Analysis of amino acids in nectar from pitchers of Sarracenia purpurea (Sarraceniaceae)

Sarracenia purpurea L. (northern pitcher plant) is an insectivorous plant with extrafloral nectar that attracts insects to a water-filled pitfall trap. We identified and quantified the amino acids in extrafloral nectar produced by pitchers of S. purpurea. Nectar samples were collected from 32 pitchers using a wick-sampling technique. Samples were analyzed for amino acids with reverse-phase high-performance liquid chromatography with phenylisothiocyanate derivatization. Detectable amounts of amino acids were found in each of the 32 nectar samples tested. Mean number of amino acids in a nectar sample was 9 (SD = 2.2). No amino acid was detected in all 32 samples. Mean amount of amino acids in a nectar sample (i.e., amount per wick) was 351.4 ng (SD = 113.2). Nine amino acids occurred in 20 of the 32 samples (aspartic acid, cysteine, glutamic acid, glycine, histidine, hydroxyproline, methionine, serine, valine) averaging 263.4 ng (SD = 94.9), and accounting for ~75% of the total amino acid content. Nectar production may constitute a significant cost of carnivory since the nectar contains amino acids. However, some insects prefer nectar with amino acids and presence of amino acids may increase visitation and capture of insect prey.     

Response of fire ants (Formicidae: Solenopsis invicta and S.gerninata) to artificial nectars with amino acids

Abstract.

• 1 The role that amino acids in extrafloral nectars play in attracting ants to plants was investigated. Workers from laboratory colonies of Solenopsis invicta Buren and S.geminafa (F). (Formicidae) fed from artificial nectaries containing mimics of the extrafloral nectar of Passiflora menispermifoh and P.caerulea; P.menispermifoh nectar contains higher levels of amino acids (1347.3 pdml) than does the nectar of P.currulea (125.2 μm /ml).
• 2 When sugar-only and sugar—amino acid nectar mimics were presented simultaneously, more S.invicta workers were counted at sugar—amino acid nectar mimics than at sugar-only nectars. S.geminatu did not discriminate between the two nectars.
• 3 When the two Pamiflora L. nectar mimics were presented simultaneously, S.invicta and S.geminata workers were more abundant at the nectaries containing high levels of amino acids (P.menispermifolia HBK mimic) than at the nectaries containing low levels of amino acids (P.cuerulea L. mimic).
• 4 The behaviour shown by S.invicta and S.gerninata suggests that plants with high levels of amino acids in their extrafloral nectars attract more ant protectors and might suffer less herbivory than plants producing nectars with low levels of amino acids. If so, ants may favour, over evolutionary time, plants that produce nectars with high levels of amino acids.
• 5 Day-to-day variability in ant behaviour was considerable even among laboratory colonies maintained on the same diet in similar environmental conditions. This variability will reduce the selective impact that ants have on plants and may help to explain why most ant-plant interactions are facultative.

     

THE EXTRAFLORAL NECTARIES OF IPOMOEA CARNEA (CONVOLVULACEAE)

Ipomoea carnea (Convolvulaceae) possesses two types of extrafloral nectaries, located on the petiole and on the pedicel. These secrete a complex nectar containing sugars and amino acids. The insects attracted to the extrafloral nectaries are predominantly ants and they are relatively abundant throughout the year. A number of incidents of plant defense as a result of the presence of extrafloral nectary visitors at the extrafloral nectaries of I. carnea were observed and are consistent with the ant-guard theory of the function of extrafloral nectaries.     

EXTRAFLORAL NECTAR OF FEROCACTUS ACANTHODES (CACTACEAE): COMPOSITION AND ITS IMPORTANCE TO ANTS

Ants are attracted to extrafloral nectaries subtending reproductive organs of Ferocactus acanthodes var. lecontei (Cactaceae) in central Arizona. Extrafloral nectar produced by these glands contained amino acids, sugars, and water. Nectar quality and composition varied temporally in relation to plant reproductive phenology. The number of nectar glands on a barrel cactus did not change significantly, however; the mass of nectar produced per gland increased significantly with immature fruit production. Of the three sugars present in extrafloral nectar (fructose, glucose, and sucrose), only glucose occurred at a higher concentration in June, when immature fruits first appeared on barrel cactus. Amino acid concentration and composition in extrafloral nectar of barrel cactus did not change significantly over time. Ant density on barrel cactus increased significantly from mid-May to mid-June at two field sites. Water availability per nectar gland increased 158% from May to June. Water plays an important role in attracting ants to barrel cacti.     

Sugar and amino acid composition of ant‐attended nectar and honeydew sources from an Australian rainforest

Abstract Ants (Hymenoptera: Formicidae) consume a broad spectrum of liquid food sources including nectar and honeydew, which play a key role in their diet especially in tropical forests. This study compares carbohydrates and amino acids from a representative spectrum of liquid sources used by ants in the canopy and understorey of a tropical rainforest in northern Queensland, Australia. Eighteen floral nectars, 16 extrafloral nectars, two wound sap and four homopteran honeydew sources were analysed using high performance liquid chromatography. Wounds comprised flower abscission scars on Normanbya normanbyi L. H. Bailey and bitemarks on Cardwellia sublimis F. Muell. where ants were actively involved in wounding. Discriminant analysis was performed to model differences between food sources in sugar and amino acid concentration and composition. All characteristics varied significantly among plant species. Honeydew contained a broader spectrum of sugars (including melezitose, raffinose, melibiose, lactose and maltose) than nectar (sucrose, glucose, fructose), but certain extrafloral nectars had similar amino acid profiles and, like honeydew sources, were often monopolized by ants. Most common amino acids across the sources were proline, alanine and threonine among 17 α‐amino acids identified. Interspecific variability concealed characteristic differences in sugar and amino acid parameters between nectar, honeydew and wound sap across all plants, but these types differed significantly when found on the same plant. Among all sources studied, only a few flower nectars were naturally not consumed by ants and they were significantly less attended than sugar controls in feeding trials. These nectars did not differ in sugars and amino acids from ant‐attended flower nectars, suggesting the activity of repellents. Apart from these exceptions, variability in amino acids and carbohydrates is proposed to play a key role in ant preferences and nutrition.     

伤害和挥发物对棉花花外蜜的诱导效应

植物花外蜜的分泌是一种植物间接防御反应。为了明确植食性昆虫、机械伤和机械伤诱导的挥发性气体在植物花外蜜诱导分泌中的作用,分析了咀嚼式口器昆虫棉铃虫Helicoverpa armigera(Hübner)、刺吸式口器昆虫棉蚜Aphis gossypii Glover取食、剪刀机械伤、剪刀机械伤+棉铃虫反吐物、针刺机械伤以及机械伤诱导挥发物、顺式-茉莉酮对棉花Gossypium hirsutum L.叶片花外蜜分泌量的影响。结果表明,棉铃虫取食、剪刀机械伤、剪刀机械伤+棉铃虫反吐物处理均显著增加了被处理叶片花外蜜的分泌量。棉花花外蜜的诱导效应在处理叶片上表现明显,并且在较幼嫩的第3片真叶上也有系统性增长。顺式-茉莉酮和机械伤挥发物处理1 d对棉花较幼嫩的第4、5片真叶花外蜜有诱导效应。棉花叶片花外蜜的诱导主要与植物组织损伤有关;不同口器类型的昆虫对棉花叶片花外蜜的诱导量不同,咀嚼式口器的棉铃虫对棉花花外蜜的诱导强度显著高于刺吸式口器的棉蚜;顺式-茉莉酮和机械伤诱导的挥发物能作为棉花植株间交流的信息物质诱导棉花幼嫩叶片花外蜜的分泌。     

Detection of amino acids in artificial nectars by two tropical ants,Leptothorax and Monomorium

Summary Many ants forage at extrafloral nectar on plants and provide the plant with some measure of protection from herbivory. These nectars contain sugars, amino acids and, often, other compounds. The role of amino acids in attracting ants to extrafloral nectars was studied by baiting with Karo-syrup-based solutions. Control (without amino acids) and experimental (with amino acids) solutions were placed in second growth forest in Trinidad, W.I. The number of ants visiting the solutions was counted at fiveminute intervals for 45 min. In tests of solutions with only one amino acid, both Leptothorax sp. and Monomorium sp. visited solutions with alanine, arginine, serine, cysteine, methionine or aspartic acid more frequently than sugaronly controls. Monomorium preferred control solutions to tyrosine solutions; Leptothorax preferred control solutions to histidine solutions. Leptothorax did not discriminate between control and tyrosine solutions; Monomorium did not discriminate between control and histidine solutions. However, in six of eight tests of combinations of amino acids, ants visited control solutions more frequently than experimental solutions. These results suggest that ants can act as selective agents, favoring plants with particular amino acids in their nectars.     

A novel indirect defence in Brassicaceae: Structure and function of extrafloral nectaries in Brassica juncea

While nectaries are commonly found in flowers, some plants also form extrafloral nectaries on stems or leaves. For the first time in the family Brassicaceae, here we report extrafloral nectaries in Brassica juncea. The extrafloral nectar (EFN) was secreted from previously amorphic sites on stems, flowering stalks and leaf axils from the onset of flowering until silique formation. Transverse sections at the point of nectar secretion revealed a pocket‐like structure whose opening was surrounded by modified stomatal guard cells. The EFN droplets were viscous and up to 50% of the total weight was sugars, 97% of which was sucrose in the five varieties of B. juncea examined. Threonine, glutamine, arginine and glutamate were the most abundant amino acids. EFN droplets also contained glucosinolates, mainly gluconapin and sinigrin. Nectar secretion was increased when the plants were damaged by chewing above‐ and belowground herbivores and sap‐sucking aphids. Parasitoids of each herbivore species were tested for their preference, of which three parasitoids preferred EFN and sucrose solutions over water. Moreover, the survival and fecundity of parasitoids were positively affected by feeding on EFN. We conclude that EFN production in B. juncea may contribute to the indirect defence of this plant species.     

Nectar chemistry is tailored for both attraction of mutualists and protection from exploiters

Plants produce nectar to attract pollinators in the case of floral nectar (FN) and defenders in the case of extrafloral nectar (EFN). Whereas nectars must function in the context of plant-animal mutualisms, their chemical composition makes them also attractive for non-mutualistic, exploiting organisms: nectar robbers and nectar-infesting microorganisms. We reviewed the chemical composition of both FNs and EFNs and found that nectar composition appears tailored to fulfil these ambivalent roles. Carbohydrates and amino acids usually function in the attraction of mutualists and appear adapted to the physiological needs of the respective mutualists. Volatiles are a further group of compounds that serves in the attractive function of nectars. By contrast, secondary compounds such as alkaloids and phenols serve the protection from nectar robbers, and most nectar proteins that have been characterised to date protect FN and EFN from microbial infestation. Nectar components serve both in attraction and the protection of nectar.Key words: extrafloral nectar, floral nectar, indirect defence, mutualism, pollination     

Short‐term proteomic dynamics reveal metabolic factory for active extrafloral nectar secretion by Acacia cornigera ant‐plants

Despite the ecological and evolutionary importance of nectar, mechanisms controlling its synthesis and secretion remain largely unknown. It is widely believed that nectar is ‘secreted phloem sap’, but current research reveals a biochemical complexity that is unlikely to stem directly from the phloem. We used the short daily peak in production of extrafloral nectar by Acacia cornigera to investigate metabolic and proteomic dynamics before, during and after 2 h of diurnal secretion. Neither hexoses nor dominating nectar proteins (nectarins) were detected in the phloem before or during nectar secretion, excluding the phloem as the direct source of major nectar components. Enzymes involved in the anabolism of sugars, amino acids, proteins, and nectarins, such as invertase, β–1,3–glucanase and thaumatin‐like protein, accumulated in the nectary directly before secretion and diminished quantitatively after the daily secretion process. The corresponding genes were expressed almost exclusively in nectaries. By contrast, protein catabolic enzymes were mainly present and active after the secretion peak, and may function in termination of the secretion process. Thus the metabolic machinery for extrafloral nectar production is synthesized and active during secretion and degraded thereafter. Knowing the key enzymes involved and the spatio‐temporal patterns in their expression will allow elucidation of mechanisms by which plants control nectar quality and quantity.     

FACULTATIVE MUTUALISM BETWEEN WEEDY VETCHES BEARING EXTRAFLORAL NECTARIES AND WEEDY ANTS IN CALIFORNIA

Vetches (Vicia spp.) were studied in the San Francisco Bay Area of California in the spring of 1978. The stipular nectaries of the vetches are visited by the Argentine ant, Iridomyrmex humilis Mayr. The nectaries were removed to exclude ants in controlled experiments to determine if these ants protect the vetches from herbivores or seed predators. Plants with excised nectaries suffered substantially greater damage to their foliage than control plants, indicating that ants protect the foliage. There was no indication that ants protect the vetches from seed predators, but fruit set was substantially lower in plants with excised nectaries. Analysis of sugar and amino acid composition of extrafloral nectar served as a basis for feeding tests with Argentine ants by using artificial nectar solutions. Ants preferred sucrose and glucose solutions over fructose. They showed no preference for any one sugar mixture over another, nor did they exhibit differential recruitment to artificial nectar solutions containing only sugars or sugars and amino acids.     

Geographic variation in a facultative mutualism: consequences for local arthropod composition and diversity

Geographic variation in the outcome of interspecific interactions may influence not only the evolutionary trajectories of species but also the structure of local communities. We investigated this community consequence of geographic variation for a facultative mutualism between ants and wild cotton (Gossypium thurberi). Ants consume wild cotton extrafloral nectar and can protect plants from herbivores. We chose three sites that differed in interaction outcome, including a mutualism (ants provided the greatest benefits to plant fitness and responded to manipulations of extrafloral nectar), a potential commensalism (ants increased plant fitness but were unresponsive to extrafloral nectar), and a neutral interaction (ants neither affected plant fitness nor responded to extrafloral nectar). At all sites, we manipulated ants and extrafloral nectar in a factorial design and monitored the abundance, diversity, and composition of other arthropods occurring on wild cotton plants. We predicted that the effects of ants and extrafloral nectar on arthropods would be largest in the location with the mutualism and weakest where the interaction was neutral. A non-metric multidimensional scaling analysis revealed that the presence of ants altered arthropod composition, but only at the two sites in which ants increased plant fitness. At the site with the mutualism, ants also suppressed detritivore/scavenger abundance and increased aphids. The presence of extrafloral nectar increased arthropod abundance where mutual benefits were the strongest, whereas both arthropod abundance and morphospecies richness declined with extrafloral nectar availability at the site with the weakest ant–plant interaction. Some responses were geographically invariable: total arthropod richness and evenness declined by approximately 20% on plants with ants, and extrafloral nectar reduced carnivore abundance when ants were excluded from plants. These results demonstrate that a facultative ant–plant mutualism can alter the composition of arthropod assemblages on plants and that these community-level consequences vary across the landscape.     

Using the stable isotope marker 13C to study extrafloral nectar uptake by parasitoids under controlled conditions and in the field

Parasitic wasps are prominent natural enemies of crop pests. They usually feed on floral resources during the adult stage (nectar, pollen, or honeydew). Extrafloral nectar is an alternative source of sugar easily accessible to adult parasitoids. We developed an original method of nectar labelling based on the injection of labelled sugar solution into the plant stem in order to analyse the nectar uptake by parasitoids (cotton wick method). This method was used to artificially enrich extrafloral cornflower, Centaurea cyanus L. (Asteraceae), nectar with the stable isotope ¹³C. We analysed (1) the transfer of ¹³C from the sugar solution into extrafloral nectaries, (2) the uptake of labelled nectar by parasitoids under laboratory conditions, and (3) the ability of the method to discriminate, in an oilseed rape (Brassica napus L., Brassicaceae) field, between labelled parasitoids (i.e., those who have fed on labelled cornflowers located adjacent to the field) and unlabelled parasitoids to track parasitoid movements from the margin into the field. The extrafloral nectar of all test plants was ¹³C‐labelled. Most (66%) of the parasitoids were identified as marked after 96 h of exposure to labelled plants in the laboratory. We could also detect labelled parasitoids inside the field, but the detection rate was only 1%. The experiments clearly demonstrate that the cotton wick method is appropriate to label extrafloral nectar and parasitoids feeding on this labelled nectar. Further research is needed on the amount of labelled extrafloral nectar required to obtain a sufficient marker level to track parasitoid movements in the field.     

Herbivory-induced extrafloral nectar increases native and invasive ant worker survival

Ascertaining the costs and benefits of mutualistic interactions is important for predicting their stability and effect on community dynamics. Despite widespread designation of the interaction between ants and extrafloral nectaries (EFNs) as a mutualism and over 100 years of studies on ant benefits to plants, the benefits to ants have never been experimentally quantified. The success of invasive ants is thought to be linked to the availability of carbohydrate-rich resources, though reports of invasive ant visits to EFNs are mixed. In two laboratory experiments, we compared worker survival of one native (Iridomyrmex chasei) and two invasive ant species (Linepithema humile and Pheidole megacephala) exposed to herbivorized or non-herbivorized EFN-bearing plants (Acacia saligna) or positive and negative controls. We found that non-herbivorized plants did not produce any measurable extrafloral nectar, and ants with access to non-herbivorized plants had the same survival as ants with access to an artificial plant and water (unfed ants). Ants given herbivorized plants had 7–11 times greater worker survival relative to unfed ants, but there were no differences in survival between native and invasive ants exposed to herbivorized plants. Our results reveal that ants cannot induce A. saligna extrafloral nectar production, but workers of both native and invasive ant species can benefit from extrafloral nectar as much as they benefit from sucrose.     

Effect of floral and extrafloral resources on predation of Aphis spiraecola by Harmonia axyridis on Apple

Flowering companion plants and plants producing extrafloral nectar are being proposed to enhance biological control in apple (Malus domestica Borkh.) orchards. This experiment evaluated the impact of floral and extrafloral resources on predation of spirea aphid (Aphis spiraecola Patch) on apple by adult Harmonia axyridis (Pallas) under greenhouse conditions. Predation of spirea aphids was not affected by the presence of flowering buckwheat (Fagopyrum esculentum) as compared with either buckwheat with flowers removed or an uninfested apple shoot. However, there was a significant reduction in predation of spirea aphids on an apple shoot in the presence of a peach shoot with extrafloral nectar glands compared with either a peach shoot of the same cultivar without nectar glands or an uninfested apple shoot. These findings demonstrate that alternative food resources potentially could interfere with rates of biological control and, therefore, need to be carefully evaluated before incorporating in an orchard design. More studies are needed to adequately gauge the net effects of adding floral or extrafloral resources in orchards for conservation biological control.     

Antibiotics,Sugars, and Amino Acids in Nectar of Rhododendron and Piptanthus Species from Nepal

Nectars were extracted in Helambu and Langtang Nepal Himalayas from Rhododendron arboreum and Rh. barbatum (Ericaceae), and from Piptanthus nepalensis (Fabaceae), and analyzed for sugars, amino acids, and antibiotic activity. Sugar content in Rhododendron nectar varied from 17 to 65%. In most nectars, G and F were present in a 1:1 ratio. In addition, from a Rh. arboreum nectar, S could be detected. Piptanthus nectar showed a very high sugar content (up to 80%), F and G, or F, G, and S, respectively, being the main sugar constituents. In all Rhododendron nectar amino acids could be found. In Piptanthus nectar there were no traceable amino acids. Neither an evolutionary significance of amino acid contents in floral nectar nor their close correlation with bees, the main pollinators of both genera, could be confirmed from our studies. Rhododendron nectar showed antibiotic activity due to the presence of acetylandromedol. The biological significance of acetylandromedol, the main toxic principle from Rhododendron nectar, seems to be its antibiotic activity, which could possibly compensate for lower sugar contents in preventing bacterial growth.     

Whitebellied sunbirds (<Emphasis Type="Italic">Nectarinia talatala</Emphasis>, Nectariniidae) do not prefer artificial nectar containing amino acids

Amino acids are the most abundant class of compounds in nectar after sugars. Like its sugar concentration, the amino acid concentration of nectar has been linked to pollinator type, and it has been suggested that amino acid concentrations are high in the floral nectars of plant species pollinated by passerine birds compared to those pollinated by hummingbirds. We investigated the feeding response of whitebellied sunbirds (Nectarinia talatala) to the inclusion of amino acids in artificial nectar (0.63 M sucrose solution). The response to asparagine, glutamine, phenylalanine, proline, serine and valine, amino acids commonly found in floral nectars, was tested individually and using a mixture of all six amino acids, at two different concentrations (2 and 15 mM). Sunbirds showed no significant preference for amino acids in nectar, or avoided them, especially at the higher concentration. We discuss these findings in the light of the nitrogen requirements of nectarivorous birds and data on amino acids in floral nectars.     

Two Mallotus species of different life histories adopt different defense strategies in relation to leaf age

Plants defend their leaves using multiple defense traits that change functions with leaf age. We examined the effects of leaf age on the development of multiple defense traits in two related Mallotus (Euphorbiaceae) species: young plants of the fast‐growing Mallotus japonicus (Spreng.) Müll. Arg. and the slow‐growing Mallotus philippensis (Lam.) Müll. Arg. Sequential leaves of the two species were measured for their leaf area, leaf mass/area, densities of trichomes and pellucid dots, extrafloral nectar volume, and the numbers of extrafloral nectaries and pearl bodies. Mallotus japonicus shifted its defense tactics from direct defense using trichomes and pellucid dots in young leaves to biotic defense using extrafloral nectar and pearl bodies in middle‐aged leaves. In contrast, M. philippensis used direct, chemical defense throughout all leaf ages, together with the shift from indirect, biotic defense using extrafloral nectar in young leaves to direct, physical defense using leaf toughness in middle‐aged leaves. These results strongly suggest that, in relation to life history, plants can alter optimal combinations of multiple defense traits with leaf age.     

Is nectar reabsorption restricted by the stalk cells of floral and extrafloral nectary trichomes?

Reabsorption is a phase of nectar dynamics that occurs concurrently with secretion; it has been described in floral nectaries that exude nectar through stomata or unicellular trichomes, but has not yet been recorded in extrafloral glands. Apparently, nectar reabsorption does not occur in multicellular secretory trichomes (MST) due to the presence of lipophilic impregnations – which resemble Casparian strips – in the anticlinal walls of the stalk cells. It has been assumed that these impregnations restrict solute movement within MST to occur unidirectionally and exclusively by the symplast, thereby preventing nectar reflux toward the underlying nectary tissues. We hypothesised that reabsorption is absent in nectaries possessing MST. The fluorochrome lucifer yellow (LYCH) was applied to standing nectar of two floral and extrafloral glands of distantly related species, and then emission spectra from nectary sections were systematically analysed using confocal microscopy. Passive uptake of LYCH via the stalk cells to the nectary tissues occurred in all MST examined. Moreover, we present evidence of nectar reabsorption in extrafloral nectaries, demonstrating that LYCH passed the stalk cells of MST, although it did not reach the deepest nectary tissues. Identical (control) experiments performed with neutral red (NR) demonstrated no uptake of this stain by actively secreting MST, whereas diffusion of NR did occur in plasmolysed MST of floral nectaries at the post‐secretory phase, indicating that nectar reabsorption by MST is governed by stalk cell physiology. Interestingly, non‐secretory trichomes failed to reabsorb nectar. The role of various nectary components is discussed in relation to the control of nectar reabsorption by secretory trichomes.