Protocols for Investigating the Host-tissue Distribution, Transmission-mode, and Effect on the Host Fitness of a Densovirus in the Cotton Bollworm

Summary

Here, we present a protocol to investigate the host-tissue distribution, transmission mode, and effect on the host fitness of a densovirus within a lepidopteran species, the cotton bollworm. This protocol can also be used for studying the interaction between other orally-transmitted viruses and their insect hosts.

Abstract

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, Helicoverpa armigera densovirus (HaDV2), in a lepidopteran species, the cotton bollworm, Helicoverpa armigera (Hubner). Here, we describe the protocols that are currently used to study the effect of HaDV2 on its host. First, we establish a HaDV2-free cotton bollworm colony from a single breeding pair. Then, we orally inoculate some neonate larval offspring with HaDV2-containing filtered liquid to produce two colonies with the same genetic background: one HaDV2-infected, the other uninfected. A protocol to compare life table parameters (e.g., larval, pupal, and adult periods and fecundity) between the HaDV2-infected and -uninfected individuals is also presented, as are the protocols for determining the host-tissue distribution and transmission efficiency of HaDV2. These protocols would also be suitable for investigating the effects of other orally transmitted viruses on their insect hosts, lepidopteran hosts in particular.

Introduction

In the last few decades, the development of sequencing technology such as next-generation sequencing (NGS) has facilitated the discovery of many novel DNA and RNA viruses, especially nonpathogenic viruses, but also novel isolates of previously known viruses^{1,2,3,4,5,6,7,8,9,10,11,}

Protocol

1. Construction of the HaDV2-free Cotton Bollworm Colony

1. Rear cotton bollworm larvae (H. armigera) on an artificial diet³² in a controlled growth room or an artificial climate chamber at 25 ± 1 °C, with 14 h light/10 h dark and 60% relative humidity.
2. Aid single-pair mating of newly eclosed moths by using a plastic cage per pair (10 cm height, 5 cm diameter) covered with cotton gauze to ensure good ventilation and to serve as an oviposition substrate. To increase the likelihood of obtaining a virus-free colony, utilize at least 30 pairs of single-pair matings. Feed adult moths with a 10% sugar and 2% vita....

Results

Progenies from the parents that were HaDV2-free (Figure 3A) were reared as the NONINF-strain. We successfully amplified the actin gene using the same DNA templates, suggesting that the DNA templates were of good quality (Figure 3B). In addition, the randomly selected eight progenies were also free of HaDV2 (Figure 3C), HaNPV (Figure 3D), and Wolbachia (Figure 3E). Again, we concluded tha.......

Discussion

In the last few decades, most studies on insect-virus interactions have focused on honey bee health^34,35,36, vectors of human disease³⁷, plant viruses³⁸, and some insect pathogenic viruses that have great potential as biological control agents³⁹. Little attention has been paid to covert viruses in insects, especially in lepidopteran pests. Here, we present a.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
24-well plate	Corning	07-200-740	Multiple suppliers available.
DNA extraction kit	TIANGEN	DP304-03	Multiple suppliers available.
thermal cycler	Veriti; Applied Biosystems	4375786
PBS	Corning	21-040-CV
0.22 µm membrane filter	Millipore	SLGS025NB
pEASY-T Cloning Vector	TransGen, Beijing, China	CT301-02
Tweezers	IDEAL-TEK	2.SA
Premix Ex Taq (Probe qPCR)	Takara	RR390A
Probes	Invitrogen	Custom order
Primers	Invitrogen	Custom order
microspectrophotometry NanoDrop 2000c	Thermo scientific	not available
7500 Real-Time PCR system	Applied Biosystems	not available
stereomicroscope SZX-16	Olympus	not available
sucrose			Multiple suppliers available.
vitamin complex			Multiple suppliers available.