Morphological, Toxicological, and Biochemical Characterization of Two Species of Gambierdiscus from Bahía de La Paz, Gulf of California

Abstract

We describe five new isolates of two Gambierdiscus species from Bahía de La Paz in the southern Gulf of California. Batch cultures of Gambierdiscus were established for morphological characterization using light microscopy (LM) and scanning electron microscopy (SEM). Pigment and amino acid profiles were also analyzed using high-performance liquid chromatography (HPLC-UV and HPLC-DAD). Finally, toxicity (CTX-like and MTX-like activity) was evaluated using the Artemia salina assay (ARTOX), mouse assay (MBA), marine fish assay (MFA), and fluorescent receptor binding assay (fRBA). These strains were identified as Gambierdiscus cf. caribaeus and Gambierdiscus cf. carpenteri. Toxicity for CTX-like and MTX-like activity was confirmed in all evaluated clones. Seven pigments were detected, with chlorophyll a, pyridine, Chl2, and diadinoxanthin being particularly noteworthy. For the first time, a screening of the amino acid profile of Gambierdiscus from the Pacific Ocean was conducted, which showed 14 amino acids for all strains except histidine, which was only present in G. cf. caribeaus. We report the presence of Gambierdiscus and Fukuyoa species in the Mexican Pacific, where ciguatera fish poisoning (CFP) cases have occurred.

Keywords: Gambierdiscus; Gulf of California; amino acids; ciguatera; ciguatoxins; pigments; toxicity.

Figure 12

Sampling sites in Bahía de La Paz used to collect Gambierdiscus species.

Figure 1

Light microscopy of Gambierdiscus cf. caribaeus (GbSa-7). (A) Live cell, (B) Epifluorescence of chloroplast, (C) Plate tabulation apical view stained with Fluorescent Brightener 28, (D) Antapical view, (E) Apical view, (F) Antapical view, and (G) Ventral view. Scale bars: (A–F) 50 µm and (G) 30 µm.

Figure 2

Scanning electron micrographs of Gambierdiscus cf. caribaeus (GbSa-7). (A) Apical view, (B) Antapical view, (C) Po plate, (D) Sulcal area in antapical view, (E) Sulcal area in ventral view, and (F) Dorsal view. Scale bars: (A,B) 50 µm, (C) 10 µm, (D) 20 µm, and (E,F) 30 µm.

Figure 3

Light microscopy of Gambierdiscus cf. carpenteri (GbSa-4). (A) Live cell, (B) Epifluorescence of chloroplast, (C) Plate tabulation apical view stained with Fluorescent Brightener 28, (D) Antapical view, (E) Apical view, (F) Antapical view, and (G) Ventral view. Scale bars: (A–F) 50 µm and (G) 30 µm.

Figure 4

SEM micrographs of Gambierdiscus cf. carpenteri (GbSa-4). (A) Apical view arrow showing an apparent rostrum, (B) Antapical view, (C) Po plate, (D) Sulcal area in antapical view, (E) Sulcal area in ventral view, and (F) Dorsal view arrow points to apparent rostrum. Scale bars: (A,B) 50 µm, (C) 10 µm, (D) 20 µm, and (E,F) 30 µm.

Figure 5

To investigate the composition and concentration of pigments, samples were collected between days 24 and 25 of culture during the early exponential growth phase. As a result of this, nine pigments were identified in Gambierdiscus strains (Figure 5 and Figure 6).

Figure 6

Principal pigments and amino acids detected in five strains of Gambierdiscus cf. caribaeus and G. cf. carpenteri in this study. (A) Pigments: (1) chlorophyll c1-c2, (2) Dinoxanthin, (3) diatoxanthin, (4) peridinin, (5) Chlorophyll a, (6) diadinoxanthin, and (7) β, β-carotene. (B) Amino acids: (1) aspartic acid, (2) glycine, (3) tyrosine, (4) leucine, (5) glutamic acid, (6) threonine, (7) valine, (8) lysine, (9) serine, (10) alanine, (11) phenylalanine, (12) proline, (13) histidine, (14) arginine, and (15) isoleucine.

Figure 7

High-performance liquid chromatography (HPLC) chromatograms registered at 338 nm. (A) STD amino acids. Peak identification: (1) aspartic acid, (2) glutamic acid, (3) serine, (4) histidine, (5) glycine, (6) threonine, (7) alanine, (8) arginine, (9) tyrosine, (10) valine, (11) phenylalanine, (12) isoleucine, (13) leucine, and (14) lysine. G. cf. caribaeus strain (B) Gbsa-2, (C) Gbsa-7, G. cf. carpenteri strain (D) Gbsa-4, (E) Gbsa-5 and (F) Gbsa-6. The chromatograms for proline are shown in Figure S10.

Figure 8

Left ring charts showed clinical signs recorded during the mouse bioassay (MBA) using CTX-like activity for Gambierdiscus cf. caribaeus strains (A) GbSa-2 and (B) GbSa-7 and Gambierdiscus cf. carpenteri strains (C) GbSa-4, (D) GbSa-5, and (E) GbSa-6. Right ring chart clinical signs recorded during the MBA using MTX-like activity of Gambierdiscus cf. caribaeus strains (A) GbSa-2 and (B) GbSa-7) and Gambierdiscus cf. carpenteri strains (C) GbSa-4, (D) GbSa-5, and (E) GbSa-6.

Figure 9

Artemia salina lethality bioassay. (A) Brine shrimp exposed to CTX-like activity of Gambierdiscus cf. caribaeus (GbSa-2 and GbSa-7) and Gambierdiscus cf. carpenteri (GbSa-4, GbSa-5, and GbSa-6). (B) Brine shrimp exposed to MTX-like activity of Gambierdiscus cf. caribaeus (GbSa-2 and GbSa-7) and Gambierdiscus cf. carpenteri (GbSa-4, GbSa-5, and GbSa-6).

Figure 10

(A) Toxicity in Seriola rivoliana larvae exposed to the CTX-like activity of Gambierdiscus cf. caribaeus (GbSa-2 and GbSa-7) and Gambierdiscus cf. carpenteri (GbSa-4, GbSa-5, and GbSa-6). (B) Toxicity in larvae of Seriola rivoliana exposed to MTX-like activity of Gambierdiscus cf. caribaeus (GbSa-2 and GbSa-7) and Gambierdiscus cf. carpenteri (GbSa-4, GbSa-5, and GbSa-6).

Figure 11

Light microscopy of typical morphology plate 1p, 1′, 2′, 3′, 4″, Po plate under present in Gambierdiscus cf. caribaeus (GbSa-2, GbSa-7) and Gambierdiscus cf. carpenteri (GbSa-4, GbSa-5, GbSa-6). Scale bar: 20 µm.