Cell-free Hemolymph Research Articles

Antimicrobial activity in the tick Rhipicephalus (Boophilus) microplus eggs: Cellular localization and temporal expression of microplusin during oogenesis and embryogenesis

Arthropods display different mechanisms to protect themselves against infections, among which antimicrobial peptides (AMPs) play an important role, acting directly against invader pathogens. We have detected several factors with inhibitory activity against Candida albicans and Micrococcus luteus on the surface and in homogenate of eggs of the tick Rhipicephalus (Boophilus) microplus. One of the anti-M. luteus factors of the egg homogenate was isolated to homogeneity. Analysis by electrospray mass spectrometry (ESI-MS) revealed that it corresponds to microplusin, an AMP previously isolated from the cell-free hemolymph of R. (B.) microplus. Reverse transcription (RT) quantitative polymerase chain reactions (qPCR) showed that the levels of microplusin mRNA gradually increase along ovary development, reaching an impressive highest value three days after the adult females have dropped from the calf and start oviposition. Interestingly, the level of microplusin mRNA is very low in recently laid eggs. An enhance of microplusin gene expression in eggs is observed only nine days after the onset of oviposition, achieving the highest level just before the larva hatching, when the level of expression decreases once again. Fluorescence microscopy analysis using an anti-microplusin serum revealed that microplusin is present among yolk granules of oocytes as well as in the connecting tube of ovaries. These results, together to our previous data, suggest that microplusin may be involved not only in protection of adult female hemocele, but also in protection of the female reproductive tract and embryos, what points this AMP as a considerable target for development of new methods to control R. (B.) microplus as well as the vector-borne pathogens.

Contamination of bivalve haemolymph samples by adductor muscle components: implications for biomarker studies

Haemolymph samples and haemocytes collected via the adductor muscles of bivalve molluscs are extensively used in ecotoxicological studies. Withdrawal of haemolymph from mussels, Mytilus edulis, via the posterior adductor muscle, may lead to contamination with the intracellular contents of adductor myocytes. Lysopine dehydrogenase (LyDH) activity, an adductor myocyte marker, was used to investigate the impact of this potential contamination on levels of total glutathione, glutathione peroxidase (GPx) and acetylcholinesterase (AChE) measured in cell-free haemolymph. The mean glutathione content of cell-free haemolymph from 28 mussels was 3.2 +/- 1.8 microM (mean +/- SD). There was a linear relationship (slope = 0.28 +/- 0.03 min; mean +/- SE; P < 0.0001, n = 28) with haemolymph LyDH levels suggesting that at least some of the glutathione measured in cell-free haemolymph had arisen from contamination. Haemolymph LyDH activity was significantly higher in samples extracted using larger diameter needles, and also in samples where there had been some difficulty in the extraction. Exposure of mussels to oxidative stress using 40 microg l(-1) Cu for 5 days resulted in a 1.7 fold increase in glutathione (P = 0.033), but no increase (P = 0.810) in LyDH activity in adductor muscle. This was reflected in a similar increase in the slope of a plot of cell-free haemolymph glutathione versus LyDH activity (P = 0.011), consistent with both of these having originated from the adductor muscle. Cell-free haemolymph GPx and AChE activities also correlated with LyDH activity (Spearman rank correlation coefficients of 0.531 (P = 0.0068) and 0.537 (P = 0.0062), respectively, n = 27) suggesting that these also arise from contamination of the haemolymph. For GPx there was a significant linear relationship (P = 0.025) with haemolymph LyDH levels consistent with both enzymes originating from the myocytes. However, there was hyperbolic relationship (P = 0.0004) between haemolymph AChE and LyDH activities. It appears that this is because the AChE originates from a different compartment to the LyDH, i.e. cholinergic neuromuscular junctions in the adductor muscle. We conclude that it would be prudent, when considering the possibility of using a biomarker in cell-free haemolymph from bivalve molluscs, to check whether contamination could be an issue.

Starvation-induced Changes of Hemocyte Parameters in the Zhikong Scallop Chlamys farreri

Substantial nutritional and energetic demands m-e associated with immune activation and the maintenance of an efficient immune system. One-year-old Chlamys farreri (Jones and Preston) scallops were maintained ill lantern nets ill different nutritional conditions (satiation and starvation) for 40 days. After the 40-day treatments, the condition index and the total hemocyte count (THC) decreased significantly in the starved group compared with the satiated and initial control groups. The percentage of phagocytic hemocytes also was significantly reduced with starvation. In contrast. no significant effect of starvation was observed oil reactive oxygen species (ROS) production. The acid phosphatase (ACP) activities in cell-free hemolymph increased significantly in scallops in starved and satiated treatments compared with the initial control. whereas ACP activity in hemocyte lysate was significantly lower ill the starved group. These results indicate that starvation stress compromises immunological activities of scallops.

Participation of cell‐free haemolymph of Biomphalaria tenagophila in the defence mechanism against Schistosoma mansoni sporocysts

Biomphalaria tenagophila of Taim strain is able to completely destroy Schistosoma mansoni sporocyst few hours after parasite penetration, although the mechanism is still not well known. In this experimental work we show that passive transference of cell-free haemolymph, especially from B. tenagophila Taim, resulted in higher resistance of B. tenagophila Cabo Frio to S. mansoni infection. This effect was demonstrated in vivo, by the reduction in the infection rate, and the significantly lower production of sporocysts and cercariae of the parasite in snails treated with Taim cell-free haemolymph compared to CBSS-inoculated snails. The protective effect of Taim cell-free haemolymph was also observed during the in vitro interaction between haemocytes and sporocysts. In this system, addition of B. tenagophila cell-free haemolymph, especially from Taim strain, was responsible for significant increase in sporocyst mortality compared to B. glabrata cell-free haemolymph or culture medium. Moreover, the combination of Taim cell-free haemolymph and Cabo Frio haemocytes increased significantly the mortality of sporocysts. The results show that Taim cell-free haemolymph would act direct and indirectly on destruction of S. mansoni sporocysts. The results also suggest that cell-free haemolymph indirectly increases parasite recognition by the circulating granulocytes and it is species specific.

Antibacterial activity in vivo and in vitro in the hemolymph of Galleria mellonella infected with Pseudomonas aeruginosa

The antibacterial activity of hemolymph from Galleria mellonella infected with entomopathogenic strain of Pseudomonas aeruginosa and non-pathogenic bacterium Escherichia coli was studied. In vivo, the antimicrobial activity appeared shortly after P. aeruginosa infection, reached the maximum level 18 h postinjection, while 30 h later only trace activity was noted. The activity induced by E. coli sustained on the high level until 48 h after infection. We also noted that the antimicrobial activity level induced by the non-pathogenic bacterium was higher in comparison to that measured in insects infected with the pathogenic strain of P. aeruginosa. The results of our in vitro studies indicated that inducible antimicrobial peptides of G. mellonella larvae were digested by P. aeruginosa elastase B. After 1 h incubation of cell-free hemolymph of immune-challenged larvae with elastase B, no antibacterial activity was observed. It was also shown that elastase B degraded synthetic cecropin B while in the presence of 6 mM EDTA antibacterial activity of cell-free hemolymph as well as cecropin B, was not changed which confirmed that the activity was abolished by the metalloprotease.

Differential involvement of mussel hemocyte sub-populations in the clearance of bacteria

Mussels are filter-feeders living in a bacteria-rich environment. We have previously found that numerous bacterial species are naturally present within the cell-free hemolymph, including several of the Vibrio genus, whereas the intra-cellular content of hemocytes was sterile. When bacteria were injected into the circulation of the mussel, the number of living intra-hemocyte bacteria dramatically increased in less than an hour, suggesting intense phagocytosis, then gradually decreased, with no viable bacteria remaining 12 h post-injection for Micrococcus lysodeikticus, 24 h for Vibrio splendidus and more than 48 h for V ibrio anguillarum. The total hemocyte count (THC) was dramatically lowered by the bacterial injections, as quantified by flow cytometry. V. splendidus induced the strongest decreases with −66% 9 h post-injection of living bacteria and −56% 3 h post-injection of heat-killed bacteria. Flow cytometry was used to identify three main sub-populations of hemocytes, namely hyalinocytes, small granulocytes and large granulocytes. When THC was minimal, i.e. within the first 9 h post-injection, proportions of the three cell categories varied dramatically, suggesting differential involvement according to the targets, but small granulocytes remained the majority. According to a decrease in their number followed by an increase (+90% at 12 h with living V. splendidus), hyalinocytes also appeared to be involved as cellular effectors of antibacterial immunity, despite possessing little capacity for phagocytosis and not containing antimicrobial peptides.

Investigation of EROD, CYP1A immunopositive proteins and SOD in haemocytes of Chamelea gallina and their role in response to B[ a

CYP1A sub-family represents the main form of cytochrome P450 involved in benzo[ a]pyrene (B[ a]P) detoxification, but there are no clear evidences about its presence in invertebrates. 7-Ethoxy resorufin O-deethylase (EROD) activity is strictly related to CYP1A presence, at the same time P450-dependent oxidative metabolism leads to reactive oxygen species (ROS) production, thought to be an important mechanism of pollutant-mediated toxicity in aquatic organisms. Superoxide dismutases (SODs), EROD and CYP1A activities and/or expressions were detected in haemocytes of pooled clams ( Chamelea gallina) and cell-free haemolymph after 24 h, 7 and 12 days of exposure to 0.5 mg/L of B[ a]P. After 24 h, B[ a]P content was maximum in whole tissues. A 61 kDa band was recognized in haemocytes and cell-free haemolymph by polyclonal anti-fish CYP1A, while 53.5 and 63.8 kDa CYP1A immunopositive proteins were discriminate without differences of expression. Differently, EROD, MnSOD activity/expression and ECSOD expression decreased in haemocytes and haemolymph. C. gallina immune system presents an interesting response dose/time exposure of B[ a]P and the 7 days condition highlights the major effects of xenobiotic action. The identification of basal EROD levels supports the possible presence of the CYP1A, never identified in C. gallina and more specifically never isolated in immune cells, as confirmed by CYP1A-immunopositive proteins identification.

Lipid mediators and vector infection: Trypanosoma rangeli inhibits Rhodnius prolixus hemocyte phagocytosis by modulation of phospholipase A 2 and PAF-acetylhydrolase activities

In this work we investigated the effects of Trypanosoma rangeli infection through a blood meal on the hemocyte phagocytosis in experiments using the 5th instar larvae of Rhodnius prolixus. Hemocyte phagocytic activity was strongly blocked by oral infection with the parasites. In contrast, hemocyte phagocytosis inhibition caused by T. rangeli infection was rescued by exogenous arachidonic acid (20 μg/insect) or platelet activating factor (PAF; 1 μg/insect) applied by hemocelic injection. Following the oral infection with the protozoan we observed significant attenuation of phospholipase A 2 (PLA 2) activities in R. prolixus hemocytes (cytosolic PLA 2: cPLA 2, secreted PLA 2: sPLA 2 and Ca +2-independent PLA 2: iPLA 2) and enhancement of sPLA 2 activities in cell-free hemolymph. At the same time, the PAF-acetyl hydrolase (PAF-AH) activity in the cell-free hemolymph increased considerably. Our results suggest that T. rangeli infection depresses eicosanoid and insect PAF analogous (iPAF) pathways giving support to the role of PLA 2 in the regulation of arachidonic acid and iPAF biosynthesis and of PAF-AH by reducing the concentration of iPAF in R. prolixus. This illustrates the ability of T. rangeli to modulate the immune responses of R. prolixus to favor its own multiplication in the hemolymph.

First Evidence of Altered Immune Responses and Resistance to Air Exposure in the Clam Chamelea gallina Exposed to Benzo(a)pyrene

Effects of benzo(a)pyrene [B(a)P] (at a nominal concentration of 0.5 mg/L) on immune responses of the clam Chamelea gallina were investigated after 1, 7, and 12 days exposure. Total hemocyte count (THC), hemocyte volume, phagocytic activity, lysozyme-like activity in both hemocyte lysate (HL) and cell-free hemolymph (CFH) were measured. As unexpected alterations in hemocyte adhesion capability were observed in short-term hemocyte cultures for phagocytosis assays after a 1-day exposure, an adhesion test (not included in the original experimental setup) was performed after 7 and 12 days of exposure only. The survival-in-air test was carried out to evaluate general stress conditions in B(a)P-exposed clams. No alterations in THC was observed, whereas exposure for 7 and 12 days to B(a)P significantly decreased phagocytic activity and adhesion capability when compared with controls. Significant decreases in lysozyme activity were observed in CFH and HL, with respect to controls. B(a)P was also shown to alter the resistance to air exposure of clams. The LT(50) values fell from 9 days in control clams to 7 days in 1-day-exposed animals, and from 6 days in control clams to 5 days in 7-day-exposed bivalves. No significant variations in LT(50) values were recorded after 12 days of exposure. Results highlight a relationship between B(a)P exposure and alterations in hemocyte functionality and suggest that the contaminant induced irreversible immunosuppression in C. gallina, by altering phagocytic activity, adhesion capability, and enzymatic activity. Conversely, reduction in resistance to air exposure was reversible, suggesting that impairment of important physiological functions of clams occurred in the first phases of exposure only.

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 IC 50 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.

Changes in Galleria mellonella lysozyme level and activity during Pseudomonas aeruginosa infection

The level of lysozyme in fat body, hemocytes and cell-free hemolymph from Galleria mellonella larvae infected with Pseudomonas aeruginosa was determined and evaluated. In the samples of fat body and hemocytes, an increase in lysozyme content was detected 1 d after infection and then a significant decrease was observed after a prolonged infection time. In the case of cell-free hemolymph, an increase in the lysozyme level was noticeable during the first 30 h post injection and stayed at a similar level for 42 h. The smaller decrease of the lysozyme level after 42 h might be associated with the development of bacteremia of P. aeruginosa in insects. In addition, the gradual increase in the content of lysozyme correlated with the increase of its activity in the hemolymph of the infected larvae as a response to injection with P. aeruginosa. The G. mellonella lysozyme appeared to be insensitive to extracellular proteinases produced in vivo by P. aeruginosa.

Clearance of Vibrio campbellii injected into the hemolymph of Callinectes sapidus, the Atlantic blue crab: The effects of prior exposure to bacteria and environmental hypoxia

The Atlantic blue crab, Callinectes sapidus (Rathbun), lives in a bacteria-rich environment that experiences daily fluctuations in water quality. In the present study, we tested the hypothesis that crustaceans with prior or ongoing exposure to bacteria in their hemolymph have an increased susceptibility to subsequent infections, and that acute exposure to low dissolved oxygen (hypoxia) and elevated carbon dioxide levels (hypercapnia) may further confound the ability of blue crabs to counter a subsequent infection. Adult male blue crabs held in well-aerated (normoxic; P O 2 = 20.7 kPa ; CO 2 < 0.06 kPa; pH 7.8–8.0) or hypercapnic hypoxic (HH; P O 2 = 4 kPa ; CO 2 = 1.8 kPa; pH 6.9–7.2) seawater received an injection (pre-challenge dose) of 1 × 10 5 Vibrio campbellii g −1 crab. Control animals were injected with an equivalent dose of HEPES-buffered saline (1 μl g −1 crab). At 2 h or 24 h after the pre-challenge injection, both Vibrio and saline-pre-challenged animals were injected with a dose of live V. campbellii (1 × 10 5 g −1 crab). This second injection will be referred to as a second injection or challenge injection. Degradation in or physical removal of intact bacteria from hemolymph was quantified using real-time PCR; bacteriostasis was quantified as the percentage of intact bacteria that could not be recovered by selective plating. We demonstrated that bacteriostasis occurs in the hemolymph of blue crabs. Furthermore, blue crabs that received a challenge injection 2 h after a pre-challenge dose of V. campbellii cleared culturable bacteria from their hemolymph more rapidly when compared to animals that received a pre-challenge dose of saline. This enhanced clearance of culturable bacteria was associated with an increase in antibacterial activity in the cell-free hemolymph. However, the enhanced clearance of culturable bacteria disappeared when the time interval between the pre-challenge and challenge dose was extended to 24 h and when crabs were held in HH seawater throughout the experiment. Neither the time interval between the pre-challenge and the challenge dose nor exposure to HH altered the pattern of intact bacterial clearance in blue crabs. These results demonstrate that prior exposure to bacteria does not increase the susceptibility of C. sapidus to a second, sublethal dose of V. campbellii. In fact, a recent exposure to V. campbellii enhances the ability of blue crabs to render bacteria non-culturable and the immune mechanisms/effectors responsible for this are short lived and appear to be sensitive to low dissolved oxygen and high carbon dioxide concentrations in the environment.

Immunotoxicity of the xenoestrogen 4-nonylphenol to the cockle Cerastoderma glaucum

The in vivo effects of 4-nonylphenol (NP) on functional responses of haemocytes from the cockle Cerastoderma glaucum were investigated after 7 days exposure to sublethal NP concentrations (0, 0+acetone, 0.0125, 0.025, 0.05 and 0.1 mg/l NP). Haemocytes from both controls and exposed cockles were collected, and the effects of NP on total haemocyte count (THC) and volume of circulating cells, intracellular superoxide anion ( O 2 - ) levels, acid phosphatase and lysozyme-like activities in both haemocyte lysate (HL) and cell-free haemolymph (CFH) were evaluated. Exposure of cockles to 0.1 mg/l NP significantly increased THC ( p < 0.05) with respect to controls. Analysis of haemocyte size frequency distribution showed that the haemocyte fraction of about 7–8 μm in diameter and 250 femtolitres in volume increased markedly in cockles exposed to the highest NP concentration tested. Apoptosis resulting in cell volume reduction in NP-exposed animals cannot be excluded. No statistically significant variation in intracellular O 2 - levels was observed. Conversely, significant increases ( p < 0.05) in acid phosphatase activity were observed in CFH from 0.05 and 0.1 mg/l NP-exposed animals; no significant differences in enzyme activity were recorded in HL. Lysozyme-like activity also increased significantly in CFH from cockles exposed to 0.05 mg/l NP ( p < 0.05) and 0.1 mg/l NP ( p < 0.001). Instead, lysozyme-like activity decreased significantly ( p < 0.05) in the HL of animals exposed to 0.05 mg/l NP. Our results suggest that NP induces variations in the functional responses of haemocytes of C. glaucum, mainly by reducing cell membrane stability and promoting cell degranulation.

The role of hemocytes in the immunity of the spider Acanthoscurria gomesiana

Invertebrates protect themselves against microbial infection through cellular and humoral immune defenses. Since the available information on the immune system of spiders is scarce, the main goal of the present study was to investigate the role of hemocytes and antimicrobial peptides (AMPs) in defense against microbes of spider Acanthoscurria gomesiana. We previously described the purification and characterization of two AMPs from the hemocytes of naïve spider A. gomesiana, gomesin and acanthoscurrin. Here we show that 57% of the hemocytes store both gomesin and acanthoscurrin, either in the same or in different granules. Progomesin labeling in hemocyte granules indicates that gomesin is addressed to those organelles as a propeptide. In vivo and in vitro experiments showed that lipopolysaccharide (LPS) and yeast caused the hemocytes to migrate. Once they have reached the infection site, hemocytes may secrete coagulation cascade components and AMPs to cell-free hemolymph. Furthermore, our results suggest that phagocytosis is not the major defense mechanism activated after microbial challenge. Therefore, the main reactions involved in the spider immune defense might be coagulation and AMP secretion.

Developmental modulation of immunity: Changes within the feeding period of the fifth larval stage in the defence reactions of Manduca sexta to infection by Photorhabdus

In insect pathogen interactions, host developmental stage is among several factors that influence the induction of immune responses. Here, we show that the effectiveness of immune reactions to a pathogen can vary markedly within a single larval stage. Pre-wandering fifth-stage (day 5) larvae of the model lepidopteran insect Manduca sexta succumb faster to infection by the insect pathogenic bacterium Photorhabdus luminescens than newly ecdysed fifth-stage (day 0) caterpillars. The decrease in insect survival of the older larvae is associated with a reduction in both humoral and cellular defence reactions compared to less developed larvae. We present evidence that older fifth-stage larvae are less able to over-transcribe microbial pattern recognition protein and antibacterial effector genes in the fat body and hemocytes. Additionally, older larvae show reduced levels of phenoloxidase (PO) activity in the cell-free hemolymph plasma as well as a dramatic decrease in the number of circulating hemocytes, reduced ability to phagocytose bacteria and fewer melanotic nodules in the infected tissues. The decline in overall immune function of older fifth-stage larvae is reflected by higher bacterial growth in the hemolymph and increased colonization of Photorhabdus on the basal surface of the insect gut. We suggest that developmentally programmed variation in immune competence may have important implications for studies of ecological immunity.

Protective effects of selenium on mercury-induced DNA damage in mussel haemocytes

Little is known of the antioxidant role of selenium (Se) in aquatic invertebrates. We investigated the effects of Se on mercury-induced DNA damage in haemocytes from Mytilus edulis using alkaline single cell gel electrophoresis, that is, the Comet assay. The basal percentage tail DNA value for mussel haemocytes was 9.8 ± 0.2% (mean ± S.E.M., n = 70). Exposing mussels to Hg 2+ (nominal concentration 20 μg L −1) for three days led to an increase in tail DNA to 61.1 ± 1.8% ( n = 10). With added Se (as selenite, nominal concentration 4 μg L −1), Hg-induced DNA damage was reduced to 39.5 ± 3.1% ( n = 10). Se pre-exposure also provided some protection against Hg-induced DNA damage (% tail DNA = 51.0 ± 2.9%, n = 10). Basal glutathione peroxidase (GPx) activity in cell-free haemolymph was 93.7 ± 3.5 nmol min −1 mg −1 (mean ± S.E.M., n = 70). Increases in GPx activity were seen when Se was added during and/or after exposure to Hg. For example, a 3–4-fold increase was seen after three days exposure to Hg in the presence of added Se. Interestingly GPx activity doubled after three days in the presence of added Se alone, but was unchanged after exposure to HgCl 2 alone. These results suggest that the availability of Se in the natural environment could affect the antioxidant status of mussels, and consequently could affect levels of DNA damage.

Immune condition of Chlamys farreri in response to acute temperature challenge

The effects of acute temperature challenge on some immune parameters of haemocyte in Zhikong scallop, Chlamys farreri, recognised as a temperature sensitive bivalve species, were evaluated over a short period of time. Scallops were suddenly transferred from 17 °C to 11 °C, 23 °C and 28 °C for a period of 72 h. Total haemocyte count (THC), percentage of phagocytic haemocytes, reactive oxygen species (ROS) production, acid phosphatase (ACP) and superoxide dismutase (SOD) activities (in both haemocyte lysate and cell-free haemolymph) were chosen as biomarkers of temperature stress. Results demonstrated that the percentage of phagocytic haemocytes and ACP activity in cell-free haemolymph of scallops challenged at 28 °C for 72 h significantly decreased. By contrast, reactive oxygen species production by haemocytes increased when compared to the initial values. It is concluded that haemocyte activities of C. farreri appear to be compromised when scallops were transferred from 17 °C to 28 °C. Meanwhile, no obvious negative effect of acute temperature stress was detected on haemocyte activities of C. farreri challenged at 11 °C, which highlighted the high tolerance of scallops to acute decrease of seawater temperatures.

Pathogenesis of a Thai strain of white spot syndrome virus (WSSV) in juvenile, specific pathogen-free Litopenaeus vannamei

White spot syndrome virus (WSSV) causes disease and mortality in cultured and wild shrimp. A standardized WSSV oral inoculation procedure was used in specific pathogen-free (SPF) Litopenaeus vannamei (also called Penaeus vannamei) to determine the primary sites of replication (portal of entry), to analyze the viral spread and to propose the cause of death. Shrimp were inoculated orally with a low (10(1.5) shrimp infectious dose 50% endpoint [SID50]) or a high (10(4) SID50) dose. Per dose, 6 shrimp were collected at 0, 6, 12, 18, 24, 36, 48 and 60 h post inoculation (hpi). WSSV-infected cells were located in tissues by immunohistochemistry and in hemolymph by indirect immunofluorescence. Cell-free hemolymph was examined for WSSV DNA using 1-step PCR. Tissues and cell-free hemolymph were first positive at 18 hpi (low dose) or at 12 hpi (high dose). With the 2 doses, primary replication was found in cells of the foregut and gills. The antennal gland was an additional primary replication site at the high dose. WSSV-infected cells were found in the hemolymph starting from 36 hpi. At 60 hpi, the percentage of WSSV-infected cells was 36 for the epithelial cells of the foregut and 27 for the epithelial cells of the integument; the number of WSSV-infected cells per mm2 was 98 for the gills, 26 for the antennal gland, 78 for the hematopoietic tissue and 49 for the lymphoid organ. Areas of necrosis were observed in infected tissues starting from 48 hpi (low dose) or 36 hpi (high dose). Since the foregut, gills, antennal gland and integument are essential for the maintenance of shrimp homeostasis, it is likely that WSSV infection leads to death due to their dysfunction.

Haemocytes of the cockle Cerastoderma glaucum: Morphological characterisation and involvement in immune responses

For the first time, morpho-functional characterisation of haemocytes from the cockle Cerastoderma glaucum was performed to identify circulating cell types and to study their involvement in immune responses. Haemocyte mean number was 5.5 (×10 5) cells/mL haemolymph. Two main haemocyte types were found in haemolymph: granulocytes (85%), about 10 μm in diameter and with evident cytoplasmic granules, and hyalinocytes (15%), 8 to 14 μm in diameter, with a few or no granules. Most of the cytoplasmic granules stained in vivo with Neutral Red, indicating that they were lysosomes. On the basis of haemocyte staining properties, granulocytes and hyalinocytes were further classified as basophils and acidophils. Acidophil hyalinocytes were the largest haemocyte type (about 14 μm in diameter) and had an eccentric nucleus and a large cytoplasmic vacuole. Both granulocytes and hyalinocytes (except acidophils) were able to phagocytise yeast cells, although the basal phagocytic index was very low (about 2%). It increased significantly (up to 26%) after pre-incubation of yeast in cell-free haemolymph, suggesting that haemolymph has opsonising properties. Haemocytes also produced superoxide anion. Moreover, both granulocytes and hyalinocytes (except acidophils) were positive for some important hydrolytic and oxidative enzymes. Lysozyme-like activity was recorded in both cell-free haemolymph and haemocyte lysate, although enzyme activity in cell lysate was significantly higher. Results indicate that haemocytes from C. glaucum are effective cells in immune responses.

Effects of salinity on the clam Chamelea gallina. Part I: alterations in immune responses

In the present study, the effects of differing salinities on some important functional responses of haemocytes from the clam, Chamelea gallina, were investigated. The animals were kept for 7 days at 28‰ (hyposalinity), 34‰ (control) and 40‰ salinity (hypersalinity), and total haemocyte count (THC), haemocyte volume, phagocytosis, lysozyme-like activity (in both haemocyte lysate and cell-free haemolymph) were measured. The survival-in-air test was also performed. Clams kept at 28‰ showed significantly increased THC with respect to animals kept at 34 and 40‰. The analysis of haemocyte size frequency distribution highlighted that in clams kept at 28‰ the haemocyte fraction of about 5 μm in diameter and 50–100 femtolitre in volume increased markedly. Conversely, in animals kept at 40‰ an increase was observed in the haemocyte fraction having about 8–10 μm diameter and 400–500 femtolitre volume. Higher phagocytic activity was recorded in haemocytes from control clams, with respect to that of clams kept at 28 and 34‰. Lysozyme-like activity in haemocyte lysate was shown to increase significantly in animals kept at 28‰ with respect to that of clams kept at 40‰, whereas enzyme activity in cell-free haemolymph from clams kept at 34‰ was significantly higher with respect to that of clams maintained at 40‰. A relationship between phagocytosis and lysozyme secretion is suggested. The resistance to air exposure of clams kept at 28 and 40‰ was shown to decrease significantly; LT50 values fell from 7 days in clams kept at 34‰ to 4 and 5 days in those kept at 28 and 40‰, respectively. Results demonstrated that salinity values far from 34‰ affects the functional responses of haemocytes and reduce the resistance of clams to exposure to air.