nutritional interactions in insect-microbial symbioses

P1: ARS/ary P2: NBL/plb QC: NBL

October 27, 1997 16:35 Annual Reviews AR048-02

Annu. Rev. Entomol. 1998. 43:17–37Copyright c© 1998 by Annual Reviews Inc. All rights reserved

NUTRITIONAL INTERACTIONS ININSECT-MICROBIAL SYMBIOSES:Aphids and Their Symbiotic BacteriaBuchnera

A. E. DouglasDepartment of Biology, University of York, PO Box 373, York, YO1 5YW, UnitedKingdom; e-mail: [email protected]

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

ABSTRACT

Most aphids possess intracellular bacteria of the genusBuchnera. The bacte-ria are transmitted vertically via the aphid ovary, and the association is obli-gate for both partners: Bacteria-free aphids grow poorly and produce few orno offspring, andBuchneraare both unknown apart from aphids and apparentlyunculturable. The symbiosis has a nutritional basis. Specifically, bacterial pro-visioning 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 sterolnutrition of aphids. Buchnerahave been implicated in various non-nutritionalfunctions. Of these, just one has strong experimental support: promotion ofaphid transmission of circulative viruses. It is argued that strong parallels mayexist between the nutritional interactions (including the underlying mechanisms)in the aphid-Buchneraassociation and other insect symbioses with intracellularmicroorganisms.

PERSPECTIVES AND OVERVIEW

The main topic of this review is the symbiosis between aphids and bacteria ofthe genusBuchnera.This association is a “mycetocyte symbiosis,” as definedby three characteristics:

170066-4170/98/0101-0017$08.00

Ann

u. R

ev. E

ntom

ol. 1

998.

43:1

7-37

. Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

Wes

tern

Mic

higa

n U

nive

rsity

on

01/1

7/12

. For

per

sona

l use

onl

y.



18 DOUGLAS

Table 1 Mycetocyte symbioses in insects1

Insect Microorganism2 Incidence

Blattaria (cockroaches) Flavobacteria (6) Universal

HeteropteraCimicidae Bacteria3 UniversalLygaeidae Irregular

Homoptera γ -Proteobacteria in aphids (73) Nearly universal4

and whitefly (18);β-Proteobacteriain mealbugs (75); pyrenomyceteyeasts in delphacid planthoppers(77) and hormaphidine aphids(42); bacteria in other groups3

Anoplura Bacteria3 Universal

Mallophaga Bacteria3 Irregular

DipteraGlossinidae γ3-Proteobacteria UniversalDiptera 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 γ3-Proteobacteria (91) UniversalFormicini 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.

(a) The microorganisms are intracellular and restricted to the cytoplasm of oneinsect 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 mi-croorganisms 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 entomologicalliterature.

Ann

u. R

ev. E

ntom

ol. 1

998.

43:1

7-37

. Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

Wes

tern

Mic

higa

n U

nive

rsity

on

01/1

7/12

. For

per

sona

l use

onl

y.



APHID SYMBIONTS AND NUTRITION 19

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 unbalanceddiets, e.g. phloem sap, vertebrate blood, and wood, and the microorganismsare believed to provide a supplementary source of essential nutrients, primarilyessential amino acids, vitamins, and lipids.

As well as being of general entomological interest, the mycetocyte sym-bioses are potentially of great applied value. Many of the insects that dependon their mycetocyte symbionts for normal growth and reproduction are pests ofagricultural or medical importance. They include crop pests, e.g. aphids andother Homoptera, weevils, grain beetles; timber pests, e.g. anobiids; insects ofpublic health importance, e.g. cockroaches; bed bugs Cimicidae; and insectvectors of pathogens of humans and domestic animals, e.g. tsetse flyGlossina.Ticks, which include important vectors of disease (71), also have symbiosesthat parallel the mycetocyte symbioses of insects (14). To date, the require-ment of these various groups for microorganisms has not been exploited inpest management. This is primarily because these symbioses are widely con-sidered to be intractable to experimental study and are, consequently, littlestudied.

As this article considers, some previously intractable aspects of mycetocytesymbioses are becoming amenable to study as a result of, first, the increasedsensitivity of many physiological and biochemical techniques and, second, theadvent of molecular techniques. Virtually all recent research, however, has beenconducted on the aphid-Buchnerasymbiosis, and there is now strong evidencethat the bacteria provide aphids with essential amino acids but do not contributeto the lipid (including sterol) nutrition of aphids.

The principal purpose of this article is to review the literature on the nutri-tional interactions that underpin the aphid-Buchnerasymbiosis. This is donein the context of the basic biology of the relationship between aphids andbothBuchneraand other microorganisms, as is considered in the first section.Undoubtedly, parallels exist between the aphid-Buchnerasymbiosis and othermicrobial associations in insects, and this review should, therefore, be relevantto insect-microbial interactions in general, especially mycetocyte symbioses ininsects other than aphids. Readers are referred to some excellent recent re-views on the allied topics of aphid biology and feeding (e.g. 67, 68, 94) and themolecular biology ofBuchnera(10).

APHIDS AND THEIR MICROBIOTA

Buchnera,The Primary Symbiont of AphidsThe bacteria in the mycetocyte symbioses of aphids were traditionally called pri-mary 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

Ann

u. R

ev. E

ntom

ol. 1

998.

43:1

7-37

. Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

Wes

tern

Mic

higa

n U

nive

rsity

on

01/1

7/12

. For

per

sona

l use

onl

y.



20 DOUGLAS

(53). Baumann and colleagues used rDNA sequences to identify the bacte-ria asγ3-Proteobacteria (73), allied to the Enterobacteriaceae (which includesEscherichia coli), and assigned them to the novel genusBuchnera(74), inrecognition of Paul Buchner’s contribution to the study of insect-microbialsymbioses. The genusBuchnerahas been identified in all members of theAphidoidea sensu Heie (49) examined, except the families Phylloxeridae andAdelgidae and some species of the Hormaphididae (14, 40, 73). [The phyllox-erids apparently lack microbial symbionts, whereas the adelgids have bacteriathat are morphologically distinct fromBuchneraand some Hormaphidids haveyeasts (14).]Buchnerais present in all aphid morphs, apart from the dwarfmales of some Lachnidae and Pemphigidae and the sterile female soldiers insome Pemphigidae (14, 41). In parthenogenetic morphs of the Aphididae, thereare∼107 bacteria per mg aphid weight, equivalent to 10% of the total aphidbiomass (8, 55).

From studies on the molecular evolution of the aphid symbiosis, the evolu-tionary history ofBuchneracan plausibly be reconstructed as follows:

1. The ancestralBuchnerawas acquired once by aphids, subsequent to thedivergence of the Phylloxeridae and Adelgidae, probably 160–280 millionyears ago (70).

2. TheBuchneragenus has diversified in parallel with its aphid hosts, asindicated by the strictly congruent phylogenies of the aphids and bacteria(73, 83). This is in contrast to some microbial associates of insects thathave occasionally been transmitted horizontally, forming multiple novelassociations over evolutionary time (69).

3. Buchnerahave been lost secondarily from some aphids of the familyHormaphididae. These aphids lack mycetocytes but have a substantialpopulation of pyrenomycete yeasts (14, 40, 41).

Buchneramay have evolved from a bacterium that inhabited the gut of ances-tral aphids (46). It is closely allied with the mycetocyte symbionts in tsetse flies(3) andCamponotusants (91), suggesting that this lineage ofγ3-Proteobacteriamay be predisposed to enter into intimate associations.

Characteristics of the aphid-BuchnerasymbiosisLOCATION OF THE BACTERIA In all aphids, theBuchneraare located in myce-tocytes in the hemocoel. The mycetocytes rarely, if ever, divide after the birthof an aphid, but they increase in size as the bacteria within them proliferate.For example, the vetch aphidMegoura viciaeis born with approximately 80mycetocytes, each of volume 104 µm3, and the cells increase in size eightfold

Ann

u. R

ev. E

ntom

ol. 1

998.

43:1

7-37

. Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

Wes

tern

Mic

higa

n U

nive

rsity

on

01/1

7/12

. For

per

sona

l use

onl

y.




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

MATERNAL INHERITANCE OF THE BACTERIA The Buchneracells are trans-mitted maternally from one aphid generation to the next via the ovaries, a processcalled transovarial transmission, and this is the basis for the strict congruence ofaphid andBuchneragene phylogenies, described above. In oviparous aphids,the bacteria are endocytosed by each ovum, and they can be observed readily asa “symbiont ball” at the posterior pole of the mature egg (12, 14). In viviparousmorphs, 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 sym-biosis has been demonstrated by studies of bacteria-free aphids, usually calledaposymbiotic aphids. These aphids can be generated experimentally by antibi-otic treatment, usually chlortetracycline or rifampicin administered either orallyor by injection into the hemolymph (e.g. 44, 59, 82). At the appropriate dosage,these antibiotics have minimal direct deleterious effect on insect metabolismor behavior (25, 44, 103). Larval aposymbiotic aphids grow very slowly, de-veloping into small adults that produce few or no offspring (30, 33, 88). Theseeffects of aposymbiosis probably reflect the nutritional contribution ofBuch-nera to aphids, and, as considered below, the use of aposymbiotic aphids hasbeen crucial to the elucidation of the nutritional interactions in the symbiosis.

Buchneraare widely accepted also to require the association, although theevidence is, as yet, entirely negative.Buchnerahave not been brought intoculture (14, 51) and have not been reported in any habitat other than aphidmycetocytes.

The Diversity of Microorganisms in AphidsBuchneraare not the sole microorganisms borne by aphids. Some membersof several aphid families, including the Aphididae, Lachnidae, Drepanosiphi-dae, and Pemphigidae have “secondary symbionts,” i.e. bacteria that are mor-phologically distinct fromBuchnerain mycetocytes and/or sheath cells of themycetome; a few lachnid species have a total of three morphological types ofbacteria in the mycetomes (14). The secondary symbionts inhabiting the sheathcells of the pea aphidAcyrthosiphon pisumhave been identified as Enterobac-teriaceae and are allied withE. coli (95), but there is no information on thenutritional significance, if any, of secondary symbionts to aphids.

Microorganisms have been identified in the gut lumen of some aphids bymicroscopical, microbiological, and molecular techniques (43, 46, 47). Manyof these microorganisms may be transient, i.e. ingested with food and lost,

Ann

u. R

ev. E

ntom

ol. 1

998.

43:1

7-37

. Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

Wes

tern

Mic

higa

n U

nive

rsity

on

01/1

7/12

. For

per

sona

l use

onl

y.



22 DOUGLAS

either with the passage of food through the gut or, for cells in the foregutand hindgut, at insect ecdysis. The absence of any gut microbiota in aphidsfeeding 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 betweencertain aphid species and their mycetocyte symbionts may be studied withoutthe complication of a substantive gut microbiota that is nutritionally importantto the insect, as occurs, for example, in leafhoppers (27). However, assessmentof the microbiota in aphid clones that are studied intensively is important, asis illustrated by the recent demonstration that nearly half of 122 clones ofA. pisumheld in one laboratory bore rickettsiae in their hemolymph (17).

HOW NUTRITIONAL INTERACTIONS IN THEAPHID-BUCHNERASYMBIOSIS ARE INVESTIGATED

Nutritional PhysiologyThe nutritional interactions between aphids andBuchnerahave been estab-lished primarily by nutritional physiology at the whole insect level. The aphidsymbiosis 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 aposym-biotic aphids and aphids bearing the bacteria (called symbiotic aphids).Differences between the two groups can be attributed to bacterial func-tion in the symbiotic aphids. However, some experiments require carefulinterpretation to distinguish between the direct effect of bacterial elimina-tion and secondary consequences of aposymbiosis, which arise from, forexample, small size or depressed embryo production.

2. Preparations of isolatedBuchneracan be obtained by techniques akin to theprotocols for the isolation of organelles, such as mitochondria (48, 54, 57).The isolatedBuchnerapreparations are viable and metabolically active forseveral 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). TheseBuchnerapreparations are not, however, metabolicallyequivalent toBuchnerain symbiosis, as is illustrated by the dramatic effectof isolation on the array of proteins synthesized by the bacteria (58).

3. Chemically defined diets, comprising sucrose, amino acids, minerals, andvitamins, buffered with phosphate, are available for rearing several aphidspecies over one to multiple generations (20). These artificial diets arecrucial to studies of the dietary requirements of aphids because it is impos-sible to manipulate the nutritional composition of the aphid’s natural diet

Ann

u. R

ev. E

ntom

ol. 1

998.

43:1

7-37

. Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

Wes

tern

Mic

higa

n U

nive

rsity

on

01/1

7/12

. For

per

sona

l use

onl

y.




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

Chemically defined diets have been used extensively to explore the nutri-tional requirements of aphids (20). Bacterial provisioning of a nutrient is indi-cated, specifically, by the dietary requirement of aposymbiotic but not symbi-otic aphids for that nutrient. This line of evidence should be complemented bydirect study of the metabolic capabilities of the two conditions with the predic-tion that a nutrient required only by aposymbiotic aphids is synthesized onlyby symbiotic aphids. Valuable confirmatory information can be obtained fromquantitation of the activity of key enzymes and/or titers of particular metabolitesin aphids, which would be anticipated to reflect the biosynthetic capabilitiesand nutritional requirements of symbiotic and aposymbiotic aphids.

As considered later in this article, these several approaches of nutritionalphysiology have been used widely to explore essential amino acid and lipidnutrition of the aphid-Buchnerasymbiosis.

Molecular BiologyIn the context of the symbiosis, molecular techniques have, to date, been ap-plied primarily to the study ofBuchneraand not the aphid partner. Analysis oftheBuchneragene content offers an excellent test for the genetic capability ofBuchnerato meet the nutritional functions identified by nutritional physiology.The sequence of these genes and flanking regions can additionally provide valu-able information on the regulation of gene expression (e.g. presence/absenceof transcription attenuation sites) or protein function (e.g. the sequence re-sponsible for allosteric inhibition of an enzyme) (10). Comparisons of genomeorganization and gene sequence betweenBuchnerain different aphid speciescan also be used to explore the evolutionary changes in the biosynthetic capa-bilities of Buchnera(e.g. 63, 64).

Approaches Not Currently AvailableAs a broad generalization (32), symbioses ideally suited to experimental studyhave the following two characteristics: (a) The participating organisms canbe maintained and studied in isolation; (b) the symbiosis can be resynthesizedfrom its constituent organisms. The characteristics of “experimental” sym-bioses between partners that do not naturally form associations or involving anorganism bearing a specific mutation (e.g. an auxotrophic mutant) are particu-larly informative. These procedures have been central to the elucidation of theinteractions between leguminous plants and nitrogen-fixing rhizobia (97) and

Ann

u. R

ev. E

ntom

ol. 1

998.

43:1

7-37

. Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

Wes

tern

Mic

higa

n U

nive

rsity

on

01/1

7/12

. For

per

sona

l use

onl

y.



24 DOUGLAS

to the recent developments in our understanding of the symbiosis between thesquidEuprymna scolopesand luminescent bacteria (85).

The main reason why the aphid-Buchneraassociation and mycetocyte sym-bioses 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 isolatedBuchnerabecausethey can be maintained for only a few hours. Also, the aphid symbiosis cannotbe synthesized from isolatedBuchneraand aposymbiotic aphids.Buchnerainjected into the hemocoel of aposymbiotic aphids are lysed (AE Douglas,unpublished observations), whereas the injection of intact mycetocytes intoaposymbionts has not, to my knowledge, been attempted. Development of themethods to synthesize, or otherwise manipulate, the aphid-Buchneraassocia-tion and other mycetocyte symbioses would transform the research on thesesystems.

NUTRITIONAL INTERACTIONS IN THE SYMBIOSIS

Essential Amino Acid ProvisioningA bacterial supply of essential amino acids would be of nutritional advantage toany insects feeding on the phloem sap of plants, which has low or undetectableconcentrations of many essential amino acids (31). Direct experimental supportfor microbial provisioning of essential amino acids is, however, available onlyfor the aphid-Buchnerasymbiosis, and such support comes from studies ofaphid dietary requirements and metabolic capabilities and the molecular biologyof Buchnera.

DIETARY STUDIES Various studies on the dietary amino acid requirements ofaphids have shown that the essential amino acid content of the diet has a greaterimpact on the performance of aposymbiotic aphids than on symbiotic aphids.In an early and important set of experiments, aposymbioticMyzus persicaewasfound to have a dietary requirement for every essential amino acid, as indicatedby poor growth and survival on all diets from which one essential amino acidwas omitted (66), whereas the growth of symbioticM. persicaewas unaffectedby the individual deletion of 7 of the 10 essential amino acids (21). Otherstudies have used diet formulations that bear some resemblance to phloem sap.In particular, the performance of aposymbioticA. pisumhas been explored ondiets of uniform nitrogen content that contain all protein amino acids but with aratio of essential:nonessential amino acids varying from 1:1 to 1:5. The larvalgrowth rate ofA. pisumwas more than twofold greater on the 1:1 diet than onthe 1:5 diet, whereas that of symbiotic aphids did not vary significantly withamino acid compositon (80).

Ann

u. R

ev. E

ntom

ol. 1

998.

43:1

7-37

. Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

Wes

tern

Mic

higa

n U

nive

rsity

on

01/1

7/12

. For

per

sona

l use

onl

y.




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

METABOLIC STUDIES Most metabolic studies on amino acid synthesis in aphidshave used14C- or 15N-labeled nonessential amino acids as precursors. One ofthe most widely metabolized amino acids is glutamate, which is a precursorof a number of essential amino acids, including isoleucine, leucine, threonine,lysine, and valine, in symbioticA. pisum(39, 89, 105). Unfortunately, sev-eral studies have omitted aposymbiotic aphids as controls, and therefore thesemetabolic capabilities cannot formally be attributed to the bacteria. As impor-tant, most studies have merely demonstrated essential amino acid synthesis inthe symbiosis, without considering whether the nutrients are made available tothe aphid tissues. In the context of the symbiosis, not only are the biosyntheticcapabilities of symbiotic bacteria of interest, but so is the access of the insectto the products of these capabilities.

To my knowledge, there has been just one definitive demonstration of nutri-ent provisioning in the aphid-Buchnerasymbiosis. This concerns the sulphur-containing essential amino acid, methionine, the synthesis of which can bequantified by the incorporation of35S from dietary inorganic sulphate. Isolatedpreparations ofBuchneracan utilize sulphate as a sulphur source for methioninesynthesis (29). Symbiotic, but not aposymbiotic,M. persicaealso incorporated35S into methionine (25), and up to 20% of35S-labeled methionine was recov-ered from the anteriorBuchnera-free tissues of the symbiotic aphids, which in-dicates that methionine synthesized byBuchnerais made available to the aphidtissues.

Three complementary experimental approaches provide strong evidence forbacterial provisioning of the essential amino acid tryptophan. The first is thataposymbiotic, but not symbiotic, aphids ofA. pisumandM. persicaehave adietary requirement for this amino acid (21, 36). Second, isolatedBuchnerapreparations and symbiotic, but not aposymbiotic,A. pisumhave apprecia-ble activity of tryptophan synthetase (36). Finally, symbiotic aphids reared ontryptophan-free diets continue to produce tryptophan in their honeydew formany days (36).

Ann

u. R

ev. E

ntom

ol. 1

998.

43:1

7-37

. Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

Wes

tern

Mic

higa

n U

nive

rsity

on

01/1

7/12

. For

per

sona

l use

onl

y.



26 DOUGLAS

MOLECULAR STUDIES Molecular studies of the gene content ofBuchnerahaveconfirmed that this bacterium has the genetic capability to synthesize essentialamino acids. There is no published report of a failure to identify any gene codingfor enzymes in amino acid synthesis, and a total of 13 genes directly involved inessential amino acid biosynthesis have been described:aroA, aroH, andaroE,in the common pathway of aromatic amino acid synthesis (61, 84);leuABCD,which encodes enzymes specific to leucine synthesis (11); andtrpEGDC(F)BA,the genes for tryptophan synthesis (62, 72).

In the Aphididae, the genesleuABCDandtrpEGare apparently absent fromtheBuchnerachromosome and located on plasmids, known as the leucine plas-mid and tryptophan plasmid, respectively (11, 62). The genetic organization ofthese plasmids is described in References 11, 62, and 64. In the context of thenutritional interactions in the symbiosis, the key issue is that the plasmid-bornegenes are amplified, relative to the chromosomal genes ofBuchnera(11, 62).This genetic organization can plausibly be interpreted in terms of increased bac-terial capacity to synthesize leucine and tryptophan (these issues are discussedfurther in Reference 34).

Both leuABCDandtrpEGare chromosomal inBuchnerafrom certain aphidspecies of the family Pemphigidae (63; R van Ham, A Moya & A Latorre,unpublished observations), and comparisons between thetrpEG sequence inBuchnerafrom Aphididae and Pemphigidae indicate that the plasmid-bornegenes are derived fromBuchnerachromosomal genes and have not been ac-quired horizontally from a different bacterium (83). The leucine and tryptophanplasmids inBuchnerafrom the Aphididae represent a remarkable confirmationof the significance of essential amino acid synthesis to the association.

Genes encoding enzymes in the synthesis of other essential amino acidsare not on small (<35 kb) plasmids inBuchneraof the Aphididae, but thepossibility that they are amplified, either on theBuchnerachromosome or onlarge plasmids, cannot be excluded.

Nitrogen Recycling and UpgradingNitrogen recycling in an animal-microbial symbiosis refers to the microbialmetabolism of animal waste nitrogen to compounds, such as essential aminoacids, that are of nutritional value to the animal (32). Microbial recyclingof nitrogen is potentially of great nutritional value to an animal because itincreases the efficiency with which the animal can utilize dietary nitrogen,enabling it to survive and grow on low nitrogen input. However, nitrogenrecycling has not been demonstrated unequivocally in any association, althoughthere is strong evidence that the mycetocyte symbionts in cockroaches (19),yeasts in planthoppers (90), and gut bacteria in termites (78) may all consumenitrogenous waste products of their insect hosts.

Ann

u. R

ev. E

ntom

ol. 1

998.

43:1

7-37

. Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

Wes

tern

Mic

higa

n U

nive

rsity

on

01/1

7/12

. For

per

sona

l use

onl

y.




Buchneraexhibit the metabolic characteristics expected of a symbiotic mi-croorganism that recycles nitrogen. As considered above, essential amino acidssynthesized byBuchneraare translocated to the aphid, and the nitrogen in theseessential amino acids must be derived ultimately from the aphid. Further, iso-lated Buchneracan take up ammonia, the chief nitrogenous waste productof aphids (102), andBuchneracells in symbiosis presumably have access toaphid-derived ammonia, a compound that diffuses freely across biologicalmembranes (except from compartments of low pH).

To date, there has been no direct investigation of nitrogen recycling in theaphid-Buchnerasymbiosis, i.e. an analysis of the incorporation of nitrogen,derived from the aphid waste ammonia, into essential amino acids. Much ofthe recent research has focused on the nutritional physiology of aposymbioticA. pisum. Relative to symbiotic aphids, aposymbioticA. pisumhave elevatedconcentrations of ammonia and glutamine in both their tissues and honeydew(79, 87, 102, 104), and they also have elevated activity of the enzyme glutaminesynthetase (104). Glutamine synthetase in animals contributes to the detoxi-fication of ammonia by catalyzing the synthesis of glutamine from ammoniaand glutamate. The link between glutamine synthetase activity and ammoniaload in aphids is confirmed by the elevated glutamine synthetase activity insymbiotic aphids maintained on ammonia-supplemented diets (104). These re-sults are compatible with the hypothesis thatBuchneraassimilate aphid-derivedammonia, i.e. they act as a sink for aphid ammonia.

Recent dietary experiments indicate that, if nitrogen recycling of aphid am-monia occurs, it is not quantitatively important to aphid growth. For symbioticA. pisumreared on a low-nitrogen diet, a dietary supplement of nonessentialamino acids, but not ammonia, promoted aphid growth (104). These resultsare compatible with the conclusion, from studies on nonessential amino aciduptake and metabolism by isolatedBuchnerapreparations, that glutamate maybe the principal nitrogenous compound utilized byBuchnera(89, 101).

Nonessential amino acids are the principal nitrogenous compounds in theaphid diet of phloem sap (31). Although much of the detail of aphid andbacterial metabolism of these compounds remains to be resolved, the broadoutline of nitrogen relations of the symbiosis inA. pisumis becoming clear.Buchneraupgrade dietary nonessential amino acids to essential amino acids,thereby contributing to the aphid’s capacity to utilize phloem sap, and bacterialrecycling of aphid nitrogen is not quantitatively important.

Lipids (Including Sterols): Nutritional Independence of Aphidsfrom Their Symbiotic BacteriaThe possibility that symbiotic bacteria may contribute to the lipid and sterolnutrition of aphids was raised by the early nutritional studies that revealed

Ann

u. R

ev. E

ntom

ol. 1

998.

43:1

7-37

. Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

Wes

tern

Mic

higa

n U

nive

rsity

on

01/1

7/12

. For

per

sona

l use

onl

y.



28 DOUGLAS

that aphids, unlike many insects, can be maintained over many generations onchemically defined diets lacking lipids (reviewed in Reference 20).

Much of the research has concerned the source of two fatty acids: linoleicacid (9,12-octadecadienoic acid), a major component of aphid phospholipids,and sorbic acid (2,4-hexadienoic acid), one of the short chain fatty acids thatcontributes to aphid triacylglycerols. The symbiotic bacteria were implicatedin their synthesis because vertebrate animals cannot synthesize linoleic acidand 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 linoleicacid (23). With respect to aphids, the crucial experiment was the demonstrationthat both symbiotic and aposymbioticA. pisumincorporate radioactivity from14C-acetate into linoleic acid (24). The principal evidence that the aphid, andnot its bacteria, synthesizes sorbic acid is that the fatty acid composition oftriacylglycerols in bothA. pisumandMacrosiphum euphorbiaeis unaffectedby rifampicin treatment (82, 99). Sorbic acid synthesis from14C-acetate byM. euphorbiaehas been demonstrated (98), but the critical experiments on thebiosynthetic 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 virtu-ally unknown in bacteria. Consistent with this generality, the aphidSchizaphisgraminummaintained on diets containing [2-14C] melavonate exhibited no de-tectable incorporation of radioactivity into sterols or metabolic intermediatesin sterol synthesis (16). It is very likely that plant-reared aphids derive theirsterol requirement from phytosterols in the phloem sap. The source of sterols indiet-reared aphids remains to be resolved. Campbell & Nes (16) suggested thatthe diets may be routinely contaminated with fungi, which could be a sourceof sterols, but no such contaminants are revealed by mycological sterility test-ing of diets. Fungal contamination cannot account for the observed promotionof performance of aposymbiotic, but not symbiotic,M. persicaeby a dietarysupply of sterols (26). It appears that the bacteria in aphids may have a sparingeffect on the insect’s requirement for sterols, but the underlying mechanismsare, at present, unknown.

Vitamin ProvisioningThe contribution of symbiotic microorganisms to the vitamin requirements ofinsects can most appropriately be explored by analysis of the insect’s dietaryrequirements. Early nutritional studies on aphids established thatM. persicaerequires all B vitamins and vitamin C (ascorbic acid) (22) and thatNeomyzuscircumflexusrequires all except riboflavin (38). Aphid requirement for some ofthe vitamins only became apparent after several insect generations were rearedon the vitamin-free diets.

Ann

u. R

ev. E

ntom

ol. 1

998.

43:1

7-37

. Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

Wes

tern

Mic

higa

n U

nive

rsity

on

01/1

7/12

. For

per

sona

l use

onl

y.




These data provide persuasive evidence that the symbiotic bacteria do notgenerally supply the vitamin requirements of aphids (20).

VARIATION AND VERSATILITY IN NUTRITIONALINTERACTIONS

The firm evidence thatBuchneracontribute essential amino acids to aphidsraises the question whether the rates at which amino acids are made availableto the insect and the profile of amino acids provided may vary, either betweenaphid taxa or in a single aphid, perhaps in accordance with aphid nutritionaldemand (as determined by the mismatch between the dietary supply of essentialamino acids and requirements for aphid growth). These issues are central tothe several proposals in the literature that symbiotic bacteria may influence thecapacity of phytophagous insects to utilize different plants and, thereby, act asone determinant of their host plant range (e.g. 7, 15, 60). Only very limiteddata are available, and they are reviewed here.

Variation Between Aphid TaxaIt would be exceptional if amino acid provisioning byBuchnerawas absolutelyuniform across the Aphidoidea. At the very least, the aphids whoseBuchnerahaveleu andtrp genes amplified on plasmids probably have greater access tobacteria-derived leucine and tryptophan, respectively, than aphid species whoseBuchneralack these plasmids (discussed in Reference 63).

The host plant affiliation of aphids of the family Aphididae can apparentlyevolve very rapidly, such that closely related aphid taxa have distinct or over-lapping host plant ranges (45). These evolutionary shifts could involve changesin the metabolic capabilities ofBuchnera, to compensate for differences in thenutritional composition of phloem sap in different plant species. Just one sys-tem has been studied to date, theAphis fabaespecies complex. An analysisof the performance of aphids deprived of their bacteria suggests that, in thissystem, insect and not bacterial function limits the capacity ofA. fabae fabaeto utilize plant species specific to the subspeciesA. fabae mordwilkoi(2). Thesymbioses in other aphid species complexes warrant investigation.

One set of experiments has been conducted on variation in the symbiosis be-tween clones of a single aphid species. Two clones ofA. pisumare independentof a dietary supply of different essential amino acids (93): clone C of isoleucine,phenylalanine, and valine and clone J of leucine, lysine, and tryptophan. Asthe authors comment (93), this can most plausibly be interpreted as differencesbetween the profiles of amino acids provided by theBuchnerain the differentaphid clones, but no direct analysis of bacterial amino acid provisioning hasbeen done on these aphids.

Ann

u. R

ev. E

ntom

ol. 1

998.

43:1

7-37

. Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

Wes

tern

Mic

higa

n U

nive

rsity

on

01/1

7/12

. For

per

sona

l use

onl

y.



30 DOUGLAS

The Metabolic Versatility ofBuchneraMetabolic versatility in symbiotic microorganisms can be defined as the ex-tent to which nutrients provided by the microorganisms to their (insect) hostmay vary, in accordance with the host nutritional demand for those nutrients.Versatility in essential amino acid provisioning byBuchneramay arise by (a)variation in the rate of supply of amino acids, through changes in the bacterialbiomass and/or rate of provisioning per bacterial cell, and (b) variation in thecomposition of amino acids provided, through shifts in the relative rates ofsynthesis and release of different amino acids.

The variation in biomass ofBuchnerawith aphid developmental age andmorph is relatively well studied (8, 35, 52, 55, 101), but the impact of host plantor dietary nutrients on the bacterial biomass has rarely been considered. Onerecent study concernedA. fabaereared on different plant species, on whichbacterial promotion of aphid growth is known to vary (2). The volume of thesymbiosis, relative to total aphid weight, was remarkably uniform. Even onplant species, on which the larval relative growth rate of symbiotic and aposym-biotic A. fabaewere not significantly different, the symbiosis was maintained(1). These data suggest that the bacterial population inA. fabaeis not regulatedaccording to its immediate nutritional value to the insect.

The versatility ofBuchnerain the composition of amino acids provided to theaphid depends critically on the mode of release of these nutrients. At present,virtually nothing is known, although the nutrients are generally accepted to bereleased from living cells of the bacteria. The amino acids may be released asfree amino acids, small peptides, or proteins, any of which would be translocatedacross the cell membrane of the bacteria and the insect membrane (symbiosomemembrane) that separates eachBuchneracell from the surrounding mycetocytecytoplasm. There is evidence for the release of one protein, the chaperoninGroEL, which attains high concentrations inBuchneracells (9) and is alsorecovered from the hemolymph ofM. persicae(96). The rapid decline in theGroEL titers in hemolymph of antibiotic-treated aphids is consistent with thepossibility that this protein is released from metabolically activeBuchneracellsand degraded rapidly by the aphid. There can be no versatility in the profileof amino acids provided via GroEL, the amino acid composition of which isdetermined genetically. Metabolic versatility inBuchneracould potentially beobtained if free amino acids were translocated from the bacteria to the aphid, viaan array of membrane transporters, specific to individual amino acids, mediatingthe export from bacteria and uptake across the insect symbiosome membrane.The density and activity of these putative transporters could be regulated veryprecisely, in accordance with aphid demand. At present, however, there is noevidence for the transport of free amino acids fromBuchnerato aphid tissues(discussed in Reference 34).

Ann

u. R

ev. E

ntom

ol. 1

998.

43:1

7-37

. Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

Wes

tern

Mic

higa

n U

nive

rsity

on

01/1

7/12

. For

per

sona

l use

onl

y.




One published data set exists that is consistent with the possibility thatBuchneraare very versatile in their nutritional interactions with the insect. Whena single clone ofA. pisumwas reared on chemically defined diets of differentamino acid composition, the free amino acid titers of symbiotic aphids werevery uniform, whereas those of aposymbiotic aphids bore distinct similarities tothe amino acid composition of the diets on which they were feeding (65). Thesedata were interpreted as evidence for a breakdown in regulation of the freeamino acid pools in aposymbiotic aphids. The implication is that the bacteriacontribute directly to amino acid homeostasis in symbiotic aphids, i.e. controlover the profile of amino acids provided by the bacteria is so fine-tuned that itmaintains the optimal amino acid titers of the aphid body fluids. A study ofclose similar design, but conducted onA. fabaereared on plants with phloem sapof very diverse amino acid compositions, obtained tightly regulated amino acidtiters in both symbiotic and aposymbiotic aphids (1). Bacterial contribution tothe amino acid homeostasis of aphids does not appear, therefore, to be general.

The sole indication that the metabolic versatility ofBuchneramay be limitedcomes from the study of tryptophan production in symbioticA. pisum(36). Asdescribed above, the bacteria in aphids reared on tryptophan-free diets continueto produce honeydew containing tryptophan. A plausible interpretation of thesedata is that the bacteria provide tryptophan at rates greater than aphid demandand the excess is excreted via honeydew, which suggests that the aphid mayhave only limited control over the metabolic activities of its bacteria.

In summary, the data on the versatility of essential amino acid provisioningby Buchnera, both with dietary supply of amino acids and between aphid taxa,are fragmentary and, in part, contradictory. There is a real need for directmetabolic study of amino acid translocation fromBuchnerain different aphidtaxa and in aphids reared under different nutritional conditions.

NON-NUTRITIONAL FUNCTIONS OF BUCHNERAIN APHIDS

The nutritional functions of symbiotic microorganisms in aphids and otherinsects arise directly from the fact that insects, like other animals, are metabol-ically impoverished, lacking the capacity to synthesize essential amino acids,vitamins, etc (32). The symbiotic microorganisms have, however, been pro-posed to contribute to the biology of their insect hosts in ways that do not relatedirectly to a match between the metabolic capabilities of the bacteria and thebasic nutritional requirements of the insect. For example,Buchnerahave beenimplicated in resistance of aphids to insecticides (97a) and aphid stylet pene-tration of plant tissues (37), and the microorganisms in other Homoptera havebeen proposed to be required for the differentiation of the insect abdomen (92).

Ann

u. R

ev. E

ntom

ol. 1

998.

43:1

7-37

. Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

Wes

tern

Mic

higa

n U

nive

rsity

on

01/1

7/12

. For

per

sona

l use

onl

y.



32 DOUGLAS

All these proposed functions were developed without proper use of controls,in particular aposymbiotic insects obtained with appropriate concentrations ofantibiotic, 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 con-trols is Buchnera-mediated promotion of viral transmission by aphids. Theviruses are circulative luteoviruses, notably the potato leafroll virus transmittedby M. persicae, whose inoculation from the aphid into a plant depends on thetransfer of the virus particles from the aphid gut to the salivary glands, via thehemolymph. The viral particles are stabilized by theBuchneraprotein GroEL,released from the bacteria (see above), and viral transmission is depressed inaphids by treatment with chlortetracycline to arrest bacterial protein synthesis.GroEL of E. coli can also stabilize the virus particles (96). This suggests thatthe interaction between GroEL ofBuchneraand luteoviruses is not an exam-ple of coevolution between the symbiosis and virus but a fortuitous molecularcompatibility, reflecting the principal function of GroEL in the stabilization ofparticular conformations of proteins.

The discovery of other non-nutritional processes mediated by the symbioticbacteria in aphids may be anticipated.

CONCLUDING REMARKS

The principal conclusion from recent research on nutritional interactions in theaphid-Buchnerasymbiosis is that the bacteria provide aphids with essentialamino acids. This interaction contributes to the capacity of aphids to utilizephloem sap, a diet that is deficient in essential amino acids. All Homopterafeeding on phloem sap have symbiotic microorganisms, and those groups thathave secondarily switched to feeding on intact plant cells have lost the symbiosis(Table 1). The implication is that the microbial symbiosis is causally linkedwith phloem feeding, and specifically that essential amino acid provisioningmay underpin all these associations, even though the symbioses in the variousHomoptera may differ in the identity of the microbial partners, anatomicallocation and structural organization of the symbiosis, developmental origin ofmycetocytes, and mode of transovarial transmission (14, 28).Buchneraareclearly not exceptional in their capacity to participate in nutritional interactionswith insect cells.

An important research area for the future is the mechanisms underlying aminoacid provisioning, in particular the identity of the compounds transferred toaphids and the regulation of translocation. In other symbiotic systems, notablysymbiotic algae in animals (32), common mechanisms of nutrient release havebeen established that are independent of the identity of the organisms, andparallels between nutritional interactions in the various mycetocyte symbioses

Ann

u. R

ev. E

ntom

ol. 1

998.

43:1

7-37

. Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

Wes

tern

Mic

higa

n U

nive

rsity

on

01/1

7/12

. For

per

sona

l use

onl

y.




may also occur. The mechanisms of nutrient release fromBuchneramay,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 radiationof insect-bacteria symbioses, e.g. aphid-Buchnera, variation and diversity areto be anticipated. In particular, the profile of the amino acids provided and themetabolic versatility ofBuchneramay vary between aphid higher taxa or be-tween aphid species that have narrow and broad host plant ranges. A coherentarray of techniques is now in place to explore these issues, and the mycetocytesymbioses are becoming increasingly amenable to study through a combinationof nutritional physiology and molecular biology.

ACKNOWLEDGMENTS

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

Visit the Annual Reviews home pageathttp://www.AnnualReviews.org.

Literature Cited

1. Adams D. 1996.Host plant effects onan aphid-bacterial symbiosis.PhD thesis.Univ. York, United Kingdom, 102 pp.

2. Adams D, Douglas AE. 1997. How sym-biotic bacteria influence plant utilizationby the polyphagous aphid,Aphis fabae.Oecologia.In press

3. Aksoy S, Pourhosseini AA, Chow A.1995. Mycetome endosymbionts of tsetseflies constitute a distinct lineage relatedto Enterobacteriaceae.Insect Mol. Biol.4:15–22

4. Deleted in proof5. Ball BV, Bailey L. 1978. The symbiotes

of Myzus persicae(Sulz.) in strains re-sistant and susceptible to demeton-S-methyl.Pest. Sci.9:522–24

6. Bandi C, Damiani G, Magrassi L, GrigoloA, Fani R, Sacchi L. 1994. Flavobacteriaas intracellular symbionts in cockroaches.Proc. R. Soc. Lond. B257:43–48

7. Barbosa P, Krischik VA, Jones CG, eds.1991. Microbial Mediation of Plant-Herbivore Interactions.New York: Wiley& Sons

8. Baumann L, Baumann P. 1994. Growthkinetics of the endosymbiontBuchn-era aphidicola in the aphid Schiza-phis graminum. Appl. Environ. Microbiol.60:3440–43

9. Baumann P, Baumann L, Clark MA. 1996.Levels of Buchnera aphidicolachapa-ronin GroEL during growth of the aphidSchizaphis graminum. Curr. Microbiol.32:279–85

10. Baumann P, Lai C-Y, Roubakhsh D,Moran NA, Clark MA. 1995. Genet-ics, physiology, and evolutionary re-lationships of the genusBuchnera—intracellular symbionts of aphids.Annu.Rev. Microbiol.49:55–94

11. Bracho AM, Martinez-Torres D, Moya A,Latorre A. 1995. Discovery and molecu-lar characterization of a plasmid localizedin Buchnerasp. bacterial endosymbiontof the aphidRhopalosiphum padi. J. Mol.Evol.41:67–73

12. Brough CN, Dixon AFG. 1990. Ultra-structural features of egg development inoviparae of the vetch aphid,Megoura vi-ciaeBuckton.Tissue Cell22:51–63

13. Brown KS. 1975. The chemistry of aphidsand scale insects.Chem. Soc. Rev.4:263–88

14. Buchner P. 1965.Endosymbiosis of An-imals with Plant Microorganisms.NewYork: Wiley & Sons

15. Campbell BC. 1990. On the role ofmicrobial symbiotes in herbivorous in-sects. InInsect-Plant Interactions, ed. EA

Ann

u. R

ev. E

ntom

ol. 1

998.

43:1

7-37

. Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

Wes

tern

Mic

higa

n U

nive

rsity

on

01/1

7/12

. For

per

sona

l use

onl

y.



34 DOUGLAS

Bernays, 2:1–43. Boca Raton, FL: CRCPress

16. Campbell BC, Nes WD. 1983. Areappraisal of sterol biosynthesis andmetabolism in aphids.J. Insect Physiol.29:149–56

17. Chen D-Q, Campbell BC, Purcell AH.1996. A new rickettsia from a herbivo-rous insect, the pea aphidAcyrthosiphonpisum(Harris).Curr. Microbiol. 33:123–28

18. Clark MA, Baumann L, Munson MA,Baumann P, Campbell BC. 1992. Theeubacterial endosymbionts of whiteflies(Homoptera: Aleyrodoidea) constitute alineage distinct from the endosymbiontsof aphids and mealybugs.Curr. Micro-biol. 25:119–23

19. Cochran DG. 1985. Nitrogen excretion incockroaches.Annu. Rev. Entomol.30:29–49

20. Dadd RH. 1985. Nutrition: organisms.In Comprehensive Insect Physiology Bio-chemistry and Pharmacology,ed. GAKerkut, LI Gilbert, 4:313–91. Oxford:Pergamon

21. Dadd RH, Krieger DL. 1968. Dietary re-quirements of the aphidMyzus persicae.J. Insect Physiol.14:741–64

22. Dadd RH, Krieger DL, Mittler TE. 1967.Studies on the artificial feeding of theaphidMyzus persicae.IV. Requirementsfor water-soluble vitamins and ascorbicacid.J. Insect Physiol.13:249–72

23. de Renobles M, Cripps C, Stanley-Samuelson DW, Jurenka RA, BlomquistGJ. 1987. Biosynthesis of linoleic acid ininsects.Trends Biochem. Sci.12:364–66

24. de Renobles M, Ryan RO, HeislerCR, McLean DL, Blomquist GJ. 1986.Linoleic acid biosynthesis in the peaaphid, Acyrthosiphon pisum(Harris).Arch. Insect Biochem. Physiol.3:193–203

25. Douglas AE. 1988. Sulphate utilisationin an aphid symbiosis.Insect Biochem.18:599–605

26. Douglas AE. 1988. On the source ofsterols in the green peach aphid,Myzuspersicae, reared on holidic diets.InsectBiochem.34:403–8

27. Douglas AE. 1988. Experimental stud-ies on the mycetome symbiosis in theleafhopper Euscelis incisus. J. InsectPhysiol.34:1043–53

28. Douglas AE. 1989. Mycetocyte symbio-sis in insects.Biol. Rev.69:409–34

29. Douglas AE. 1990. Nutritional interac-tions betweenMyzus persicaeand itssymbionts. InAphid-Plant Genotype In-teractions,ed. RK Campbell, RD Eiken-bary, pp. 319–27. Amsterdam: Elsevier

30. Douglas AE. 1992. Requirement of peaaphids (Acyrthosiphon pisum) for theirsymbiotic bacteria.Entomol. Exp. Appl.65:195–98

31. Douglas AE. 1993. The nutritional qual-ity of phloem sap utilized by natural aphidpopulations.Ecol. Entomol.18:31–38

32. Douglas AE. 1994.Symbiotic Interac-tions.Cambridge: Oxford Univ. Press

33. Douglas AE. 1996. Reproductive failureand the amino acid pools in pea aphids(Acyrthosiphon pisum) lacking symbioticbacteria.J. Insect Physiol.42:247–55

34. Douglas AE. 1997. Parallels and contrastsbetween symbiotic bacteria and bacterial-derived organelles: evidence fromBuch-nera, the bacterial symbiont of aphids.FEMS Microbiol. Ecol.In press

35. Douglas AE, Dixon AFG. 1987. Themycetocyte symbiosis in aphids: varia-tion with age and morph in virginoparaeof Megoura viciaeand Acyrthosiphonpisum. J. Insect Physiol.33:109–13

36. Douglas AE, Prosser WA. 1992. Synthe-sis of the essential amino acid tryptophanin the pea aphid (Acyrthosiphon pisum)symbiosis.J. Insect Physiol.38:565–8

37. Dreyer DL, Campbell BC. 1987. Chem-ical basis of host-plant resistance toaphids.Plant Cell Environ.10:353–61

38. Ehrhardt P. 1968. Die Wirking ver-scheidener Spurenelemente auf Wachs-tum. Reproduktion und Symionten vonNeomyzus circumflexus Buckt. (Aphi-dae: Homoptera: Insecta) bei kunstlicherErnahrung.Z. Vgl. Physiol.58:47–75

39. Febvay G, Liadouze I, Guillaud J, BonnotG. 1995. Analysis of energetic amino acidmetabolism inAcyrthosiphon pisum—a multidimensional approach to aminoacid metabolism in aphids.Arch. InsectBiochem. Physiol.29:45–69

40. Fukatsu T, Aoki S, Kurosu U, Ishikawa H.1994. Phylogeny of Cerataphidini aphidsrevealed by their symbiotic microorgan-isms and basic structure of their galls:implications for host-symbiont coevolu-tion and evolution of sterile soldier castes.Zool. Sci.11:613–23

41. Fukatsu T, Ishikawa H. 1992. Soldierand male of an eusocial aphidColophinaarma lack endosymbiont: implicationsfor physiological and evolutionary inter-action between host and symbiont.J. In-sect Physiol.38:1033–42

42. Fukatsu T, Ishikawa H. 1996. Phy-logenetic position of yeast-like sym-biont of Hamiltonaphis styraci (Ho-moptera, Aphididae) based on 18S rDNAsequence.Insect Biochem. Mol. Biol.26:383–88

Ann

u. R

ev. E

ntom

ol. 1

998.

43:1

7-37

. Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

Wes

tern

Mic

higa

n U

nive

rsity

on

01/1

7/12

. For

per

sona

l use

onl

y.




43. Grenier A-M, Nardon C, Rahbe Y.1994. Observations on the microorgan-isms occurring in the gut of the peaaphid Acyrthosiphon pisum. Entomol.Exp. Appl.70:91–96

44. Griffiths GW, Beck SD. 1974. Effects ofantibiotics on intracellular symbiotes inthe pea aphidAcyrthosiphon pisum. CellTissue Res.48:287–300

45. Guldemond JA. 1990. Evolutionary ge-netics of the aphidCryptomyzuswith apreliminary analysis of host plant prefer-ence, reproductive performance and hostalternation.Entomol. Exp. Appl.57:65–76

46. Harada H, Ishikawa H. 1993. Gut microbeof aphid closely related to its intracellularsymbiont.BioSystems31:185–91

47. Harada H, Oyaizu H, Ishikawa H. 1996.A consideration about the origin of aphidintracellular symbiont in connection withgut bacterial flora.J. Gen. Appl. Micro-biol. 42:17–26

48. Harrison CP, Douglas AE, Dixon AFG.1989. A rapid method to isolate symbioticbacteria from aphids.J. Invert. Pathol.53:427–28

49. Heie OE. 1980. The Aphidoidea ofFennoscandia and Denmark. I. Intro-duction and the families Mindarideae,Hormaphididae, Thelaxidae, Anoeciidae,and Pemphigidae.Fauna Entomol. Scand.9:1–236

50. Hinde R. 197l. The control of the myceto-cyte symbiotes of the aphidsBrevicorynebrassicae, Myzus persicaeandMacrosi-phum rosae. J. Insect Physiol.17:1791–800

51. Hinde R. 1971. Maintenance of aphidcells and the intracellular symbiotes ofaphids in vitro.J. Invert. Pathol.17:333–38

52. Hongoh Y, Ishikawa H. 1994. Changesin the mycetocyte symbiosis in responseto flying behaviour of alatiform aphid(Acyrthosiphon pisum) Zool. Sci.11:731–35

53. Houk EJ, Griffiths GW. 1980. Intracel-lular symbiotes of the Homoptera.Annu.Rev. Entomol.25:161–87

54. Houk EJ, McLean DL. 1974. Isolation ofthe primary intracellular symbiote in thepea aphid,Acyrthosiphon pisum. J. Invert.Pathol.23:237–41

55. Humphreys NJ. 1996.Symbiosis and em-bryo production in the aphid symbiosis.PhD thesis. Univ. York, United Kingdom.162 pp.

56. Iimura Y, Sasaki T, Fukatsu T, IshikawaH. 1995. Diminution of intracellular sym-biont of aphid maintained on artificial

diet—a morphological study.Zool. Sci.12:795–99

57. Ishikawa H. 1981. Isolation of the intra-cellular symbionts and partial character-isation of their RNA species of the el-der aphidAcyrthosiphon pisum. Comp.Biochem. Physiol.72B:239–47

58. Ishikawa H. 1984. Characterization of theprotein species synthesizedin vivoandinvitro by an aphid endosymbiont.InsectBiochem.14:417–25

59. Ishikawa H, Yamaji M. 1985. Sym-bionin, an aphid endosymbiont-specificprotein—I. Production of insects deficientin symbiont.Insect Biochem.16:155–63

60. Jones CG. 1984. Microorganisms as me-diators of plant resource exploitation byinsect herbivores. InA New Ecology:Novel Approaches to Interactive Systems,ed. PW Price, CN Slobodchikoff, WSGand, pp. 53–99. New York: Wiley &Sons

61. Kolibachuk D, Baumann P. 1995. Ar-monatic amino acid biosynthesis inBuchnera aphidicola(endosymbiont ofaphids): cloning and sequencing ofa DNA fragment containing aroH-thrS-infC-rpmL-rplT. Curr. Microbiol.30:313–16

62. Lai C-Y, Baumann L, Baumann P. 1994.Amplification of trpEG; adaptation ofBuchnera aphidicolato an endosymbioticassociation with aphids.Proc. Natl. Acad.Sci. USA91:3819–23

63. Lai C-Y, Baumann P, Moran NA. 1995.Genetics of the tryptophan biosyntheticpathway of the prokaryotic endosymbiont(Buchnera) of the aphidSchlechtendaliachinensis. Insect Mol. Biol.4:47–59

64. Lai C-Y, Baumann P, Moran NA. 1996.The endosymbiont (Buchnera sp.) of theaphidDiuraphis noxiacontains plasmidsconsisting oftrpEG and tandem repeatsof trpEGpseudogenes.Appl. Environ. Mi-crobiol. 62:332–39

65. Liadouze I, Febvay G, Guillaud J, BonnotG. 1995. Effect of diet on the free aminoacid pools of symbiotic and aposymbioticpea aphids,Acyrthosiphon pisum. J. In-sect Physiol.41:33–40

66. Mittler TE. 1971. Dietary amino acidrequirement of the aphidMyzus persi-caeaffected by antibiotic uptake.J. Nutr.101:1023–28

67. Montllor CB. 1991. The influence ofplant chemistry on aphid feeding be-havior. In Insect-Plant Interactions,ed.EA Bernays, 3:125–73. Boca Raton, FL:CRC Press

68. Moran NA. 1992. Evolution of aphid lifecycles.Annu. Rev. Entomol.37:321–28

Ann

u. R

ev. E

ntom

ol. 1

998.

43:1

7-37

. Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

Wes

tern

Mic

higa

n U

nive

rsity

on

01/1

7/12

. For

per

sona

l use

onl

y.



36 DOUGLAS

69. Moran NA, Baumann P. 1994. Phylo-genetics of cytoplasmically-inherited mi-croorganisms of arthropods.Trends Ecol.Evol.9:15–20

70. Moran NA, Munson MA, Baumann P,Ishikawa H. 1993. A molecular clock inendosymbiotic bacteria is calibrated us-ing the insect hosts.Proc. R. Soc. Lond. B253:167–71

71. Munderloh UG, Kurtti TJ. 1995. Cellu-lar and molecular interrelationships be-tween ticks and prokaryotic tick-bornepathogens.Annu. Rev. Entomol.40:221–43

72. Munson MA, Baumann P. 1993. Molec-ular cloning and nucleotide sequence ofa putativetrpDC(F)BA operon inBuch-nera aphidicola (endosymbiont of theaphid Schizaphis graminum.J. Bacteriol.175:6426–32

73. Munson MA, Baumann P, Clark MA,Baumann L, Moran NA, et al. 1991. Ev-idence for the establishment of aphid-eubacterium endosymbiosis in an ances-tor of four aphid families.J. Bacteriol.173:6321–24

74. Munson MA, Baumann P, Kinsey MG.1991.Buchneragen. nov. andBuchneraaphidicola sp. nov., a taxon consistingof the mycetocyte-associated, primary en-dosymbionts of aphids.Int. J. Syst. Bac-teriol. 41:566–68

75. Munson MA, Baumann P, Moran NA.1992. Phylogenetic relationships ofthe endosymbionts of mealybugs (Ho-moptera: Pseudococcidae) based on 16SrDNA sequences.Mol. Phylogen. Evol.1:26–30

76. Noda H, Kodama K. 1996. Phylogeneticposition of yeastlike endosymbionts ofanobiid beetles.Appl. Environ. Microbiol.62:162–67

77. Noda H, Nakashima N, Koizumi M.1995. Phylogenetic position of yeast-like symbiotes of rice planthoppers basedon partial 18S rDNA sequences.InsectBiochem. Mol. Biol.25:639–36

78. Potrikus CJ, Breznak JA. 1981. Gut bacte-ria recycle uric acid nitrogen in termites:a strategy for nutrient conservation.Proc.Natl. Acad. Sci USA78:4601–5

79. Prosser WA, Douglas AE. 1991. Theaposymbiotic aphid: an analysis ofchlortetracycline-treated pea aphid,Acyrthosiphon pisum. J. Insect Physiol.37:713–19

80. Prosser WA, Douglas AE. 1992. A test ofthe hypotheses that nitrogen is upgradedand recycled in an aphid (Acyrthosiphonpisum) symbiosis. J. Insect Physiol.38:93–99

81. Prosser WA, Simpson SJ, Douglas AE.1992. How an aphid (Acyrthosiphonpisum) symbiosis responds to variationin dietary nitrogen.J. Insect Physiol.38:301–7

82. Rahbe Y, Delobel B, Febvay G, Chante-grel B. 1994. Aphid-specific triglyc-erides in symbiotic and aposymbioticAcyrthosiphon pisum. Insect Biochem.Mol. Biol. 24:95–101

83. Rouhbakhsh D, Lai C-Y, von Dohlen C,Clark MA, Baumann L, et al. 1996. Thrtryptophan biosynthetic pathway of aphidendosymbionts (Buchnera): genetics andevolution of plasmid-associated anthrani-late synthase (trpEG) within the Aphidi-dae.J. Mol. Evol.42:414–21

84. Roubakhsh D, Moran NA, Baumann L,Voegtlin DJ, Baumann P. 1994. Poly-merase chain reaction-based identifica-tion of Buchnera: the primary prokary-otic endosymbiont of aphids.Insect Mol.Biol. 3:213–17

85. Ruby EG. 1996. Lessons from a coop-erative bacterial-animal association: theVibrio fischeri–Euprymna scolopeslightorgan symbiosis.Annu. Rev. Microbiol.50:591–624

86. Sander K. 1977. Anomalien der Oogeneseund Embryogenese symbiontenfuhrenderKleinzikaden unter Tetracyclin-Einfluss.Verhand. Deutsch. Zool. Gesell.1977:306

87. Sasaki T, Aoki T, Hayashi H, IshikawaH. 1990. Amino acid composition of thehoneydew of symbiotic and aposymbioticpea aphidsAcyrthosiphon pisum. J. InsectPhysiol.36:35–40

88. Sasaki T, Hayashi H, Ishikawa H. 1991.Growth and reproduction of the sym-biotic and aposymbiotic pea aphids,Acyrthosiphon pisummaintained on ar-tificial diets.J. Insect Physiol.37:749–56

89. Sasaki T, Ishikawa H. 1995. Production ofessential amino acids from glutamate bymycetocyte symbionts of the pea aphid,Acyrthosiphon pisum. J. Insect Physiol.41:41–46

90. Sasaki T, Kawamura M, Ishikawa H.1996. Nitrogen recycling in the brownplanthopper, Nilaparvata lugens: in-volvement of yeast-like endosymbionts inuric acid metabolism.J. Insect Physiol.42:125–30

91. Schroeder D, Deppisch H, ObermayerM, Krohne G, Stackbrandt E, et al.1996. Intracellular endosymbiotic bac-teria of Camponotusspecies (carpenterants): systematics, evolution and ultra-structural characterization.Mol. Micro-biol. 21:479–89

92. Schwemmler W, Duthoit JJ, Kuhl G,

Ann

u. R

ev. E

ntom

ol. 1

998.

43:1

7-37

. Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

Wes

tern

Mic

higa

n U

nive

rsity

on

01/1

7/12

. For

per

sona

l use

onl

y.




Vago C. 1974. Endosymbionts: factors ofegg pattern formation.J. Insect Physiol.20:1467–74

93. Srivastava PN, Gao Y, Levesque J, Au-clair JL. 1985. Differences in amino acidrequirement between two biotypes of thepea aphid,Acyrthosiphon pisum. Can. J.Zool.63:603–6

94. Tjallingii WF. 1995. Regulation ofphloem feeding by aphids. InRegulatoryMechanisms in Insect Feeding,ed. RFChapman, G de Boer, pp. 190–209. NewYork: Chapman & Hall

95. Unterman BM, Baumann P, McLean DL.1989. Pea aphid symbiont relationshipsestablished by analysis of 16S rRNAs.J.Bacteriol.171:2970–74

96. van den Heuvel JFJM, Verbeek M, van derWilk F. 1994. Endosymbiotic bacteria as-sociated with circulative transmission ofpotato virus byMyzus persicae. J. Gen.Virol. 75:2559–65

97. van Rhijn P, Vanderleyden J. 1995. TheRhizobium-plant symbiosis.Microbiol.Rev.59:124–42

97a. von Amiressami M, Petzold H. 1976.Symbioseforschung und Insektizidresis-tenz. Ein licht- und elektronenmikro-scopisher Beitrag zur Klarung der Insek-tizidresistenz von Aphiden unter Beruck-sichtigung der Mycetom-Symbionten beiMyzus persicaeSulz. Angew. Entomol.82:252–59

98. Walters FS, Mullin CA. 1994. Biosyn-thetic alternatives for acetogenic produc-

tion of sorbic acid in the potato aphid(Homoptera: Aphididae).Arch. InsectBiochem. Physiol.27:249–64

99. Walters FS, Mullin CA, Gildow FE. 1994.Biosynthesis of sorbic acid in aphids: aninvestigation into symbiont involvementand potential relationships with aphid pig-ments. Arch. Insect Biochem. Physiol.26:49–67

100. Whitehead LF, Douglas AE. 1993. Po-pulations of symbiotic bacteria in theparthenogenetic pea aphid (Acyrthosi-phon pisum) symbiosis.Proc. R. Soc.Lond. B254:29–32

101. Whitehead LF, Douglas AE. 1993. Ametabolic study ofBuchnera, the intracel-lular bacterial symbionts of the pea aphid,Acyrthosiphon pisum. J. Gen. Microbiol.139:821–26

102. Whitehead LF, Wilkinson TL, DouglasAE. 1992. Nitrogen recycling in the peaaphid (Acyrthosiphon pisum) symbiosis.Proc. R. Soc. Lond. B250:115–17

103. Wilkinson TL, Douglas AE. 1995. Aphidfeeding, as influenced by the disruption ofthe symbiotic bacteria.J. Insect Physiol.41:635–40

104. Wilkinson TL, Douglas AE. 1995. Whyaphids lacking symbiotic bacteria have el-evated levels of the amino acid glutamine.J. Insect Physiol.41:921–27

105. Wilkinson TL, Douglas AE. 1996. Theimpact of aposymbiosis on amino acidmetabolism of pea aphids.Entomol. Exp.Appl.80:279–82

Ann

u. R

ev. E

ntom

ol. 1

998.

43:1

7-37

. Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

Wes

tern

Mic

higa

n U

nive

rsity

on

01/1

7/12

. For

per

sona

l use

onl

y.

nutritional interactions in insect-microbial symbioses

Documents