The probe used in this study, MTC849 probe (5` –
CGTTAGCTCCACCACTAAG – 3`), was designed with ARB [35 (link)] to target the 16S rRNA gene sequences
of the symbiotic MOX of both Campeche sponge species. This probe is a
modification of MTC850 probe, designed to target Marine Methylotrophic Group
(MMG) 2 MOX [36 (link)]. Apart from the
symbiotic and MMG2 MOX, the MTC849 probe targets the closely-related
Methylomonas and Methylomarinum clades.
The MTC849 oligonucleotide was double-labeled with Atto594 dye (Biomers, Ulm,
Germany), and applied to 8 μm sections of sponge tissue using
hybridization buffer with 20% formamide as described previously [37 (link)]. These hybridization conditions are
assumed to ensure specificity, given the three mismatches that the MTC849 probe
had to the 16S rRNA gene sequences of all other Campeche sponge bacteria [38 (link)].The general bacterial probe EUB338
[39 (link)] was used as a positive control
and the NON338 probe was used as a control for background autofluorescence
[40 (link)]. Photomicrographs were acquired
with a Zeiss Axioplan 2 epifluorescence microscope (Zeiss, Jena, Germany) or
with a confocal laser-scanning microscope (LSM 780, Carl Zeiss, Germany).
Brightness and contrast of the images were adjusted with Adobe Photoshop (Adobe
Systems, Inc., USA).
CGTTAGCTCCACCACTAAG – 3`), was designed with ARB [35 (link)] to target the 16S rRNA gene sequences
of the symbiotic MOX of both Campeche sponge species. This probe is a
modification of MTC850 probe, designed to target Marine Methylotrophic Group
(MMG) 2 MOX [36 (link)]. Apart from the
symbiotic and MMG2 MOX, the MTC849 probe targets the closely-related
Methylomonas and Methylomarinum clades.
The MTC849 oligonucleotide was double-labeled with Atto594 dye (Biomers, Ulm,
Germany), and applied to 8 μm sections of sponge tissue using
hybridization buffer with 20% formamide as described previously [37 (link)]. These hybridization conditions are
assumed to ensure specificity, given the three mismatches that the MTC849 probe
had to the 16S rRNA gene sequences of all other Campeche sponge bacteria [38 (link)].The general bacterial probe EUB338
[39 (link)] was used as a positive control
and the NON338 probe was used as a control for background autofluorescence
[40 (link)]. Photomicrographs were acquired
with a Zeiss Axioplan 2 epifluorescence microscope (Zeiss, Jena, Germany) or
with a confocal laser-scanning microscope (LSM 780, Carl Zeiss, Germany).
Brightness and contrast of the images were adjusted with Adobe Photoshop (Adobe
Systems, Inc., USA).