Four of the orchards contained wild bees such as Bombus vosnesenskii Rad. and Bombus melanopygus Nyl., and the fifth contained the managed native blue orchard bee O. lignaria.In 2009, we categorized 14 almond orchards either as having non-Apis bees or lacking non-Apis bees , based on standard observations of flower visitors . In each orchard, we covered a set of almond branches with mesh bags before flowering to exclude pollinator visits. Once anorchard was in bloom, the bags were removed, and the branches with the previously unvisited flowers were removed from the trees and immediately presented to foraging A. mellifera. After an A. mellifera had visited one of these flowers, the flower was removed from the branch, its petals and anthers were removed, and it was placed in a 1.5 ml microcentrifuge tube containing 0.5 ml of water, such that the stigma did not touch the tube’s surface and the pedicle was in water. These flowers were left at room temperature, away from direct sunlight for 72 h to allow pollen tubes to grow. After 72 h, the pistils were fixed in FAA and stored at 48C until further processing. From each of the 14 orchards, an average of 30 stigmas were processed after a single honey bee visit .
To examine pollen tube growth, pistils were removed from the FAA and the tissue softened by boiling in 5 per cent sodium sulphite for 30 min. They were then soaked in tap water for 20 min,drainage planter pot and incubated for 24 h in a decolourized solution of 0.1 per cent aniline blue dye, dissolved in 0.1 N K3PO4. The softened stained pistils were squashed onto a microscope slide to reveal pollen tubes. The slides were examined using a fluorescent microscope . For each slide, the numbers of pollen tubes initiating growth on the stigma and reaching the base of the style were scored. In 2009, we measured fruit set in each of nine orchards with nonApis bees and nine without non-Apis bees. We marked 1 m lengths of branches on five trees on the outer row of each orchard. To calculate fruit set, the number of flowers on each marked branch section was counted and in July the number of developing fruits on the same section of branch was recorded. In 2008, standardized observations of flower visitors were conducted in the same orchards. In the nine orchards with non-Apis bees , overall 18 different non-Apis bee species/morphospecies were observed visiting almond flowers, with Andrena cerasifolii being the most common non-Apis visitor .In 2011, three large cages were set up in one of the study orchards. Each cage contained two rows of four trees .
One row was the Monterey variety and the other the Carmel variety. The cages were stocked with bees at the initiation of bloom . One cage received a four-frame nucleus A. mellifera colony with two entrances, set up so that one entrance opened into the cage and one entrance to the open orchard. One cage received 32 female and 32 male individuals of O. lignaria. Wooden nesting blocks, water and loose soil were provided for nesting. In the third cage both A. mellifera and O. lignaria were stocked at opposite ends of the cage as described previously. Observational scans were made of the frequency of flower visits in the cages to assess the number of foraging bees within each cage. In each scan, a group of flowers was observed for 20 s, and the number of flowers observed and the frequency of flower visits recorded. Scans were repeated in different sections of each tree on different days . Many of the same methods as detailed above for the open orchards were used in the cages, with the following differences. The movement between adjacent trees was recorded during 1 min observations. Three observations were made down one row, three down the other row and four between the rows. These observations were conducted on 5 days in the A. mellifera cage and 4 days in the mixed A. mellifera/O. lignaria cage . Single-visit pollination effectiveness was measured for A. mellifera in the A. mellifera cage and the mixed cage .
For each visit, the number of pollen grains on the stigma, the number of pollen tubes initiating growth and the number of pollen tubes reaching the base of the style were counted. The fruit set was estimated as above by marking two branches on each of the eight trees per cage. Per visit fruit set was estimated by dividing the fruit set by the average flower visitation rate in the cage.Data from the open orchards on A. mellifera movement , pollen tube growth and fruit set were analysed using generalized linear mixed models . All models were simplified by stepwise deletion. Analysis of variance was used to compare the loss of explanatory power from the removal of an explanatory variable, and if p 0.05 the variable was dropped. A Mann –Whitney U-test was performed on the data from flower visitor observations in the orchards where fruit set was recorded. The visitation rate of A. mellifera at the orchard edge was compared between the orchards with non-Apis bees present and those without. For the cage data, means and standard errors were calculated for informal comparison between the cages, as replications at the cage level were not possible. All analyses were carried out in R v. 2.14.1 .Our findings show that increased pollinator diversity can synergistically increase pollination service through species interactions that alter the behaviour and resulting functional quality of a dominant pollinator species. This highlights a largely unexplored facilitative component of the benefit of biodiversity to ecosystem services. Total bee visitation rates were similar between orchard types, and the visitation rate of A. mellifera was lower in orchards with non-Apis bees. However, A. mellifera pollination effectiveness was greater and fruit set was higher when non-Apis bees were present. Apis mellifera increased their proportion of movement between tree rows when non-Apis bees were present, thereby improving pollination effectiveness. More successful pollen tube growth translated into significantly higher fruit set in the orchards where non-Apis bees were present. The greater proportion of between row movements by A. mellifera individuals most probably resulted in the deposition of more compatible pollen, since pollen from the same variety generally does not set fruit . This synergistic effect of the presence of non-Apis bees suggests that maintaining biodiversity in agricultural ecosystems could provide unrecognized benefits, and it offers exciting opportunities for the integration of more diverse pollination systems to improve the longer-term sustainability of crop production for almond and similar crops. Unlike in sunflower, very few direct interactions were observed between A. mellifera and non-Apis bees. One potential mechanism for the increased proportion of inter-row movement of A. mellifera in the presence of other non-Apis bees is linked to resource depletion. Because some non-Apis bees can fly at lower temperatures than A. mellifera, and therefore earlier in the day, it may be that if A. mellifera begin foraging and find flowers have already been depleted then they increase the distance of their foraging flights. Alternatively, it may be related to the scent marks left by non-Apis bees on the flowers. Apis mellifera have been shown to avoid visiting flowers that have been marked by bumble bees, and it may be that as well as avoiding the flowers they also shift their foraging location. Given that A. mellifera tend to move down the same row, if a response to perceived resource competition is to shift foraging locations, this could be expected to involve movement across rows.
However, at present the mechanism for the increased between-row movements when non-Apis bees are present is unknown. In the controlled cage environment, the steeper increase in the number of pollen tubes initiated with increasing pollen deposition in the mixed cage and the higher fruit set supports the findings from the open orchards that more compatible pollen is being moved when pollinator communities are diverse. Because there was only one cage per treatment,plant pot with drainage the data from the cages are only descriptive, but they do support the findings from the open orchards. Future work should attempt to replicate similar treatments. The greater fruit set in the mixed cage when the visitation rate was lower than in the A. mellifera cage may be due to the slightly higher proportion of between-row movements in the mixed cage. Although the differences in movement were not great, the pollen tube data suggest that more pollen was moving between varieties in the mixed cage. The size of the cages limited the opportunity for between-tree flights. This and the relatively low visitation rate of O. lignaria in the cages compared with the open orchards may explain why the difference in pollen tube formation and fruit set between the cages was less than in the open orchards. The price of California almond in 2011 was approximately $1.79 per pound and production was estimated at 2670 lbs per acre . If a 5 per cent increase in fruit set as calculated from the controlled cage environment translated into an equivalent increase in production, farmers would make $239 more per acre . This estimate from cages may be a lower bound compared with open orchards. Sampling effects and complementarity have been the primary explanations for a positive relationship between biodiversity and ecosystem function. Here, we show a different mechanism, possibly due to interspecific competition, where community composition alters the behaviour of a service-providing organism with a positive knock-on effect for the ecosystem service. Our results show alterations in A. mellifera foraging behaviour when a diverse community of other bees are present, and suggest almond yield can be increased by encouraging wild bees in the orchards. As such, natural habitat near almond orchards should be conserved to protect wild bee communities. The availability of A. mellifera is not predicted to increase at the same rate as demand for their services in agriculture. Thus, increasing the pollination effectiveness of A. mellifera and conserving wild pollinator communities could help increase crop yields.
The synergistic combination of A. mellifera and non-Apis bees represents a sustainable way to improve crop pollination services, but the generality of such effects still need to be tested across multiple crop systems.Mosquitoes rely on the olfactory system to find plants as a source of carbohydrates, hosts for blood meals, and oviposition sites. Because pathogens might be transmitted during a bite by an infected mosquito, there is understandably a great deal of interest in unraveling the olfactory aspects of human-mosquito interactions to explore ways of reducing mosquito bites. However, plant nectar sources are often essential for mosquitoes because they increase mosquito life span and reproductive capacity and long-living mosquitoes are more dangerous . Therefore, understanding how mosquitoes find plants/flowers is also important for reducing the transmission of vector-borne diseases. Previously, we have identified generic and plant kairomone sensitive odorant receptors from the Southern house mosquito, Culex quinquefasciatus . One of these ORs, CquiOR1, belongs to a cluster of 6 ORs from the Southern house mosquito and 2 ORs from the yellow fever mosquito, Aedesaegypti . Specifically, CquiOR2, CquiOR4, AaegOR14, AaegOR15, CquiOR5, CquiOR84, and CquiOR85. Of note, CquiOR2 is not the previously reported oviposition attractant-detecting OR2 , which has been renamed CquiOR121 . We cloned CquiOR2 and the other ORs in this cluster . We then deorphanized these receptors using the Xenopus oocyte recording system and a panel of odorants with physiologically and behaviorally relevant compounds, including oviposition attractants, mosquito repellents, and plant-derived compounds . Here, we report that these receptors, particularly CquiOR4, CquiOR5, and AaegOR15, are very sensitive to plant-derived compounds, including repellents. CquiOR4, for example, which is very specific to female antennae, with high and low transcript levels in non-blood fed and blood-fed mosquitoes, respectively, showed a robust response to the natural repellent 2-phenylethanol. Repellency activity elicited by 2-phenylethanol reduced significantly in CquiOR4-dsRNA-treated mosquitoes, but it was unchanged when these mosquitoes were tested against DEET, which is detected with another receptor . Mosquitoes used in this study were from a laboratory colony of Cx. quinquefasciatus originating from adult mosquitoes collected in Merced, CA in the 1950s and kept at the Kearney Agricultural Research Center, University of California, Parlier, CA. Specifically, we used mosquitoes from the Davis colony, which was initiated about eight years ago with mosquitoes from the Kearney colony. In Davis, mosquitoes were maintained at 27 ± 1 °C, 75 ± 5% relative humidity, and under a photoperiod of 12:12 h.