On the Functional Significance of Symbiotic Microorganisms in The

Physiological Entomology (2001) 26, 86±93 On the functional signi®cance of symbiotic microorganisms in the Homoptera: a comparative study of Acyrthosiphon pisum and Nilaparvata lugens T. L. WILKINSON andH. ISHIKAWA Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Bunkyo ku, Tokyo 113, Japan Abstract. All phloem-feeding Homoptera possess symbiotic microorganisms. Although the phylogenetic position and anatomical location of the microorganisms differ, the underlying theme of the symbiosis is the same; the microorganisms improve the nutritional quality of the diet through the provision of essential amino acids. The symbiosis has been well documented in aphids, but little information is available from other homopteran groups. The impact of the loss of bacterial symbionts in the pea aphid Acyrthosiphon pisum Harris and eukaryotic yeast-like symbionts in the Asian rice brown planthopper Nilaparvata lugens StaÊl was examined in parallel. The weight and relative growth rate of aphids and planthoppers was signi®cantly reduced by symbiont loss, and characteristic features of aposymbiotic pea aphids, so-called `metabolic signatures', were, for the ®rst time, observed in aposymbiotic N. lugens. For example, the amount of protein per unit fresh weight was reduced by 26 and 10%, and the free amino acid levels increased 1.8- and 1.4-fold, in aposymbiotic A. pisum and N. lugens, respectively. In addition, the concentration of the amino acid glutamine was elevated in the tissues of aposymbiotic insects. The data are discussed in the context of our current understanding of the nutritional role of the symbiosis and the mechanisms of nitrogen metabolism in the two insect species. It is concluded that the metabolic adjustments of the insects to symbiont loss are broadly equivalent. Key words. Amino acids, aphid, aposymbiotic, bacteria, insect, planthopper, symbiosis, yeast-like symbionts. Introduction cells, a diet with a more balanced nutritional content, have secondarily lost the symbiosis. The implication is clear. To All phloem-feeding members of the Homoptera possess quote Douglas (1998): `the microbial symbiosis is causally symbiotic microorganisms. Although the diversity, location linked with phloem feeding'. and phylogenetic position of the symbionts varies (see The symbiosis in aphids (Sternorrhyncha: Aphidoidea) has Buchner, 1965; Douglas, 1989), the underlying theme of the been extensively studied. In these insects the symbiotic symbiosis is the provision of essential amino acids. Phloem sap partners are bacteria located in specialized cells, called has an unbalanced amino acid composition dominated by non- mycetocytes or bacteriocytes, loosely aggregated through the essential amino acids, and there is now substantial evidence abdominal haemolymph (see Douglas, 1998, for full review). that the microorganisms in phloem-feeding insects synthesize The bacteria have been assigned to the genus Buchnera essential amino acids that are made available to the insect host (Munson et al., 1991). The impact of symbiont loss has also (see Douglas, 1989, 1998; Baumann et al., 1995, for full been well documented (e.g. Wilkinson, 1998). For example, reviews). Interestingly, those Homoptera, such as typhlocybine bacteria-free or aposymbiotic pea aphids Acyrthosiphon pisum, leafhoppers, which have switched to feeding on intact plant produced by mild antibiotic therapy, have a reduced growth rate, attain a lower adult size, and their reproductive output is Correspondence: School of Biosciences, University of Birmingham, curtailed dramatically (Sasaki et al., 1991; Douglas, 1992). In Edgbaston, Birmingham B15 2TT, U.K. Tel.: + 44 (0)121 4145481; addition, they exhibit so-called metabolic `signatures' which fax: + 44 (0)121 4145925; e-mail: [email protected] can be attributed directly to the loss of their symbiotic partners. 86 # 2001 Blackwell Science Ltd Symbiotic microorganisms in aphids and planthoppers 87 In particular, the concentration of free amino acids in the impact of symbiont loss in A. pisum and N. lugens, tissues of aposymbiotic aphids is elevated, but the levels of respectively. protein are reduced, suggesting that protein synthesis is limited by a lack of essential amino acids (Prosser & Douglas, 1991; Liadouze et al., 1995; Wilkinson & Douglas, 1996). The amino Materials and Methods acid glutamine is also signi®cantly elevated in the tissues of aposymbiotic aphids, even when the insects are reared on diets Insect material which contain no glutamine. Loss of the symbiosis induces an elevated ammonia load in aposymbiotic aphids, and the The insects used in this study were derived from long-term accumulation of glutamine has been linked to the detoxi®ca- cultures maintained at the University of Tokyo. Acyrthosiphon tion of ammonia through its incorporation into glutamate pisum clone TLW97/TU9 was maintained as asexual viviparae (Wilkinson & Douglas, 1995a). The robust nature of these on Vicia faba seedlings at 20°C, in LD 18 : 6 h regime, and metabolic signatures in aphids has been con®rmed by analysis N. lugens was maintained as a sexually reproducing colony on of similar responses in the black bean aphid Aphis fabae Oryza sativa seedlings at 25°C, in LD 18 : 6 h conditions. (Adams et al., 1996; Adams, 1996) Although there is little detailed information from other homopteran groups concerning the functional signi®cance of Production of aposymbiotic insects the symbiotic microorganisms and the response of the insect host to symbiont loss, it is clear that the Buchnera in pea The bacterial symbionts in A. pisum were disrupted with an aphids and other members of the Aphididae are not exceptional oral dose of the antibiotic rifampicin, administered via in their ability to improve the nutritional quality of phloem sap chemically de®ned diets (at 50 mg/mL diet) over the ®rst for their insect hosts. The study reported here addresses this 2 days of nymphal development. The diet was formulation A of issue in two phylogenetically distant insects, the pea aphid Prosser & Douglas (1992), and full details of the antibiotic Acyrthosiphon pisum Harris (Sternorrhyncha: Aphididae) and technique are given in RahbeÂ et al. (1994). The yeast-like the Asian rice brown planthopper Nilaparvata lugens StaÊl symbionts in N. lugens were disrupted by rearing newly (Auchenorryncha: Delphacidae), and represents, to our know- hatched nymphs at 35°C for 3 days (Noda & Saito, 1979; Chen ledge, the ®rst direct comparison of the requirement for et al., 1981; Sasaki et al., 1996). These methods have symbiotic microorganisms in insects from different suborders previously been demonstrated as effective in targeting the within the Homoptera. Both A. pisum and N. lugens feed on symbiotic microorganisms with little or no deleterious effects phloem sap containing low concentrations of essential amino on the insects themselves (see Wilkinson, 1998, for a review of acids (Hayashi & Chino, 1990; Douglas, 1993; SandstroÈm & antibiotic-treatment in aphids, and Chen et al., 1981 and Sasaki Pettersson, 1994) and possess symbiotic microorganisms that et al., 1996, for studies on the impact of heat-treatment in supplement the diet through the provision of essential amino eliminating the yeast-like symbionts from N. lugens). In both acids (Douglas, 1998; Koyama, 1985). However, the identity cases, symbiotic control insects were maintained under parallel of the microorganisms differs considerably. Acyrthosiphon conditions as aposymbiotic insects, but without the disrupting pisum contains well-documented populations of bacteria factor, i.e. symbiotic aphids were reared on chemically de®ned (Buchnera; see above), whereas the symbionts in N. lugens diets lacking antibiotic for the ®rst 2 days of development, and are eukaryotic and have historically been termed `yeast-like symbiotic planthoppers were reared under standard culture symbionts' (e.g. Noda, 1974). Recent molecular studies have re®ned this classi®cation and have placed the symbionts of conditions. N. lugens (and symbionts from other members of the To obtain insects of uniform age, two approaches were Delphacidae) in the class Pyrenomycetes in the subphylum adopted, re¯ecting the different biology of the two species. For Ascomycotina (Noda et al., 1995; this study will refer to these aphids, adult apterae were isolated on fresh plants and any microorganisms as yeast-like symbionts). In addition, the offspring produced after 16 h were collected and transferred to yeast-like symbionts in the small brown planthopper chemically de®ned diets (see above). For planthoppers, adult Laodelphax striatellus have been implicated in the production insects were allowed to oviposit on clean rice seedlings for of 24-methylenecholesterol (Noda et al., 1979), whereas there 3 days. Preliminary experiments indicated that maximum egg is de®nitive evidence only for essential amino acid provision- hatch occurred approximately 10 days after oviposition; any ing by the Buchnera in aphids. insects that hatched prior to the main collection day were The diversity of the microorganisms in the two species removed to ensure uniformity of age within the experimental dictated the experimental design adopted in this study because animals. Insects hatching within a 16-h `window' on the tenth the symbionts in N. lugens are not susceptible to antibiotic day after oviposition were transferred to clean seedlings at therapy. Disruption of the eukaryotic symbionts can be either 25°C (symbiotic insects) or 35°C (aposymbiotic

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