L-Canavanine, a Dietary Nitrogen Source for the Seed Predator Caryedes brasiliensis (Bruchidae)
Gerald A. Rosenthal; Charlie G. Hughes; Daniel H. Janzen
Science, New Series, Vol. 217, No. 4557. (Jul. 23, 1982), pp. 353-355.
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http://www.jstor.org Fri May 11 15:48:10 2007 oceans over the past 20 million years, but their everi!s (D. V. Kent and D. Spariosu, Init. Rep: standing of the basic biochemical inter data were too limited to allow the history of Deep Sea Drill. Pro)., iit press). Consistent A"C to be determined for intervals of I million paleomagnetic reversals were only recognized in action between higher plants arid their or 2 million years. Pliocene-Pleistocene sediments at sites 502 and insect predators. 24. M. L. Bender and L. D. Keigwin, Jr.. Earth 503. Planet. Sci. Lett. 45, 383 (1979); ..... , D. W. 29. L. G. Marshall, R. F. Butler, R. E. Drake; To test this hypothesis, living terminal Graham, Mar. Micropaleontol. 6,451 (1981). G. H. Curtis, R. H. Tedford, Science 204, 272 25. J. L. Reid, Deep-Sea Res. 26, 1199 (1979). (1979). stadium larvae (9 g, fresh weight) Were 26. Although isolation and evaporatioh of the Medi 30. C. Emiliani, S. Gartner, S. Lidz, Palaeof?eogr. washed thoroughly with deionized water terranean Sea probably decreased world ocean Palaeoclimatol. Palaeoecol. 11, I (1972). ~alihity by I per mil [W. B. F. Ryan, M. B. Cita, 31. C. A. Brunner, thesis, University of Rhode to remove adhering plant material; each M. D. Rawson, L. J:t. Burckle, T. Saiio, Riv; Island, Kingstoh (1978). larva was then injected with I iJ-1 of Ital. Paleontol. Stratigr. 80; 631 (1974)), there 32. A. G. Kaneps, Science 204, 297 (1979). . was probably no oceanwide oxygen isotopic 33. w. F. Ruddiman and A. McIntyre, ibid., p. 173. 150 mM 15N-labeled urea (5). The treat" effect because the evaporated water returned to 34. I. thank M. L. Bender, B. H. Corliss., W. B. ed larvae were maintained in the dark for the sea. Curry, D. W. Graham, D. A. Johnson, P. 27. P. O. Weyl, Meteorol. Monogr. 8, 37 (1968). Kroopnick, M. T. Ledbetter, ~. M. Savin, and 48 hours at 28°C before storage at 28. Ages of latest Miocene samples are interpolated D. F. Williams for their comments. I also thank between the carbon isotopic shift (6 million the Deep Sea Drilling Project staff for inviting -60~C. The frozen larvae were ground years ago) and the Miocene-Pliocene boundary me to participate in DSDP leg 68. This research for 60 seconds with 50 ml of freshly (5.26 million years ago). Samples from the inter was supported by NSF grant OCE79-14594 val in the late Miocene preceding the carbon (CENOP) and by OCE81-17590. This is Woods distilled acetone (Sorvall Omnimixer, shift are not considered in Fig. 3 because of Hole Oceanographic Insiitution Contribution fuiI power). The larval acetone powder uncertainties. in assigning absolute ages from No. 5101. biostratigraphy (15). Ages of Pliocene samples was collected by vacuum fiitratioit, and are interpolated between magnetostratigraphic 20 April 1982 reground as above for 120 seconds and dried in air overnight at 22°C. The free amino acids of the acetone powder (2.7 g) were obtained by mechanical stirring at 3°C for 20 hours with 250 ml of 50 L-Canavanine, a Dietary Nitrogen Source for the Seed Predator percent . aqueous ethanol containing Caryedes brasiliensis (Bruchidae) 0.15N HCI. The ethanolic extract was clarified by centrifugation at 18,OOOg for Abstract. Larvae of the bruchid beetle Caryedes brasiliensis (Bruchidae) develop 20 minutes, adjusted to pH 7.0 with IN entirely within the seed of the neotropical legume DiocIea megacarpa. The seed NaOH, and concentrated by rotary contains an appreciable concentration of L-canavanine, a potent arttimetabolite and evaporation at reduced pressure. Partic structural analog of L-arginine. Tnis bruchid beetle uses the nitrogen stored iii this ulate materials. deposited from the ex toxic allelochemical as an effective dietary nitrogen source for amino acid biosynthe tract during concentration were removed sis. by centrifugation as above. Finally, the ethanolicextract was reduced to 35 ml, Maturing larvae of the bruchid beetle a toxic higher plant allelochemical in adjusted to pH 2.5, and fiitered (Mill i Caryedes brasiliensis (Bruchidae) devel amino acid synthesis. The ease with pore as membra'ne)~ op entirely within the seed of the neo which this insect can convert canavanine The clarified larval extract was placed tropical legume Dioclea megacarpa (I) to canaline and urea and the availability on a column (20 by 495 mm) of Dowex-50 (Fig. 1). More than 95 percent df the free of 15N-labeled urea have made possible (H+) arid washed with 3 liters of deion amino acid nitrogen of this leguminous an experimental verification of this hy ized H20 at 3°C. Amino acids were eluF seed accumulates as L-canavanine, an pothesis which is important to our under- ed fully with 1.2 liters of 0.3N NH40H insectidd.al structural analog of L-argi nine (2). This seed predator is distinctive ih its synthesis of appreciable urease Table 1. Percent 15N incorporation into the free amino acids of the larvae of the bruchid beetle Caryedes brasiliensis. The values presented for percent of 15N ineorporation were obtained by (E.C. 3.5.1.5) which makes possible three independent determinations of the same sample. The fragment structures are based on the effective mobilization of the nitrogen work of Gelpi et al. (8). The amino acids were analyzed as their N-TFA-n-butyl ester stored in canavanine via. hydrolysis of derivatives. See Fig. 2 legend for the ainino acid designations.
--~.------urea; the latter is derived from canavan Fragment used in calculations __• ______• ____ 0____ ine by the action of arginase (E.C. 15N 3.5.3.1). Rela- Amino incor- tive acid poration m/e* abun- Structure (%) dance (%) L-canavanine Ala 35, 33, 33 140 100 M - C4H9COO H2N-O-CH2-CH2-CH(NH2)COOH + Gly 18, 18, 16 126 98.9 M - C4 H9COO [,-canaline 2-ABA 30,31, 28 154 100 M - C4 H9COO Thr 0,0,0 152 33.5 CF3CONH ::: C ::: Ci-ICH3 Ser 16,20,19 138 49.2 CF3CONH ::: C ::: CH2 urea Val 7, 5, 7 168 100 M - C4H9COO H2N-C(=O)-NH2~Cb2 + 2NH3 3-Ala 23, -,21 168 100 M - OC4H9 Leu 0,0,0 182 59.6 M - C4 H9C()O It has been proposed that these hydrolyt Ile 0,0,0 182 SO.5 M - C4 H9COO ic reactions not only detoxify canavan H-Ser 11,6,7 152 100 CF3CONH::: C ::: CHCH3 ine, but also make ammonia available as Pro 25, 25, 25 166 100 M - C4H9COO a dietary nitrogen source for the devel Hy-Pro 0,0,0 164 100 (M - C4H9COO) - CFJCOOH Met 11,9, 10 227 64.8 M - (CH2CH - S - CH3) oping larva (3). If this hypothesis is Cor Asp/Asn 24,22,25 240 79.7 M - C4H9COO rect, it would represent a significant find Lys 0,0,0 293 14.4 M - C4H"COO ing since, with the possible exception of Glu/Gln 44,49,48 198 100 (M - C4H9COO) - C4Hs cyanide incorporation into L-asparagine His 0,0,0 206 75.8 M - C4H9COO (4), no instance is known of insects using 'Ratio of mass to charge. SCIENCE, VOL. 217, 23 JULY 1982 0036-8075/82/0723-0353$01.00/0 Copyright © 1982 AAAS 353 (6). The effluent was rapidly dried at -60°C. The thawed insect extract was developed with deionized water and 1A reduced pressure, the residue dissolved concentrated at reduced pressure to 1.5 mI fractions were collected every 1.5 in to ml of deionized H20, filtered ml and placed on a column (20 by 1350 minutes; fractions 70 through llO were (Whatman 50 paper), and stored at mm) of Sephadex G-I0. The column was pooled, concentrated at reduced pres sure to 8.1 ml, and stored at -60°C (7). Amino acid analyses ,of the purified lar val extract were conducted by automat ed procedures that relied on ninhydrin detection at 440 and 570 nm. The N triftuoroacetyl-n-butyl ester derivatives were analyzed by gas chromatography mass spectroscopy (8). Some 25 free amino acids occur in the larva of C. brasiliensis but the principal components are canavanine, lysine, pro line, glutamic acid, glutamine, alanine, and glycine. Analysis by mass spectrom etry of the ISN incorporation of these larval amino acids revealed incorpo ration of the heavier nitrogen isotope amounting to more than 25 percent ofthe total for glutamic acid (or glutamine), aspartic acid (or asparagine), proline, 3- alanine, 2-aminobutyric acid, and ala nine (Table 1 and Fig. 2). Lesser but readily detectable amounts of ISN were found in glycine, serine, valine, homo serine, and methionine (Table 1). There was no evidence for nitrogen transfer from urea to threonine, leucine, isoleucine, lysine, histidine, and hydroxyproline (9). The above findings are consistent with Fig. I. (A-D) Release of the adult bruchid beetle Caryedes brasiliensis from the seed of Dioclea a primary role for glutamic acid and megacarpa. The larvae, developing within the seed, use the nitrogen of canavanine as a dietary glutamine in the assimilation of urea's nitrogen source. The newly formed adult emergesthtOdlh an escape lid bored in the seed coat. nitrogen by this seed predator. Amino [Photographs by James E. Mattler) transferases that yield L-alanine from reaction of pyruvic acid and L-glutamic acid and that provide L-aspartic acid from oxaloacetic acid and L-glutamic a" acid are among the most common and + c catalytically active of this diverse group a of enzymes in higher animals and insects >. (/0, 11). I- + In addition, glutamic acid and aspartic fII fII :>. C oJ acid can be reductively aminated with .2 ammonia to yield their amide-containing -; c o fII derivatives: glutamine and asparagine I- « + (12). Insects oxidize proline to glutamic i:> «II) aci<,l as a means of providing 2-oxogluta ..> rate (via transamination) to generate en () ergy by the reactions of the Krebs cycle (13). This reaction is known to be revers ible, presumably via 4-glutamyl semial dehyde and ~ l-pyrroline-5-carboxylic o 15 20 30 35 45 50 55 ~cid. Serine biosynthesis is also linked to Time (mlnl glutamic acid since transamination of Fig. 2. Total)on chromatogram of the N-trifluoroacetyl-n-butyi ester derivatives of the free gIutamic acid to 2-oxogJutanite supplies amino acids of the larvae of the bruchid beetle Caryedes brasiliensis. [Glass capillary column: the nitrogen for 3-phosphoserine, the im (50 m by 0.5 mm, inside diameter), coated with OV-IOI; temperature from 100° to 250°C at 3° mediate; precursor of serine. Glycine can per minute; heiium carrier gas flow rate, 4.5 ml per minute.) The amino acid abbreviations are: Ala, alanine; Gly , glycine; 2-ABA, 2-aminobutyric acid; Thr, threonine; Ser, serine; 3.-Ala, 3- form directly from serine in insects by an alanine; Val, valine; Leu, leucine; tIe, isoleucine; H-Ser, homoserine; Pro, proline; Hy-Pro, aminotransfer between glyoxylic acid hydroxyproline; Unk-unknown; Met, methionine; Asp, aspartic acid; Asn, asparagine; am, and alanine (10). The appreciable ISN ornithine; Phe, phenylalanine; Cav, canavanine; Lys, lysine; Tyr, tyrosine ; GIn, glutamine: label found in 2-amino-n-butyric acid (2- Glu, glutamic acid; His, histidine; Trp, tryptophan. 4-Aminobutyric acid (GABA; elution time, 25.5 minuteS) may not be present in the larva since it was not detected by gas chromatography ABA) may, on first cOrisideration, be mass spectrometry procedures that disclosed authentic material. Automated amino acid surprising; however, it is generated froin analysis disclosed a ninhydrin-positive substance with the column retention time of GABA. 2-oxobutyric acid, an oxo-containing 354 SCIENCE, VOL. 217 acid that forms readily from threonine offive buffers and requires 5 hours to complete. 10. W. Chefurka, in The Physiology of Insecta, M. The amino acid derivatives used for gas chroma Rockstein, Ed. (Academic Pn;ss,. New York, and methionine. Homoserine, which can tography-mass spectrometry were prepared by 1965), vol. 2. pp. 669-768. form either from cystathionine or more the method of D. L. Stalling and C. W. Gehrke II. S. S. Tate and A. Meister, in The Enzymes of [Biochem, Biophys. Res. Commun. 22, 329 Glutamine Metabolism, ~. prusiner and E. R. importantly by reductive deamination of (1966)]. Mass fragment identification was based Stadtman, Eds. (Academic Press, New York, on the work of E. Gelpi et al. [J. Chromatogr. 1969), pp.77-127; P. S. Chen, Adv. Insect canaline (14), is subject to a deamination Sci. 7, 604 (1969)] and R. E. Summons et at. Physiol. 3, 53 (1966). . that also forms 2-oxobutyric acid. While [Anal. Ch~m. 46, 582 (1974)]. Gas chromatogra 12. Our enzymatic analyses of C. brasiliensis larvae phy-mass spectrometry analyses were con
SCIENCE, VOL. 217, 23 JULY 1982 0036-8075/82/0723-0355$QI.00/0 Copyright © 1982 AAAS 355