Do Bumble Bees Produce Brood Pheromones?

Reproductive division of labor, a defining feature of social insects, is often regulated by a combination of behavioral and chemical means. It is hypothesized that behavioral interactions play a more important role in regulating reproduction of primitive eusocial species, while pheromones are typically used by large sized, advanced eusocial species. Here we examined if worker reproduction in the primitively eusocial species Bombus impatiens is regulated by brood pheromones. We recently demonstrated that worker egg laying in this species is inhibited by young larvae and triggered by pupae. However, the mechanism by which the brood communicates its presence and whether brood or hunger pheromones are involved remain unknown. We found that workers were behaviorally attracted to pupae over larvae or control in a choice experiment, in line with their reproductive interests. However, odors from larvae or pupae were insufficient to inhibit worker reproduction. We further show that the youngest larvae are particularly vulnerable to starvation, however, despite a slight attraction and fewer eggs laid by workers in the presence of starved compared with fed larvae, these effects were insignificant. Our study demonstrates that workers can differentiate between larvae and pupae, but not between starved and fed larvae based on olfactory information. However, these signals alone do not explain the reduction in worker egg laying previously found. Bumble bee workers may use information from multiple sources or rely solely on behavioral interactions with brood and other females to make decisions about reproduction, in line with their small colony size and simple social organization.

Maculatic Acids-Sex Attractant Pheromone Components of Bald-Faced Hornets.

Yellowjackets in the genera Vespula and Dolichovespula are prevalent eusocial insects of great ecological and economic significance, but the chemical signals of their sexual communication systems have defied structural elucidation. Herein, we report the identification of sex attractant pheromone components of virgin bald-faced hornet queens (Dolichovespula maculata). We analyzed body surface extracts of queens by coupled gas chromatographic-electroantennographic detection (GC-EAD), isolated the compounds that elicited responses from male antennae by high-performance liquid chromatography (HPLC), and identified these components by GC mass spectrometry (MS), HPLC-MS, and NMR spectroscopy. In laboratory olfactometer experiments, synthetic (2Z,7E)-3,7-dimethyldeca-2,7-diendioic acid (termed here maculatic acid A) and (2Z,7E)-10-methoxy-3,7-dimethyldeca-10-oxo-deca-2,7-dienoic acid (termed here maculatic acid C) in binary combination significantly attracted bald-faced hornet males. These are the first sex attractant pheromone components identified in yellowjackets.

Evidence for a Nest Defense Pheromone in Bald-Faced Hornets, Dolichovespula Maculata, and Identification of Components.

In eusocial insects like Bald-faced hornets, Dolichovespula maculata, nest defense is essential because nests contain a large number of protein-rich larvae and pupae, and thus are attractive to nest predators. Our objectives were to investigate whether D. maculata exhibit pheromone-mediated nest defense, and to identify and field test any pheromone components. We tested for pheromone-mediated nest defense behavior of D. maculata by placing a paired box-apparatus near the entrance of D. maculata nests, and treating both boxes with a solvent control, or one of the two boxes with a solvent control and the other with either venom sac extract, the putative source of nest defense pheromone, or synthetic pheromone. The sound impulses caused by nest mates attempting to sting or strike the boxes were recorded for 3 min. Compared to the double-control treatment, the number of strikes increased 27-fold when one of the two boxes was treated with venom sac extract, providing evidence for an alarm response. The box treated with venom sac extract also induced a significantly greater proportion of strikes than the corresponding control box, providing evidence for a target-oriented response. Analyzing venom sac extract by gas chromatographic-electroantennographic detection (GC-EAD) and GC-mass spectrometry resulted in the identification of seven candidate pheromone components: (a) dimethylaminoethanol, (b) dimethylamino ethyl acetate, (c) 2,5-dimethylpyrazine, (d) N-3-methylbutylacetamide, (e) 2-heptadecanone, (f) (Z)-8-heptadecen-2-one, and (g) (Z)-10-nonadecen-2-one. Testing in paired-box bioassays blends of the nitrogen-containing volatile components a-d, the less volatile ketones e-g, or both (a-g), indicated that a-d primarily have an alarm function. The ketones e-g, in contrast, induced target-oriented responses, possibly marking the box, or potential nest predators, for guided and concerted attacks, or enhancing the alarm-inducing effect of the volatile pheromone components, as shown in honey bees. Comparing the behavioral effects of venom sac extract, blends a-d, e-g, and a-g, venom sac extract was most effective in triggering the full complement of alarm and target-oriented responses. These comparisons further suggested that a component is missing in the group of components that triggers the alarm rather than the target-oriented response.

Disruption of Darna pallivitta (Lepidoptera: Limacodidae) by Conventional and Mobile Pheromone Deployment.

Identification of the Darna pallivitta (Moore) pheromone component n-butyl (E)-7,9-decadienoate (E7,9-10:COOn-Bu) has made it possible to investigate communication disruption to control this lepidopteran pest. Conventional communication disruption trials showed marked decreases in the mean number of male moths captured in E7,9-10:COOnBu-treated fields compared with control fields. For traps baited with E7,9-10:COOnBu, percent disruptions were 94.4% and 92.1% for septa (1 g pheromone/ha, 1-wk trial duration) and spirals (6 g pheromone/ha, 8-wk trial duration) respectively. For traps baited with virgin female moths, percent disruption was 73.3% using septa disruptors (1 g pheromone/ha, 1-wk trial duration). Mobile communication disruption using Bactrocera cucurbitae (Coquillett) as carriers for E7,9-10:COOn-Bu was evaluated in the following three areas: fly survivorship, attraction of male moths to treated flies, and moth disruption in a small-scale field trial. Topical application of E7,9-10:COOnBu showed no significant decrease in survivorship at 50 and 80 µg/fly. However, decreased survivorship was observed at 100 µg/fly and linear regression showed E7,9-10:COOnBu dose was significantly correlated with B. cucurbitae survivorship. Traps containing honey-pheromone-fed flies attracted and caught D. pallivitta over a 1-wk period, demonstrating the attractiveness of the carrier. Releasing E7,9-10:COOnBu-fed B. cucurbitae (∼2 g pheromone/ha, 1-wk trial duration) resulted in significantly reduced trap catches in treatment fields compared with control fields on the first 2 d of the field trial. Percent disruptions were 84.7% (day 1) and 56.0% (day 2). These results suggest that both conventional communication disruption and mobile communication disruption have potential to control D. pallivitta.

Differential gene expression underlying the biosynthesis of Dufour's gland signals in Bombus impatiens.

Pheromones regulating social behavior are one of the most explored phenomena in social insects. However, compound identity, biosynthesis and their genetic basis are known in only a handful of species. Here we examined the gene expression associated with pheromone biosynthesis of two main chemical classes: esters and terpenes, using the social bee Bombus impatiens. We conducted chemical and RNA-seq analyses of the Dufour's gland, an exocrine gland producing a plethora of pheromones regulating social behavior in hymenopteran species. The Dufour's gland contains mostly long-chained hydrocarbons, terpenes and esters that signal reproductive and social status in several bee species. In bumble bees, the Dufour's gland contains queen- and worker-specific esters, in addition to terpenes and terpene-esters only found in gynes and queens. These compounds are assumed to be synthesized de novo in the gland, however, their genetic basis is unknown. A whole transcriptome gene expression analysis of the gland in queens, gynes, queenless and queenright workers showed distinct transcriptomic profiles, with thousands of differentially expressed genes between the groups. Workers and queens express genes associated with key enzymes in the biosynthesis of wax esters, while queens and gynes preferentially express key genes in terpene biosynthesis. Overall, our data demonstrate gland-specific regulation of chemical signals associated with social behavior and identifies candidate genes and pathways regulating caste-specific chemical signals in social insects.

Field trapping the little fire ant, Wasmannia auropunctata.

Two detection methods for the little fire ant, Wasmannia auropunctata (Roger) (Hymenoptera: Formicidae), both employing the pheromone attractant 2,5-dimethyl-3-(2-methylbutyl)pyrazine (2-MeBu-diMePy), were compared with peanut butter based detection, in order to evaluate differences in species specificity and detection reliability. Trapping was conducted using a transect through a macadamia orchard on the island of Hawaii. The transect consisted of a series of three-tree plots, each plot contained a peanut butter coated stick (the most common detection method used for W. auropunctata in Hawaii), a one-way trap treated with 2-MeBu-diMePy, and a piece of double-sided tape treated with 2-MeBu-diMePy. While there were no differences in the number of W. auropunctata counted with each detection method, and no differences in detection reliability (detecting the known presence of W. auropunctata in a plot), the pheromone-incorporating methods showed greater species specificity, retaining W. auropunctata almost exclusively. These results demonstrate the potential of pheromone-detection methods to selectively capture target ant species even when other ants are present and abundant. Combined data from all three detection methods and a previous visual survey along the transect showed a marked difference in the frequency of cohabitation among ant species. Of the 10 ant species collected, W. auropunctata was found as the sole ant species on a given tree at a significantly higher frequency than all other ant species except Pheidole fervens. 94% percent of the trees with W. auropunctata had only W. auropunctata, supporting previous observations that this species tends to displace other ant species. In addition, W. auropunctata microhabitat preferences were investigated using one-way traps containing 2-MeBu-diMePy, which were placed in three arboreal and three non-arboreal locations. While the number of ants captured did not differ by trap placement, when grouped, captures were significantly higher in arboreal than non-arboreal microhabitats.

Dufour's gland analysis reveals caste and physiology specific signals in Bombus impatiens.

Reproductive division of labor in insect societies is regulated through multiple concurrent mechanisms, primarily chemical and behavioral. Here, we examined if the Dufour's gland secretion in the primitively eusocial bumble bee Bombus impatiens signals information about caste, social condition, and reproductive status. We chemically analyzed Dufour's gland contents across castes, age groups, social and reproductive conditions, and examined worker behavioral and antennal responses to gland extracts. We found that workers and queens each possess caste-specific compounds in their Dufour's glands. Queens and gynes differed from workers based on the presence of diterpene compounds which were absent in workers, whereas four esters were exclusive to workers. These esters, as well as the total amounts of hydrocarbons in the gland, provided a separation between castes and also between fertile and sterile workers. Olfactometer bioassays demonstrated attraction of workers to Dufour's gland extracts that did not represent a reproductive conflict, while electroantennogram recordings showed higher overall antennal sensitivity in queenless workers. Our results demonstrate that compounds in the Dufour's gland act as caste- and physiology-specific signals and are used by workers to discriminate between workers of different social and reproductive status.

Optimizing Laboratory Rearing of a Key Pollinator, Bombus impatiens.

Bumble bees are key pollinators for wild and managed plants and serve as a model system in various research fields, largely due to their commercial availability. Despite their extensive use, laboratory rearing of bumble bees is often challenging, particularly during the solitary phase queens undergo before founding a colony. Using a literature survey, we demonstrate that most studies rely on commercially available species that are provided during the colony's social phase, limiting study on early phases of the life cycle and the ability to control for colony age and relatedness. Laboratory rearing is challenging since the queen solitary phase is less understood compared to the social phase. To overcome this barrier, we examined several aspects related to the queen solitary phase: the effect of age on likelihood of mating, how the timing of CO2 narcosis post-mating (a technique to bypass diapause) affects egg-laying, and whether different social cues affect the success of colony initiation. Our data show an optimum age for mating in both sexuals and decreased egg-laying latency in the presence of workers and pupae. The timing of CO2 narcosis did not significantly affect egg laying in queens. These findings can be incorporated to improve bumble bee rearing for research purposes.

Plant Volatiles Help Mediate Host Plant Selection and Attraction of the Spotted Lanternfly (Hemiptera: Fulgoridae): a Generalist With a Preferred Host.

Host plant volatiles play a key role in mediating plant-herbivore interactions. How an array of host plant volatiles guides host preference and attraction in the invasive polyphagous Lycorma delicatula (White), the spotted lanternfly (SLF), is largely unknown. A pernicious phloem feeder, SLF feeds on over 70 species of plants, some with high economic impact. To aid the development of detection and monitoring tools for SLF, we used a two-choice olfactometer to compare 14 host plant species for attraction, first to a blank control, and then to their preferred host Ailanthus altissima (Mill.) Swingle (Sapindales: Simaroubaceae), tree-of-heaven. SLF were significantly attracted to seven host plants compared to a blank control, but no host plant was more attractive than tree-of-heaven. We then used electroantennographic detection (EAD) to screen select host plants for EAD active compounds, hypothesizing that EAD-active plant volatiles act as kairomones and mediate SLF attraction to host plants. Out of 43 unique antennal responses, 18 compounds were identified and tested individually for attraction in a two-choice olfactometer against a blank control and then against methyl salicylate, the current best attractant. Eleven compounds were significantly attractive, and one, sulcatone, was more attractive than methyl salicylate. Blends of kairomones were then tested for attraction, revealing five blends that were significantly more attractive than methyl salicylate, and could be developed into lures for field testing. The presence of these kairomones in volatile profiles of 17 plant species is described. These findings support the hypothesis that the identified volatiles act as kairomones and function in attraction to host plants.

Distribution, Survival, and Development of Spotted Lanternfly on Host Plants Found in North America.

Studies were conducted from 2015 to 2018 to evaluate spotted lanternfly (SLF) distribution and developmental suitability of different plant species in the U.S. Tree bands on 283 trees spanning 33 species captured 21,006 SLF in 2 yr. More SLF per tree were trapped on tree-of-heaven Ailanthus altissima (Mill.) Swingle (Sapindales: Simaroubaceae) than on other species, on average, and most adults were captured on tree-of-heaven. Frequency of detection of adult SLF was higher on tree-of-heaven than on other species but was actually equal or lower on tree-of-heaven than on all other species combined for younger SLF stages in 2015. An enclosed choice test between tree-of-heaven and black walnut Juglans nigra L. (Fagales: Juglandaceae) revealed nymphs showed little consistent preference, whereas adults consistently and significantly preferred tree-of-heaven. No-choice field sleeve studies evaluated SLF survivorship on 26 host plant species in 17 families. Ten plant species supported SLF for an average of ≥45 d, with the rest unable to support SLF for >30 d. Eight species were able to support development from first instar to adult: black walnut, chinaberry Melia azedarach L. (Sapindales: Meliaceae), oriental bittersweet Celastrus orbiculatus Thunb. (Celastrales: Celastraceae), tree-of-heaven, hops Humulus lupulus L. (Rosales: Cannabaceae), sawtooth oak Quercus acutissima Carruthers (Fagales: Fagaceae), butternut Juglans cinerea L, and tulip tree Liriodendron tulipifiera L. (Magnoliales: Magnoliaceae). The ability of SLF to develop to adult on hosts other than tree-of-heaven may impact pest management decisions.

Discovery of Three Kairomones in Relation to Trap and Lure Development for Spotted Lanternfly (Hemiptera: Fulgoridae).

The spotted lanternfly, Lycorma delicatula (White), is an invasive phloem feeder recently introduced into North America that attacks a broad range of woody plants. When feeding in large numbers, they can seriously damage or kill a tree. Their preferred host is the invasive tree-of-heaven, Ailanthus altissima Swingle (Sapindales: Simaroubaceae), but they are serious pests of grape, Vitis vinifera L. (Vitales: Vitaceae) and a number of other commercially important host plants. Volatile collections were conducted on tree-of-heaven and grape, and the most abundant compounds from these plants present in samples and indicated in the literature were tested for attraction in the laboratory and field. Three compounds, methyl salicylate, (Z)-3-hexenol, and (E,E)-α-farnesene, were found to be highly attractive in laboratory behavioral bioassays. Methyl salicylate was attractive to all stages of L. delicatula, whereas the youngest nymphs were not as attracted to (Z)-3-hexenol or (E,E)-α-farnesene in laboratory bioassays. When comparing individual compounds, methyl salicylate attracted the most L. delicatula. Methyl salicylate lures in the field produced a two- to four-fold increase in captures compared with unbaited controls, and field testing also revealed a significant positive dose response. Of the several types of sticky bands tested, Web-Cote Industries sticky bands were found to be most efficient at trapping L. delicatula adults and nymphs.

Yeasts Harbored by Vespine Wasps in the Pacific Northwest.

The ecological role of social wasps has been extensively studied, but little is known about symbiotic relationships of these wasps with microbes. Recently, it was shown that vespid wasps in Europe carry yeasts, predominantly Saccharomyces cerevisiae, in their gastrointestinal (GI) tract. Interestingly, this niche allowed for sexual recombination of yeasts to occur and the formation of novel hybrid species. Our goals were 1) to survey the GI tract of eusocial wasps in the Pacific Northwest for the presence of yeasts and 2) to compare the diversity of such yeasts to that described for wasps in Europe. The GI tracts of 19 individual wasps from five species were plated, and 27 yeast-like colonies were identified to the species level. Yeasts in the genera Lachancea and Hanseniaspora each comprised ∼30% of the isolates; ∼25% were identified as Metschnikowia spp., with the remaining 10% belonging to Rhodotorula. Four bacterial isolates were identified as Escherichia coli, Enterococcus faecalis, and two isolates of Stenotrophomonas maltophilia. Yeasts were present at all life stages of the wasps except for two unfed gynes of Dolichovespula maculata (L.) that contained only bacteria. The presence of a particular yeast species was not correlated with any wasp species. Furthermore, S. cerevisiae was not found in any wasp species. This highlights an interesting difference in the life cycle of both S. cerevisiae and wasps in Europe and the Pacific Northwest, and prompts further studies on the interactions of these microbes with their host wasps.