BIOTROPIA No. 4, 1990/1991: 19-30 STRAIN DIFFERENCES IN TWO SPECIES OF CALLOSOBRUCHUS (COLEOPTERA: BRUCHIDAE) DEVELOPING ON SEEDS OF COWPEA {VIGNA UNGUICULATA (L.)} AND GREEN GRAM {V. RADIATA (L.)} RENNIE ROESLI Department of Agronomy, Faculty of Agriculture Bogor Agricultural University, Bogor, Indonesia PHIL DOBIE Storage Department, ODNRI, Slough, UK BRIAN M. GERARD School of Agriculture, Faculty of Science University of Edinburgh, Edinburgh, UK ABSTRACT Ovipositional behaviour, development period, and density effect on adult survival of C. maculatus strains from Indonesia, Nigeria, and Yemen, and C. chinensis strains from Indonesia and Kenya on cowpea and green gram were studied at 20°C and 70% relative humidity. Variations on ovipositional behaviour were found among C. maculatus as well as among C. chinensis strains. Variations on developmental period were found only among C. maculatus strains. The developmental period of Callosobruchus spp. was shorter on green gram than that on cowpea. Density effect was remarkably found only on adult survival of C. maculatus Yemen strain. These results make useful contribution to the species biology, and have important implication if strains of these species are accidentally imported to countries, or when new legume crops are introduced. INTRODUCTION Beetles belonging to the family Bruchidae are the most important insect pests of stored legumes. Infestation by bruchids causes losses of weight, nutritional value and germination potential, and therefore the commercial value of the commodity may be reduced (Southgate 1978; Dick and Credland 1986). The most economically important and widespread bruchids species are the cowpea seed beetle, Callosobruchus maculatus (Fabricius), and the Adzuki bean beetle, C. chinensis (Linnaeus) (Southgate 1978; TDRI 1984). The use of resistant varieties of cultivated legumes is one of the recommended control methods of bruchid infestations. Varietal resistance against Callosobruchus has been reported in cowpeas and chickpea (Dobie 1981; Raina 1971; Singh 1978). 19 BIOTROPIA No. 4, 1990/1991 However, there were variations reported in the response of geographically different strains of C. maculatus to a resistant variety of cowpea (Dick 1984; Dick and Credland 1986). Further studies on variation on geographically different strains of C. maculatus revealed the occurrence of differences in their biology and behaviour (Credland et al. 1986; Credland and Dick 1987; Credland 1986). This study was made to seek more information on the occurrence of geographical variations among C. maculatus and C. chinensis strains, especially to compare strains from Indonesia (Asia) with those from other tropical countries. MATERIALS AND METHOD Three strains of C. maculatus viz. strain from the IITA cultures, Nigeria (labeled as IITA), Yemen, and Indonesia; and two strains of C. chinensis viz. strains from Indonesia and Kenya were used. They were obtained from the culture of ODNRI, Slough, UK. Two types of seeds were used as hosts i.e. Californian black eyed cowpea (Vigna unguiculata (L.)) and Australian green gram (Vigna radiata (L.)). Daily egg production The surface area of cowpea seeds is larger than that of green gram. As oviposition of the species is assumed to be influenced by the surface area, the cowpea seed number used should therefore be different from that of green gram. After measuring the surface areas of both seeds, it was decided that the surface area of one cowpea seed was approximately equal to the surface area of three green gram seeds. One kernel of cowpea or three kernels of green gram were introduced into a 2.5 cm diameter and 5 cm high glass tube. A pair of adults (age < 1 day) of each strain was added into the tube, and the tubes were covered with foam bungs. Twenty five replicates were made on both types of seed and all insect strains. The whole set of experiment was kept in the laboratory at 27°C and the R.H. at 70%. The following day, the number of eggs laid by each female was counted and recorded, and egg-laden seeds were replaced by fresh seeds. The observation and seed replacement were done daily until the 10th day. Effect of seed availability on egg production Different numbers of seeds i.e. 1 (low), 3 (medium), and 10 (high) kernels of cowpea; and 3 (low), 9 (medium), and 30 (high) kernels of green gram were introduced into the tubes. One pair of adults (age < 1 day) of each strain was placed 20 Strain differences in two species of Callosobruchus-TH. Roesli, P. Dobie & B.M. Gerard in each tube and the tube was covered with foam bungs. Twenty replicates were made of all treatments and all insect strains, and kept in the experimental room. Observation on the number of eggs laid by each female was done on the 7th day after treatment. Developmental period Eight to 10 adults of each insect strain were introduced into each petri dish containing cowpea or green gram seeds. The dishes were covered with the lids and kept in the experimental room for one night The adults were removed the following day. Under a binocular microscope, seeds bearing a single egg were taken out and individually put into tubes. The tubes were covered with foam bungs and kept in the experimental room. Twenty five replicates were made of all insect strains. Observation on adult emergence were started on the 20th day after treatment. Emergence of fresh adults was recorded daily until no more adults emerged. Adult survival from seeds bearing different number of eggs To obtain cowpea seeds bearing 1, 2, 3, 4, and 5 eggs, the same procedure as in the developmental period experiment was applied. However, selection was done not only of seeds bearing 1 egg, but of those bearing 2, 3, 4, and 5 eggs as well. The replicate number of each treatment was designed to be inversely proportional to the egg density (Giga 1982). Thus, the number of eggs used were approximately the same in all treatments (Table 1). Table 1. The design of the experiment on adult survival from seed bearing different number of eggs No. of eggs/seed 1 2 3 4 5 No. of replicates 20 10 7 5 4 No. of eggs 20 20 21 20 20 Emergence of adults was observed and recorded daily starting from the 20th day after treatment. RESULT Daily egg production Some females were found to lay eggs on the seeds and on the tube walls and so both were recorded and analyzed separately. 21 BIOTROPIA No. 4, 1990/1991 The general pattern of daily egg production of all strains on seed was the same. High number of eggs were laid during the early period of the female's life. The maximum number was reached either on the second or third day, and then it gradually decreased (Fig. 1). The sum of 10-day egg production per female of all strains was analyzed by two factors analysis of variance. Oviposition on seed, on tube wall, or the total, over the period of 10 days was influenced by insect strain (Table 2). C. maculatus IITA strain was found to have the highest fecundity followed by C. chinensis Indonesian, C. maculatus Yemen, C. chinensis Kenyan, and C. maculatus Indonesian strain (Table 2, column "Total"). Differences in the fecundity were found between each strain of C. maculatus, as well as between each strain of C. chinensis (Table 2 and Fig. 2). C. chinensis Indonesian strain laid the highest number of eggs on seed, followed by C. maculatus IITA strain. Lower numbers were laid by C. maculatus Yemen and C. chinensis Kenyan strains with the lowest number laid by C. maculatus Indonesian strain (Table 2). Large number of eggs on the tube wall were especially laid by C. maculatus IITA and Yemen strains. The rest of the strains laid very small number of eggs on the tube. The mean egg number was not significantly different from one another, but they differ significantly from those laid by C. maculatus IITA and Yemen strains. Seed species did not affect oviposition of all strains over 10 days, either on seed, on tube wall or the total (Table 3). Table 2. Means and standard errors of egg product ion of five strains of Callosobruchus maculatus over 10 days on seed, on tube wall, and the total Insect strains On seed On tube wall Total Cm* IITA 63 .90 a ±4.57 17.02 a ±2.55 80.92 a ±4.98 Cm Yemen 45.90 b ±4.11 6.06 b ±1.35 51.96b ±4.67 Cm Indonesia 32.08 c ±2.08 0.50 c ± 0.30 32.55 c ±2.15 Cc* Indonesia 68.24 a ±1.86 0.47 c ±0.25 68.74 a ±1.91 Cc Kenya 38.33 bc ±3.20 0.02 c ± 0.02 38. 35 be ±3.20 a, b and c indicate level of significance at 5%; relevant only down the column. * Cm for Callosobruchus maculatus Cc for Callosobruchus chinensis. Table 3. Means and standard errors of insect oviposition over 10 days on seeds, on tube wall, and the total Seed species On seed On tube wall Total Cowpea 48.91 NS ± 2.56 4.89 NS ± 0.96 53.80 NS ± 2.86 Green gram 51.08 NS ± 2.38 4.98 NS ± 1.09 56.06 NS ± 2.77 NS : Non significant. 22 Strain differences in two speciess of Callosobruchus – R. Roesli, P. Dobie & B.M. Gerard Figure 1. Oviposition over 10 days of C. Maculatus IITA (1), Yemen (2), Indonesia (3) and C. Chinensis on cowpea (Cp) and green gram (Gg) 23 BIOTROPIA No. 4, 1990/1991 Figure 2. Effect of seed availability on the fecundities of C. maculatus IITA (1), Yemen (2), Indonesia (3) and C. chinensis on cowpea (Cp) and green gram (Gg). 24 Strain differences in two species of Callosobruchus-R. Roesli, P. Dobie & B.M. Gerard Effect of seed availability on egg production Some females were also found to lay eggs on the tube wall. Eggs laid on seeds and on tube walls were analyzed separately. Tables 4, 5, and 6 show that the oviposition of Callosobruchus over 7 days was influenced by seed availability and insect strain, but not by the seed species. The total number of eggs increased significantly with the increase of seed availability. C. maculatus IITA produced the highest total number of eggs, followed by C. chinensis Indonesia. Lower number was produced by C. chinensis Kenya, and the lowest number was produced by C. maculatus Yemen and Indonesian strains. The number of eggs laid on the seed increased significantly with the increase of seed availability (Table 4). In contrast, the number of eggs laid on the tube wall decreased with the increase of seed availability (Table 4), however the number of eggs laid on tubes with low seed number did not significantly differ from that laid on tube with medium number of seeds. Table 4. Mean and standard errors of Callosobruchus spp. oviposition on seed, on tube wall, and the total at different seed availabilities Seed number On seed On tube wall Total Low Medium High 35.42 c±1.50 50.22 b±1.56 59.88 a ±1.46 8.55 b±1.14 .31 b±1.03 3. 63 a ±0.62 43.96 c± 1.78 57.51 b±1.81 63.51 a±1.54 a, b, and c indicate level of significance at 5%; relevant only down the column. Table 5. Mean and the standard errors of five strains of Callosobruchus spp. ovipositions on seed, on tube wall and the total Insect strains On seed On tube wall Total Cm* IITA 58.98 a ±2.52 18.68 a ±1.85 77.64 a ±2.39 Cm Yemen 36.54 c ± 1.85 11.50 b ± 1.36 48.04 c ± 2.21 Cm Indonesia 34.10 c ± 1.48 1.21 c ±0.24 35. 29 d ± 1.48 Cc* Indonesia 63 .05 a ±1.92 0.84 c ± 0.32 63. 89 b ± 1.90 Cc Kenya 49.85 b ±1.31 0.24 c ± 0.07 50.09 c ± 1.33 a, b, c, and d indicate level of significance at 5%; relevant only down the column. * Cm : C. maculatus CC : C. chinensis Table 6. Mean and standard errors of Callosobruchus spp. oviposition on seed, on tube wall, and the total of two kinds of seed. Seed kind On seed On tube wall Total Cowpea 49.29 NS ± 1.43 4 . 39 b ± 0.56 53 . .67 NS ± 1.51 Green gram 47.72 NS ± 1.30 8 . .60 a ± 0.95 56 . .32 NS ± 1.45 a and b indicate the level of significance at 5%; relevant only down the column. 25 BIOTROPIA No. 4, 1990/1991 C. maculatus IITA laid the highest number of eggs on tube wall followed by C. maculatus Yemen. The rest of the three strains laid only a small number of eggs on tube wall and they did not differ significantly (Table 5). The number of eggs laid on the tube containing green gram was higher than that on tube containing cowpea (Table 6). The effect of seed availability on egg production of individual strains on cowpea and green gram is shown in Figure 2. The effect of seed availability is remarkably seen in C. maculatus Yemen and Indonesian strains. Development period The results indicated that there were differences in the development rate among the insect strains (Table 7). The development period of C. maculatus IITA strain was the longest, and was significantly longer than the others. Development periods of C. maculatus Yemen and Indonesian strains did not differ significantly. The difference in the development period of the two strains of C. chinensis was not significant, however they were significantly shorter than the development periods of all C. maculatus strains (Table 7). Seed species significantly influenced C. maculatus and C. chinensis development periods. Most of the insect strains developed faster on green gram than on cowpea, except the C. maculatus strain from Yemen (Table 8). Table 7. Mean and the standard errors of development periods of 5 Callosobruchus strains Table 8. Mean and standard errors of development period of Callosobruchus on cowpea and green gram a and b indicate the level of significance at 5%. 26 Strain differences in two species of Callosobruchus-R. Roesli, P. Dobie & B.M. Gerard Adult survival from seeds bearing different number of eggs The results indicated that egg density, from 1 to 5 eggs per seed, did not influence significantly the number of adult survival of most strains, except on C. maculatus Yemen strain (Fig. 3). Remarkable decreases were noted on adult survival means of seeds bearing 4 and 5 eggs. Figure 3. Adult survival from seeds bearing different number of eggs of 5 different Callosobruchus strains. DISCUSSION Some females, especially those of C. maculatus females, were found to lay eggs on tube surface. However, there seemed to be less preference for oviposition on tube wall because the number of eggs laid on the tube wall was usually large only when there was a shortage of seeds for oviposition, such as during the peak days of the ovipositional period (Fig. 1), or when there were few seeds for oviposition (Fig. 2). Females of Callosobruchus spp. have been reported to control the successful development of their progenies by choosing an appropriate site for oviposition and distributing their eggs more or less equally over the available seeds. They avoid laying eggs on seeds with a rough surface and on seeds already bearing bruchid eggs if noninfested seeds are still available. If noninfested seeds are unavailable, the 27 BIOTROPIA No. 4, 1990/1991 females will lay eggs on seeds already bearing eggs, but will choose the bigger ones first. The females can also detect small differences in egg density, and prefer to oviposit on seeds bearing smaller number of eggs (Yoshida 1961; Avidov et al. 1965; Nwanze et al. 1975; Messina and Renwick 1985a, b). The discriminating ability of female Callosobruchus in uniformly distributing their eggs is highly developed, however, in some species the discrimination in choice of seeds suitable for larval growth is not developed. In other words, the ovipositional behaviour was not related to the suitability of seeds for the development of larvae (Avidov et al. 1965; Bhattacharya et al. 1977). Therefore, the apparent preference for oviposition on the tube wall under these conditions could possibly be more accurately described as a strong repellence to ovipositing on seeds that are already bearing numerous eggs. It is difficult to say if the differences in the number of eggs laid on the tube wall by different strains of C. maculatus were due to differences in the ability of females of different strains to discriminate between suitable sites for larval development or simply because they have different fecundities. For example, the fecundity of C. maculatus IITA female was the highest, and that strain female also laid the highest number of eggs on the tube wall. Probably the female of that strain had relatively greater seed shortage problem than the other two strains. In contrast, C. chinensis appears to have more developed discriminating ability to choose suitable sites for larval development. It was found that although females of the C. chinensis Indonesia had higher fecundity than those of the C. maculatus Yemen and Indonesian strains, fewer eggs were laid on the tube by C. chinensis Indonesia. The number of eggs oviposited by females C. maculatus Indonesian and Yemen strain was suppressed when only a small number of seed was available. However, those oviposited by the other strains were not remarkably suppressed with the reduction of seed availability. Credland (1986) stated that the conditions that determine the maximum fecundity differ within and between strains. The reduction in female fecundity as a response to low seed availability is perhaps due to deterrence effects, chemically or physically, of eggs already laid on the seed (Messina and Renwick 1985a). The development period of Callosobruchus, in this experiment was slightly shorter on green gram than that on cowpea. This suggests that green gram is a slightly better host for Callosobruchus. Giga and Smith (1978) reported that of the several pulses tested, including green gram and cowpea, green gram was the most favourable food species for oviposition, speed of development and survival of C. maculatus. When suitable host seeds are infested at numbers above the population's optimum density, there is a reduction in the number emerging of adults due to mortality which primarily took place during the larval stage (Utida 1941; Mitchel 28 Strain differences in two species of Callosobruchus-R. Roesli, P. Dobie & B.M. Gerard 1975; Giga 1982; Dick 1984). The reduction in the number of adults produced due to high density was more pronounced in the C. maculatus Yemen strain than those of other strains used in this experiment. Variations in the effect of density on the number of adults produced have also been reported to occur among strains of C. maculatus. Strains from Brazil and Nigeria (IITA) can produce more than ten adults from a seed with numerous eggs, whereas a strain from Yemen rarely produced more than three (Dick 1984; Dick and Credland 1984; Credland et al. 1986). The density effect on the number of adults observed in C. maculatus Indonesian strain, C. chinensis Indonesian and Kenyan strains seems to be similar to that in C. maculatus IITA. However, higher densities than the maximum density recorded in this experiment should have been used to be able to see the effect more clearly. The geographical variations on the biology and behaviour of C. maculatus and C. chinensis found in this experiment are possibly the result of either genetic evolution of a population which occupies a particular environment and is therefore subjected to that environment selection pressures; or genetic divergence among populations which is caused by chance fluctuations in its allele frequency; or the change in the gene pool (Dick 1984; Credland 1986). The occurrence of geographical variations among populations of an insect species should be noted when studying or referring to the species biology or behavioral characteristics. Attention should also be given to the possibility that a less important species population might become a serious pest if a better plant host species or variety were introduced to the area. ACKNOWLEDGEMENT The authors would like to thank Dr. C.P. Haines for his assistance and advice, Mr. D.J.B. Calverley, Head of Storage Department, ODNRI, Slough, UK. for providing laboratory space and facilities, and the British Council for the research funds. REFERENCE AVIDOV, Z., S.W. APPLEBAUM, and M.J. BERLINGER, 1965. Physiological aspects of host specificity in the Bruchidae II: Ovipositional preference and behavior of Callosobruchus chinensis (L.). Ent. Exp. & Appl. 8: 96-106. BHATACHARYA, A.K., P.K. PATHAK, and S.P. SHAH, 1977. Oviposition and development of Callosobruchus chinensis (Linn) (Coleoptera : Bruchidae) on several host species. Bull. Grain Technol. 15: 38-41. 29 BIOTROPIA No. 4, 1990/1991 CREDLAND, P.P. 1986. Effect of host availability on reproductive performance in Callosobruchus maculatus (F) (Coleoptera : Bruchidae). J. Stored Prod. Res. 22: 49-54. CREDLAND, P.P. and K.M. DICK. 1987. Food consumption by larvae of three strains of Callosobruchus maculatus Coleoptera : Bruchidae). J. Stored Prod. Res. 23: 31-40. DICK, K.M. 1984. 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