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

Abstract

A unique symbiosis occurs between embryos of the spotted salamander (Ambystoma maculatum) and a green alga (Oophila amblystomatis). Unlike most vertebrate host-symbiont relationships, which are ectosymbiotic, A. maculatum exhibits both an ecto-and an endo-symbiosis, where some of the green algal cells living inside egg capsules enter embryonic tissues as well as individual salamander cells. Past research has consistently categorized this symbiosis as a mutualism, making this the first example of a Bbeneficial^microbe entering vertebrate cells. Another closely related species of salamander, Ambystoma gracile, also harbors beneficial Oophila algae in its egg capsules. However, our sampling within the A. gracile range consistently shows this to be a strict ectosymbiotic interaction-with no sign of tissue or presumably cellular entry. In this study we swapped cultured algae derived from intracapsular fluid of different salamander hosts to test the fidelity of tissue entry in these symbioses. Both A. maculatum and A. gracile embryos were raised in cultures with their own algae or algae cultured from the other host. Under these in vitro culture conditions A. maculatum algae will enter embryonic A. maculatum tissues. Additionally, although at a much lower frequency, A. gracile derived algae will also enter A. maculatum host tissues. However, neither Oophila strain enters A. gracile hosts in these co-culture conditions. These data reveal a potential host-symbiont fidelity that allows the unique endosymbiosis to occur in A. maculatum, but not in A. gracile. However, preliminary trials in our study found that persistent endogenous A. maculatum algae, as opposed to the cultured algae used in subsequent trials, enters host tissues at a higher frequency. An analysis of previously published Oophila transcriptomes revealed dramatic differences in gene expression between cultured and intracapsular Oophila. These include a suite of genes in protein and cell wall synthesis, photosynthesis, central carbon metabolism suggesting the intracapsular algae are assimilating ammonia for nitrogen metabolism and may be undergoing a life-cycle transition. Further refinements of these coculture conditions could help determine physiological differences between cultured and endogenous algae, as well as rate-limiting cues provided for the alga by the salamander.

Heterotrophic Carbon Fixation in a Salamander-Alga Symbiosis

“…The result suggests that some algae persist through the washes but living A. maculatum embryos do not actively accrue additional measurable photosynthate from lab-cultured algae in these conditions. We note that although cultured algae are in a different transcriptional state from intracapsular algae (Kerney et al, 2019), lab-cultured algae are able to interact with and invade A. maculatum embryos ex situ, similar to algae in the intracapsular environment (Kerney et al, 2019). In this study, decapsulated embryos incubated in the dark accumulated significantly more fixed carbon ( Figure 2B) compared to embryos incubated with pre-labeled algae (p < 0.001) or embryos killed with glutaraldehyde prior to incubation with bicarbonate (p < 0.001).…”

“…Particular attention has been given to the conspicuous association between the common spotted salamander of North America ( Ambystoma maculatum ) and its O. amblystomatis symbiont ( Orr, 1888 ; Gilbert, 1942 ). While recent studies have focused on the unique facultative endosymbiotic association of algal cells inside the embryo host ( Kerney et al, 2011 , 2019 ; Burns et al, 2017 ), there is a long history of research into the ecto-symbiotic association between free-living Oophila inside the embryonic egg capsule ( Kerney, 2011 ). This intracapsular Oophila has a role in oxygenating the egg capsule microenvironment ( Gilbert, 1942 , 1944 ; Bachmann et al, 1986 ; Pinder and Friet, 1994 ; Mills and Barnhart, 1999 ; Bianchini et al, 2012 ) and potentially in removal of nitrogenous waste from the host ( Goff and Stein, 1978 ; Bianchini et al, 2012 ; Small et al, 2014 ).…”

Batrachochytrium salamandrivorans elicits acute stress response in spotted salamanders but not infection or mortality

“…Oophila sp. ; Kerney et al, 2019) in disease resistance. Skin pH did not differ among these species and therefore does not appear to contribute to the susceptibility differences among these species.…”

Diversity and substrate-specificity of green algae and other micro-eukaryotes colonizing amphibian clutches in Germany, revealed by DNA metabarcoding

“…This clade had a somewhat isolated (phylogenetic) position within Chlamydomonadales, and besides, the clutch-associated algae contained only three isolates of free-living algae. Isolates of this clade have been consistently considered as O. amblystomatis, which is regarded as the numerically most abundant alga in A. maculatum (Jurga et al 2020) and A. gracile (Kerney et al 2019;Marco and Blaustein 2000) egg capsule chambers. These O. amblystomatis isolates have been used in studies of gene expression (Burns et al 2017;Kerney et al 2019), carbon fixation (Burns et al 2020), and host-symbiont fidelity (Kerney et al 2019).…”