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ISJ 3: 1-3, 2006                            ISSN 1824-307X 
 

 
Short Communication  

 
 

Monitoring of the immune efficiency of Mytilus galloprovincialis in Adriatic sea mussel 

farms in 2005 

 
 
D Malagoli, L Casarini, E Ottaviani 

 
Department of Animal Biology, University of Modena and Reggio Emilia, Modena,  Italy  

 
 

Accepted January 13, 2006 
 

 
Abstract 

The monthly evaluation of the cytotoxicity of hemolymph from the mussel Mytilus galloprovincialis 
revealed some variations in the percentage of cytotoxic animals during the year. Cytotoxicity is confirmed 
to be a dynamic parameter that can be used as an indicator of immune efficiency and, therefore, of the 
state of health of the animals. 
 
Key words: Mytilus galloprovincialis; cytotoxicity assay; cytotoxic activity; Adriatic sea mussel farms 
 
 
 
Introduction 

 
The state of health of Mytilus galloprovincialis 

mussels was evaluated in terms of cytotoxic activity in 
the hemolymph. It is well-known that molluscs, as all 
invertebrates, possess only innate or natural immunity 
and recognize and eliminate non-self material mainly 
through cellular and humoral components. Among the 
latter, cytotoxicity is one of the most important immune 
functions (Franceschi et al., 1991). This function has 
been well conserved during evolution and described 
both in invertebrates and vertebrates (Ratcliffe et al., 
1985; Savary and Lotzová, 1986). Wittke and Renwrantz 
(1984) have demonstrated that circulating cells 
(immunocytes) from Mytilus edulis are able to produce 
cytotoxic substances, which lyse human erythrocytes. A 
cytotoxic protein complex has also been found in M. 
galloprovincialis hemolymph (Hubert et al., 1997). 
Recently, we have designed an easy-to-use and low-
cost cytotoxicity test (Malagoli and Ottaviani, 2005) that 
has been used here to evaluate the cytotoxic activity of 
M. galloprovincialis collected each month during 2005 
from Adriatic sea mussel farms. 
 
 
Corresponding Author: 
Enzo Ottaviani 

Department of Animal Biology 
via Campi 213/D 
41100 Modena, Italy 
E-mail: ottaviani.enzo@unimore.it 

Materials and Methods 
 
Animals 

Specimens of the mussel Mytilus galloprovincialis 
were obtained each month from local fishermen in the 
Cesenatico area (FC, Italy) immediately after being 
caught. Forty animals were sacrificed to collect 
hemolymph on the spot, while the remaining samples 
were taken to the Department of Animal Biology 
(Modena, Italy) and maintained in the laboratory 
aquarium (artificial seawater, temperature 19 ± 1 °C, pH 
8.0 ± 0.2, salinity 35 ± 2 psu) for at least 10 days before 
control experiments. No animals could be obtained in 
February because of adverse weather conditions. 
 
Hemolymph preparation and cytotoxicity assay 

The detailed procedure for the cytotoxicity assay has 
been described elsewhere (Malagoli and Ottaviani, 
2005). Briefly, the hemolymph was collected from the 
posterior adductor muscle using a sterile syringe, filtered 
into sterile tubes with 0.2 µm filters, aliquoted, 
immediately frozen and maintained at –80 °C for at least 
12 h before use. The cytotoxic activity was assessed by 
checking the cytolysis of human A positive erythrocytes. 
In order to eliminate damaged erythrocytes, the whole 
blood sample was washed at least three times in 9 vol. of 
sterile NaCl 0.9 %, and the erythrocytes were then re-
suspended in sterile TBS (50 mM Tris-HCl, 200 mM 
NaCl, 10 mM CaCl2, pH 8.5) at a final concentration of 
2x109 cells/ml. 500 µl of filtered hemolymph were added  



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Fig. 1 Mussel cytotoxicity during 2005 in the Cesenatico sea area. The red line represents the mean percentage.
 
 
 
to 500 µl of erythrocyte suspension and incubated for 

1h at 25 °C (Hubert et al., 1997; Malagoli and Ottaviani, 
2005). After incubation, samples were centrifuged at 
3.000xg for 5 min at 4 °C, and the optical density (OD) of 
supernatants was measured by evaluating the 
absorbance at 541 nm with a Helios β 
spectrophotometer (Spectronic Unicam, Cambidge, UK). 
It should be noted here that a threshold OD level of 0,5 
was fixed as the minimum for a positive test, while 
samples that clearly exceeded this value were 
considered positive without further measurement 
(Malagoli and Ottaviani, 2005). The experiment was 
repeated in duplicate two times for each animal. 
 
Statistical analysis 

Statistical analysis was performed using the χ2 test 
with p< 0.05 taken as significant. 
 
 

Results and Discussion 
 

The periodic evaluation of the cytotoxicity of 
hemolymph from the mussel M. galloprovincialis reveals 
variations in the percentage of cytotoxic animals during 
the year (Fig. 1). The mean percentage of mussels 
positive to the cytotoxicity assay was around 53 %, but 
there were various peaks. It is interesting to note that the 
minimum and the maximum values were during the 
periods in which the water temperature reached its 
minimum and maximum levels, respectively, as indicated 
in the annual report on inshore water conditions of the 
Italian region Emilia-Romagna in 2003. In a previous 
study, we observed that rapid changes in water 
temperature, salinity and pH resulted in a significant 
decrease in the mean cytotoxic activity. A rapid increase 
in water temperature resulted in a significant drop in the 
number of animals positive to the cytotoxicity assay 
(Malagoli and Ottaviani, 2005). Conversely, the 
observations reported here seem to indicate a positive 
correlation between water temperature and the 
percentage of cytotoxic molluscs.  However, no sudden 

 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Fig. 2 Comparison between mussel cytotoxicity 
immediately after being caught (A) or after a period of 
maintenance in the aquarium (B). The mean values of six 
independent experiments ± SD are shown. 

 
 

modifications in environmental parameters were seen in 
the present study. It should be underlined that the mean 
percentages of cytotoxic animals did not differ among 
mussels maintained in the laboratory aquarium for at 
least 10 days prior to control experiments (Fig. 2). As far 
as the peak registered in September is concerned, it 
should be noted that the samples were collected after a 
period in which many animals had been died as a result 
of a loss of byssus. This pathology is still of uncertain 
origin, but a fungal infection has been observed to be 
involved in serious damage to the byssus apparatus 
(Franchini et al., 2005). Even if more detailed studies are 
needed to clarify the question, the large number of 
cytotoxic specimens in the survivor population could 
suggest a link between cytotoxic activity and the animal's 
ability to overcome epidemics. 

Overall, cytotoxicity is confirmed as a dynamic 
parameter that can be used as an indicator of immune 
efficiency and, therefore, of the good state of health of 
the mussels. 

 

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Acknowledgement 
 

We are grateful to the Centro di Ricerche Marine 
(Cesenatico, FC, Italy) for financial support for this study. 
The Authors also wish to thank Mr M Marangoni who 
kindly provided the mussels and the Centro 
Trasfusionale Policlinico (Modena, Italy) for providing the 
blood. 
 
 
References 
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Natural cytotoxicity in a freshwater polmonate mollusc: an 
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Franchini A, Malagoli D, Ottaviani E. Investigation of the loss of 
byssus in Mytilus galloprovincialis from mussel farms in  

 the Adriatic Sea. Cell Biol. Int. 29: 857-860, 2005. 
Hubert F, Cooper EL, Roch Ph. Structure and differential target 

sensitivity of the stimulable cytotoxic complex from 
hemolymph of the Mediterranean mussel Mytilus 
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 Malagoli D, Ottaviani E. Cytotoxicity as a marker of mussel 
health status. J. Mar. Biol. Ass. UK 85: 359-362, 2005. 

Ratcliffe NA, Rowley AF, Fitzgerald SW, Rhodes CP. 
Invertebrate immunity: basic concepts and recent 
advances. Int. Rev. Cytol.  97: 183–350, 1985. 

Savary CA, Lotzová E. Phylogeny and ontogeny of NK cells. In: 
Lotzová E, Heberman RB (eds), Immunobiology of natural 
killer cells, vol 1, FLA: CRC Press, Boca Raton,  pp 45–61, 
1986. 

Wittke M, Renwrantz L. Quantification of cytotoxic hemocytes of 
Mytilus edulis using a cytotoxicity assay in agar. J. 
Invertebr. Pathol. 43: 248-253, 1984.

 

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