Paulo Milet-Pinheiro

Do euglossine males (Apidae, Euglossini) leave tropical rainforest to collect fragrances in sugarcane monocultures?

Euglossine bees are known to be long-distance pollinators in tropical rainforests. But there is controversy concerning to the flight ranges of these bees between forest fragments. In an isolated fragment of Atlantic Rainforest in Pernambuco, NE Brazil, surrounded by sugarcane monocultures, it was examined if euglossine males leave closed rainforest to collect fragrances. In a straight-line transect leading from forest into a sugarcane plantation, euglossine males were simultaneously captured by scent baits at seven distinct points: inside the forest, forest edge, outside the forest in the sugarcane fields at distances of 10 m, 50 m, 100 m, 250 m and 500 m from the forest edge. A total of 945 euglossine bees of 16 species were recorded. The results demonstrate different relations of the Euglossini species to the closed forest. Males of 11 species did not leave the forest. Such species, together with the plants they are linked to, seem to be the most threatened by habitat fragmentation. Only bees of five species were found at the scent baits in the sugarcane fields. Already the 10 m sampling point outside the forest showed a drastic reduction in species richness, indicating that the forest edge functions as a barrier for most euglossine species.

The Chemical Basis of Host-Plant Recognition in a Specialized Bee Pollinator

Many pollinators specialize on a few plants as food sources and rely on flower scents to recognize their hosts. However, the specific compounds mediating this recognition are mostly unknown. We investigated the chemical basis of host location/recognition in the Campanula-specialist bee Chelostoma rapunculi using chemical, electrophysiological, and behavioral approaches. Our findings show that Ca. trachelium flowers emit a weak scent consisting of both widespread and rare (i.e., spiroacetals) volatiles. In electroantennographic analyses, the antennae of bees responded to aliphatics, terpenes, aromatics, and spiroacetals; however, the bioassays revealed a more complex response picture. Spiroacetals attracted host-naive bees, whereas spiroacetals together with aliphatics and terpenes were used for host finding by host-experienced bees. On the intrafloral level, different flower parts of Ca. trachelium showed differences in the absolute and relative amounts of scent, including spiroacetals. Scent from pollen-presenting flower parts elicited more feeding responses in host-naive bees as compared to a scentless control, whereas host-experienced bees responded more to the nectar-presenting parts. Our study demonstrates the occurrence of learning (i.e., change in the bee’s innate chemical search-image) after bees gain foraging experience on host flowers. We conclude that highly specific floral volatiles play a key role in host-flower recognition by this pollen-specialist bee, and discuss our findings into the broader context of host-recognition in oligolectic bees.

Pollination in Jacaranda rugosa (Bignoniaceae): euglossine pollinators, nectar robbers and low fruit set

Nectar robbers access floral nectar in illegitimate flower visits without, in general, performing a pollination service. Nevertheless, their effect on fruit set can be indirectly positive if the nectar removal causes an incremental increase in the frequency of legitimate flower visits of effective pollinators, especially in obligate outcrossers. We studied pollination and the effect of nectar robbers on the reproductive fitness of Jacaranda rugosa, an endemic shrub of the National Park of Catimbau, in the Caatinga of Pernambuco, Brazil. Xenogamous J. rugosa flowers continuously produced nectar during the day at a rate of 1 mul.h(-1). Female and male Euglossa melanotricha were the main pollinators. Early morning flower visits substantially contributed to fruit set because stigmas with open lobes were almost absent in the afternoon. Ninety-nine per cent of the flowers showed damage caused by nectar robbers. Artificial addition of sugar water prolonged the duration of flower visits of legitimate flower visitors. Removal of nectar, simulating the impact of nectar robbers, resulted in shorter flower visits of euglossine bees. While flower visits of nectar-robbing carpenter bees (Xylocopa frontalis, X. grisescens, X. ordinaria) produced only a longitudinal slit in the corolla tube in the region of the nectar chamber, worker bees of Trigona spinipes damaged the gynoecium in 92% of the flowers. This explains the outstandingly low fruit set (1.5%) of J. rugosa in the National Park of Catimbau.

Identification of male-borne attractants in Anastrepha fraterculus (Diptera: Tephritidae)

The South American fruit fly is one of the most destructive polyphagous pests in South America. In this species, males gathered in aggregations emit volatiles that attract females; however, the compounds involved in this task remain unknown. In this study, we investigated the chemical composition of the volatile blend emitted by males aiming to identify the specific compounds within this blend that elicit behavioral responses in conspecific females. For this purpose, we performed chemical and electrophysiological analyses and bioassays. The chemical analyses revealed the presence of 29 compounds in headspace samples of A. fraterculus males, of which six compounds, i.e. α-pinene, limonene, (Z)-3-nonen-1-ol, (E,Z)-3,6-nonadien-1-ol, α-farnesene and (S,S)-(−)-epianastrephin, triggered antennal depolarization in conspecific females. In laboratory bioassays, five out of eight synthetic compounds tested individually elicited more behavioral responses than a hexane control, but only the synthetic mixture composed of all EAD-active compounds triggered behavioral responses in females similar to the responses to the headspace samples of conspecific males. In an experiment under semi-natural conditions, the synthetic mixture was more attractive to females than a hexane control and equally attractive to headspace extracts of males. This study reports the identification of male volatile compounds that act as attractant for A. fraterculus females, which may be useful for the control of this pest in infested orchards.

(E)-Caryophyllene and α-Humulene: Aedes aegypti Oviposition Deterrents Elucidated by Gas Chromatography-Electrophysiological Assay of Commiphora leptophloeos Leaf Oil.

Aedes aegypti is responsible for the transmission of dengue, a disease that infects millions of people each year. Although essential oils are well recognized as sources of compounds with repellent and larvicidal activities against the dengue mosquito, much less is known about their oviposition deterrent effects. Commiphora leptophloeos, a tree native to South America, has important pharmacological properties, but the chemical profile and applicability of its essential oil in controlling the spread of the dengue mosquito have not been investigated. The aim of this study was to determine the composition of C. leptophloeos leaf oil and to evaluate its larvicidal and oviposition deterrent effects against A. aegypti. Fifty-five components of the essential oil were detected by gas chromatography (GC)—mass spectrometry, with α-phellandrene (26.3%), (E)-caryophyllene (18.0%) and β-phellandrene (12.9%) identified as the major constituents. Bioassays showed that the oil exhibited strong oviposition deterrent effects against A. aegypti at concentrations between 25 and 100 ppm, and possessed good larvicidal activity (LC50 = 99.4 ppm). Analysis of the oil by GC coupled with electroantennographic detection established that seven constituents could trigger antennal depolarization in A. aegypti gravid females. Two of these components, namely (E)-caryophyllene and α-humulene, were present in substantial proportions in the oil, and oviposition deterrence assays confirmed that both were significantly active at concentrations equivalent to those present in the oil. It is concluded that these sesquiterpenes are responsible, at least in part, for the deterrent effect of the oil. The oviposition deterrent activity of the leaf oil of C. leptophloeos is one of the most potent reported so far, suggesting that it could represent an interesting alternative to synthetic insecticides. The results of this study highlight the importance of integrating chemical and electrophysiological methods for screening natural compounds for their potential in combating vectors of insect-borne diseases.

Comunidade de abelhas (Hymenoptera, Apoidea) e plantas em uma área do Agreste pernambucano, Brasil

The Agreste is a transition region of tropical rainforest and Caatinga in northeastern Brazil. In this region, the majority of the native Atlantic Rainforest was destroyed to give place to livestock farming. It is not known whether degraded areas maintain a diversified bee-plant community or not and which kinds of bee-plant interactions occur. The vegetation at the study site comprises pastures, ruderal vegetation and minute remanents of rainforest vegetation. Both bees and associated plants were collected monthly, from August 2001 to July 2002, sampling during two consecutive days from 5:30 am to 5:30 pm. We collected a total of 1004 bees from 79 species. Apidae was the most abundant and species-rich bee family (732 individuals, 43 species) followed by Halictidae (194 individuals and 20 spp.), Megachilidae (47 individuals and 13 spp.), Colletidae (16 individuals and 2 spp.) and Andrenidae (15 individuals and 1 sp.). Only three species of eusocial bees and five of euglossine bees were recorded, even though both groups are diversified in Neotropical rainforests. The absence of native stingless bees of the genera Plebeia, Frieseomelitta, Partamona, Scaptotrigona and Trigonisca, as well as of other euglossine bee species, is probably due to the lack of nesting sites and to the paucity of pollen and nectar resources in this disturbed area. The bees visited flowers of 87 plant species, mainly of herbs and small shrubs. Isolated rainforest trees in the pasture as well as cultivated fruit crops contributed to some extent to maintain a diversified native bee community.

Pollination biology in the dioecious orchid Catasetum uncatum: How does floral scent influence the behaviour of pollinators?

Catasetum is a neotropical orchid genus that comprises about 160 dioecious species with a remarkable sexual dimorphism in floral morphology. Flowers of Catasetum produce perfumes as rewards, which are collected only by male euglossine bees. Currently, floral scents are known to be involved in the selective attraction of specific euglossine species. However, sexual dimorphism in floral scent and its eventual role in the pollination of Catasetum species have never been investigated. Here, we have investigated the pollination of Catasetum uncatum and asked: (1) Is floral scent a sexual dimorphic trait? (2) Does pollinarium removal/deposition affect scent emission? (3) Does sexual dimorphism in floral scent and changed scent emission have implications with regard to the behaviour of the pollinators? The frequency and behaviour of floral visitors were observed in non-manipulated flowers (both flower sexes) and in manipulated flowers (pistillate only) in which pollinaria were deposited. Scents of staminate and pistillate flowers (both manipulated and non-manipulated) were collected by using dynamic headspace methods and analysed chemically. Electrophysiological analyses were performed to detect compounds triggering antennal depolarisation in the euglossine species. C. uncatum is pollinated mainly by males of Euglossa nanomelanotricha. Pollinators were more frequent in pistillate than in staminate inflorescences. Bees approaching staminate flowers frequently flew away without visiting them, a behavioural pattern not observed in pistillate flowers. In the chemical analyses, we recorded 99 compounds, 31 of which triggered antennal depolarisation in pollinators. Multivariate analyses with the electrophysiological-active compounds did not detect differences between the scent composition of staminate and pistillate flowers. Pollinarium removal or deposition resulted in diminished scent emission within 24h in staminate and pistillate flowers, respectively. Surprisingly, bees discriminated pollinated from non-pollinated pistillate flowers as early as 2h after pollination. The rapid loss in the attractiveness of flowers following pollinarium removal/deposition can be interpreted as a strategy to direct pollinators to non-pollinated flowers. We have found no evidence that euglossine males discriminate staminate from pistillate flowers by means of floral scent. Instead, we speculate that bees use visual cues, such as sex dimorphic traits, to discriminate flowers of different sexes. Together, our results provide interesting insights into the evolution of floral signals in gender-dimorphic species and into its significance in plant reproductive biology.

Visual and Olfactory Floral Cues of Campanula (Campanulaceae) and Their Significance for Host Recognition by an Oligolectic Bee Pollinator

Oligolectic bees collect pollen from a few plants within a genus or family to rear their offspring, and are known to rely on visual and olfactory floral cues to recognize host plants. However, studies investigating whether oligolectic bees recognize distinct host plants by using shared floral cues are scarce. In the present study, we investigated in a comparative approach the visual and olfactory floral cues of six Campanula species, of which only Campanula lactiflora has never been reported as a pollen source of the oligolectic bee Ch. rapunculi. We hypothesized that the flowers of Campanula species visited by Ch. rapunculi share visual (i.e. color) and/or olfactory cues (scents) that give them a host-specific signature. To test this hypothesis, floral color and scent were studied by spectrophotometric and chemical analyses, respectively. Additionally, we performed bioassays within a flight cage to test the innate color preference of Ch. rapunculi. Our results show that Campanula flowers reflect the light predominantly in the UV-blue/blue bee-color space and that Ch. rapunculi displays a strong innate preference for these two colors. Furthermore, we recorded spiroacetals in the floral scent of all Campanula species, but Ca. lactiflora. Spiroacetals, rarely found as floral scent constituents but quite common among Campanula species, were recently shown to play a key function for host-flower recognition by Ch. rapunculi. We conclude that Campanula species share some visual and olfactory floral cues, and that neurological adaptations (i.e. vision and olfaction) of Ch. rapunculi innately drive their foraging flights toward host flowers. The significance of our findings for the evolution of pollen diet breadth in bees is discussed.

Mutual reproductive dependence of distylic Cordia leucocephala (Cordiaceae) and oligolectic Ceblurgus longipalpis (Halictidae, Rophitinae) in the Caatinga

BACKGROUND AND AIMS The close relationship between distylic Cordia leucocephala and the bee Ceblurgus longipalpis, both endemic to the Caatinga, north-east Brazil, was investigated, emphasizing reproductive dependence, morphological adaptations of the partners, and pollen flow. METHODS In the municipality of Pedra, in the Caatinga of Pernambuco, the breeding system and reproductive success of C. leucocephala, its interaction with flower visitors and inter- and intramorph pollen flow were determined. KEY RESULTS The bee Ceblurgus longipalpis, the unique flower visitor and effective pollinator of self-incompatible Cordia leucocephala, presents morphological features adapted to exploit hidden pollen and nectar in the long and narrow corolla tubes. Pollen of low-level anthers is collected with hairs on prolonged mouthparts and pollen of high-level anthers with clypeus, mandibles, and labrum, showing pollen removal from both levels with the same effectiveness. In both morphs, this results in similar legitimate, i.e. intermorph cross-pollen flow. Illegitimate pollen flow to stigmas of pin flowers, however, was much higher than to stigmas of thrum flowers. Moreover, more illegitimate pollen was transported to stigmas of pin and less to those of thrum flowers when compared with legitimate pollen flow. CONCLUSIONS The study reveals a one-to-one reproductive inter-dependence between both partners. Data indicate that this relationship between bee species and plant species is one of the rare cases of monolecty among bees. Monotypic Ceblurgus longipalpis, the only rophitine species of Brazil, evolved prolonged mouthparts rare among short-tongued bees that enable them to access pollen from flowers with short-level anthers hidden for bees of other species, and nectar at the base of the flower tube.

Evaluation of the Activity of the Essential Oil from an Ornamental Flower against Aedes aegypti: Electrophysiology, Molecular Dynamics and Behavioral Assays.

Dengue fever has spread worldwide and affects millions of people every year in tropical and subtropical regions of Africa, Asia, Europe and America. Since there is no effective vaccine against the dengue virus, prevention of disease transmission depends entirely on regulating the vector (Aedes aegypti) or interrupting human-vector contact. The aim of this study was to assess the oviposition deterrent activity of essential oils of three cultivars of torch ginger (Etlingera elatior, Zingiberaceae) against the dengue mosquito. Analysis of the oils by gas chromatography (GC)—mass spectrometry revealed the presence of 43 constituents, of which α-pinene, dodecanal and n-dodecanol were the major components in all cultivars. Solutions containing 100 ppm of the oils exhibited oviposition deterrent activities against gravid Ae. aegypti females. GC analysis with electroantennographic detection indicated that the oil constituents n-decanol, 2-undecanone, undecanal, dodecanal, trans-caryophyllene, (E)-β-farnesene, α-humulene, n-dodecanol, isodaucene and dodecanoic acid were able to trigger antennal depolarization in Ae. aegypti females. Bioassays confirmed that solutions containing 50 ppm of n-dodecanol or dodecanal exhibited oviposition deterrent activities, while a solution containing the alcohol and aldehyde in admixture at concentrations representative of the oil presented an activity similar to that of the 100 ppm oil solution. Docking and molecular dynamics simulations verified that the interaction energies of the long-chain oil components and Ae. aegypti odorant binding protein 1 were quite favorable, indicating that the protein is a possible oviposition deterrent receptor in the antenna of Ae. aegypti.