Honeydew feeding in the solitary bee Osmia bicornis as affected by aphid species and nectar availability

Like honey bees (Apis mellifera), non-Apis bees could exploit honeydew as a carbohydrate source. In addition to providing carbohydrates, this may expose them to potentially harmful plant products secreted in honeydew. However, knowledge on honeydew feeding by solitary bees is very scarce. Here we determine whether the polylectic solitary bee Osmia bicornis (=O. rufa) collects honeydew under semi-field conditions, and whether this is affected by aphid species and presence of floral nectar. Bees were provided with oilseed rape plants containing flowers and/or colonies of either Myzus persicae or Brevicoryne brassicae. We used the total sugar level of the bee crop as a measure of the individual's nutritional state and the oligosaccharide erlose as indicator for honeydew consumption. Erlose was present in honeydews from both aphid species, while absent in oilseed rape nectar, nor being synthesized by O. bicornis. When bees were confined to a single honeydew type as the only carbohydrate source, consumption of M. persicae honeydew was confirmed for 47% of the bees and consumption of B. brassicae honeydew for only 3%. Increased mortality in the latter treatment provided further evidence that B. brassicae honeydew is an unsuitable food source for O. bicornis. All bees that were given the choice between honeydew and floral nectar showed significantly increased total sugar levels. However, the fact that no erlose was detected in these bees indicates that honeydew was not consumed when suitable floral nectar was available. This study demonstrates that honeydew exploitation by O. bicornis is dependent on honeydew type and the presence of floral nectar.     

Genetic manipulation in cultivars of oilseed rape (Brassica napus) using Agrobacterium

Summary The response of oilseed rape cultivars to infection with Agrobacterium tumefaciens and A. rhizogenes and the possibility of regenerating genetically transformed oilseed rape plants were examined. The frequency at which Agrobacterium induced galls or hairy-roots on in vitro cultured plants ranged from 10% to 70%, depending on the cultivar. From galls induced by the tumorigenic strain T37, known to be strongly shoot inducing on tobacco, roots developed frequently. Occasionally, shoots formed and some of these produced tumour cell specific nopaline. Attempts to grow the transformed shoots into plants have so far been unsuccessful. Whole plants transformed with Ri-T-DNA, however, were regenerated. These had crinkled leaves and abundant, frequently branching roots that showed reduced geotropism, similar to previously isolated Ri T-DNA transformed tobacco and potato plants. The transformed oilseed rape plants flowered, but failed to form seeds.     

Presence of Unintended Agrobacterium tumefaciens Cloning Vector Sequences in Genetically Modified Plants

Agrobacterium transformation was used in the production of genetically modified plants from oilseed rape (Brassica napus) and tobacco (Nicotiana tabacum). After inoculation stop with the antibiotic timentin, a subsequent one-week treatment eliminated the vector bacterium from the oilseed rape plate explant cultures. From the tobacco, however, we recorded vector-derived signals one week after potting the regenerants in the greenhouse and still 10 weeks later. Genetically modified plants produced through Agrobacterium-transformation therefore cannot be guaranteed to be completely free of unintended vector sequences after antibiotic treatment.     

A Four-Year Field Program Investigating Long-Term Effects of Repeated Exposure of Honey Bee Colonies to Flowering Crops Treated with Thiamethoxam

Neonicotinoid residues in nectar and pollen from crop plants have been implicated as one of the potential factors causing the declines of honey bee populations. Median residues of thiamethoxam in pollen collected from honey bees after foraging on flowering seed treated maize were found to be between 1 and 7 µg/kg, median residues of the metabolite CGA322704 (clothianidin) in the pollen were between 1 and 4 µg/kg. In oilseed rape, median residues of thiamethoxam found in pollen collected from bees were between <1 and 3.5 µg/kg and in nectar from foraging bees were between 0.65 and 2.4 µg/kg. Median residues of CGA322704 in pollen and nectar in the oilseed rape trials were all below the limit of quantification (1 µg/kg). Residues in the hive were even lower in both the maize and oilseed rape trials, being at or below the level of detection of 1 µg/kg for bee bread in the hive and at or below the level of detection of 0.5 µg/kg for hive nectar, honey and royal jelly samples. The long-term risk to honey bee colonies in the field was also investigated, including the sensitive overwintering stage, from four years consecutive single treatment crop exposures to flowering maize and oilseed rape grown from thiamethoxam treated seeds at rates recommended for insect control. Throughout the study, mortality, foraging behavior, colony strength, colony weight, brood development and food storage levels were similar between treatment and control colonies. Detailed examination of brood development throughout the year demonstrated that colonies exposed to the treated crop were able to successfully overwinter and had a similar health status to the control colonies in the following spring. We conclude that these data demonstrate there is a low risk to honey bees from systemic residues in nectar and pollen following the use of thiamethoxam as a seed treatment on oilseed rape and maize.     

Pollinator foraging behavior and pollen collection on the floral morphs of tristylous Pontederia cordata L.

Summary The foraging behavior of the pollinators of tristylous Pontederia cordata was studied to determine if differences in floral morphology would lead to preferential visitation of the floral morphs. Although nectar production is not different in the three floral morphs, differences in the production and size of pollen grains produced by the three anther levels results in the morphs offering variable amounts of resources to pollen-collecting insects. Bumblebees (Bombus spp.) and the solitary bee Melissodes apicata used P. cordata primarily as a nectar source and therefore did not seem to exhibit any morph preference. In contrast, honeybees visited flowers mainly for pollen and preferred to forage on long-level anthers of the short-and mid-styled morphs. An analysis of the composition of corbicular pollen loads indicated that, relative to the frequency of production in the population: 1) honeybees collected an excess of pollen from long-level anthers; 2) bumblebees collected the three types of pollen without any apparent preference; and 3) M. apicata preferentially collected pollen from the short-level anthers — presumably because their proboscides are modified by the presence of tiny hairs. The results suggest that P. cordata in Ontario is serviced by a diverse, unspecialized pollinator fauna which is not co-adapted to the tristylous floral polymorphism.     

Transfer and segregation of triazine tolerant chloroplasts in Brassica napus L.

Summary Hypocotyl protoplasts of 45 different genotypes of German winter oilseed rape Brassica napus L. (double zero quality: high in yield, seeds low in erucic acid and glucosinolate content) were regenerated to plants. Triazine/triazinone (tri)-tolerant chloroplasts of the Canadian spring oilseed rape variety OAC Triton were introduced into some winter oilseed rapes by means of protoplast fusion. X-ray irradiation was used to limit the transfer of nuclear DNA of Triton protoplasts and to promote the selective transfer of tri-tolerant chloroplasts. Regenerated cybrid plants survived a treatment rate of 1000 g/ha metribuzin. The presence and segregation of the tri-tolerant chloroplasts in winter oilseed rape plants, regenerated from fusion products and their progeny, was investigated by restriction fragment length polymorphism (RFLP). Our results indicate that chloroplast segregation was not completed in plants regnerated from fusion products derived from X-irradiated OAC Triton mesophyll protoplasts and German winter oilseed rape hypocotyl protoplasts. In regenerants and their progeny both chloroplast types can still be present. Chloroplasts derived from wintertype protoplasts can outcompete tritolerant chloroplasts during plant development. In some instances, even progeny plants not kept under selective conditions (metribuzin) lost tri-tolerant chloroplasts. A homogenous population of tri-tolerant chloroplasts was necessary to obtain stable tri-tolerant winter oilseed rape plants.     

Thermal effects on activity patterns and behavioural switching in a concourse of foragers on Stachytarpheta mutabilis (Verbenaceae) in Papua New Guinea

Summary 1. The foraging activities of the papilionid butterflies Ornithoptera priamus poseidon and Papilio ulysses, and the solitary bee Amegilla sapiens (Apoidea, Anthophoridae) on the shrub Stachytarpheta mutabilis were studied in highland Papua New Guinea. 2. The insects' activity patterns were analysed at three sites with differing diurnal microclimate variation. O. priamus and A. sapiens foraged in the morning (after a period of basking and wing-whirring) and late afternoon when temperatures were well below daily maxima, whereas P. ulysses showed foraging peaks during the hottest part of the day. 3. Site choice by all 3 species appeared to be determined primarily by temperature, but within the limits imposed by temperature, nectar supplies probably determined which site was visited. 4. P. ulysses showed interspersed foraging and courtship behaviour, and no behavioural switching was observed for this species. At high temperatures, both O. priamus and A. sapiens ceased foraging and showed territorial and courtship behaviour. This behavioural change allowed avoidance of heat stress, and occurred even when nectar supplies were maintained at high levels. 5. Thermal effects on behavioural switching in these insects are compared with related phenomena in other bees and butterflies.     

Effect of a turnip rape <Emphasis Type="Italic">(Brassica rapa)</Emphasis> trap crop on stem-mining pests and their parasitoids in winter oilseed rape <Emphasis Type="Italic">(Brassica napus)</Emphasis>

Concerns about the negative effects of chemical control of oilseed rape (Brassica napus L.) pests on non-target species, human safety, and development of insecticide resistance, require alternative control strategies such as the use of trap crops and biocontrol to be developed. Psylliodes chrysocephala(L.) (Coleoptera: Chrysomelidae) (cabbage stem flea beetle) and Ceutorhynchus pallidactylus (Marsh.) (Coleoptera: Curculionidae) (cabbage stem weevil) are two major stem-mining pests of oilseed rape. This study investigated the phenology of these pests and their main parasitoids in the UK, the potential use of turnip rape (Brassica rapa L.) as a trap crop to reduce oilseed rape infestation, and the effects of insecticide treatment on pest incidence and larval parasitism. Water trap samples, plant dissections and pest larval dissections were done to determine: the incidence of adult pests and their parasitoids, the level of plant infestation by the pests and percentage larval parasitism, respectively. The turnip rape trap crop borders reduced P. chrysocephalabut not C. pallidactylus infestation of oilseed rape plots. Treatment of the trap crop with insecticide had little effect on either pest or parasitoid incidence in the oilseed rape. TersilochusmicrogasterSzép. andT. obscurator Aub. (Hymenoptera: Ichneumonidae) were the main larval parasitoids of P. chrysocephalaand C. pallidactylus, respectively. Tersilochus microgasteris reported for the first time in the UK. The implications for integrated pest management are discussed.     

Interspecific hybrids between Brassica napus L. and B. oleracea L. developed by embryo culture

Summary Interspecific hybrids between Brassica napus and B. oleracea are difficult to produce, and previous attempts to transfer economic characters from one species to the other have largely been unsuccessful. In these studies, oilseed rape cv. Tower (2n38) (B. napus) was crossed with broccoli and kale (2n18) (B. oleracea), and hybrid plants were developed from embryos in culture by either organogenesis or somatic embryogenesis. In rape × broccoli, F₁ plants were regenerated from hybrid embryos and the plants produced viable selfed seeds. F₅ plants (2n38) homozygous for white flower colour were selected for high oil content (47%) and Line 15; a selection from these plants produced fertile hybrids with rape, broccoli and kale without embryo culture. In reciprocal crosses between oilseed rape cv. Tower and an aphid resistant diploid kale, 28 and 56 chromosome F₁ hybrid plants were regenerated from somatic embryos. The 56 chromosome plants were self-fertile and it was concluded from F₂ segregation ratios that a single dominant gene controls resistance to cabbage aphid in kale. The 28 chromosome F₁'s were self-sterile, but these and the 56 chromosome F₁'s could be backcrossed to rape and kale. A cross between the F₁ (2n56) and a forage rape resulted in the selection of a cabbage aphid (Brevicoryne brassicae L.) resistant line (Line 3). Both Line 15 and Line 3 can serve as bridges for gene interchange between B. campestris, B. napus and B. oleracea, which has not been possible hitherto. Hybridisations between rape and tetraploid kale produced F₁ plants with 37 chromosomes. One F₂ plant possessed coronal scales and the inheritance was shown to be controlled by a single recessive gene unlinked to petal colour.This paper is dedicated to Mr. T. P. Palmer, a colleague and close friend who retired from the DSIR as Assistant Director of the Crop Research Division in September 1984     

Unexpected Diversity of Feral Genetically Modified Oilseed Rape (Brassica napus L.) Despite a Cultivation and Import Ban in Switzerland

Despite cultivation and seed import bans of genetically modified (GM) oilseed rape (Brassica napus L.), feral GM plants were found growing along railway lines and in port areas at four sites in Switzerland in 2011 and 2012. All GM plants were identified as glyphosate-resistant GM event GT73 (Roundup Ready, Monsanto). The most affected sites were the Rhine port of Basel and the St. Johann freight railway station in Basel. To assess the distribution and intra- and interspecific outcrossing of GM oilseed rape in more detail, we monitored these two sites in 2013. Leaves and seed pods of feral oilseed rape plants, their possible hybridization partners and putative hybrid plants were sampled in monthly intervals and analysed for the presence of transgenes by real-time PCR. Using flow cytometry, we measured DNA contents of cell nuclei to confirm putative hybrids. In total, 2787 plants were sampled. The presence of GT73 oilseed rape could be confirmed at all previously documented sampling locations and was additionally detected at one new sampling location within the Rhine port. Furthermore, we found the glufosinate-resistant GM events MS8xRF3, MS8 and RF3 (all traded as InVigor, Bayer) at five sampling locations in the Rhine port. To our knowledge, this is the first time that feral MS8xRF3, MS8 or RF3 plants were detected in Europe. Real-time PCR analyses of seeds showed outcrossing of GT73 into two non-GM oilseed rape plants, but no outcrossing of transgenes into related wild species was observed. We found no hybrids between oilseed rape and related species. GM plants most frequently occurred at unloading sites for ships, indicating that ship cargo traffic is the main entry pathway for GM oilseed rape. In the future, it will be of major interest to determine the source of GM oilseed rape seeds.     

Melliferous potential of Brassica napus L. subsp. napus (Cruciferae)

Morphophysiological characteristics of oilseed rape flowers, such as features of the nectaries, nectar production, and observations on honey bee visits and honey and seed yield were studied with the aim to evaluate the melliferous potential of this crop as well as its attractiveness to pollinators. Calculation of the theoretical maximal honey yield revealed that the actual amount of extracted honey was much lower than the potential yield, indicating that this bee pasture is underutilized. We found that honey bee pollination increased oilseed rape yield, i.e., seed production, by 12 % compared with the treatment in which pollinators were excluded.     

Competitiveness of transgenic oilseed rape

The competitiveness of two transgenic oilseed rape (Brassica napus ssp.napus) lines and their fertile transgenic hybrid was tested in field trials in Belgium and Denmark. The lines contained genes for male sterility, restoration of fertility and herbicide resistance. The competitiveness of the three transgenic lines was related to three non-transformed commercially-grown oilseed rape varieties: Drakkar, Topas and Line. As a reference of a more aggressive crucifer, white mustard (Sinapis alba) was also included in the experiment. The experimental design was a complete block design with two locations, monocultures and mixtures with barley (Hordeum vulgare), three plant densities, four harvest times and four blocks. The yield density relationship of the transgenic oilseed rape lines was not different from that of the non-transgenic varieties in either location. The first harvest times showed a vigorous biomass production of white mustard, which in turn produced a significant difference in the competitive ability between oilseed rape and white mustard. Later, this difference decreased, and in Belgium there was no difference at the last harvest time. Variations within populations may blur actual differences between lines and varieties, and it is argued that unless the experimental design covers a range of competitiveness for which it is possible to detect significant differences, test results reporting a lack of difference between transgenic and non-transgenic plants are of little value.     

Tritrophic Choice Experiments with Bt Plants,the Diamondback Moth (Plutella xylostella) and the parasitoid Cotesia plutellae

Parasitoids are important natural enemies of many pest species and are used extensively in biological and integrated control programmes. Crop plants transformed to express toxin genes derived from Bacillus thuringiensis (Bt) provide high levels of resistance to certain pest species, which is likely to have consequent effects on parasitoids specialising on such pests. A better understanding of the interaction between transgenic plants, pests and parasitoids is important to limit disruption of biological control and to provide background knowledge essential for implementing measures for the conservation of parasitoid populations. It is also essential for investigations into the potential role of parasitoids in delaying the build-up of Bt-resistant pest populations. The diamondback moth (Plutella xylostella), a major pest of brassica crops, is normally highly susceptible to a range of Bt toxins. However, extensive use of microbial Bt sprays has led to the selection of resistance to Bt toxins in P. xylostella. Cotesia plutellae is an important endoparasitoid of P. xylostella larvae. Although unable to survive in Bt-susceptible P. xylostella larvae on highly resistant Bt oilseed rape plants due to premature host mortality, C. plutellae is able to complete its larval development in Bt-resistant P. xylostella larvae. Experiments of parasitoid flight and foraging behaviour presented in this paper showed that adult C. plutellae females do not distinguish between Bt and wildtype oilseed rape plants, and are more attracted to Bt plants damaged by Bt-resistant hosts than by susceptible hosts. This stronger attraction to Bt plants damaged by resistant hosts was due to more extensive feeding damage. Population scale experiments with mixtures of Bt and wildtype plants demonstrated that the parasitoid is as effective in controlling Bt-resistant P. xylostella larvae on Bt plants as on wildtype plants. In these experiments equal or higher numbers of parasitoid adults emerged per transgenic as per wildtype plant. The implications for integrated pest management and the evolution of resistance to Bt in P. xylostella are discussed.     

Flower choice by honey bees (Apis mellifera L.): sex-phase of flowers and preferences among nectar and pollen foragers

Bees foraging for nectar should choose different inflorescences from those foraging for both pollen and nectar, if inflorescences consist of differing proportions of male and female flowers, particularly if the sex phases of the flowers differ in nectar content as well as the occurrence of pollen. This study tested this prediction using worker honey bees (Apis mellifera L.) foraging on inflorescences of Lavandula stoechas. Female flowers contained about twice the volume of nectar of male flowers. As one would predict, bees foraging for nectar only chose inflorescences with disproportionately more female flowers: time spent on the inflorescence was correlated with the number of female flowers, but not with the number of male flowers. Inflorescence size was inversely correlated with the number of female flowers, and could be used as a morphological cue by these bees. Also as predicted, workers foraging for both pollen and nectar chose inflorescences with relatively greater numbers of both male and female flowers: time spent on these inflorescences was correlated with the number of male flowers, but not with the number of females flowers. A morphological cue inversely associated with such inflorescences is the size of the bract display. Choice of flowers within inflorescences was also influenced predictably, but preferences appeared to be based upon corolla size rather than directly on sex phase.     

The role of insect water balance in pollination ecology: Xylocopa and Calotropis

Summary Two carpenter bees (Xylocopa spp.) in southern Israel both use the asclepiad Calotropis procera as a primary nectar source. This plant genus is coevolved with carpenter bees, and aspects of the insect-flower interaction in Israel suggest that the smaller bee, X. sulcatipes, is the natural co-adapted pollinator, a view borne out by the geographical distributions of the species concerned. There are significant mismatches between the plant and the larger X. pubescens, involving physical fit and behaviour. These mismatches are particularly evident when the physiologies of the bees and the plant are considered. The different sizes and colours of the two bees lead to different daily activity patterns, only X. sulcatipes being thermally suited to, and thus abundant at, times of maximum nectar production by Calotropis. Similarly the water requirements of X. sulcatipes are finely balanced with the water production in the floral nectar; this bee gains just enough water when foraging to restore its blood concentration and production in the floral nectar; for deposition in the nest. X. pubescens does not incur net water loss in flight and gains too much water from Calotropis flowers, necessitating copious urination and tonguelashing. Hence physiological information can be of use in deciphering insect-plant coevolutionary patterns, and the water component of nectar is confirmed as a potentially major determinant of foraging activities. The circumstances where this will be particularly true, and cases where it may not apply, are discussed.     

Endophytic Bacteria Induce Growth Promotion and Wilt Disease Suppression in Oilseed Rape and Tomato

To determine whether bacteria isolated from within plant tissue can have plant growth-promotion potential and provide biological control against soilborne diseases, seeds and young plants of oilseed rape (Brassica napus L. cv. Casino) and tomato (Lycopersicon lycopersicum L. cv. Dansk export) were inoculated with individual bacterial isolates or mixtures of bacteria that originated from symptomless oilseed rape, wild and cultivated. They were isolated after surface sterilization of living roots and stems. The effects of these isolates on plant growth and soilborne diseases for oilseed rape and tomato were evaluated in greenhouse experiments. We found isolates that not only significantly improved seed germination, seedling length, and plant growth of oilseed rape and tomato but also, when used for seed treatment, significantly reduced disease symptoms caused by their vascular wilt pathogens Verticillium dahliae Kleb and Fusarium oxysporum f. sp. lycopersici (Sacc.), respectively.     

Stability of transgene integration and expression in subsequent generations of doubled haploid oilseed rape transformed with chitinase and β-1,3-glucanase genes in a double-gene construct

A double-gene construct with one chitinase and one β-1,3-glucanase gene from barley, both driven by enhanced 35S promoters, was transformed into oilseed rape. From six primary transformants expressing both transgenes 10 doubled haploid lines were produced and studied for five generations. The number of inserted copies for both the genes was determined by Southern blotting and real-time PCR with full agreement between the two methods. When copy numbers were analysed in different generations, discrepancies were found, indicating that at least part of the inserted sequences were lost in one of the alleles of some doubled haploids. Chitinase and β-1,3-glucanase expression was analysed by Western blotting in all five doubled haploid generations. Despite that both the genes were present on the same T-DNA and directed by the same promoter their expression pattern between generations was different. The β-1,3-glucanase was expressed at high and stable levels in all generations, while the chitinase displayed lower expression that varied between generations. The transgenic plants did not show any major impact on fungal resistance when assayed in greenhouse, although purified β-1,3-glucanase and chitinase caused retardment of fungal growth in vitro.     

A method for quantifying the gene flow that results from a single bumblebee visit using transgenic oilseed rape,Brassica napus L. cv. Westar

Genetically modified plants containing selectable markers offer a unique opportunity for pollination biologists to investigate some of the major, but intractable questions about paternity distributions and their causes. Here, a method is reported that uses transgenic plants to enable the quantification of the outcrossed fertilizations that result from a single pollinator visit. Gene flow mediated by worker bumblebees (Bombus terrestris) was studied among plants of oilseed rape (Brassica napus L. cv. Westar) where transgenic paternity in seeds of a non-transgenic plant was manifested as herbicide resistance. Overall, 91% of the resistant seeds resulted from the first four flowers that were visited after the bumblebee left the transgenic plant, and none was found beyond the 14th successively visited flower. The possibilities for developing the method to address various questions in pollination biology are discussed.     

Energetics of nectar production in some strawberry cultivars as a predictor of floral choice by honeybees

Nectar productivity of thirteen cultivars of strawberry (Fragariq sp.) was examined in relation to the foraging activity ofApis cerana indica F. andApis mellifera L. The cultivars were found to vary in the quantity of nectar produced (0·020 to 0·735 ul/flower/day), nectar-sugar concentration (30 to 42%) and amount of sugar (0.006 to 0·2898 mg/flower/day). Consequently energy reward varied from 0·1037 to 4·851 joules/ flower/day. Foraging of bothApis cerana indicq andApis melliferq correlated with energy yields. The results suggest that cultivars with higher caloric rewards had a competitive edge over the others in attracting foraging populations of both species of the bee.