Effect of pea lectin expressed transgenically in oilseed rape on pollen beetle life-history parameters

When investigating resistance to herbivorous insects in transgenic plants, it is primarily the damaging stage of the insect that is targeted. In our study, we showed that small effects of the transgenic plant on the pest performance might lead to a clear effect on the pest population level. Pollen beetle, Meligethes aeneus (Fabricius) (Coleoptera: Nitidulidae), larvae were reared on three transgenic lines of Brassica napus L. (Brassicaceae) expressing pea (Pisum sativum L.) lectin at 0.2, 0.6, and 0.7% of total soluble protein in anthers, and then monitored until the adult stage. Two lines without the gene construct and consequently containing no pea lectin were used as controls. The egg viability, larval weight, development time, and survival rate were recorded. In a separate experiment, the development time from egg to adult, adult weight, and winter survival of pollen beetles developed on intact plants were recorded. Survival of larvae to adult stage was significantly lower on the transgenic plant lines, independent of lectin level. Survival during hibernation was only 2.4% and was not affected by plant line. Combining previously published results from adult feeding and oviposition assays with the new data on larval development we summarize the effect of the transgenic oilseed rape on the pollen beetle, from oviposition in spring to overwintered adults of the new generation. Net reproductive rate was then shown to be reduced by half on the transgenic oilseed rape compared to the control plants.     

Pea Lectin Expressed Transgenically in Oilseed Rape Reduces Growth Rate of Pollen Beetle Larvae

In several studies plant lectins have shown promise as transgenic resistance factors against various insect pests. We have here shown that pea seed lectin is a potential candidate for use against pollen beetle, a serious pest of Brassica oilseeds. In feeding assays where pollen beetle larvae were fed oilseed rape anthers soaked in a 1% solution of pea lectin there was a reduction in survival of 84% compared to larvae on control treatment and the weight of surviving larvae was reduced by 79%. When a 10% solution of pea lectin was used all larvae were dead after 4 days of testing. To further evaluate the potential use of pea lectin, transgenic plants of oilseed rape (Brassica napus cv. Westar) were produced in which the pea lectin gene under control of the pollen-specific promoter Sta44-4 was introduced. In 11 out of 20 tested plants of the T₀-generation there was a significant reduction in larval weight, which ranged up to 46% compared to the control. A small but significant reduction in larval survival rate was also observed. In the T₂-generation significant weight reductions, with a maximum of 32%, were obtained in 10 out of 33 comparisons between transgenic plants and their controls. Pea lectin concentrations in anthers of transgenic T₂-plants ranged up to 1.5% of total soluble protein. There was a negative correlation between lectin concentration and larval growth. Plants from test groups with significant differences in larval weights had a significantly higher mean pea lectin concentration, 0.64% compared to 0.15% for plants from test groups without effect on larval weight. These results support the conclusion that pea lectin is a promising resistance factor for use in Brassica oilseeds against pollen beetles.     

Impact of herbivory and pollination on performance and competitive ability of oilseed rape transformed for pollen beetle resistance

Competitive ability of transgenic oilseed rape transformed with a pea lectin gene was estimated by comparisons of its performance when grown in a mixture with its non-transgenic counterpart and when grown singly, with and without pollen beetles present. The experiments were carried out in cages, once with bumblebees as pollinators and once without. In the absence of herbivory but with the presence of bumblebees, singly grown plant lines without lectin generally performed better than lines with lectin. Pollen beetles affected plant growth and reproduction, but there were no consistent differences between the lectin and non-lectin plant lines indicating that the transgenic trait did not protect plants from pest attack. Herbivory reduced the number of seeds when bumblebees were present. In the absence of bumblebees, however, plants produced more seeds with pollen beetles than without, indicating that some pollination was carried out by the beetles. Efficient pollination affected the competitive abilities of the lines; lectin lines were more competitive with bumblebees present and the reverse was true when bumblebees were absent. In the presence of bumblebees, lectin lines gained from being grown mixed with its non-transgenic counterpart. Because the transgenic plants expressed pea lectin in developing pollen it is possible that pollen quality in those plants was reduced, which may explain why the lectin lines had an advantage over non-lectin lines when exchange of pollen between the two plant types was facilitated by bumblebees.     

Potato proteins, and other plant proteins, as potential transgenic resistance factors to pollen beetles in oilseed rape

Proteinase inhibitors (PIs), lectin and patatin purified from potato tubers were tested in no‐choice feeding assays with pollen beetle larvae (Meligethes spp.). The idea was to search for resistance factors possible to introduce into oilseed rape (Brassica napus) by genetic engineering. The larval diet was prepared by soaking oilseed rape anthers in protein solutions of known concentrations. Potato lectin was the most potent in that it was the only of these proteins that reduced both larval survival and growth rate, while cysteine, aspartic and metallo PIs and patatin only reduced larval growth rate. Serine PIs had no significant effect on larval performance. Subsequently, the effect of potato lectin was compared to that of lectins from other food or feed crops, resulting in the following mortality‐ based ranking of activity: Con A from jackbean > wheat germ lectin > potato lectin > peanut lectin. In choice tests, larvae did not discriminate between Con A‐ and control‐diets. These results suggest that the effect of Con A on larvae is toxic, not deterrent. Adult response was stronger to Con A than to potato lectin in no‐choice tests, just as it was in larvae. However, adult survival rate and weight was not affected by Con A but the lectin significantly reduced adult feeding as well as oviposition rates. A resistance factor that suppresses adult feeding on flower buds is important for reduced impact of the pollen beetle on the Brassica oilseed crop.     

Pollen digestion by flower-feeding Scarabaeidae: protea beetles (Cetoniini) and monkey beetles (Hopliini)

Pollen protoplasm is very nutritious, but the hard and highly resistant outer wall (exine) of the pollen grain presents an obstacle that pollen-feeders must overcome to benefit from the valuable protoplasm. Pollen digestion in three pollen-eating scarab beetles, the green protea beetle Trichostetha fascicularis and two monkey beetle species, Peritrichia cinerea and Pachynema flavolineata, was investigated. Adult beetles were collected for observations of feeding mechanisms, scanning electron microscopy of mouthparts, and histological examination of gut contents. Serial sections of different regions along the gut were stained for different nutrients and examined to determine the appearance of the pollen grains and the removal of carbohydrates, proteins and lipids from the pollen. During feeding, beetles used dense brushes of setae on the tips of their maxillae to mop up nectar and sweep pollen into their mouths. Grains were ingested intact. Nutrients were effectively removed from the grains as they passed along the gut and most pollen grains in the hindgut were empty, with broken exines. All the beetles excreted the undigested exine. Comparison with other studies on pollen digestion by a variety of insect and vertebrate pollen-feeders suggests that digestive enzyme penetration of ingested pollen grains and exudation of the products of digestion is the most likely method used by these beetles.     

Floral reward presentation favored the expression of male function in the pollen‐only flower Melastoma malabathricum

Abstract Animal‐pollinated plant species modulate the presentation of pollinator rewards to maximize reproductive success. In plants providing pollen as the only reward for pollinators, it is usually difficult to unravel the dual roles of reward presentation and the realization of male and female functions (pollen removal and deposition). Exploiting the two types of anther in the androecia of Melastoma malabathricum L., we examined whether the removal of pollen for reward is regulated primarily to favor male function or female function. Pollen removal by carpenter bees from the feeding and pollination anthers, as well as pollen deposition on the stigmas, were quantified during anthesis of M. malabathricum. There was no significant difference in pollen removal rates from the feeding and pollination anthers of M. malabathricum between the onset of anthesis and flower wilting. The stigmatic pollen loads exceeded the ovule number after three sonication bouts, and female function was satisfied earlier than male function. The results support the hypothesis that the presentation of pollination reward in this species is regulated primarily to favor the expression of male function, rather than female function, in agreement with the pollen‐donation hypothesis. A cooperative relationship between the feeding and pollination anthers was demonstrated in heterantherous flowers, which optimizes the balance in investments between pollinator rewards and “functional pollen” for gene transfer.     

Development of Biosafe Genetically Modified <Emphasis Type="Italic">Solanum tuberosum</Emphasis> (Potato) Cultivars for Growth within a Centre of Origin of the Crop

Male sterile cv Desiree has been generated for use with other transgenic traits where hybridisation of a potato crop with wild relatives might occur. A ribosomal inactivating protein from maize (b32RIP) was expressed under the control of a tapetal specific promoter (TA29). Its known low activity against plant ribosomes was not correlated with either a lower incidence or less complete male sterility than occurred for similar lines expressing an RNAse (barnase) from Bacillus amyloliquefaciens. The b32RIP lines had a lower incidence of abnormal vegetative growth than barnase lines. Pollen grains of eight b32RIP lines and two barnase lines rarely showed uptake of the fluorophore fluorescein diacetate unlike the wild type Desiree. Pollen tubes formed very infrequently for sterile lines in conditions where this occurred for >80% of pollen of an untransformed control. Hand crossing with pollen from wild type but not the male sterile lines achieved fertilisation. Histological section of anthers for one b32RIP line confirmed its pollen grains had not matured in contrast to control plants. This is the first report of b32RIP having activity in planta against plant cells.     

Evaluation of transgenic Bt corn for resistance to the Asian corn borer (Lepidoptera: Pyralidae)

The Asian corn borer, Ostrinia furnacalis (Guenée), is the most important insect pest on corn in China. Bt transgenic corn provides a new tool for Asian corn borer control. Monsanto's YieldGard Bt transgenic corn expressing Cry1Ab protein, and a non-Bt control, were evaluated in Beijing. Laboratory bioassays were carried out by exposing neonates to an agar-free diet containing Bt corn whorl leaves, tassels, and anthers, or by exposing neonates directly to fresh silk and pollen. These are the tissues initially attacked by neonates in the field. All of these tissues, with the exception of pollen, contained sufficient insecticidal protein to kill > or = 95% of larvae within 7 d. Surviving larvae had also not grown beyond first instar and weighed < or = 0.1 mg. Although larvae feeding on Bt corn pollen were significantly smaller than those on non-Bt corn pollen, there was no significant difference in mortality. Field trials were also conducted with artificial infestations of Asian corn borer at mid whorl, late whorl, and silking stages. Damage ratings and number of larvae surviving per plant indicated that Bt corn was highly resistant to Asian corn borer. Therefore, YieldGard offers the potential for season-long protection against first- and second-generation Asian corn borer.     

Heteranthery as a solution to the demand for pollen as food and for pollination – Legitimate flower visitors reject flowers without feeding anthers

• Heteranthery, the presence of feeding and pollinating anthers in the same flower, seems to mediate the evolutionary dilemma for plants to protect their gametes and yet provide food for pollinators. This study aims to elucidate the role of heteranthery in the buzz‐pollinated Senna reniformis.
• The fecundity of pollen from long‐, medium‐ and short‐sized anthers was determined by hand cross‐pollination experiments, and the quantity, size, ornamentation and viability of pollen of different anthers were compared. Rates of flower rejection by bees were measured in anther removal experiments to assess the preferences of flower visitors for feeding or pollinating anthers.
• Large bees, which were the effective pollinators of self‐incompatible S. reniformis, avoided flowers without short feeding anthers, but not those without medium or long anthers. Illegitimate small and medium‐sized bees were unresponsive to anther exclusion experiments. Long anthers deposited pollen on the back and short anthers on the venter of large bees. Pollen from long anthers had higher in vitro viability and higher fruit and seed set after cross‐pollination than pollen from other sized anthers.
• Short anthers produce feeding pollen to effective pollinators and long anthers are related to pollination of S. reniformis. Bee behaviour and size was found to directly influence the role of anthers in the ‘division of labour’. Only large bee pollinators that carry the pollinating pollen from long anthers in ‘safe sites’ associated short anthers with the presence of food. In the absence of these larger bee pollinators, the role of heteranthery in S. reniformis would be strongly compromised and its function would be lost.

     

Morphology and biology of the flower‐visiting water scavenger beetle genus Rygmodus (Coleoptera: Hydrophilidae)

Hydrophilidae (water scavenger beetles) is well known as an aquatic beetle family; however, it contains ca. 1,000 secondarily terrestrial species derived from aquatic ancestors. The New Zealand endemic genus Rygmodus White is a member of the hydrophilid subfamily Cylominae, which is the early‐diverging taxon of the largest terrestrial lineage (Cylominae + Sphaeridiinae) within the Hydrophilidae. In this paper we report that Rygmodus beetles are pollen‐feeding flower visitors as adults, but aquatic predators as larvae. Based on analyses of gut contents and a summary of collecting records reported on museum specimen labels, adult Rygmodus beetles are generalists feeding on pollen of at least 13 plant families. Rygmodus adult mouthparts differ from those of other (saprophagous) hydrophilid beetles in having the simple scoop‐like apex and mola with roughly denticulate surface, resembling the morphology found in pollen‐feeding staphylinid beetles. Larvae were found along the sides of streams, under stones and in algal mats and water‐soaked moss; one collected larval specimen was identified using DNA barcoding of two molecular markers, mitochondrial cytochrome oxidase 1 (cox1) and nuclear histone 3 (H3). Larvae of two species, Rygmodus modestus and Rygmodus sp., are described in detail and illustrated; they closely resemble ambush‐type predatory larvae of the hydrophilid tribe Hydrophilini in the head morphology. Rygmodus is the only known hydrophilid beetle with adults and larvae inhabiting different environments.     

TRACKING POLLEN FLOW OF SOLANUM ROSTRATUM (SOLANACEAE) USING BACKSCATTER SCANNING ELECTRON MICROSCOPY AND X-RAY MICROANALYSIS

A technique using micronized metal powders was developed for both general labeling of pollen and marking of individual pollen grains. After labeling, pollen flow is analyzed by the use of backscatter scanning electron microscopy and X-ray microanalysis. To test the efficiency and efficacy of the technique, we assessed differences in pollen distribution in Solanum rostratum, an enantiostylous species with dimorphic anthers which are putatively feeding and pollinating anthers. Pollen from each set of anthers was labeled using different micronized metal powders. We could not confirm the differentiation of functional anthers in S. rostratum. This technique provides an efficient and convenient method for tracking pollen movement within and between flowers, and anthers within a single blossom can be differentially marked.     

Pollen beetle (Meligethes aeneus) oviposition and feeding preference on different host plant species

The oviposition and feeding preferences of the pollen beetle, Meligethes aeneus, were determined in choice and no-choice tests in field, semi-field and greenhouse trials. Plant species used were Brassica napus, B. campestris, B. juncea, B. nigra, B. carinata, Sinapis alba and Crambe abyssinica. With respect to number of eggs laid, S. alba and C. abyssinica were inferior to the other species. Pollen beetles laid fewer eggs on B. nigra than on the other Brassica spp. in no-choice tests, however this difference was partly due to fewer eggs laid per bud rather than fewer buds used for oviposition. Most eggs, for all plant species, were deposited in buds sized 2–3 mm. Feeding damage on all plant species was relatively similar. Pollen beetles seem to have a wider host range for feeding than for oviposition. There was good agreement in plant species ranking as oviposition hosts between the field, semi-field and greenhouse trials.     

Competition between the oligolectic beePtilothrix plumata (Anthophoridae) and the flower closing beetlePristimerus calcaratus (Curculionidae) for floral resources ofPavonia cancellata (Malvaceae)

The flowers ofPavonia cancellata, a creeping ruderal half-shrub of northeastern Brazil, open synchronously at 6:00 h with all anthers already dehisced. The oligolectic beePtilothrix plumata was the most effective pollinator. During 90—180 min, female bees make up to 40 brief pollen collection trips to provision their brood cells. The pollen of about 40 flowers ofP. cancellata is needed to feed one bee larva. The most frequent flower visitors, however, are the specialized curculionid beetlesPristimerus calcaratus, which do not crosspollinate the flowers. They perforate the epidermis with their mouthparts, provoking dehydration, and then actively close the loose petals with their legs. Two hours after opening, half of the flowers had already been closed by the beetles. We interpret the fast, uninterrupted pollen foraging ofPtilothrix plumata bees as a strategy adapted to synchronous pollen presentation ofPavonia and to competition withPristimerus calcaratus: the female bees have to provision their brood cells before the beetles succeed in closing the flowers.     

Acceptance of low-saponin lines of alfalfa with varied phenolic concentrations by pea aphid (Homoptera: Aphididae)

This research aims to examine the effect of phenolics on pea aphid (Acyrthosiphon pisum) (Homoptera: Aphididae) development and feeding behaviour, on leaves of selected low-saponin lines of Radius alfalfa (Medicago sativa). There was a slight, negative correlation (Spearman rank correlation r _s = −0.80) between concentrations of saponins and phenols. Lines with higher concentrations of saponins had less phenolics. Levels of phenolics in low-saponin lines of alfalfa cv. Radius were related to their acceptance by the pea aphid. Our data revealed an inverse relationship between level of phenolics and the aphid abundance and its biology on studied alfalfa lines. Larval development of the pea aphid was longer, reproduction period was shorter, and the fecundity was lower on low-saponin lines with higher level of phenolics. There were observed some tendencies in the pea aphid feeding behaviour on these lines: prolonging the probing of the peripheral tissues (epidermis and mesophyll) and shortening the period of phloem sap ingestion. The better hosts for the pea aphid were low-saponin lines with low levels of phenolic compounds.     

Tree colonization by the Asian longhorn beetle,Anoplophora glabripennis (Coleoptera: Cerambycidae): effect of habitat and tree suitability

Tree colonization and feeding activity of the invasive wood‐borer Asian longhorn beetle (Anoplophora glabripennis), an Asian pest introduced into North America and Europe, was studied in a newly invaded area in Italy. The hypothesis being tested was that the reproductive success of the insect depend on habitat type and tree suitability. Adult beetles were caged on branches of host and nonhost species, in both urban and forest habitats. Two months later, number and size of feeding patches on plant tissues, eggs laid, and surviving larvae were assessed. Bark concentration of C and N was also measured from the same trees. Results indicated that the mean area of plant tissues consumed by adult feeding was significantly larger on trees growing in forest than in urban habitat, although within the same habitat there were no differences between susceptible and nonsusceptible trees. ALB tree colonization, in terms of number of eggs laid and young larvae survival, was not affected by habitat while it was higher on susceptible trees. Although trees growing in forests had a lower nitrogen concentration, they allowed colonization rates similar to those of trees growing in the urban habitat. Hence, the amount of carbon and nitrogen did not fully explain tree suitability or habitat selection. We suggest compensatory feeding as a potential mechanism that might explain this peculiar situation, as supported by a more intensive feeding activity recorded on trees in the forest. Suitability of different trees may be due to other factors, such as secondary chemical compounds.     

The structure of feeding behavior in the colorado potato beetle,Leptinotarsa decemlineata (Coleoptera: Chrysomelidae)

Feeding behavior, in an ad libitum situation on potato plants in the laboratory, was continuously observed for approximately 7 h/day on 2 successive days for 18 adult femaleLeptinotarsa decemlineata. Additional behaviors were also recorded including resting, walking, biting, local movements, grooming, defecating, and regurgitating. These data were used to calculate a time budget for the various behaviors. The feeding data were analyzed to describe the structure of feeding for young adult females on their normal host plant. The criterion for a meal (minimum intermeal interval) was determined to be 286 s. This criterion was used to distinguish between intra- and intermeal interruptions in feeding for all subsequent analyses. Meals taken from leaves that were young, medium aged, or old did not differ, but on average beetles took 60% of their meals from young leaves. Meal size and meal duration were equally good predictors of when a meal would end. Feeding from stems was a prominent feature for most beetles. The stem meals had much longer durations than leaf meals, but stem feeding did not affect subsequent leaf feeding. The structure of feeding by these beetles is compared with that found in other insects, especiallyLocusta migratoria.     

Visits to Manipulated Flowers by Episyrphus balteatus (Diptera: Syrphidae): Partitioning the Signals of Petals and Anthers

In a number of previous studies attention has been directed to the selection on corolla dimensions by pollinator preference, but anthers may also be a signal. This experiment examined the relative importance of petals and anthers in the attraction of male Episyrphus balteatus (Diptera, Syrphidae) to individual oil-seed rape flowers (Bracus rapae oleifera). Rape flowers have four petals and six anthers. Nine treatments were created, of flowers with four, two, or zero petals and six, three, or zero anthers. Twenty males were tested for preference among the experimental flowers by recording the sequence and duration of their visits (which were all for feeding) to the flowers. The number of first visits and the duration of visits on each flower were significantly related to the number of anthers but not the number of petals. There was no correlation between petal area and pollen load of control flowers. It is suggested that Syrphidae may not always attend to petals, and variation in preference of different pollinator taxa should be taken into account when discussing the evolution of floral characters.     

转Bt-cry1Ab玉米花粉对异色瓢虫生长发育及体内三种代谢酶活性的影响

用转Bt-cry1Ab基因玉米花粉饲喂异色瓢虫,初步研究了转基因玉米花粉对瓢虫的影响。结果显示,异色瓢虫取食混有适量蚜虫的转Bt-cry1Ab基因玉米花粉时与取食混有适量蚜虫的非转基因亲本玉米花粉时相比,各虫态发育历期没有显著差异;取食转Bt-cry1Ab基因玉米花粉对异色瓢虫的体重增加无明显影响。多数龄期内取食转基因玉米花粉的异色瓢虫体内的α-乙酸萘酯酶活性、乙酰胆碱酯酶活性以及谷光甘肽-S-转移酶活性与对照组相比没有显著差异。用酶联免疫（ELISA）方法在取食转Bt-cry1Ab基因玉米花粉的瓢虫体内未检测到Bt杀虫蛋白。转Bt-cry1Ab玉米花粉对异色瓢虫生长发育没有显著负面影响,初步证明Bt玉米MON810花粉对异色瓢虫是安全的。     

Pollen flow inPrimula veris (Primulaceae)

Observations on two natural and one artificial distylous populations ofPrimula veris in Britain revealed very high proportions of intramorph pollen on pin stigmas and moderate proportions of intermorph pollen on thrum stigmas. The average ratio of pin: thrum pollen production per flower was 2.4:1. Large amounts of pollen remained in dehisced anthers, perhaps reflecting low levels of pollinator activity. Population size and density had no clear effect on pollen flow patterns, which are asymmetrical for the two morphs.     

The importance of non-pollen plant parts as food sources for the common blossom thrips, Frankliniella schultzei

Like other flower thrips, Frankliniella schultzei Trybom (Thysanoptera: Thripidae) feeds on pollen. However, the influence of a pollen diet on the life history of F. schultzei may not be as significant as reported for other thrips species. Frankliniella schultzei was reared successfully and with low mortalities (20%) on Wax Mallow, (Malvaviscus arboreus Cav.) plant part diets. Development times and fecundity on a petal diet were not significantly different from that on a pollen diet. Fecundity on a diet combining M. arboreus pollen, petal and leaf tissues was significantly higher than those individually containing these tissues. In laboratory choice tests, F. schultzei females encountered petal most often of the three plant parts. Pollen and leaf were encountered with similar frequencies. A significantly higher proportion of petal encounters (0.8) resulted in feeding than did pollen encounters (0.5). Few leaf encounters (0.1) were followed by feeding. Adult and larval F. schultzei were found in M. arboreus flowers but not on leaves. All parts of the flower were inhabited and not just the pollen-bearing petal apices and anthers. We propose that feeding on pollen within M. arboreus flowers is just one of many influences on the life history of F. schultzei and suggest that this may extend to other thrips species/host-plant combinations.