Green Eggs and Solar Powered Salamanders

The relationship between the Spotted Salamander and the green algae Oophila amblystomatis is very interesting. Most literature supports the idea of a symbiotic relationship between the two organisms, despite there not being much research into the benefits for the algae. However, a recent study found genes in the algae responsible for fermentation and suggests that the algae is stressed and the relationship is parasitic. (Burns et al, 2017)

The project aimed to better understand the relationship between algae and salamander and focus on the algae and its role in the relationship.

Individual eggs were kept in cuvettes in four groups: high light, medium light, low light, eggs completely in the dark. Using a fluorometer, chlorophyll a fluorescence was measured in light treated eggs to determine photosynthetic efficiency by creating OJIP curves. This was done at least twice per week. All eggs were imaged at least twice a week and embryonic development was determined using a chart and measuring gill length, tail development, etc.

Some egg masses were kept intact and left in water in plastic boxes, in two groups: dark and light. Ammonia levels of the water were measured once per week.

Embryos were imaged two to three times per week to determine relative size and developmental speed. Images were taken with SharpCap on a laptop, connected to a microscope.

OJIP curves were created using a fluorometer. The fluorometer measures photosynthetic efficiency by measuring bottlenecks in the electron transport chain. Shapes of OJIP curves are specific to different algae species. Our graphs had two main peaks, and based on their heights in different treatments we were able determine how light treatments affect different protein complexes in the light reactions of photosynthesis.

Figure 1: Because embryos with more algae were greater in size, we hypothesize that increased oxygen production from algae increases embryo mass. In the next round of experiments, dry salamander biomass will be measured after hatching. Similarly, embryos with more algae developed faster. We also attribute this to increased oxygen supply.

Figure 2: Development of embryos was fastest at high light conditions and where algae was most abundant.

Figure 3: In high light, oxygen production by the algae was three times higher than basal embryo oxygen consumption. However, in low light, oxygen production was lower than embryo requirements. It seems likely that algae in low light would not produce enough oxygen for it's normal metabolic pathways, and that it would do fermentation when not under sufficient light conditions.

Figure 4: OJIP curves showed a decrease in photosynthetic efficiency in highlight conditions. Because of the absence of a K band on the curve, this is likely due to dynamic photoinhibition due to downregulation of Photosystem II activity.

Figure 5: Ammonia levels in dark treated egg masses were 16 times higher than light treated egg masses. It is hypothesized that algae recycles ammonia, a toxic byproduct produced by the salamanders, into less toxic compounds that it can use as fertilizer.

This is a three year project, so the experiment will be repeated this spring and next spring. This year, light variable experiments will be repeated the same, except each light treatment will have one egg from each egg mass collected, to control for genetic effects from salamander mothers. Also, further ammonia experiments will be done by using a type of bacteria commonly used in fish tanks that recycles ammonia to see if it has similar effects to egg masses with algae.

I'm also developing synthetic eggs made of alginate and agarose that can be used for more controlled, year-round experiments.

Credit statement

Conceptualization: Alonso Zavafer, Glenn Tattersall; Methodology: Alonso Zavafer; Software: Alonso Zavafer; Validation: Laura Palumbo, Alonso Zavafer; Formal analysis: Alonso Zavafer; Investigation: Laura Palumbo, Alonso Zavafer, Glenn Tattersall; Resources: Patrick Moldowan, Alonso Zavafer, Glenn Tattersall; Data Curation: Alonso Zavafer, Laura Palumbo; Writing - Original Draft: Laura Palumbo, Alonso Zavafer; Writing - Review & Editing: Laura Palumbo, Patrick Moldowan, Glenn Tattersall; Visualization: Laura Palumbo, Alonso Zavafer; Supervision: Alonso Zavafer; Project administration: Alonso Zavafer; Funding acquisition: Alonso Zavafer, Glenn Tattersall.

Acknowledgement

Laura Palumbo and Alonso Zavafer thank Dr. Jae Jung for the support in the preparations for the field work in Algonquin Park.

Alonso Zavafer and Glenn Tattersall would like to thank the staff of the Algonquin Park Research Station for their continuous and selfless support.

Funding

Alonso Zavafer and Glenn Tattersall were financially supported by an NSERC Discovery Program.

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