We would like to thank Dr. Julia Fine for her ideas and discussion during the protocol creation process, as well as Dr. Cassondra Vernier for her helpful comments throughout editing. These materials contributed towards projects that were supported in part by the Foundation for Food and Agriculture Research, under grant ID 549025.

The authors have nothing to disclose.

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 (Figure 5). Following confirmation of infective ability and/or pathology, the generated particles can then be used in bioassays to elucidate the interactions between honey bees and honey bee viruses.
One of the most distinctive benefits of the described protocols is that each is easily scaled to a large volume, whether it be pupae harvested, particles produced, or bioassays performed. Using pupal excision methods33, one can expect to remove ~100 pupae per hour, though this number will scale with proficiency. However, the larval self-removal method, reported here, while requiring different planning and scheduling procedures, can easily generate 10-20 times that number of removed bees overnight while involving comparatively little manual effort and less mechanical strain on the bees.
The main limitation of this method is that there is no way of guaranteeing that the self-removed larvae were not previously Varroa-infested. Regular mite treatment and monitoring of source hives can minimize this risk, but some larvae still may have had some level of Varroa parasitization. Manually removing pupae individually, however, allows the user to observe if any mites are present in the cell of a given pupa. Additionally, because the self-removal process relies on the food-seeking behavior of the larvae, the frames containing these larvae must be removed before the final feeding that normally occurs. Only due to this lack of provisioning by the workers do the larvae crawl from their cells. Therefore, the pupae derived from this method experience a very short window of nutritional stress compared to larvae that developed completely inside a colony and can appear slightly smaller. This is particularly notable in the youngest larvae in the cohort present on the frame; because the queen has laid eggs over a 24 h window, these larvae are missing proportionally more feeding time. These are usually clearly notable during pupation for their very small size compared to those removed by manual excision. However, the volume of larvae produced by self-removal more than compensates for their diminished individual biomass. Furthermore, the manual excision method also can cause substantial mechanical stress to the pupae as they are pulled from their cells. If either method is to be used as part of an experiment, and not just to produce virus particles, care should be taken to ensure proper controls.
Regardless of the extraction method, the virus propagation protocol can generate large quantities of virus particles using the pupae, which minimizes the variability induced by other currently practiced virus inoculation methods35,36,37 when testing for honey bee viral response. It is important to note that this protocol was optimized and tested using non-enveloped viruses in the order Picornavirales (e.g., Israeli acute paralysis virus, deformed wing virus). Different strategies to isolate viral particles should be followed when working with enveloped viruses44. As a rough approximation, each of the 16 samples involved in the virus harvesting effort (which included the 4 colonies of Figure 4) were generated from 200-300 injected pupae and yielded between 2-2.5 mL of concentrated virus particle preparations. Assuming a virus inoculum concentration of 0.1% and a standard 35-bee cage, each of the 16 virus preparations would provide sufficient particles for 3,300-4,200 cages. This surplus of infective material reduces experimental restrictions and enables high-throughput bioassays. It is important to note that although the virus particle concentrate can remain viable for months or years when stored at -80 &#176;C, it can slowly decrease in infectivity, even if subjected to few freeze-thaw cycles. It is, therefore, recommended to store the viral preparation stock in small aliquots, several of which can then be used to quantify viral titers and verify the treatment dose at the time of the experiment. Additionally, the inter-year variability demonstrated in Figure 5, further reinforces the need for periodic dose-response testing, thereby minimizing the chances of unexpected loss in virus viability.
The cage bioassays themselves also have limitations, or at least caveats necessary to take into consideration, the primary one being the artificial nature of the cage bioassay environment (Figure 3). Grouping bees into enclosures allows for viral testing beyond the individual level; the cage becomes the experimental unit rather than the bee itself. Although this is more similar to an actual colony than a single bee being treated in isolation, it is still far from a realistic hive environment. Removed from the social environment, including queen and brood pheromones, bees of different ages, and other cues, these bees may not respond as a full-sized colony might. These are primarily considerations for larger-scale experiments using the cage system, however. The results gathered from cage viral bioassays should primarily be treated as baseline information establishment that can be used to inform future virus testing decisions scaled to a more field-realistic setting as desired by the user.
The methods described here provide a standardized process for the mass production of virus particles for use in honey bee viral assays. Such assays have already been implemented to examine various aspects of honey bee-virus interactions, including multi-virus infection and how diet quality and nutritional supplements affect survivorship in the face of viral infection11,18,45,46. They have been scaled up for use in colony-wide infection experiments11,47 and to study the effects of infection on behavior47 and gene expression48. Overall, these methods provide a baseline in tools that can be used to produce and evaluate honey bee virus inocula.

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 pathogens1,2. 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 both before and after overwintering periods3,4,5. Although there have been promising advances in the development of technologies used to combat virus infection6,7,8,9, the dynamics by which many viruses propagate, spread, and interact within a honey bee or colony are still poorly understood5,10. Understanding the basic biology of honey bee and virus interactions and their relationships with other environmental factors is critical for developing effective virus management techniques.
However, studying honey bee-virus interactions poses challenges with numerous known and unknown factors complicating the process. These include interactions with diet11,12, pesticide exposure13, and bee genetic background14,15. Even when focusing on virus infection alone, complications are common because honey bee populations, both managed and wild, always have some degree of background virus infection, though often without manifesting acute symptoms16,17, and the effects of virus coinfection are not well understood18. This has made the study of honey bee virus effects difficult to disentangle.
Many honey bee virus studies have used circumstantial virus infections to look for interactions with other stressors, observing how background infections change with other treatments12,19,20,21. While this approach has been successful at identifying important effects, especially discovering how pesticide or dietary treatments affect virus levels and replication, inoculation with a virus treatment of known content and concentration is critical for experimental testing of virus infection dynamics. Even so, separating experimental treatment from background infection can also pose challenges. In field studies, researchers have differentiated strains of deformed wing virus (DWV) to provide evidence for virus transmission from honey bees to bumble bees22, but using this approach would be difficult within honey bees alone. Virus infectious clones are a powerful tool, not just for tracking infection23,24,25 but for reverse genetics studies of honey bee viruses and for virus-host interaction research26,27,28. However, in most instances, infectious clones are still required to fulfill the infection cycle inside cells to produce particles. Such particles are preferred as inocula for experimental treatments because their infectivity is higher than the naked viral RNA and inoculation with encapsidated genomes mimics a natural infection.
The production of pure, uncontaminated honey bee virus inocula (wild-type virus strains or those derived from infectious clones) also pose challenges. These are primarily due to the difficulties in obtaining a reliable, continuously-replicating, virus-free honey bee cell line to produce pure-strain viruses29,30. While some cell lines have been produced, these systems remain imperfect; still, there is hope a viable cell line can be produced29, which would allow for finer control of virus production and investigation. Until such a line becomes widely available, most virus production protocols will continue to rely on the use of in vivo virus production and purification18,31,32,33,34. These approaches involve identifying and purifying virus particles of interest (or producing an infectious clone) and using them to infect honey bees, usually as pupae. The pupae are injected with the target virus and then sacrificed, and further particles are extracted and purified. However, because no bees are virus-free to begin with, there is always some degree of contamination from traces of other viruses in any such concentrate, and, therefore, great care must be taken in choosing bees with a low likelihood of background infections. Further, methods for removing the pupae from the comb cells for use in these protocols33 are very labor intensive and can induce stress in the bees, limiting production by these means18,32. Here, we report an alternative method that allows for large scale removal of larvae with little labor and less mechanical stress on the bees.
Once pupae are obtained and injected with the starting virus inoculum, they must be incubated to provide the virus time to replicate. Subsequently, produced virus particles can be processed into a form usable to infect experimental bees. There are several simple methods to achieve this, including using a crude homogenate35,36 or hemolymph generated from virally infected bees as a source of infection37. These methods are effective but run into a greater chance of introducing unknown variables from the background substrate (e.g., other factors in the dead bee homogenates). Additionally, it is desirable to concentrate the particles if an experiment requires giving a large, known dose of a virus in a short period of time. Therefore, for better control, it is preferable to use methods that allow for some level of purification and concentration of the virus particles. Generally, a series of precipitation and centrifugation steps will result in the removal of almost all possible non-target virus material33.
After producing this concentrated inoculum, it is beneficial to quantify the viral titers (qPCR) and characterize it with in vivo bioassays to test its viability and ability to cause mortality, as well as to corroborate that increased virus titers are obtained after infection. This can be achieved through injection experiments (either into pupae or adults) or feeding experiments (into larvae or adults). While all these approaches are possible, feeding to groups of adult bees in a cage is often the fastest and simplest. The cage assay method is also widely used for testing various other treatments on bees including pesticide toxicity38, ovary development39, and nutritional influence on behavior40,41 and, therefore, can form a good basis for experiments linking virus infection with other factors42.
Here we describe a reliable method for producing large quantities of semi-pure, highly-enriched virus particles without using an expensive ultracentrifuge, including a method for removing pupae that reduces labor and mechanical stress on the bees and a highly repeatable, high-throughput bioassay for testing viral infection and effects. By tightly controlling the purity of the viral inocula, investigators are able to reduce variation in honey bee viral response relative to other viral inoculation methods. Furthermore, the bioassay can screen for viral effects at a small groups level using highly repeatable experimental units before scaling to field-realistic settings, which is far more labor intensive to manage. In combination, these two methods provide the necessary tools for studies that can help improve our overall understanding of honey bee-virus physiological interactions.

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

preparation of virus-enriched inoculum for oral infection of honey bees (apis mellifera)

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.

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 injected (desired) virus will become the dominant in any given pupa, even if the pupae were sampled from the same original colony. Figure 4 demonstrates the potential range of viral proportions that one could expect to see following extraction. The four displayed colonies represent a subset of a larger virus harvesting effort in which every pupa was initially injected with a ~95% Israeli acute paralysis virus (IAPV) inoculum. Although 10 out of the 16 colony samples involved in these extractions contained highly pure IAPV (&#62; 95%), including some &#62; 99% (e.g., Colony 1), other samples varied in their IAPV proportion (e.g., Colony 2-3), with some even being dominated by other viruses such as deformed wing virus (DWV) (e.g., Colony 4).
Table 1 provides additional context for the amplification level of the four viruses (BQCV, DWV, IAPV, SBV) shown in Figure 4 in the form of RT-qPCR threshold cycle (Ct) values (the point at which a PCR target reaches the threshold of detection) and total virus genome equivalents (ge) per 100 ng RNA. Ct values can be used as a predictor of proportion, but ge values need to be calculated using a standard-curve based method17. Notice that the actual quantity of particles (i.e., ge) produced is dependent on the number of pupae processed and the filtering stringency during the extraction process.
Virus particle preparations (amplified inoculum) should be stored at -80 &#176;C and it is recommended to aliquot them, as they will degrade significantly if subjected to multiple freeze-thaw cycles18. Additionally, dose-response assays should always be conducted prior to experimentation, as a multitude of external factors (hive genetics, bee health, etc.) can lead to highly variable viral response. Using data derived from experimental cages (Figure 3), Figure 5 demonstrates such variability by comparing the dose-response survival curves of honey bees, fed the same IAPV particles during two different years. Despite identical testing parameters, including the same viral inocula and testing concentrations ranging from 1% to 0.01% IAPV, the trials conducted in 2018 (Figure 5A) and 2019 (Figure 5B) produced noticeably different survival responses in all but the control treatment, which received no virus in its sucrose inoculum. Note that, if desired, LD50 calculations can also be performed at this point to obtain more precise mortality measurements43, but this is usually not necessary as approximations are generally sufficient. In 2018, a 1% dose resulted in approximately 50% survival at 72 hours post-infection (hpi), thereby making it the default concentration for most viral cage experiments conducted that year. However, that same dose achieved near total mortality in 2019 at the same timepoint, and as a result, most viral cage experiments conducted that year received a 0.01% IAPV inoculum instead. This significantly lower concentration reached the same levels of mortality as a 1% IAPV inoculum in 2018 while also using 100 times fewer virus particles.
These data were produced with bioassays using cages similar to the one diagrammed in Figure 3. The feeder holes in the top and side allow for easy diet control and the sliding cage door makes it simple to add or remove objects from the cage environment, such as inoculum trays or dead bees. However, the generalized viral assay protocol is not restricted to these types of cages nor diet choices and should be modified to suit experimental needs.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61725/61725fig01.jpg" /> 
Figure 1: Representative images of various stages during the larval self-removal protocol.&#160;(A) Example larval mass that was expected following the overnight self-removal period (1.6). (B) Larvae spaced apart from one another on separate injection/growth trays (1.6.3) (C,D) White-eye pupae ready for viral injection (1.9). Brown spots are dried larval frass, which do not need to be removed. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61725/61725fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61725/61725fig02.jpg" /> 
Figure 2: Example injector apparatus created by combining a hypodermic needle with a multi-dispenser tip.&#160;The needle and tip were joined using liquid adhesive to consistently deliver 1 &#181;L fluid injections when attached to a repeating pipetter. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61725/61725fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61725/61725fig03.jpg" /> 
Figure 3: Example cage used in virus bioassay.&#160;Sucrose solution and pollen were provided ad libitum though feeder holes during the duration of the trial. Virus inocula could be easily delivered using a tray inserted through the bottom of the cage. Note that the cage type and feeder content could be adjusted as necessary to suit experimental parameters. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61725/61725fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61725/61725fig04.jpg" /> 
Figure 4: Average total virus loads and proportions across viral preparations from four sample colonies.&#160;Virus loads were measured by RT-qPCR as black queen cell virus (BQCV) + deformed wing virus (DWV) + Israeli acute paralysis virus (IAPV) + sacbrood virus (SBV) genome equivalents (ge) in 100 ng RNA. Each sample colony consisted of 150+ homogenized pupae originally injected with IAPV and represents the typical range of virus proportions generated from the pupal virus injection protocol. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61725/61725fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61725/61725fig05.jpg" /> 
Figure 5: Dose-response survival curves of honey bee bioassay cages.&#160;Cages from both 2018 (A) and 2019 (B) were inoculated with IAPV and fed 30% sucrose solution ad libitum throughout the duration of the trial. Treatments represent IAPV inocula concentrations with 10-2 to 10-4 denoting 1% to 0.1% IAPV particle preparations mixed in 30% sucrose solution; control sucrose inoculations contained no virus. n = 56 total cages in 2018; n = 77 total cages in 2019. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61725/61725fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Average threshold cycle (Ct) values and genome equivalents per 100 ng RNA of virus mixtures in four viral preparations from four sample colonies. Each sample colony consists of 150+ homogenized pupae generated from the pupal virus injection protocol. Viruses were detected by RT-qPCR using specific primers for each virus. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61725/Table_1.xlsx" target="_blank">Please click here to download this table.</a>

Watch this Scientific Journal Video about Preparation of Virus-Enriched Inoculum for Oral Infection of Honey Bees Apis mellifera at JoVE.com

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

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.

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

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 ...

preparation-virus-enriched-inoculum-for-oral-infection-honey-bees

Nanyang Technological University

Research

JoVE Journal

Biology

2.8K Views.  University of Illinois. 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.

Video: Preparation of Virus-Enriched Inoculum for Oral Infection of Honey Bees Apis mellifera

Behavioural Pharmacology in Classical Conditioning of the Proboscis Extension Response in Honeybees (Apis mellifera)

Honeybees (Apis mellifera) are well known for their communication and orientation skills and for their impressive learning capability1,2. Because the survival of a honeybee colony depends on the exploitation of food sources, forager bees learn and memorize variable flower sites as well as their profitability. Forager bees can be easily trained in natural settings where they forage at a feeding site and learn the related signals such as odor or color. Appetitive associative learning can also be studied under controlled conditions in the laboratory by conditioning the proboscis extension response (PER) of individually harnessed honeybees3,4. This learning paradigm enables the study of the neuronal and molecular mechanisms that underlie learning and memory formation in a simple and highly reliable way5-12. A behavioral pharmacology approach is used to study molecular mechanisms. Drugs are injected systemically to interfere with the function of specific molecules during or after learning and memory formation13-16.
Here we demonstrate how to train harnessed honeybees in PER conditioning and how to apply drugs systemically by injection into the bee flight muscle.

Behavioural Pharmacology in Classical Conditioning of the Proboscis Extension Response in Honeybees Apis mellifera

We demonstrate how to implement a behavioral pharmacology method in an appetitive olfactory conditioning paradigm in honeybees (Apis mellifera) by systemic application of drugs. This method allows investigation of the mechanisms underlying learning and memory formation in a simple and reliable way.

Honeybees (Apis mellifera) are well known for their communication and orientation skills and for their impressive learning capability1,2. Because the ...

Neuroscience

Empirical, Metagenomic, and Computational Techniques Illuminate the Mechanisms by which Fungicides Compromise Bee Health

Growers often use fungicide sprays during bloom to protect crops against disease, which exposes bees to fungicide residues. Although considered &#34;bee-safe,&#34; there is mounting evidence that fungicide residues in pollen are associated with bee declines (for both honey and bumble bee species). While the mechanisms remain relatively unknown, researchers have speculated that bee-microbe symbioses are involved. Microbes play a pivotal role in the preservation and/or processing of pollen, which serves as nutrition for larval bees. By altering the microbial community, it is likely that fungicides disrupt these microbe-mediated services, and thereby compromise bee health. This manuscript describes the protocols used to investigate the indirect mechanism(s) by which fungicides may be causing colony decline. Cage experiments exposing bees to fungicide-treated flowers have already provided the first evidence that fungicides cause profound colony losses in a native bumble bee (Bombus impatiens). Using field-relevant doses of fungicides, a series of experiments have been developed to provide a finer description of microbial community dynamics of fungicide-exposed pollen. Shifts in the structural composition of fungal and bacterial assemblages within the pollen microbiome are investigated by next-generation sequencing and metagenomic analysis. Experiments developed herein have been designed to provide a mechanistic understanding of how fungicides affect the microbiome of pollen-provisions. Ultimately, these findings should shed light on the indirect pathway through which fungicides may be causing colony declines.

Microbial consortia within bumble bee hives enrich and preserve pollen for bee larvae. Using next generation sequencing, along with laboratory and field-based experiments, this manuscript describes protocols used to test the hypothesis that fungicide residues alter the pollen microbiome, and colony demographics, ultimately leading to colony loss.

Growers often use fungicide sprays during bloom to protect crops against disease, which exposes bees to fungicide residues. Although considered ...

Environment

Evaluating the Effect of Environmental Chemicals on Honey Bee Development from the Individual to Colony Level

The presence of pesticides in the beekeeping environment is one of the most serious problems that impacts the life of a honey bee. Pesticides can be brought back to the beehive after the bees have foraged on flowers that have been sprayed with pesticides. Pesticide contaminated food can be exchanged between workers which then feed larvae and therefore can potentially affect the development of honey bees. Thus, residual pesticides in the environment can become a chronic damaging factor to honey bee populations and gradually lead to colony collapse. In the presented protocol, honey bee feeding methods are described and applied to either an individual honey bee or to a colony. Here, the insect growth regulator (IGR) pyriproxyfen (PPN), which is widely used to control pest insects and is harmful to the development of honey bee larvae and pupae, is used as the pesticide. The presenting procedure can be applied to other potentially harmful chemicals or honeybee pathogens for further studies.

Herein we present a method to feed pesticide contaminated food to both an individual honey bee and a beehive colony. The procedure evaluates the pesticide effect on individual honey bees by in vivo feeding of basic larval diet and also on the natural condition of beehive colony.

The presence of pesticides in the beekeeping environment is one of the most serious problems that impacts the life of a honey bee. Pesticides can be ...

Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees (Apis mellifera L.)

Honey bees (Apis mellifera L.) are eusocial insects and well known for their complex division of labor and associative learning capability1, 2. The worker bees spend the first half of their life inside the dark hive, where they are nursing the larvae or building the regular hexagonal combs for food (e.g. pollen or nectar) and brood3. The antennae are extraordinary multisensory feelers and play a pivotal role in various tactile mediated tasks4, including hive building5 and pattern recognition6. Later in life, each single bee leaves the hive to forage for food. Then a bee has to learn to discriminate profitable food sources, memorize their location, and communicate it to its nest mates7. Bees use different floral signals like colors or odors7, 8, but also tactile cues from the petal surface9 to form multisensory memories of the food source. Under laboratory conditions, bees can be trained in an appetitive learning paradigm to discriminate tactile object features, such as edges or grooves with their antennae10, 11, 12, 13. This learning paradigm is closely related to the classical olfactory conditioning of the proboscis extension response (PER) in harnessed bees14. The advantage of the tactile learning paradigm in the laboratory is the possibility of combining behavioral experiments on learning with various physiological measurements, including the analysis of the antennal movement pattern.

Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees Apis mellifera L.

In this protocol we show how to condition harnessed honey bees to tactile stimuli and introduce a 2D motion capture technique for analyzing the kinematics of fine-scale antennal sampling pattern.

Honey bees (Apis mellifera L.) are eusocial insects and well known for their complex division of labor and associative learning capability1, 2. The worker ...

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees (Apis mellifera)

This video demonstrates novel techniques of RNA interference (RNAi) which downregulate two genes simultaneously in honey bees using double-stranded RNA (dsRNA) injections. It also presents a protocol of proboscis extension response (PER) assay for measuring gustatory perception.	
	RNAi-mediated gene knockdown is an effective technique downregulating target gene expression. This technique is usually used for single gene manipulation, but it has limitations to detect interactions and joint effects between genes. In the first part of this video, we present two strategies to simultaneously knock down two genes (called double gene knockdown). We show both strategies are able to effectively suppress two genes, vitellogenin (vg) and ultraspiracle (usp), which are in a regulatory feedback loop. This double gene knockdown approach can be used to dissect interrelationships between genes and can be readily applied in different insect species.	
	The second part of this video is a demonstration of proboscis extension response (PER) assay in honey bees after the treatment of double gene knockdown. The PER assay is a standard test for measuring gustatory perception in honey bees, which is a key predictor for how fast a honey bee's behavioral maturation is. Greater gustatory perception of nest bees indicates increased behavioral development which is often associated with an earlier age at onset of foraging and foraging specialization in pollen. In addition, PER assay can be applied to identify metabolic states of satiation or hunger in honey bees. Finally, PER assay combined with pairing different odor stimuli for conditioning the bees is also widely used for learning and memory studies in honey bees.

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees Apis mellifera

In this protocol, we describe two strategies that simultaneously suppress two genes (double gene knockdown) in honey bees. Then we present how to use the proboscis extension response (PER) assay to study the effect of double gene knockdown on honey bee gustatory perception.

This video demonstrates novel techniques of RNA  interference (RNAi) which downregulate two genes simultaneously in honey bees  using double-stranded RNA ...

Propagation of Homalodisca coagulata virus-01 via Homalodisca vitripennis Cell Culture

The glassy-winged sharpshooter (Homalodisca vitripennis) is a highly vagile and polyphagous insect found throughout the southwestern United States. These insects are the predominant vectors of Xylella fastidiosa (X. fastidiosa), a xylem-limited bacterium that is the causal agent of Pierce&#39;s disease (PD) of grapevine. Pierce&#8217;s disease is economically damaging; thus, H. vitripennis have become a target for pathogen management strategies. A dicistrovirus identified as Homalodisca coagulata virus-01 (HoCV-01) has been associated with an increased mortality in H. vitripennis populations. Because a host cell is required for HoCV-01 replication, cell culture provides a uniform environment for targeted replication that is logistically and economically valuable for biopesticide production. In this study, a system for large-scale propagation of H. vitripennis cells via tissue culture was developed, providing a viral replication mechanism. HoCV-01 was extracted from whole body insects and used to inoculate cultured H. vitripennis cells at varying levels. The culture medium was removed every 24 hr for 168 hr, RNA extracted and analyzed with qRT-PCR. Cells were stained with trypan blue and counted to quantify cell survivability using light microscopy. Whole virus particles were extracted up to 96 hr after infection, which was the time point determined to be before total cell culture collapse occurred. Cells were also subjected to fluorescent staining and viewed using confocal microscopy to investigate viral activity on F-actin attachment and nuclei integrity. The conclusion of this study is that H. vitripennis cells are capable of being cultured and used for mass production of HoCV-01 at a suitable level to allow production of a biopesticide.

Propagation of Homalodisca coagulata virus-01 via Homalodisca vitripennis Cell Culture

Here we present a protocol to propagate Homalodisca vitripennis cells and HoCV-1 in vitro. Medium was removed from HoCV-1 positive cultures and RNA extracted every 24 hr for 168 hr. Cell survivability was quantified by trypan blue staining. Whole virus particles were extracted post-infection. Extracted RNA was quantified by qRT-PCR.

The glassy-winged sharpshooter (Homalodisca vitripennis) is a highly vagile and polyphagous insect found throughout the southwestern United States. These ...

Immunology and Infection

Preparation of Single-cohort Colonies and Hormone Treatment of Worker Honeybees to Analyze Physiology Associated with Role and/or Endocrine System

Honeybee workers are engaged in various tasks related to maintaining colony activity. The tasks of the workers change according to their age (age-related division of labor). Young workers are engaged in nursing the brood (nurse bees), while older workers are engaged in foraging for nectar and pollen (foragers). The physiology of the workers changes in association with this role shift. For example, the main function of the hypopharyngeal glands (HPGs) changes from the secretion of major royal jelly proteins (MRJPs) to the secretion of carbohydrate-metabolizing enzymes. Because worker tasks change as the workers age in typical colonies, it is difficult to discriminate the physiological changes that occur with aging from those that occur with the role shift. To study the physiological changes in worker tissues, including the HPGs, in association with the role shift, it would be useful to manipulate the honeybee colony population by preparing single-cohort colonies in which workers of almost the same age perform different tasks. Here we describe a detailed protocol for preparing single-cohort colonies for this analysis. Six to eight days after single-cohort colony preparation, precocious foragers that perform foraging tasks earlier than usual appear in the colony. Representative results indicated role-associated changes in HPG gene expression, suggesting role-associated HPG function. In addition to manipulating the colony population, analysis of the endocrine system is important for investigating role-associated physiology. Here, we also describe a detailed protocol for treating workers with 20-hydroxyecdysone (20E), an active form of ecdysone, and methoprene, a juvenile hormone analogue. The survival rate of treated bees was sufficient to examine gene expression in the HPGs. Gene expression changes were observed in response to 20E- and/or methoprene-treatment, suggesting that hormone treatments induce physiological changes of the HPGs. The protocol for hormone treatment described here is appropriate for examining hormonal effects on worker physiology.

Here we describe our detailed protocol for the preparation of single-cohort honeybee colonies&#160;&#8211; a useful tool for analyzing the role-associated worker physiology. We also describe detailed protocols for treating workers with juvenile hormone and ecdysone to evaluate the involvement of these hormones in the regulation of worker behavior and/or physiology.

Honeybee workers are engaged in various tasks related to maintaining colony activity. The tasks of the workers change according to their age (age-related ...

Neuropharmacological Manipulation of Restrained and Free-flying Honey Bees, Apis mellifera

Honey bees demonstrate astonishing learning abilities and advanced social behavior and communication. In addition, their brain is small, easy to visualize and to study. Therefore, bees have long been a favored model amongst neurobiologists and neuroethologists for studying the neural basis of social and natural behavior. It is important, however, that the experimental techniques used to study bees do not interfere with the behaviors being studied. Because of this, it has been necessary to develop a range of techniques for pharmacological manipulation of honey bees. In this paper we demonstrate methods for treating restrained or free-flying honey bees with a wide range of pharmacological agents. These include both noninvasive methods such as oral and topical treatments, as well as more invasive methods that allow for precise drug delivery in either systemic or localized fashion. Finally, we discuss the advantages and disadvantages of each method and&#160;describe common hurdles and how to best overcome them. We conclude with a discussion on the importance of adapting the experimental method to the biological questions rather than the other way around.

Neuropharmacological Manipulation of Restrained and Free-flying Honey Bees, Apis mellifera

This manuscript describes several protocols for administering pharmacological agents to honey bees, including simple noninvasive methods for free-flying bees, as well as more invasive variants that allow precise localized treatment of restrained bees.

Honey bees demonstrate astonishing learning abilities and advanced social behavior and communication. In addition, their brain is small, easy to visualize ...

Anti-RDL and Anti-mGlutR1 Receptors Antibody Testing in Honeybee Brain Sections using CRISPR-Cas9

Cluster Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is a gene editing technique widely used in studies of gene function. We use this method in this study to check for the specificity of antibodies developed against the insect GABAA receptor subunit Resistance to Dieldrin (RDL) and a metabotropic glutamate receptor mGlutR1 (mGluRA). The antibodies were generated in rabbits against the conjugated peptides specific to fruit flies (Drosophila melanogaster) as well to honeybees (Apis mellifera). We used these antibodies in honeybee brain sections to study the distribution of the receptors in honeybee brains. The antibodies were affinity purified against the peptide and tested with immunoblotting and the classical method of preadsorption with peptide conjugates to show that the antibodies are specific to the corresponding peptide conjugates against which they were raised. Here we developed the CRISPR-Cas9 technique to test for the reduction of protein targets in the brain 48 h after CRISPR-Cas9 injection with guide RNAs designed for the corresponding receptor. The CRISPR-Cas9 method can also be used in behavioral analyses in the adult bees when one or multiple genes need to be modified.

Presented here is a protocol to use the CRISPR-Cas9 system for reducing the production of a protein in the adult honeybee brain to test antibody specificity.

Cluster Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is a gene editing technique widely used in studies of ...

Experimental Viral Infection in Adult Mosquitoes by Oral Feeding and Microinjection

Mosquito-borne viruses (MBVs), which are infectious pathogens to vertebrates, are spread by many mosquito species, posing a severe threat to public health. Once ingested, the viruses must overcome the mosquito midgut barrier to reach the hemolymph, from where they might potentially spread to the salivary glands. When a mosquito bites, these viruses are spread to new vertebrate hosts. Similarly, the mosquito may pick up different viruses. In general, only a tiny portion of viruses may enter the salivary glands via the gut. The transmission efficiency of these viruses to the glands is be affected by the two physical barriers found in different mosquito species: midgut barriers and salivary glands barriers. This protocol presents a method for virus detection in salivary glands of Aedes aegypti&#39;s&#160;following oral feeding and intrathoracic injection infection. Furthermore, determining whether the guts and/or salivary glands hinder viral spread can aid in the risk assessments of MBVs transmitted by Aedes aegypti.

This methodology, which included oral feeding and intrathoracic injection infection, could effectively assess the influence of midgut and/or salivary gland barriers on arbovirus infection.

Mosquito-borne viruses (MBVs), which are infectious pathogens to vertebrates, are spread by many mosquito species, posing a severe threat to public ...

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

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Propagation of Homalodisca coagulata virus-01 via Homalodisca vitripennis Cell Culture

Preparation of Single-cohort Colonies and Hormone Treatment of Worker Honeybees to Analyze Physiology Associated with Role and/or Endocrine System

Neuropharmacological Manipulation of Restrained and Free-flying Honey Bees, Apis mellifera

Anti-RDL and Anti-mGlutR1 Receptors Antibody Testing in Honeybee Brain Sections using CRISPR-Cas9

Experimental Viral Infection in Adult Mosquitoes by Oral Feeding and Microinjection