Immunological and cytotoxicological responses of the Asian clam,Corbicula fluminea (M.), experimentally exposed to cadmium

Abstract

Bivalve molluscs, as filter-feeding organisms, are known to accumulate metals that can produce deleterious effects on organisms. The phagocytic activity of haemocytes and lysosomal alterations in the digestive gland cells were measured in the freshwater Asian clam exposed to cadmium, in order to assess the possible use of immunocompetence and lysosomal responses as biomarkers of freshwater quality. Clams were exposed in the laboratory to nominal concentrations of 3, 10, 21.4, 46.5 and 100 µg l^?1 of cadmium and sampled after 7, 15 and 30 days of exposure. The results show a decrease of phagocytic activity after only 7 days of exposure to 10 µg l^?1 of cadmium. This response was also observed as the exposure time was increased. Lysosomes in the digestive cells increased in size and number after 7 days of exposure as cadmium concentration increased. After 30 days of exposure, a decrease in size and number indicated a change in the response to the metal from concentrations of 46.5 µg l^?1 of cadmium. A dose and time response both in phagocytic activity of haemocytes and lysosomal structure demonstrated a possible use of these biomarkers in freshwater biomonitoring.     

Experimental exposure of the blue mussel (Mytilus edulis, L.) to the toxic dinoflagellate Alexandrium fundyense: Histopathology, immune responses, and recovery

Mussels (Mytilus edulis) were exposed to cultures of the toxic dinoflagellate Alexandrium fundyense or the non-toxic alga Rhodomonas sp. to evaluate the effects of the harmful alga on the mussels and to study recovery after discontinuation of the A. fundyense exposure. Mussels were exposed for 9 days to the different algae and then all were fed Rhodomonas sp. for 6 more days. Samples of hemolymph for hemocyte analyses and tissues for histology were collected before the exposure and periodically during exposure and recovery periods.Mussels filtered and ingested both microalgal cultures, producing fecal pellets containing degraded, partially degraded, and intact cells of both algae. Mussels exposed to A. fundyense had an inflammatory response consisting of degranulation and diapedesis of hemocytes into the alimentary canal and, as the exposure continued, hemocyte migration into the connective tissue between the gonadal follicles. Evidence of lipid peroxidation, similar to the detoxification pathway described for various xenobiotics, was found; insoluble lipofuchsin granules formed (ceroidosis), and hemocytes carried the granules to the alimentary canal, thus eliminating putative dinoflagellate toxins in feces. As the number of circulating hemocytes in A. fundyense-exposed mussels became depleted, mussels were immunocompromised, and pathological changes followed, i.e., increased prevalences of ceroidosis and trematodes after 9 days of exposure. Moreover, the total number of pathological changes increased from the beginning of the exposure until the last day (day 9). After 6 days of the exposure, mussels in one of the three tanks exposed to A. fundyense mass spawned; these mussels showed more severe effects of the toxic algae than non-spawning mussels exposed to A. fundyense.No significant differences were found between the two treatments during the recovery period, indicating rapid homeostatic processes in tissues and circulating hemocytes.     

Vibrio–bivalve interactions in health and disease

In the marine environment, bivalve mollusks constitute habitats for bacteria of the Vibrionaceae family. Vibrios belong to the microbiota of healthy oysters and mussels, which have the ability to concentrate bacteria in their tissues and body fluids, including the hemolymph. Remarkably, these important aquaculture species respond differently to infectious diseases. While oysters are the subject of recurrent mass mortalities at different life stages, mussels appear rather resistant to infections. Thus, Vibrio species are associated with the main diseases affecting the worldwide oyster production. Here, we review the current knowledge on Vibrio–bivalve interaction in oysters (Crassostrea sp.) and mussels (Mytilus sp.). We discuss the transient versus stable associations of vibrios with their bivalve hosts as well as technical issues limiting the monitoring of these bacteria in bivalve health and disease. Based on the current knowledge of oyster/mussel immunity and their interactions with Vibrio species pathogenic for oyster, we discuss how differences in immune effectors could contribute to the higher resistance of mussels to infections. Finally, we review the multiple strategies evolved by pathogenic vibrios to circumvent the potent immune defences of bivalves and how key virulence mechanisms could have been positively or negatively selected in the marine environment through interactions with predators.     

Ontogeny of protein concentration,haemocyte concentration and antimicrobial activity against Escherichia coli in haemolymph of the invasive harlequin ladybird Harmonia axyridis (Coleoptera: Coccinellidae)

The harlequin ladybird is considered to be one of the most successful invasive insect species. Among other traits, its invasive success is considered to be caused by a powerful immune system. In the present study, we investigate the ontogenetic profile of protein concentration, concentration of circulating haemocytes and constitutive antimicrobial activity against Escherichia coli in Harmonia axyridis haemolymph during late larval development and early adult life. Protein concentration increases during the first 32 days of adult life from 45 to 100 mg per mL of haemolymph and reaches intermediate values during larval stages. The concentration of circulating haemocytes is very low (5000 haemocytes per μL of haemolymph) in late larval stages and increases strongly during first 8 days of adult life to values of approximately 30 000 haemocytes per μL of haemolymph. The killing efficiency of haemolymph against E. coli is lowest in larval stages, rapidly increases in the prepupal stage and then steadily grows during the whole period of adult life. There are no significant effects of sex on any of the investigated physiological or immune parameters. In general, the patterns observed for H. axyridis contrast with many results that are reported for other insects (e.g. bees, fruit flies, crickets or mosquitoes). One possible explanation is the contrasting life history of H. axyridis, with a fast preimaginal development and a long adult lifespan being linked to a long reproductive period. Substantial variation in physiological and immune parameters during ontogeny also has important methodological implications because individuals of exactly the same stage/age have to be employed for comparative studies.     

Seasonal dependence of cadmium molecular effects on Mytilus galloprovincialis (Lamarck, 1819) protamine‐like protein properties

Mussels have a seasonal reproduction and cadmium is a common stressor in estuarine and coastal environments. In previous studies, we have shown that exposure to subtoxic doses of cadmium produced alterations in the properties of winter Mytilus galloprovincialis sperm protamine‐like (PL) proteins. In this study, it was analyzed the possibility that these cadmium effects may be seasonal. Winter and summer mussels were exposed to CdCl₂, and it was tested the PL‐proteins for cadmium bioaccumulation, electrophoretic pattern, DNA binding, and potentiality to induce DNA oxidative damage. It was found that cadmium exposure did not produce the same effects on PL‐proteins of summer mussels that were produced on PL‐proteins of winter mussels, that is: cadmium bioaccumulation, alterations in the acetic acid‐urea polyacrylamide gels (AU‐PAGE) and sodium dodecyl sulfate‐PAGE pattern, a reduced DNA binding affinity and the ability to induce DNA oxidative damage. PL‐proteins from summer mussels, apart from not being affected by all the abovementioned effects of cadmium, also showed a very low DNA binding affinity, independent of cadmium exposure. This study reveals clock‐associated seasonal responses to cadmium in M. galloprovincialis. Understanding the mechanisms through which environmental signals guide biological rhythms is fundamental to understanding the seasonal sensitivity of this bioindicator, to use M. galloprovincialis in appropriate seasonal periods.     

Detection of DNA damage in haemocytes of zebra mussel using comet assay

The aim of the study was to use the comet assay on haemocytes of freshwater mussel, Dreissena polymorpha Pallas, for detection of possible DNA damage after exposure to pentachlorophenol (PCP) and to evaluate the potential application of the comet assay on mussel haemocytes for genotoxicity monitoring of freshwater environment. Zebra mussels were exposed for seven days to different concentrations (10, 80, 100, 150 μg/l) of PCP and in the river Sava downstream from Zagreb municipal wastewater outlet. Significant increase in DNA damage was observed after exposure to PCP at doses of 80 μg/l and higher and after in situ exposure in the river Sava as well. This study confirmed that the comet assay applied on zebra mussel haemocytes may be a useful tool in determining the potential genotoxicity of water pollutants.     

Effects of repeated sampling on the haemocytes and haemolymph of Eledone cirrhosa (Lam.)

Sampling blood has an effect on both the number of circulating haemocytes and the concentration of copper present in the haemolymph of Eledone cirrhosa. Haemocytes were sampled over 4 h and over 7, 10 and 24 days. The number of haemocytes per millilitre significantly increases (P<0.05) within 2 h of the 4-h sample period and within 24 h of the 7-, 10- and 24-day sampling periods. Haemocyte counts, however, significantly (P<0.05) decreased within 4 h of the 4-h sample and within 3 days of the 7- and 10-day sampling periods. Over the 24-day sampling periods, haemocyte counts per millilitre decreased significantly (P<0.05) by day 5 but had significantly (P<0.05) increased again by day 17. The percentage of haemocytes with Giemsa-positive cytoplasmic granules significantly increased (P<0.05) over the 4-h sampling period and over 24 h for both the 10- and 24-day sampling period. Methods for acid phosphatase, peroxidase, protein and carbohydrate gave variable staining results over 10 and 24 days. Increased staining was observed on the second and third sampling times for both protein and acid phosphatase over the 10-day sampling period whereas increased positive results were observed for all stains over the 24-day sampling period. The concentration of copper in the haemolymph decreases within 4 h of the 4-h sampling period and continues to decrease over an 11-day sampling period. Protein values decreased over the 4-h sampling period but showed no significant change between the first and last samples over 7 days.     

Changes of Blue Mussels Mytilus edulis L. Lipid Composition Under Cadmium and Copper Toxic Effect

The lipid and fatty acid composition of the blue mussels Mytilus edulis L. gills and digestive glands was evaluated after 24 and 72 h of cadmium (Cd) and copper (Cu) exposure. Mussels were exposed to different cadmium (10, 100, and 500 μg/L) and copper (5, 50, and 250 μg/L) concentrations. Similar stress response of predominant membrane phospholipids level as well as polyenoic and non-methylene interrupted (NMI) fatty acids content was observed in mussel gills under both cadmium and copper effects. Increased NMI fatty acids level after 24 h, the metal ions treatment suggests that these acids contribute to the protective response to the membrane oxidative stress caused by accumulation of the metals. The content of cholesterol, some minor membrane phospholipids, and storage lipids (triacylglycerols, TAG) in the mussels’ organs alter significantly under the cadmium and copper effect. A two-step response at the digestive glands TAG level depends on the duration of the cadmium and copper treatments (24 and 72 h) on the mussels. The results demonstrate that Cd and Cu impact has adverse effects on gills and digestive glands lipid and fatty acids composition. The type of observed effects varies with the nature and concentration of the metal ions and depends on the role of the metals in the mussels’ life activity.     

Immunological and cytotoxicological responses of the Asian clam, Corbicula fluminea (M.), experimentally exposed to cadmium

Bivalve molluscs, as filter-feeding organisms, are known to accumulate metals that can produce deleterious effects on organisms. The phagocytic activity of haemocytes and lysosomal alterations in the digestive gland cells were measured in the freshwater Asian clam exposed to cadmium, in order to assess the possible use of immunocompetence and lysosomal responses as biomarkers of freshwater quality. Clams were exposed in the laboratory to nominal concentrations of 3, 10, 21.4, 46.5 and 100 µg l^-1 of cadmium and sampled after 7, 15 and 30 days of exposure. The results show a decrease of phagocytic activity after only 7 days of exposure to 10 µg l^-1 of cadmium. This response was also observed as the exposure time was increased. Lysosomes in the digestive cells increased in size and number after 7 days of exposure as cadmium concentration increased. After 30 days of exposure, a decrease in size and number indicated a change in the response to the metal from concentrations of 46.5 µg l^-1 of cadmium. A dose and time response both in phagocytic activity of haemocytes and lysosomal structure demonstrated a possible use of these biomarkers in freshwater biomonitoring.     

Mitogenicity and Adjuvanticity of a Marine Bacterium,Vibrio anguillarum,in Mice

The effects of whole cells of three different O serotypes of Vibrio anguillarum on the murine immune response were studied. The addition of different doses (1–100/ig/ml) of V. anguillarum cells, as well as Salmonella typhimurium lipopolysaccharide, markedly increased the incorporation of [³H] thymidine into in vitro cultured spleen cells of C57BL/6 mice. All three serotype strains of V. anguillarum were able to induce the mitogenic effect at 10 μg/ml and 100 μg/ml, but serotype I strains were more potent than the others. Since pretreatment of spleen cells with rabbit anti-mouse thymocyte antiserum did not affect the mitogenic activity of V. anguillarum, Vibrio cells may be a B-lymphocyte mitogen. When sheep or horse erythrocytes and Vibrio cells were injected intraperitoneally into ddY mice, Vibrio cells exhibited an enhancing effect on antibody response in vivo, regardless of the different serotypes. Vibrio cells, when injected intraperitoneally into mice before the antigen, markedly suppressed the antibody response. Several days after the injection of Vibrio cells, these mice showed an enhanced carbon clearance activity. Acid phosphatase activity in their peritoneal cells was also augmented, suggesting that Vibrio cells activated macrophages in the mice.     

Cadmium accumulation and interactions with zinc, copper, and manganese, analysed by ICP-MS in a long-term Caco-2 TC7 cell model

The influence of long-term exposure to cadmium (Cd) on essential minerals was investigated using a Caco-2 TC7 cells and a multi-analytical tool: microwave digestion and inductively coupled plasma mass spectrometry. Intracellular levels, effects on cadmium accumulation, distribution, and reference concentration ranges of the following elements were determined: Na, Mg, Ca, Cr, Fe, Mn, Co, Ni, Cu, Zn, Mo, and Cd. Results showed that Caco-2 TC7 cells incubated long-term with cadmium concentrations ranging from 0 to 10 μmol Cd/l for 5 weeks exhibited a significant increase in cadmium accumulation. Furthermore, this accumulation was more marked in cells exposed long-term to cadmium compared with controls, and that this exposure resulted in a significant accumulation of copper and zinc but not of the other elements measured. Interactions of Cd with three elements: zinc, copper, and manganese were particularly studied. Exposed to 30 μmol/l of the element, manganese showed the highest inhibition and copper the lowest on cadmium intracellular accumulation but Zn, Cu, and Mn behave differently in terms of their mutual competition with Cd. Indeed, increasing cadmium in the culture medium resulted in a gradual and significant increase in the accumulation of zinc. There was a significant decrease in manganese from 5 μmol Cd/l exposure, and no variation was observed with copper.     

Generation of free radicals in haemocytes of mussels after exposure to low molecular weight PAH components: immune activation, oxidative and genotoxic effects

Polycyclic aromatic hydrocarbons (PAHs) constituents, such as phenanthrene (PH) and anthracene (AN), are considered toxic for marine organisms, including bivalve mollusks. The present study showed that the perturbation of health status in mussels, as well as their inability to survive in air (stress on stress response), following exposure to either PH or AN (at a final concentration of 0.1 mg/L respectively), and a mixture of them (ration 1:1, at a final concentration of 0.2 mg/L) for 7 days, is probably related with alterations occurred in their haemocytes. According to the present study, PH and AN, either alone or in a mixture, could induce elevated levels of superoxide anions (^•O₂⁻) within haemocytes of mussels, thus leading to immune susceptibility as indicated by the increased levels of cell death and the elevated levels of lysosomal membrane acid phosphatase (AcP) activity, probably occurred via its overproduction or its release into the cytosol after lysosomal membrane destabilisation. PAH-mediated oxidative and genotoxic effects, indicated by the increased levels of both lipid peroxidation (MDA content) and nuclear abnormalities (MN test) could be related with haemocytes' inability to overcome free radical generation, thus leading to attenuation of general health status, before other disturbances, such as death, occur.     

Pathology and immune response of the blue mussel (Mytilus edulis L.) after an exposure to the harmful dinoflagellate Prorocentrum minimum

The harmful dinoflagellate Prorocentrum minimum has different effects upon various species of grazing bivalves, and these effects also vary with life-history stage. Possible effects of this dinoflagellate upon mussels have not been reported; therefore, experiments exposing adult blue mussels, Mytilus edulis, to P. minimum were conducted. Mussels were exposed to cultures of toxic P. minimum or benign Rhodomonas sp. in glass aquaria. After a short period of acclimation, samples were collected on day 0 (before the exposure) and after 3, 6, and 9 days of continuous-exposure experiment. Hemolymph was extracted for flow-cytometric analyses of hemocyte, immune-response functions, and soft tissues were excised for histopathology. Mussels responded to P. minimum exposure with diapedesis of hemocytes into the intestine, presumably to isolate P. minimum cells within the gut, thereby minimizing damage to other tissues. This immune response appeared to have been sustained throughout the 9-day exposure period, as circulating hemocytes retained hematological and functional properties. Bacteria proliferated in the intestines of the P. minimum-exposed mussels. Hemocytes within the intestine appeared to be either overwhelmed by the large number of bacteria or fully occupied in the encapsulating response to P. minimum cells; when hemocytes reached the intestine lumina, they underwent apoptosis and bacterial degradation. This experiment demonstrated that M. edulis is affected by ingestion of toxic P. minimum; however, the specific responses observed in the blue mussel differed from those reported for other bivalve species. This finding highlights the need to study effects of HABs on different bivalve species, rather than inferring that results from one species reflect the exposure responses of all bivalves.     

Haemocyte response associated with induction of shell regeneration in the deep-sea vent mussel Bathymodiolus azoricus (Bivalvia: Mytilidae)

This study reports on the haemocyte responses after induction of shell regeneration in the hydrothermal mussel Bathymodiolus azoricus. Haemolymph was drawn from live mussels collected at Menez Gwen hydrothermal vent site (850 m depth) at the Mid Atlantic Ridge (MAR) and was compared with those collected following laboratory acclimatisation (1 atm and Ca-rich algal diet) and also with induced specimen for up to 30 days. Simultaneously, histological changes in mantle micro-morphology with the histochemical detection of Ca mobilisation in tissues were conducted.On the basis of light- and transmission electron microscopy, it is concluded that the physiological equipment involved in shell regeneration in the deep sea bivalve closely resembles that in littoral mytilids, a group that B. azoricus is closely related. This in spite of previously alleged molecular and cellular adaptations to extreme conditions typical at deep sea hydrothermal vents. Three types of blood cells were identified sharing various morphological similarities with those in many non-vent bivalves. Significant increase in the number of circulating haemocytes was detected from day 5 after induction shell regeneration. It is suggested that the increase may be a result of migration of haemocytes from the connective tissue, probably to the shell growth frontline. It is alleged that a first peak in haemocyte number is a non-specific immune response related wound healing, which renders changes in the pallial fluid that are favourable for CaCO₃ deposition. The conspicuous presence of an unidentified, acid soluble, highly refractive structure in the haemolymph of induced mussels was detected, which may play a role in Ca nucleation.This study has set the stage for investigations underway on the influence of hydrostatic pressure on shell biomineralisation in B. azoricus subjected to post-capture hyperbaric simulations.     

The activity parameters,cDNA cloning and mRNA expression of lysozyme in Cyclina sinensis (Bivalvia,Veneridae) responding to the pathogenic bacterium Vibrio anguillarum

ABSTRACT

Lysozyme is an important component of the innate immune response against pathogen infection. The functional forms have been well-studied in the c-type lysozymes of vertebrates but less so in i-type lysozymes prevalent in most invertebrate animals. Cyclina sinensis is a commercially important marine venerid bivalve that is abundant and widely distributed around the maritime coasts of Asia. We obtained an i-type lysozyme (i-lyz) gene in C. sinensis by large scale EST sequencing of a SMART-cDNA library. In C. sinensis, the i-lyz gene encodes 181 amino acids. The lysozyme activities and the mRNA levels of the i-lyz gene in haemocytes were upregulated during the infection by a bacterium, Vibrio anguillarum. The lysozyme activity in haemocytes was increased after 12?h infection by V. anguillarum, and reached the highest level at 24?h post-infection, which was significantly higher than that of the control group (P?<?0.01). The expression level of the i-lyz gene in haemocytes was increased after 3?h infection of Vibrio, and reached the highest level at 6?h post-infection. A recombinant i-lyz was obtained through prokaryotic expression which, when isolated and purified, had a molecular weight of approximately 19?kDa. Western blotting was used to validate the expression of the fusion protein. Furthermore, we demonstrate that the recombinant i-lyz protein has obvious bacteriostatic activity upon Escherichia coli whereby the K value of the curve was significantly lower than control and blank groups. Our study shows that the C. sinensis i-type lysozyme plays an important role in the immune defence against V . anguillarum and provides a theoretical reference for clarifying the mechanism of immune response against pathogen invasion in this bivalve.     

The effects of feeding Karlodinium veneficum (PLY # 103; Gymnodinium veneficum Ballantine) to the blue mussel Mytilus edulis

The effects of exposure to the type species for Karlodinium veneficum (PLY # 103) on immune function and histopathology in the blue mussel Mytilus edulis were investigated. Mussels from Whitsand Bay, Cornwall (UK) were exposed to K. veneficum (PLY # 103) for 3 and 6 days. Assays for immune function included total and differential cells counts, phagocytosis and release of extra cellular reactive oxygen species. Histology was carried out on digestive gland and mantle tissues. The toxin cell quota for K. veneficum (PLY # 103) was measured by liquid chromatography–mass spectrometry detecting two separable toxins KvTx1 (11.6 ± 5.4 ng/ml) and KvTx2 (47.7 ± 4.2 ng/ml). There were significant effects of K. veneficum exposure with increasing phagocytosis and release of reactive oxygen species following 6 days exposure. There were no significant effects on total cell counts. However, differential cell counts did show significant effects after 3 days exposure to the toxic alga. All mussels produced faeces but not pseudofaeces indicating that algae were not rejected prior to ingestion. Digestive glands showed ingestion of the algae and hemocyte infiltration after 3 days of exposure, whereas mantle tissue did not show differences between treatments. As the effects of K. veneficum were not observed in the mantle tissue it can be hypothesized that the algal concentration was not high enough, or exposure long enough, to affect all the tissues. Despite being in culture for more than 50 years the original K. veneficum isolate obtained by Mary Parke still showed toxic effects on mussels.     

Combined effects of temperature and cadmium exposure on haemocyte apoptosis and cadmium accumulation in the eastern oyster Crassostrea virginica (Gmelin)

Combined effects of acclimation temperature (12, 20 and 28 °C) and exposure to a toxic metal cadmium (Cd, 50 μg L⁻¹) on haemolymph parameters related to immune defense and metal transport were studied in a model marine bivalve, Crassostrea virginica. Acclimation to elevated temperatures resulted in higher plasma protein concentrations and increased Cd levels in oyster haemolymph plasma and haemocytes. Cd accumulation in haemocytes was linear over the 45 days of Cd exposure and accumulation rates were 0.10, 0.53 and 0.56 μg Cd g⁻¹ dry mass at 12, 20 and 28 °C, respectively. Percentage of blood Cd burden associated with haemocytes increased with increasing temperatures from 13–20% at 12 °C to 26–47% at 20 and 28 °C suggesting a higher role for cellular Cd transport at elevated temperatures. Cd levels in gills and hepatopancreas were positively correlated with Cd concentration in haemocytes, but accumulation rates were considerably faster, so that after 45 days of exposure Cd levels in gills and hepatopancreas were >10–20 times higher than in haemocytes. As a result of slow Cd accumulation possibly reflecting fast haemocyte turnover rates and/or exocytosis of Cd-containing granules, haemocytes in Cd-exposed oysters did not reach threshold Cd burdens required to trigger apoptosis. This suggests that haemocyte viability is not likely to contribute to immunosuppression in the environmentally relevant Cd range. In contrast, elevated temperature (28 °C) resulted in a significant increase in the percentage of apoptotic haemocytes compared to 12 or 20 °C supporting the notion that 28 °C is physiologically stressful for C. virginica. Overall, our study demonstrates strong effects of environmental temperature on haemocyte viability and other important blood parameters such as plasma protein content and metal transport capability which may mask potential Cd effects at environmentally relevant exposure levels.     

Detection of serpins involved in cellular immune response of Rhipicephalus microplus challenged with fungi

The understanding of tick physiology and immune system is important to improve the effective control of this ectoparasite. Invertebrates' innate immune response is activated when the organism is challenged with pathogens. The present study describes the changes of serine proteinase inhibitors (serpins) and in the number of circulating haemocytes involved in cellular immune defence of Rhipicephalus microplus engorged females challenged with the entomopathogenic fungi Metarhizium anisopliae or Beauveria bassiana, or with the non-entomopathogenic fungus Fusarium oxysporum. The cell-free haemolymph was separated from haemocytes by centrifugation and cells were re-suspended in phosphate buffer pH 7.2. The proteins of haemocytes were analysed by SDS-PAGE and the segments of the 1D gel were submitted to protein digestion with trypsin. The peptides were analysed by liquid chromatography coupled with electrospray tandem mass spectrometry (LC-ESI-MS/MS). The analysis by mass spectrometry allowed the identification of several proteins through the search in the database built based on public banks of Ixodidae and Argasidae. In haemocytes, many proteins were identified highlighting serpins. The results showed that the entomopathogenic fungi M. anisopliae or B. bassiana reduced the amount of serpins, while F. oxysporum increased. The present study reports, for the first time, the variation of serpins in haemocytes of R. microplus engorged females infected by fungi.