Preparation of Virus-Enriched Inoculum for Oral Infection of Honey Bees (Apis mellifera)

Summary

Here we describe two protocols: first to propagate, extract, purify, and quantify large quantities of honey bee non-enveloped virus particles, including a method for removing honey bee pupae and second to test the effects of viral infection using a highly repeatable, high-throughput cage bioassay.

Abstract

Honey bees are of great ecological and agricultural importance around the world but are also subject to a variety of pressures that negatively affect bee health, including exposure to viral pathogens. Such viruses can cause a wide variety of devastating effects and can often be challenging to study due to multiple factors that make it difficult to separate the effects of experimental treatments from preexisting background infection. Here we present a method to mass produce large quantities of virus particles along with a high throughput bioassay to test viral infection and effects. Necessitated by the current lack of a continuous, virus-free honey bee cell line, viral particles are amplified in vivo using honey bee pupae, which are extracted from the hive in large volumes using minimally stressful methodology. These virus particles can then be used in honey bee cage bioassays to test inocula viability, as well as various other virus infection dynamics, including interactions with nutrition, pesticides, and other pathogens. A major advantage of using such particles is that it greatly reduces the chances of introducing unknown variables in subsequent experimentation when compared to current alternatives, such as infection via infected bee hemolymph or homogenate, though care should still be taken when sourcing the bees, to minimize background virus contamination. The cage assays are not a substitute for large-scale, field-realistic experiments testing virus infection effects at a colony level, but instead function as a method to establish baseline viral responses that, in combination with the semi-pure virus particles, can serve as important tools to examine various dimensions of honey bee-virus physiological interactions.

Introduction

Honey bees (Apis mellifera) play a critical role in the modern global agricultural landscape but are currently suffering from a combination of biotic and abiotic stressors, including pesticide exposure, poor forage, parasites, and pathogens^1,2. One of the most important pathogens of concern are viruses, many of which are vectored by another of the major honey bee stressors, the parasitic Varroa mite (Varroa destructor). These viruses can cause an array of negative effects in honey bees including reduced brood survival, developmental defects, and paralysis that can lead to total hive collapse ....

Protocol

1. Mass bee extraction option 1: larval self-removal

1. Cage a honey bee queen on an empty, drawn-out Langstroth frame and return her to her colony. Allow the queen to lay eggs on this frame for 24 h.
   1. Check the frame after 24 h to ensure most of the comb cells contain newly laid eggs. Depending on the queen and colony, eggs are sometimes not laid very well in the first 24 h. If this occurs, allow for an additional 24 h and adjust the time as necessary.
2. After the 24 h egg-laying period, release the queen. Mark the frame clearly and return it to the colony.
3. Exactly 192 h (8 days) after caging the queen (assuming nor....

Results

Successfully following the protocols (Figures 1) for pupal injection and viral extraction should produce large quantities of virus particles. However, sampling and injecting pupae sourced from a variety of colonies at multiple time points maximizes the chances of acquiring target virus with low contamination. The dynamics by which viruses replicate and interact with one another within a honey bee is not well understood; coupled with the likelihood for preexisting infection, there is no guarantee that the.......

Discussion

Here we have outlined methods detailing every step of the virus amplification and inoculum stock preparation process, including larvae collection and virus propagation, extraction, and concentration, as well as viral treatment in the form of cage-feeding experiments. These methods allow for production of semi-pure virus particles (Figure 4), the effectiveness of which can be consistently be quantified by dose-response mortality testing for viruses that are lethal to adults (

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10% bleach solution
24:1 chloroform:isoamyl alcohol	SigmaAldrich	C0549
70% ethanol solution
Cages for bioassay			Dependent on experimental setup
Combitips Advanced 0.1 mL	Eppendorf	30089405	Optional (if no injector appartus is available)
Containers for larval self-removal			Should measure roughly 19" x 9-1/8" (Langstroth deep frame dimensions)
Forceps			Blunt, soft forceps for larval separationl; blunt, hard forceps for pupal excision
Fume hood
Incubator			Capable of maintaining 34 ºC and 50% relative humidity
Kimwipes	Fisher Scientific	06-666	Any absorbent wipe will work
Medium-sized weight boats			Serve as inoculum trays
Microcon-100kDa with Biomax membrane	MilliporeSigma	MPE100025
NaCl
Nitrile gloves
Phosphate buffered saline (PBS)	SigmaAldrich	P5119
Polyethylene glycol 8000 (PEG)	SigmaAldrich	1546605
Refrigerated benchtop centrifuge			Capable of 15,000 x g
Refrigerated centrifuge			Capable of 21,000 x g
Repeater M4 Multipipette	Eppendorf	4982000322	Optional (if no injector appartus is available)
RNAse Away	ThermoFisher	7000TS1
RNAse-free water	SigmaAldrich	W4502
Sucrose
TES	SigmaAldrich	T1375