Advances in Environmental Biology Donax trunculus

Advances in Environmental Biology, 8(13) August 2014, Pages: 740-749
AENSI Journals
Advances in Environmental Biology
ISSN-1995-0756
EISSN-1998-1066
Journal home page: http://www.aensiweb.com/AEB/
Quantitative and Qualitative Analysis of Proteins in Gonads of Donax
trunculusfrom the Annaba Bay: Effects of Site, Season and Sex
Hamdani A., Soltani Mazouni N. and Soltani N.
Laboratory of Applied Animal Biology, Department of Biology, Faculty of Sciences, University Badji Mokhtar of Annaba 23000-Annaba,
Algeria
ARTICLE INFO
Article history:
Received 25 June 2014
Received in revised form
8 July 2014
Accepted 14 September 2014
Available online 10 October 2014
Keywords:
Donax trunculus, Marine pollution,
Gonads, Proteins, Electrophoresis
ABSTRACT
The city of Annaba is a major economic hub and tourism center located on the east
coast of Algeria. Previous works have highlighted the importance of the industrial
complex Fertial in the contamination of air and water of the Bay of Annaba. In addition,
various heavy metals were detected in sediments, but also in tissues of an edible bivalve
bioindicator of pollution, Donax trunculus L. (Mollusca, Bivalvia). This study aims to
determine quantitative and qualitative changes in male and female gonad proteins
during an annual cycle. Quantification and electrophoretic separation of proteins from
gonads were performed during the year 2011. Samples were collected seasonally at two
sites in the bay: El Battah a relatively clean site and Sidi Salem a polluted site. Assays
revealed a reduction in the amounts of proteins from male and female gonads in
individuals sampled at Sidi Salem compared to those of El Battah. The analysis of
variance indicates a highly significant effect of sites, seasons and sexes. Moreover, the
electrophoretic separation of proteins on sodium dodecyl sulfate polyacrylamide slab
gels showed differences in the number of protein fractions varying with the times, sites
and sexes. These quantitative and qualitative differences in proteins recorded in males
and female gonads are discussed in relation to the reproductive events and the pollution
level characterizing the two study sites.
© 2014 AENSI Publisher All rights reserved.
To Cite This Article: Hamdani A., Soltani Mazouni N. and Soltani N., Quantitative and Qualitative Analysis of Proteins in Gonads of
Donax trunculusfrom the Annaba Bay: Effects of Site, Season and Sex. Adv. Environ. Biol., 8(13), 740-749, 2014.
INTRODUCTION
The environmental monitoring projects have always played an important role to provide scientific
knowledge in assessing health and sustainability of ecosystems [23, 24, 69, 37]. Several mollusk species are
used as bioindicators for ecosystemhealth assessment becausetheir high bioaccumulation capacities [25, 26, 28,
31, 32, 36, 70]. Furthermore, this accumulation is influenced by several factors [53] such as seasonality [55],
location [13] salinity [20], organic matter [51], sex [19, 65], food acquisition capability [56] or stage of gonad
development [16]. The regional coastal environments have been subjected to various forms of degradation [1,
7,41, 58]. Thus, elevated levels of heavy metals were detected in a locally prevalent edible mollusk Donax
trunculus L. (Mollusca, Bivalvia) [7]. This species has been successfully used as a bioindicator of marine
pollution in the Bay of Annaba through the direct measurement of several biomarkers[4, 12, 61, 67].
In a previous report, the main components (lipids, proteins, carbohydrates) of gonads from D. trunculus
were determined monthly during the reproductive period (from January to August 2009) in the Bay of Annaba
[34]. The results showed changes correlated to the reproductive events in the Bay. Moreover, additional
biochemical analyzes made in mantles revealed a significant decrease in protein, lipid and carbohydrate amounts
in individuals collected at Sidi Salem, a polluted site, comparatively with those from El Battah, a relatively
clean site [60].
Recently, a growing interest is devoted to the development of new biomarkers of exposure and effect to
pollutants in various aquatic ecosystems and protein biomarkers were used as bioindicator for water
pollution[39].Therefore, the present study, in continuation to previous works[4, 6, 7, 12, 60, 61, 67], aimed the
quantitative and qualitative analysis of gonad proteins in an edible mollusk species D. trunculus since
biochemical indicators provide more accurate information about gonad performance and environmental
stress[63].Samples are collected during the four seasons in 2011 at two sites in Annaba Bay, El Battah, a
Corresponding Author: Soltani N., Laboratory of Applied Animal Biology, Department of Biology, Faculty of Sciences,
University Badji Mokhtar of Annaba 23000-Annaba, Algeria
E-mail: [email protected]
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relatively clean site and Sidi Salem, a polluted site. The data collected indicated significant effects of time, sex,
and site and allowed additional information on the impact of pollution on the protein composition of gonads.
MATERIALS AND METHODS
Presentation of sampling sites:
The Bay of Annaba is located in East Algeria. El Battah site (36° 50' N - 7° 50' E) is situated about 30 km
to the East of Annaba. It is far from any source of pollution and expected as a relatively clean site away from
pollution sources. The second site Sidi Salem (36° 50' N - 7° 47' E), situated about 1 km to the East of Annaba
city, received through the river Seybouse urban waste, and other releases from the port and an industrial zone
specialized in fertilizer and pesticide production (Fig. 1).
Fig. 1: Location of the two sampling sites in the Annaba Bay (Sidi Salem and El Battah).
Collection of samples:
D. trunculusof standardized shell size (27 ± 1 mm) were collected monthly during 2011, from the two
selected sites in Annaba Bay. Sex was separated by macroscopic inspection according to the color of gonads
[30]. Gonads were dissected and subjected to extraction of main biochemical components.
Protein quantification:
A total of 73 specimens of D. trunculus(4 to 6 specimens in each season from each site and each sex) were
dissected. Proteins from gonads were extracted following the procedure of Shibko et al. [59]. Each gonad
sample (weight: 45 ± 2 mg) was individually homogenized in 1 ml of trichloroacetic acid (20%) and then
centrifuged (5.000 g for 10 min). The pellet added with a mixture of ether and chloroform (1V/1V) was
subjected to a second centrifugation (5.000 g for 10 min). The second resultingpellet added with NaOH (0.1N)
serves fortotal protein quantificationin accordance with Bradford [15] usingblue brilliant of coomassie (G 250,
Merck) as a reagent and bovine serum albumin (Sigma) as standard. Absorbances were read at 595 nm
wavelength. Protein contents were expressed in μg.mg-1 of wet weight of tissues (w.w).
Electrophoresis:
For electrophoretic analysis of gonadal proteins, 12.5% sodium dodecyl sulfate–polyacrylamide slab gels
(SDS–PAGE) were prepared following the procedure of Laemmli [41] under native conditions. Briefly, gonads
were dissected at appropriate times from males and females. Each pooled sample (each containing 2 to 3
gonads) was placed in phenyl methyl sulfonyl fuoride or PMSF (45mg/ml ethanol) at 0.1% (500 µl) and stored
at -20°C until analysis. Proteins were extracted from samples as previously described [66]. The gels were
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stained in 0.025% Coomassie brillant blue R 250 (Merck), 10% acetic acid, and 25% propanol 2 and destined in
10% acetic acid in water. Molecular weight of bands was estimated using a mixture of 10 blue-stained protein
standards (10–250 kDa) (reference 161-0373, Bio-Rad, France).
Statistics:
Data were expressed by the mean ± standard deviation (SD). The normality of data was verified using the
Kolmogorov-Smirnov test, and the homogeneity of variances was checked by Bartlett and Levene's test. The
comparison of mean values between sites was estimated by Student’s t-test. The effects of seasons (time),
sampling sites (space) and sexes (gonads) were tested by a three-way analysis of variance (ANOVA).All
statistical analyses were performed usingMINITAB Software (Version 16, PA State College, USA). The
significant level was at p<0.05.
Results:
Quantitative evaluation of proteins from male and female gonads:
Seasonal changes in protein amounts were determined in male and female gonads of D. trunculus collected
from two sites (El Battah, Sidi Salem) in the Bay of Annaba are summarized in figure 2.One-way ANOVA
showed significant effects of season in both sexesin El Battah (males F=253.21; df= 3, 18; p<0.001 ; females
F=693.47; df= 3, 16; p< 0.001) and also in Sidi Salem (males F=171.20; df= 3, 15; p< 0.001; females
F=118.36; df= 3, 18; p< 0.001). The Tukey HSD test revealed that El Battahsite presents three groups for males
(thefirst group contains summer, the second contains winter and autumn and the third contains spring) and four
groups for females (the first is represented by summer, the second by winter, the third by autumn, and the fourth
by spring).
The site of Sidi Salem showed three groups of season for males (the first contains summer; the second
contains autumn, and the third contains winter and spring) and two groups for females (the first group contains
summer; autumn and winter, while the second group contains only spring). In addition, the lowest values were
recorded in spring while the highest values were recorded in summer in both sexes and sites.
The comparison of protein amounts recorded in gonads between the two sites using Student’s t-test revealed
a significant differences (p<0.05) for all seasons in both sexes.Lower amounts of proteins were recorded in the
gonads from Sidi Salem individuals compared to those of El Battah in both sexes(Fig. 2).The comparison of
protein levels recorded in the same site and season between the sexes using Student’s t-test revealed significant
difference(p< 0.001) in summer and autumnin individuals from El Battah, and in spring and summerfor
individuals from Sidi Salem, respectively.Three-wayANOVAindicated significant effects (p< 0.001) of season
(F= 1040.13; df= 3, 55; p= 0.000), sex (F=52.00; df= 1, 55; p=<0.001) and site (F=876.65; df= 1, 55;
p=<0.001).
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Fig. 2: Seasonal variation of protein amounts (µg/mg of tissue) in male (A) and female (B) gonads of Donax
trunculus collected from two sites in Annaba Bay during 2011(mean ± SD; n= 12-14). Asterisks above
values from the same season indicate a significant difference between the two sites (***: P< 0.001, **:
P< 0.01, *: P< 0.05).
Qualitative analysis of proteins from male and female gonads:
Protein extracts from gonads were analyzed by SDS-PAGE and electrophoretic patterns were shown in
figures 3 and 4. The number of protein bands varies as a function the seasons in both sexes. Indeed, we have
observed a slight reduction in the number (1 to 3 protein bands/season) and the intensity of some protein bands
in individuals from Sidi Salem as compared to data from the relatively clean site (El Battah) (Fig. 3).
For males, the molecular weights of detected bands ranged from 13 to 270 kDa in individuals from El
Battah and from 13 to 257 kDa in individuals from Sidi Salem. The following numbers of bands were recorded
by season: in winter, 7protein bands at El Battah vs. 6protein bandsat Sidi Salem (absence of the following
bands 200 and 110 kDaat Sidi Salem and 257 kDa at El Battah), in spring,13 protein bands at El Battah vs 11 at
Sidi Salem(30 and 13 kDa bands were absentat Sidi Salem), in summer 5 protein bands at El Battah vs. 4 at Sidi
Salem (absence of the 98 kDa band), and in autumn 10protein bands at El Battah vs7at Sidi Salem ( absence of
170, 140 and 97 kDa).
Fig. 3: Electrophoretic separation on SDS-PAGE (12.5%) of proteins in male gonads in Donax trunculus
collected from two sites in Annaba Bay during four seasons in the year 2011. [A]1: Protein markers;
2:Winter El Battah; 3:Winter Sidi Salem; 4: Spring El Battah; 5: Spring Sidi Salem. [B]1: Protein
markers; 6: Summer El Battah; 7: Summer Sidi Salem; 8: AutumnEl Battah; 9: Autumn Sidi Salem).
(Arrow head: position of missing bands).
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In females,the number of protein bands also varies as function the site and season: in winter4 protein bands
at El Battah vs 7 at Sidi Salem (absence of 200, 158, 124 and 67 kDabands at El Battah). in spring2protein
bands at El Battah vs 4 at Sidi Salem (absence of 47 and 13 kDa bands at El Battah), in summer3protein bands
at El Battah vs 4 at Sidi Salem (absence of the 47 kDa band at El Battah), and in autumn3protein bands at El
Battah vs4 at Sidi Salem (absenceof the bands 57 kDa at El Battah).
Fig 4: Electrophoretic separation by SDS-PAGE (12,5 %) of proteins in female gonads inDonax trunculus
collected from two sites in Annaba Bay during four season on year 2011. 1: Protein markers; 2: Winter
El Battah; 3:Winter Sidi Salem; 4: Spring El Battah; 5: Spring Sidi Salem; 6: Summer El Battah; 7:
Summer Sidi Salem; 8: AutumnEl Battah; 9: Autumn Sidi Salem (Arrow head: position of missing
bands).
Discussion:
Aquatic organisms can accumulate harmful substances and achieve very high concentrations in their tissues.
The contamination of waters affects aquatic biota and poses considerable environmental risks and concerns
and human health[17, 48, 50, 54]. Recent biomonitoring programs are now involving biomarkers, which are
measurable parameters at different levels of biological organization [37]. The area of Annaba has observed
markedly increased levels of pollution in recent decades [1, 2,7, 27,42, 58].This pollutioncauses oxidative
stresses measured by several selected biomarkers. It has been reported a significant inhibition of AChE activity
and a significant increase in the activity of the GST in D. trunculus collected from Sidi Salem as compared to El
Battah [12, 61]. Concerning the Bay of Annaba, the reproductive period is observed from January to August,
the resting period occurs from September to November and two periods of recruitment are observed: the first in
spring and the second in autumn [8, 35]. The changes in biochemical composition of gonads are related to the
reproductive cycle of D. trunculus in the Bay of Annaba. Thus, an elevation in protein and lipid amounts during
the gametogenesis, and a decrease of these components related to spawning phase, while decreases in
carbohydrate during the reproductive period were respectively observed [34].
Proteins play important functional and structural roles during the reproductive cycle [10]. In addition, the
expressed proteome in mussel tissues is important to understand the biological effect of environmental pollution
and climate change. Consequently,a putative set of protein biomarkers that may be significantly correlated to
environmental factors were identified [39]. Our results indicated significant effects of season, sex, and site. The
seasonal variationofamounts ofprotein ingonadsare related to the reproductive cycle of D. trunculusconfirming
previous studies made in the same species in the Annaba Bay [35, 60] and in the Algiers Bay [5], or in other
mollusk species such as Mytillus edulis from the Dutch Wadden Sea [74], Pecten maximus from the Ria de
Arousa(Spain)[52], Macoma balthica from the gulf of Gdansk [21]. Moreover, our results indicated higher
amounts of proteins in females of D. trunculus. This could be explained by a difference in the energy demand
between male and female gametes [9]. Lastly, male bivalves produce small spermatozoa with few energy
reserves by comparison with females which elaborate vitellin for developing oocytes [10].
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Several studies reported significant differences in metal concentrations between sexes like Perna perna
from Yemen [64], Choromytilus meridionalis from the South Africa, Mytilus californianus from Southern
California Bight [33], Perna viridis from the Bay of Bengal [19] or Mytilus trossulus and Crenomytilus
graganus from Japan [40]. The study of Mauri [45] showed an effect of sex in the accumulation of zinc, since
females present higher concentrations than males in D. trunculus. The seasonal variation in metal concentration
is influenced by the stage of gonad development [7].The accumulation of contaminants in tissues of aquatic
organisms depends on several endogenous factors, namely physiological condition, geographic habitat, fat
content, adaptive capacity, and biotope characteristics [46, 62,14] and affected the population exposed [71].
Concerning effect of site, our results globally indicated a decrease in theamounts of proteins in individuals
fromSidi Salem in both sexes comparatively to individuals from El Battah. This agrees with previous reports in
the same species in the bay of Annaba[34, 60]. These resultsconfirm thelevel of pollution in this site. In a
previous report, heavy metal levels were determined in D. trunculus sampled from the same studied sites [9].
These authors showed a significant effect of seasons for all metals measured and there was a significant
difference in Cd concentrations between sites. Similar observations werereported in other clams such as
Indonaia caerufeus from Paithan in India [43], Mya arenaria from the Saguenay Fjord [29], Perna perna from
south-western Morocco [11].
Gametogenesis in bivalve represents a period of particularly high demand of energy and a transfert of
proteins, lipids, and carbohydrates from the adductor muscle and the digestive gland to the gonad occurred
[49].Sublethal concentrations of metals (silver, cadmium, copper, mercury and zinc)caused a quantitative
modification of proteins in the gland digestive from of Mytilus edulis and Crassostrea gigas[30], while
tributyltin chloride induced a significant depletion in protein metabolic profiles in gonad, gill and digestive
gland in the freshwater bivalve Lamellidens marginalis, suggesting an increased proteolysis and a possible
utilization of their degration products for metabolic purpose [38].
The highest protein amounts observed in males during the spring period at Sidi Salem comparatively at El
Battah, can be explained by a reduction in intensity of spawning period at Sidi Salem. Indeed,it has been
reported that pollution reduce the intensity of spawning in the same speciesin Tunisia[68]. The electrophoretic
separation of proteins reveals changes in male and female gonad proteins since effects of bothseason, sex and
site were observed.The Seasonal variation in qualities of proteins in gonads males and females on D.
trunculuscould be due depended with the stage of the reproductive cycle in the Annaba Bay and these energy
needs. This hypothesis is confirmed in Elliptio complanat[73].In addition, the seasonal changes in
environmental conditions can beaffected the biological functions of organisms such as respiration, growth or
diet; they largely determine the intensity of the relationship with the environment and the quality of filtered
water[3, 22].
Concerning the effect of sex and site, our results indicated that female gonads showedan increase in the
number of protein bands in Sidi Salem as compared to individuals from El Battah. This agrees with a report of
Christopher[21] made in femalesof Macoma balthicaexposed to an anthropogenic pollution. Theincrease in the
number of protein bands at Sidi Salem a polluted site[7, 8],can be explained by asubsequent production of stress
proteins [21].For males,we found a reduction in the number of protein bands at Sidi Salem as compared
withinfrom El Battah. This difference may be due to variations in responses to pollutants between the two sexes
[7, 64]. A decrease in the number of protein bands was also reportedin hepatopancreas of clam Macrobrachium
malcolmsoniiexposed to carbaryl toxicity[57], while the environmental stress can cause a polar attenuation of
protein bands[72].
Conclusion:
The quantitative and qualitative analysis of proteins in gonads of Donax trunculusfrom the gulf of
Annabarevealsdifferences between sites, season and sexes.Globally, individuals from Sidi salem are more
affected than those from El battah. Moreover, males are more sensitive to environmental stress than females.
Finally, the current study gives additional data onthe pollution in the Bay of Annaba. According to Ielmini et
al.[39], proteins are useful as biomarker to assess the environmental effects of pollution.
ACKNOWLEDGEMENTS
The authors wish to thank two anonymous referees for useful discussion and critical reading of the
manuscript.This study was supported by the National Fund for Scientific Research to Pr. N. Soltani and by the
Ministry of High Education and Scientific Research of Algeria (CNEPRU and PNR projects to Pr. N. SoltaniMazouni).
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