Earthworm, Lumbricus Terrestris: A Novel Microinjection Vasculature In vivo Invertebrate Model

Asis Lopez; Yaswitha Mikkilineni; Shayna Berman; Damir Khismatullin; Gregory T. Clement; Matthew R. Myers

doi:10.3791/62449

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Method Article

Earthworm, Lumbricus Terrestris: A Novel Microinjection Vasculature In vivo Invertebrate Model

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DOI:

10.3791/62449

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April 1st, 2021

Asis Lopez¹^,⁵ , Yaswitha Mikkilineni² , Shayna Berman³ , Damir Khismatullin¹^,⁴ , Gregory T. Clement⁵ , Matthew R. Myers⁵

¹Bioinnovation PhD Program, Tulane University, ²Biology Department, Virginia Commonwealth University, ³Mechanical Engineering Department, Montgomery College, ⁴Biomedical Engineering Department, Tulane University, ⁵US Food and Drug Administration

Summary

Earthworms are a novel invertebrate in vivo bench-top model for vasculature studies. We present techniques and equipment that allow efficient surgery and microinjection into the earthworm vasculature. Surgical protocols, microinjection techniques and the procedure for producing custom-made micropipettes are described.

Abstract

Although vertebrates are indispensable to biomedical research, studies are often limited by factors such as cost, lengthy internal review, and ethical considerations. We present the earthworm as an alternative, low-cost, invertebrate applicable to certain preliminary vasculature studies. Due to the surgical availability of the earthworm's dorsal vessels, ventral vessels, and five pairs of pseudo hearts, earthworms are readily accessible, offer low-cost maintenance, and require administration of only small doses of a given compound. The earthworm model provides a simple closed vascular circulatory system with a hemoglobin structure similar to human blood. A protocol is provided for anaesthetizing the earthworms and performing surgical incisions to expose relevant blood vessels. Micropipettes for compound administration are formed by heating and pulling glass with a pipette puller and using a beveling system to create a micron-scale fine needle tip. The tips are then used with a micropositioner and microinjector to inject arbitrary compounds into the vascular system of an earthworm, repeatably, with the availability of large sample sizes and small compound volumes. Details on the intricacies of injection procedure are provided. The small vessel size of the earthworm is challenging, particularly in the case of the ventral vessel; however, mastery of the techniques presented offers high repeatability as a low-cost solution, making studies of very large sample size practical.

Introduction

The earthworm has been used as an important bioindicator and bioassay for previous scientific applications¹^,²^,³^,⁴^,⁵^,⁶; it is an ideal organism for assessing biological risks from hazardous and toxic waste in terrestrial environments for in situ and bioaccumulation studies, such as biocides (insecticides) in soil and adverse ecotoxicological effects⁷^,⁸^,⁹^,