Olfactory specialization for perfume collection in male orchid bees.

Insects rely on the olfactory system to detect a vast diversity of airborne molecules in their environment. Highly sensitive olfactory tuning is expected to evolve when detection of a particular chemical with great precision is required in the context of foraging and/or finding mates. Male neotropical orchid bees (Euglossini) collect odoriferous substances from multiple sources, store them in specialized tibial pouches and later expose them at display sites, presumably as mating signals to females. Previous analysis of tibial compounds among sympatric species revealed substantial chemical disparity in chemical composition among lineages with outstanding divergence between closely related species. Here, we tested whether specific perfume phenotypes coevolve with matching olfactory adaptations in male orchid bees to facilitate the location and harvest of species-specific perfume compounds. We conducted electroantennographic (EAG) measurements on males of 15 sympatric species in the genus Euglossa that were stimulated with 18 compounds present in variable proportions in male hind tibiae. Antennal response profiles were species-specific across all 15 species, but there was no conspicuous differentiation between closely related species. Instead, we found that the observed variation in EAG activity follows a Brownian motion model of trait evolution, where the probability of differentiation increases proportionally with lineage divergence time. However, we identified strong antennal responses for some chemicals that are present as major compounds in the perfume of the same species, thus suggesting that sensory specialization has occurred within multiple lineages. This sensory specialization was particularly apparent for semi-volatile molecules ('base note' compounds), thus supporting the idea that such compounds play an important role in chemical signaling of euglossine bees. Overall, our study found no close correspondence between antennal responses and behavioral preferences/tibial contents, but confirms the utility of EAG profiling for discovering certain behaviorally active compounds.

Antagonistic activity of endo-ß-1,3-glucanase from a novel isolate, Streptomyces sp. 9X166, against black rot in orchids.

A total of 123 actinomycetes was isolated from 12 varieties of wild orchids and screened for potential antagonistic activity against Phytophthora, which causes black rot disease in orchids. In vitro and in vivo experimental results revealed that Streptomyces sp. strain 9X166 showed the highest antagonistic activity; its ß-1,3-glucanase production ability was a key mechanism for growth inhibition of the pathogen. PCR amplification and DNA sequencing of the 16S ribosomal RNA gene allowed the identification of this strain, with high similarity (99.93%) to the novel species Streptomyces similaensis. The glucanase enzyme, purified to homogeneity by anion exchange and gel filtration chromatography, showed a specific activity of 58 U mg(-1) (a 3.9-fold increase) and yield of 6.4%. The molecular weight, as determined by SDS-PAGE and gel filtration, was approximately 99 and 80 kDa, respectively, suggesting that the enzyme was a monomer. The purified enzyme showed the highest substrate specificity to laminarin, indicating that it was ß-1,3-glucanase. The hydrolyzed products of cello-oligosaccharides suggested that this enzyme was endo-type ß-1,3-glucanase. Streptomyces sp. 9X166 culture filtrate, possessing ß-1,3-glucanase activity, could degrade both freeze-dried and living mycelium. This is the first report on a ß-1,3-glucanase-producing Streptomyces sp. that could be an effective biocontrol agent for black rot disease in orchids.

The effect of mealybug Pseudococcus longispinus (Targioni Tozzetti) infestation of different density on physiological responses of Phalaenopsis × hybridum 'Innocence'.

Cultivated orchids are the most abundantly attacked by polyphagous mealybugs. This study documented how different density of mealybug Pseudococcus longispinus (Targioni Tozzetti) infestation is associated with a response of antioxidative systems of Phalaenopsis × hybridum 'Innocence'. The degree of cell damage, estimated by electrolyte leakage measurement and the level of thiobarbituric acid reactive substances (TBARS), the content of pigments as well as the activity of antioxidative enzymes and proline level, as measurements of stress and stress compensation in moth orchid were examined. The highest electrolyte leakage (E L) value among samples from colonized plants was found in the orchids from series III (50 individuals/plant), whereas the lowest in the plants from series II (20 individuals/plant). The TBARS content reached the highest level at the lowest number of feeding insects (series I). Peroxidase activity toward guaiacol was significantly increased in series I (5 individuals/plant). The highest catalase activity was recorded in plants colonized by the highest number of scale insects (series III). Whereas, the highest value of proline was in series II. The content of individual photosynthetic pigments (chlorophyll a, chlorophyll b and carotenoids) in plant tissues did not vary significantly between control and colonized orchids. The results have not confirmed hypothesis that the increasing number of mealybugs occurring on plant enhanced plant physiological response. The degree of longtailed mealybug infestation on plants was positively correlated only with electrolyte leakage and catalase activity in leaf tissues.

The evolution of floral deception in Epipactis veratrifolia (Orchidaceae): from indirect defense to pollination.

BACKGROUND: It is estimated that floral deception has evolved in at least 7500 species of angiosperms, of which two thirds are orchids. Epipactis veratrifolia (Orchidaceae) is a model system of aphid mimicry as aphidophagous hoverflies lay eggs on false brood sites on their flowers. To understand the evolutionary ecology of floral deception, we investigated the pollination biology of E. veratrifolia across 10 populations in the Eastern Himalayas. We reconstructed the phylogeny of Epipactis and mapped the known pollination systems of previously studied species onto the tree. RESULTS: Some inflorescences of E. veratrifolia were so infested with aphids while they were still in bud that the some larvae of hoverflies developed to the third instar while flower buds opened. This indicated that adult female hoverflies were partly rewarded for oviposition. Although flowers failed to secrete nectar, they mimicked both alarm pheromones and aphid coloring of to attract female hoverflies as their exclusive pollinators. Phylogenetic mapping indicate that pollination by aphidophagous hoverflies is likely an ancestral condition in the genus Epipactis. We suggest that the biological interaction of aphid (prey), orchid (primary producer) and hoverfly (predator) may represent an intermediate stage between mutualism and deception in the evolution of pollination-by-deceit in E. veratrifolia. CONCLUSIONS: Our analyses indicate that this intermediate stage may be used as a model system to interpret the origin of oviposition (brood site) mimicry in Epipactis. We propose the hypothesis that some deceptive pollination systems evolved directly from earlier (partly) mutualistic systems that maintained the fidelity of the original pollinator(s) even though rewards (nectar/ brood site) were lost.

Pollination and protection against herbivory of Nepalese Coelogyninae (Orchidaceae).

PREMISE OF THE STUDY: Although many species in the orchid genus Coelogyne are horticulturally popular, hardly anything is known about their pollination. Pollinators of three species were observed in the field in Nepal. This information is urgently needed because many orchid species in Nepal are endangered. Whether the exudates produced by extrafloral nectaries played a role in protection against herbivory was also investigated. METHODS: Pollinators of C. flaccida, C. nitida, and Otochilus albus were filmed, captured, and identified. Ant surveys and exclusion experiments were carried out. To investigate whether pollinators are needed for fruit set, plants were wrapped in mesh wire bags. Inflorescence stems were examined with microscopy. Fehling's reagent was used to detect sugars in extrafloral exudates. KEY RESULTS: Coelogyne flaccida and C. nitida need pollinators to set fruit and are pollinated by wild bees identified as Apis cerana. Otochilus albus was found to be pollinated by Bombus kashmirensis. Extrafloral nectar was found to be exuded by nectary-modified stomata and contained high amounts of sugars. Different species of ants were observed collecting these exudates. A significant difference was found in damage inflicted by flower and leaf-eating beetles between C. nitida plants living in trees with ant nests and those in ant-free trees. CONCLUSIONS: Floral syndromes include scented and colored trap flowers without reward to their pollinators. All orchids investigated exude extrafloral nectar by nectary-modified stomata. This nectar was found to flow from the phloem to the stomata through intercellular spaces in the outer parenchymatous layer of the inflorescence.

Pollinator convergence and the nature of species' boundaries in sympatric Sardinian Ophrys (Orchidaceae).

BACKGROUND AND AIMS: In the sexually deceptive Ophrys genus, species isolation is generally considered ethological and occurs via different, specific pollinators, but there are cases in which Ophrys species can share a common pollinator and differ in pollen placement on the body of the insect. In that condition, species are expected to be reproductively isolated through a pre-mating mechanical barrier. Here, the relative contribution of pre- vs. post-mating barriers to gene flow among two Ophrys species that share a common pollinator and can occur in sympatry is studied. METHODS: A natural hybrid zone on Sardinia between O. iricolor and O. incubacea, sharing Andrena morio as pollinator, was investigated by analysing floral traits involved in pollinator attraction as odour extracts both for non-active and active compounds and for labellum morphology. The genetic architecture of the hybrid zone was also estimated with amplified fragment length polymorphism (AFLP) markers, and pollination fitness and seed set of both parental species and their hybrids in the sympatric zone were estimated by controlled crosses. KEY RESULTS: Although hybrids were intermediate between parental species in labellum morphology and non-active odour compounds, both parental species and hybrids produced a similar odour bouquet for active compounds. However, hybrids produced significantly lower fruit and seed set than parental species, and the genetic architecture of the hybrid zone suggests that they were mostly first-generation hybrids. CONCLUSIONS: The two parental species hybridize in sympatry as a consequence of pollinator overlap and weak mechanical isolation, but post-zygotic barriers reduce hybrid frequency and fitness, and prevent extensive introgression. These results highlight a significant contribution of late post-mating barriers, such as chromosomal divergence, for maintaining reproductive isolation, in an orchid group for which pre-mating barriers are often considered predominant.

Validation of the Berlese-funnel technique for thrips extraction.

In order to get the accreditation EN ISO/IEC 17025 for Thrips palmi the Berlese-funnel technique, which is used for the isolation of quarantine insects out of plant material, was validated. Following parameters were investigated: cleaning of the funnel, temperature during isolation, detection limit and duration of the isolation period. Thrips fuscipennis was collected from heavily infected rosehip and used as target organism. Besides orchids, artificially contaminated maple leaves (Acer pseudoplatanus) were used for the validation. Results showed that thrips and other organisms can be present alive or dead in the funnel after removing the treated plants and can contaminate the next sample or isolate. Cleaning of the funnel with a vacuum cleaner and compressed-air apparatus is necessary before running a new extraction. Contamination of the recipient is also possible from the environment. This can be avoided by closing the opening between the funnel and the recipient. To reach an optimal temperature for isolation of the thrips a 60 Watt bulb is necessary. The results showed that the maximum temperature doesn't reach a temperature above 51 degrees C, the average temperatures were situated between 35, 74 degrees C and 39, 38 degrees C. A 40 Watt bulb doesn't create enough heat to guarantee an efficient isolation of the thrips; the average temperature was 34, 74 degrees C and the maximum temperature 36, 80 degrees C. Based on the results we can conclude that an isolation time of 20 hours is necessary to obtain accurate data. Dependent on the number of thrips in the artificially infected samples 87 to 95% is isolated after 20 hours. The detection limit is 1 thrips with a probability of 95% being isolated after 20 hours.

Genetic structure of Pseudococcus microcirculus (Hemiptera: Pseudococcidae) populations on epiphytic orchids in south Florida.

In 2012, the orchid mealy bug Pseudococcus microcirculus was first detected in situ in North America's more diverse orchid region, the Big Cypress Basin (Collier Co FL). A follow-up survey showed that the mealy bug is more widespread and found on epiphytic orchids in two locations, in both the Fakahatchee Strand State Preserve (sites B and F) and the Florida Panther National Wildlife Refuge (sites M and C). There, we collected mealy bugs (n = 54) from 35 orchid individuals and screened allelic variation at seven microsatellite loci. We estimated genetic diversity and differentiation among all sites and compared the variation among individuals collected on the same plant. Genetic differentiation between sites M and C (FST = 0.03, P < 0.01) and,Mand B (FST = 0.04, P < 0.01) was detected.We also detected significantly lower mean pairwise relatedness among individuals from site B compared to all the other locations, and this population had the lowest inbreeding coefficient. Genetic diversity and mean pairwise relatedness were highly variable among plants with multiple individuals; however, plants from sites F and M tend to have collections of individuals with higher mean pairwise relatedness compared to sites B and C. Our results indicate that there is genetic diversity and differentiation among mealy bugs in these locations, and that collections of individuals on the same plant are genetically diverse. As such, the mealy bugs throughout these areas are likely to be genetically diverse and exist in multiple distinct populations.

Adding Biotic Interactions into Paleodistribution Models: A Host-Cleptoparasite Complex of Neotropical Orchid Bees.

Orchid bees compose an exclusive Neotropical pollinators group, with bright body coloration. Several of those species build their own nests, while others are reported as nest cleptoparasites. Here, the objective was to evaluate whether the inclusion of a strong biotic interaction, such as the presence of a host species, improved the ability of species distribution models (SDMs) to predict the geographic range of the cleptoparasite species. The target species were Aglae caerulea and its host species Eulaema nigrita. Additionally, since A. caerulea is more frequently found in the Amazon rather than the Cerrado areas, a secondary objective was to evaluate whether this species is increasing or decreasing its distribution given South American past and current climatic conditions. SDMs methods (Maxent and Bioclim), in addition with current and past South American climatic conditions, as well as the occurrences for A. caerulea and E. nigrita were used to generate the distribution models. The distribution of A. caerulea was generated with and without the inclusion of the distribution of E. nigrita as a predictor variable. The results indicate A. caerulea was barely affected by past climatic conditions and the populations from the Cerrado savanna could be at least 21,000 years old (the last glacial maximum), as well as the Amazonian ones. On the other hand, in this study, the inclusion of the host-cleptoparasite interaction complex did not statistically improve the quality of the produced models, which means that the geographic range of this cleptoparasite species is mainly constrained by climate and not by the presence of the host species. Nonetheless, this could also be caused by unknown complexes of other Euglossini hosts with A. caerulea, which still are still needed to be described by science.

Ants visit nectaries of Epidendrum denticulatum (Orchidaceae) in a Brazilian rainforest: effects on herbivory and pollination.

Epidendrum denticulatum (Orchidaceae) produces nectar on the petioles of buds, flowers, and fruits (extrafloral nectaries) but no nectar is found on its flowers, and it is probably a deceptive species. In the Brazilian Atlantic rainforest, some aspects of both the ecology and behavior of Camponotus sericeiventris (Formicinae) and Ectatomma tuberculatum (Ponerinae), two ant species foraging on E. denticulatum extrafloral nectaries, were investigated. Both experiments, using termites as baits and field observations, suggest that these ant species are able to prevent reproductive organ herbivory, without affecting pollinator behaviour. Since a low fruit set is often cited as a characteristic of the family, especially for deceptive species, ants attracted to orchid inflorescences protect reproductive structures and increase the probability of pollination success. Epidendrum denticulatum flowers were visited and probably pollinated by Heliconius erato (Nymphalidae) and Euphyes leptosema (Hesperiidae).

Effect of seven new orchard pesticides on Galendromus occidentalis in laboratory studies.

BACKGROUND: Biological control of phytophagous mites in orchards requires that pesticides used to manage other arthropod pests or diseases are harmless to predacious mites, as these are essential to keep phytophagous mites at non-injurious population levels. This study evaluates the possible toxic attributes of acetamiprid, imidacloprid, thiacloprid, thiamethoxam, spirodiclofen, spinosad and methoxyfenoxide currently used in western Canadian orchards. RESULTS: None of these pesticides has any ovicidal properties against Galendromus occidentalis (Nesbitt). Imidacloprid and acetamiprid were highly toxic to the adults and reduced fecundity significantly. Thiamethoxam and spirodiclofen were non-toxic to adults, but they slightly reduced fecundity. Thiacloprid, spinosad and methoxyfenoxide were harmless to adults and had no effect on fecundity. All compounds showed some repellence at 24 h intervals for 72 h. CONCLUSIONS: Imidacloprid and acetamiprid are incompatible with IPM programs because they are toxic to adults and negatively affect fecundity. Thiamethoxam and spirodiclofen need further field evaluation to determine if they are compatible with IPM programs because they slightly reduced fecundity. Thiacloprid, spinosad and methoxyfenoxide are harmless to adults, but they are slightly repellent. Therefore, with the exception of imidacloprid and acetamiprid, all these compounds should be field tested for compatibility in an IPM program.

Resource competition triggers the co-evolution of long tongues and deep corolla tubes.

BACKGROUND: It is normally thought that deep corolla tubes evolve when a plant's successful reproduction is contingent on having a corolla tube longer than the tongue of the flower's pollinators, and that pollinators evolve ever-longer tongues because individuals with longer tongues can obtain more nectar from flowers. A recent model shows that, in the presence of pollinators with long and short tongues that experience resource competition, coexisting plant species can diverge in corolla-tube depth, because this increases the proportion of pollen grains that lands on co-specific flowers. METHODOLOGY/PRINCIPAL FINDINGS: We have extended the model to study whether resource competition can trigger the co-evolution of tongue length and corolla-tube depth. Starting with two plant and two pollinator species, all of them having the same distribution of tongue length or corolla-tube depth, we show that variability in corolla-tube depth leads to divergence in tongue length, provided that increasing tongue length is not equally costly for both species. Once the two pollinator species differ in tongue length, divergence in corolla-tube depth between the two plant species ensues. CONCLUSIONS/SIGNIFICANCE: Co-evolution between tongue length and corolla-tube depth is a robust outcome of the model, obtained for a wide range of parameter values, but it requires that tongue elongation is substantially easier for one pollinator species than for the other, that pollinators follow a near-optimal foraging strategy, that pollinators experience competition for resources and that plants experience pollination limitation.

Confirmation by DNA analysis that Contarinia maculipennis (Diptera: Cecidomyiidae) is a polyphagous pest of orchids and other unrelated cultivated plants.

The cytochrome oxidase subunit I gene in mitochondrial DNA of 53 larvae of Contarinia maculipennis Felt from flower buds of various host plants, collected from Hawaii, Japan and Thailand was analysed. Monophyly of the clade including C. maculipennis from Hawaii, Thailand and Japan was supported. There was no sequential variation within the specimens from Hawaii and Japan, which differed from one another by 6 bp (1.37%). Three haplotypes were recognized in specimens from Thailand but differences from Hawaiian and Japanese specimens were small. Overall, there were no differences in the 146 deduced amino acid residues. It is therefore concluded that C. maculipennis is a polyphagous species that can develop on plant hosts representing at least seven botanical families. This pest of Dendrobium flower buds in glasshouses is considered to have entered Hawaii, Florida and Japan from Southeast Asia, and was recently intercepted in the Netherlands. Infestations have established and spread in orchid glasshouses, causing concern about the possibility of more extensive damage to orchids and to crops, such as bitter gourd, grown in close proximity to orchid glasshouses in Japan. The potential usefulness of DNA analysis in determining host plant ranges of morphologically identical cecidomyiid species that are currently identified solely on differences of host plant is emphasized.