Provision of riboflavin to the host aphid, Acyrthosiphon pisum, by endosymbiotic bacteria, Buchnera

Journal of Insect Physiology 45 (1999) 1–6

Provision of riboflavin to the host aphid, Acyrthosiphon pisum, by endosymbiotic bacteria, Buchnera Atsushi Nakabachi, Hajime Ishikawa

*

Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan Received 30 March 1998; received in revised form 3 June 1998; accepted 3 June 1998

Abstract Differential cDNA display and quantitative RT-PCR suggested that the riboflavin synthase complex of the aphid endosymbiont, Buchnera, is active only when the symbiotic system is maintained and well organized in young hosts. Since this finding suggested the provision of riboflavin by Buchnera, we examined the effect of dietary riboflavin on the performance of symbiotic and aposymbiotic aphids using chemically-defined diets. Our results indicate: (1) dietary riboflavin is slightly detrimental to young, symbiotic aphids; (2) dietary riboflavin is essential to aposymbiotic aphids; (3) dietary riboflavin remarkably improves the performance of aposymbiotic aphids. These results strongly suggest that young, symbiotic aphids are provided with riboflavin by their endosymbionts, Buchnera.  1998 Elsevier Science Ltd. All rights reserved. Keywords: Pea aphid; Endosymbiont; Buchnera; Differential cDNA display; Riboflavin

1. Introduction The intracellular symbiosis in the aphid bacteriocyte represents one of the most intimate interactions between a bacterium and the eukaryotic cell (Buchner, 1965; Ishikawa, 1989; Baumann et al., 1995). The endosymbionts Buchnera (Munson et al., 1991) are maternally transmitted between generations of the host insect, just like eukaryotic cell organelles. However, this intimate symbiosis tends to disintegrate as the host ages (Ishikawa, 1984). In a previous study, taking note of this phenomenon, we compared mRNA populations in the bacteriocytes of young and old pea aphids, Acyrthosiphon pisum (Nakabachi and Ishikawa, 1997), using differential cDNA display (Liang and Pardee, 1992) and quantitative RT-PCR (Noonan et al., 1990). As a result, we were successful in demonstrating that several mRNA species related to nitrogen recycling (Sasaki and Ishikawa, 1995) are specifically detected in the symbiotic system that is well organized in young insects. In this study, we further compare mRNA populations of young and old bacteriocytes by the same methods,

* Corresponding author. [email protected]

Fax:

+

81-3-5800-3553;

E-mail:

and demonstrate that mRNA for putative riboflavin synthase ␤ chain of Buchnera is also abundant only in young bacteriocytes. Since all animals, including insects, are unable to synthesize the isoalloxazine ring (Miller and Silhacek, 1995), it is possible that riboflavin is synthesized and supplied to aphids by Buchnera. To test this possibility, the effect of dietary riboflavin on the growth of aposymbiotic aphids was examined using chemicallydefined synthetic diets. The results clearly indicate that riboflavin, which is essential to the host’s growth, is provided by Buchnera.

2. Materials and methods 2.1. Insects A long-established parthenogenetic clone of the pea aphid, Acyrthosiphon pisum Harris, was maintained on young broad bean plants, Vicia faba L. at 15°C in a longday regime of 16 h light and 8 h dark (Ishikawa, 1982). The insects for differential cDNA display (DD) analysis were collected within 24 h after larviposition by apterous mothers. These nymphs were described as 0-day aphids. Once the aphids reached adulthood, they were trans-

0022–1910/98/$ - see front matter  1998 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 2 - 1 9 1 0 ( 9 8 ) 0 0 1 0 4 - 8

2

A. Nakabachi, H. Ishikawa / Journal of Insect Physiology 45 (1999) 1–6

ferred twice a week to fresh plants in order to keep the nutritional conditions constant. Aposymbiotic aphids were obtained by rifampicin injection (Ishikawa, 1982). The adult aphids were anaesthetized in a stream of carbon dioxide and injected with 0.1 ␮l of rifampicin at 200 ␮g/ml (about 5 ng/mg body weight). After injection, they were transferred to synthetic diets with or without riboflavin, and allowed to deposit offspring for 2 days. Larvae born during this period were discarded, since short-term treatment with rifampicin may not eliminate Buchnera completely. Offspring produced during the next 24 h were described as 0-day aposymbiotic aphids and were used for nutritional studies. Elimination of Buchnera was verified by genomic PCR using primers specific to Buchnera 16S rRNA gene (Nakabachi and Ishikawa, 1997). 2.2. Synthetic diets Synthetic diets used in this study were prepared according to Sasaki et al. (1991) except that riboflavin was omitted from the riboflavin-free diet. The diet solution was filtered through MILLEX GV 0.22 ␮m filter units (MILLIPORE) and aseptically enclosed in stretched Parafilm (American National Can) as described by Srivastava and Auclair (1971). The cages used for experiments were plastic dishes (35 mm dia, 10 mm high). 2.3. Isolation of bacteriocytes The aphids were dissected in a drop of buffer A [35 mM Tris–HCl (pH 7.5), 25 mM KCl, 10 mM MgCl2, 250 mM sucrose; Ishikawa, 1982] on a glass slide. Bacteriocytes, freed from the insect body were collected by suction with a thin glass capillary connected to a peristaltic pump (Sasaki and Ishikawa, 1995), and immediately transferred into the RNA extraction reagent.

facturer’s instructions (Nakabachi and Ishikawa, 1997). Primers used in this study are listed in Table 1. Reverse transcription was performed in a mixture of 2 ␮l of 0.1 ␮g/␮l total RNA, 2 ␮l of 2 ␮M 3⬘-anchored oligo-dT primer, 1.6 ␮l of 250 ␮M dNTP, 4 ␮l of 5X RT buffer [125 mM Tris–HCl (pH 8.3), 188 mM KCl, 7.5 mM MgCl2, 25 mM DTT], 9.4 ␮l of RNase-free dH2O, and 1 ␮l of 100 unit/␮l Moloney murine leukemia virus reverse transcriptase. The reaction was performed using a PCR thermal cycler (Takara Shuzo). The PCR mixture was prepared using 2 ␮l of reversetranscribed sample from the preceding step, 2 ␮l of 10X PCR buffer [100 mM Tris–HCl (pH 8.4), 500 mM KCl, 15 mM MgCl2, 0.01% gelatin], 1.6 ␮l of 25 ␮M dNTP, 2 ␮l of 2 ␮M 5⬘-arbitrary primer, 2 ␮l of 2 ␮M 3⬘-anchored oligo-dT primer, 1 ␮l of [␣-35S] dATP(1000– 1500 Ci/mmole, New England Nuclear), 0.2 ␮l of 5 unit/␮l AmpliTaq DNA polymerase (Perkin–Elmer), and 9.2 ␮l of dH2O. The cycling parameters were as follows: 94°C for 30 s, 40°C for 2 min, 72°C for 30 s for 40 cycles followed by 72°C for 5 min. The denatured products were separated by electrophoresis on 6% polyacrylamide DNA sequencing gel containing 7 M urea. The gel was dried under vacuum at 80°C on filter paper, and exposed to X-ray film. 2.6. Recovery and reamplification of cDNA fragments After developing the X-ray film, cDNA bands of interest were cut from the gel. The gel slice, together with the filter paper, was incubated in dH2O, and cDNA was diffused out by boiling and recovered by ethanol precipitation. Reamplification of cDNA was performed using the same primer set and PCR conditions as used in differential display, except that the dNTP concentration was raised and no isotope was added.

2.4. RNA preparation

Table 1 Sequence of the primers used in DD

Total RNA extraction from bacteriocytes was performed using TRIzol Reagent (GIBCO BRL). Since the reagent was a mono-phasic solution containing phenol and guanidine isothiocyanate, the extraction procedure used was based on the single-step method of total RNA extraction developed by Chomczynski and Sacchi (1987). To remove chromosomal DNA contamination, RNA samples were treated with DNase I.

Primers

2.5. Differential cDNA display Differential display of cDNA was performed using the RNA image Kit (GenHunter), according to the manu-

3⬘-anchored oligo-dT primers 5⬘-AAGCTTTTTTTTTTTA-3⬘ H-T11A: 5⬘-AAGCTTTTTTTTTTTC-3⬘ H-T11C: 5⬘-AAGCTTTTTTTTTTTG-3⬘ H-T11G: 5⬘-arbitrary pimers H-AP1: 5⬘-AAGCTTGATTGCC-3⬘ H-AP2: 5⬘-AAGDTTCGACTGT-3⬘ H-AP3: 5⬘-AAGCTTTGGTCAG-3 H-AP4: 5⬘-AAGDTTCTCAACG-3⬘ H-AP5: 5⬘-AAGCTTAGTAGGC-3⬘ H-AP6: 5⬘-AAGCTTGCACCAT-3⬘ H-AP7: 5⬘-AAGCTTAACGAGG-3⬘ H-AP8: 5⬘-AAGCTTTTACCGC-3⬘

A. Nakabachi, H. Ishikawa / Journal of Insect Physiology 45 (1999) 1–6

2.7. Cloning and sequencing of cDNA fragments Reamplified cDNA fragments were ligated into pCR 2.1 vector and cloned in INV␣F⬘ One Shot competent cells using TA Cloning Kit (Invitrogen). Plasmid DNA sequencing of cloned cDNA with M13 primer was carried out using the Thermo Sequenase Kit (Amersham).

3

ment was extremely AT-rich (70%), which was characteristic of Buchnera gene (Ishikawa, 1987; Ohtaka and Ishikawa, 1993; Baumann et al., 1995). The amino acid sequence corresponding to bases 1–339 was 78% similar and 54% identical to C-terminal region of 6,7-dimethyl8-ribityllumazine synthase (EC 2.5.1.9, riboflavin synthase ␤ chain) of Escherichia coli (sp: RISB–ECOLI). Alignment of the amino acid sequences is shown in Fig. 1(b).

2.8. Quantitative RT-PCR Total RNAs were prepared from bacteriocytes as described above, and the cDNAs were synthesized with 1 ␮g of total RNA, 1 ␮l of 200 mM DTT, and 1 ␮l of 0.2 ␮g/␮l pd(N)6 primer using the First-Strand cDNA Synthesis Kit (Pharmacia). PCRs were carried out using 0.25 unit of Ex Taq DNA polymerase (Takara), 1 ␮l of 10X Ex Taq buffer, 1 ␮l of 2.5 mM dNTP, 0.5 ␮l each of 5 ␮M primers (RISB-1, 5⬘-TGTTCCTGGAACATATGAAATACC-3⬘; and RISB-2, 5⬘TCTAATGCGGCTAAAGCGGC-3⬘), and 0.1 ␮l of [␣32 P]dCTP (3000 Ci/mmole, ICN) in a final volume of 10 ␮l. PCR products were resolved on polyacrylamide gels, the radioactivity was determined using a Bas-2500 bioimaging analyzer (Fuji Film). 2.9. Nutritional experiments Symbiotic and aposymbiotic aphids were reared on synthetic diets (Sasaki et al., 1991) with or without riboflavin, and their performance was assessed in terms of change in body weight, length of the nymphal period, and survival rate during development. All the experiments were performed at 15°C with a photoperiod of 16 h, and the diet sachets were changed every third day.

3. Results 3.1. Detection of mRNA for riboflavin synthase ␤ chain by differential cDNA display Bacteriocytes from young aphids (20 days) and old aphids (50 days) were compared with respect to their mRNA populations by the differential cDNA display (DD) technique, using 24 primer combinations with three 3⬘-anchored oligo-dT primers and eight 5⬘-arbitrary primers (Table 1). The cDNA bands that were detected solely in the young bacteriocytes were cut from the gels and the cDNA fragments were reamplified by PCR, cloned and sequenced. A computer search for homology via the BLAST network server characterized a cDNA fragment (Fig. 1(a)) with GenBank accession number AB011407 (431 bp fragment obtained by DD with H-T11G primer and H-AP3 primer). The putative protein coding region (bases 1–351) of this cDNA frag-

3.2. Confirmation of a decrease in RISB mRNA with age by quantitative RT-PCR Total RNAs were extracted from bacteriocytes of 10, 20, 30, 40, and 50 day-old insects, and reverse transcribed. After calibration of the initial amount of target cDNA using Buchnera 16S rRNA as an internal standard (Nakabachi and Ishikawa, 1997), we examined yields of PCR product from RISB cDNA after various numbers of cycles, and defined the maximum cycle number, where the yield was proportional to the amount of the initial template, as the optimal cycle number. The amounts of PCR product at the optimal cycle number were compared in terms of age of the bacteriocyte from which RNA was extracted (Fig. 2). The results demonstrate that mRNA for putative RISB becomes less abundant as aphids age, confirming the results obtained by DD. 3.3. Effect of dietary riboflavin on symbiotic and aposymbiotic aphids Symbiotic and aposymbiotic aphids were maintained on chemically-defined synthetic diets with or without riboflavin (Fig. 3). Fig. 3(a) shows change in body weight of aphids during post-embryonic development. Symbiotic aphids reared on riboflavin-free diets were significantly heavier than those reared on diets with riboflavin [see ANOVA in legend to Fig. 3(a)]. The maximal weight attained on day 27 by symbiotic aphids reared on diets with and without riboflavin was 2.47 ± 0.11 and 2.51 ± 0.09 mg per individual, respectively. Aposymbiotic aphids invariably weighed less than symbiotic aphids. The maximal weight attained by aposymbiotic aphids reared on diets with and without riboflavin was 1.39 ± 0.06 mg (day 27) and 0.38 ± 0.03 mg (day 12) per individual, respectively, indicating that the growth of aposymbiotic aphids reared on riboflavin-free diets was considerably inferior to that of aposymbiotic aphids reared on diets containing riboflavin ( p ⬍ 0.001, using Students t-test). Length of the nymphal period and survival rate are shown in Fig. 3(b) and (c), respectively. Symbiotic aphids reared on diets without riboflavin reached adulthood sometime between day 14 and 17. The development of symbiotic aphids that received dietary riboflavin

4

A. Nakabachi, H. Ishikawa / Journal of Insect Physiology 45 (1999) 1–6

Fig. 1. Detection of putative riboflavin synthase ␤ chain (RISB) mRNA of Buchnera. (a) Differentially displayed band in DD analysis of young (Y, 20 days) and old (O, 50 days) bacteriocytes using a primer combination of H-T11G and H-AP3. Total RNAs were isolated from young and old bacteriocytes, reverse-transcribed with 3⬘-anchored oligo-dT primers, followed by PCR amplification using 3⬘-anchored oligo-dT primers, 5⬘arbitrary primers, dNTP, [␣-35S]dATP, and AmpliTaq DNA polymerase. PCR products were separated by electrophoresis on DNA sequencing gel, which was then dried on filter paper, and exposed to X-ray film. The arrowhead indicates the band due to RISB mRNA. (b) Amino acid sequence of part of Buchnera RISB deduced from the nucleotide sequence of the DD fragment shown in (a). The sequence is aligned with that of the corresponding part of RISB of Escherchia coli (156 amino acids). Identical and similar amino acid residues in the two sequences are denoted by asterisks and dots, respectively. Numbering of Buchnera RISB is based on that of its nucleotide sequence registered in GenBank while E. coli RISB is numbered from its N-terminus.

was delayed by about 1 day. This was consistent with the finding that symbiotic aphids reared on riboflavinfree diets weighed more than those reared on diets containing riboflavin. It took 17–27 days for 86.1% of aposymbiotic aphids that received dietary riboflavin to reach adulthood. The remaining aphids (13.9%) died before reaching adulthood. No aposymbiotic aphid attained adulthood on a diet lacking riboflavin. These aphids died before day 18, while most aphids in other groups survived until day 30.

4. Discussion Comparison of young and old bacteriocytes by DD suggested that RISB mRNA of Buchnera is abundant only in the symbiotic system of young aphids (Fig. 1), which was further confirmed by quantitative RT-PCR (Fig. 2). Since RISB is a component of the riboflavin synthase complex which catalyzes two final reactions in the riboflavin synthetic pathway, this finding suggested that riboflavin is actively synthesized by Buchnera in young aphids. This was reminiscent of early reports describing several insect species which contained sym-

biotic microorganisms and could grow without dietary riboflavin (Pant and Fraenkel, 1950; Wicker, 1983; Grenier et al., 1994) though evidently no insect is able to synthesize this vitamin (Sang, 1956; Niijima, 1993; Bruins et al., 1997). As for aphids, while the effect of dietary riboflavin has been studied repeatedly, the results are inconsistent. Dadd et al. (1967) reported that the green peach aphid, Myzus persicae had a dietary need for riboflavin, as well as ascorbic acid and other water-soluble vitamins such as thiamin, nicotinic acid, pyridoxine, folic acid, calcium pantothenate, meso-inositol, choline, and biotin. Contrary to this, riboflavin was reported to have a detrimental effect on the growth of A. pisum and M. persicae, and it was suggested that the deleterious effect may be, at least in part, due to the formation of stable and insoluble, nutritionally unavailable complexes with trace minerals included in the diet (Markkula and Laurema, 1967; Mittler, 1976). Boisvert and Auclair (1981) also reported that the omission of riboflavin from the diet significantly increased the growth, reproduction, survival and ingestion rate of two biotypes of A. pisum. In this context, it should be reminded that these results were obtained from experiments using symbiotic aphids.

A. Nakabachi, H. Ishikawa / Journal of Insect Physiology 45 (1999) 1–6

Fig. 2. Relative amount of RISB mRNA detected by quantitative RTPCR. Total RNA was prepared from bacteriocytes of aphids at the indicated age (days). cDNA was synthesized with pd(N)6 primer using the First-Strand cDNA Synthesis Kit (Pharmacia), and amplified by PCR with ExTaq (Takara), dNTP, [␣-32P]dCTP, and RISB-specific primers (RISB-1, 5⬘-TGTTCCTGGAACATATGAAATACC-3⬘; and RISB-2, 5⬘-TCTAATGCGGCTAAAGCGGC-3⬘). Each cycle of PCR included 30 s of denaturation at 94°C, 30 s of primer annealing at 55°C, and 1 min of extension at 72°C. PCR products were run on polyacrylamide gel. The radioactivity of the band corresponding to RISB mRNA was determined using a Bas-2500 bioimaging analyzer (Fuji Film).

With all these results in mind, in this study we examined in detail the effect of dietary riboflavin on the performance of symbiotic and aposymbiotic aphids (Fig. 3). The results clearly indicate that: (1) dietary riboflavin is slightly detrimental to symbiotic aphids; (2) dietary riboflavin is essential to aposymbiotic aphids; and (3) dietary riboflavin significantly improves the performance of aposymbiotic aphids. These results, taken together with the finding that RISB mRNA is abundant only in Buchnera from young bacteriocytes (Figs. 1 and 2) strongly suggest that young, symbiotic aphids are provided with sufficient amounts of riboflavin by their endosymbionts, Buchnera. In this context, since no dietary riboflavin is required by symbiotic aphids, its supplementation is detrimental, in that it is liable to form stable complexes with trace amounts of minerals included in Fig. 3. Effects of dietary riboflavin on the performance of symbiotic and aposymbiotic aphids. All experiments were carried out at 15°C in a long-day regime of 16 h light and 8 h dark. 䊏, Symbiotic aphids reared on diets with riboflavin (0.5 mg/100 ml diet); 䊐, symbiotic aphids reared on diets without riboflavin; 쎲, aposymbiotic aphids reared on diets with riboflavin (0.5 mg/100 ml diet); 䊊, aposymbiotic aphids reared on diets without riboflavin. (a) Body weight of aphids. Each data point is the mean ± s.e. of six replicate groups of six individual aphids. ANOVA on symbiotic aphids: age F(9,100) = 357.8, P ⬍ 0.001; riboflavin F(1,100) = 16.4, P ⬍ 0.001; interaction F(9,100) = 0.8, P > 0.05. (b) Length of the nymphal period. Emergence of the cauda was taken as an indication of reaching adulthood. (c) Survival rate during development.

5

6

A. Nakabachi, H. Ishikawa / Journal of Insect Physiology 45 (1999) 1–6

the diet (Markkula and Laurema, 1967). For aposymbiotic aphids, which do not enjoy the supply of this vitamin by Buchnera, dietary riboflavin must be a life line even though it decreases the amount of available minerals in the diet (Fig. 3). Fig. 2 suggests that the riboflavin synthase complex of Buchnera is fully active only when symbiotic aphids are young and the production of progeny is nearly maximal. It is also known that young aphids tend to temporarily sacrifice their symbiotic system when they encounter physiologically adverse conditions (Hongoh and Ishikawa, 1994). This may account for the inconsistent results obtained so far with respect to the effect of dietary riboflavin on the performance of symbiotic aphids (Dadd et al., 1967). It is conceivable that the aphid’s demand for dietary riboflavin is sensitive to the physiological conditions of their symbiotic system.

Acknowledgements This work was supported in part by a Grant from the Program for Promotion of Basic Research Activities for Innovation Biosciences (ProBRAIN) of the Bio-oriented Technology Research Advancement Institution, and Grants-in-Aid for Scientific Research from the Japanese Ministry of Education, Science, Sports and Culture.

References Baumann, P., Baumann, L., Lai, C.-Y., Rouhbakhsh, D., Moran, N.A., Clark, M.A., 1995. Genetics, physiology, and evolutionary relationships of the genus Buchnera: intracellular symbionts of aphids. Annual Review of Microbiology 49, 55–94. Boisvert, J.M., Auclair, J.L., 1981. Influence de la riboflavine sur les besoins globaux en vitamines hydrosolubles chez le puceron du pois, Acyrthosiphon pisum. Canadian Journal of Zoology 59, 164–169. Bruins, B.G., Scharloo, W., Tho¨rig, G.E.W., 1997. Light-induced vitamin deficiency in Drosophila melanogaster. Archives of Insect Biochemistry and Physiology 36, 51–67. Buchner, P., 1965. Endosymbiosis of Animals with Plant Micro-organisms. Interscience, New York. Chomczynski, P., Sacchi, N., 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction. Analytical Biochemistry 162, 156–159. Dadd, R.H., Krieger, D.L., Mittler, T.E., 1967. Studies on the artificial feeding of the aphid Myzus persicae (Sulzer)—IV. Requirements for water-soluble vitamins and ascorbic acid. Journal of Insect Physiology 13, 249–272. Grenier, A.M., Nardon, C., Nardon, P., 1994. The role of symbiotes in flight activity of Sitophilus weevils. Entomologia Experimentalis et Applicata 70, 201–208. Hongoh, Y., Ishikawa, H., 1994. Changes of mycetocyte symbiosis in

response to flying behavior of alatiform aphid (Acyrthosiphon pisum). Zoological Science 11, 731–735. Ishikawa, H., 1982. Host-symbiont interactions in the protein synthesis in the pea aphid, Acyrthosiphon pisum. Insect Biochemistry 12, 613–622. Ishikawa, H., 1984. Age-dependent regulation of protein synthesis in an aphid endosymbiont by the host insect. Insect Biochemistry 14, 427–433. Ishikawa, H., 1987. Nucleotide composition and kinetic complexity of the genomic DNA of an intracellular symbiont in the pea aphid Acyrthosiphon pisum. Journal of Molecular Evolution 24, 205–211. Ishikawa, H., 1989. Biochemical and molecular aspects of endosymbiosis in insects. International Review of Cytology 116, 1–45. Liang, P., Pardee, A.B., 1992. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257, 967–971. Markkula, M., Laurema, S., 1967. The effect of amino acids, vitamins, and trace elements on the development of Acyrthosiphon pisum Harris (Hom., Aphididae). Annales Agriculturae Fenniae 6, 77–80. Miller, S.G., Silhacek, D.L., 1995. Riboflavin binding proteins and flavin assimilation in insects. Comparative Biochemistry and Physiology 110B, 467–475. Mittler, T.E., 1976. Ascorbic acid and other chelating agents in the trace-mineral nutrition of the aphid Myzus persicae on artificial diets. Entomologia Experimentalis et Applicata 20, 81–98. Munson, M.A., Baumann, P., Kinsey, M.G., 1991. Buchnera gen. nov. and Buchnera aphidicola sp. nov., a taxon consisting of the mycetocyte associated primary endosymbionts of aphids. International Journal of Systematic Bacteriology 174, 1869–1874. Nakabachi, A., Ishikawa, H., 1997. Differential display of mRNAs related to amino acid metabolism in the endosymbiotic system of aphids. Insect Biochemistry and Molecular Biology 27, 1057–1062. Niijima, K., 1993. Nutritional studies on an aphidophagous chrysopid, Chrysopa septempunctata Wesmael (Neuroptera: Chrysopidae) III. Vitamin requirement for larval development. Applied Entomology and Zoology 28, 89–95. Noonan, K.E., Beck, C., Holzmayer, T.A., Chin, J.E., Wunder, J.S., Andrulis, I.L., Gazdar, A.F., Willman, C.L., Griffith, B., Von Hoff, D.D., Roninson, I.B., 1990. Quantitative analysis of MDR1 (multidrug resistance) gene expression in human tumors by polymerase chain reaction. Proceedings of the National Academy of Sciences of the United States of America 87, 7160–7164. Ohtaka, C., Ishikawa, H., 1993. Accumulation of adenine and thymine in a groE-homologous operon of an intracellular symbiont. Journal of Molecular Evolution 36, 121–126. Pant, N.C., Fraenkel, G., 1950. The function of the symbiotic yeasts of two insect species, Lasioderma serricorne F. and Stegobium (Sitodrepa) paniceum L. Science 112, 498–500. Sang, J.H., 1956. The quantitative nutritional requirements of Drosophila melanogaster. Journal of Experimental Biology 33, 45–72. Sasaki, T., Hayashi, H., Ishikawa, H., 1991. Growth and reproduction of the symbiotic and aposymbiotic pea aphids, Acyrthosiphon pisum maintained on artificial diets. Journal of Insect Physiology 37, 749–756. Sasaki, T., Ishikawa, H., 1995. Production of essential amino acids from glutamate by mycetocyte symbionts of the pea aphid, Acyrthosiphon pisum. Journal of Insect Physiology 41, 41–46. Srivastava, P.N., Auclair, J.L., 1971. Influence of sucrose concentration on diet uptake and performance by the pea aphid, Acyrthosiphon pisum. Annals of the Entomological Society of America 64, 739–743. Wicker, C., 1983. Differential vitamin and choline requirements of symbiotic and aposymbiotic S. oryzae (Coleoptera: Curculionidae). Comparative Biochemistry and Physiology 76A, 177–182.