Embryo Injection Technique for Gene Editing in the Black-Legged Tick, Ixodes scapularis

Summary

The present protocol describes a method for injecting tick embryos. Embryo injection is the preferred technique for genetic manipulation to generate transgenic lines.

Abstract

Ticks can transmit various viral, bacterial, and protozoan pathogens and are therefore considered vectors of medical and veterinary importance. Despite the growing burden of tick-borne diseases, research on ticks has lagged behind insect disease vectors due to challenges in applying genetic transformation tools for functional studies to the unique biology of ticks. Genetic interventions have been gaining attention to reduce mosquito-borne diseases. However, the development of such interventions requires stable germline transformation by injecting embryos. Such an embryo injection technique is lacking for chelicerates, including ticks. Several factors, such as an external thick wax layer on tick embryos, hard chorion, and high intra-oval pressure, are some obstacles that previously prevented embryo injection protocol development in ticks. The present work has overcome these obstacles, and an embryo injection technique for the black-legged tick, Ixodes scapularis, is described here. This technique can be used to deliver components, such as CRISPR/Cas9, for stable germline transformations.

Introduction

Ticks are vectors of medical and veterinary importance, capable of transmitting a variety of viral, bacterial, protozoan pathogens and nematodes^1,2. In the eastern United States, the black-legged tick, Ixodes scapularis, is an important vector of the Lyme disease (LD) pathogen, the spirochete Borrelia burgdorferi. Over 400,000 cases of LD are reported each year in the United States, making it the top vector-borne infectious disease in the US¹. In addition to B. burgdorferi, six other microorganisms are transmitted by I. scapularis- including four bact....

Protocol

Ixodes scapularis adults were either purchased from Oklahoma State University (OSU) or reared at the University of Nevada, Reno (UNR) (IACUC protocol #21-001-1118).

1. Preparation of female ticks for embryo collection

NOTE: To collect eggs of appropriate age, it is important to synchronize egg-laying. Although egg-laying cues in ticks remain unclear, under the standard insectary conditions (27 °C temperature and >90% relative humidit.......

Discussion

This is the first protocol developed to inject early tick embryos successfully. A survival rate of ~4%-8% has been achieved, which is comparable to embryo injection in other well-established insect models⁵.

As this is the initial protocol, it is anticipated that this protocol will be further refined and specialized to individual tick species. In particular, injection timing will vary from species to species, dependent upon embryogenesis, especially the timing of cellula.......

Materials

Name	Company	Catalog Number	Comments
Aluminum silicate capillaries, with filament	Sutter instruments	AF100-64-10	Embryo injection
Benzalkonium chloride 50% in water, 25 g	TCI-America	B0414	Embryo treatment, 25 g is approximately 25 mL
Filter paper	Whatman	1001-090	Post-injection care
Forceps	Thomas Scientific	300-101	Gene`s organ manipulation
Lab Wipes	Genesee Scientific	88-115
Microloader tips	Eppendorf	930001007	Loading the pulled needles
Micromanipulator	Sutter instruments	ROE-200	Embryo injection
Microscopic slides- plain, ground edges	Genesee Scientific	29-100	Embryo alignment, ground edges are preferred, beveled edges could obscure the eggs from view
NaCl	Research Products International	S23020-500.0	Embryo treatment
Needle Puller	Sutter Instruments	P-1000
Permanent Double sided tape	Scotch	34-8716-3417-5	Embryo alignment
Petri plates	Genesee Scientific	32-107G	Post-injection care
Tegaderm/ Transparent film dressing	3M Healthcare	1628	Embryo alignment
Tungsten needles	Fine Science Tools	10130-10	Gene`s organ manipulation
Tungsten Wire	Amazon	B08DNT7ZK3	Gene`s organ manipulation
XenoWorks Digital Microinjector	Sutter instruments	MPC-200	Embryo injection