NUTRITIONAL INTERACTIONS in INSECT-MICROBIAL SYMBIOSES: Aphids and Their Symbiotic Bacteria Buchnera

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NUTRITIONAL INTERACTIONS IN INSECT-MICROBIAL SYMBIOSES: Aphids and Their Symbiotic Bacteria Buchnera

A. E. Douglas Department of Biology, University of York, PO Box 373, York, YO1 5YW, United Kingdom; e-mail: [email protected]

KEY WORDS: mycetocyte, nutrition, amino acids, endosymbiosis, arthropod

ABSTRACT Most aphids possess intracellular bacteria of the genus Buchnera. The bacteria are transmitted vertically via the aphid ovary, and the association is obli- gate for both partners: Bacteria-free aphids grow poorly and produce few or no offspring, and Buchnera are both unknown apart from aphids and apparently unculturable. The symbiosis has a nutritional basis. Speciﬁcally, bacterial provisioning of essential amino acids has been demonstrated. Nitrogen recycling, however, is not quantitatively important to the nutrition of aphid species studied, and there is strong evidence against bacterial involvement in the lipid and sterol nutrition of aphids. Buchnera have been implicated in various non-nutritional functions. Of these, just one has strong experimental support: promotion of

by Western Michigan University on 01/17/12. For personal use only. aphid transmission of circulative viruses. It is argued that strong parallels may exist between the nutritional interactions (including the underlying mechanisms) Annu. Rev. Entomol. 1998.43:17-37. Downloaded from www.annualreviews.org in the aphid-Buchnera association and other insect symbioses with intracellular microorganisms.

PERSPECTIVES AND OVERVIEW The main topic of this review is the symbiosis between aphids and bacteria of the genus Buchnera. This association is a “mycetocyte symbiosis,” as deﬁned by three characteristics:

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Table 1 Mycetocyte symbioses in insects1

Insect Microorganism2 Incidence

Blattaria (cockroaches) Flavobacteria (6) Universal Heteroptera Cimicidae Bacteria3 Universal Lygaeidae Irregular Homoptera γ -Proteobacteria in aphids (73) Nearly universal4 and whitefly (18); β-Proteobacteria in mealbugs (75); pyrenomycete yeasts in delphacid planthoppers (77) and hormaphidine aphids (42); bacteria in other groups3 Anoplura Bacteria3 Universal Mallophaga Bacteria3 Irregular Diptera Glossinidae γ 3-Proteobacteria Universal Diptera Pupipera Bacteria3 Universal Coleoptera Yeasts “close to Discomycetes” Reported in 10 families, in anobiids (76); yeasts in including Bostrychidae, Cerambycidae3; otherwise Lyctidae (universal); bacteria3 Anobiidae, Chrysomelidae, Cuculionidae (widespread) Formicidae (ants) Camponoti γ 3-Proteobacteria (91) Universal Formicini Bacteria3 Irregular 1Based on Buchner (14) and references in review by Douglas (28). 2Microorganisms identified by 16S/18S rDNA sequence analysis are referenced. 3Bacteria/yeasts that have not been identified by molecular methods. 4Absent from typhlocybine leafhoppers, phylloxerid aphids, and apiomorphine scale insects. by Western Michigan University on 01/17/12. For personal use only. (a) The microorganisms are intracellular and restricted to the cytoplasm of one Annu. Rev. Entomol. 1998.43:17-37. Downloaded from www.annualreviews.org insect cell type, called a mycetocyte1;

(b) the microorganisms are maternally inherited;

Mycetocyte symbioses have evolved independently between various microorganisms and insect groups (Table 1) (14, 28). It is widely accepted that

1The cells bearing these microorganisms are described as “bacteriocytes” by some authors. This review will use the term mycetocyte, following the established practice in the entomological literature.

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these symbioses have a nutritional basis (32, 53). Nearly all of the insects pos- sessing them live through the life cycle on nutritionally poor or unbalanced diets, e.g. phloem sap, vertebrate blood, and wood, and the microorganisms are believed to provide a supplementary source of essential nutrients, primarily essential amino acids, vitamins, and lipids. As well as being of general entomological interest, the mycetocyte symbioses are potentially of great applied value. Many of the insects that depend on their mycetocyte symbionts for normal growth and reproduction are pests of agricultural or medical importance. They include crop pests, e.g. aphids and other Homoptera, weevils, grain beetles; timber pests, e.g. anobiids; insects of public health importance, e.g. cockroaches; bed bugs Cimicidae; and insect vectors of pathogens of humans and domestic animals, e.g. tsetse fly Glossina. Ticks, which include important vectors of disease (71), also have symbioses that parallel the mycetocyte symbioses of insects (14). To date, the requirement of these various groups for microorganisms has not been exploited in pest management. This is primarily because these symbioses are widely considered to be intractable to experimental study and are, consequently, little studied. As this article considers, some previously intractable aspects of mycetocyte symbioses are becoming amenable to study as a result of, first, the increased sensitivity of many physiological and biochemical techniques and, second, the advent of molecular techniques. Virtually all recent research, however, has been conducted on the aphid-Buchnera symbiosis, and there is now strong evidence that the bacteria provide aphids with essential amino acids but do not contribute to the lipid (including sterol) nutrition of aphids. The principal purpose of this article is to review the literature on the nutritional interactions that underpin the aphid-Buchnera symbiosis. This is done in the context of the basic biology of the relationship between aphids and both Buchnera and other microorganisms, as is considered in the first section.

by Western Michigan University on 01/17/12. For personal use only. Undoubtedly, parallels exist between the aphid-Buchnera symbiosis and other microbial associations in insects, and this review should, therefore, be relevant Annu. Rev. Entomol. 1998.43:17-37. Downloaded from www.annualreviews.org to insect-microbial interactions in general, especially mycetocyte symbioses in insects other than aphids. Readers are referred to some excellent recent reviews on the allied topics of aphid biology and feeding (e.g. 67, 68, 94) and the molecular biology of Buchnera (10).

APHIDS AND THEIR MICROBIOTA Buchnera, The Primary Symbiont of Aphids The bacteria in the mycetocyte symbioses of aphids were traditionally called primary symbionts, as deﬁned by the morphological criteria of coccoid form, 2.5– 4 m in diameter, thin Gram-negative cell wall, and division by constriction

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(53). Baumann and colleagues used rDNA sequences to identify the bacteria as γ 3-Proteobacteria (73), allied to the Enterobacteriaceae (which includes Escherichia coli), and assigned them to the novel genus Buchnera (74), in recognition of Paul Buchner’s contribution to the study of insect-microbial symbioses. The genus Buchnera has been identiﬁed in all members of the Aphidoidea sensu Heie (49) examined, except the families Phylloxeridae and Adelgidae and some species of the Hormaphididae (14, 40, 73). [The phyllox- erids apparently lack microbial symbionts, whereas the adelgids have bacteria that are morphologically distinct from Buchnera and some Hormaphidids have yeasts (14).] Buchnera is present in all aphid morphs, apart from the dwarf males of some Lachnidae and Pemphigidae and the sterile female soldiers in some Pemphigidae (14, 41). In parthenogenetic morphs of the Aphididae, there are 107 bacteria per mg aphid weight, equivalent to 10% of the total aphid biomass∼ (8, 55). From studies on the molecular evolution of the aphid symbiosis, the evolutionary history of Buchnera can plausibly be reconstructed as follows:

1. The ancestral Buchnera was acquired once by aphids, subsequent to the divergence of the Phylloxeridae and Adelgidae, probably 160–280 million years ago (70).

2. The Buchnera genus has diversiﬁed in parallel with its aphid hosts, as indicated by the strictly congruent phylogenies of the aphids and bacteria (73, 83). This is in contrast to some microbial associates of insects that have occasionally been transmitted horizontally, forming multiple novel associations over evolutionary time (69).

3. Buchnera have been lost secondarily from some aphids of the family Hormaphididae. These aphids lack mycetocytes but have a substantial population of pyrenomycete yeasts (14, 40, 41). by Western Michigan University on 01/17/12. For personal use only. Buchnera may have evolved from a bacterium that inhabited the gut of ances- Annu. Rev. Entomol. 1998.43:17-37. Downloaded from www.annualreviews.org tral aphids (46). It is closely allied with the mycetocyte symbionts in tsetse ﬂies (3) and Camponotus ants (91), suggesting that this lineage of γ 3-Proteobacteria may be predisposed to enter into intimate associations. Characteristics of the aphid-Buchnera symbiosis LOCATION OF THE BACTERIA In all aphids, the Buchnera are located in mycetocytes in the hemocoel. The mycetocytes rarely, if ever, divide after the birth of an aphid, but they increase in size as the bacteria within them proliferate. For example, the vetch aphid Megoura viciae is born with approximately 80 mycetocytes, each of volume 104 m3, and the cells increase in size eightfold

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over larval development (35). The bacteria occupy 60% of the mycetocyte cytoplasm (100). ∼

MATERNAL INHERITANCE OF THE BACTERIA The Buchnera cells are transmitted maternally from one aphid generation to the next via the ovaries, a process called transovarial transmission, and this is the basis for the strict congruence of aphid and Buchnera gene phylogenies, described above. In oviparous aphids, the bacteria are endocytosed by each ovum, and they can be observed readily as a “symbiont ball” at the posterior pole of the mature egg (12, 14). In viviparous morphs, the bacteria pass into the blastocoel of young embryos and are subse- quently endocytosed by the embryo’s mycetocytes, as these cells differentiate (14, 50).

MUTUAL DEPENDENCE ON THE SYMBIOSIS Aphid requirement for the symbiosis has been demonstrated by studies of bacteria-free aphids, usually called aposymbiotic aphids. These aphids can be generated experimentally by antibiotic treatment, usually chlortetracycline or rifampicin administered either orally or by injection into the hemolymph (e.g. 44, 59, 82). At the appropriate dosage, these antibiotics have minimal direct deleterious effect on insect metabolism or behavior (25, 44, 103). Larval aposymbiotic aphids grow very slowly, de- veloping into small adults that produce few or no offspring (30, 33, 88). These effects of aposymbiosis probably reﬂect the nutritional contribution of Buch- nera to aphids, and, as considered below, the use of aposymbiotic aphids has been crucial to the elucidation of the nutritional interactions in the symbiosis. Buchnera are widely accepted also to require the association, although the evidence is, as yet, entirely negative. Buchnera have not been brought into culture (14, 51) and have not been reported in any habitat other than aphid mycetocytes. The Diversity of Microorganisms in Aphids

by Western Michigan University on 01/17/12. For personal use only. Buchnera are not the sole microorganisms borne by aphids. Some members of several aphid families, including the Aphididae, Lachnidae, Drepanosiphi- Annu. Rev. Entomol. 1998.43:17-37. Downloaded from www.annualreviews.org dae, and Pemphigidae have “secondary symbionts,” i.e. bacteria that are morphologically distinct from Buchnera in mycetocytes and/or sheath cells of the mycetome; a few lachnid species have a total of three morphological types of bacteria in the mycetomes (14). The secondary symbionts inhabiting the sheath cells of the pea aphid Acyrthosiphon pisum have been identified as Enterobac- teriaceae and are allied with E. coli (95), but there is no information on the nutritional significance, if any, of secondary symbionts to aphids. Microorganisms have been identified in the gut lumen of some aphids by microscopical, microbiological, and molecular techniques (43, 46, 47). Many of these microorganisms may be transient, i.e. ingested with food and lost,

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either with the passage of food through the gut or, for cells in the foregut and hindgut, at insect ecdysis. The absence of any gut microbiota in aphids feeding on sterile diets and clean plants (AE Douglas, unpublished observations) suggests that aphids may not generally possess a resident gut microbiota. With appropriate rearing techniques, the nutritional interactions between certain aphid species and their mycetocyte symbionts may be studied without the complication of a substantive gut microbiota that is nutritionally important to the insect, as occurs, for example, in leafhoppers (27). However, assessment of the microbiota in aphid clones that are studied intensively is important, as is illustrated by the recent demonstration that nearly half of 122 clones of A. pisum held in one laboratory bore rickettsiae in their hemolymph (17).

HOW NUTRITIONAL INTERACTIONS IN THE APHID-BUCHNERA SYMBIOSIS ARE INVESTIGATED Nutritional Physiology The nutritional interactions between aphids and Buchnera have been established primarily by nutritional physiology at the whole insect level. The aphid symbiosis is particularly amenable to this approach, for three technical reasons. 1. Aposymbiotic aphids can be generated routinely by antibiotic treatment. Most experimental designs involve direct comparisons between aposymbiotic aphids and aphids bearing the bacteria (called symbiotic aphids). Differences between the two groups can be attributed to bacterial function in the symbiotic aphids. However, some experiments require careful interpretation to distinguish between the direct effect of bacterial elimina- tion and secondary consequences of aposymbiosis, which arise from, for example, small size or depressed embryo production. 2. Preparations of isolated Buchnera can be obtained by techniques akin to the by Western Michigan University on 01/17/12. For personal use only. protocols for the isolation of organelles, such as mitochondria (48, 54, 57). The isolated Buchnera preparations are viable and metabolically active for Annu. Rev. Entomol. 1998.43:17-37. Downloaded from www.annualreviews.org several hours, and they can be used to establish basic metabolic capabilities, such as capacity for aerobic respiration and DNA and protein synthesis (e.g. 57, 101). These Buchnera preparations are not, however, metabolically equivalent to Buchnera in symbiosis, as is illustrated by the dramatic effect of isolation on the array of proteins synthesized by the bacteria (58). 3. Chemically deﬁned diets, comprising sucrose, amino acids, minerals, and vitamins, buffered with phosphate, are available for rearing several aphid species over one to multiple generations (20). These artiﬁcial diets are crucial to studies of the dietary requirements of aphids because it is impos- sible to manipulate the nutritional composition of the aphid’s natural diet

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of plant phloem sap. Interpretation of experiments with aphids reared over many generations on diets is, however, complicated by the characteristi- cally low reproductive output of some aphid species reared this way and by the recent evidence that the symbiosis is depressed in pea aphids A. pisum maintained on diets for three to four generations (56).

Chemically defined diets have been used extensively to explore the nutritional requirements of aphids (20). Bacterial provisioning of a nutrient is indicated, specifically, by the dietary requirement of aposymbiotic but not symbiotic aphids for that nutrient. This line of evidence should be complemented by direct study of the metabolic capabilities of the two conditions with the predic- tion that a nutrient required only by aposymbiotic aphids is synthesized only by symbiotic aphids. Valuable confirmatory information can be obtained from quantitation of the activity of key enzymes and/or titers of particular metabolites in aphids, which would be anticipated to reflect the biosynthetic capabilities and nutritional requirements of symbiotic and aposymbiotic aphids. As considered later in this article, these several approaches of nutritional physiology have been used widely to explore essential amino acid and lipid nutrition of the aphid-Buchnera symbiosis. Molecular Biology In the context of the symbiosis, molecular techniques have, to date, been applied primarily to the study of Buchnera and not the aphid partner. Analysis of the Buchnera gene content offers an excellent test for the genetic capability of Buchnera to meet the nutritional functions identified by nutritional physiology. The sequence of these genes and flanking regions can additionally provide valuable information on the regulation of gene expression (e.g. presence/absence of transcription attenuation sites) or protein function (e.g. the sequence re- sponsible for allosteric inhibition of an enzyme) (10). Comparisons of genome

by Western Michigan University on 01/17/12. For personal use only. organization and gene sequence between Buchnera in different aphid species can also be used to explore the evolutionary changes in the biosynthetic capa- Annu. Rev. Entomol. 1998.43:17-37. Downloaded from www.annualreviews.org bilities of Buchnera (e.g. 63, 64). Approaches Not Currently Available As a broad generalization (32), symbioses ideally suited to experimental study have the following two characteristics: (a) The participating organisms can be maintained and studied in isolation; (b) the symbiosis can be resynthesized from its constituent organisms. The characteristics of “experimental” symbioses between partners that do not naturally form associations or involving an organism bearing a speciﬁc mutation (e.g. an auxotrophic mutant) are particularly informative. These procedures have been central to the elucidation of the interactions between leguminous plants and nitrogen-ﬁxing rhizobia (97) and

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to the recent developments in our understanding of the symbiosis between the squid Euprymna scolopes and luminescent bacteria (85). The main reason why the aphid-Buchnera association and mycetocyte symbioses in general have traditionally been considered as intractable systems (see “Perspectives and Overview”) is that these procedures are not fully available. Only very limited information can be gleaned from isolated Buchnera because they can be maintained for only a few hours. Also, the aphid symbiosis cannot be synthesized from isolated Buchnera and aposymbiotic aphids. Buchnera injected into the hemocoel of aposymbiotic aphids are lysed (AE Douglas, unpublished observations), whereas the injection of intact mycetocytes into aposymbionts has not, to my knowledge, been attempted. Development of the methods to synthesize, or otherwise manipulate, the aphid-Buchnera association and other mycetocyte symbioses would transform the research on these systems.

NUTRITIONAL INTERACTIONS IN THE SYMBIOSIS Essential Amino Acid Provisioning A bacterial supply of essential amino acids would be of nutritional advantage to any insects feeding on the phloem sap of plants, which has low or undetectable concentrations of many essential amino acids (31). Direct experimental support for microbial provisioning of essential amino acids is, however, available only for the aphid-Buchnera symbiosis, and such support comes from studies of aphid dietary requirements and metabolic capabilities and the molecular biology of Buchnera.

DIETARY STUDIES Various studies on the dietary amino acid requirements of aphids have shown that the essential amino acid content of the diet has a greater impact on the performance of aposymbiotic aphids than on symbiotic aphids.

by Western Michigan University on 01/17/12. For personal use only. In an early and important set of experiments, aposymbiotic Myzus persicae was found to have a dietary requirement for every essential amino acid, as indicated Annu. Rev. Entomol. 1998.43:17-37. Downloaded from www.annualreviews.org by poor growth and survival on all diets from which one essential amino acid was omitted (66), whereas the growth of symbiotic M. persicae was unaffected by the individual deletion of 7 of the 10 essential amino acids (21). Other studies have used diet formulations that bear some resemblance to phloem sap. In particular, the performance of aposymbiotic A. pisum has been explored on diets of uniform nitrogen content that contain all protein amino acids but with a ratio of essential:nonessential amino acids varying from 1:1 to 1:5. The larval growth rate of A. pisum was more than twofold greater on the 1:1 diet than on the 1:5 diet, whereas that of symbiotic aphids did not vary signiﬁcantly with amino acid compositon (80).

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The most plausible explanation for the dependence of aposymbiotic, but not symbiotic, aphids on the dietary supply of all essential amino acids is that Buchnera provide symbiotic aphids with these nutrients. However, on no diet (or plant) is the performance of aposymbiotic aphids comparable to that of symbiotic aphids, which suggests that the dietary supply of essential amino acids cannot replace Buchnera. Perhaps Buchnera have functions required by the aphid in addition to essential amino acid provisioning (see below). Al- ternatively, the aphids may not be able to assimilate dietary essential amino acids across the gut wall as rapidly as the bacteria-derived nutrients are made available to the aphid tissues (81).

METABOLIC STUDIES Most metabolic studies on amino acid synthesis in aphids have used 14C- or 15N-labeled nonessential amino acids as precursors. One of the most widely metabolized amino acids is glutamate, which is a precursor of a number of essential amino acids, including isoleucine, leucine, threonine, lysine, and valine, in symbiotic A. pisum (39, 89, 105). Unfortunately, several studies have omitted aposymbiotic aphids as controls, and therefore these metabolic capabilities cannot formally be attributed to the bacteria. As important, most studies have merely demonstrated essential amino acid synthesis in the symbiosis, without considering whether the nutrients are made available to the aphid tissues. In the context of the symbiosis, not only are the biosynthetic capabilities of symbiotic bacteria of interest, but so is the access of the insect to the products of these capabilities. To my knowledge, there has been just one deﬁnitive demonstration of nutrient provisioning in the aphid-Buchnera symbiosis. This concerns the sulphur- containing essential amino acid, methionine, the synthesis of which can be quantiﬁed by the incorporation of 35S from dietary inorganic sulphate. Isolated preparations of Buchnera can utilize sulphate as a sulphur source for methionine synthesis (29). Symbiotic, but not aposymbiotic, M. persicae also incorporated 35 35

by Western Michigan University on 01/17/12. For personal use only. S into methionine (25), and up to 20% of S-labeled methionine was recovered from the anterior Buchnera-free tissues of the symbiotic aphids, which in- Annu. Rev. Entomol. 1998.43:17-37. Downloaded from www.annualreviews.org dicates that methionine synthesized by Buchnera is made available to the aphid tissues. Three complementary experimental approaches provide strong evidence for bacterial provisioning of the essential amino acid tryptophan. The ﬁrst is that aposymbiotic, but not symbiotic, aphids of A. pisum and M. persicae have a dietary requirement for this amino acid (21, 36). Second, isolated Buchnera preparations and symbiotic, but not aposymbiotic, A. pisum have apprecia- ble activity of tryptophan synthetase (36). Finally, symbiotic aphids reared on tryptophan-free diets continue to produce tryptophan in their honeydew for many days (36).

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MOLECULAR STUDIES Molecular studies of the gene content of Buchnera have confirmed that this bacterium has the genetic capability to synthesize essential amino acids. There is no published report of a failure to identify any gene coding for enzymes in amino acid synthesis, and a total of 13 genes directly involved in essential amino acid biosynthesis have been described: aroA, aroH, and aroE, in the common pathway of aromatic amino acid synthesis (61, 84); leuABCD, which encodes enzymes specific to leucine synthesis (11); and trpEGDC(F)BA, the genes for tryptophan synthesis (62, 72). In the Aphididae, the genes leuABCD and trpEG are apparently absent from the Buchnera chromosome and located on plasmids, known as the leucine plasmid and tryptophan plasmid, respectively (11, 62). The genetic organization of these plasmids is described in References 11, 62, and 64. In the context of the nutritional interactions in the symbiosis, the key issue is that the plasmid-borne genes are amplified, relative to the chromosomal genes of Buchnera (11, 62). This genetic organization can plausibly be interpreted in terms of increased bacterial capacity to synthesize leucine and tryptophan (these issues are discussed further in Reference 34). Both leuABCD and trpEG are chromosomal in Buchnera from certain aphid species of the family Pemphigidae (63; R van Ham, A Moya & A Latorre, unpublished observations), and comparisons between the trpEG sequence in Buchnera from Aphididae and Pemphigidae indicate that the plasmid-borne genes are derived from Buchnera chromosomal genes and have not been acquired horizontally from a different bacterium (83). The leucine and tryptophan plasmids in Buchnera from the Aphididae represent a remarkable confirmation of the significance of essential amino acid synthesis to the association. Genes encoding enzymes in the synthesis of other essential amino acids are not on small (<35 kb) plasmids in Buchnera of the Aphididae, but the possibility that they are amplified, either on the Buchnera chromosome or on large plasmids, cannot be excluded. by Western Michigan University on 01/17/12. For personal use only. Nitrogen Recycling and Upgrading

Annu. Rev. Entomol. 1998.43:17-37. Downloaded from www.annualreviews.org Nitrogen recycling in an animal-microbial symbiosis refers to the microbial metabolism of animal waste nitrogen to compounds, such as essential amino acids, that are of nutritional value to the animal (32). Microbial recycling of nitrogen is potentially of great nutritional value to an animal because it increases the efﬁciency with which the animal can utilize dietary nitrogen, enabling it to survive and grow on low nitrogen input. However, nitrogen recycling has not been demonstrated unequivocally in any association, although there is strong evidence that the mycetocyte symbionts in cockroaches (19), yeasts in planthoppers (90), and gut bacteria in termites (78) may all consume nitrogenous waste products of their insect hosts.

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Buchnera exhibit the metabolic characteristics expected of a symbiotic microorganism that recycles nitrogen. As considered above, essential amino acids synthesized by Buchnera are translocated to the aphid, and the nitrogen in these essential amino acids must be derived ultimately from the aphid. Further, isolated Buchnera can take up ammonia, the chief nitrogenous waste product of aphids (102), and Buchnera cells in symbiosis presumably have access to aphid-derived ammonia, a compound that diffuses freely across biological membranes (except from compartments of low pH). To date, there has been no direct investigation of nitrogen recycling in the aphid-Buchnera symbiosis, i.e. an analysis of the incorporation of nitrogen, derived from the aphid waste ammonia, into essential amino acids. Much of the recent research has focused on the nutritional physiology of aposymbiotic A. pisum. Relative to symbiotic aphids, aposymbiotic A. pisum have elevated concentrations of ammonia and glutamine in both their tissues and honeydew (79, 87, 102, 104), and they also have elevated activity of the enzyme glutamine synthetase (104). Glutamine synthetase in animals contributes to the detoxi- ﬁcation of ammonia by catalyzing the synthesis of glutamine from ammonia and glutamate. The link between glutamine synthetase activity and ammonia load in aphids is conﬁrmed by the elevated glutamine synthetase activity in symbiotic aphids maintained on ammonia-supplemented diets (104). These results are compatible with the hypothesis that Buchnera assimilate aphid-derived ammonia, i.e. they act as a sink for aphid ammonia. Recent dietary experiments indicate that, if nitrogen recycling of aphid ammonia occurs, it is not quantitatively important to aphid growth. For symbiotic A. pisum reared on a low-nitrogen diet, a dietary supplement of nonessential amino acids, but not ammonia, promoted aphid growth (104). These results are compatible with the conclusion, from studies on nonessential amino acid uptake and metabolism by isolated Buchnera preparations, that glutamate may be the principal nitrogenous compound utilized by Buchnera (89, 101).

by Western Michigan University on 01/17/12. For personal use only. Nonessential amino acids are the principal nitrogenous compounds in the aphid diet of phloem sap (31). Although much of the detail of aphid and

Annu. Rev. Entomol. 1998.43:17-37. Downloaded from www.annualreviews.org bacterial metabolism of these compounds remains to be resolved, the broad outline of nitrogen relations of the symbiosis in A. pisum is becoming clear. Buchnera upgrade dietary nonessential amino acids to essential amino acids, thereby contributing to the aphid’s capacity to utilize phloem sap, and bacterial recycling of aphid nitrogen is not quantitatively important. Lipids (Including Sterols): Nutritional Independence of Aphids from Their Symbiotic Bacteria The possibility that symbiotic bacteria may contribute to the lipid and sterol nutrition of aphids was raised by the early nutritional studies that revealed

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that aphids, unlike many insects, can be maintained over many generations on chemically deﬁned diets lacking lipids (reviewed in Reference 20). Much of the research has concerned the source of two fatty acids: linoleic acid (9,12-octadecadienoic acid), a major component of aphid phospholipids, and sorbic acid (2,4-hexadienoic acid), one of the short chain fatty acids that contributes to aphid triacylglycerols. The symbiotic bacteria were implicated in their synthesis because vertebrate animals cannot synthesize linoleic acid and sorbic acid is unknown in triacylglycerols of any animals apart from aphids (13). It is now appreciated that many, but not all, insects can synthesize linoleic acid (23). With respect to aphids, the crucial experiment was the demonstration that both symbiotic and aposymbiotic A. pisum incorporate radioactivity from 14C-acetate into linoleic acid (24). The principal evidence that the aphid, and not its bacteria, synthesizes sorbic acid is that the fatty acid composition of triacylglycerols in both A. pisum and Macrosiphum euphorbiae is unaffected by rifampicin treatment (82, 99). Sorbic acid synthesis from 14C-acetate by M. euphorbiae has been demonstrated (98), but the critical experiments on the biosynthetic capabilities of aposymbiotic aphids have not been explored. Bacterial contribution to the sterol requirements of aphids is inherently im- probable because sterol synthesis is a characteristic of eukaryotes and is virtually unknown in bacteria. Consistent with this generality, the aphid Schizaphis graminum maintained on diets containing [2-14C] melavonate exhibited no de- tectable incorporation of radioactivity into sterols or metabolic intermediates in sterol synthesis (16). It is very likely that plant-reared aphids derive their sterol requirement from phytosterols in the phloem sap. The source of sterols in diet-reared aphids remains to be resolved. Campbell & Nes (16) suggested that the diets may be routinely contaminated with fungi, which could be a source of sterols, but no such contaminants are revealed by mycological sterility test- ing of diets. Fungal contamination cannot account for the observed promotion of performance of aposymbiotic, but not symbiotic, M. persicae by a dietary

by Western Michigan University on 01/17/12. For personal use only. supply of sterols (26). It appears that the bacteria in aphids may have a sparing effect on the insect’s requirement for sterols, but the underlying mechanisms Annu. Rev. Entomol. 1998.43:17-37. Downloaded from www.annualreviews.org are, at present, unknown. Vitamin Provisioning The contribution of symbiotic microorganisms to the vitamin requirements of insects can most appropriately be explored by analysis of the insect’s dietary requirements. Early nutritional studies on aphids established that M. persicae requires all B vitamins and vitamin C (ascorbic acid) (22) and that Neomyzus circumﬂexus requires all except riboﬂavin (38). Aphid requirement for some of the vitamins only became apparent after several insect generations were reared on the vitamin-free diets.

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These data provide persuasive evidence that the symbiotic bacteria do not generally supply the vitamin requirements of aphids (20).

VARIATION AND VERSATILITY IN NUTRITIONAL INTERACTIONS The firm evidence that Buchnera contribute essential amino acids to aphids raises the question whether the rates at which amino acids are made available to the insect and the profile of amino acids provided may vary, either between aphid taxa or in a single aphid, perhaps in accordance with aphid nutritional demand (as determined by the mismatch between the dietary supply of essential amino acids and requirements for aphid growth). These issues are central to the several proposals in the literature that symbiotic bacteria may influence the capacity of phytophagous insects to utilize different plants and, thereby, act as one determinant of their host plant range (e.g. 7, 15, 60). Only very limited data are available, and they are reviewed here. Variation Between Aphid Taxa It would be exceptional if amino acid provisioning by Buchnera was absolutely uniform across the Aphidoidea. At the very least, the aphids whose Buchnera have leu and trp genes amplified on plasmids probably have greater access to bacteria-derived leucine and tryptophan, respectively, than aphid species whose Buchnera lack these plasmids (discussed in Reference 63). The host plant affiliation of aphids of the family Aphididae can apparently evolve very rapidly, such that closely related aphid taxa have distinct or over- lapping host plant ranges (45). These evolutionary shifts could involve changes in the metabolic capabilities of Buchnera, to compensate for differences in the nutritional composition of phloem sap in different plant species. Just one system has been studied to date, the Aphis fabae species complex. An analysis

by Western Michigan University on 01/17/12. For personal use only. of the performance of aphids deprived of their bacteria suggests that, in this system, insect and not bacterial function limits the capacity of A. fabae fabae Annu. Rev. Entomol. 1998.43:17-37. Downloaded from www.annualreviews.org to utilize plant species speciﬁc to the subspecies A. fabae mordwilkoi (2). The symbioses in other aphid species complexes warrant investigation. One set of experiments has been conducted on variation in the symbiosis between clones of a single aphid species. Two clones of A. pisum are independent of a dietary supply of different essential amino acids (93): clone C of isoleucine, phenylalanine, and valine and clone J of leucine, lysine, and tryptophan. As the authors comment (93), this can most plausibly be interpreted as differences between the proﬁles of amino acids provided by the Buchnera in the different aphid clones, but no direct analysis of bacterial amino acid provisioning has been done on these aphids.

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The Metabolic Versatility of Buchnera Metabolic versatility in symbiotic microorganisms can be deﬁned as the ex- tent to which nutrients provided by the microorganisms to their (insect) host may vary, in accordance with the host nutritional demand for those nutrients. Versatility in essential amino acid provisioning by Buchnera may arise by (a) variation in the rate of supply of amino acids, through changes in the bacterial biomass and/or rate of provisioning per bacterial cell, and (b) variation in the composition of amino acids provided, through shifts in the relative rates of synthesis and release of different amino acids. The variation in biomass of Buchnera with aphid developmental age and morph is relatively well studied (8, 35, 52, 55, 101), but the impact of host plant or dietary nutrients on the bacterial biomass has rarely been considered. One recent study concerned A. fabae reared on different plant species, on which bacterial promotion of aphid growth is known to vary (2). The volume of the symbiosis, relative to total aphid weight, was remarkably uniform. Even on plant species, on which the larval relative growth rate of symbiotic and aposymbiotic A. fabae were not signiﬁcantly different, the symbiosis was maintained (1). These data suggest that the bacterial population in A. fabae is not regulated according to its immediate nutritional value to the insect. The versatility of Buchnera in the composition of amino acids provided to the aphid depends critically on the mode of release of these nutrients. At present, virtually nothing is known, although the nutrients are generally accepted to be released from living cells of the bacteria. The amino acids may be released as free amino acids, small peptides, or proteins, any of which would be translocated across the cell membrane of the bacteria and the insect membrane (symbiosome membrane) that separates each Buchnera cell from the surrounding mycetocyte cytoplasm. There is evidence for the release of one protein, the chaperonin GroEL, which attains high concentrations in Buchnera cells (9) and is also recovered from the hemolymph of M. persicae (96). The rapid decline in the

by Western Michigan University on 01/17/12. For personal use only. GroEL titers in hemolymph of antibiotic-treated aphids is consistent with the possibility that this protein is released from metabolically active Buchnera cells Annu. Rev. Entomol. 1998.43:17-37. Downloaded from www.annualreviews.org and degraded rapidly by the aphid. There can be no versatility in the proﬁle of amino acids provided via GroEL, the amino acid composition of which is determined genetically. Metabolic versatility in Buchnera could potentially be obtained if free amino acids were translocated from the bacteria to the aphid, via an array of membrane transporters, speciﬁc to individual amino acids, mediating the export from bacteria and uptake across the insect symbiosome membrane. The density and activity of these putative transporters could be regulated very precisely, in accordance with aphid demand. At present, however, there is no evidence for the transport of free amino acids from Buchnera to aphid tissues (discussed in Reference 34).

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One published data set exists that is consistent with the possibility that Buchnera are very versatile in their nutritional interactions with the insect. When a single clone of A. pisum was reared on chemically defined diets of different amino acid composition, the free amino acid titers of symbiotic aphids were very uniform, whereas those of aposymbiotic aphids bore distinct similarities to the amino acid composition of the diets on which they were feeding (65). These data were interpreted as evidence for a breakdown in regulation of the free amino acid pools in aposymbiotic aphids. The implication is that the bacteria contribute directly to amino acid homeostasis in symbiotic aphids, i.e. control over the profile of amino acids provided by the bacteria is so fine-tuned that it maintains the optimal amino acid titers of the aphid body fluids. A study of close similar design, but conducted on A. fabae reared on plants with phloem sap of very diverse amino acid compositions, obtained tightly regulated amino acid titers in both symbiotic and aposymbiotic aphids (1). Bacterial contribution to the amino acid homeostasis of aphids does not appear, therefore, to be general. The sole indication that the metabolic versatility of Buchnera may be limited comes from the study of tryptophan production in symbiotic A. pisum (36). As described above, the bacteria in aphids reared on tryptophan-free diets continue to produce honeydew containing tryptophan. A plausible interpretation of these data is that the bacteria provide tryptophan at rates greater than aphid demand and the excess is excreted via honeydew, which suggests that the aphid may have only limited control over the metabolic activities of its bacteria. In summary, the data on the versatility of essential amino acid provisioning by Buchnera, both with dietary supply of amino acids and between aphid taxa, are fragmentary and, in part, contradictory. There is a real need for direct metabolic study of amino acid translocation from Buchnera in different aphid taxa and in aphids reared under different nutritional conditions.

NON-NUTRITIONAL FUNCTIONS OF BUCHNERA by Western Michigan University on 01/17/12. For personal use only. IN APHIDS Annu. Rev. Entomol. 1998.43:17-37. Downloaded from www.annualreviews.org The nutritional functions of symbiotic microorganisms in aphids and other insects arise directly from the fact that insects, like other animals, are metabolically impoverished, lacking the capacity to synthesize essential amino acids, vitamins, etc (32). The symbiotic microorganisms have, however, been proposed to contribute to the biology of their insect hosts in ways that do not relate directly to a match between the metabolic capabilities of the bacteria and the basic nutritional requirements of the insect. For example, Buchnera have been implicated in resistance of aphids to insecticides (97a) and aphid stylet pene- tration of plant tissues (37), and the microorganisms in other Homoptera have been proposed to be required for the differentiation of the insect abdomen (92).

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All these proposed functions were developed without proper use of controls, in particular aposymbiotic insects obtained with appropriate concentrations of antibiotic, and all have been discredited by detailed experimental analysis: in- secticide resistance (5), aphid feeding (103), and embryogenesis (86). One non-nutritional interaction that has been investigated with adequate controls is Buchnera-mediated promotion of viral transmission by aphids. The viruses are circulative luteoviruses, notably the potato leafroll virus transmitted by M. persicae, whose inoculation from the aphid into a plant depends on the transfer of the virus particles from the aphid gut to the salivary glands, via the hemolymph. The viral particles are stabilized by the Buchnera protein GroEL, released from the bacteria (see above), and viral transmission is depressed in aphids by treatment with chlortetracycline to arrest bacterial protein synthesis. GroEL of E. coli can also stabilize the virus particles (96). This suggests that the interaction between GroEL of Buchnera and luteoviruses is not an example of coevolution between the symbiosis and virus but a fortuitous molecular compatibility, reﬂecting the principal function of GroEL in the stabilization of particular conformations of proteins. The discovery of other non-nutritional processes mediated by the symbiotic bacteria in aphids may be anticipated.

CONCLUDING REMARKS The principal conclusion from recent research on nutritional interactions in the aphid-Buchnera symbiosis is that the bacteria provide aphids with essential amino acids. This interaction contributes to the capacity of aphids to utilize phloem sap, a diet that is deﬁcient in essential amino acids. All Homoptera feeding on phloem sap have symbiotic microorganisms, and those groups that have secondarily switched to feeding on intact plant cells have lost the symbiosis (Table 1). The implication is that the microbial symbiosis is causally linked

by Western Michigan University on 01/17/12. For personal use only. with phloem feeding, and speciﬁcally that essential amino acid provisioning may underpin all these associations, even though the symbioses in the various Annu. Rev. Entomol. 1998.43:17-37. Downloaded from www.annualreviews.org Homoptera may differ in the identity of the microbial partners, anatomical location and structural organization of the symbiosis, developmental origin of mycetocytes, and mode of transovarial transmission (14, 28). Buchnera are clearly not exceptional in their capacity to participate in nutritional interactions with insect cells. An important research area for the future is the mechanisms underlying amino acid provisioning, in particular the identity of the compounds transferred to aphids and the regulation of translocation. In other symbiotic systems, notably symbiotic algae in animals (32), common mechanisms of nutrient release have been established that are independent of the identity of the organisms, and parallels between nutritional interactions in the various mycetocyte symbioses

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may also occur. The mechanisms of nutrient release from Buchnera may, therefore, be immediately relevant to other mycetocyte symbioses. These considerations should not, however, be taken as an indication of uni- formity among mycetocyte symbioses in insects. Even within a given radiation of insect-bacteria symbioses, e.g. aphid-Buchnera, variation and diversity are to be anticipated. In particular, the proﬁle of the amino acids provided and the metabolic versatility of Buchnera may vary between aphid higher taxa or between aphid species that have narrow and broad host plant ranges. A coherent array of techniques is now in place to explore these issues, and the mycetocyte symbioses are becoming increasingly amenable to study through a combination of nutritional physiology and molecular biology.

ACKNOWLEDGMENTS I thank Dr. H van den Heuvel, Dr. R van Ham, Professor A Moya, and Dr. A Latorre for their constructive comments on a draft of this review.

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