# ASSESSING RESOURCE LIMITATION IN BUMBLEBEE ECOLOGY

Fabrice Requier ID, Kim K. Jowanowitsch, Katharina Kallnik, and Ingolf Steffan-Dewenter

## Study Description

Flower abundance and diversity benefit bee populations. However, flower resource availability is disturbed in farmlands and little is known about which protein or carbohydrate resources may limit bee fitness. Here, we test the hypothesis of complementary resource limitation using a novel experimental approach of supplemental feeding in differently structured landscapes. We applied this assumption to bumblebees. We found positive effects of seminatural habitats, as a foraging resource of pollen and nectar, on reproductive performance and behavior of bumblebees. Moreover, the results suggest co-limitation in pollen and nectar in mixed landscapes, providing new perspectives on improving pollinator-friendly practices for the conservation of pollinators in farmlands.

<footnote>Requier, F., K. K. Jowanowitsch, K. Kallnik, and I. Steffan-Dewenter. 2020. Assessing Resource Limitation in Bumblebee Ecology. Bull Ecol Soc Am 101(2):e01675. https://doi.org/10.1002/bes2.1675</footnote>
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Photo 1. We performed supplemental feeding as an indicator of landscape resource limitation in bumblebees (Bombus terrestris), assuming that colony growth and reproductive performance should depend on the continuous supply of carbohydrates and proteins, through the foraging for nectar and pollen, respectively. We used a factorial design of supplemental feeding consisting of the addition of sugar water (carbohydrate, supplemented or not) crossed by pollen (protein, supplemented or not). Two types of tanks were placed at the entrance of wooden nesting box. Horizontal tanks were fulfilled with freeze-dried pollen, or nothing, according to the treatment allocated. A vertical tank (Greiner 250-mL tube) was fulfilled with sugar water, or nothing, according to the treatment allocated. In the field (see lower row), the tanks were refilled every week and covered with gray polystyrene cylinders for isolation. Whenever bumblebees wanted to leave the nest to forage, they had the choice to fly and collect nectar and pollen on the flower resources surrounding the colony nest, or they could collect sugar water and pollen (depending on the experimental treatment) in the two feeders at the colony entrance. Photo credit: Kim K. Jowanowitsch.
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Photo 2. Establishing *Bombus terrestris* colonies through the rearing of wild-caught queens. In spring, foraging queens were caught from the wild in the neighborhood of the Hubland campus at the University of Würzburg (Germany). Wild-caught queens were then placed in individual rearing boxes and kept in climate chambers. Temperature, light, and humidity were controlled, and queens were fed with carbohydrates and proteins. After 4–5 weeks of wild-caught queen rearing in climate chambers (from the first date of egg-laying), each starting colony was placed in a cardboard box with addition of moss and kapok for isolation and nesting facility (see upper row) and moved to the field. The colonies were brought back to the laboratory at the end of the experiment (see lower row) to quantify reproductive performance. Photo credit: Kim K. Jowanowitsch.
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Photo 3. The study was carried out in a German region characterized by a mixed agricultural landscape, mainly composed of arable land, seminatural habitats, and settlement areas. We selected eight sites to cover a gradient in the amount of seminatural habitats in the surrounding landscape. Four colonies of *B. terrestris* were placed at each site, totaling 32 colonies. The colonies were distinguished at each site by supplemental feeding of (1) sugar water (carbohydrate), (2) pollen (protein), (3) both sugar water and pollen, or (4) none as a control. During the field experiment, the 32 colonies were monitored once a week. In the field, we registered the mass of each colony and recorded the number of returning foragers. We also recorded the presence (or absence) of pollen loads on their legs as an indicator of the colonies' investments to pollen collection. Photo credit: Kim K. Jowanowitsch.
<figure><img src="image_4.png" /><figcaption>Step 2</figcaption></figure>

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Photo 4. At the end of the experiment, the 32 colonies were collected to quantify reproductive performance. First, in the field, the entrance of the colony was closed after sunset to ensure that no bumblebees were outside the nest. Then, colonies were brought back to the laboratory, weighed, and frozen in their cardboard box. The number of adults was counted (step 1) as well as the number of larvae and pupae (step 2) for workers, males, and queens. Males and young queens produced (brood cells and newly emerged queens) were used to estimate reproductive performance of the colony. Photo credit: Kim K. Jowanowitsch.

These photographs illustrate the article “Limitation of complementary resources affects colony growth, foraging behavior, and reproduction in bumble bees” by Requier, F., Jowanowitsch, K. K., Kallnik, K., Steffan-Dewenter, I., published in *Ecology*. https://doi.org/10.1002/ecy.2946