Progress 07/01/24 to 06/30/25

Outputs
Target Audience:Presentations were given to scientific audiences by graduate student Carrie Ganote, Dr. Lílian Caesar and Dr. Irene Newton. · ASM Ohio Chapter, "Getting bywith a little help fromyour friends: protective symbionts in the honeybee" Spring 2024 · Rutgers University, Microbiome Seminar Series, "Getting bywith a little help fromyour friends: protective symbionts in the honeybee" host: Lily Khadempour, Spring 2024 · Newton, ILG. 2024. EMBO Molecular mechanisms of evolution workshop. Heidelberg, Germany · Newton, ILG. 2024. Keynote at Beneficial Microbes, Madison, WI · Newton, ILG. 2024. Keynote at Lake Arrowhead Microbial Genomics conference Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project trained two graduate students at Indiana University, Carrie Ganote and Joselyn Molinar. Also, postdoctoral fellow Lílian Caesar was trained on this award. How have the results been disseminated to communities of interest?Results have been disseminated in posters and talks at conferences (ASMicrobe 2025) and at departmental seminars by Dr. Irene Newton. What do you plan to do during the next reporting period to accomplish the goals?Work is presently underway in Summer 2025 to repeat our analyses with more divergent queens of more distinct physiologies. These queens will be generated by the Tarpy lab and sent to both the Newton and Mattila labs for microbiome and behavioral assessments in June 2025. ? With our current data, we are performing additional coassemblies of treatments and assemblies of individuals to try to recover more MAGs.

Impacts
What was accomplished under these goals? For this reporting period, our project advanced in Objective 1 and 3, detailed below. To look at the relative abundances of bacteria across treatments, we mapped reads from each sample to the BEEXACT 16S database. For worker samples, we found that the microbiome is dominated mostly by Lactobacillus. We also found several other common worker associates such as Gilliamella, Snodgrassella, Frischella, Apilactobacillus, and Bombilactobacillus.For the queen samples, we found that a microbiome that is more stochastic than the workers. Many samples have a high abundance of Commensalibacter, but there wasn't one single group that consistently abundant across samples. We found no obvious clustering of the microbiome by treatment group, indicating that our high and low quality queens may not actually be sufficiently different. We then performed coassemblies of workers and queens separately using MEGAHIT, calculated sequencing depth, and binned contigs using MetaBAT2. From this, we recovered 156 bins for workers and 52 bins for queens. Of this, only 40 bins were medium quality or above (medium quality being >50% complete and <5% contamination by CheckM2). Bins were assigned taxonomy by GTDB-TK. From the queen coassembly, we recovered several representative MAGs several known bee-associated bacteria including Bombilactobacillus, Apilactobacillus, Bombella, Commensalibacter. From the workers, we recovered 4 Bombella species, 2 Commensalibacter, and a Bombilactobacillus. However, we are missing representative genomes for several abundant honey bee associates including Gilliamella, Snodgrassella, Frischella, Apilactobacillus. This indicates to us that there are issues in our assembly process. Using genomad, we recovered 58 high quality phage genomes from the coassemblies (7 of which were recovered in both worker and queen coassemblies). The vast majority of these contigs were classified as Caudoviricetes, but 2 were classified as filamentous phages. I calculated relative abundances across all worker and queen samples and found that workers tend to have an overall higher virus load as well as diversity than queens.We performed a clustering analysis using the relative virus abundances by calculating the Bray-Curtis index. We found clustering by sample caste, but not treatment. At Wellesley College (co-PI Heather Mattia), we worked on evaluating metrics of queen performance for high-quality and low-quality queens. The queens were produced at North Carolina State University (David Tarpy's lab, a co-PI), we monitored their in-colony performance at Wellesley, and then the queens were shipped to Indiana University for gut microbiome analysis (PI Irene Newton). Our goal was to link markers of high-quality queens with potential gut microbiome markers of good health and productivity (in contrast to a low-quality queen comparison group). At Wellesley, we assessed queen activity level, oviposition rate, laying pattern, and ability to attract worker attention (trophallaxis bouts and size of queen retinue). Observation hive and field colony research was conducted throughout the summer of 2024 and analysis of the data (video and image analysis) was completed by Spring 2025. A PCA of our summer work revealed that we needed to repeat the experiment (increase our sample size and have more strongly discriminated queen treatment groups), but that our methodology was solid and repeatable. Below is the PCA analysis of the performance metrics assessed at Wellesley and an abstract that was submitted for a planned presentation at the November 2025 Entomological Society of America. The royal treatment: does delayed entry of honey bee (Apis mellifera) larvae into the queen developmental pathway influence their performance as adults? Delilah Hammarlund1, Arianna Groover-Landis1, Caroline Goodwin1, Emily Cruz1, Chloe McGibbon1, Irene Newton2, David Tarpy3, Heather Mattila1 1 Department of Biological Sciences, Wellesley College, Wellesley, MA, 02481 2 Department of Applied Ecology, North Carolina State University, Raleigh, NC 27695 3 Department of Biology, Indiana University Bloomington, Bloomington, IN, 47405 The vitality of a honey bee (Apis mellifera, Hymenoptera: Apidae) queen is a crucial determinant of her colony's long-term success. In the past, a honey bee queen was expected to live for years and lay thousands of eggs per day. However, queen failure has become a pervasive concern in recent years, with reports of shortened lifespans, supersedure of young queens, and queen loss leading to a heightened focus on the link between queen quality and colony productivity. The rapid development of female larvae into adult queens is fueled by consistent provisions throughout the larval stage of abundant quantities of royal jelly, a nutrient-rich food that worker-destined larvae receive in limited quantities during the early days of larval development only. Ideally, female larvae enter the queen-specific developmental pathway as first instars. While it is possible for late-instar larvae to be redirected from a worker to a queen pathway, as might occur during emergency queen rearing, such queens often show markers of low quality as adults, including smaller size, reduced mating success, and altered pheromone production. This study explores the effect of delaying the switch to a queen-specific developmental trajectory on the behavior of queens as adults. High-quality and low-quality queens, produced by entry into the queen-developmental pathway as either first-instar or third-instar larvae, were monitored in observation hives for traits including oviposition rate, activity level, and the ability to harness worker care. Our results explore factors that may explain recent reports of declining queen performance in managed hives.

Publications