Pollen-feeding behavior of diverse insects on Geranium delavayi, a flower with large, accessible pollen grains.

PREMISE: Why have pollen grains evolved to be exceptionally large in some species? Pollen-feeding hypothesis suggests that if the proportion of pollen amounts for feeding is reduced in a flower, the low allocation to pollen number would allow pollen grains to be larger. METHODS: To examine whether species with large pollen grains experience low pollen consumption, the behavior of insects feeding on nectar and pollen was observed and pollen transfer efficiency was estimated for four visitor types in Geranium delavayi. To see whether bees actively collected pollen, the numbers of grains in pollen baskets and on the body were compared. Both nutritional value (total protein and lipid) and chemical defense (phenolic metabolites) in pollen against pollen feeders were measured. RESULTS: Bumblebees and honeybees foraged for nectar, rarely groomed pollen into corbiculae, and had >5× higher pollen transfer efficiency than smaller solitary bees and flies, which were pollen eaters that removed more pollen but deposited less. Pollen grains were characterized by low protein and high lipid content with a low protein-lipid ratio, an unfavorable combination for bumblebees. Three secondary metabolites were significantly higher in pollen grains (7.77 mg/g) than in petals (1.08 mg/g) or in nectar (0.44 mg/g), suggesting stronger chemical defense in pollen. CONCLUSIONS: Our results indicated that large bees took nectar but little of the nutritionally poor and highly toxic pollen. These data support one prediction of the pollen-feeding hypothesis, that species with few and large pollen grains would also have low pollen-consumption rates.

No evidence of social learning in a socially roosting butterfly in an associative learning task.

Insects may acquire social information by active communication and through inadvertent social cues. In a foraging setting, the latter may indicate the presence and quality of resources. Although social learning in foraging contexts is prevalent in eusocial species, this behaviour has been hypothesized to also exist between conspecifics in non-social species with sophisticated behaviours, including Heliconius butterflies. Heliconius are the only butterfly genus with active pollen feeding, a dietary innovation associated with a specialized, spatially faithful foraging behaviour known as trap-lining. Long-standing hypotheses suggest that Heliconius may acquire trap-line information by following experienced individuals. Indeed, Heliconius often aggregate in social roosts, which could act as 'information centres', and present conspecific following behaviour, enhancing opportunities for social learning. Here, we provide a direct test of social learning ability in Heliconius using an associative learning task in which naive individuals completed a colour preference test in the presence of demonstrators trained to feed randomly or with a strong colour preference. We found no evidence that Heliconius erato, which roost socially, used social information in this task. Combined with existing field studies, our results add to data which contradict the hypothesized role of social learning in Heliconius foraging behaviour.

Male pheromone composition depends on larval but not adult diet in Heliconius melpomene.

1. Condition-dependent traits can act as honest signals of mate quality, with fitter individuals being able to display preferred phenotypes. Nutrition is known to be an important determinant of individual condition, with diet known to affect many secondary sexual traits. 2. In Heliconius butterflies, male chemical signalling plays an important role in female mate choice. Potential male sex pheromone components have been identified previously, although it is unclear what information they convey to the female. 3. In the present study, the effect of diet on androconial and genital compound production is tested in male Heliconius melpomene rosina. To manipulate larval diet, larvae are reared on three different Passiflora host plants: Passiflora menispermifolia, the preferred host plant, Passiflora vitifolia and Passiflora platyloba. To manipulate adult diet, adult butterflies are reared with and without access to pollen, a key component of their diet. 4. No evidence is found to suggest that adult pollen consumption affects compound production in the first 10 days after eclosion. There is also a strong overlap in the chemical profiles of individuals reared on different larval host plants. The most abundant compounds produced by the butterflies do not differ between host plant groups. However, some compounds found in small amounts differ both qualitatively and quantitatively. Some of these compounds are predicted to be of plant origin and the others synthesised by the butterfly. Further electrophysiological and behavioural experiments will be needed to determine the biological significance of these differences.

Evolutionary and structural analyses uncover a role for solvent interactions in the diversification of cocoonases in butterflies.

Multi-omic approaches promise to supply the power to detect genes underlying disease and fitness-related phenotypes. Optimal use of the resulting profusion of data requires detailed investigation of individual candidate genes, a challenging proposition. Here, we combine transcriptomic and genomic data with molecular modelling of candidate enzymes to characterize the evolutionary history and function of the serine protease cocoonase. Heliconius butterflies possess the unique ability to feed on pollen; recent work has identified cocoonase as a candidate gene in pollen digestion. Cocoonase was first described in moths, where it aids in eclosure from the cocoon and is present as a single copy gene. In heliconiine butterflies it is duplicated and highly expressed in the mouthparts of adults. At least six copies of cocoonase are present in Heliconius melpomene and copy number varies across H. melpomene sub-populations. Most cocoonase genes are under purifying selection, however branch-site analyses suggest cocoonase 3 genes may have evolved under episodic diversifying selection. Molecular modelling of cocoonase proteins and examination of their predicted structures revealed that the active site region of each type has a similar structure to trypsin, with the same predicted substrate specificity across types. Variation among heliconiine cocoonases instead lies in the outward-facing residues involved in solvent interaction. Thus, the neofunctionalization of cocoonase duplicates appears to have resulted from the need for these serine proteases to operate in diverse biochemical environments. We suggest that cocoonase may have played a buffering role in feeding during the diversification of Heliconius across the neotropics by enabling these butterflies to digest protein from a range of biochemical milieux.

On the ecological significance of pollen color: a case study in American trout lily (Erythronium americanum).

Evolutionary ecologists seek to explain the processes that maintain variation within populations. In plants, petal color variation can affect pollinator visitation, environmental tolerance, and herbivore deterrence. Variation in sexual organs may similarly affect plant performance. Within-population variation in pollen color, as occurs in the eastern North American spring ephemeral Erythronium americanum, provides an excellent opportunity to investigate the maintenance of variation in this trait. Although the red/yellow pollen-color polymorphism of E. americanum is widely recognized, it has been poorly documented. Our goals were thus (1) to determine the geographic distribution of the color morphs and (2) to test the effects of pollen color on components of pollen performance. Data provided by citizen scientists indicated that populations range from monomorphic red, to polymorphic, to monomorphic yellow, but there was no detectable geographic pattern in morph distribution, suggesting morph occurrence cannot be explained by a broad-scale ecological cline. In field experiments, we found no effect of pollen color on the probability of predation by the pollen-feeding beetle Asclera ruficollis, on the ability of pollen to tolerate UV-B radiation, or on siring success (as measured by the fruit set of hand-pollinated flowers). Pollinators, however, exhibited site-specific pollen-color preferences, suggesting they may act as agents of selection on this trait, and, depending on the constancy of their preferences, could contribute to the maintenance of variation. Collectively, our results eliminate some hypothesized ecological effects of pollen color in E. americanum, and identify effects of pollen color on pollinator attraction as a promising direction for future investigation.

Evolutionary history of tyrosine-supplementing endosymbionts in pollen-feeding beetles.

Many insects feeding on nutritionally challenging diets like plant sap, leaves, or wood engage in ancient associations with bacterial symbionts that supplement limiting nutrients or produce digestive or detoxifying enzymes. However, the distribution, function, and evolutionary dynamics of microbial symbionts in insects exploiting other plant tissues or relying on a predacious diet remain poorly understood. Here, we investigated the evolutionary history and function of the intracellular gamma-proteobacterial symbiont "Candidatus Dasytiphilus stammeri" in soft-winged flower beetles (Coleoptera, Melyridae, Dasytinae) that transition from saprophagy or carnivory to palynivory (pollen-feeding) between larval and adult stage. Reconstructing the distribution of the symbiont within the Dasytinae phylogeny unraveled not only a long-term coevolution, originating from a single acquisition event with subsequent host-symbiont codiversification, but also several independent symbiont losses. The analysis of 20 different symbiont genomes revealed that their genomes are severely eroded. However, the universally retained shikimate pathway indicates that the core metabolic contribution to their hosts is the provisioning of tyrosine for cuticle sclerotization and melanization. Despite the high degree of similarity in gene content and order across symbiont strains, the capacity to synthesize additional essential amino acids and vitamins and to recycle urea is retained in some but not all symbionts, suggesting ecological differences among host lineages. This report of tyrosine-provisioning symbionts in insects with saprophagous or carnivorous larvae and pollen-feeding adults expands our understanding of tyrosine supplementation as an important symbiont-provided benefit across a broad range of insects with diverse feeding ecologies.

Assessment of the impacts of microbial plant protection products containing Bacillus thuringiensis on the survival of adults and larvae of the honeybee (Apis mellifera).

This study was aimed at evaluating the effect of a microbial pest-controlling product (MPCP) with the active substance Bacillus thuringiensis ssp. aizawai (strain: ABTS-1857) on adults and larvae of honeybees. To determine the contamination levels of Bt spores in different matrices, a colony-feeding study under semi-field conditions was performed. Furthermore, two chronic adult trials and a chronic larval study were conducted under laboratory conditions to test the effects of different concentrations of the plant protection product (PPP) on the development and mortality. Possible modifications of the chronic oral toxicity test were assessed by additional pollen feeding. Our results showed that Bt spores were detected in all matrices over the entire test duration in different concentrations, decreasing over time. The survival of adult bees and larvae was negatively affected in laboratory conditions after a chronic exposure to the MPCP depending on the tested concentrations. Moreover, the earliest sign of bee mortality, resulting from exposure to ABTS-1857, was recorded only after 96 h at the highest tested concentration. Pollen feeding to adults significantly increased the survival of the treated bees. In conclusion, the PPP with the Bt strain ABTS-1857 showed an effect on the mortality of adults and larvae under laboratory conditions. Further studies with Bt-based PPPs under realistic field conditions are necessary to evaluate the potential risk of those MPCPs on honeybees.

Bacteriome-Localized Intracellular Symbionts in Pollen-Feeding Beetles of the Genus Dasytes (Coleoptera, Dasytidae).

Several insect taxa are associated with intracellular symbionts that provision limiting nutrients to their hosts. Such tightly integrated symbioses are especially common in insects feeding on nutritionally challenging diets like phloem sap or vertebrate blood, but also occur in seed-eating and omnivorous taxa. Here, we characterize an intracellular symbiosis in pollen-feeding beetles of the genus Dasytes (Coleoptera, Dasytidae). High-throughput tag-encoded 16S amplicon pyrosequencing of adult D. plumbeus and D. virens revealed a single gamma-proteobacterial symbiont ('Candidatus Dasytiphilus stammeri') that amounts to 52.4-98.7% of the adult beetles' entire microbial community. Almost complete 16S rRNA sequences phylogenetically placed the symbiont into a clade comprising Buchnera and other insect endosymbionts, but sequence similarities to these closest relatives were surprisingly low (83.4-87.4%). Using histological examination, three-dimensional reconstructions, and fluorescence in situ hybridization, we localized the symbionts in three mulberry-shaped bacteriomes that are associated with the mid- to hind-gut transition in adult male and female beetles. Given the specialized pollen-feeding habits of the adults that contrasts with the larvae's carnivorous lifestyle, the symbionts may provision limiting essential amino acids or vitamins as in other intracellular symbioses, or they might produce digestive enzymes that break up the fastidious pollen walls and thereby contribute to the host's nutrition. In either case, the presence of gamma-proteobacterial symbionts in pollen-feeding beetles indicates that intracellular mutualists are more widely distributed across insects with diverse feeding habits than previously recognized.

Pollen feeding proteomics: Salivary proteins of the passion flower butterfly, Heliconius melpomene.

While most adult Lepidoptera use flower nectar as their primary food source, butterflies in the genus Heliconius have evolved the novel ability to acquire amino acids from consuming pollen. Heliconius butterflies collect pollen on their proboscis, moisten the pollen with saliva, and use a combination of mechanical disruption and chemical degradation to release free amino acids that are subsequently re-ingested in the saliva. Little is known about the molecular mechanisms of this complex pollen feeding adaptation. Here we report an initial shotgun proteomic analysis of saliva from Heliconius melpomene. Results from liquid-chromatography tandem mass-spectrometry confidently identified 31 salivary proteins, most of which contained predicted signal peptides, consistent with extracellular secretion. Further bioinformatic annotation of these salivary proteins indicated the presence of four distinct functional classes: proteolysis (10 proteins), carbohydrate hydrolysis (5), immunity (6), and "housekeeping" (4). Additionally, six proteins could not be functionally annotated beyond containing a predicted signal sequence. The presence of several salivary proteases is consistent with previous demonstrations that Heliconius saliva has proteolytic capacity. It is likely that these proteins play a key role in generating free amino acids during pollen digestion. The identification of proteins functioning in carbohydrate hydrolysis is consistent with Heliconius butterflies consuming nectar, like other lepidopterans, as well as pollen. Immune-related proteins in saliva are also expected, given that ingestion of pathogens is a likely route to infection. The few "housekeeping" proteins are likely not true salivary proteins and reflect a modest level of contamination that occurred during saliva collection. Among the unannotated proteins were two sets of paralogs, each seemingly the result of a relatively recent tandem duplication. These results offer a first glimpse into the molecular foundation of Heliconius pollen feeding and provide a substantial advance towards comprehensively understanding this striking evolutionary novelty.

Mechanical damage to pollen aids nutrient acquisition in Heliconius butterflies (Nymphalidae).

Neotropical Heliconius and Laparus butterflies actively collect pollen onto the proboscis and extract nutrients from it. This study investigates the impact of the processing behaviour on the condition of the pollen grains. Pollen samples (n = 72) were collected from proboscides of various Heliconius species and Laparus doris in surrounding habitats of the Tropical Research Station La Gamba (Costa Rica). Examination using a light microscope revealed that pollen loads contained 74.88 ± 53.67% of damaged Psychotria pollen, 72.04 ± 23.4% of damaged Psiguria/Gurania pollen, and 21.35 ± 14.5% of damaged Lantana pollen (numbers represent median ± first quartile). Damaged pollen grains showed deformed contours, inhomogeneous and/or leaking contents, or they were empty. Experiments with Heliconius and Laparus doris from a natural population in Costa Rica demonstrated that 200 min of pollen processing behaviour significantly increased the percentage of damaged pollen of Psychotria compared to pollen from anthers (P = 0.015, Z = -2.44, Mann-Whitney U-test). Examination of pollen loads from green house reared Heliconius butterflies resulted in significantly greater amounts of damaged Psiguria pollen after 200 min of processing behaviour compared to pollen from flowers (P < 0.001, Z = -4.583, Mann-Whitney U-test). These results indicate that pollen processing functions as extra oral digestion whereby pollen grains are ruptured to make the content available for ingestion.

Pode Euseius alatus DeLeon (Acari: Phytoseiidae) predar Aceria guerreronis Keifer (Acari: Eriophyidae) em coqueiro? / Can Euseius alatus DeLeon (Acari: Phytoseiidae) prey on Aceria guerreronis Keifer (Acari: Eriophyidae) in coconut palm?

Ácaros do gênero Euseius são geralmente considerados especialistas na alimentação de pólen. Euseius alatus DeLeon é uma das seis espécies de ácaros fitoseídeos mais comumente encontrados em plantas de coqueiro no Nordeste do Brasil, associado com Aceria guerreronis Keifer. Apesar de a morfologia de E. alatus não favorecer a exploração da área meristemática do fruto habitada por A. guerreronis, o predador pode ter algum papel no controle do eriofídeo durante o processo de dispersão. O objetivo deste trabalho foi avaliar o desenvolvimento e a reprodução de E. alatus nas seguintes dietas: A. guerreronis, pólen de Ricinus communis (Euphorbiaceae); e Tetranychus urticae Koch (Tetranychidae) + pólen de R. communis + mel a 10 por cento. Euseius alatus desenvolveu-se mais rapidamente e ovipositou mais quando alimentada em dieta composta por T. urticae + pólen + mel. Contudo, os parâmetros da tabela de vida foram muito semelhantes em todas as dietas, sugerindo que E. alatus pode contribuir na redução da população de A. guerreronis no campo.

Mites of the genus Euseius are generally considered specialist as pollen feeders. Euseius alatus DeLeon is one of the six species of phytoseiid mites most commonly found on coconut plants in northeast Brazil associated with Aceria guerreronis Keifer. Although the morphology of E. alatus does not favor the exploitation of the meristematic area of the fruit inhabited by A. guerreronis, the predator may have some role in the control of this eriophyid during the dispersion process. The objective of this work was to evaluate the development and reproduction of E. alatus on the following diets: A. guerreronis, Ricinus communis pollen (Euphorbiaceae), and Tetranychus urticae Koch (Tetranychidae) + R. communis pollen + honey solution 10 percent. Euseius alatus developed slightly faster and had slightly higher oviposition rate when feeding on the diet composed of T. urticae + pollen + honey. However, life table parameters were very similar on all diets, suggesting that E. alatus may contribute in reducing the population of A. guerreronis in the field.