Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Summary

Here, we present a protocol to isolate endosymbionts from the whitefly Bemisia tabaci through dissection and filtration. After amplification, the DNA samples are suitable for subsequent sequencing and study of the mutualism between endosymbionts and whitefly.

Abstract

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to study the functions and evolution of bacterial symbionts in their host. As most symbionts cannot be cultured in vitro, methods to isolate an adequate quantity of bacteria for genome sequencing are very important. In the whitefly Bemisia tabaci, a number of endosymbionts have been identified and are predicted to be of importance in the development and reproduction of the pests through multiple approaches. However, the mechanism underpinning the associations remains largely unknown. The obstacle partially comes from the fact that the endosymbionts in whitefly, mostly restrained in bacteriocytes, are hard to separate from the host cells. Here we report a step-by-step protocol for the identification, extraction and purification of endosymbionts from the whitefly B. tabaci mainly by dissection and filtration. Endosymbiont samples prepared by this method, although still a mixture of different endosymbiont species, are suitable for subsequent genome sequencing and analysis of the possible roles of endosymbionts in B. tabaci. This method may also be used to isolate endosymbionts from other insects.

Introduction

Bacteria forming an intimate symbiotic relationship with relative hosts are widespread in arthropods¹. The endosymbionts have been demonstrated to affect aspects of hosts, such as nutrition metabolism, reproduction, responses to environmental stresses^2,3,4 etc., in almost every developmental stage⁵. However, the mechanism underpinning the associations still remains largely unknown. Genomics is of priority and importance when studying the potential functions and roles of bacteria. Some fundamental information, i.e.

Protocol

1. Whitefly Rearing and Cryptic Species Identification

1. Maintain the whitefly species on cotton Gossypium hirsutum (Malvaceae) (cv. Zhe-Mian 1973) in cages under standard conditions of 27 ± 1 °C, 70 ± 10% humidity and 14 h light: 10 h dark regime.
2. Collect an individual adult whitefly and homogenize in 30 µL of lysis buffer (10 mM Tris, pH 8.4, 50 mM KCl, 0.45% [wt/vol] Tween-20, 0.2% [wt/vol] gelatin, 0.45% [vol/vol] Nonidet P 40, 60 g/mL Proteinase K).
3. Incubate the homogenate at 65 °C for 1 h and then 100 °C for 10 min.
   NOTE: The incubation time at 65 °C can be increased if necessary. T....

Results

The Middle East Asia Minor 1 (MEAM1) species of the B. tabaci complex was taken as an example here for description. Cotton for rearing whiteflies and several developmental stages of whiteflies are shown in Figure 1 including a cotton plant, adult whitefly and the 1^st, 2^nd and 4^th instar nymphs of whitefly (the 3^rd instar nymph looks similarly as the 4^th instar nymph). It was obvious that the 4

Discussion

Since the endosymbionts within whiteflies cannot be cultured in vitro, dissection and assembling bacteriocytes is an effective way to obtain enough genetic material of endosymbionts. Before dissection, the species of whitefly and endosymbionts involved should be explicitly confirmed. The whitefly B. tabaci is a species complex with more than 35 morphologically indistinguishable species and different cryptic species may contain different endosymbionts. Portiera is uniformly harbored as an obliga.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Taq DNA polymerase	Takara	R001A	including rTaq, 10×Buffer and dNTP
Gel DNA extraction kit	Qiagen	28704
DNA sample sequencing system	ABI	ABI-3730XL
Microtome	Leica	EM UC7
Transmission electron microscopy	Hitachi	H-7650 TEM
Stereo microscope	Zeiss	Stemi 2000-C
20 μL microloader	Eppendorf	F2771951
Filter holder	Millipore	SX0001300
Filter membrane filter	Millipore	SMWP001300	5.0 μm SMWP
REPLI-g UltraFast Mini Kit	Qiagen	150033	DNA amlification kit
NanoDrop	Thermo Scientific	NanoDrop 2000
Qubit Fluorometer	Thermo Fisher Scientific	Q33216
Genome Sequencer	Illumina	Hiseq 2000