Medición inducida glutatión-Alimentación Respuesta en 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 y medición de la respuesta de alimentación Mantener pólipos Hydra en cultivo mediante la alimentación diariamente con artemia y mantenerlos en un medio (mM de tampón Tris-HCl 1, pH 7,6, NaCl 1 mM, 1 mM CaCl 2, 0,1 mM KCl, y 0,1 mM MgSO 4) contenida en una tazón de vidrio a 18 ° C por debajo de 12 hr hr luz-12 ciclos oscuros como se describe anteriormente 12. Para la medición de la respuesta de alimentación, transferir uno maduro pólipo hidr…

Representative Results

El glutatión provoca hydra exponer rizado de los tentáculos hacia la boca con el propósito de que envuelve la presa. Tal rizado de tentáculos trae extremos apicales de los tentáculos más cerca de la hipostoma. Esto resulta en la reducción de la propagación tentáculo, o la distancia lineal entre el extremo apical del tentáculo y hipostoma (Figura 1). La propagación tentáculo relativa, o la relación de tentáculo promedio propagan antes y después de la adición de glutatión, promediados a t…

Discussion

La alimentación en el comportamiento hidra representa uno de los sistemas quimiosensoriales más ancestrales en los metazoos. Aunque la presencia de GSH en el fluido crustáceo en libertad después de la captura de presas nematocisto asistida se detectó hace mucho tiempo ^1, ni la proteína GSHR ni los genes de codificación putativo / s se han caracterizado desde hidra hasta la fecha. Pocos intentos se han realizado para caracterizar las proteínas de unión GSH en Hydra ^{8, 14, 15.} Sin embargo, l…

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