Mark J. Carroll

Cowpea Chloroplastic ATP Synthase Is the Source of Multiple Plant Defense Elicitors during Insect Herbivory

In cowpea (Vigna unguiculata), fall armyworm (Spodoptera frugiperda) herbivory and oral secretions (OS) elicit phytohormone production and volatile emission due to inceptin [Vu-In; +ICDINGVCVDA−], a peptide derived from chloroplastic ATP synthase γ-subunit (cATPC) proteins. Elicitor-induced plant volatiles can function as attractants for natural enemies of insect herbivores. We hypothesized that inceptins are gut proteolysis products and that larval OS should contain a mixture of related peptides. In this study, we identified three additional cATPC fragments, namely Vu-GE+In [+GEICDINGVCVDA−], Vu-E+In [+EICDINGVCVDA−], and Vu-In−A [+ICDINGVCVD−]. Leaf bioassays for induced ethylene (E) production demonstrated similar effective concentration50 values of 68, 45, and 87 fmol leaf−1 for Vu-In, Vu-E+In, and Vu-GE+In, respectively; however, Vu-In−A proved inactive. Shortly following ingestion of recombinant proteins harboring cATPC sequences, larval OS revealed similar concentrations of the three elicitors with 80% of the potential inceptin-related peptides recovered. Rapidly shifting peptide ratios over time were consistent with continued proteolysis and preferential stability of inceptin. Likewise, larvae ingesting host plants with inceptin precursors containing an internal trypsin cleavage site rapidly lost OS-based elicitor activity. OS containing inceptin elicited a rapid and sequential induction of defense-related phytohormones jasmonic acid, E, and salicylic acid at 30, 120, and 240 min, respectively, and also the volatile (E)-4,8-dimethyl-1,3,7-nonatriene. Similar to established peptide signals such as systemin and flg22, amino acid substitutions of Vu-In demonstrate an essential role for aspartic acid residues and an unaltered C terminus. In cowpea, insect gut proteolysis following herbivory generates inappropriate fragments of an essential metabolic enzyme enabling plant non-self-recognition.

The Attraction of Spodoptera frugiperda Neonates to Cowpea Seedlings is Mediated by Volatiles Induced by Conspecific Herbivory and the Elicitor Inceptin

Neonate fall armyworms [FAW; Spodoptera frugiperda (Smith)] often encounter conspecific herbivore damage as they disperse from an egg mass to an initial feeding site. We investigated the orientation responses of dispersing neonates to herbivore damage in cowpea seedlings, specifically examining whether neonate behaviors were affected by inceptin, the primary elicitor of FAW-induced defenses in cowpea leaves. We focused on responses to damage caused by conspecific first instars, as might occur during the dispersal of siblings from an egg mass. Inceptin contents of damaging first instar FAW were controlled through their diets, with leaf-fed FAW producing inceptins in their oral secretions, and root-fed or starved FAW lacking these elicitors. In a bioassay designed to evaluate neonate dispersal off a host plant, a higher percentage of neonates remained on herbivore-induced or inceptin-treated plants than on undamaged plants, mechanically damaged plants, freshly damaged plants, or on plants damaged by FAW lacking inceptins. Further investigations of neonate responses to plant odors with a four-arm olfactometer demonstrated that neonate attraction to odors from 4-h old FAW damage was strongly dependent on previous diet of the damaging larvae. Neonates were attracted to odors from 4-h old FAW damage over odors from undamaged plants or fresh FAW damage, provided that the damaging larvae had previously ingested leaf material. In a direct comparison of odors from induced plants, plants damaged by leaf-fed FAW were as attractive as plants treated with synthetic inceptin. GC-MS analysis confirmed that (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT) was the major volatile induced by FAW herbivory. While both DMNT and undamaged plant odors were more attractive than air, neonates preferred DMNT-supplemented plant odors. These results suggest that neonate FAW exploit herbivore-induced plant volatiles as host plant location and recognition cues.

Honey bee colonies provided with natural forage have lower pathogen loads and higher overwinter survival than those fed protein supplements

Malnutrition is a major cause of colony losses. In managed hives, bees are fed protein supplements (PS) during pollen shortages. If bees were provided with natural forage instead of PS, would they have lower pathogen levels and higher queen and colony survival? We addressed this question by either providing colonies with forage (Brassica rapa—rapini) or feeding them PS from November to February. Soluble protein concentrations in the PS were lower than the rapini pollen as were levels of most amino acids. Nurse bees digested less of the protein in PS than the pollen. Hemolymph protein titers in nurse bees and colony growth did not differ between those fed PS or foraging on rapini. However, colonies fed PS had higher levels of black queen cell virus and Nosema and greater queen losses, indicating that natural forage might improve overwintering survival.

An amino acid substitution inhibits specialist herbivore production of an antagonist effector and recovers insect-induced plant defenses

Plants respond to insect herbivory through the production of biochemicals that function as either direct defenses or indirect defenses via the attraction of natural enemies. While attack by closely related insect pests can result in distinctive levels of induced plant defenses, precise biochemical mechanisms responsible for differing responses remain largely unknown. Cowpea (Vigna unguiculata) responds to Fall armyworm (Spodoptera frugiperda) herbivory through the detection of fragments of chloroplastic ATP synthase γ-subunit proteins, termed inceptin-related peptides, present in larval oral secretions (OS). In contrast to generalists like Fall armyworm, OS of the legume-specializing velvetbean caterpillar (VBC; Anticarsia gemmatalis) do not elicit ethylene production and demonstrate significantly lower induced volatile emission in direct herbivory comparisons. Unlike all other Lepidoptera OS examined, which preferentially contain inceptin (Vu-In; +ICDINGVCVDA−), VBC OS contain predominantly a C-terminal truncated peptide, Vu-In−A (+ICDINGVCVD−). Vu-In−A is both inactive and functions as a potent naturally occurring antagonist of Vu-In-induced responses. To block antagonist production, amino acid substitutions at the C terminus were screened for differences in VBC gut proteolysis. A valine-substituted peptide (Vu-InΔV; +ICDINGVCVDV−) retaining full elicitor activity was found to accumulate in VBC OS. Compared with the native polypeptide, VBC that previously ingested 500 pmol of the valine-modified chloroplastic ATP synthase γ-subunit precursor elicited significantly stronger plant responses in herbivory assays. We demonstrate that a specialist herbivore minimizes the activation of defenses by converting an elicitor into an antagonist effector and identify an amino acid substitution that recovers these induced plant defenses to a level observed with generalist herbivores.

LUTEIN SEQUESTRATION AND FURANOCOUMARIN METABOLISM IN PARSNIP WEBWORMS UNDER DIFFERENT ULTRAVIOLET LIGHT REGIMES IN THE MONTANE WEST

Both biotic and abiotic selection pressures can contribute to geographic variation in allelochemical production in plants. We examined furanocoumarin production in western North American populations of Heracleum lanatum and Pastinaca sativa that, at different latitudes and altitudes, experience different ultraviolet (UV) light regimes. Total furanocoumarins and linear furanocoumarins of fruits were negatively correlated with UV irradiance, whereas amounts of angular furanocoumarins, which are generally less phototoxic, were not. Another factor potentially influencing furanocoumarin production is the presence of the parsnip webworm Depressaria pastinacella, (Lepidoptera: Oecophoridae), an herbivore that feeds on reproductive structures of both plant species. These insects sequester lutein from their host plants; this carotenoid acts to ameliorate furanocoumarin toxicity. Although the concentration of lutein in fruits did not vary with UV irradiance, lutein sequestration by sixth instars was positively correlated with UV irradiance. Webworm populations are variably infested with the polyembryonic webworm parasitoid Copidosoma sosares Walker (Hymenoptera: Encyrtidae). H. lanatum fruits from populations with webworms parasitized by C. sosares had lower concentrations of furanocoumarins, with the exception of sphondin, than fruits from plants infested with webworms free from parasitism. Lower levels of these furanocoumarins may reduce negative effects on the fitness of this parasitoid. In contrast with the variation in furanocoumarin content, the ability of webworms to metabolize furanocoumarins by cytochrome P450 did not differ significantly among populations from New Mexico to Alberta.

Sublethal Effects of Imidacloprid on Honey Bee Colony Growth and Activity at Three Sites in the U.S.

Imidacloprid is a neonicotinoid pesticide heavily used by the agricultural industry and shown to have negative impacts on honey bees above certain concentrations. We evaluated the effects of different imidacloprid concentrations in sugar syrup using cage and field studies, and across different environments. Honey bee colonies fed sublethal concentrations of imidicloprid (0, 5, 20 and 100 ppb) over 6 weeks in field trials at a desert site (Arizona), a site near intensive agriculture (Arkansas) and a site with little nearby agriculture but abundant natural forage (Mississippi) were monitored with respect to colony metrics, such as adult bee and brood population sizes, as well as pesticide residues. Hive weight and internal hive temperature were monitored continuously over two trials in Arizona. Colonies fed 100 ppb imidacloprid in Arizona had significantly lower adult bee populations, brood surface areas and average frame weights, and reduced temperature control, compared to colonies in one or more of the other treatment groups, and consumption rates of those colonies were lower compared to other colonies in Arizona and Arkansas, although no differences in capped brood or average frame weight were observed among treatments in Arkansas. At the Mississippi site, also rich in alternative forage, colonies fed 5 ppb imidacloprid had less capped brood than control colonies, but contamination of control colonies was detected. In contrast, significantly higher daily hive weight variability among colonies fed 5 ppb imidacloprid in Arizona suggested greater foraging activity during a nectar flow post treatment, than any other treatment group. Imidacloprid concentrations in stored honey corresponded well with the respective syrup concentrations fed to the colonies and remained stable within the hive for at least 7 months after the end of treatment.

Standard methods for chemical ecology research in Apis mellifera

Summary This paper describes basic methods essential in elucidating chemically-mediated behavioural interactions among honey bees, and between honey bees and other arthropods. These range from bioassay methods used to demonstrate the role of specific behaviours, techniques and equipment used to collect and analyse semiochemicals (both volatiles and non-volatiles e.g. cuticular hydrocarbons) from individual honey bees, groups of bees or an entire colony in its native environments. This paper covers: collection and analysis of honey bee volatiles in the natural environment, collection and analysis of bee volatiles out of their natural environment and their antennal detection, collection and analysis of non-volatile cuticular hydrocarbons, bioassays with queen pheromone and finally a section focusing on in vitro bioassays as a tool for elucidation of mechanisms regulating pheromone gland activity.

Honey bees preferentially consume freshly-stored pollen

Honey bees (Apis mellifera) collect and store both honey and pollen in preserved forms. Pollen storage involves the addition of honey or nectar and oral secretions to pollen granules. It is controversial whether the duration of pollen storage alters the palatability or nutritive value of the pollen storage medium. We examined how bees utilize different-aged stored pollen during an extended pollen flow. The deposition of pollen into wax cells and subsequent consumption were monitored daily on 18 brood frames from 6 colonies over an 8d observation period. Despite a greater abundance of older stored pollen cells on brood frames, bees showed a marked preference for the consumption of freshly-stored pollen. Two to four day-old pollen cell contents were significantly more likely to be consumed, while pollen cell contents more than seven days old were eaten at much lower rates. Similar experiments that controlled for cell abundance and spatial effects using cage assays yielded the same result. One day-old stored pollen was consumed approximately three times more often than 10d-old stored pollen, and two times more often than 5d-old stored pollen. These consumption preferences for freshly-stored pollen occurred despite a lack of clear developmental advantages. Young adult workers reared for 7 days on 1d-, 5d-, or 10d-old stored pollen showed no difference in body mass, stored pollen consumption, hindgut fecal material accumulation, or hypopharyngeal gland (HPG) protein titers, suggesting that different-aged pollen stores did not vary in their nutritional value to adult bees. These findings are inconsistent with the hypothesis promoting a period of microbially-mediated, “beebread maturation” that results in greater palatability or nutritive value for aged pollen stores. Rather, stored pollen that is not eaten in the first few days accumulates as excess stores preserved in a less preferred, but nutritionally-similar state.

Collection of volatiles from honeybee larvae and adults enclosed on brood frames

We describe a system for the in situ collection of volatiles from bees enclosed on a standard Langstroth frame face. The system includes an observation frame consisting of a glass plate and an aluminum frame that encloses a single frame face. A push–pull airflow system and an in-line volatile collection filter allow for air exchange and headspace volatile capture. This system can provide insight into colony chemical communication. The emissions of four compounds (2-heptanone, methyl benzoate, decanal, and 3-carene) associated with adult bees or colony materials remained steady or increased slightly in repeated collections from frames with maturing larvae. The emissions of the larval food component octanoic acid reflected changes in food consumption patterns by differently aged larvae. The production of the primer pheromone E-β-ocimene was greatest in comb containing young larvae and recently capped brood, but was lower on comb with capping larvae.

Aethina tumida (Coleoptera: Nitidulidae) attraction to volatiles produced by Apis mellifera (Hymenoptera: Apidae) and Bombus impatiens (Hymenoptera: Apidae) colonies

In this study, small hive beetle (SHB) attraction to whole honey bee and bumble bee colony volatiles as well as volatiles from individual colony components was investigated using four-way olfactometer choice tests. This was done to determine the role olfactory cues play in SHB host location and differentiation. Results from the bumble bee bioassays suggest that SHBs are attracted to adult bumble bees, stored pollen, brood, wax, and whole colony volatiles though not to honey volatiles. The honey bee bioassay results suggest that SHBs are attracted to adult honey bees, brood, honey, stored pollen, wax, and whole colony volatiles. SHBs did not exhibit a preference for honey bee or bumble bee component volatiles in the honey bee vs. bumble bee bioassays. Collectively, the data suggest that SHB attraction to bumble and honey bee colony volatiles is mediated chemically.