Project Number: 2080-21000-017-17-R
Project Type: Reimbursable Cooperative Agreement
Start Date: Feb 1, 2018
End Date: Jan 31, 2019
The long-term goal is to understand and share how the health of one generation of pollinators influences the pollination potential of the next generation. Accordingly, we will elucidate factors that mediate variation in development of Megachile rotundata, a solitary alfalfa leafcutting bee (ALCB), with high economic and agricultural value. Specifically, we will investigate how variation in maternal health interacts with genotype to influence progeny development, and, thus, the quantity and quality of bees available to pollinate the following year’s crop. We aim to achieve this long-term goal by meeting two objectives with experiments involving ALCBs. 1. Determine how maternal condition (as mediated by nutritional manipulations) influences reproductive strategy, offspring developmental patterns, and offspring survival; 2. Identify the mechanisms for maternal effects on offspring phenotype, including variation in offspring provisioning and maternal inheritance of RNA transcripts in oocytes.
The two objectives will involve field and laboratory experiments with ALCBs, molecular biology applications, and computational analyses. We will subject newly emerged female ALCBs to nutritional treatments designed to manipulate maternal condition, and determine the effects on reproductive strategy and progeny phenotypes. We will determine the mechanisms by which maternal condition and genotype influence offspring development. We will consider effects on provision size, maternal transcripts deposited in eggs, and genetic variants. Mated females will be randomly assigned to one of four treatment groups: (1) sugar water only, (2) sugar water + pollen (protein), (3) sugar water + pollen + fatty-acid synthesis inhibitor (2% TOFA), (4) sugar water + pollen + fatty acid supplement (0.5% oleic acid). These treatments will be administered daily in in individual cages for 3 d. Food will be replaced, and the amount consumed will be recorded daily. We anticipate recruiting at least 20 surviving females from each of the 3 source populations into each treatment group for a total of 240 females. After the 3 d treatment period, females will be released into field cages to track reproductive activity. Reproductive activity will be tracked using established methods. At the end of the nesting season, all of the nests will be recovered and stored. Bee development and survival will be tracked, and the adult progeny number, weight, intertegular length, and sex will be recorded for summer-emerging and overwintered bees. We will subject newly emerged females to nutritional treatments as before, using only those treatments that yield the most significant differences in diapause fates among progeny. After the treatment periods, females will be paint-marked, released, and monitored for nesting activities in field cages. Rather than allowing eggs to develop in the field, we will collect them upon oviposition for two purposes: (a) lab-rearing on different provision sizes to parse the effects of maternal condition and larval diet on offspring development, and (b) mRNA and miR sequencing to characterize differences in maternal RNAs associated with maternal condition and their influence on offspring development. In the laboratory, eggs will be reared on large or small natural, uniform provisions. Development under constant temperature will be monitored through cocoon spinning. Emerged adults will be weighed and sexed and subsequently be sacrificed for lipid analysis. For another set of experiments, once a female has begun laying eggs, we collect 1 freshly laid (24 h) egg and 1 mature oocyte; these samples will be frozen. We will rear out the next laid egg to get an indicator of what the diapause fate of the egg we collected would have been. Frozen eggs and oocytes will be measured, maternal RNA will be profiled, and genetic variants associated with diapause will be identified. For RNA analysis, total RNA will be sent to University of Illinois Roy J. Carver Biotechnology Center for mRNA enrichment, library preparation, and sequencing on the Illumina HiSeq 4000 platform.