Harmful microalgal blooms of the genus Karlodinium are responsible for mortalities of wild and cultured aquatic species worldwide. (Deeds et al. 2002; Kempton et al. 2002; Adolf et al. 2007; Place et al. 2012) Karlodinium are small marine dinoflagellates (~ 8-12 µm) belonging to the family Kareniaceae and commonly found in coastal aquatic ecosystems (Goshorn et al. 2004; Bachvaroff et al. 2008; Place et al. 2012) where they produce outbreaks regularly (reviewed in Place et al. (Place et al. 2012)). Frequently Karlodinium is present at relatively low cell abundances (102-103 cell mL−1) but can form dense blooms (Deeds et al. 2002, 2006) (104-105 cell mL−1). Exposure to high Karlodinium's cell densities has been shown to elicit a response in fish gills including increased ionic permeability, oedema, hyperplasia, and epithelial necrosis. (Ulitzor and Shilo 1966; Jones K. et al. 1982)
Some species of the genus Karlodinium produce a class of ichthyotoxins called karlotoxins (KmTxs). Several of them have been identified. (Takeshita et al. 2000; Van Wagoner et al. 2008, 2010; Place et al. 2012; Waters et al. 2015; Cai et al. 2016; Krock et al. 2017) These are large (molecular weight >1000 Da), lipophilic compounds whose ecological role could be related to chemical defence against grazing and/or their use for prey acquisition's. (Adolf et al. 2007; Waggett et al. 2008; Place et al. 2012) In the Table 1, the isolated congeners studied to date have been included with their corresponding bioactivities. KmTxs have been characterized either by their hemolytic in vivo or in vitro activity and have been claimed to kill fish by damaging their gill epithelia. (Deeds et al. 2002, 2006, 2015; Kempton et al. 2002; Bachvaroff et al. 2009) The potency of the KmTx congeners, regarding hemolytic activity, depends greatly on the congener although different studies have also shown significant variability. For instance, the range of EC50 is 47-5245 ng mL−1 for KmTx-1 and sulfo-KmTx-10, respectively. It should be noted that comparisons based on these EC50s must be carefully made since different species and methodology were used. In addition, these studies were performed using toxin preparations at varying stages of purification (no reference standards are available for this toxin group). In the case of KmTx-2 (as in others), the observed EC50 range for the same hemolytic assay was 368-1768 ng mL−1 (erythrocytes of Oncorhynchus mykiss). The most studied analogues are KmTx-1 and KmTx-2. (Deeds et al. 2002; Kempton et al. 2002) In vivo studies have been conducted almost exclusively with these two congeners, with KmTx-2-1 being the only exception. (Adolf et al. 2007; Mooney et al. 2010) Rapid morphological changes in zebrafish larval epithelia have been evidenced when exposed to high doses of KmTx-2 (4 µg mL−1) presenting mortality within the first 15 minutes of exposure, and they also manifest in intense cell swelling and epithelial detachment in exposed surfaces. (Deeds et al. 2006) However, epithelial damage can be observed at a much lower concentration (Deeds et al. 2006) (EC50 = 800 ng mL−1). The specific action mechanisms of KmTxs are not completely known but it has been suggested that KmTxs could act by forming pores in the cell membranes by binding to membrane lipids. Since different cell lines and animals are sensitive to these toxins, the pore-formation process is probably not initiated by a very specific lipid-binding phenomenon. Notwithstanding, it has been shown that the lytic effect (and self-protection) is dependent on the content or type of sterols in the target membranes. (Mooney et al. 2009; Rasmussen et al. 2017) Previous studies showed that KmTx-2 sub-lethal doses produce an in vitro increase in the permeability of the plasma membrane to certain cations (Na2+, Ca2+, and Mn2+). (Deeds et al. 2015) This pre-lytic action could initiate an apoptotic-like pathway leading to severe damage in the gill´s epithelia. (Rasmussen et al. 2017) The secondary necrosis of apoptotic cells could be regulated by the increase of cations such as calcium and the formation of pores in the plasma membrane. Since some ichthytoxins have been shown to trigger apoptosis (Qi et al. 2016), the molecular mechanisms underlying the alteration of the osmotic balance, oxidative stress or the loss of membrane’s functionality, among others, are relevant for the understanding of the risk of sub-lethal exposure. (Zhang et al. 2018)
Table 1
Bioactivities of the isolated karlotoxins.
KmTx congener | Strain | Geographic origin | Hemolytic activity EC50 | in vivo effects | in vitro EC50 cancer cell | Reference |
KmTx-1 | CCMP 1974 CCMP 1975 | Chesapeake Bay, USA | 284 ng mL−1 (EF1) | D. rerio, Cyprinodon variegatus (post-hatched larvae); Mortality and epithelial damage. | 2000 ng mL−1 | 2 Deeds et al., 2002 |
KmTx-1 | CCMP 1974 | Chesapeake Bay, USA | na | Danio rerio (post-hatched larvae) EC50= 800 ng mL−1, epithelial damage. | na | 7 Deeds et al., 2006 |
KmTx-1 | CCMP 2936 | Delaware, USA. | 47 ng mL−1 (EH) | na | na | 15 Van Wagoner et al., 2008 |
KmTx-1 | | Chesapeake Bay, USA | 82 ng mL−1 (EF1) | na | na | 4 Place et al., 2012 |
KmTx-2 | CCMP 2282 | South Carolina, USA | 368 ng mL−1 (EF1) | Danio rerio (post-hatched larvae EC50= 800 ng mL−1 - juvenile EC50 ≈ 400 ng mL−1, gill damage) | na | 7 Deeds et al., 2006 |
KmTx-2 | KVHU01 | Huon River, Australia | 343 ng mL−1 (EF1) | na | na | 20 Mooney et al., 2009 |
KmTx-2 | KVHU01 | Huon River, Australia | na | Cyprinodon variegatus (larvae) EC50= 508.2 ng mL−1 | na | 19 Mooney et al., 2010 |
KmTx-2 | CCMP 2064 | Georgia, USA | na | Danio rerio, Cyprinodon variegatus (juvenile), mortality, and gill damage. | na | 27 Peng et al., 2010 |
KmTx-2 | | Chesapeake Bay, USA | 1768 ng mL−1 (EF1) | na | | 4 Place et al., 2012 |
KmTx-2 | 010410-C6 | South Carolina, USA | EF1 (hemolytic activity) | na | na | 18 Deeds et al., 2015 |
KmTx-2 | CCMP2778 | Longboat Key, Florida USA | 1988 ng mL−1 (EF2) | na | na | 10 Krock et al., 2017 |
KmTx-2-1 | KVSR01 | Swan River, Australia | 66 ng mL−1 (EF1) | na | na | 20 Mooney et al., 2009 |
KmTx-2-2 | KVSR01 | Swan River, Australia | 63 ng mL−1 (EF1) | na | na | 19 Mooney et al., 2010 |
KmTx-2-1, KmTx-2-2 | AUS#7 | Swan River, Australia. | EF1 (hemolytic activity) | Cyprinodon variegatus (juvenile) mortality. | na | 1 Adolf et al., 2015 |
KmTx-2-1 | KVSR01 | Swan River, Australia | na | Cyprinodon variegatus (larvae) EC50= 563.2 ng mL−1 | na | 19 Mooney et al., 2010 |
KmTx-3 | CCMP 2936 | Delaware, USA. | 158 ng mL−1 (200 nM) (EH) | na | na | 11 Van Wagoner et al., 2010 |
KmTx-3 | | Chesapeake Bay, USA | 188 ng mL−1 (EF1) | na | na | 4 Place et al., 2012 |
KmTx-8 | AUS#7 | Swan River, Australia, | 49 nM (EF1) | na | 1064 nM. | 14 Waters et al., 2015 |
KmTx-9 | KDCSO15* | Southern Ocean, Australia | 3000 nM (EF1) | na | na | 14 Waters et al., 2015 |
65-E-chloro-KmTx-1 | CCMP 2936 | Delaware, USA. | 56 nM (EH) | na | na | 11 Van Wagoner et al., 2010 |
10-0-sulfo-KmTx-1 | CCMP 2936 | Delaware, USA. | 30 nM (EH) | na | na | 11 Van Wagoner et al., 2010 |
64-E-chloro-KmTx-3 | CCMP 2936 | Delaware, USA. | 110 nM (EH) | na | na | 11 Van Wagoner et al., 2010 |
10-0-sulfo-KmTx-3 | CCMP 2936 | Delaware, USA. | 2400 nM (EH) | na | na | 11 Van Wagoner et al., 2010 |
4,5-dihydro-KmTx-2 | GM2 | East China Sea | 997 ng mL−1 (EF2) | na | 15mM | 13 Cai et al., 2016 |
4,5-dihydro-dechloro-KmTx-2 | GM2 | East China Sea | 943 ng mL−1 (EF2) | na | 36mM | 13 Cai et al., 2016 |
sulfo KmTx-10 | K10, E11 | Ebro Delta Bays, Spain | 5245 ng mL−1 (EF2) | na | na | 10 Krock et al., 2017 |
EF1: Erythrocytes of Fish (Oncorhynchus mykiss) / EF2: Erythrocytes of Fish (Sparus aurata) / EH: Erythrocytes of Human / na: data not available / *K. conicum |
Strains of Karlodinium veneficum showed high chemical variability regarding KmTxs composition, although in some cases it could be influenced by growth conditions as well. (Krock et al. 2017) In the cited work, genetic comparison was carried out for species and strain identification based on ITS and LSU rDNA sequences. (Krock et al. 2017) Most of the characterized strains showed higher toxicity in their culture supernatants than in the cell extracts. This would suggest that KmTxs are released to the environment in natural conditions. However, certain amounts of toxins can also be released due to mechanical stress during filtering or centrifugation. Thus, it appears that KmTxs may act as allelochemicals related to chemical defense against grazing and/or in prey acquisition. It has been suggested that the length of the lipophilic arm is an important determinant of potency, and that sulfation and chlorination could also influence the damage on biological membranes. (Place et al. 2012; Adolf et al. 2015) There is little information however on the effects of KmTxs at sublethal concentrations on species that cohabit in the water column, their potential bioaccumulation in these, and on human health (due to consumption of contaminated fish).
Recent investigations have strengthened the utility of early-stages Danio rerio in marine microalgae toxicity's evaluation; either to evaluate changes in toxicity induced by different nutrients conditions (Alexandrium tamarense (Guan et al. 2018)) or in response to a future climate change conditions such as ocean acidification and solar ultraviolet radiation (Karenia mikimotoi (Wang et al. 2019)). In addition, as mentioned above, this animal model had already been used to evaluate Karlodinium extracts toxicity (employing early-larvae and juveniles). (Deeds et al. 2002, 2006; Peng et al. 2010) In this study, our interest was to evaluate and compare the toxic effect of sublethal concentrations of K. veneficum K10 supernatants' extracts containing five novel putative karlotoxins (cand. KmTx-10, cand. KmTx-12, cand. KmTx-13, cand. KmTx-11 and cand. sulfo-KmTx-10; “cand.” stands for candidate) and contribute to the knowledge of the potential effects of this type of ichthyotoxins on the development of fish species in the natural environment.