In spite of the great environmental and sanitary importance of cyanobacteria, their biodiversity is little known in Tunisia. In this work, a review was carried out, based on literature data, of potentially toxic cyanobacteria occurrence in Tunisia. Microcystis, one of the most widely distributed toxic bloom-forming cyanobacteria genera, was represented by Microcytis wesenbergii, found only in Lebna water reservoir, and Microcytis aeruginosa recorded in different water bodies. The invasive potentially toxic cyanobacterium Cylindrospermopsis raciborskii, reported for the first time in Tunisia in October 2004, was observed in the semi-arid to arid areas. The harmful genus Planktothrix was represented in Tunisian freshwater by the green-pigmented species P. agardhii. The filamentous cyanobacteria dominance is increasingly reported in Tunisia in eutrophic water bodies. This dominance increases especially during the summer–autumn period. Recently, potentially toxic cyanobacteria blooms have been reported in some reservoirs in the north east of the country. These blooms were generated by the potentially toxic Chroococcale Microcystis aeruginosa. Harmful cyanobacteria tend to spatio-temporal expansion in the Tunisian inland waters. The toxicological potential evaluated by several methods showed that none of the Tunisian strains were proved to be cylindrospermopsin nor saxitoxin producers. However, the majority of Microcystis were able to synthesize microcystin.

INTRODUCTION

Cyanobacteria are a major group of prokaryotes that occur throughout the world (WHO 1998). They are the Earth's oldest known oxygen-producing organisms, with fossil remains dating back 3500 million years (Schopf 2000). Through their photosynthetic activity, they were largely responsible for the modern-day oxygen-enriched atmosphere, and subsequent evolution of our planet's higher plant and animal life (Schopf 2000; Whitton & Potts 2000). Cyanobacteria have many unique features among phytoplankton, such as buoyancy and nitrogen fixation, and the production of a wide variety of bioactive compounds. Several species of cyanobacteria form blooms that are frequently toxic, and thus pose a health risk for humans and animals (Sivonen & Jones 1999). They can produce toxic secondary metabolites including hepatotoxins that have carcinogenic potential, neurotoxins and lipopolysaccaride endotoxins (Carmichael & Falconer 1993; Codd 2000; Carmichael 2001). The tragic deaths of 70 of 131 patients exposed to the hepatotoxins microcystins (MC) through renal dialysis in Brazil are the only well known substantiated human fatalities due to cyanotoxins (Jochimsen et al. 1998). Nevertheless, some illnesses reported previously were life-threatening (Ressom et al. 1994), such as the poisoning of 138 children and 10 adults due to hepatotoxin cylindrospermopsin (CYN) in Palm Island (Australia) (Hawkins et al. 1985).

While cyanobacterial harmful algal blooms have been reported in the scientific literature for more than 130 years (Francis 1878), in recent decades, the incidence and intensity of these blooms, as well as economic loss associated with these events, has increased in both fresh and marine waters (Chorus & Bartram 1999; Carmichael 2001; Hoagland et al. 2002; Teneva et al. 2005; Hudnell 2008; Heisler et al. 2008; Paerl 2008; Paul 2008; Paerl & Huisman 2008). The most common cyanobacterial genera known for their potential ability to produce toxins include Anabaena, Aphanizomenon, Cylindrospermopsis, Lyngbya, Microcystis, Nodularia, Nostoc and Planktothrix. However, the number of identified toxic cyanobacteria is still increasing as a result of new detections (Sivonen 1998; Aboal & Puig 2005; Humpage et al. 2012). In fact, Ballot et al. (2005) reported that the hepatotoxin MC-YR and the neurotoxin anatoxin-a were produced, in a monocyanobacterial strain of Arthrospira fusiformis, isolated from Lake Sonachi (Kenya). It is important to note that the cyanobacterium Arthrospira (Spirulina), having a long history of use as food for humans (Vonshak 1997), is used as a nutritive supplement for its beneficial effects, including detoxication, increased energy, weight loss and therapeutic applications (Ciferri 1983; Belay 1997; Mei Li & Zao Qi 1997). Limnothrix, a commonly occurring cyanobacterial genus, was recently shown to produce a novel toxin ‘Limnothrixi’ (Torres-Arińo & Mora-Heredia 2010; Bernard et al. 2011).

The dominance of cyanobacteria in aquatic ecosystems, in different geographical locations, with considerable variability in terms of size, morphology, salinity and hydrologic conditions, indicates that climate change can help synergistically with anthropogenic nutrient enrichment on the growth of these micro-organisms across the globe (Paerl & Paul 2012). Several studies reported that freshwater cyanobacteria blooms are typically associated with eutrophic and poorly flushed waters (Paerl 1988; Carmichael 1995; Lee et al. 2000; Albay et al. 2003). Kosten et al. (2012) conducted monitoring in 143 lakes of different trophic situations, localized according to the latitudinal gradient from north of Europe to South America. They found that the percentage contribution of cyanobacteria in total phytoplankton biomass increases significantly with temperature. These authors noted also that the rise in temperature can reduce the levels of nutrient uptake that cause the initiation and maintenance of blooms. Recent studies in laboratory scale as well as field observations have clearly demonstrated that the combination of anthropogenic inputs of nutrients, rising temperatures, increased stratification and elevated atmospheric CO2 concentrations, favored the dominance of cyanobacteria in a wide range of aquatic ecosystems (Paerl & Paul 2012). The warm Mediterranean climate favors the occurrence and extended duration of the cyanobacterial blooms in eutrophic freshwaters, which may start in spring and persist until December, or in hypertrophic lakes, may even be continuous throughout the year (Moustaka-Gouni et al. 2007). Although toxic and non-toxic strains usually co-exist in a water body, distinguishing toxic strains from non-toxic strains is impossible under a microscope. Therefore, many studies have focused on the discriminative detection of toxin-producing strains (Baker et al. 2001; Via-Ordorika et al. 2004; Ouahid et al. 2005). Researchers have already described the MC-producing mechanism on the genetic level (Dittmann et al. 1997; Tillett et al. 2000). MC are produced by the enzyme complex MC synthetase encoded in the mcy gene cluster, which code for both non-ribosomal peptide synthetases (PSs) and polyketide synthases (PKSs) (Dittmann et al. 2001). Microcystis mcy genes are organized in a cluster of two operons. One operon contains PS genes (mcyA, mcyB, mcyC), responsible for the incorporation and activation of the amino acid [MDha-Ala-X-MAsp-Z] constituents of MC. The second operon includes one PKS gene (mcyD), two hybrid genes corresponding to PS and PKS (mcyE and mcyG), and additional genes (Nishizawa et al. 2000). McyD, -G and -E genes code for the Adda group synthesis. The association of glutamic acid with the Adda group and its activation are encoded by the mcyE gene (Sivonen & Börner 2008). Kellmann & Neilan (2007) have proposed the saxitoxin (STX) biosynthesis pathway. The sxt gene clusters within each organism all contain a core set of genes putatively responsible for the biosynthesis of STX. The production cylindrospermopsin (CYN) implicates enzymes coded by the PS and the PKS genes. Schembri et al. (2001) described a novel amidinotransferase (AMT) gene within the genomic region encoding PS and PKS. Fergusson & Saint (2003) developed a multiplex-polymerase chain reaction (PCR) test to amplify PS and PKS determinants associated with CYN production and to distinguish Cylindrospermopsis raciborskii strains from other CYN-producing cyanobacteria. The discovery of these genes will allow the rapid and accurate detection of harmful paralytic shellfish poisoning (PSP)-producing species in water (Kellmann et al. 2008).

TUNISIAN CONTEXT

Tunisia is a Mediterranean country located in the northeastern extremity of Africa. With Sicily, it defines both eastern and western basins of the Mediterranean. It covers an area of 164,150 km². Being under the disruptive climatic influence of the temperate region in the north and the Saharan region in the south, Tunisia is characterized by relative aridity on the major part of its territory. Indeed, two-thirds of the country receives rainfall amounts between 50 and 350 mm/year (Ben Boubaker et al. 2003). To this aridity is added the variability of the Mediterranean climate, with erratic and unpredictable periods of drought and violent floods, to make water an often limited resource and distributed unequally in time and space (Benzarti 2003). Bergaoui & Louati (2010) showed the temperature increase with an inflow reduction even for relatively wet hydrological years. Tunisia's hydric potential, mainly formed by surface water, is very modest. It was estimated at 4,630 thousand million m3. It is the lowest value of the Maghreb countries (Ben Boubaker et al. 2003). In fact, the important evaporation which is the cause of the climatic water balance to be in deficit, and the reduced size of the catchment areas make Tunisia one of the Mediterranean countries less endowed with hydraulic resources. The water resource volume available per capita is 450 m3/year against 556 m3/year in Morocco, 776 m3/year in Syria and 2,200 m3/year in Turkey (Cherif 2003).

Tunisia's freshwater resources are under increasing stress from a growing population and an expanding economy. In addition, almost all of the country's freshwater resources have been fully allocated, while the water quality of these resources has declined due to increased pollution caused by industry, urbanization and agriculture. The reservoirs have become progressively more enriched during recent decades. The importance and current extent of eutrophication in Tunisian water bodies has been highlighted in previous studies, showing that these ecosystems present an increasing productivity stimulated continually by fertilizing contributions owing to the important anthropisation and the drier climate (Mouelhi 2000; Turki 2002; Fathalli et al. 2006; El Herry et al. 2008a). Eutrophication is generally indicated by high values of nutrients, accumulation of metabolic products (e.g., hydrogen sulfide in deep waters), discoloration or turbidity of water (resulting in low or poor light penetration), deterioration in the taste of water, depletion of dissolved oxygen and an enhanced occurrence of toxic cyanobacterial bloom-forming species (Dauta & Feuillade 1995). However, in spite of its great environmental and sanitary importance, the biodiversity of cyanobacteria is little known in Tunisia as a consequence of a small number of publications. In fact, the first reference to Tunisian freshwater toxic cyanobacreria was published only in 2006 (Ben Rejeb Jenhani et al. 2006).

In this review, we report the available information on the presence of potentially toxic cyanobacteria in Tunisian water storage reservoirs that have already been subject of limnological studies.

OCCURRENCE OF POTENTIALLY TOXIC CYANOBACTERIA IN TUNISIAN FRESHWATER

Data were obtained from scientific articles, reports, theses and communications at congresses. The water bodies are located in the four most important Tunisian hydrological basins (Figure 1, Table 1).

Table 1

Characteristics and uses of some water bodies in Tunisia

       Min-max or means
 
  
Dams Geographic coordinates Impound-ment Main supply catchment area (Km2Volume (106 m3Area (Ha) T (°C) NO3 (mg/l) PO43− (mg/l) Uses 
N37°10′42″ 1994 Séjnène river 376 138 790 18.56 2.25 – 5.,88 Drinking water; irrigation; fish farming 
E09°28′25″ 
N36°59′23″ 1983 Joumine river 418 130 660 19.3 2.52 – 5.34 0.01 – 0.29 
E09°36′59″ 
N36°52′ 1996 El Hjar river 61 254 12.3–28.8 7.05 0.031 Irrigation 
E11°02′ 
N 36°43′16″ 1959 Bezirk river 84 6.5 102 13.2–31.2 3.02–8.24 0.00–0.08 
E 10°37′57″ 
Ml N 36° 49′ 27″ 1965 Mlaabi river 35 3.7   13.2–29.4 0.42–4.23 0.00-0.09 
N10° 59′ 26″ 
Ch N 36°41′52″ 1963 Chiba river 63 6.9 66 12.3–28.3 1.35–6.86 0.00–0.08 
E 10°46′17″ 
 M N 36°31′50″ 1968 Masri river 53 6.9 66 12.1–30 2.35–9.60 0.00–0.07 Drinking water; irrigation 
E 10°29′08″ 
BMt N 36°43′10″ 1953 El lil river 103 57.6 310.6 Drinking water irrigation; fish farming electric energy 
E 08°44′10″ 
 K N 36°45′30″ 1968 Kasseb river 101 81 437 21,4 2,22 0,02 
E 09°00′50″ 
SSM N36°35′27″ 1981 Medjerda river 18,000 555 4,300 10.2 – 27.4 0.12 – 2.88 0.001 – 0.056 
E 09°23′51″ 
GG N 36° 44′ 1968 Kasseb reservoir Medjerda/Cap Bon channel 40 12.5–29.5 0.35–5.96 0.005–0.042 Drinking water 
E 09° 58′ 50″ 
Mo N 36° 44′ 1982 Kasseb reservoir Medjerda/Cap Bon channel 15 150 12.5–29.5 0.22–6.35 0.00–0.035 
E 09° 58′ 50″ 
N36°44′01″ 1986 Lebna river 189 30.2 650 19,7 0.72 0.09 Irrigation; fish farming 
E10°55′08″ 
LK N 35°59′58″ 1966 Lakhmess river 127 102 
E 09°28′31″ 
BM N36°30′36″ 1971 Méliane river 1,260 209 2,000 20.3 1.69 0.16 
E10°00′38″ 
N36°03′34″ 1965 Nebhana river 855 87.2 532 22.04 2.34 0.09 
E09°52′34″ 
SS N35°25′31″ 1981 Zeroud river 8,950 53 110 8.7 – 29 0.04 – 0.43 0.03 
E09°41′50″ 
       Min-max or means
 
  
Dams Geographic coordinates Impound-ment Main supply catchment area (Km2Volume (106 m3Area (Ha) T (°C) NO3 (mg/l) PO43− (mg/l) Uses 
N37°10′42″ 1994 Séjnène river 376 138 790 18.56 2.25 – 5.,88 Drinking water; irrigation; fish farming 
E09°28′25″ 
N36°59′23″ 1983 Joumine river 418 130 660 19.3 2.52 – 5.34 0.01 – 0.29 
E09°36′59″ 
N36°52′ 1996 El Hjar river 61 254 12.3–28.8 7.05 0.031 Irrigation 
E11°02′ 
N 36°43′16″ 1959 Bezirk river 84 6.5 102 13.2–31.2 3.02–8.24 0.00–0.08 
E 10°37′57″ 
Ml N 36° 49′ 27″ 1965 Mlaabi river 35 3.7   13.2–29.4 0.42–4.23 0.00-0.09 
N10° 59′ 26″ 
Ch N 36°41′52″ 1963 Chiba river 63 6.9 66 12.3–28.3 1.35–6.86 0.00–0.08 
E 10°46′17″ 
 M N 36°31′50″ 1968 Masri river 53 6.9 66 12.1–30 2.35–9.60 0.00–0.07 Drinking water; irrigation 
E 10°29′08″ 
BMt N 36°43′10″ 1953 El lil river 103 57.6 310.6 Drinking water irrigation; fish farming electric energy 
E 08°44′10″ 
 K N 36°45′30″ 1968 Kasseb river 101 81 437 21,4 2,22 0,02 
E 09°00′50″ 
SSM N36°35′27″ 1981 Medjerda river 18,000 555 4,300 10.2 – 27.4 0.12 – 2.88 0.001 – 0.056 
E 09°23′51″ 
GG N 36° 44′ 1968 Kasseb reservoir Medjerda/Cap Bon channel 40 12.5–29.5 0.35–5.96 0.005–0.042 Drinking water 
E 09° 58′ 50″ 
Mo N 36° 44′ 1982 Kasseb reservoir Medjerda/Cap Bon channel 15 150 12.5–29.5 0.22–6.35 0.00–0.035 
E 09° 58′ 50″ 
N36°44′01″ 1986 Lebna river 189 30.2 650 19,7 0.72 0.09 Irrigation; fish farming 
E10°55′08″ 
LK N 35°59′58″ 1966 Lakhmess river 127 102 
E 09°28′31″ 
BM N36°30′36″ 1971 Méliane river 1,260 209 2,000 20.3 1.69 0.16 
E10°00′38″ 
N36°03′34″ 1965 Nebhana river 855 87.2 532 22.04 2.34 0.09 
E09°52′34″ 
SS N35°25′31″ 1981 Zeroud river 8,950 53 110 8.7 – 29 0.04 – 0.43 0.03 
E09°41′50″ 

S: Séjnène; J: Joumine ; H: Hjar; L: Lebna; B: Bezirk ; M: Masri; Ml: Mlaabi; Ch: Chiba; BMt: Beni Mtir; K: Kasseb; SSM: Sidi Salem; GG: Gdir el golla; Mo: Mornaguia; LK: Lakhmess; BM: Bir M'cherga; NB: Nebhana; SS: Sidi Saâ.

Figure 1

Tunisian hydrological basins and the location of major dams (Ben Mammou and Louati 2007).

Figure 1

Tunisian hydrological basins and the location of major dams (Ben Mammou and Louati 2007).

Investigations conducted in Tunisian reservoirs revealed the presence of 55 species of cyanobacteria belonging mainly to filamentous strains. In fact, these species are represented by 34 Oscillatoriales, 16 Chroococcales, three Nostocales and two Pleurocapsales. Some of them have been described in the literature as potentially toxic (Table 2). The identification of the majority raw cyanobacterial samples was performed using UTERMÖHL technique based on classic inverted microscopy. However, some of them are confirmed by molecular biology tools using isolated and cultured strains (El Herry et al. 2008a; 2008b; Fathalli et al. 2010, 2011a, 2011b). In fact, molecular taxonomy proved necessary in order to avoid confusion between morphologically similar species.

Table 2

Inventory cyanobacteria species in Tunisian reservoirs

Order Species Ml Ch BMt SSM GG Mo LK BM NB SS 
Chroococcales Aphanothece c.v. brevis                 
Merismopedia glauca         
Merismopedia elegans               
Merismopedia miniata                 
Merismopedia sp.                 
Microcytis aeruginasa x⬤ x⬤ x⬤ x⬤        x⬤ x⬤ 
Microcytis wesenbergii    x⬤              
Chroococcus sp.         
Chroococcus minitus                 
Chrococcus turgidus                 
Aphanothece sp.                 
Aphanocapsa muscicola                 
Snowella sp.                 
Snowella atomus                 
Synechococcus elongatus                
Synechocystis aqualis                 
Nostocales Anabaena sp.             
Aphanizomenon sp.                 
Cylindrospermopsis raciborskii               x⬤ x⬤ x⬤ 
Oscillatoriales Borzia trilocularis               
Limnothrix sp.               x⬤   
Limnothrix redekei                 x⬤ 
Leptolyngbya sp.                x⬤  
Lyngbya sp.               
Lyngbya limnetica                
Lyngbya rubida                 
Oscillatoria acutissima                 
Oscillatoria amphibia                 
Oscillatoria articulata                
Oscillatoria chlorina           
Oscillatoria geminata                 
Oscillatoria homogenea            
Oscillatoria lacustris              
Oscillatoria limnitica               
Oscillatoria planctonica          
Oscillatoria pseudogeminata                
Oscillatori simplissima                 
Oscillatoria splendida                
Oscillatoria spp.     
Oscillatoria tenuis    x⬤           
Phormidium cf incrustatum                
  Phormidium frigidum                 
Phormidium luridum                
Phormidium olivascens                
Phormidium rezii                 
Phormidium sp.              
Phormidium tenue                
Planktolyngbya subtilis                 
Planktothrix agardhii  x⬤             x⬤ x⬤ x⬤ 
Planktothrix mougeotii                 
Pseudanabaena catenata       
Pseudanabaena constricta             
Romaria sp.                 
Pleurocapsales Hydrococcus sp.                 
Hydrococcus rivularis                
Order Species Ml Ch BMt SSM GG Mo LK BM NB SS 
Chroococcales Aphanothece c.v. brevis                 
Merismopedia glauca         
Merismopedia elegans               
Merismopedia miniata                 
Merismopedia sp.                 
Microcytis aeruginasa x⬤ x⬤ x⬤ x⬤        x⬤ x⬤ 
Microcytis wesenbergii    x⬤              
Chroococcus sp.         
Chroococcus minitus                 
Chrococcus turgidus                 
Aphanothece sp.                 
Aphanocapsa muscicola                 
Snowella sp.                 
Snowella atomus                 
Synechococcus elongatus                
Synechocystis aqualis                 
Nostocales Anabaena sp.             
Aphanizomenon sp.                 
Cylindrospermopsis raciborskii               x⬤ x⬤ x⬤ 
Oscillatoriales Borzia trilocularis               
Limnothrix sp.               x⬤   
Limnothrix redekei                 x⬤ 
Leptolyngbya sp.                x⬤  
Lyngbya sp.               
Lyngbya limnetica                
Lyngbya rubida                 
Oscillatoria acutissima                 
Oscillatoria amphibia                 
Oscillatoria articulata                
Oscillatoria chlorina           
Oscillatoria geminata                 
Oscillatoria homogenea            
Oscillatoria lacustris              
Oscillatoria limnitica               
Oscillatoria planctonica          
Oscillatoria pseudogeminata                
Oscillatori simplissima                 
Oscillatoria splendida                
Oscillatoria spp.     
Oscillatoria tenuis    x⬤           
Phormidium cf incrustatum                
  Phormidium frigidum                 
Phormidium luridum                
Phormidium olivascens                
Phormidium rezii                 
Phormidium sp.              
Phormidium tenue                
Planktolyngbya subtilis                 
Planktothrix agardhii  x⬤             x⬤ x⬤ x⬤ 
Planktothrix mougeotii                 
Pseudanabaena catenata       
Pseudanabaena constricta             
Romaria sp.                 
Pleurocapsales Hydrococcus sp.                 
Hydrococcus rivularis                

x: species identified by the classical method of inverted microscopy; ⬤: species confirmed by the molecular biology tools.

S: Séjnène; J: Joumine ; H: Hjar; L: Lebna; B: Bezirk ; M: Masri; Ml: Mlaabi; Ch: Chiba; BMt: Beni Mtir; K: Kasseb; SSM: Sidi Salem; GG: Gdir el golla; Mo: Mornaguia; LK: Lakhmess; BM: Bir M'cherga; NB: Nebhana; SS: Sidi Saâd.

Microcystis, one of the most widely distributed toxic bloom-forming cyanobacteria genera, was observed in 58% of studied Tunisian reservoirs. It was represented by Microcytis wesenbergii, found only in the Lebna dam, and Microcytis aeruginosa recorded in different water bodies from different geographic situations across the country (Figure 2) (El Herry et al. 2008a; Fathalli et al., 2011a; Sellami et al. 2012). This latter species was the most incriminated cyanobacterium in incidents of human and animal poisoning over the world by production of the hepatotoxins MC that present over 70 natural structural variants, and are potent and specific inhibitors of protein phosphatases (Chorus & Bartram 1999; Codd et al. 2005). Based on morphological criteria, 10 species have been distinguished in Europe, the other side of the Mediterranean: Microcystis aeruginosa, M. viridis, M. wesenbergii, M. novacekii, M. ichthyoblabe, M. flos-aquae, M. natans, M. firma, M. smithii and M. botrys (Komárek & Anagnostidis 1999). In North African freshwater bodies, four species are found: Microcystis aeruginosa, M. wesenbergii, M. ichthyoblabe and M. novacekii (Abdel-Rahman et al. 1993; Oudra et al. 2001; Oudra et al. 2002; Nasri et al. 2004; Ben Rejeb Jenhani et al. 2006; El Herry et al. 2009).

Figure 2

Toxic strains of Microcystis observed in Tunisian freshwater (El Herry et al. 2008a; Fathalli 2012). M1, M2: M. aeruginosa occurred in Lebna reservoir; M3: M. wesenbergii occurred in Lebna reservoir; Micro-NB-01: M. aeruginosa isolated from Nabhena reservoir; Micro-JM-02: M. aeruginosa isolated from Joumine reservoir; Micro-HJ-02: M. aeruginosa isolated from Hjar reservoir.

Figure 2

Toxic strains of Microcystis observed in Tunisian freshwater (El Herry et al. 2008a; Fathalli 2012). M1, M2: M. aeruginosa occurred in Lebna reservoir; M3: M. wesenbergii occurred in Lebna reservoir; Micro-NB-01: M. aeruginosa isolated from Nabhena reservoir; Micro-JM-02: M. aeruginosa isolated from Joumine reservoir; Micro-HJ-02: M. aeruginosa isolated from Hjar reservoir.

The invasive potentially toxic freshwater cyanobacterium Cylindrospermopsis raciborskii, which was firstly recorded in tropical to subtropical climate regions, was reported for the first time in Tunisia in October 2004 (Fathalli et al. 2010). It was observed in freshwater Bir M'cherga reservoir as pale blue-green, straight trichomes without mucilaginous sheaths, bearing terminal drop-shaped heterocysts with pointed ends. Coiled trichomes were never observed in Tunisian freshwater. This species was observed, later, in Nebhana and Sidi Saâd reservoirs, located in the center of the country (Fathalli et al., 2011a). These reservoirs are characterized by relatively high trophic levels (Fathalli et al. 2010; Sellami et al. 2010, 2012) and their environmental conditions seemed to be favorable for the species appearance. Fathalli et al. (2010) showed that the Cylindrospermopsis density was correlated positively with temperature, transparency and salinity. In fact, the occurrence of C. raciborskii was recorded in summer when the temperature values were higher than 20 °C. This confirms previous observations (Padisák 1997; Briand et al. 2002) showing that a water temperature ranging between 22 and 23.5 °C is a key factor for the germination of akinetes. The presence of this species during this period was also supported by the decrease of nitrate values (Fathalli et al. 2010). The same result was found by Briand et al. (2002), who observed a low nitrate concentration during the summer proliferation. Chorus & Bartram (1999) reported that the lack of nitrate was considered to be the main reason for the proliferation of heterocystic species, such as C. raciborskii.

Hepatotoxin-producing strains of C. raciborskii have been found in Europe, Australia, Asia and Africa (Hawkins et al. 1997; Saker et al. 1999, 2003; Li et al. 2001; Fastner et al. 2003; Bernard et al. 2003; Mohamed 2007), while neurotoxin-producing strains have been reported in Brazil, where it produced PSP toxin (Lagos et al. 1999). Incidents where C. raciborskii has been suspected to cause human sickness (Bourke et al. 1983; Hawkins et al. 1985) and cattle mortality (Saker et al. 1999) have been restricted to Australia. This toxic effect is now known to be due to the alkaloid CYN (Terao et al. 1994).

In Africa, the occurrence of C. raciborskii has also been recorded in Algeria, Egypt, Uganda, Senegal and South Africa (Bouaıcha & Nasri 2004; Mohamed 2007; Haande et al. 2008; Janse van Vuuren & Kriel 2008). From all these strains, only the Egyptian one was reported as hepatotoxic by the mouse bioassay (Mohamed 2007). C. raciborskii has been observed in other Mediterranean countries, including Spain, France, Italy, Greece and Israel (Romo & Miracle 1994; Bernard et al. 2003; Vardaka et al. 2005; Moustaka-Gouni et al. 2007; Messineo et al. 2010; Alster et al. 2010).

Planktothrix, considered to be an important genus of harmful cyanobacteria since it is one of the most frequent MC producers (Kurmayer et al. 2005), was represented in Tunisian freshwater by the green-pigmented species P. agardhii. This genus may contain a higher concentration of MC per cell than Microcystis. So, the risk level is uppermost when Planktothrix is dominant (Codd et al. 2005). The species P. agardhii was recorded at four different reservoirs (Joumine, Bir m'cherga, Nebhana and Sidi Saâd). Fathalli (2012) reports that, from 1 year to another, there is a considerable development increase of the species. Indeed the average density, in Bir M'cherga dam, changes from 0.017 × 106 filaments/l in 2005 to 0.5 × 106 filaments/l in 2006. Moreover, the same author notes a very high density of P. agardhii at the beginning of 2007 reaching 6.69 × 106 filaments/l. The species P. agardhii has generally presented a relatively homogeneous vertical distribution. It is endowed with dispersed development ecostrategy (Chorus & Bartram 1999). Despite its preference for warm waters (Berger & Sweers 1988), this species can occur in high densities throughout the year thanks to its tolerance for low temperatures and low light intensities (Dokulil & Teubner 2000; Scheffer, 1998). Thus, a competitive advantage is expressed in taxa that tolerate low light intensities, i.e., Planktothrix, Cylindrospermopsis raciborskii and Limnothrix, which thrive better than the species susceptible to loss of light, i.e., Anabaena spp. and Aphanizomenon spp. (Reynolds et al. 2002). In fact, an assemblage of these three filamentous cyanobacteria (Figure 3) (Planktothrix agardhii, Cylindrospermopsis raciborskii and Limnothrix sp.) was reported in the freshwater Bir M'cherga dam (Fathalli 2012).

Figure 3

Some filamentous cyanobacteria strains observed in Tunisian freshwater (El Herry et al. 2008a; Fathalli 2012). (a): Limnothrix sp. isolated from Bir M'cherga reservoir; (b): Leptolyngbya sp. isolated from Nabhena reservoir; (c): Planktothrix agardhii isolated from Bir M'cherga reservoir; (d): P. agardhii isolated from Joumine reservoir; (e): Cylindrospermopsis raciborskii isolated from Bir M'cherga reservoir; (f): Oscillatoria tenuis occurred in Lebna reservoir.

Figure 3

Some filamentous cyanobacteria strains observed in Tunisian freshwater (El Herry et al. 2008a; Fathalli 2012). (a): Limnothrix sp. isolated from Bir M'cherga reservoir; (b): Leptolyngbya sp. isolated from Nabhena reservoir; (c): Planktothrix agardhii isolated from Bir M'cherga reservoir; (d): P. agardhii isolated from Joumine reservoir; (e): Cylindrospermopsis raciborskii isolated from Bir M'cherga reservoir; (f): Oscillatoria tenuis occurred in Lebna reservoir.

The filamentous cyanobacteria dominance is increasingly reported in Tunisia in eutrophic water bodies such as Sidi Saâd (Sellami et al. 2012) and Bir M'cherga dams (Fathalli et al. 2010). This dominance increases especially during the summer–autumn period. Indeed, Fathalli (2012) showed the persistence of high cyanobacteria density in freshwater Bir M'cherga reservoir throughout the year. Upper densities of 6.14 × 106 and 9.58 × 106 individuals per liter were recorded during July 2005 and 2006, respectively. Recently, potentially toxic cyanobacteria blooms have been reported in some reservoirs in the north (Hjar and Lebna dams). These blooms were generated by the potentially toxic Chroococcale Microcystis aeruginosa (Ben Rejeb Jenhani et al. 2010). Oudra et al. (2001), Sabour et al. (2002), Nasri et al. (2004, 2008) and Douma et al. (2010) showed that in Morocco and in Algeria, a neighboring country with close climatic conditions, natural cyanobacterial blooms containing MC were dominated by the genus Microcystis. Thus, harmful cyanobacteria tend to spatio-temporal expansion in the Tunisian inland waters. They are consequently considered as precursors of freshwater-quality degradation.

OCCURRENCE OF CYANOBACTERIAL TOXINS IN TUNISIAN FRESHWATER

While the occurrence of cyanotoxins was reported worldwide since the beginning of the last century (Sivonen & Jones 1999), in Tunisian freshwater, their presence has been confirmed only in 2003, from Hjar Reservoir in the Cap Bon region, North-East of the country, using high performance liquid chromatography coupled to a diode array detector and tandem mass spectrometry technique that revealed three variants of the hepatotoxins MC: MC-LR, MC-RR, MC-YR (El Herry et al. 2007). The cyanotoxicity monitoring of the Tunisian freshwater was performed mainly using the protein phosphatase (PP2A) inhibition assay (Table 3). Ben Rejeb Jenhani et al. (2006) demonstrated that, compared to other untreated water, those of Hjar, Mlaabi and Lebna dams are distinguished by their higher levels of toxins, often observed in the particulate fraction. The same authors report that the maxima observed in these water bodies have coincided with a relatively massive development of Oscillatoria and Pseudoanabaena genera in the Hjar dam and the species Oscillatoria chlorina in Mlaabi freshwater. El Herry et al. (2008a) noted that toxin concentrations reached a value of 5.57 μg MC-LR equivalent per liter in the Lebna reservoir suggesting that the three morphospecies of Microcystis and the filamentous species O. tenuis that occurred in this dam are potentially toxigenic. In this water body two variants of MC (MC-LR and MC-YR), were identified. This finding is certainly consistent with prior studies from Mediterranean countries, which have shown that MC-LR is the major toxin in cyanobacterial blooms from France (Vezie et al. 1998), and Morocco (Oudra et al. 2002) and frequently co-occurs with MC-YR and/or MC-RR in Morocco (Oudra et al. 2001), in Algeria (Nasri et al. 2004; 2008), and in Turkey (Albay et al. 2005). The cyanotoxins concentrations detected in the three above-mentioned dams, used mainly for irrigation, was higher than the guideline value for MC-LR (1 μg L−1) established by the WHO (1998) for drinking water. However, they remain lower than those found in Algeria (Nasri et al. 2004) and in Morocco (Oudra et al. 2001), where the MC concentrations were estimated to be 29,163 μg MC-LR equivalent L−1 and >500 μg MC-LR equivalent L−1. Saqrane & Oudra (2009) reported that cyanotoxicity impact on both aquatic and terrestrial crop plants irrigated by water containing these toxins has become more and more available. This fact is gaining importance since plants could in a direct or indirect manner contribute to cyanotoxin transfer through the trophic network, and thus constitutes a potent health risk source. The use of this contaminated irrigation water can also have an economic impact which appears as a reduction in the germination rate of seeds, and alteration of the quality and the productivity of crop plants (Silva & Vasconcelos 2010).

Table 3

Extreme values of cynotoxins concentrations in Tunisian freshwaters (using PP2A inhibition assay)

  Total cyanotoxins (μg MC-LR equivalent L−1References 
Ghdir el Golla 0.001–0.028 Ben Rejeb Jenhani et al. (2006)  
Mornaguia 0–0.024 
Sejnene 0.001–0.837 
Joumine 0.001–0.168 
Hjar 0.023–7.455 
Bezirk 0.011–0.500 
Masri 0.032–0.752 
Mlaabi 0.010–1.040 
Chiba 0.003–0.495 
Kasseb 0–0.034 Fathalli et al. (2006)  
Lebna 0.021–5.485 El Herry et al. (2008a)  
Bir M'cherga 0.002–0.931 Fathalli et al. (2010)  
  Total cyanotoxins (μg MC-LR equivalent L−1References 
Ghdir el Golla 0.001–0.028 Ben Rejeb Jenhani et al. (2006)  
Mornaguia 0–0.024 
Sejnene 0.001–0.837 
Joumine 0.001–0.168 
Hjar 0.023–7.455 
Bezirk 0.011–0.500 
Masri 0.032–0.752 
Mlaabi 0.010–1.040 
Chiba 0.003–0.495 
Kasseb 0–0.034 Fathalli et al. (2006)  
Lebna 0.021–5.485 El Herry et al. (2008a)  
Bir M'cherga 0.002–0.931 Fathalli et al. (2010)  

The Tunisian freshwaters studied were classified according to three levels of toxicity: <0.1 μg MC-LR equivalent L−1, 0.1–1 μg MC-LR equivalent L−1 and >1 μg MC-LR equivalent L−1. All Tunisian reservoirs used for drinking water production have been characterized by toxicity values less than 1 μg MC-LR equivalent L−1 (Ben Rejeb Jenhani et al. 2006). The highest values of toxicity were recorded at dams situated in the north-east hydrologic basin characterized by high anthropogenic activity. In fact, this region which represents 8.5% of the whole area of Tunisia, concentrates paradoxically 45% of urban population, 49% of industrial employment, 36% of tourism capacity and 30% of irrigable surface (Cherif 2003).

GENOMIC POTENTIALITY OF CYANOTOXINS SYNTHESIS

The cyanotoxins have been reported in almost the whole regions where cyanobacteria were studied, thus making the cyanotoxicose a public health problem of global concern. Cyanobacterial toxins are usually classified according to their mode of action, into hepatotoxins (MC, nodularins and cylindrospermopsins), neurotoxins such as the STXs, and dermatotoxins which is characterized by their lipopolysaccharide nature (Chorus & Bartram 1999). The toxic and non-toxic character of the same species may vary between different strains (Carmichael 1992). Recent development of techniques in biotechnology has enabled the development of new methods of molecular detection of cyanobacteria and their toxins. Detection methods based on PCR amplification of DNA of Cyanobacteria are interesting because of their potential selectivity with respect to genes involved in the biosynthesis of toxins, their sensitivity and timeliness (Ouellette & Wilhelm 2003). In Tunisia, few studies have been conducted on the biodiversity of cyanobacteria and their molecular characterizations (Table 4). El Herry et al. (2008a) showed the expression of the NMT domain of the MC synthetase genes mcyA, -B and -C in the genome of three morphospecies of Microcystis and the filamentous species Oscillatoria tenuis collected from the natural samples of Lebna reservoir. The authors indicated that these cyanobacterial species were responsible for MC production in this dam. They confirmed this result through the detection of MC-LR and MC-YR, in the cyanobacterial sample containing the three morphospecies of Microcystis and the filamentous species O. tenuis, via LC/MS/MS technique. Fathalli et al. (2011a) evaluated, also by molecular biology tools, the toxicological potential of 27 cyanobacterial strains isolated from seven reservoirs located in the north and center of Tunisia and belonged mainly to Microcystis aeruginosa, Cylindrospermopsis raciborskii and Planktothrix agardhii species. This study showed that none of the isolated strains carried segments of the gene cluster responsible for the production of cylindrospermopsin and STX, while the majority of Microcystis isolates were able to synthesize MC, since they presented the six characteristic segments of the MC synthetase mcy cluster (mcyA,-B,-C,-D,-E and-G). This was further confirmed by MALDI-TOF analysis that showed the presence of eight MC variants, including MC-LR. Microcystis strains isolated from Lebna (Micro-LB-01) and Bir M'cherga (Micro-BM-02) reservoirs did not produce any kind of MC. In fact, these strains showed the absence of either the gene mcyA or mcyA,-C,-D and-E. In another study, Fathalli et al. (2011b) assessed the toxicity of four C. raciborskii strains isolated from the Bir M'cherga Tunisian reservoir. They concluded that all the isolated strains were not producing cylindrospermopsin, STX or MC. This result was further confirmed by HPLC and MALDI-TOF analyses. However, these authors reported for the first time in C. raciborskii the presence of mcy A and mcy E, two segments of the MC synthetase mcy cluster in the strain Cyl-BM-07. The strain C. raciborskii (MG) that was confirmed not producing cylindrospermopsin present only the PKS gene (Fathalli et al. 2010). Schembri et al. (2001) confirmed that both the PS and the PKS genes were associated with the ability to produce cylindrospermopsins.

Table 4

Toxicity assessment of cyanobacteria in Tunisian freshwater

Cyanobacteria Origins Samples Tested genes numbera Expressed genes (PCR+ sequencing) Toxins Methods references 
Oscillatoria spp. + Pseudoanabaena spp. Hjar natural     MC-LR, MC-RR, MC-YR HPLC/MS/MS El Herry et al. (2007)  
Microcystis aeruginosa Lebna natural 3 mcyA, -B and -C MC PP2A El Herry et al. (2008b)  
Microcystis aeruginosa Lebna Culture 11 mcyB,-C,-D,-E and-G No toxins MALDI-TOFMS Fathalli et al. (2011a), Fathalli (2012)  
Microcystis aeruginosa Bir M'cherga Culture 11 mcy B and-G No toxins MALDI-TOFMS Fathalli et al. (2011a), Fathalli, (2012)  
Microcystis aeruginosa Hjar Culture 11 mcyA,-B,-C,-D,-E and-G MC-LR, MC-FR, MC-RR MC-WR, Demethyl-MC-LR MALDI-TOFMS Fathalli et al. (2011a), Fathalli (2012)  
Microcystis aeruginosa Nebhana Culture 11 mcyA,-B,-C,-D,-E and-G MC-LR, MC-YR MALDI-TOFMS Fathalli et al. (2011a), Fathalli (2012)  
Microcystis aeruginosa Joumine Culture 11 mcyA,-B,-C,-D,-E and-G MC-LR, MC-YR, Demethyl-MC-LR, Demethyl-MC-YR MALDI-TOFMS Fathalli et al. (2011a), Fathalli (2012)  
Microcystis aeruginosa Séjnène Culture 11 mcyA,-B,-C,-D,-E and-G MC-LR, MC-RR, MC-YR, Demethyl-MC-LR, Demethyl-MC-RR, Demethyl-MC-YR MALDI-TOFMS Fathalli et al. (2011a), Fathalli (2012)  
Microcystis wesenbergii. Lebna Natural mcyA, -B and -C MC PP2A El Herry et al. (2008b)  
Oscillatoria tenuis Lebna Natural mcyA, -B and -C MC PP2A El Herry et al. (2008a)  
Planktothrix agardhii Joumine Culture 11 mcyA No toxins MALDI-TOFMS Fathalli et al. (2011a), Fathalli (2012)  
Leptolyngbya sp. Nebhana Culture 11 mcyA No toxins MALDI-TOFMS Fathalli et al. (2011a), Fathalli (2012)  
Cylindrospermopsis raciborskii Bir M'cherga Culture 11 PKS (MG strain) No toxins Elisa , HPLC MALDI-TOFMS Fathalli et al. (2010, 2011b): Fathalli (2012)  
mcyA and mcyE (CYL-BM-07 strain) 
Cyanobacteria Origins Samples Tested genes numbera Expressed genes (PCR+ sequencing) Toxins Methods references 
Oscillatoria spp. + Pseudoanabaena spp. Hjar natural     MC-LR, MC-RR, MC-YR HPLC/MS/MS El Herry et al. (2007)  
Microcystis aeruginosa Lebna natural 3 mcyA, -B and -C MC PP2A El Herry et al. (2008b)  
Microcystis aeruginosa Lebna Culture 11 mcyB,-C,-D,-E and-G No toxins MALDI-TOFMS Fathalli et al. (2011a), Fathalli (2012)  
Microcystis aeruginosa Bir M'cherga Culture 11 mcy B and-G No toxins MALDI-TOFMS Fathalli et al. (2011a), Fathalli, (2012)  
Microcystis aeruginosa Hjar Culture 11 mcyA,-B,-C,-D,-E and-G MC-LR, MC-FR, MC-RR MC-WR, Demethyl-MC-LR MALDI-TOFMS Fathalli et al. (2011a), Fathalli (2012)  
Microcystis aeruginosa Nebhana Culture 11 mcyA,-B,-C,-D,-E and-G MC-LR, MC-YR MALDI-TOFMS Fathalli et al. (2011a), Fathalli (2012)  
Microcystis aeruginosa Joumine Culture 11 mcyA,-B,-C,-D,-E and-G MC-LR, MC-YR, Demethyl-MC-LR, Demethyl-MC-YR MALDI-TOFMS Fathalli et al. (2011a), Fathalli (2012)  
Microcystis aeruginosa Séjnène Culture 11 mcyA,-B,-C,-D,-E and-G MC-LR, MC-RR, MC-YR, Demethyl-MC-LR, Demethyl-MC-RR, Demethyl-MC-YR MALDI-TOFMS Fathalli et al. (2011a), Fathalli (2012)  
Microcystis wesenbergii. Lebna Natural mcyA, -B and -C MC PP2A El Herry et al. (2008b)  
Oscillatoria tenuis Lebna Natural mcyA, -B and -C MC PP2A El Herry et al. (2008a)  
Planktothrix agardhii Joumine Culture 11 mcyA No toxins MALDI-TOFMS Fathalli et al. (2011a), Fathalli (2012)  
Leptolyngbya sp. Nebhana Culture 11 mcyA No toxins MALDI-TOFMS Fathalli et al. (2011a), Fathalli (2012)  
Cylindrospermopsis raciborskii Bir M'cherga Culture 11 PKS (MG strain) No toxins Elisa , HPLC MALDI-TOFMS Fathalli et al. (2010, 2011b): Fathalli (2012)  
mcyA and mcyE (CYL-BM-07 strain) 

aNumber of genes tested; 3: mcyA, -B and -C (NMT domain of the MC synthetase gene)

11: mcyA,-B,-C,-D,-E,-G (MC synthetase gene cluster); AMT (hepatotoxins); GAMT, PS, PKS (cylindrospermopsin synthase); sxt1 (STXs).

PHYLOGENY

As previously reported in the literature, the research performed on the phylogeny of Tunisian cyanobacteria strains confirmed that the 16S rRNA, which is commonly used to distinguish broad phylogenetic relationships among cyanobacteria because of availability of many sequences of this gene for different species of cyanobacteria, may not fully discriminate closely related species even when they are morphologically distinct. In fact, the phylogenic tree based on 16S rDNA sequences showed that three morphospecies of Microcystis, isolated from Lebna reservoir and assigned to Microcystis aeruginosa and Microcystis wesenbergii, are indistinguishable from each other, from the reference strain PCC 7806 and from many other known Microcystis species and, therefore, this tree did not necessarily correlate to the distinctions between morphospecies (El Herry et al. 2008b). To elucidate the phylogenetic relationships of these strains, El Herry et al. (2008b) used the PCR amplification and restriction fragment length polymorphism (RFLP) analysis of the 16S-23S rRNA spacer region (ITS) of these three morphospecies of Microcystis, that provided a similar pattern type for the whole strains. However, it was shown that this region is a good tool to discriminate bacteria strains at the species level and interspecific (Jensen et al. 1993; Neilan 1995). Phylogenetic study based on ITS sequences of Microcystis aeruginosa strains isolated from five Tunisian waters bodies with several other morphospecies listed in the NCBI database, revealed the existence of two clusters. The first one was formed by most of the Tunisian isolates that were highly similar to M. aeruginosa from the continents of Africa, Europe and America. The second one was composed of one toxic Microcystis strain that also clustered with European strains (Fathalli et al. 2011a). None of the Tunisian M. aeruginosa strains were similar to those found in Asia. No clustering was observed between the toxic and non-toxic Microcystis strains; although a well defined sub-group was formed by two non-toxic Tunisian Microcystis strains (Figure 4) (Fathalli et al. 2011a). Otsuka et al. (1999) reported that the same cluster included all the Microcystis novacekii and M. ichthyoblabe strains and most M. aeruginosa strains. These authors also noted that MC-synthesizing and non-producing genotypes were closely related. Tillett et al. (2001) confirmed that the analysis of the ITS region was not helpful in distinguishing between MC-producing and non-producing.

Figure 4

Neighbour-joining phylogenetic trees based on ITS gene for Microcystis aeruginosa species and based on rpoC1 gene for Cylindrospermopsis raciborskii species. Bootstrap values are placed at each branch point (Fathalli et al. 2011a).

Figure 4

Neighbour-joining phylogenetic trees based on ITS gene for Microcystis aeruginosa species and based on rpoC1 gene for Cylindrospermopsis raciborskii species. Bootstrap values are placed at each branch point (Fathalli et al. 2011a).

Tunisian isolates of C. raciborskii were highly similar to each other and they formed a distinct cluster based on rpoC1 sequences, which was separate from other African strains collected in Senegal and Uganda. In fact, the phylogenetic tree based on rpoC1 sequences separated our species from American, African and European/Australian clusters (Figure 4) (Fathalli et al. 2011a). The rpoC1 gene was reported to be more discriminatory at the species level than the 16S rRNA, for this species (Wilson et al. 2000). This different clustering of the African strains demonstrates that the population structure in this continent is somewhat heterogeneous, supporting the uniqueness of the Tunisian isolates relative to C. raciborskii strains from other geographical locations (Fathalli et al. 2011b). Moreira et al. (2011) showed, based on a concatenated fragment of 2.9 Kb encompassing the four genetic markers 16S rRNA, ITS longer spacer, ITS shorter spacer and rpoC1 sequences, that the Tunisian C. raciborskii strains were clustered with the American strain. In fact, this study revealed that the C. raciborskii strains grouped in to three well-supported distinct clusters: (i) European, (ii) African (Tunisian)/American (Brazilian) and (iii) Asian/Australian. This method provided a better phylogeographical distinction of the strains, with higher statistical significance than if considering the information of each gene alone. In fact, several authors attribute the recent dispersion of C. raciborskii to migration of water birds, like gulls, that carry these organisms in their intestinal tract or even owing to the importation of items from tropical countries that bring resistant cyanobacterial cells that can later grow when suitable conditions are found (Manti et al. 2005; Vasconcelos 2006).

CONCLUSION

In Tunisia, the increase in cyanobacterial occurrence, observed in some water bodies, poses a potential risk to the environment and public health. Microcystis, one of the most widely distributed toxic bloom-forming cyanobacteria genera, was represented mainly by Microcytis aeruginosa recorded in different water bodies. The invasive potentially toxic cyanobacterium Cylindrospermopsis raciborskii was observed in the semi-arid to arid areas. The harmful genus Planktothrix was represented in Tunisian freshwater by the green-pigmented specie P. agardhii. The filamentous cyanobacteria dominance is increasingly reported in Tunisia in eutrophic water bodies. These cyanobacteria which synthesize secondary metabolites responsible for the deterioration of water quality and their uses did not form real blooms at all prospected water bodies, except for el Hjar and Lebna reservoirs. Thus, the question remains, ‘How long will these environments still be safe?’ knowing that neighboring countries (Morocco and Algeria) currently are faced with the complexity of this phenomenon.

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