Variation in the response of Callosobruchus maculatus (F.) to a resistant veriety of cowpea

J. stored Prod. Res. Vol. 22, No. I, pp. 4348, Printed in Great Britain

1986

0022-474X/86 $3.00 + 0.00 Pergamon Press Ltd

VARIATION

IN THE RESPONSE OF CALLOSOBRUCHUS MACULATUS (F.) TO A RESISTANT VARIETY OF COWPEA K. M. DICK* and P. F. CREDLAND~ Department of Zoology, Royal Holloway and Bedford New College, University of London, Alderhurst, Bakeham Lane, Englefield Green, Surrey TW20 9TY, England (Received

26 September

1985)

Abstract-A Nigeriancultivar of the cowpea has previously been identified as being less suitable than conventional varieties for the development of a local strain of C. macularus (F.). The survival and duration of development of the immature stages of three strains of C. maculatus in the dried seeds of this cultivar were compared with the performance of the same strains of beetle on a susceptible variety. In all beetle strains, fewer larvae survived to produce adults, and the duration of development was significantly extended, in the resistant cowpea cultivar. A Brazilian strain produced fewest adults and a Nigerian strain had the longest mean development period. Combining all the data, it appeared that a strain from the Yemen Arab Republic was least influenced and that from Brazil most adversely affected by the resistant cowpea. Evidence is presented indicating that factors in both the cowpea seeds and the beetles themselves may contribute to the poorer performance of bruchids in the resistant as compared to the susceptible variety. INTRODUCTION

The cowpea, Vigna unguiculala (L.) Walp., is an impoFtant food crop in many parts of the tropics and subtropics. In common with many other leguminous crops, stored cowpeas are attacked by insects, all of the major pests belonging to the family Bruchidae (Coleoptera) (Centre for Overseas Pest Research, 1981). The most important species attacking cowpeas is Callosobruchus maculutus (F.), which causes weight loss, decreased germination potential and a reduction in commercial value (Booker, 1967; Caswell, 1968,198 1). In an attempt to reduce the level of damage, the Grain Legume Improvement Programme at the International Institute of Tropical Agriculture (IITA) in Nigeria has sought to breed cowpea lines resistant to bruchid attack. After screening approx. 7000 cowpea varieties, only one, TVu 2027, was found to markedly increase mortality among immature beetles of a Nigerian strain of C. maculutus (IITA, 1981). Further work (Redden et al., 1983) suggested that a different population of the beetles, from Brazil, responded rather differently to TVu 2027, raising the question of whether that cultivar offered prospects for control of the beetles outside the area from which it was first obtained. It has already been shown that three strains of C. maculutus differ in a number of basic bionomic characteristics (Dick and Credland, 1984). We have now studied the responses of these same three strains to TVu 2027, looking particularly at the survival of the larval stages through to adulthood and the duration of development from oviposition to adult emergence. These parameters are of special importance since it is already known that neither egg production nor entry of larvae into the seeds are reduced on TVu 2027 (Gatehouse et al., 1979). MATERIALS

AND

METHODS

insects

The term “strain” is used here to describe populations of C. macularus collected from widely separated geographical areas. Three strains have been used and they are referred to as “Yemen”, “IITA” and “Campinas”. They were collected from the Yemen Arab Republic, Nigeria and Brazil, respectively. Details of their collection and culture procedures have been given previously (Dick

*Present address: ICRISAT Patancheru P.O., Andrha Pradesh 502324, India. tTo whom all correspondence should be addressed. 43

44

K. M. DICK and P. F. CREDLAND

and Credland, 1984). The cultures were maintained and all experiments 70 + 10% r.h. in a constant temperature (CT) room.

conducted

at 27 f 1°C and

The cowpeas used for the maintenance of insect stocks were of the type known as black-eyed peas or beans from California. Three varieties “Cowpea 3”, “Cowpea 5” and “Magnolia”, are marketed by the California Bean Growers Association (C. Fellows, personal communication) but no indication is provided on which varieties are present in any particular batch. On arrival in the laboratory. the cowpeas were stored in a deep freeze. Use of stocks from this source has indicated that they are readily susceptible to bruchid attack (Dick and Credland, 1984) and they have been used as a control when assessing the relative resistance of TVu 2027. Cowpeas of the variety TVu 2027 were received directly from IITA and arrived in two separate batches in 1982. As no information was provided on the growing conditions, number of parent plants or dates of harvest, the two batches were thoroughly mixed before use and were subsequently treated as a single sample. Like the Californian cowpeas, the TVu 2027 were stored in a deep freeze prior to use. Before use in experiments, cowpeas were spread evenly and thinly on trays, covered with muslin, and left for a minimum of 7 days at the standard temperature and relative humidity to standardize their moisture content (Pixton and Warburton, 1968). At the end of this period, the moisture content of the cowpeas was approx. 11.5%, as determined by the “one-stage oven determination” method (Anon. 1980). Samples of 100 Californian black-eyed cowpeas and 100 TVu 2027 were weighed after conditioning. The mean wet weights (+95% C.I.) of individual cowpeas of the two varieties were 266.8 f 4.6 and 317.3 f 5.7 mg. respectively. E.uperimental protocol

Single Californian cowpeas were placed in each of 50 glass tubes, 2.5 cm in diameter and 5 cm length and single seeds of TVu 2027 were each placed in a further 50 identical tubes. Females of the Yemen strain were taken from stock cultures and one was placed in each of the tubes which were then closed with a porous foam bung. An identical procedure was followed for the IITA strain but for the Campinas strain 100 replicate tubes were used for each variety of cowpea since preliminary experiments had indicated a lower survival rate of this strain on the resistant cowpeas. After 24 h. the females were removed from the tubes and the number of eggs on each cowpea was reduced to one by scraping off excess eggs with a scalpel blade. The cowpeas, in their tubes, were then returned to the CT room. After a further 14 days the cowpeas were examined again and the failure of any eggs to hatch or the failure of the first-instar larvae to penetrate the testa of the seed was noted. These two conditions were not subsequently distinguished in the analysis of results. The cowpeas were again returned to the CT room in their tubes. The emergence of adult insects from the cowpeas was noted daily and continued until no further adults had emerged for a period of 72 h. The tubes containing those seeds infested by IITA or Campinas individuals were than placed in a deep freeze for 3 days to kill any insects which may still have been present. They were then returned to the CT room for a further 7 days. Individual females from the stock cultures of the same strains as had previously oviposited on the seeds, were then placed in the tubes and the procedure described above was repeated.

The percentage of hatched eggs, or larvae entering the seeds, which had produced an adult after a fixed time interval of 45 days, the total percentage survival to emergence and the mean development period from oviposition to adult emergence were calculated for each strain on Californian cowpeas and TVu 2027. Howe’s Index of Suitability for Development (Howe, 1971), which is obtained by dividing the common logarithm of the total percentage survival by the mean development period (in days), was also calculated as an additional indicator of the suitability of the different cowpea varieties as hosts for the three strains of beetles.

Response

of C. maculatus

to resistant

45

cowpea

Table I. Performance of Yemen, IITA and Campinas on susceptible and TV112027 cowpeas at a larval density of one per seed IITA

Yemen

Campinas

Control (Californian)

TV” 2027

Control (Californian)

TVu 2027

Control (Californian)

TV” 2027

adult emergence

91.8

63.1

97.9 (91.7)

70.8 (70.4)

100.0 (91.7)

9.3 (9.8)

Mean development period (days)

25.3

39.2

25.1 (26.5)

49.1 (43.3)

25.8 (26.4)

39.5 (34.0)

% Adult emergence by day 45

97.8

50.0

97.9 (91.7)

21. I (34.1)

100.0

6.2 (7.6)

Cowpea cultivar

_

% Survival to

Howe’s Index (log SIT)

0.079

0.045

0.079 (0.074)

0.038 (0.043)

(91.7) 0.077 (0.074)

0.024 (0.029)

Values from a second infestation of the same seeds are given m parentheses

RESULTS

In all strains, the proportion of first-instar larvae which survived to adult emergence from the cowpea was significantly lower (P < 0.05) on TVu 2027 than on the Californian cowpeas (xl = 19.5, 15.5 and 107.0 for the Yemen, IITA and Campinas strains, respectively). This difference was greatest in the Campinas strain in which only 9.3% of the first-instar larvae survived to emerge as adults from TVu 2027, compared with the 100% survival in the Californian cowpeas (Table 1). There was no significant difference between the total percentage survival of the Yemen and IITA strains in TVu 2027 (63.1 and 70.8%, respectively). In all strains, the mean development period was longer in TVu 2027 than in the Californian cowpeas (Table l), that of the IITA strain being the most extended. However, the value for the Campinas strain on TVu 2027 is based on only 9 surviving insects and is therefore less reliable than those for the other strains. Almost twice as many Yemen individuals as IOTA insects had emerged from seeds of TVu 2027 by day 45. This difference in the temporal pattern of emergences from TVu 2027 (Fig. 1) accounts IO

(a)

Yemen

(b)

IITA

(c1

Campmos

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a 6 4 2 LlBlJl& O 10 8 6 4 2 0 L

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:

Days after Fig.

I. Temporal

oviposition

distribution of adult emergences from TV112027 for (a) Yemen, Campinas strains. Each seed was infested by one insect.

(b) IrTA and (c)

46

K. M. DICK

and P. F.

CREDLAND

for the difference in the value of Howe’s Index for the two strains. In this calculation, short development periods produce larger Index values which in turn indicate greater suitability of the host seed. Thus, although the total number of Yemen and IITA adults produced was statistically similar, Howe’s Index indicates that Yemen insects found TVu 2027 more suitable for development. Regardless of whether they were reared on susceptible or resistant varieties, the performance of the first and second “infestations” of IITA and Campinas beetles was very similar in terms of both larval survival and development times. For the TITA strain only, the adult emergence data for both infestations of TVu 2027 were pooled, and a coefficient calculated to determine whether any correlation existed between the fate of the larvae in the two infestations. Only seeds on which eggs of both infestations had hatched were considered. For each infestation, those cowpeas from which an adult had emerged were scored as positive and those which failed to produce an adult were scored as negative. The number of seeds with the same score in the two infestations (either + / + or - / - ) and the number with different scores ( + / - or - / + ) were then counted. The Binomial test was used to analyse the results. If the survival of the insects depended solely on the seed then the number of seeds (23) with two like scores should have exceeded the number with two unlike scores (19) but the test revealed no significant difference in the frequency with which the two types of score occurred. There was therefore no correlation between the fates of larvae in the same seed. Insufficient adults of the Campinas strain emerged for a similar test to be conducted. No departure from a 1: 1 sex ratio was observed for any of the strains emerging from either the Californian or TVu 2027 cowpeas. DISCUSSION This study confirms that the cowpea cultivar TVu 2027 exhibits a significant level of resistance to attack by C. macularus. It also demonstrates that different strains of the beetle vary in their response to the resistant variety. All strains exhibited >90% survival to adult emergence in Californian cowpeas used as a control, whereas only 60-70% of the Yemen and IITA larvae survived in TVu 2027 and less than 10% of the Campinas strain. These data are somewhat at variance with the only comparable information. Redden et al. (1983) used multiple infestations of seeds of both control and resistant cultivars of cowpeas in their study of the inheritance of resistance to attack by bruchids, and in their work on the IITA strain, fluctuating ambient rather than constant temperatures and relative humidities. It is therefore not surprising that whilst their data on the Campinas strain, which revealed survival of 95% on the control cowpeas and 11% on TVu 2027, were similar to ours, their results with the IITA strain, which indicated survival of only 79% on the control cowpeas and 41% on TVu 2027, were markedly lower. No work has previously been undertaken to examine the performance of the Yemen strain on TVu 2027. For a strain that rarely produced more than 2 adults from a single cowpea regardless of how many eggs were initially present compared with the 12 or more which may emerge from a cowpea bearing eggs of the IITA or Campinas strains (Dick and Credland, 1984) reliable total percentage survival data relevant to the resistance of the cowpeas can be obtained only from cowpeas which bear one initial egg, since survival in seeds bearing numerous eggs is low even on a susceptible variety. Redden and McGuire (1983) determined that the mean, as opposed to median, day of emergence is the most discriminating variable for the measurement of seed resistance. Using this criterion alone, it would appear that TVu 2027 is more resistant to the IITA strain (mean day of emergence 43-49) than either the Yemen or Campinas strains, where the mean emergence day was prior to day 40 after oviposition. Redden and McGuire (1983) found the mean emergence day of their IITA strain on TVu 2027 to be day 51 which, bearing in mind the different experimental conditions that they employed, is not greatly dissimilar from the value obtained in our experiments. Percentage adult emergence to an optimum time was the second best criterion identified for the measurement of seed resistance by Redden and McGuire (1983). Day 45 was used by them since it ensured that no second-generation adults could be produced by adults emerging early from seeds bearing several eggs, mating and ovipositing before their removal from the proximity of the

Response of C. macularus to resistant cowpea

47

Although no such possibility existed in our experiments with seeds bearing single eggs, day 45 is near the mean day of emergence for all three strains and was retained. In practice, this parameter separated the three strains most clearly. Whereas 50% of the Yemen adults had emerged, comparable figures for the IITA and Campinas strains were only 30 and 7%, respectively. Thus, the assessment of resistance by this criterion suggested that the Campinas strain was most adversely affected on TVu 2027. Comparison of our data on the IITA strain with that of Redden and McGuire (1983) revealed a major discrepancy since they obtained only 11% emergence by day 45. As the two parameters selected as being most discriminating in the measurement of seed resistance gave conflicting results, the use of Howe’s Index which incorporates both total survival and mean emergence periods is an appropriate way of combining the two kinds of information. The values obtained supported the subjective assessment that Campinas was the least and Yemen the most successful strain of TVu 2027 and, reassuringly, did not discriminate between the three strains on the control cowpeas. Furthermore, it very conveniently provided a simple numerical estimate of the performance of the strains which was more valid than using any single parameter which could be measured. Previous experiments have indicated that there is significant seed-to-seed variation in the expression of resistance to C. maculalus in TVu 2027 (Dick, 1984). This suggested that the level of trypsin inhibitor, which is known to play a major part in the resistant properties of the cultivar (Gatehouse et al., 1979; Gatehouse and Boulter, 1983) may vary among the seeds. The results of the Binomial test carried out on two generations of the IITA strain infesting the same seeds indicated that additional factors are involved in influencing the development of larvae within each cowpea. At least some of these factors are probably present in the insects and not the cowpeas but it should be recognized that the trypsin inhibitor may not be distributed uniformly around the seed and this may have contributed to the variable performance of individual larvae within a single seed. A most important outcome of the experiments of wider significance than simply in the context of TVu 2027 and C. maculatus, is that it is clear that the measurement of different parameters will suggest different answers to the question of which strain(s) are most and least affected by resistant hosts. This has been recognized previously (Redden and McGuire, 1983) but has been underlined in this study. It must also be accepted that other parameters will undoubtedly be involved in the longer-term effects of maintaining beetles on different varieties of their hosts. For example, it may be suggested that larval development in TVu 2027 could impair the fecundity of the emergent adults. In this particular case, no such effect was found (Dick, 1984). Assuming a genetic basis for the variation among the strains of C. maculutus employed in this study, it is reasonable to think that the breeding of beetles in resistant cultivars may result in a progressive increase in the frequency of resistance genes and, hence, an improvement in the performance of the strains on the cowpeas. Such a situation has already arisen in other pest species (Pimentel et al., 1984). Nevertheless, geographical variation in C. maculutus has important short-term implications for the success of resistant cowpea varieties produced for widespread distribution, where the bruchid-resistant properties are derived from a single resistant cultivar, TVu 2027. cowpea.

would like to thank the Storage Department of the Tropical Development and Research Institute for the provision of facilities and a research grant, Dr S. R. Singh of IITA for the stocks of TVu 2027 and SERC for their contribution to a CASE studentship with TDRI. We are indebted to Dr P. Dobie for his comments on the manuscript and Ann Edwards for secretarial assistance. Acknowledgements-We

REFERENCES Anon. (1980) A Manual of Crop Storage Technology. Ministry of Agriculture & Natural Resources, Malawi. Booker R. H. (1967) Observations on three bruchids associated with cowpea in northern Nigeria. J. stored Prod. Res. 3, l-15.

Caswell G. H. (1968) The storage of cowpeas in the northern states of Nigeria. Proc. agric. Sot. Nigeria 5, 44. Caswell G. H. (1981) Damage to stored cowpea in the northern part of Nigeria. Samaru J. agric. Rex 1, 11-19. Centre for Overseas Pest Research (1981) Pest Control in Tropical Grain Legumes. COPR, London. Dick K. M. (1984) Bionomic variation among three populations of the Southern cowpea weevil, Callosobruchus maculatus, and their responses to resistant varieties of the primary host. Ph.D. thesis, University of London.

48

K. M. DlrK and P. F. CREDLAND

Dick K. M. and Credland P. F. (1984) Egg production and development of three strains of Cullosobruchus maculafus (Fab.) (Coleoptera: Bruchidae). J. stored Prod. Res. 20, 221-221. Gatehouse A. M. R. and Boulter D. (1983) Assessment of the antimetabolic effects of trypsin inhibitors from cowpea (Vigna unguiculata) and other legumes on development of the bruchid beetle Callosobruchus maculatus. J. Sci. Fd Agric. 34, 345-350. Gatehouse A. M. R., Gatehouse J. A., Dobie P.. Kilminster A. M. and Boulter D. (1979) Biochemical basis of insect resistance in Vigna unguiculukz. J. Sci. Fd Agric. 30, 948-958. Howe R. W. (1971) A parameter for expressing the suitability of an environment for insect development. J. srored Prod. Res. 7, K--65. International Institute of Tropical Agriculture (1981) Reseurch Highlights ,fbr 1980. Ibadan, Nigeria. Pimentel D.. Glenister C.. Fast S. and Gallahan D. (1984) Environmental risks of biological pest controls, O&OS 42, 2833290. Pixton S. W. and Warburton S. (1968) The time required for conditioning grain to equilibrium with specific relative humidities. J. .stored Prod. Res. 4, 261 26.5. Redden R. J. and McGuire J. (1983) The genetic evaluation of bruchid resistance in seed of cowpea. .4ust. J. ugric. Res. 34, 707 715. Redden R. J.. Dobie P. and Gatehouse A. M. R. (1983) The inheritance of seed resistance to Callosobruchus mnculufus Aust. F. in cowpea (Vigna unguiculufa L. Walp.) I. Analyses of parental, F, , FL, F,, and backcross seed generations. J. agric. Res. 34, 681-695.