Meten-Glutathion geïnduceerde Feeding Response in 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 Cultuur en waardering van de Feeding Response Handhaaf hydra poliepen in kweek door ze dagelijks met artemia en bewaar ze in een medium (1 mM Tris-HCl buffer, pH 7,6, 1 mM NaCl, 1 mM CaCl2, 0,1 mM KCl en 0,1 mM MgSO4) in een glazen schaal bij 18 ° C onder 12 uur licht 12 uur donker cycli zoals eerder 12 beschreven. Voor het meten van de voedingsreactie transfer een volwassen hydra poliep met 5-6 tentakels een enkel putje van een 24-wells plaat. Verwijder de…

Representative Results

Glutathion veroorzaakt hydra tot krullen van de tentakels vertonen op de bek ten behoeve van overspoelt de prooi. Dergelijke curling tentakels brengt apicale uiteinden van de tentakels dichter bij de hypostoom. Dit resulteert in afname van de tentakel spread of de lineaire afstand tussen apicale uiteinde van de tentakel en hypostoom (figuur 1). De relatieve tentakel spread of de verhouding van gemiddelde tentakel voor verspreiden en na het toevoegen van glutathion, gemiddeld over meerdere poliepen vermi…

Discussion

Voedingsgedrag in hydra is een van de meest voorouderlijke chemosensory systemen in de Metazoa. Hoewel de aanwezigheid van GSH in de schaaldieren vloeistof vrijgelaten na-nematocyst bijgestaan ​​hun prooi vangen werd lang geleden ontdekt ^1, hebben noch de GSHR eiwit, noch de vermeende coderende gen / s gekarakteriseerd van hydra-to-date. Weinig pogingen gedaan om karakteriseren GSH bindingseiwitten hydra ^{8, 14, 15.} De identiteit van deze vermeende receptor eiwitten blijft onduidelijk en weinig …

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