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J. srored Prod. Rex Vol. 31, No. I, pp. 83-89, 1995Coovrieht 0 1995 Elsevier Science Ltd

Printedih &ea;Britain. All rights reserved0022-474X/95 $9.50 -t 0.000022-474X(94)00028-X

Development of Callosobruchus chinensis (L.)and C. maculatus (F.)(Coleoptera: Bruchidae)L. Differing inand Convicinein Seeds oftheir Tannin,ContentsVicia fabaVicine

P. DESROCHES, E. EL SHAZLY,2 N. MANDON, G. DUC3 andJ. HUIGNARD

IBEAS, URA CNR S 1298, Av enue Mange, Paw Grandm ont , 37200 Tours, France,Departm ent of Plant Protection, Cairo University, Egypt and Stat ion de Ghkt ique

et dAm6liorat ion des plan tes, IN RA, BV 1540, 21034 Dijon cedex, France(Received 5 July 1994)

Abstract-The seeds of the tested genotypes of Viciafaba L. diiered by the presence or absenceof tannins in the seed coat and in the level of vicine and convicine in the cotyledons. In vertebrates,these two glycosides can be transformed into divicine and isouramil which influence the enzymaticactivity of glucose-u-phosphate dehydrogenase (G-6-PDH). Callosobruchus chinensis (L.) devel-ops in V.faba seeds and causes high losses during storage. C. muculatus (F.) is less able to developin the seeds of V. fiba. For both bruchids the seed coat represents a barrier that only 45-60%of larvae overcome. The presence of tannins did not affect the perforation rate. The texture ofthe seed coat, its hardness or the presence of other toxic compounds could explain these results.The larvae of C. chinensis were able to develop in the seeds of P.faba whatever their vicine andconvicine contents and the survival rate during the post-embryonic development was higher than70%. The larvae of C. maculutus could only develop well in seeds poor in vicine and in convicine.The Ll larvae died as soon as they began to consume the cotyledons of the seeds containing highlevels of glycosides. The presence of vicine in the seeds seemed to be the main mortality factorfor C. tnuculutus larvae. Variations in the convicine content had a more limited influence. Theenzymatic activity of G-6-PDH in haemolymph depended on the seed vicine and convicinecontents and the bruchid species. This activity was low when larvae developed in seeds rich invicine and convicine. Whatever the genotype of the seeds, the enzymatic activity of the G-CPDHwas always higher in C. chinensis adults than in C. muculutus adults. Varietal selection, throughthe reduction of the vicine and convicine content of the seeds, can favour the colonization of Y.faba by other phytophagous insects such as C. maculatus.Key words-Bruchidae, Vi& faba, vicine, convicine, tannins, development, glucose-6-phos-phate dehydrogenase, enzymatic activity.

INTRODUCTIONThe seeds of Vicia faba (Leguminosae) are rich in proteins (30% of their dry weight) andcontain most of the amino acids necessary for human and animal nutrition (Smartt, 1976).However the concentration of sulphur amino acids is low. This plant, which probablyoriginated in Afghanistan and Ethiopia, is widely cultivated around the Mediterranean area andin China.

83

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84 P. Desroches t al.Several species of Bruchidae develop in V. faba seeds and cause high seed losses. Bruchus

rz&manus (Boh.) is a univoltine species. Females oviposit on the green pods and the adults emergingfrom the seeds are in reproductive diapause from autumn to next spring. This species cannotreproduce in stores. Bruchus dentipes (Baudi) and Bruchus quinqueguttatus (Oliv.), developing onV. fuba in the Middle East, have the same biology (Hariri, 1981). Cullosobruchus chinensis (L.) andBruchidius incarnatus (Boh.) are polyvoltine and in the stores, the successive generations cause highlosses (Shomar, 1963; Hariri, 1981).These bruchid species, developing during their post-embryonic life in V. fuba seeds, probablydetoxify the secondary compounds that they contain. During the perforation of the seed coat, thelarvae must consume the phenolic compounds and condensed tannins present (Griffiths, 1989). Insome animals, these tannins can combine with alimentary proteins and prevent their digestion andsubsequent assimilation (Marquardt, 1989a; Marquardt et al., 1978). Two pyrimidine glucosides,vicine and convicine (Fig. l), are present in the cotyledons of most genotypes of V. faba. In manand in monogastric animals, these glycosides are hydrolysed by the intestinal microflora to highlyreactive free radicals, generating two compounds, divicine and isouramil. These compounds caninhibit the activity of G-6-PDH and cause adverse effects including lipid peroxidation, altered fatand mitochondrial metabolism (Marquardt, 1989b). In order to limit the antinutritional effect ofthese secondary metabolites, new V. faba genotypes have been selected. In these genotypes, thetannins are absent in the seed coat (zero-tannin variety) or (and) there is very little vicine andconvicine in the cotyledons (Due et al., 1989).During this study we analysed the post-embryonic development of C. chinensis in differentgenotypes of V. faba. Its developmental capability was compared to that of C. maculutus. This fairlygeneralist tropical bruchid, often sympatric with C. chinensis, does not often develop in seeds ofV. faba (Boughdad et al., 1986a, b). The presence of these secondary compounds mentioned abovecould explain these low developmental capacities of C. maculatus. Following larval developmentin seeds differing by their vicine and convicine content, the effect on enzymatic activity of G-6-PDHin the adult haemolymph was examined.

MATERIALS AND METHODSRearing conditions

The strain of C. chinensis used for this study was collected in broad bean stores in the Cairoregion of Egypt in 1989. The strain of C. maculutus was collected in cowpea fields in the regionof Ouagadougou (Burkina Faso) in October 1992. The two species of bruchids were reared in thelaboratory for several generations in seeds of Vigna unguiculata in conditions of 40 :25C 12: 12 h,12: 12 h LD. In these climatic conditions, the post-embryonic development lasted between 25 and35 days.

Vicine Convicine

Divicine IsouramilFig. 1. Structure of vicine and convicine and their respective aglycone.

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Development of Callosobruchus in Vicia 85Table I. Characteristics of the tested genotypes: presence ( + ) or absence ( - ) of tannins in the seed coat; vicine and convicine

contents (% of the seed dry matter)Genotmes Tannins Vicine Convicine Genotvoes Tannins Vicine ConvicineEK.OTEK.T106AEC1665.0T1665.TI660cv. ALFREDcv. BLANDINEcv. ALBATROS16551656cv. ASCOTT1268

-gene 2 0.65+ 0.59+ 0.63+ 0.60-gene I 0.53+ 0.48+ 0.44+ 0.71

-gene I 0.67-gene I 0.59

+ 0.54+ 0.49+ 0.48+ 0.06

0.330.300.200.210.130.100.100.320.360.210.300.320.160.006

EB.T.V + 0.49 0.20EB.0T.V -gene I 0.59 0.28EB.T.OV + 0.08 0.03EB.OT.OV -gene I 0.05 0.01EE.T.V + 0.62 0.19EE.0T.V -gene 2 0.50 0.16EE.T.OV + 0.08 0.009EE.OT.OV -gene 2 0.06 0.004EQ + 0.40 0.12EC + EC + 0.12 0.02EA + EA + 0.05 0.003ET + 0.04 0.005

The tested V. faha genot ypes (Table 1)The V. faba bean genotypes were obtained from Station de Genetique et dAmClioration desplantes, INRA, Dijon. The absence of tannins in the seed coat is monogenic in origin and twodistinct genes controlling a zero tannin character were determined by Picard (1976). The low level

of vicine and convicine in the cotyledons (15 times lower than normal) is determined by a singlegene (Due et al., 1989). The genotypes used in this study were a collection of cultivars and linescovering a wide range of tannins and glycoside contents (Table 1). Additionally, near isogenicpopulations were selected in segregating F3 that allowed comparisons of the same geneticbackgrounds (EK, 1665. EB, EE) with different possible combinations (OT/T = zero-tannin/tanninand/or OVjV = low in glycosides/rich in glycosides). The structure of vicine (2,6-diamino-4,5-dihy-droxypyrimidine 5-( b-D-glucopyranoside) and convicine (2,4,5-trihydroxy-6-aminopyrimidine 5-(b-D-glucopyranoside) and their respective aglycone, divicine and isouramil are presented inFig. 1.The vicine and convicine levels of the seeds were determined by high pressure liquid chromatog-raphy according to the technique described by Quemener (1988). The glycosides were water-extracted from flour, separated on Lichrosorb Hibar RP 18250-4 (10 ,um) column of 10 pm, anddetected under UV (273 nm wavelength), using uridine as the internal standard.

Bruchid development on the direrent genot ypesThe inseminated females of C. maculatus and of C. chinensis were placed for 24 h on seeds ofeach genotype of V. faba from which the seed coat had or had not been removed. Only seeds whichreceived between one and four eggs were kept as increased larval density causes high mortality

(Yoshida, 1990). The following parameters were determined: the number of eggs laid, the numberof Ll larvae having perforated the seed coat and reached the cotyledons, and number of adultsemerging from the seeds. It was thus possible to determine the penetration rates (number of Llhaving penetrated/number of eggs laid) and the survival rates (number of emerging adults/totalnumber of Ll larvae having penetrated).

Enzym atic activ ity qf glucose-&phosphate dehydrogenase in haemolym phThe effect of consumption of vicine and convicine by the bruchid larvae was measured by theactivity of G-6-PDH in the haemolymph of adults emerging from the seeds. The haemolymph wasdrawn from the abdomen using a Pasteur micropipette. The enzyme activity was determined bya Sigma Diagnostics procedure. This procedure is a modification of the method of Kornberg andHorecker (1955) and Lohr and Waller (1974); it allows quantitative determination of the G-6-PDHactivity. The rate of formation of NADPH is proportional to the G-6-PDH activity and is measuredspectrophotometrically as an increase in absorbance NADPH at 340 nm UV. For each test, 1 ,ulof haemolymph was used.

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86 P. Desroches et al.Table 2. Analysis of the penetration rates in the seeds of different V. a babean enotypes by the two bruchidsoecies

Genotypes

C. chintw sisNo. of Penetrationhatching ratesems (%)

C. macularusNo. of Penetrationhatching rates

em (%)Tannins present cv. ALFRED 491 49.9 330 45. I1655 535 47.1 94 56.31656 322 55.9 177 55.8cv. ASCOTT 490 55.1 219 49.31268 366 49.4 553 58.2Zero-tannin cv. BLANDINE 253 56.2 137 46.1cv. ALBATROS 512 49.6 166 46.31655.0T 379 54.3 143 56.6EK.OT 469 55.2 235 52.1x28df,P

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Development of Callosobruchus in Vicia 87100

r

100. (A) r

. (B)

0 0.1 0.5 0.8 0 0.05 0.25 0.4Vicine concentration (% dry matter) Convicine concentration (% dry matter)

Fig. 3. Survival rates of C. maculatus in genotypes of V. faba presented in Table 1differing by their contentin vicine (A) and convicine (B).

When the vicine content of the seeds varied between 0.4 and 0.7% of dry matter and that ofconvicine between 0.10 and 0.35%, the survival rates were significantly different (x = 48.8, 10 &P < 0.05) and depended on the seed vicine and convicine contents (Fig. 3). The development of52 larvae emerging from eggs laid on decorticated seeds of the genotype EE.T.V. was analysed.When emerging from the eggs, the neonate larvae tried to feed on the cotyledons but 89.5% ofthem died during this phase. The other larvae were able to develop in the seeds of this genotypeand gave rise to adults.

Relat ive importance of vicine and conv icine on the surviv al rates of C. maculatus (Table 3)In these experiments, the seed coat was removed in order to reduce the mortality during the firstphase of development and to estimate only the influence of the two glycosides.When the larvae developed in seeds with similar vicine contents (0.48-0.50% of the dry matter)

but differing in the convicine content (0.16 and 0.32% of the dry matter), the survival rates werenot significantly different.In the presence of seeds with the same convicine content (0.32% of the dry matter) but differingin their vicine content (0.49-0.7 1% of the dry matter), the survival rates were significantly different.The vicine content of the seeds was probably the main mortality factor for larvae of C. maculatusand the variations in the convicine content had little influence. Nevertheless, convicine concen-trations were always lower than those of vicine and perhaps lower than the threshold of sensitivity.It would be necessary to incorporate the pure glycosides at different concentrations in artificialmedia (pellets of cowpea flour) and then to analyse the development of the bruchids.

Table 3. Analysis of the survival rates of C. macularus larvae in seeds of genotypes differing eitherin their vicine convent (V) or in their convicine content (C). The survival rates in cv. ASCOTT,EE.0T.V. and 1656 were not significantly different (x2 = 0.10 NS 2 d/i P < 0.05). The differenceswere sieniticant after development in cv. ALFRED and I656 (Y 2 = 6.3 S I& P C 0.05)

GenotypesLevel of vicineand convicine

(% MS)No. of

penetratinglarvaeNo. of

emergingadults

Survivalrates

CY. ASCOTT V = 0.48 I08 I6 14.8C =O.l6

EE.0T.V v = 0.50 164 27 16.5C=O.l6

1656 v = 0.49 106 I7 I6C = 0.32cv. ALFRED v=o.71 III 6 5.4C=n77

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88 P. Desroches et al.Table 4. Enzymatic activity of the G-6-PDH (U/ml) in the haemo-lymph of adults in the two species developing in the seeds of threeeenotvws of V. faba

C. chinensisGenotypes Males Females Males Femalescv. ALFRED 3.84 3.31 1.24 4.52cv. BLANDINE 4.59 3.40 1.95 5.25I268 6.42 4.98 8.85 6.60

Eflect of larval development in diflerent varieties of seeds of V. faba on the G-bPDH activit yThis enzymatic activity was tested in the haemolymph of C. chinensis and C. maculatus adultsdeveloping in seeds of the genotypes cv. ALFRED, cv. BLANDINE, and 1268 (Table 4). In thetwo species, the enzymatic activities of G-6-PDH were always higher in males than in females. InC. chinensis the enzymatic activity of G-6-PDH, in males and in females, depended on the larvaldevelopment conditions. The highest activities were observed when the larvae developed in seeds

of the genotype 1268. In C. maculatus haemolymph, the G-6-PDH activity was always lower thanthat observed in C. chinensis. As in C. chinensis this enzymatic activity depended on the vicine andconvicine contents and was always lower after development of the C. maculatus larvae in seeds witha high vicine and convicine content than in seeds with a low glycoside content.

DISCUSSION

When the neonate larvae of the two species of bruchids encounter broad bean seeds, the seedcoat represents a mechanical or a chemical barrier and only 4558% of them penetrate. Theneonate larvae probably consume some fragments of the seed coat during the penetration asobserved by Boughdad et al. (1986a, b), but in our experiments the presence of tannins does notseem to be responsible for the observed mortality of 42-55% of larvae. These results are verydifferent to those of Janzen et al. (1977) and Boughdad et al. (1986a, b) which demonstrated thatthe tannins were highly toxic for bruchid larvae. However, these authors analysed the larvaldevelopment of C. maculatus in cowpea flour pellets containing high concentrations of tannins (5%of dry matter). In these pellets, the larvae consumed tannins during all their post-embryonicdevelopment. On the other hand in the presence of seeds, the neonate larvae probably consumedsmall quantities of tannins.

Janzen (1977) analysed the perforation capacity of C. maculatus larvae in seeds of 73 species ofLeguminosae and observed the importance of the seed coat in the relationships between thebruchids and their host plants. The seed coat prevented the penetration of larvae in 69.5% of theseeds tested. Once again the texture of the seed coat, its thickness and its chemical compositionmay explain these results.C. chinensis can develop in the seeds of I. faba whatever their vicine and convicine contents.Our experiments confirm the low capacities of C. maculatus larvae to develop in I. aba seeds withhigh vicine and convicine contents.Biochemical studies can explain the mechanisms allowing the survival of the two species in seedswith high vicine and convicine contents. The two glycosides could have been transformed intodivicine and isouramil in the digestive tract of the larvae by the intestinal microflora as observedin vertebrates (Marquardt, 1989a, b). The presence of divicine and isouramil in the haemolymphof the larvae could influence the activity of G-6-PDH. This enzymatic activity was low when thetwo bruchid species developed in seeds with high vicine and convicine contents. The consequencesof this reduction of the G-6-PDH activity were limited in C. chinensis. In this species, twomechanisms could explain these results:-High concentrations of G-6-PDH could be present in haemolymph of the beetles. The inhibitionof the enzymatic activity by divicine and isouramil would not be complete and the beetles wouldhave a sufficient quantity of enzymes to continue their respiratory metabolism and survive.-The enzymatic systems of C. chinensis could detoxify the two ingested glycosides. The bruchids

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Development of Callosobruchus in Vicia 89present high capacities for adaptation and are capable of detoxifying antinutritional factors as hasbeen shown by Rosenthal et al . (1977) on Caryedes brasiliensis.In C. maculatus the enzymatic activity of the G-6-PDH was analysed only in the haemolymphof adult survivors. Nevertheless the activity of G-6-PDH was always lower than in C. chinensis.C. maculatus may produce lower quantities of G-6-PDH and the presence of divicine and isouramilin haemolymph might inhibit the activity of this enzyme and reduce the metabolism thus bringingabout the death of the larvae as soon as they begin to consume the cotyledons. The survivors shouldbe able to produce higher levels of G-6-PDH and to maintain the activity of this enzyme at asufficient level to ensure their survival. These survivors might also possess other enzymatic systemsdetoxifying the two glycosides and their respective aglycone. Complementary studies based on ananalysis of the activity of G-6-PDH in haemolymph of neonate larvae, and on selection experimentscarried out on individuals able to develop in seeds rich in vicine and convicine, are now necessary.In the coevolutional relationships between plants and bruchids (Janzen, 1977; Johnson, 1981)vicine and convicine could represent defence substances developed by the plant against phy-tophagous pests. By eliminating these substances, plant breeding makes these seeds more digestibleand more nutritive for man and domestic livestock. It may also make them more susceptible toinsect attack and can enable the colonization of these plants by other insects such as C. maculatus.AcknoM,Ldgemenrs-The authors wish to thank: C. Thibeaudeau and G. Tortay for their technical assistance during thisstudy. The work was carried out within a programme financed by the Centre Regional dbrnovation TechnologiqueValicentre.

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faba L. A first report of a gene which codes for nearly zero vicine and zero convicine contents. In Recent Advances ofResearch in Ant inut ritional Factors in Legumes Seeds (Edited by Huisman J., van der Poe1 T. F. B. and Liener I. E.).Pudoc, Wageningen.Griffiths D. W. (1989) Polyphenolics and their possible effect on nutritive value. Asp. Appl. Biol. 19, 93-103.Hariri G. (1981) Distribution and importance of bruchid attacks on different species of pulses consumed in the near east.In The Ecology of Bruchids attacking Legumes (Pulses) (Edited by Labeyrie V.). W. Junk Publishers, London.Janzen D. H. (1977) How southern cowpea weevil larvae (Bruchidae: Callosobruchus macularus) die on nonhost seeds.Ecology 58, 921-927.Janzen D. H., Juster J. B. and Bell E. A. (1977) Toxicity of secondary compounds to the seed-eating larvae of the bruchidbeetle Callosobruchus maculat us. Phyt ochemist ry 16, 223-227.Johnson C. D. (1981) Relations of Acanthoscelides with their plant hosts. In The Ecology of Bruchids attacking Legumes(Pulses) (Edited by Labeyrie V.). W. Junk Publishers, London.Kornberg A. and Horecker B. L. (1955) Glucose-6-phosphate dehydrogenase. In Methods in Enzymology (Edited byColowick S. P. and Kaplan N. 0.). Academic Press, New York.Lohr G. W. and Waller H. D. (1974) Glucose-6-phosphate dehydrogenase. In Methods of Enzymatic Analysis (Edited byBergmeyer). Academic Press, New York.Marquardt R. R. (1989a) Dietary effects of tannins, vicine and convicine. In Recent Advances of Research in AntinutritionalFactors in Legume Seeds (Edited by Huisman J., van der Poe1 T. F. B. and Liener I. E.). Pudoc, Wageningen.Marquardt R. R. (1989b) Vicine, convicine and their aglycones-divicine and isouramil. In Toxicants of planf origin: IIGlycosides (Edited by Checke P. R.). CRC Press, Boca Raton, Florida.Marquardt R. R., Ward A. T. and Evans L. E. (1978) Comparative properties of tannin-free and tannin-containing cultivarsof faba beans (Vicia faba ). Can. J. Pl ant . Sci. 58, 7533760.Picard J. (1976) Apercu sur Ihtrtdite du caractere absence de tanins dans les graines de feverole (Vicia faba L.). Ann.Am elior. Pla ntes 26, 101-106.Quemener B. (1988) Improvements in the high-pressure liquid chromatographic determination of amino sugars ongalactosides in,faba bean, lupin and pea. J. Agric. Food Chem. 36, 754-759.Rosenthal G. A.. Janzen D. H. and Dahlman D. L. (1977) Degradation and detoxification of canavanine by a specializedseed predator. Science 196, 6588660.Shomar N. F. H. (1963) A monographic revision of the Bruchidae of Egypt (U.A.R.). Bull. Sot. em. Egypte 47, 141-196.Smartt J. (1976) Tropical Pulses. Longman, London.Yoshida T. (1990) Historical review of bruchid studies in Japan. In Bruchids and Legumes: Econom ics, Ecology andCoevolurion (Edited by Fujii K., Gatehouse A. M. R., Johnson C. D., Mitchel R. and Yoshida T.), pp. 1-24. KluwerAcademic Publishers, London.