Experimental Procedure (updated version)

Independent Variable: Impaired honey bees

Dependent Variable: Foraging rates and health of the nicotinic acetylcholine receptors

Control Variable:  Non- impaired honey bees                                                                                                                                                           

  1. Preparing Bee Restraining Harness:

Make simple restraining harnesses for the honey bees out of plastic soda straws, hard plastic tubing, or machined metal tubing ( approximately 0.9 cm inner diameter and have 1-2 mm wall thickness).Cut 3 cm sections of the tubing. Next, partially trim away approximately half of the upper 1 cm of the harness to make, this makes it easier to hold the bee in place while fastening the harness.Cut a 0.2 by 6 cm strip of duct tape and attach one end to one side of the bee harness, leaving the other end free.

  1. Collecting, sample worker bees:  Use vials to hold the bees. Collect the bees quickly to lessen the time they spend in the vials. Keep the vials with bees in them in a small box in the shade. Transfer the box with the vials and bees to the lab, and place the vials into an ice-water “slurry” until the bees stop moving. Once a bee is immobile, remove the vial from the ice immediately to avoid overexposure to cold, and place the bee into a restraining harness.  

3.Restraing the bees:                                                                                                       Place the bee in the harness with its dorsal thorax facing the cut-out portion of the tubing and its head just above the top of the tubing. Gently press the bee close to the tubing so its mouth extends beyond the edge of the tubing. Slide the strip of duct tape in between the head and the thorax on the dorsal side of the bee and firmly attach the free end to the side of the harness. The bees should be placed in two different areas. One conditioning area, is the control bees without Imidacloprid, and the other is the experimental conditioning are, the bees subjected to Imidacloprid.

  1. Feeding the bees:                                                                                                         About 30 min after harnessing, after the bees have recovered from the initial setup, feed the bees 3-4 μl 0.5 M sucrose (mixed in water). This amount will be sufficient for a feeding-to-conditioning interval of 3-4 hr. Feed 1 μl 0.5 M sucrose for approximately each hour of wait time between feeding and conditioning to ensure the bees survive the interval, but are hungry enough at the beginning of conditioning to be motivated to enough to stick the PER out. NOTE: “With a feeding to conditioning interval of 24 hr, make sure all the bees are fed to satiation with 0.5 or 1 M sucrose at least 24 hr before conditioning will begin. At room temperature, bees require approximately 24 hr before they are motivated enough to respond well in a conditioning protocol.” During the time between feeding and conditioning place the bees on the countertop in a quiet area of the lab to avoid unnecessary disturbance. If the laboratory has low “ambient humidity”, place bees in a container with wet paper towels during the time interval. This prevents desiccation, because the bees may die if they are exposed to low humidity for long time periods.
  2. Conditioning:                                                                                                                     A few minutes before beginning conditioning, test the bee’s motivation to feed by touching their antennae with a small droplet of 0.5 M sucrose solution (the same concentration used during feeding). Don’t allow them to feed during this test. If they respond by extending their proboscis, they are probably motivated enough to learn and can be used for the protocol.

6.Set up the Odor Delivery System, and the Airflow Source and Flow Rate

  1. Administering the Imidacloprid:                                                                                       Using, most likely a syringe or test tube, 12 ng of Imidacloprid will be placed on the back of the HoneyBees thorax.It is vital to the test that the bees with Imidacloprid are placed very far away from the control bees so there is no contamination of the control bees.

7.5 Delivering the Sucrose:                                                                                                      Use a toothpick to deliver the sucrose. NOTE: It is best to use a plastic toothpick since the wooden toothpicks can have an odor that will influence the bee’s responses to the conditioned stimulus. Dip the tip of the toothpick into the sucrose solution, and  when it is time to present the unconditioned stimulus, hold the toothpick in front of the bee and let the bee lick the sucrose from the toothpick for 1 sec. Replace the toothpick every single time to avoid buildup of sucrose.

  1. Presenting the Sucrose:                                                                                                      Hold the tip of the needle approximately 2 cm from the bee while the odor is presented. As soon as the feeding signal sounds, lightly touch the antenna until the bee extends its proboscis, then feed the bee. Use a reward amount between 0.2 and 0.8 μl. The bee will consume the entire droplet within the time period  for the unconditioned stimulus (~1-2 sec). Watch for sucrose build up on the antenna. Modify the feeding procedure if this occurs. To ensure accurate pairing of the odor cue and the sucrose reward, make sure to present the droplet of sucrose solution to the bee as soon as possible after the tone.
  2. The testing:                                                                                                                      After conditioning use unreinforced test trials to assess how well the bees learned and/or remember the conditioned association. NOTE: Hopefully the bees with the Imidacloprid won’t remember the and or didn’t learn the scent of the sucrose, proving our hypothesis.
  3. Recording the data:                                                                                                         We will most likely use “Video Analysis” , and record the responses of each group, compare them, and go from there.

Once Again, another hive update!!!

Langstroth-Hive-PartsWOOOOOO, HOOOOOOOO! Well, I am pretty positive that my bees definitely made it through the harsh spring, and they are doing good. Last Thursday, on 5/3/18, I switched the top box on the hive to the bottom. This was done to give the bees more space to produce more food and to also prevent swarming. Here is a photo of what a normal two deep hive would look like —————————->

Overall Outlook on CCD

Colony Collapse Disorder is a very important, and serious disease. For example, the USDA has poured millions of dollars into finding a solution to the disease (Bodnar 2008). At this point, we are just beginning to understand the possible mitigating factors to CCD and how they may interplay with each other.  The coming few years will likely be hard ones on the apiary and agricultural industries (Bodnar 2008). Even though since, 2006, CCD has been killing about 31.1% of honeybee colonies each year, there is still some hope. So far, in the year 2017, there has only been a loss percentage of about 22-25%. This is most likely due to the increased levels of pollen from the spring. These increased levels help the honeybees, because when they are just getting back outside of the hive after the long harsh winter, and they would normally have to look really hard to find fresh pollen, but due to the increased levels the pollen can be found almost anywhere. That being said, for the next year or so CCD will most likely be held back to a minimum and as long as we help the bees through this epidemic, the bees should be just fine, and they will recover from this horrible disease called, Colony Collapse Disorder.

Neonicotinoidsssssss:}|

Neonicotinoids are a relatively new class of insecticides, which are chemically related to nicotine. There are seven types of Neonicotinoids, Imidacloprid, Acetamiprid, Clothianidin, Dinotefuran, Thiamethoxam, Nithiazine, and Thiacloprid. Imidacloprid is the most widely used neonicotinoid in the world (Yamamoto, Izuru, 1999). Although less toxic to mammals and other vertebrates, neonicotinoids are highly toxic in small quantities to many invertebrates, including beneficial insects such as bees. Honeybees come into contact with at least six toxic chemicals or Neonicotinoids every single day (EPA 2013). Therefore making them very susceptible to these harmful neonicotinoids. Neonicotinoid residues in plants only become of importance for bees once they are exposed. The most important measures of exposure are the concentrations in bee-collected plant materials, such as pollen, bee products like bee bread, honey and beeswax, and in the bees themselves (Gestel 2012).

 

Here is some common methodology used in PER assays

Relative, to testing the Proboscis Extension Reflex(PER), there are many common methodologies, to test it. Such as the PER conditioning protocol, developed for the honeybee, provides an ecologically-relevant and easily quantifiable means for studying several different mechanisms of learning in many insect species (Smith 2014).  The PER conditioning protocol was designed over fifty years ago and is still very common. The PER procedure provides a robust and easy-to-employ framework for studying several different ecologically relevant mechanisms of behavioral plasticity. It is easily adaptable for use with several other insect species and other behavioral reflexes (Smith 2014).  Honey bees will readily exhibit simple and easily quantifiable behaviors in the laboratory. This makes it possible to study, in a controlled setting, the mechanisms that influence this ecologically relevant behavior. PER can also be used within a conditioning protocol to investigate stimulus perception and learning and memory under different treatment conditions, which are designed to reveal the behavioral and neural mechanisms (Smith 2014). PER protocols have also provided an important way to evaluate the sublethal effects of environmental conditions as well as toxins on health and foraging efficiency of honey bees. Due to the various ways to test the PER, Imidacloprid could be substituted in and used to test the impairment of the PER, in the protocol experiment.

Hive Update!!!!

Hello followers!!!! So yesterday, April 9th, I decided I would go outside and check on the hive. To do this I leaned up against the hive, tapped on it and listened. I heard a lot of buzzing, which is a good sign because that means the bees are still alive!!!! Woooo hooooo, but anyways hopefully the bees will be valid, in good health, and ready for the experiment in mid-may!

I’m currently writing an Abstract for my final paper

 So far this is what I have……..If any of my followers are reading this blog, please comment and let me know what you think of it so far! Also, don’t forget to drop a like on this post!

As general pollinators, honey bees or Apis Mellifera add an estimated fifteen billion dollars of value to the crops they pollinate in the United States alone (Fine, 2017). Due to this, honey bees are arguably one of the most important pollinators in the world. Unfortunately, these vital creatures are dying from Colony Collapse Disorder (CCD). Multiple factors including pesticides, pathogens, parasitism, and poor nutrition have been implicated; but at this time, no single element has been successfully shown to cause colony failure (Fine, 2017). Sub-lethal exposure to agricultural- or beekeeper-applied neonicotinoids can weaken honey bee immune systems (vanEngelsdorp, 2009). These harmful neonicotinoids can seriously damage the Proboscis Extension Reflex, which is part of the honey bees feeding, and foraging behaviors.