# Review on Cyanobacterial Studies in Portugal: Current Impacts and Research Needs
## 2 Cyanobacterial Studies
### Molecular Studies  
Molecular studies were initiated in the mid 2000s (Figure 2) with a first study applying _C. raciborskii_ Portuguese isolated strains to determine their potential to produce either cylindrospermopsins or saxitoxins. Results from this analysis were negative for both cyanotoxins and englobe the application of peptide synthetase (PS) and polyktetide synthase (PKS) genes through simple PCR analysis [24]. Recent studies have shown that environmental DNA belonging to _C. raciborskii_ can be already found in the north and center regions of Portugal demonstrating its invasive nature but without forming any blooms and with no association with toxicity genes [14]. Other studies used the multiplex PCR technique in uncovering the toxigenic nature of dietary food supplements of cyanobacter  
Figure 2: Graphical representation of the percentage of each assay published in Portugal.  
rial origin. In fact, toxicigenic _Microcytis_ were found to be present in all analyzed dietary supplements produced from the nontoxic cyanobacterium _Chrysosporum flos-aquae_[25]. PCR was further considered an early warning technique in cyanobacterial investigations since it correlated well with ELISA assays when applying Portuguese water samples [26]. [PERSON] et al. [27] developed a multiplex PCR that simultaneously amplified _mcyA_-cd, _mcyAB_, and _mcyB_ amplicons of the microccyst gene cluster in 124 cyanobacterial isolates and in 37 environmental samples. The toxicological status of the isolates was assessed by high-performance liquid chromatography where the multiplex PCR developed showed a sensitivity of 92.3% and a specificity of 100%. In environmental samples, ELISA was used and results for the presence of microcystins in these samples gave a sensitivity of 80% and a specificity of 100% [27]. Studies applying Real-Time PCR analysis were initiated by [PERSON] et al. [28] with the variation of _C. raciborskii_ and cylindrospermopsins in field samples gathered from Vela Lake with primers previously described characterizing total cyanobacteria (16S rRNA), _C. raciborskii_ (_rpoC1_), and cylindrospermopsin synthetase gene (_cyrC_). The results report the high abundance of both cyanobacteria and _C. raciborskii_ in Vela Lake, with _C. raciborskii_ representing 0.4% to 58% of the total cyanobacteria population. Cylindrospermopsin synthetase gene was detected in one of the samples [28]. In the same year, [PERSON] et al. [29] quantified _Microcytis_ and MC-producing _Microcytis_ to determine the genotypic composition of the natural _Microcytis_ population. Results from his study further highlighted that a negative significant correlation was observed between toxic (with _mcy_ genes) to non-toxic (without _mcy_ genes) genotypes ratio and the overall _Microcytis_ density [29]. More recently, [PERSON] et al., [30] developed primers for the Real-Time PCR amplification of _Planktonix agandhi_ based on the _rpoC1_ gene fragment. Cell concentration determined by the Real-Time PCR method showed a linear correlation with the cell concentration determined from direct microscopic counts and detection limit for cell quantification was 8 cells/\(\upmu\)L. More recently [PERSON] et al. [7] carried an extensive surveillance study that included the application of representative genetic markers for the amplification in water samples of the four main cyanotoxins in the North and Center freshwater systems. Amplification results include that 79% of the samples had microcystins (_mcyA_), 40% had cylindrospermopsins (_cyrC_), 40% had anatoxin-a (_anaC_) and finally 50% had saxitoxins (_sxtl_) [13].