Location: Pollinating Insect-Biology, Management, Systematics Research
Project Number: 2080-21000-019-015-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Apr 15, 2018
End Date: Aug 30, 2021
Objective:
Objective 1. Determine how alkali bees adjust their investment in reproductive effort and survivorship in response to immune challenge.
Objective 2. Identify mechanisms underlying tradeoffs between reproductive effort, survivorship, body condition, and susceptibility to viral infection in alkali bees.
Objective 3. Evaluate the frequency of viral infections and mechanisms of vertical transmission of viruses in alkali bees.
Objective 4: Identify via sequencing the viral genotypes infecting the bees and compare to known genotypes in honeybees.
Objective 5: Measure the levels of genes involved in viral immunity in the bees from the various treatments.
Approach:
Experiments will be performed both in the field and in the lab.
In Touchet, Washington, bees will used from local bee beds. A field laboratory will be created at a local facility. In June-July 2018, we will capture newly emerged bees from bee beds. Upon capture, females will be randomly assigned to one of the following treatments:
1. Immune challenge injections of heat-killed E. coli. This is a technique commonly used to cause an upregulation of the immune system without having pathogenic effects.
2. Vehicle control injection of Ringer’s solution. This will serve as a control for the injection of foreign substance into the body, but should not elicit an immune response.
3. Handling control Sham injection. This will serve as a control for the stress of handling, without eliciting an immune response.
All bees will be provided sugar water and pollen ad libitum and be randomly assigned to one of four experimental periods: 1, 5, 10, or 25 days. We will include 25 bees in each treatment group for each experimental period, for a total of 300 experimental bees.
To assess longevity, we will perform mortality. We will flash freeze dead bees immediately upon discovery by submerging them in liquid nitrogen. Bees that survive the entire experimental period will also be flash-frozen in liquid nitrogen to preserve tissue and RNA. We will use these bees to measure response to treatment in reproductive effort (egg development), body condition (fat stores), viral loads (RNA-based detection of known viruses), and the mechanisms underlying these tradeoffs (RNA-based quantification of bee genes involved in immune response and reproduction).
We will also collect newly emerged females and actively nesting females to identify the viruses most common in alkali bees and better describe the dynamics of viral transmission.
Upon return to USU, we will dissect these bees to measure condition (lipid stores), reproductive potential (oocyte development), and viral loads. We will perform all dissections to maintain integrity of the viral RNA while preserving tissue structure.
We will separate ovaries and measure the stage of oogenesis using previously established methods. We will qualitatively score the size of the fat body as a measure of nutritional stores and body condition.
After abdomen dissections, the hindgut, fat body, and ovaries will be independently homogenized to isolate RNA. We will use quantitative real-time PCR (qRT-PCR) to measure viral loads in the gut and in the ovaries. We will therefore, quantify gene expression of bee genes in the reproduction, nutrient-sensing, and immunity pathways in the fat body to characterize the mechanisms underlying the observed life history tradeoffs. We anticipate including n = 8 bees from each of our experimental groups and newly emerged and actively nesting females in this part of the study.
