Role of Host Nutrition in Symbiont Regulation: Impact of Dietary Nitrogen on Proliferation of Obligate and Facultative Bacterial Endosymbionts of the Pea Aphid Acyrthosiphon pisum

The impact of host nutrition on symbiont regulation in the peaaphid Acyrthosiphon pisum was investigated. The population densityof the obligate symbiont Buchnera aphidicola positively correlatedwith dietary nitrogen levels. In contrast, the population densityof the facultative symbiont Serratia symbiotica increased inaphids reared on low-nitrogen diets, indicating distinct regulatorymechanisms in the same insect host.

INTRODUCTION

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Introduction

The endosymbiotic bacterial partners of aphids (Insecta: Sternorrhyncha)fall into two categories: the obligate "primary" symbiont Buchnerasp. found in almost all aphids and the facultative "secondary"bacteria whose presence is not universal (3, 6, 16). The associationbetween aphids and Buchnera sp. is well documented: the bacteriaare housed in specialized host cells, the bacteriocytes or mycetocytes,and they supplement the insects' diet through the provisionof essential amino acids (see reference 6 for a full review).In contrast, the association between aphids and their secondarysymbionts is less well understood, although the presence ofsecondary bacteria in symbiosis alongside Buchnera sp. has beenknown for many years (3, 13). The secondary bacteria are transmittedvertically between host generations (3, 4), but their distributionpatterns within and between aphid populations suggest that occasionalhorizontal transmission must have occurred (5, 19).

The infection density of endosymbionts is one of the most importantfactors for understanding their biological effects. A reductionin infection density may result in imperfect vertical transmissionand attenuated phenotypic effects, which could lead to lossof the infection in host populations. Excessive infection densitymay lead to enhanced negative or positive phenotypic effectson the host that could significantly influence host fitnessand, at their extreme, could cause pathological damage. Theproliferation of endosymbionts relies on the consumption ofresources from the insect body; consequently, infection densitiesmay be significantly influenced by the nutritional conditionof the host. To date, however, no studies have investigatedthe impact of nutrition on the population density of insectendosymbionts.

In this study, the impact of nutrition on the population densityof the obligate symbiont Buchnera aphidicola (17) and the facultativesymbiont Serratia symbiotica (16) was investigated using thepea aphid Acyrthosiphon pisum. Two clonal lineages of pea aphidwere used: (i) clone IS, a naturally S. symbiotica-infectedline (9), and (ii) clone AIST^IS, in which the S. symbioticainfection was generated artificially by hemolymph injection(10). Both of these aphid clones also contain B. aphidicola.Continuous infection with S. symbiotica for all aphid generationsand the absence of any other facultative symbionts were confirmedby PCR assay and observation by light microscopy. The nutritionalcondition of the insects was altered by rearing cohorts of geneticallyidentical aphids for the first 7 days of nymphal developmenton chemically defined artificial diets containing final totalnitrogen concentrations of 25, 50, 100, and 150 mM. Diet preparationand composition were identical to those described previously(21), with 50 mol percent essential amino acid content and 500mM sucrose.

Insect performance.

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Insect performance.

Both aphid clones IS and AIST^IS settled and fed on the diets,as indicated by the excretion of honeydew and regular productionof exuviae. After 7 days feeding on the diets a small numberof aphids on the 150 mM nitrogen diet had molted to the adultstadium. The other aphids feeding on the 150 mM nitrogen dietand all aphids reared on the 100 mM nitrogen diet were in thefourth nymphal stadium. Aphids on the 25 mM and 50 mM nitrogendiets were noticeably smaller and had not molted beyond thethird nymphal stadium. The weight gain of the insects positivelycorrelated with the total nitrogen concentration in the diet(Fig. 1) such that aphids reared on the 150 mM diet were approximatelythreefold larger than aphids reared on the 25 mM diet. In addition,clone AIST^IS was slightly larger than clone IS at all nitrogenconcentrations (see "clone" data in the legend to Fig. 1). Thesedata highlight the importance of nitrogen as a limiting factorin the nutrition of aphids and other phytophagous insects (2,12, 18). The remainder of this paper examines how the nutritionalcondition of the host, as influenced by dietary nitrogen, impactspopulations of obligate and facultative endosymbiotic bacteria.

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FIG. 1. The impact of total dietary nitrogen on the body weight of fresh aphids reared on chemically defined diets for 7 days. Open bars, clone IS; closed bars, clone AIST^IS; all data represent means ± standard errors (n = 5). ANOVA results: clone, F_(1,32) = 7.8 (0.01 > P > 0.001); diet, F_(3,32) = 175.8 (P < 0.001); interaction, F_(3,32) = 1.4 (P > 0.05).

Symbiont populations in the aphids.

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Symbiont populations in the...

The bacteria in the aphids were quantified by quantitative real-timePCR using a TaqMan PCR core reagent kit and an ABI 7700 system(Applied Biosystems) as previously described (10), and the resultsare reported in Table 1. This approach quantifies the numberof bacterial genomes within an insect rather than the absolutenumber of bacteria: the B. aphidicola genome (but not the S.symbiotica genome) is thought to be remarkably polyploid (11).The microbiota were quantified from aphids of the same chronologicalage (7 days) to limit such confounding effects of ploidy anddevelopmental time on the different diets (see above), and individualaphid fresh weight was used to convert absolute numbers of bacterialgenomes to genome densities (preliminary results indicated thatinsect cell number, as quantified in terms of elongation factor1 ${alpha}$ , was not a reliable indicator of aphid size, probably dueto differences in the rate of ovariole development on the differentdiets).

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TABLE 1. Bacterial populations in 7-day-old pea aphids reared on chemically-defined diets of different total nitrogen concentrations^a

B. aphidicola genome number was quantified in terms of groELgene copies by use of primers BuchGroEL-AF1 (5'-CAGCAACATTATTAGCACAATCTATAGTAAAT-3')and BuchGroEL-AR1 (5'-TGATAACAGCTTTATCAATTCCACGT-3') in combinationwith the fluorescently labeled probe BuchGroEL-TP1 (5'-AAGCAGTAGCAGCTGGTATGAATCCAATGG-3').The titers of B. aphidicola were not significantly differentin the aphid clones and positively correlated with the totalnitrogen concentration in the diet (see "clone" and "diet" data,respectively, in analysis of variance [ANOVA] results), althougha clone-dependent difference was observed in aphids reared onthe 150 mM diet. The general consistency of results obtainedfor the aphid clones indicates that variability in the degreeof ploidy of the B. aphidicola genome is unlikely to be theunderlying cause of the observed patterns, although this possibilitycannot be excluded. The genome density of B. aphidicola in bothclones IS and AIST^IS also tended to increase with dietary nitrogenconcentration, but the significant interaction term in the ANOVAagain highlights the reduction in B. aphidicola density in cloneIS on the 150 mM diet. Although little is known about the regulatorymechanisms that maintain B. aphidicola densities within aphids,these results demonstrate that (i) the proliferation of B. aphidicolais linked to the availability of nitrogen and (ii) the densityof B. aphidicola is not maintained at a fixed level and canvary, possibly to meet the nutritional demands of the host.In particular, there is likely to be a high demand for the metabolicrepertoire of the bacteria in the rapidly growing aphids rearedon diets containing 150 mM nitrogen compared to the resultsseen with aphids reared on diets containing 25 mM nitrogen thatgrow very slowly. An additional factor is the increasing contributionof the reproductive tissues to aphid size with both developmentaland chronological age. The proliferation of B. aphidicola islinked to reproductive condition—the density of B. aphidicolareaches a peak in actively reproducing young adults and declinesin postreproductive adults (10). This relationship may explainthe decrease in B. aphidicola density in clone IS reared onthe 150 mM diet, although degrees of reproductive investmentdid not differ significantly between aphids reared on the differentdiets (T. L. Wilkinson, unpublished results). The symbiosisis known to disintegrate in postadult aphids through a reductionin the number of B. aphidicola cells and in the number of bacteriocytes(1, 8) and a decrease in the bacterial division rate in fourthinstar larvae (20). Whatever the mechanisms involved, the regulationof B. aphidicola density on a day-to-day basis appears to befine tuned to meet the metabolic demands of the host, as wouldbe expected given the long evolutionary history of the host-symbiontassociation (15).

S. symbiotica is considered a facultative bacterial symbiontbecause its presence is not required for the survival and reproductionof the host insect (16). The population dynamics of S. symbioticafollow a simple logistic growth pattern during aphid development(10), suggesting a lack of strict control over the proliferationof the facultative symbiont. In the present study, S. symbioticagenome numbers were quantified in terms of groEL gene copiesby use of primers PASSGroE-AF1 (5'-CCTCAAGGCTGTGGCCG-3') andPASSGroE-AR1 (5'-GAGTTTGCAGAGATGGTGCCTA-3') in combination withthe fluorescently labeled probe PASSGroE-TP1 (5'-AAGCAGTTGTTGCGGCGGTTGAA-3').The titer of S. symbiotica in the insects was an order of magnitudelower than that of B. aphidicola, reflecting both the numericaldominance and the genome polyploidy of B. aphidicola (11). WhileS. symbiotica titers were variable, particularly in clone AIST^IS,there was no consistent pattern with respect to total nitrogenconcentration in the diet. However, the number of S. symbioticain clone AIST^IS was significantly higher than in clone IS (seemain effect "clone" in ANOVA). The population density of S.symbiotica in clone AIST^IS was also significantly higher thanin clone IS, and in both clones the density of S. symbioticatended to decrease as dietary nitrogen concentration was elevated.The high population density of S. symbiotica in aphids rearedon diets containing low concentrations of nitrogen was reflectedin the contribution of S. symbiotica to total bacterial load.In clone IS, S. symbiotica represented approximately 4% of thetotal endosymbiont population when the aphids were reared ondiets containing 25 mM total nitrogen, equivalent to a 3.2-foldincrease compared to aphids reared on the 150 mM diet. Similarly,S. symbiotica contributed over 8% of the endosymbiont populationin clone AIST^IS reared on a diet containing 25 mM total nitrogen,a 5.5-fold increase over aphids reared on the 150 mM diet. Theincrease in S. symbiotica when aphid performance is limitedby dietary nitrogen is surprising, since intuitively the proliferationof S. symbiotica must be dependent on nutritional resourcesfrom the aphid, the availability of which will be strongly affectedby the quality of the diet. The most plausible explanation isthat the proliferation rate of S. symbiotica is constant andindependent of insect body size or nutritional condition, suchthat the density of S. symbiotica is higher in smaller insectsof the same chronological age reared on the nutritionally restricteddiets, as was observed. Similarly, one would expect newly emergedadult aphids (i.e., of the same developmental age) reared onthe 25 mM diet to contain larger populations of S. symbioticathan aphids reared on the 150 mM diet, since development timeis extended by approximately 6 days on the nitrogen-restricteddiet. However, this was not observed in supplemental experiments(data not shown) in which there was no significant differencebetween the titers of S. symbiotica from adult aphids from eitherclone reared on the 25 mM and 150 mM nitrogen diets, suggestingthat the proliferation of S. symbiotica is not independent ofregulatory controls. Further experiments are required to determine(i) whether the response of other S. symbiotica strains to dietarynitrogen, from A. pisum and from other aphid species, is thesame as reported here and (ii) whether other factors linkedto nutritional stress, such as an impaired immune response,may be responsible for the proliferation of S. symbiotica.

Endosymbiont transmission.

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Endosymbiont transmission.

The density of S. symbiotica, but not that of B. aphidicola,was higher in the aphid clone AIST^IS than in clone IS. The differencebetween the clones was consistent in aphids reared on the chemicallydefined diets (Table 1) and was also observed in 7-day-old aphidsreared on the host plant Vicia faba (Table 2), indicating thatthe higher density of S. symbiotica arises from a differencebetween the aphid clones. To explore possible mechanisms thatmight give rise to this interclonal variation, the transmissionof S. symbiotica to the parthenogenetic embryo was investigated.Early embryos were dissected from adult aphids reared on theplant and subjected to whole-mount fluorescent in situ hybridizationas previously described (16). B. aphidicola and S. symbioticawere detected by specific probes targeting their 16S rRNA: Cy5-ApisP2a(Cy5-5'-CCTCTTTTGGGTAGATCC-3') and Cy3-PASSisR (Cy3-5'-CCCGACTTTATCGCTGGC-3'),respectively. Host cell nuclei were stained with SYTOX Green(Molecular Probes). The specimens were mounted in SlowFade antifadesolution (Molecular Probes) and observed under a laser confocalmicroscope (Pascal 5; Carl Zeiss). The localizations and densitiesof B. aphidicola were generally similar between the aphid clones(see Fig. 2), but in contrast, the number of S. symbiotica cellsinfecting embryos of the clone AIST^IS was much greater thanthat infecting clone IS. At later stages of embryonic development,the S. symbiotica infection in clone IS remained localized tothe interstitial areas between bacteriocytes and was at a muchlower density than the infection in clone AIST^IS, in which S.symbiotica occupied the same tissue location as B. aphidicola.These findings suggest that efficient transmission and rapidproliferation of S. symbiotica in the clone AIST^IS may be thecause of the higher bacterial density observed in this study,a result which is probably linked to the history of the symbioticassociation and/or the genetic background of the host insect.

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TABLE 2. Bacterial populations in 7-day-old pea aphids Acyrthosiphon pisum reared from birth on the host plant Vicia faba cv. The Sutton^a

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FIG. 2. Symbiont infection in early embryos of the clones IS and AIST^IS. (A) A stage 8 embryo of clone IS; (B) a stage 8 embryo of clone AIST^IS; (C) a stage 12 embryo of clone IS; (D) a stage 12 embryo of clone AIST^IS. Green, red, and blue signals indicate B. aphidicola, S. symbiotica, and nuclei of host cells, respectively. Bars, 50 µm. At stage 8, embryos are infected with the symbionts, and at stage 12, a large syncytial cytoplasm harboring the symbionts cellularizes into many bacteriocytes. Developmental staging of A. pisum is according to reference 14.

In this study, we demonstrated that, although cohabiting inthe same insect, the obligate symbiont B. aphidicola and thefacultative symbiont S. symbiotica respond to nutritional stressimposed on the host in different ways. This finding highlightsthe complex host-symbiont and symbiont-symbiont interactionsin this endosymbiotic ecosystem and indicates the nutritional,physiological, and ecological relevance of endosymbiotic associatesin natural insect populations. The natural diet of aphids isphloem sap, the availability and quality of which may vary temporallyfrom diurnal changes through to seasonal changes and spatiallybetween different parts of the plant and even different sieveelements (see reference 7 for a review). In polyphagous aphids,host plants of different taxonomic affiliation provide furthervariations in the quality and quantity of the food source. Undernatural conditions, therefore, the endosymbiotic microbiotain aphids must be influenced by these nutritional fluctuations,possibly as observed in this study. Of particular relevanceis the observation that the infection density of an unidentifiedfacultative symbiont in the polyphagous aphid Aphis fabae wassignificantly higher on a host plant on which the aphid exhibitedthe poorest performance (22).

The biological consequences of such up- or down-regulation ofendosymbiont populations are difficult to predict a priori,since a number of factors are affected simultaneously. For example,increasing the population density of a facultative symbiontcould enhance both negative and positive aspects of the symbiontinfection by acting directly on the host insect or at the sametime indirectly by influencing the host via B. aphidicola orother microbial associates. These considerations are relevantto virtually all aphid species; B. aphidicola is ubiquitousin aphids that feed on phloem sap, and many aphids, but notall, possess facultative secondary bacteria (16). Indeed, thereis also relevance for the approximately 10% of insect speciesthat utilize the metabolic capabilities of symbiotic microorganismsin their nutrition. In summary, to improve our understandingof the physiology and ecology of aphids and other insects, wemust take into account the complex interplay between the hostinsect, obligate and facultative symbionts, and the environment.

ACKNOWLEDGMENTS

Financial support was provided by the Japan Society for thePromotion of Science.

FOOTNOTES

* Corresponding author. Mailing address: UCD School of Biology and Environmental Science, University College Dublin, Belfield, Dublin 4, Ireland. Phone: 353 1 7162264. Fax: 353 1 7161152. E-mail: tom.wilkinson{at}ucd.ie.

Published ahead of print on 8 December 2006.

REFERENCES

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