Optimization of culturing conditions for toxicity testing with the algaOophilasp. (Chlorophyceae), an amphibian endosymbiont

Abstract

Eggs of the yellow-spotted salamander (Ambystoma maculatum) have a symbiotic relationship with green algae. It has been suggested that contaminants that are preferentially toxic to algae, such as herbicides, may impair the symbiont and, hence, indirectly affect the development of the salamander embryo. To enable testing under near-standard conditions for first-tier toxicity screening, the authors isolated the alga from field-collected eggs and identified conditions providing exponential growth rates in the apparent asexual phase of the alga. This approach provided a uniform, single-species culture, facilitating assessment of common toxicity end points and comparison of sensitivity relative to other species. Sequencing of the 18s ribosomal DNA indicated that the isolated alga is closely related to the recently described Oophila amblystomatis but is more similar to other known Chlamydomonas species, suggesting possible biogeographical variability in the genetic identity of the algal symbiont. After a tiered approach to culturing method refinement, a modified Bristol's media with 1 mM NH4 (+) as nitrogen source was found to provide suitable conditions for toxicity testing at 18 °C and 200 µmol m(-2) s(-1) photosynthetically active radiation (PAR) on a 24-h light cycle. The validity of the approach was demonstrated with Zn(2+) as a reference toxicant. Overall, the present study shows that screening for direct effects of contaminants on the algal symbiont without the presence of the host salamander is possible under certain laboratory conditions.

Aquatic hazard assessment of MON 0818, a commercial mixture of alkylamine ethoxylates commonly used in glyphosate‐containing herbicide formulations. Part 2: Roles of sediment, temperature, and capacity for recovery following a pulsed exposure

“…The amphibian algal symbiont Oophila sp. (GenBank accession number KJ394433) was field‐collected from eggs of Ambystoma maculatum (the yellow‐spotted salamander) as described in Rodriguez‐Gil et al . For the tests with R. subcapitata and C. vulgaris , liquid cultures were inoculated from the original agar slants into ASTM International (ASTM) liquid growth media and renewed at least 3 times before test initiation.…”

“…For Oophila sp. a stock liquid culture in modified Bristol's growth media was already available. Reference toxicant tests with ZnSO 4 were conducted on all 3 species prior to testing with MON 0818, and the results were within the accepted range.…”

Co-cultures of Oophila amblystomatis between Ambystoma maculatum and Ambystoma gracile hosts show host-symbiont fidelity

“…Pioneering embryologists developed protocols for Ambystoma maculatum embryo rearing (then Amblystoma punctatum) for embryonic tissue ablation and transplant experiments (Burr 1916;Twitty 1932), while established culturing protocols for Oophila sp. (Cliburn and Ward 1963) have recently been refined for taxonomy and cultured toxicology studies (Kim et al 2014;Rodriguez-Gil et al 2014).…”

“…All algae were maintained in AF6 media (Wantanabe et al 2000) in a Conviron Environmental chamber set at 18°C on a 12 h light/ 12 h dark timer with 59 μmol/m 2 /s (PAR) broadspectrum fluorescent light. We also experimented with the NH 4 + supplemented Bristol's media described in (Rodriguez-Gil et al 2014) however in our experience Oophila grew better in AF6. Importantly, these algal lines were established without plating the cultures on agar solidified media.…”

Effects of atrazine on egg masses of the yellow-spotted salamander (Ambystoma maculatum) and its endosymbiotic alga (Oophila amblystomatis)

“…Several closelyrelated strains of this chlamydomonad have been identified in A. maculatum egg capsules (Kerney et al, 2011;Graham et al, 2013;Kim et al, 2014;Rodriguez-Gil et al, 2014), and as well as from eggs of A. gracile (Northwestern salamander), Lithobates sylvatica (wood frog) and L. aurora (red-legged frog) (Kim et al, 2014). To date, most observations on the occurrence and life stages of this alga, and most experimental research into the embryo-alga relationship have focussed on A. maculatum.…”