Unlike phloem-feeding insects, prokaryotes cannot actively enter the phloem; therefore, all known phloem-associated prokaryotes are passively delivered into the phloem by phloem-feeding insects. Innovative culturing approaches are also required for future research, in order to remove one of the most formidable barriers to the study of phloem-pathogen interactions.
Journal Proceedings of the National Academy of Sciences. DOI These considerations raise two questions: in what ways are hemipteran insects uniquely predisposed to use phloem sap as food; and why is symbiosis with micro-organisms linked to this habit?
In this article, as one contributory factor, it is suggested that phloem sap poses nutritional barriers to utilization by animals that only the hemipterans have overcome, partly through the nutritional contribution from their symbiotic micro-organisms. Other hemipteran traits important to phloem feeding include the anatomy and function of the insects mouthparts and gut, and these are addressed by Goodchild and Douglas Although phloem feeding is restricted to hemipterans, other animals use phloem sap by proxy.
Honeydew is phloem sap modified in composition by passage through the hemipteran gut and released via the anus. In the latter part of this article, the phloem sap and honeydew are compared as food. In some respects, phloem sap is an excellent diet for animals. It is also generally free of toxins and feeding deterrents, a consequence of its being a highly specialized cytoplasm plant secondary compounds tend to be localized in the apoplast and cell vacuole, and not the cytoplasmic compartment.
These exceptions notwithstanding, phloem sap remains a poorly-defended, nutrient-rich food source for those animals that can access it. Despite this, phloem sap poses two major nutritional problems for animals. The nature of these barriers and the response of phloem-feeding insects to them will be considered.
The growth and fecundity of phytophagous insects are generally limited by nitrogen, in two ways: the quantity of nitrogen, i. The issue of quality arises because animals are metabolically impoverished, lacking the ability to synthesize nine of the 20 amino acids that make up protein. If the concentration of just one of these essential amino acids is in short supply, protein synthesis and growth of an animal are constrained. The nitrogen barrier to phloem sap utilization: amino acid relations of the pea aphid Acyrthosiphon pisum line LL01 feeding on Vicia faba.
A Amino acid content of V. B Amino acids derived from Buchnera symbionts during the 2 d of the final larval stadium, as calculated from the difference between amino acids required for protein growth and acquired by feeding on plant phloem sap. The nitrogen barrier to phloem sap utilization is its low nitrogen quality. Broadly speaking, the ratio of essential amino acids:non-essential amino acids in plant phloem sap is —, considerably lower than the ratio of in animal protein.
Data for the phloem sap of the broad bean Vicia faba and the legume-feeding pea aphid Acyrthosiphon pisum illustrate this mismatch Fig.
The amino acids in the phloem sap of V. All the essential amino acids are detectable in the phloem sap samples, but their combined concentration represents just 8. The essential amino acid content of phloem sap is insufficient to support the observed growth rate of the aphids. This can be illustrated for final instar larvae of A. The shortfall varies from 0. The shortfall in the dietary supply of amino acids is met by an endogenous source. As described below, there is overwhelming evidence that the endogenous source is symbiotic bacteria, Buchnera sp.
In other words, Buchnera cells enable aphids to overcome the nitrogen barrier to phloem sap utilization. The biology of Buchnera has been reviewed recently in Douglas The key information required in the present context is that Buchnera sp. Buchnera is obligately intracellular, restricted to the cytoplasm of specialized insect cells, known as bacteriocytes, in the aphid haemocoel body cavity and transferred vertically to eggs or early embryos for viviparous morphs in the female reproductive organs.
The evidence that Buchnera provide aphids with essential amino acids is 3-fold: nutritional, physiological, and genomic. The nutritional and physiological approaches have depended on the development of two sets of techniques: to eliminate the Buchnera from the aphids using antibiotics, generating aphids known as aposymbiotic aphids Wilkinson, ; and to rear the aphids on chemically-defined diets that can be manipulated Dadd, The key results of nutritional research over many years are that aphids with their normal complement of Buchnera can be reared on diets from which individual essential amino acids are eliminated, but aposymbiotic aphids have an absolute requirement for all the essential amino acids reviewed in Douglas, The complementary physiological experiments demonstrate that aphids with Buchnera , but not aposymbiotic aphids, can synthesize essential amino acids from dietary precursors such as sucrose and aspartate Douglas, ; Febvay et al.
Together, these experiments indicate that bacteria are responsible for the capacity of aphids to utilize phloem sap poor in essential amino acids by providing the aphid tissues with these nutrients. The genomic evidence supporting the central role of Buchnera in providing essential amino acids to aphids comes from annotation of the complete genome sequences of Buchnera , now available for isolates from A.
The Buchnera in all these aphid species have a small genome 0. Most unusually for bacteria, nearly all the genes have orthologues in other bacteria, including Escherichia coli.
In other words, the Buchnera genome approximates to a subset of the E. Exceptionally, Buchnera have retained genes coding for most enzymes in the biosynthetic pathways for essential amino acids, even though they have lost many other metabolic capabilities, including the capacity to synthesize most non-essential amino acids Table 1.
The biosynthetic pathway is truncated in Buchnera relative to E. Buchnera has metH , suggesting that it can synthesize methionine from homocysteine, but lacks metABC required for the conversion of homoserine to homocysteine. Animals including most insects can synthesize arginine from ornithine.
Arginine is a non-essential amino acid by the definition used here, i. Present in Buchnera from Acyrthosiphon pisum and Schizaphis graminum , absent from Buchnera from Baizongia pistacea. Present in Buchnera from Acyrthosiphon pisum and Baizongia pistacea , absent from Buchnera from Schizaphis graminum. These studies suggest strongly that aphids overcome the nitrogen barrier to phloem sap utilization by their acquisition of essential amino acid-overproducing bacterial symbionts.
As considered in the Introduction, all phloem-feeding hemipterans bear symbiotic micro-organisms, a minority of which have been studied phylogenetically. Thao et al. By analogy with the aphid— Buchnera relationship, these micro-organisms can plausibly be argued to provide their insect hosts with essential amino acids, but direct evidence is lacking. The dominant compounds in phloem sap are sugars derived from photosynthetic carbon fixation. In many plants, most of the sugar is in the form of sucrose, a chemically-stable disaccharide of low viscosity.
The phloem-mobile sugars in some plants, including many labiates, include oligosaccharides of the raffinose series, especially raffinose and stachyose with one or two galactose units, respectively, transferred to the glucose moiety of sucrose ; and some plants have appreciable levels of sugar alcohols, for example, mannitol in the Apiaceae, sorbitol in the Rosaceae Ziegler, The basis of the sugar barrier to phloem sap feeding is its very high concentration in phloem sap, up to and often exceeding 1 M sugar, and a resultant osmotic pressure 2—5 times greater than the osmotic pressure of the insect's body fluids.
The insects ingest the phloem sap at a high rate, partly because phloem sap has high hydrostatic pressure and partly to ensure a sufficient supply of other phloem nutrients at low concentrations. The predicted consequence of the continuous flow of fluid at high osmotic pressure into the gut is the transfer of water from the body fluids to the gut contents and osmotic collapse of the insect.
In other words, phloem-feeding insects are expected to shrivel as they feed. The key evidence that aphids overcome the sugar barrier by osmoregulation is that the osmotic pressure of the honeydew, the egesta voided from the gut, is comparable to that of the body fluids and is lower than the ingested food Fig. Equivalent data for other hemipterans are lacking. Analysis of honeydew sugar composition provides a clue as to how the osmotic barrier is overcome.
When aphids are reared on chemically-defined diets with sucrose as the sole sugar, the dominant honeydew sugars are the monosaccharides, glucose and fructose, at low dietary sucrose concentrations 0. It is proposed that the transformation of ingested sucrose to oligosaccharides would tend to reduce the osmotic pressure of the gut contents because the osmotic pressure exerted by solutes is determined by their molality and not their weight.
The sugar barrier to phloem sap utilization: osmotic relations of the pea aphid Acyrthosiphon pisum. A The osmotic pressure of aphid honeydew is similar to that of aphid haemolymph body fluids and lower than that of the phloem sap of Vicia faba plants on which they were feeding. B The oligosaccharide content of aphid honeydew is strongly dependent on the sucrose concentration ingested from the chemically-defined diet.
Data from Wilkinson et al. These data suggest that the sugar relations of phloem-feeding insects are intimately linked with osmoregulation.
At an enzymological level, the fate of ingested sugars is best-understood in aphids. For example, pea aphids have very high sucrase activity localized in the gut distal to the stomach Ashford et al. It has been proposed that the sucrase enzyme may also mediate the synthesis of oligosaccharides by transglucosidation, i. Transglucosidation activity has also been demonstrated in the whitefly Bemisia , generating an array of honeydew sugars Byrne et al.
An important implication of these results is that the microbiota play no part in the capacity of insects to utilize sugar-rich phloem sap.
Although some early literature has suggested a role of micro-organisms in osmoregulation and in the sugar relations of phloem-feeding insects reviewed in Douglas, , the balance of current evidence is that micro-organisms have no direct role. In particular, aphids treated with antibiotics to eliminate Buchnera maintain their capacity to osmoregulate their body fluids to a constant value over a wide range of dietary sucrose Wilkinson et al.
Phloem sap varies in composition over multiple timescales, from the diurnal cycle to the season, with the developmental age of the plant, and with abiotic factors such as temperature and water availability Douglas, ; Geiger and Servaites, ; Kehr et al. The capacity of phloem-feeding insects to respond to long-term changes, for example, associated with plant development or season, has been amply demonstrated for aphids.
This has been achieved by the use of chemically-defined diets with a range of different compositions that reflect different phloem sap compositions Karley et al.
By contrast, there is very little information on the response of insects to diurnal variation in phloem sap composition, even though this variation can be considerable. This short-term variation cannot be mimicked readily using chemically defined diets, which are of fixed composition. Much of the insect response may be behavioural, through variation in the rate of food uptake according to the nutrient content and osmotic pressure. Where compositional changes are very rapid or large, the insect has the option to withdraw the stylets and probe for a different sieve element.
There is a real possibility that the capacity of an insect to utilize a plant is influenced by the scale of diurnal variation in phloem sap composition, as well as by the composition quantified at any one point in the diurnal cycle. In this context, datasets on phloem sap composition obtained during the light period, and usually at a fixed time, may not reflect accurately the total nutritional inputs to phloem-feeding insects. After digestion and assimilation of ingested phloem sap in the hemipteran gut, the residue is voided via the anus as honeydew.
Honeydew is often produced in copious amounts. For example, first instar larvae of the willow aphid Tuberolachnus salignus produce more honeydew per hour than their body weight Mittler, Honeydew is used as a food by various animals which can be considered as secondary or proxy phloem feeders that exploit the capacity of hemipterans to access plant sieve elements. Many insects, including flies, wasps, bees, beetles, butterflies, and moths, as well as nectarivorous birds and flying foxes, consume honeydew that has fallen onto plants or other surfaces; and some animals take honeydew droplets directly from the anus of the hemipterans.
This behaviour, called tending, is widely displayed among ants, especially among the dolichoderines and formicines, and also by polybiine wasps, silvanid beetles and, remarkably, some Madagascan geckos that specifically tend planthoppers Folling et al.
Most research has concerned ant-tending relationships. They are mutualistic: the tending ants gain food and the tended phloem-feeding hemipteran is protected from natural enemies by the ants. The 14 N isotope is lost preferentially in catabolic reactions and therefore herbivores are predicted to be enriched in 15 N relative to plants, their predators enriched relative to herbivores, and so on through the trophic levels Griffiths, In a detailed analysis of ants in tropical rainforests of Peru and Brunei, Davidson et al.
In ecological terms, tending ants are herbivores, gaining access to phloem sap through mutualism with hemipterans. Explaining the abundance of ants in lowland tropical rainforest canopies. Science , — Copyright AAAS omitting species with mixed and uncertain foraging strategies.
The conclusion that hemipteran honeydew is a quantitatively important component of the diet of many ants raises the issue of the nutritional suitability of honeydew as a food for animals. Honeydew is nutritionally distinct from phloem sap because of the enzymatic and assimilatory capabilities of the hemipteran gut.
In particular, the sugars are modified by hydrolysis and transglucosidation, and the amino acid profile is altered by differential assimilation. With respect to the sugar barrier to phloem feeding, the osmotic challenge posed by high phloem sugar is negated by the osmoregulatory capabilities of phloem feeders, making honeydew an osmotically-neutral foodstuff of complex and variable sugar composition.
The osmotic pressure of honeydew is, however, expected to rise with increasing time after deposition through the evaporative loss of water, posing osmotic problems for animals feeding on honeydew on plant surfaces etc.
Furthermore, the capacity of ants and other honeydew feeders to digest the complex honeydew sugars, and its enzymological basis, remain to be investigated in detail. The significance of the nitrogen barrier to honeydew feeding depends critically on the amino acid assimilation patterns of hemipterans. In the aphid species studied in this laboratory, the amino acid composition of honeydew is generally more balanced than in phloem sap because aphids preferentially assimilate non-essential amino acids Adams, In this dataset, all of the essential amino acids and just two of the non-essential amino acids serine and aspartic acid are proportionately enriched in honeydew, relative to phloem sap.
However, it would be inappropriate to generalize from these data because there could be considerable variation among plant—hemipteran relationships. Existing techniques for the identification of phloem cells rely on the presence of phloem-specific proteins called forisomes. However, forisomes are only found in the phloem cells of plants in the bean family, limiting the application of this method. The technique presented here takes advantage of the distinctive anatomy of phloem cells by using organelle-specific dyes and fluorescent microscopy.
For example, phloem cells called sieve element lack a nucleus and vacuole, but possess parietal mitochondria, so these cells become apparent when tissue is stained with the organelle-specific Hoechst , Neutral Red, and MitoTracker Green and visualized with a fluorescent microscope. This method is applicable well beyond citrus, because it relies on the anatomy of phloem cells, rather than protein markers that vary from species to species.
That means it could be used to understand not only citrus greening but a wide variety of phloem diseases such as cucurbit yellow vine disease, corn stunt disease, and onion yellow dwarf disease. While studying phloem diseases is by far the most pressing application for this technique, and was the motivation for this study, identification of phloem cells could also help with the study of other botanical questions.
Additionally, because it involves digestion of the cell wall, "this method can be used to study membrane properties such as ion fluxes, membrane electrical properties, and biophysics of membrane elasticity -- a study not possible with intact cells.
Using established biological methods like cell wall digestion, organelle-specific staining, and fluorescent microscopy, Dr. Etxeberria and colleagues have developed a technique to accurately isolate phloem cells across plants. While this technique has wide applications, it will immediately be put into service in understanding and fighting the devastating phloem diseases causing crop failures worldwide.
Materials provided by Botanical Society of America. Note: Content may be edited for style and length. Science News.
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