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Copyright c 1998 by Annual Reviews Inc. All rights reserved
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: aed2@york.ac.uk
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 obligate 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
aphid transmission of circulative viruses. It is argued that strong parallels may
exist between the nutritional interactions (including the underlying mechanisms)
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
Microorganism2
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Insect
Incidence
Blattaria (cockroaches)
Flavobacteria (6)
Universal
Heteroptera
Cimicidae
Lygaeidae
Bacteria3
Universal
Irregular
Homoptera
γ -Proteobacteria in aphids (73)
and whitefly (18); β-Proteobacteria
in mealbugs (75); pyrenomycete
yeasts in delphacid planthoppers
(77) and hormaphidine aphids
(42); bacteria in other groups3
Nearly universal4
Anoplura
Bacteria3
Universal
Mallophaga
Bacteria3
Irregular
Diptera
Glossinidae
Diptera Pupipera
γ 3-Proteobacteria
Bacteria3
Universal
Universal
Yeasts “close to Discomycetes”
in anobiids (76); yeasts in
Cerambycidae3; otherwise
bacteria3
Reported in 10 families,
including Bostrychidae,
Lyctidae (universal);
Anobiidae, Chrysomelidae,
Cuculionidae
(widespread)
γ 3-Proteobacteria (91)
Bacteria3
Universal
Irregular
Coleoptera
Formicidae (ants)
Camponoti
Formicini
1Based
on Buchner (14) and references in review by Douglas (28).
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.
2Microorganisms
(a) The microorganisms are intracellular and restricted to the cytoplasm of one
insect cell type, called a mycetocyte1 ;
(b) the microorganisms are maternally inherited;
(c) the association is required by both the insect and microbial partners.
Mycetocyte symbioses have evolved independently between various microorganisms and insect groups (Table 1) (14, 28). It is widely accepted that
1 The 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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APHID SYMBIONTS AND NUTRITION
19
these symbioses have a nutritional basis (32, 53). Nearly all of the insects possessing 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.
Undoubtedly, parallels exist between the aphid-Buchnera symbiosis and other
microbial associations in insects, and this review should, therefore, be relevant
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 µm 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 phylloxerids 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).
Buchnera may have evolved from a bacterium that inhabited the gut of ancestral 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 µm3, 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 subsequently 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, developing into small adults that produce few or no offspring (30, 33, 88). These
effects of aposymbiosis probably reflect the nutritional contribution of Buchnera 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
Buchnera are not the sole microorganisms borne by aphids. Some members
of several aphid families, including the Aphididae, Lachnidae, Drepanosiphidae, 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 Enterobacteriaceae 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 elimination 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
protocols for the isolation of organelles, such as mitochondria (48, 54, 57).
The isolated Buchnera preparations are viable and metabolically active for
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 impossible 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 characteristically 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 prediction 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 responsible for allosteric inhibition of an enzyme) (10). Comparisons of genome
organization and gene sequence between Buchnera in different aphid species
can also be used to explore the evolutionary changes in the biosynthetic capabilities 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.
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
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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APHID SYMBIONTS AND NUTRITION
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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). Alternatively, 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 sulphurcontaining 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
S into methionine (25), and up to 20% of 35S-labeled methionine was recovered from the anterior Buchnera-free tissues of the symbiotic aphids, which indicates 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 appreciable 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.
Nitrogen Recycling and Upgrading
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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APHID SYMBIONTS AND NUTRITION
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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 detoxification 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).
Nonessential amino acids are the principal nitrogenous compounds in the
aphid diet of phloem sap (31). Although much of the detail of aphid and
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
14
C-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 improbable 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 detectable 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 testing of diets. Fungal contamination cannot account for the observed promotion
of performance of aposymbiotic, but not symbiotic, M. persicae by a dietary
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
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).
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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 overlapping 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
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
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 extent 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
GroEL titers in hemolymph of antibiotic-treated aphids is consistent with the
possibility that this protein is released from metabolically active Buchnera cells
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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APHID SYMBIONTS AND NUTRITION
31
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
IN APHIDS
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 penetration 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: insecticide 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
with phloem feeding, and specifically that essential amino acid provisioning
may underpin all these associations, even though the symbioses in the various
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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APHID SYMBIONTS AND NUTRITION
33
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 uniformity 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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