructural study of adult H. axyridis hemocytes under electron and light microscopy identified five different cellular types: plasmatocytes, granular hemocytes I, granular hemocytes II, oenocytoids, and spherule cells. Only granular hemocytes II and plasmatocytes are involved in the nodulation of bacteria and encapsulation of parasitoid eggs. Injection of Sephadex beads in H. axyridis adults induces an innate immune response and results in complete encapsulation of ≈28% of the beads after 24 h and 81% after 5 d. When H. axyridis was parasitized by D. coccinellae, we observed a decrease in the encapsulation response, and this pattern was inversely related to the number of parasitoid eggs in the H. axyridis hemolymph. The injection of polydnaviruses by D. coccinellae females and the multiple target hypotheses could both explain the encapsulation rate decreasing with increasing parasitoid egg load. Our results suggest that the immune system of H. axyridis could favor its escape from internal developing natural enemies indigenous to North America.

An MBL-like protein may interfere with the activation of the proPO-system, an important innate immune reaction in invertebrates

An important characteristic of the innate immune systems of crayfish and other arthropods is the activation of a serine proteinase cascade in the hemolymph, which results in the activation of prophenoloxidase and subsequently leading to the formation of toxic quinones and melanin. Although no true complement homologues have been detected in crayfish or crustaceans, several proteins with similarities to vertebrate pattern recognition receptors (PRRs), which are involved in the lectin pathway of complement activation in vertebrates, are present. One is a C-type lectin, a mannose-binding lectin (Pl-MBL), which is secreted from granular hemocytes. Here we report that Pl-MBL has LPS-binding capacity and is dependent upon high Ca2+ for its solubility and Pl-MBL interferes with proPO activation in vitro when HLS is prepared at high Ca2+. The proPO-activating system is efficiently activated by microbial polysaccharides and it has to be neatly regulated to avoid activation in places where it is inappropriate and the active enzyme PO should be prevented from spreading throughout the body of the animal. This may be particularly important during molting when proPO is involved in hardening of a new cuticle and the animal is vulnerable to microbes. The presence of high amount of Pl-MBL in the granular hemocytes may play a role in this process. Since a hemocyte lysate supernatant (HLS) prepared at 100mM Ca2+ could become activated when the concentration of LPS was increased up to 3mg/ml, this may indicate that Pl-MBL acts as a scavenger for LPS to prevent spreading of LPS in the hemolymph to avoid further activation of the proPO-system.