Misurazione glutatione indotta risposta alimentazione a Hydra

Summary

Here we describe a simple assay for the quantification of the feeding response in hydra induced by the reduced form of glutathione. This assay relies on measuring the distance between the apical end of the tentacle and mouth of hydra.

Abstract

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. The movement of prey organisms causes mechanosensory discharge of the stinging cells called nematocysts from hydra, which are inserted into the prey. The feeding response in hydra, which includes curling of the tentacles to bring the prey towards the mouth, opening of the mouth and consequent engulfing of the prey, is triggered by GSH present in the fluid released from the injured prey. To be able to identify the molecular mechanism of the feeding response in hydra which is unknown to date, it is necessary to establish an assay to measure the feeding response. Here, we describe a simple method for the quantitation of the feeding response in which the distance between the apical end of the tentacle and mouth of hydra is measured and the ratio of such distance before and after the addition of GSH is determined. The ratio, called the relative tentacle spread, was found to give a measure of the feeding response. This assay was validated using a starvation model in which starved hydra show an enhanced feeding response in comparison with daily fed hydra.

Introduction

Hydra is the most primitive organism possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey¹. It feeds on a variety of animals such as nematode, crustacea, insect larvae, tadpoles and newly hatched fish¹. The movement of these prey organisms causes mechanosensory discharge of the stinging capsules called nematocysts from hydra, which are inserted into the prey². GSH present in the fluid released from the injured prey subsequently activates the feeding response in hydra which includes curling of the tentacles to bring the prey towards the mouth, opening of the mouth, and consequent engulfing of the prey. Multiple molecules, such as dopamine³, glutamate⁴, GABA, glycine⁵, NMDA receptors⁶, and allatotropin⁷, have been shown to be involved in the feeding response in hydra. It has also been shown that the chemosensory response induced by GSH is modulated by the feeding status of the animal such that starved hydra exhibited enhanced feeding response¹. Such an increase in the GSH sensitivity is biologically relevant since under starvation hydra need to find its prey at higher sensitivity.

Although the feeding response induced by GSH can be clearly observed under microscope, the methods typically used for measuring the feeding response observations are non-quantitative. In most of the cases, the time during which the mouth of the hydra remains open was taken as a measure of the feeding response^8,9; whereas in another case, quantitation was based on the number of hydra out of a population showing the feeding response¹⁰. However, observing the mouth opening time of the hydra polyps is cumbersome and subject to variation induced by uncontrollable parameters such as the direction of the mouth orientation during observations. Similarly, since the feeding response is a quantitative parameter, population-based approaches are subject to variations/errors caused by the opinion or observational bias of the individual observer. To circumvent these issues we have developed a method for the relative quantification of the feeding response in hydra (Hydra vulgaris Ind-Pune¹¹) based on the distance of the apical end of the tentacle from the mouth of the hydra polyp.

Protocol

1. Hydra Cultura e misura della risposta alimentazione Mantenere polipi idra in coltura somministrando loro giornalmente con artemia e tenerli in un mezzo (buffer 1 mM Tris-HCl, pH 7,6, 1 mM NaCl, 1 mM CaCl 2, 0.1 mM KCl, e 0,1 mM MgSO4) contenuta in un ciotola di vetro a 18 ° C in 12 hr luce-12 hr cicli scuri come descritto in precedenza 12. Per misurare la risposta di alimentazione, trasferire matura polipo Hydra avere da 5 a 6 tentacoli di un unico pozzetto di un…

Representative Results

Glutatione provoca idra esporre arricciamento dei tentacoli verso la bocca al fine di inghiotte la preda. Tale arricciatura di tentacoli porta estremità apicale dei tentacoli più vicino alla ipostoma. Ciò comporta la riduzione dello spread tentacolo, o la distanza lineare tra l'estremità apicale del tentacolo e ipostoma (Figura 1). La diffusione tentacolo relativa, o il rapporto tra tentacolo media si sviluppa prima e dopo l'aggiunta di glutatione, la media su più polipi riduce nel tempo. L…

Discussion

Comportamento alimentare in idra rappresenta uno dei sistemi chemosensoriali più ancestrali dei metazoi. Sebbene la presenza di GSH nel fluido crostaceo rilasciato dopo la cattura la preda nematocisti-assistita è stato rilevato da tempo ^1, né la proteina GSHR né le putativo gene codifica / s sono stati caratterizzati da idra ad oggi. Alcuni tentativi sono stati fatti per caratterizzare GSH proteine ​​leganti in idra ^{8, 14, 15.} Tuttavia, l'identità di queste proteine ​​recettori put…

Materials

Cooled Incubator	Panasonic	MIR-254-PE
Microscope	Leica	S8AP0
Camera for the microscope	Leica	EC3
Reduced glutathione	Sigma	G4251	Stored at 4°C. Bring the bottle to room temperature before opening to avoid oxidation
Image editing program	GIMP	Version 2.8