Exploring the applicability of SYBR stains for whole-mount high-resolution confocal microscopy of chemosymbiotic invertebrates.

Localization of bacterial symbionts within host tissues and determination of their morphology are essential for unraveling the biology of chemosymbiotic marine invertebrates. Confocal microscopy offers the potential to visualize bacterial symbionts in whole-mount preparations of host animals or tissues with minimal sample preparation. Here, we investigated the applicability of SYBR stains for visualizing the distribution of symbionts in the gills of thyasirid bivalves and in the trophosome of the siboglinid Siboglinum fiordicum. We demonstrate that SYBR stains, particularly SYBR Gold and SYBR Safe, are suitable for visualizing extracellular symbionts in whole mounts of gills, as well as intracellular endosymbionts in cells of the trophosome in whole mounts of S. fiordicum. SYBR stains show potential for confocal microscopy of invertebrates with chemosynthetic symbionts, but their utility may depend on specific experimental design; for instance, in S. fiordicum, counterstaining with DAPI improved the visibility of symbionts. Contrary to previous descriptions, we show that in male S. fiordicum the trophosome is not confined to the posterior trunk but extends anteriorly to envelop the testes. We also document the gill structure of several thyasirid species, including the first published description of gill structure of Parathyasira dunbari. In general, SYBR Gold/Safe staining can be used as a quick screen for potential symbiont-bearing species in chemosynthetic environments or for monitoring symbiont populations in laboratory-maintained symbiont-dependent hosts.