DOI: 10.5281/zenodo.14708781
title: Symbiodiniaceae sequences accompanying: The influence of symbiont identity on the proteomic and metabolomic responses of the model cnidarian Aiptasia to thermal stress

description: These data are the ITS2 Symbiodiniaceae sequencing results accompanying the manuscript "The influence of symbiont identity on the proteomic and metabolomics responses of the model cnidarian Aiptasia to thermal stress", by B Lust et al. The manuscript abstract and sequencing details (from Appendix I) are below.

Abstract

We examined the effects of symbiont identity and heat stress on the host metabolome and proteome in the cnidarian-dinoflagellate symbiosis. Exaiptasia diaphana (‘Aiptasia’) was inoculated with its native symbiont (Breviolum minutum) or a heterologous (i.e., non-native) symbiont (Symbiodinium microadriaticum; Durusdinium trenchii) and thermally stressed. Integrated metabolome and proteome analyses characterised helpful and unhelpful host thermal responses between symbioses, with clear evidence of enhanced nutritional deprivation and cellular stress in hosts harbouring heterologous symbionts following temperature stress. Host metabolomes were partially distinct at the control temperature, however thermal stress caused metabolomes of anemones containing the two heterologous symbionts to become more alike and more distinct from those containing B. minutum. While these patterns could be partly explained by innate symbiont-specific differences, they may also reflect differences in symbiont density, as under control conditions D. trenchii attained 60% and S. microadriaticum 15% of the density attained by B. minutum, and at elevated temperature only D. trenchii-colonised anemones bleached (60% loss). Our findings add to a growing literature that highlights the physiological limits of partner switching as a means of adaptation to global warming. However, we also provide tentative evidence for improved metabolic functioning with a heterologous symbiont (D. trenchii) after sustained symbiosis.

Sequencing methods

Before inoculation, all algal cultures were genotyped. DNA was extracted using a bead-beat protocol according to Gabay et al. (3).  At each sampling time-point, anemones were checked for symbiont genotype. DNA was extracted according to the CTAB-chloroform protocol of Baker & Cunning (4). Briefly, 100 µL of symbiont cells in DNAB from each replicate sample were digested overnight at 37 °C using 10 µL 10 mg/mL proteinase K. Thereafter, 200 µL CTAB were added to the digested samples, which were then incubated at 65 °C for 30-60 min. After cooling the samples to room temperature, 300 µL chloroform were added, and the samples incubated at room temperature on a rotating platform for 2-3 h followed by centrifugation at 10,000 x g for 10 min, and the transfer of 250 µL of the supernatant to a new tube. 100% ethanol at twice the sample volume was added to the extracted DNA sample, which was then placed at -20 °C for a minimum of 2 h to promote DNA precipitation. Samples were then centrifuged at 10,000 x g, the ethanol decanted and the pelleted DNA dried under vacuum (Eppendorf Concentrator 5301) before redissolving it in 0.3 M sodium acetate (NaOAc). Samples were then washed with 100% ethanol and again with 70% ethanol, dried again under vacuum, and resuspended in 20 µL TE buffer.

 

PCR was performed with 2 µL extracted DNA for each replicate sample, using the primers ITSD (forward, 5’-GTGAATTGCAGAACTCCGTG-3’) and ITS2rev2 (reverse; 5’-CCTCCGCTTACTTATATGCTT-3’). The PCR amplification protocol consisted of an initial stage of 3 min at 95 °C, and then 35 rounds of 15 s at 95 °C, 15 s at 56 °C, and 10 s at 72 °C, with a final elongation phase of 5 min at 72 °C, followed by a resting period at 4 °C. PCR products were stored at 4 °C. The extracted PCR products were sent to Macrogen (Seoul, Korea) for purification and sequencing. Sequence results were checked for quality and then used for BLAST searches against the GeoSymbio and NCBI databases to confirm the symbiont species.

 

File Identifier Key

Following "GM", the following are used to identify each sample:

0/1: Timepoint

C/H: Control/Heated

A/B/D: Symbiodiniaceae genus (Symbiodinium/Breviolum/Durusdinium)

#: Biological replicate