Name: preparation of virus-enriched inoculum for oral infection of honey bees (apis mellifera)
Uploaded: 2020-08-26T15:00:00
Description: Watch this Scientific Journal Video about Preparation of Virus-Enriched Inoculum for Oral Infection of Honey Bees Apis mellifera at JoVE.com

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.

1. Mass bee extraction option 1: larval self-removal
<ol>
	<li>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.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>After the 24 h egg-layin...

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 (<strong class="xf...

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&#34; x 9-1/8&#34; (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 &#186;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...

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 ...

Our protocol demonstrates how to produce honey bee virus particles en masse for their use in controlled, high-throughput bioassays for rapid screening of viral treatment effectiveness. The assay uses easy-to-culture bee pupae rather than cell culture, reducing the labor of obtaining experimental subjects. The virus can then be used to produce predictable mortality curves and repeatable bioassays.There's been substantial interest in understanding how so-called honey bee viruses affect a larger host range. This virus production technique can be paired with other pollinator systems to test the infectivity. For mass bee extraction by larval self-removal, cage a honey bee queen on an empty, drawn-out Langstroth frame and return her to her colony.After allowing the queen to lay eggs on the frame for 24 hours, check the frame to ensure that most of the comb cells contain newly laid eggs and release the queen. Exactly 192 hours after caging the queen, remove the frame from the colony and brush off any adult bees. Upon return to the laboratory, place the frame in an incubator set to the internal conditions of a hive and thoroughly clean containers of the same height and width of the larva-filled frame with sequential soap and water, bleach, and ethanol washes.Line the bottom of the containers with several layers of paper towels and add several overlapping layers of thinner absorbent material. When the containers are ready, place the frame onto the container's focal larvae side down and use a piece of tented aluminum foil to cover the container and frame. After overnight incubation, carefully lift individual wipes from the top layer of each container to allow the larvae to be gently poured onto a transfer tray.Then, use blunt, soft forceps to spread the larvae across the surface of the trays without touching and place the tray covered with tented foil into the incubator until the larvae pupate and mature to the wide-eye stage. For virus injection of the pupae, mix the virus particles of interest at the appropriate concentration in sterile PBS in a 15-milliliter tube and attach an injector apparatus to a manual multipipette. To test the injector efficacy, load the injector with 100 microliters of water and dispense the water in one-microliter doses.If the injections are accurate, load 100 microliters of the virus particle solution into the injector and gently grasp one pupa along the thorax, applying just enough pressure to force internal fluids into the abdomen to make the tergite divisions more apparent. Stabilizing the arm holding the syringe against the lab bench for support, insert the needle just into the cuticle between the third and fourth abdominal tergites and inject one microliter of virus solution. When all the pupae have been injected, re-cover the tray with tented foil before returning the pupae to the incubator for another three to five days.At the end of the incubation, pool the pupae by colony source in individual 50-milliliter conical centrifuge tubes and vortex to homogenize the contents. To perform a viral feeding bioassay, plug the feeder holes of clean cages with an appropriately sized centrifuge tube and partially fill 15-milliliter centrifuge tubes with freshly prepared feeding solution. Then, invert the tubes and use a thumbtack to poke one to two holes around the tips.Next, brush all of the newly emerged bees from their emergence boxes into a collection receptacle lightly coated around the edges with vegetable oil and gently mix the bees by hand. Transfer 35 individual bees into a single, greased cup and place the contents of the cup into an acrylic cage. To prepare the virus inoculum, mix an appropriate quantity of thawed, concentrated virus particles with 30%sucrose solution in one sterile container per treatment type and add 600 microliters of virus solution onto individual inoculum trays.Then, carefully insert each inoculum tray into their corresponding cages, taking care to not accidentally release any bees. When all of the inocula have been consumed, replace the centrifuge tubes from the top feeder hole with the prepared feeder tubes and record the mortality within each cage at 12 hour intervals for the first 72 hours of the experiment and every 24 hours thereafter. To remove dead bees, slide the cage door up just far enough to allow a pair of alcohol-sterilized forceps to be inserted to scoop out the dead bees.Depending on the design of the experiment, place live specimens from each cage into individual centrifuge tubes on dry ice to allow sampling for viral titer measurements. In this representative larger-virus harvesting effort, every pupa was initially injected with an approximately 95%Israeli acute paralysis virus inoculum. Although 10 out of the 16 colony samples involved in these extractions contained highly pure virus, other samples varied in their virus proportion, with some even being dominated by other viruses, such as deformed wing virus.As illustrated in the table, threshold cycle values can be used as a predictor of proportion. Comparing the dose response-survival curves of honeybees fed the same virus particles during two different years reveals that, despite identical testing parameters, the trials conducted in 2018 and 2019 produced noticeably different survival responses in all but the control treatment groups, which received no virus in their sucrose inoculum. Honeybee responses to viral infection can vary greatly depending on the season, hive, or even inoculum distribution within individual cages.Therefore, large sample sizes are critical for confirming treatment effects. Using stocks of virus particles, the experiment can be expanded to include injections of adult honeybee workers, queens, or drones, or to other species of pollinators to assess the host range. Infectious stocks of virus can be used to test how the infection interacts with other factors, like diet, chemical exposure, or social environments.

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Protocols for Oral Infection of Lepidopteran Larvae with Baculovirus

Baculoviruses are widely used both as protein expression vectors and as insect pest control agents. This video shows how lepidopteran larvae can be infected with polyhedra by droplet feeding and diet plug-based bioassays. This accompanying Springer Protocols section provides an overview of the baculovirus lifecycle and use of baculoviruses as insecticidal agents, including discussion of the pros and cons for use of baculoviruses as insecticides, and progress made in genetic enhancement of baculoviruses for improved insecticidal efficacy.

In this video, we demonstrate oral infection techniques of lepidopteran larvae with baculovirus in order to determine insecticidal efficiency.

Baculoviruses are widely used both as protein expression vectors and as insect pest control agents. This video shows how lepidopteran larvae can be ...

Preparation of Pancreatic Acinar Cells for the Purpose of Calcium Imaging, Cell Injury Measurements, and Adenoviral Infection

The pancreatic acinar cell is the main parenchymal cell of the exocrine pancreas and plays a primary role in the secretion of pancreatic enzymes into the pancreatic duct. It is also the site for the initiation of pancreatitis. Here we describe how acinar cells are isolated from whole pancreas tissue and intracellular calcium signals are measured. In addition, we describe the techniques of transfecting these cells with adenoviral constructs, and subsequently measuring the leakage of lactate dehydrogenase, a marker of cell injury, during conditions that induce acinar cell injury in vitro. These techniques provide a powerful tool to characterize acinar cell physiology and pathology.

We describe a reproducible method of preparing mouse pancreatic acinar cells from a mouse for the purpose of examining acinar cell calcium signals and cellular injury with physiologically and pathologically relevant stimuli. A method for adenoviral infection of these cells is also provided.

The pancreatic acinar  cell is the main parenchymal cell of the exocrine pancreas and plays a primary  role in the secretion of pancreatic enzymes into ...

Methods for Comparing Nutrients in Beebread Made by Africanized and European Honey Bees and the Effects on Hemolymph Protein Titers

Honey bees obtain nutrients from pollen they collect and store in the hive as beebread. We developed methods to control the pollen source that bees collect and convert to beebread by placing colonies in a specially constructed enclosed flight area. Methods were developed to analyze the protein and amino acid composition of the pollen and beebread. We also describe how consumption of the beebread was measured and methods used to determine adult worker bee hemolymph protein titers after feeding on beebread for 4, 7 and 11 days after emergence. Methods were applied to determine if genotype affects the conversion of pollen to beebread and the rate that bees consume and acquire protein from it. Two subspecies (European and Africanized honey bees; EHB and AHB respectively) were provided with the same pollen source. Based on the developed methods, beebread made by both subspecies had lower protein concentrations and pH values than the pollen. In general, amino acid concentrations in beebread made by either EHB or AHB were similar and occurred at higher levels in beebread than in pollen. Both AHB and EHB consumed significantly more of the beebread made by AHB than by EHB. Though EHB and AHB consumed similar amounts of each type of beebread, hemolymph protein concentrations in AHB were higher than in EHB. Differences in protein acquisition between AHB and EHB might reflect environmental adaptations related to the geographic region where each subspecies evolved. These differences could contribute to the successful establishment of AHB populations in the New World because of the effects on brood rearing and colony growth.

Here are methods to quantify nutrients in pollen before and after its conversion to beebread by two subspecies of honeybees. We describe techniques to measure beebread consumption and resulting protein titers in both subspecies.

Honey bees obtain nutrients from pollen they collect and store in the hive as beebread. We developed methods to control the pollen source that bees ...

Bone Conditioned Medium: Preparation and Bioassay

Autologous bone grafts are widely used in oral and maxillofacial surgery, orthopedics, and traumatology. Autologous bone grafts not only replace missing bone, they also support the complex process of bone regeneration. This favorable behavior of autografts is attributed to the three characteristics: osteoconductivity, osteogenicity, and osteoinductivity. However, there is another aspect: Bone grafts release a myriad of molecules, including growth factors, which can target mesenchymal cells involved in bone regeneration. The paracrine properties of bone grafts can be studied in vitro by the use of bone-conditioned medium (BCM). Here we present a protocol on how to prepare bone-conditioned medium from native pig cortical bone, and bone that underwent thermal processing or demineralization. Cells can be directly exposed to BCM or seeded onto biomaterials, such as collagen membranes, previously soaked with BCM. We give examples for in vitro bioassays with mesenchymal cells on the expression of TGF-&#946; regulated genes. The presented protocols should encourage to further reveal the paracrine effects of bone grafts during bone regeneration and open a path for translational research in the broad field of reconstructive surgery.

We describe here how to prepare bone-conditioned medium (BCM) and test its activity in vitro.

Autologous bone grafts are widely used in oral and maxillofacial surgery, orthopedics, and traumatology. Autologous bone grafts not only replace missing ...

Dissection and Observation of Honey Bee Dorsal Vessel for Studies of Cardiac Function

The European honey bee, Apis mellifera L., is a valuable agricultural and commercial resource noted for producing honey and providing crop pollination services, as well as an important model social insect used to study memory and learning, aging, and more. Here we describe a detailed protocol for the dissection of the dorsal abdominal wall of a bee in order to visualize its dorsal vessel, which serves the role of the heart in the insect. A successful dissection will expose a functional heart that, under the proper conditions, can maintain a steady heartbeat for an extended period of time. This allows the investigator to manipulate heart rate through the application of cardiomodulatory compounds to the dorsal vessel. By using either a digital microscope or a microscope equipped with a digital camera, the investigator can make video recordings of the dorsal vessel before and after treatment with test compounds. The videos can then be scored at a time convenient to the user in order to determine changes in heart rate, as well as changes in the pattern of heartbeats, following treatment. The advantages of this protocol are that it is relatively inexpensive to set up, easy to learn, requires little space or equipment, and takes very little time to conduct.

The abdominal dorsal vessel of the honey bee and other insects serves as the functional equivalent of the mammalian heart and plays an important role in nutrient transport, waste removal, immune function, and more. Here we describe a protocol for the visualization and pharmacological manipulation of bee heart rate.

The European honey bee, Apis mellifera L., is a valuable agricultural and commercial resource noted for producing honey and providing crop pollination ...

Monitoring Colony-level Effects of Sublethal Pesticide Exposure on Honey Bees

The effects of sublethal pesticide exposure to honey bee colonies may be significant but difficult to detect in the field using standard visual assessment methods. Here we describe methods to measure the quantities of adult bees, brood, and food resources by weighing hives and hive parts, by photographing frames, and by installing hives on scales and with internal sensors. Data from these periodic evaluations are then combined with running average and daily detrended data on hive weight and internal hive temperature. The resulting datasets have been used to detect colony-level effects of imidacloprid applied in a sugar syrup as low as 5 parts per billion. The methods are objective, require little training, and provide permanent records in the form of sensor output and photographs.

Sublethal doses of pesticides may affect colonies in ways that are difficult to detect using only periodic or visual methods. Methods to measure total adult bee mass, brood, and food resources by weighing hives and hive parts, photographing frames, and installing sensors, are provided. Data analysis is also addressed.

The effects of sublethal pesticide exposure to honey bee colonies may be significant but difficult to detect in the field using standard visual assessment ...

Micromanipulation Techniques Allowing Analysis of Morphogenetic Dynamics and Turnover of Cytoskeletal Regulators

Examining the spatiotemporal dynamics of proteins can reveal their functional importance in various contexts. In this article, it is discussed how fluorescent recovery after photobleaching (FRAP) and photoactivation techniques can be used to study the spatiotemporal dynamics of proteins in subcellular locations. We also show how these techniques enable straightforward determination of various parameters linked to actin cytoskeletal regulation and cell motility. Moreover, the microinjection of cells is additionally described as an alternative treatment (potentially preceding or complementing the aforementioned photomanipulation techniques) to trigger instantaneous effects of translocated proteins on cell morphology and function. Micromanipulation such as protein injection or local application of plasma membrane-permeable drugs or cytoskeletal inhibitors can serve as powerful tool to record immediate consequences of a given treatment on cell behavior at the single cell and subcellular level. This is exemplified here by immediate induction of lamellipodial cell edge protrusion by the injection of recombinant Rac1 protein, as established a quarter-century ago. In addition, we provide a protocol for determining the turnover of enhanced green fluorescent protein (EGFP)-VASP, an actin filament polymerase prominently accumulating at lamellipodial tips of B16-F1 cells, employing FRAP and including associated data analysis and curve fitting. We also present guidelines for estimating the rates of lamellipodial actin network polymerization, as exemplified by cells expressing EGFP-tagged &#946;-actin. Finally, instructions are given for how to investigate the rates of actin monomer mobility within the cell cytoplasm, followed by actin incorporation at sites of rapid filament assembly, such as the tips of protruding lamellipodia, using photoactivation approaches. None of these protocols is&#160;restricted to components or regulators of the actin cytoskeleton, but can easily be extended to explore in analogous fashion the spatiotemporal dynamics and function of proteins in various different subcellular structures or functional contexts.

We describe how micro- and photomanipulation techniques such as FRAP and photoactivation enable the determination of motility parameters and the spatiotemporal dynamics of proteins within migrating cells. Experimental readouts include subcellular dynamics and turnover of motility regulators or of the underlying actin cytoskeleton.

Examining the spatiotemporal dynamics of proteins can reveal their functional importance in various contexts. In this article, it is discussed how ...

Oral Bacterial Infection and Shedding in Drosophila melanogaster

The fruit fly Drosophila melanogaster is one of the best developed model systems of infection and innate immunity. While most work has focused on systemic infections, there has been a recent increase of interest in the mechanisms of gut immunocompetence to pathogens, which require methods to orally infect flies. Here we present a protocol to orally expose individual flies to an opportunistic bacterial pathogen (Pseudomonas aeruginosa) and a natural bacterial pathogen of D. melanogaster (Pseudomonas entomophila). The goal of this protocol is to provide a robust method to expose male and female flies to these pathogens. We provide representative results showing survival phenotypes, microbe loads, and bacterial shedding, which is relevant for the study of heterogeneity in pathogen transmission. Finally, we confirm that Dcy mutants (lacking the protective peritrophic matrix in the gut epithelium) and Relish mutants (lacking a functional immune deficiency (IMD) pathway), show increased susceptibility to bacterial oral infection. This protocol, therefore, describes a robust method to infect flies using the oral route of infection, which can be extended to the study of a variety genetic and environmental sources of variation in gut infection outcomes and bacterial transmission.

Oral Bacterial Infection and Shedding in Drosophila melanogaster

This protocol describes methods to orally expose and infect the fruit fly Drosophila melanogaster with bacterial pathogens, and to measure the number of infectious bacteria shed following gut infection. We further describe the effect of immune mutants on fly survival following oral bacterial infection.

The fruit fly Drosophila melanogaster is one of the best developed model systems of infection and innate immunity. While most work has focused on systemic ...

Assessing Agrochemical Risk to Mated Honey Bee Queens

Current risk assessment strategies for honey bees rely heavily upon laboratory tests performed on adult or immature worker bees, but these methods may not accurately capture the effects of agrochemical exposure on honey bee queens. As the sole producer of fertilized eggs inside a honeybee colony, the queen is arguably the most important single member of a functioning colony unit. Therefore, understanding how agrochemicals affect queen health and productivity should be considered a critical aspect of pesticide risk assessment. Here, an adapted method is presented to expose honey bee queens and worker queen attendants to agrochemical stressors administered through a worker diet, followed by tracking egg production in the laboratory and assessing first instar eclosion using a specialized cage, referred to as a Queen Monitoring Cage. To illustrate the method&#39;s intended use, results of an experiment in which worker queen attendants were fed diet containing sublethal doses of imidacloprid and effects on queens were monitored are described.

This protocol was developed to enhance the understanding of how agrochemicals affect honey bee (Apis mellifera) reproduction by establishing methods to expose honey bee queens and their worker caretakers to agrochemicals in a controlled, laboratory setting and carefully monitoring their relevant responses.

Current risk assessment strategies for honey bees rely heavily upon laboratory tests performed on adult or immature worker bees, but these methods may not ...

An Improved Chemotaxis Assay for the Rapid Identification of Rhizobacterial Chemoattractants in Root Exudates

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward method to quickly identify the chemoattractants that could induce the chemotactic movement of rhizosphere growth-promoting bacteria on sterile glass slides via simple steps. Bacteria solution (OD600 = 0.5) and sterile chemoattractant aqueous solution were added dropwise on the glass slide at an interval of 1 cm. An inoculating loop was used to connect the chemoattractant aqueous solution to the bacterial solution. The slide was kept at room temperature for 20 min on the clean bench. Finally, the chemoattractant aqueous solution was collected for bacterial counting and microscopic observation. In this study, through multiple comparisons of experimental results, the method overcame multiple shortcomings of traditional bacterial chemotaxis methods. The method reduced the error of plate counting and shortened the experimental cycle. For the identification of chemoattractant substances, this new method can save 2-3 days compared to the traditional method. Additionally, this method allows any researcher to systematically complete a bacterial chemotaxis experiment within 1-2 days. The protocol can be considered a valuable resource for understanding plant-microbe interactions.

Here, we present an improved chemotaxis assay protocol. The goal of this protocol is to reduce the steps and costs of traditional bacterial chemotaxis methods and to serve as a valuable resource for understanding plant-microbe interactions.

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward ...