Bees Without Flowers: Before Peak Bloom, Diverse Native Bees Visit Insect-produced Honeydew Sugars

Bee foragers respond to complex visual, olfactory, and extrasensory cues to optimize searches for floral rewards. Their abilities to detect and distinguish floral colors, shapes, volatiles, and ultraviolet signals, and even gauge nectar availability from changes in floral humidity or electric fields are well studied. Bee foraging behaviors in the absence of floral cues, however, are rarely considered. We observed forty-four species of wild bees visiting inconspicuous, non-flowering shrubs during early spring in a protected, Mediterranean habitat. We determined experimentally that these bees were accessing sugary honeydew secretions from scale insects without the aid of standard cues. While honeydew use is known among some social Hymenoptera, its use across a diverse community of mostly solitary bees is a novel observation. The widespread ability of native bees to locate and use unadvertised, non-floral sugars suggests unappreciated sensory mechanisms and/or the existence of a social foraging network among solitary bees that may influence how native bee communities cope with increasing environmental change.


Abstract: 21
Bee foragers respond to complex visual, olfactory, and extrasensory cues to optimize 22 searches for floral rewards. Their abilities to detect and distinguish floral colors, shapes, 23 volatiles, and ultraviolet signals, and even gauge nectar availability from changes in floral 24 humidity or electric fields are well studied. Bee foraging behaviors in the absence of floral cues, 25 however, are rarely considered. We observed forty-four species of wild bees visiting 26 inconspicuous, non-flowering shrubs during early spring in a protected, Mediterranean habitat. 27 We determined experimentally that these bees were accessing sugary honeydew secretions from 28 scale insects without the aid of standard cues. While honeydew use is known among some social 29 Hymenoptera, its use across a diverse community of mostly solitary bees is a novel observation. 30 The widespread ability of native bees to locate and use unadvertised, non-floral sugars suggests 31 unappreciated sensory mechanisms and/or the existence of a social foraging network among 32 Introduction 35 Bees and flowers are inextricably linked. Their mutualistic relationship has been a 36 timeless focus for poets, artists and naturalists, as well as field ecologists, behavioral scientists, 37 and evolutionary biologists. The obsession is not without merit. Bee visits to flowers for nectar 38 and pollen are so crucial to angiosperm reproduction that bee preferences for floral colors, 39 shapes, and scents have been credited with driving floral trait evolution, a radiation in 40 angiosperm species diversity during the Late Cretaceous, and current plant community 41 composition (Regal 1977 Bees require sugar, usually as floral nectar, and protein, typically from pollen, for energy 50 and reproduction (Michener 2007). While specialist bee species are particular about their pollen 51 sources, bee visits to flowers for nectar sugars are usually indiscriminate (Linsley 1958). 52 Honeydew is a nectar-like carbohydrate-rich excretion produced as a feeding by-product by 53 phytophagous Hemipterans, such as scale insects (Hemiptera: Coccoidea) and aphids 54 (Hemiptera: Aphididae), that can sometimes be more nutrient-rich than floral nectar (Batra 55 1993). Some insects, most notably ants, attain increased fitness and longevity by using honeydew 56 as an additional sugar source (Zoebelein 1957

Experimental Design 111
We designed seven experimental treatments to differentiate the possible mechanisms and 112 causes of bee attraction to sooty mold, and randomly assigned them to 'naturally moldy' and 113 'mold-free' Adenostoma fasciculatum shrubs at three distinct 1-hectare experimental sites in 114 natural areas within Pinnacles National Park in San Benito County, California. Each selected site 115 was dominated by the large, hardy, allelopathic A. fasciculatum shrubs and included a mixture of 116 shrubs of similar stature that we could designate as either 'mold-free' (absent of sooty mold and 117 scale insects) or 'naturally moldy' (visibly covered on more than 50% of branches by sooty 118 mold). We applied each treatment (outlined below and in Table A1) to three woody shrubs of 119 pre-bloom A. fasciculatum at each of the three sites, for a total of nine shrub replicates for each 120 of seven treatments. 121 To control for any attraction, reflectance, or humidity signal of moisture, all seven 122 treatments consisted of 5 ounces of fluid sprayed on the assigned shrub, as follows: 123 Naturally-moldy plants were sprayed with either (i) water to assess baseline bee visitation 124 to moldy plants, or (ii) a natural, short-residual insecticide (Orange Guard® Water Based 125 Indoor/Outdoor Home Pest Control, active ingredient d-Limonene, 5.8%) to evaluate the 126 influence of live scale insects on bee visitation by halting their activity, while leaving sugars and 127 mold intact. 128 Mold-free plants were sprayed with either (i) water to quantify random bee visitation, (ii) 129 insecticide to test for an effect of this chemical on bee activity, (iii) non-toxic black paint to test 130 for an attraction to either the dark visual cue of mold or to potentially higher branch surface 131 temperatures, which recent research has found to be attractive to bees (Dyer et al. 2006), (iv) a 132 colorless, odorless 20% 1:1 Sucrose:Fructose solution mixed from chemical-grade sugars to 133 mimic the composition of insect honeydew (Wäckers et al. 2008), or (v) a combination of both 134 the black paint and the sugar mixture to simulate the attraction of natural mold and examine 135 interaction effects (treatments summarized in Table A1). 136

Sampling Protocol 137
Because the pilot study indicated that bee visits to honeydew were restricted to the early 138 season (Fig. 2), we concentrated our experiment in the period before peak bloom. We visited 139 each site three times between late February and late April, when native bee activity has begun at 140 Pinnacles National Park but prior to peak bloom of the plant community. Sampling was 141 conducted at one of the three sites per week on calm, sunny days over 15°C to ensure adequate 142 bee activity. At 9am on each sampling day, we began by refreshing all plants with 5oz of their 143 assigned treatment spray, which remained the same throughout the experiment. After waiting an 144 hour for the effect of the short-residual insecticide to take place and subside, and for bee activity 145 to approach peak levels for the day, a randomly ordered shrub list was divided between two 146 collectors, who spent five minutes sequentially netting all bees visiting each respective plant. 147 Temperature, wind speed, humidity, barometric pressure, and an estimate of cloud cover were 148 recorded every thirty minutes during sampling. We sampled all twenty-one plants at a site once 149 in the morning, beginning around 10am, and once in the afternoon, around 1pm, to capture bees 150 across the spectrum of diurnal activity. On sampling days, we recorded all flowering species in 151 bloom within the site, approximately a hectare in size, to provide an estimate of floral richness 152 and seasonal bloom progression. We also used an infrared thermometer to record surface 153 temperatures of three different external branches of each plant at noon on sampling days to test 154 for effects of potentially warmer, darker plants. 155

Specimens Processing & Data Management 156
All bees were labeled and pinned into field boxes each evening, then frozen for 48 hours 157 to protect from insect infestations, and transported to Utah where they were identified to 158 described species or unique morphospecies by experts at the USDA-ARS Pollinating Insect 159 Research Unit ("Logan Bee Lab"). Bee identifications were completed using high quality 160 'Leica' dissecting microscopes, the appropriate taxonomic keys where available, and confirmed 161 by comparison with the Logan Bee Lab's extensive reference collection of approximately 1.5 162 million specimens. Bees were assigned unique matrix code numbers that were included with 163 standard insect label data printed on labels affixed to each specimen pin. The unique identifier 164 and specimen field data were captured in a mySQL relational database, which was then managed 165 and queried for statistical analyses using Microsoft Access front end software. 166

Statistical Analyses 167
We employed a generalized linear mixed effects model with a negative binomial 168 distribution to assess differences in bee visitation rates among the different plant treatments, 169 which were modeled as fixed effects. We also used fixed effects to control for linear changes in 170 visitation rates over the course of the day and differences in average visitation rates among the 171 three sites. Variation in average visitation rates among the 63 plants and among the 9 sampling 172 dates were each accounted for as a random effect. Because we intentionally collected on warm, 173 sunny, calm days, the variation in environmental variables was minimal and their inclusion in the 174 statistical model did not change treatment significance. They were therefore omitted from the 175 final model for clarity. 176 Differences among treatments were estimated by comparing the number of bees that 177 would be expected to visit a given plant in a given five-minute observation window under 178 different conditions according to the negative binomial model. Ninety-five percent confidence 179 intervals for the effect of each treatment versus the control were calculated using the model's 180 variance-covariance matrix (Lawless 1987

Bee Collection 193
Despite a lack of floral cues, our 378 plant samples yielded 308 bees from forty-four 194 different species across nine genera and five of the six North American bee families (Table 1). 195 Approximately three-quarters of this bee abundance and diversity came from the two sprayed 196 sugar treatments (N=220, Species=38). Shrubs with naturally-occurring mold, and hence  Note: Bee counts are grouped into treatment ("trt") categories based on availability of natural 203 sugars from honeydew (Natural Mold), sprayed honeydew-mimic sugars (Sugar, Sugar + Paint), 204 or no known sugars accessible (Control, Insecticide, Mold + Insecticide, Paint). 205

Seasonal and Spatial Context 206
Floral richness increased linearly across the season and across 1-hectare experimental 207 sites as expected, from zero to thirteen species recorded in bloom during sampling, confirming 208 that sampling captured bee activity during the relatively nectar-depauperate period leading up to 209 peak bloom (which typically occurs at much higher richness and abundance at Pinnacles than 210 was observed during the study). Likewise, total bee specimens collected increased over the nine-211 week duration of the study at all three sites, from the first sampling round (N=85) to approach 212 peak bee activity during the third and final sampling round (N=146). Bee abundance differed 213 somewhat between sites, with bee activity at sites C (N=125) and B (N=115) consistently higher 214 than bee activity at site A (N=68). None of these temporal or site variables, however, nor any of 215 the environmental variables recorded (e.g. cloud cover, ambient temperature, wind speed, 216 humidity) influenced the modeled significance of treatment results. 217

Treatment Significance 218
Our model results confirmed our original observation that native bee visitation to pre-219 bloom Adenostoma fascicultum is significantly elevated on plants with sooty mold, despite this 220 resource lacking any floral cue (p=0.02). Model results also revealed our unadvertised 221 honeydew-mimic solution to be significantly more attractive to bees than mold (p<0.001; Fig. 3), 222 identifying simple sugars as the resource of interest in these nectar-poor landscapes. 223 Furthermore, though there was no base effect of the Insecticide treatment on bee visitation rate 224 compared to the Control (p=0.38, Fig. 4

), a significant interaction between the Mold and 225
Insecticide treatments reflects lower bee counts on moldy plants on which insecticide was 226 applied to stop the production of honeydew (p=0.04; Fig. 4), indicating that active sugar 227 production by live scale insects was a greater attraction to bees than residual sugars on branches 228 or any visual or olfactory cue from scale insect carapaces. Bees were also not using the dark 229 color of mold as a cue to locate honeydew, as evidenced by the lack of significant bee visitation 230 effects of the Paint treatment (p=0.44; Fig. 3) or any interaction between the Sugar and Paint 231 treatments (p=0.91; Fig. 4). Finally, since branch infrared thermometer readings did not differ 232 between treatments (p=0.55; Fig. A1), observed bee behaviors can also not be explained by a 233 response to thermal cues. From this experiment, we conclude that a highly diverse array of 234 native, mostly non-social bees are visiting pre-bloom Adenostoma fasciculatum shrubs for sugars 235 gleaned from honeydew, and are able to do so using foraging strategies outside the current 236 framework centered around floral displays. 237

No insecticide Insecticide
On mold-free plants

No paint Paint
No sugar spray Sugar spray being available during that time (Linsley 1958). For bees that emerge during the early season 300 into a habitat of unexpectedly poor floral resources, the ability to subsist on alternate sugar 301 sources that would extend longevity until nectar and pollens can be located could be critical to 302 survival and production of offspring. 303 In conclusion, the occurrence of over forty different species of native bees on an 304 unadvertised, non-floral sugar resource suggests widespread, previously undocumented plasticity 305 in bee foraging behaviors and diet breadth that may become increasingly relevant to bee 306 conservation with continued disruptions in floral bloom. This discovery represents not only a 307 novel behavioral phenomenon and notable departure from the historical focus on bee use of 308 visual, olfactory, and floral cues, but may also have implications related to both the resilience of 309 bee communities to temporary habitat perturbations and the social complexity of their foraging 310 dynamics. Our finding that diverse solitary bees use nontraditional resources and foraging 311 strategies during times of low bloom suggests that bee use of honeydew may be only one 312 example of adaptive bee foraging strategies that have yet to be described.   mechanism, and encourage further research in this area (Lawless 1987). 456