On the track of cellular ecology. Methodological improvements and contributions of single-cell phosphatase activity on the ecology of phytoplankton in Pyrenean lakes

Abstract

Comparative studies between different enzyme labelled fluorescence (ELF) protocols and fluorescence quantification methods were performed to improve the quantification of single-cell phosphatase activity (PA) measurements in plankton. Unfixed and liquid ELF incubations were devised, and intracellular labelling was found to be unavoidable independently of the protocol. This observation arose the question whether our measurements of phosphatase and/or other extracellular enzyme activities based on ELF and other bulk substrates has been biased by their interaction with intracellular located enzymes. The comparison of fluorescence intensity quantifications in 2D and 3D widefield and restoration microscopy showed basically a linear relationship between methods across a wide range of fluorescence intensities. Measurements of fluorescence intensity based on 2D images were significantly biased by out-of-focus light, and to a lesser extent by object size, whereas deconvolved 3D microscopy resolved this problems. We, therefore, devised restorative deconvolution 3D microscopy for most applications and we discussed how 2D image-based quantifications should be interpreted.

A direct trade-off relationship between PA and bacterivory was found in oligotrophic high mountain lakes phytoplankton, both at bulk and species level, thanks to the quantification of single-cell PA. In general, PA and bacterivory were widely used by the phytoplankton of high mountain lakes, increased along a gradient of nutrient demand, and the selection of one or another depended on the resources availability, which was determined by the lake type: Phosphatase activity was preferred in acidic lakes, whereas bacterivory was favoured in deep stratified lakes, with shallow lakes in an intermediate position. Communities with relatively high levels of investment in P scavenging activities mobilized up to 3.6 or 26.1 times more P by PA than by bacterivory. No allometric relationship was found between surface-to-volume ratio (S/V) and the selection of PA or bacterivory, but we detected a phytoplankton cell S/V threshold (1.24 μm-1) above which rates and percentage of cells active in PA and bacterivory clearly decreased indicating lower demand on nutrient scavenging activities in small cells (i.e. approximately in cells below 5 μm of diameter).

Finally, a quantitative study of PA in Cyclotella spp. from high mountain lakes described these diatoms as the main phosphatase strategist in most of the phytoplankton communities where it was present. The adaptive advantage that this functional trait confers in P limited environments could explain the ~150-years-old expansion of Cyclotella in nutrient-poor, non-acidified, and non-shallow lakes from the Northern Hemisphere, as a response to a combination of climate warming and P limiting conditions partially enhanced by atmospheric nitrogen deposition.

Aquesta tesi es consta d’una part metodològica i una d’ecològica. Vàrem comparar diferents protocols d’enzyme labelled fluorescence (ELF) per a analitzar activitat fosfatasa (PA) a nivell unicel•lular en organismes del plàncton. Es va concloure que els protocols que incubaven sense fixar i en líquid proporcionaven millors resultats. Malauradament cap protocol no va aconseguir eliminar completament la senyal intracel•lular, la qual cosa fa pensar que tant amb ELF com amb altres substractes pel conjunt de la fosfatasa (i possiblement d’altres activitats enzimàtiques extracel•lulars) podrien haver estat contaminades per part d’enzims intracel•lulars. També vàrem concloure que la quantificació de la fluorescència generada pel substracte ELF en reaccionar amb les fosfatases es pot quantificar de manera més fidedigna emprant microscòpia deconvolutiva de restauració, amb imatges 3D, que no pas amb imatges 2D. N’assenyalem les aplicacions on és altament recomanable i debatem com interpretar les dades de 2D.

D’altra banda es va estudiar la relació entre dos trets funcionals, la PA i la bacterivoria, en el fitoplàncton d’estanys oligotròfics del Pirineu. Es va observar una relació de compromís (trade-off) entre les dues activitats tant a nivell del conjunt de l’estany com a nivell específic. Les comunitats amb nivells alts d’esforç en activitats per a l’obtenció de P mobilitzaven fins a 3,6 o 26,1 vegades més P amb la PA que amb la bacterivoria. Cal destacar que es va detectar un llindar de relació superfície volum de 1.24 μm-1 per sobre del qual les taxes i percentatges de cèl•lules actives en PA i bacterivoria disminuïa.

Finalment, es va quantificar la PA en Cyclotella spp., demostrant que es troba entre els estrategues més forts de la fosfatasa en els estanys estudiats. Per això proposem que l’avantatge competitiu que li confereix aquest tret funcional podria contribuir a explicar l’expansió del gènere Cyclotella durant els darrers c. 150 anys en estanys oligotròfics, no acidificats i suficientment profunds de l’hemisferi nord, com a resposta a la combinació de l’escalfament global i a condicions limitants en P, potenciades pel dipòsit atmosfèric de N.