Interactions between Karlodinium veneficum and Prorocentrum donghaiense from the East China Sea

Abstract

The dinoflagellate Prorocentrum donghaiense is a dominant harmful algal bloom (HAB) species on the East China Sea (ECS) coast. The co-occurrence of Karlodinium veneficum with P. donghaiense is often observed and can later develop into dense blooms. However, the role of K. veneficum in P. donghaiense population dynamics is unknown. In the current study, three K. veneficum (GM1, GM2, and GM3) strains were isolated from the ECS with one (GM1) from a mixed, dense bloom of P. donghaiense and other HAB species. All three isolates had identical ITS sequences that were concordant with the species designation. Unique karlotoxin congeners were isolated from one strain (GM2). The sterol compositions of P. donghaiense and K. veneficum were consistent with sensitivity to karlotoxin in the former and insensitivity in the latter. Additional experimentation showed that: (1)in monocultures, higher growth rate of P. donghaiense than K. veneficum is observed in nutrient-enriched and nutrient-depleted media. In co-cultures, the growth of P. donghaiense is inhibited; (2) feeding on P. donghaiense by K. veneficum is clearly demonstrated by fluorescent dye tracking; and (3) the isolated karlotoxin is lethal to P. donghaiense in a concentration-dependent manner. From these studies we propose that K. veneficum may play a negative role in P. donghaiense bloom maintenance and that P. donghaiense may in turn be a bloom initiator as a prey item for K. veneficum.

Keywords: Co-culture; Karlodinium veneficum; Karlotoxin; Phagotrophy; Prorocentrum donghaiense; Sterol.

Fig. 1.

The population growth of Karlodinium veneficum (K) and Prorocentrum donghaiense (P) in different media cultured alone and co-cultured at different initial cell densities (K/P). The data are the means of triplicates. Error bars (SD) have been omitted for the sake of clarity. (A) Enriched clean natural seawater (f/2-Si), inoculating cell number proportion K/P = 1:10.(B) Enriched clean natural seawater (f/2-Si), inoculating cell number proportion K/P = 1:1.(C) Oligotrophic media of diatom filtrates, inoculating cell number proportion K/P =1:10. (D) Oligotrophic media ofdiatom filtrates, inoculating cell number proportion K/P =1:1. Solid triangles (▲): K. veneficum growth in co-culture. Solid circles (⚫): P. donghaiense growth in co-culture. Hollow triangles (△): monoculture of K. veneficum as control. Hollow circles (⚪): monoculture of P. donghaiense as control.

Fig. 2.

Feeding on P. donghaiense cells by K. veneficum. (A)K. veneficum (solid arrows➨) actively attached P. donghaiense (hollow arrow ➬). Arrow head indicates an empty P. donghaiense theca. (B) CFSE-labeled P. donghaiense cell. (C) K. veneficum and CFSE-labeled P. donghaiense. (D) Same view of (C) observed under fluorescence microscope, showing CFSE-labeled P. donghaiense (hollow arrows) and K. veneficum (solid arrows) without CFSE fluorescence. (E) K veneficum cell with CFSE-labeled P. donghaiense components (arrows). (F) K. veneficum cell with CFSE-labeled P. donghaiense components. E or F was assembled images taken by a differential interference contrast (DIC) microscope and a fluorescence microscope.

Fig. 3.

Reverse phase chromatograms (LiChrosphere 125 mm × 4 mm × 5 μm bead size RP-8) of the 60% (A) and 80% (B) methanol fractions from strain NMB jah047–1 culture filtrates fractionated with a Sep-Pak tC-18 cartridge prior to chromatography. Injected toxin was equivalent to 3.5 × 10⁷ cells. The UV spectra of the peaks (1,2, and 3) for each fraction were identical and are shown as an inset in Fig. 2B. The 235-nm UV maximum is characteristic of a terminal chlorine (Bachvaroff et al., 2008).

Fig. 4.

Mass spectra of the UV peaks (1,2, and 3) shown in Fig. 3. Putative identifications of the different ions (sodiated ions assumed) are shown. The karlotoxins in peak 1 appeared to be the sulfated forms of those in peaks 2 and 3.

Fig. 5.

Variations in Prorocentrum donghaiense cells after exposure to different amounts ofkarlotoxin in 24 h. Error bars are standard deviations. Control M: P. donghaiense culture exposed to 20% methanol. Control: Original P. donghaiense culture.