Technical Note: Determination of the metabolically active fraction of benthic foraminifera by means of Fluorescent In Situ Hybridization (FISH)

Abstract

Abstract. Benthic foraminifera are an important component of the marine biota, but protocols for investigating their viability and metabolism are still extremely limited. Classical studies on benthic foraminifera have been based on direct counting under light microscopy. Typically, these organisms are stained with Rose Bengal, which binds proteins and other macromolecules, but does not allow discrimination between viable and recently dead organisms. The fluorescent in situ hybridization technique (FISH) represents a new and useful approach to identify living cells possessing an active metabolism. Our work is the first test of the suitability of the FISH technique, based on fluorescent probes targeting the 18S rRNA, to detect live benthic foraminifera. The protocol was applied on Ammonia group and Miliolids, as well as on agglutinated polythalamous (i.e., Leptohalysis scottii and Eggerella scabra) and soft-shelled monothalamous (i.e., Psammophaga sp. and saccamminid morphotypes) taxa. The results from FISH analyses were compared with those obtained, on the same specimens assayed with FISH, from microscopic analysis of the cytoplasm colour, presence of pigments and pseudopodial activity. Our results indicate that FISH targets only metabolically active foraminifera, and allows discerning from low to high cellular activity, validating the hypothesis that the intensity of the fluorescent signal emitted by the probe is dependent upon the physiological status of cells. These findings support the usefulness of this molecular approach as a key tool for obtaining information on the physiology of living foraminifera, both in field and experimental settings.

Foraminiferal Ultrastructure: A perspective From Fluorescent and Fluorogenic Probes

“…There have been many attempts to develop a reliable method for distinguishing living foraminifera from dead ones. Methods can be classified as “terminal” and “nonterminal.” Included in the first group are the Rose Bengal ( RB ), Sudan Black B , ATP assay, and ultrastructural observations, while “nonterminal” methods include direct observations of pseudopodial activity, negative geotaxis (reviewed in Bernhard, ), and fluorescent probes such as CTG (Bernhard et al, ; Bernhard & Bowser, ), FISH (Borrelli et al, ), or Calcein AM (Ohno et al, ). So far, the RB and the CTG are the most widely used dye/probe.…”

“…Bernhard and Bowser (1996) developed a method based on the CellTracker TM Green CMFDA, as a vital fluorogenic probe that demonstrates the enzymatic activity, and consequently the vitality of the cell (Hintz et al, 2004;Pucci et al, 2009). Another fluorescent probe-based method for identifying living foraminifera was developed by Borrelli et al (2011), where fluorescent in situ hybridization (FISH) oligonucleotides (EUK 1209R and S17) tagged with the fluorescent probe Cy TM -3 are specific to foraminiferal ribosomal RNA. This approach can also be used as an indicator of the cellular metabolic activity.…”

Biomineralization ofSchlumbergerella floresiana, a significant carbonate‐producing benthic foraminifer

“…Therefore, we cannot exclude the alternative that some residual cytoplasm (endoplasm) or digested remains of food organisms were still present (Langer & Bell, 1995). We used this washing protocol because it has been tested on other living benthic foraminifera (Borrelli et al, 2011) and NaOCl is often used in the preparation of mollusk shells (Marie et al, , 2011Ramos-Silva et al, 2013). To avoid contamination by cellular debris, the organic film present on the outer surface (i.e., bacterial biofilm) and symbionts, the first extraction step was performed using denaturing guanidine thiocyanate HCl.…”

CellTracker Green labelling vs. rose bengal staining: CTG wins by points in distinguishing living from dead anoxia-impacted copepods and nematodes

“…The CTG technique, however, can have certain peculiarities. The first involves the duration of post-mortem enzymatic activity (Borrelli et al, 2011). The residual esterase activity in microbes produced around 25 % of fluorescence after heat killing, still 48 h after treatment (Kaneshiro et al, 1993).…”