928 Journal of Applied Sciences Research, 9(1): 928-936, 2013 ISSN 1819-544X This is a refereed journal and all articles are professionally screened and reviewed

ORIGINAL ARTICLES

Survey and molecular identification of pests infesting stored leguminous seeds and their associated parasitoids

Emam, Azza K., Hanafy, H.E., Salama, Shadia I. and Badoor, I.M.

Plant Protection Department, Fac. Agric, Ain Shams Univ., Shoubra El-Kheima, Cairo, Egypt.

ABSTRACT

A total of 446 adult individuals (per 12 kilograms) representing four different insect pest species belonging to Bruchidae, order Coleoptera were counted from the residue samples collected from the seed warehouses located at El-Sahel stores, during 12 months of survey work. The four bruchid insect species detected were: (Fab.), Callosobruchus chinensis (L), Bruchidius incarnatus (Boheman) and Acanthoscelides obtectus (Say). Generally, the lowest number of adult detected in the monthly seed residue samples was during January, meanwhile, higher number of insects was found in summer months. From the conducted survey, the encountered coleopteran species Callosobruchus maculatus and Callosobruchus chinensis presented the highest percentage of collected insects 45.92 and 30.90 %, respectively. These were followed by a lower percentage of 15.45 % for Bruchidius incarnates. Meanwhile, Acanthoscelides obtectus were presented by 7.73%...Three hymenopterous parasitoids were detected in the samples. Two of them were belonging to i.e. Eupelmus vuilleti (Crawford) and Dinarmus basalis (Rond.), meanwhile the third Uscana lariophaga Steffan was belonging to Trichogrammatidae. Samples of bruchid insect pests and their associated parasitoids detected in the present work were subjected to RAPD-PCR with eight random primers. All RAPD primers used in the present study allowed for enough distinction among the seven species under study except both primers F04 and B17 (for parasitoid species), while the primer F04 was the only exception for insect pests species. Overall comparison among species across the eight primers revealed the ability of RAPD- PCR in distinguishing among closely related species. C.maculatus and C.chinensis pests were shown to be the most genetically-close species (similarity index of about 81.50%). B.incarnatus was similar to C. chinensis with 81.10% and to C.maculatus with 77.30% which indicates that B.incarnatus was closer to C.chinensis than to C.maculatus. A.obtectus was closer to C.chinensis and C.maculatus (73.30% and 72.50%, respectively) than to B.incarnatus with 66.70%. The parasitoid species E.vuilleti and D.basalis were shown to be the most genetically-similar species (a similarity index of about 81.30%), meanwhile, the similarity between U.lariophaga and also D.basalis was 81.50 % , while with E.vuilleti was 79.60.

Key words: Bruchidae, Leguminous seeds, Parasitoids, RAPD-PCR Marker.

Introduction

Legume seeds are considered a main source of protein for human and nutrition (Smartt, 1976). Bruchids are serious insect pests and cause considerable damage to seeds and grains (Shomar, 1963). Although numerous insect pests attacked all parts of bean plants, bruchids were the most important field and storage pests (Abat and Ampofo, 1996). Bruchid seed beetles are important parasites of legume seeds, but their effect on germination can be unpredictable. Beetles deplete seed resources and can kill the embryo but also scarify seeds (Fox et al., 2012). Weight losses caused by the bruchids and other factors after 7 months of storage averaged 8.5% in dry beans stored by small-scale farmers. The storage losses caused by insects and factors other than insects were estimated as 6.9 and 1.6%, respectively, and bruchids accounted for 24.5% of the combined losses (Espinal et al., 2004). Entomophagous insects including predators and parasitoids play a reasonable role in the biological control of stored grain and seed pets in warehouses. Bruchids destroying cowpea seeds are always associated with several entomophagous species of . The most common entomophagous species listed were an egg parasitoid, Uscana lariophaga and three solitary larval and pupal ectoparasitoids, Dinarmus basalis, Eupelmus vuilleti and E.orientalis (Antoine et al., 2005; Huignard et al., 2005; Sanon et al., 2005 and Amevoin et al., 2006). RAPD- PCR technique has been employed to identify and detect interspecific and intraspecific genetic variations in different species which were not easily identified by morphological characters (Purves et al., 1997

Corresponding Author: Hamdy E.M. Hanafy, Department of Plant Protection, Faculty of Agriculture, Ain Shams University, Shoubra El-Kheima, Cairo, Egypt E-mail: [email protected] 929 J. Appl. Sci. Res., 9(1): 928-936, 2013 and Vijay et al., 2006). DNA differences between species are measurable with confidence and can be identified from DNA sequences alone (Whiting et al., 1997). The present work aimed to survey the stored leguminous seeds insect pests, and their associated parasitoids, in seeds residues taken from warehouses located in Cairo government. Furthermore, a molecular study was carried out to identify the detected bruchid beetle species and also their associated hymenopteran parasitoids to determine the genetic relationships among them using molecular markers in RAPD- PCR technique.

Materials and Methods

1-Survey of insect pests infesting stored leguminous seeds and their associated parasitoids:

One- year survey was carried out in an open grain warehouse at El Sahel, Rod Elfarag in Cairo Governorate to distinguish insect pests that infested stored leguminous seeds as encountered in seed residues. The leguminous seeds stored in this warehouse were cowpeas (Vigna unguiculata L.) , faba beans (Vicia faba L.), chickpeas (Cicer arietinum L.), common beans (Phaseolus vulgaris L.) and soybeans (Glycine max Merrill). Seed residues refer to all left over the floor of the warehouse, damaged, mechanically broken, waste seeds or those mixed with dust and sweepings was inspected for determine the presence of any insect pests. Only adult insects were considered and identified according to the taxonomic keys provided by Shomar (1963) and their number was recorded. At each monthly collection of the samples, the maximum and minimum temperatures as well as relative humidity were obtained from ''Ten days agro meteorological weather report, Ministry of Civil Aviation, Meteorological Authority, Cairo, Egypt. This was important to correlate the abundance of any insect species with its surrounding environment. Also, from the collected seed residues samples, the presence of parasitoids associated with the insect pests were surveyed. The mounted parasitoids were identified according to the taxonomic keys provided by Graham (1969). Obtained data were statistically analyzed by using Bivariate correlation procedure of the SPSS for windows version 10.0 and SAS statistical computer program software.

2-Molecular studies:

The collected samples of bruchid insect pests and their associated parasitoids were identified according to the taxonomic keys provided by Shomar (1963) and Graham (1969), respectively. Four insect species and three species of parasitoids were investigated and listed in Table 1.

Table 1: Insect and parasitoids species used for DNA analysis. Sample type Scientific name Family Bruchidius incarnatus (Boh.) Bruchidae Callosobruchus maculates (Fab.) Bruchidae Insect Callosobruchus chinensis (L.) Bruchidae species Acanthoscelides obtectus (Say) Bruchidae

Eupelmus vuilleti (Craw.) Pteromalidae Parasitoids Dinarmus basalis (Rond.) Pteromalidae species Uscana lariophaga Steffan Trichogrammatidae

2-1- DNA extraction:

The DNA extraction of four insect species and three parasitoid species was carried out according to Cenis et al., 1993.

2.2. The electrophoresis conditions of the polymerase chain reaction (PCR):

Thirty random 10 bp primers were tested with the considered coleopteran insect pest and their hymenopterans' parasitoid species using Random Amplification Polymorphic DNA (RAPD) approach based on polymerase chain reaction (PCR) technology. Only eight of them were successful to detect amplified fragments in the tested species. Table 2 listed names and sequences of the successful random primers.

2.3. Data analysis:

Data of polymorphic, monomorphic and unique bands for both the considered coleopteran insect pests and hymenopteran parasitoid samples were scored together and analyzed using the UVP gel documentation system. The analysis was carried out according to Gurney et al., 2000.

930 J. Appl. Sci. Res., 9(1): 928-936, 2013

Table 2: Names and sequences of the successful random primers. Name Sequence A07 5'-TGCCGAGCTG-3 B14 5'-TCCGCTCTGG-3' B17 5'-AGGGAACGAG-3' B20 5'-GGACCCTTAC-3' F01 5'-ACGGATCCTG-3' F04 5'-GGTGATCAGG-3' F07 5'-CCGATATCCC-3' F09 5'-CCAAGCTTCC-3'

Results:

1. Survey of insect pests infesting stored leguminous seeds:

As presented in Table 3 a total of 466 adult individuals per 12 kilograms representing four different insect pest species belonging to Bruchidae, order Coleoptera were counted in the residue samples collected from the seed warehouses located at El-Sahel stores during 12 months of survey. The four bruchid insect species detected were: (1) The mung bean or cowpea beetle Callosobruchus maculatus (Fab.) (2) The azuki bean or southern cowpea beetle Callosobruchus chinensis (L.) (3) The small broad bean beetle Bruchidius incarnatus (Boheman). (4) The common (pinto) bean beetle Acanthoscelides obtectus (Say) It is worth mentioning that this survey was only concerned with the encountered adult stage of the insect pests. The coleopteran species Callosobruchus maculatus and Callosobruchus chinensis presented the highest percentage of the total number of collected insects being 45.92 and 30.9%, respectively. This is followed by a lower percentage of 15.45% for Bruchidius incarnates. Meanwhile, Acanthoscelides obtectus were presented by only 7.73%.

Stored leguminous crop insect pests Associated parasitoids Insect

Species

i .basalis .basalis .incarnatus .incarnatus .obtectus .vuillet Total U.lariophaga C.chinensis B A E D C.maculates Total Total 144.00 72.00 36.00 214.00 466.00 44.0 36.00 24.00 104.00 Mean 12.00 6.00 3.00 17.83 38.83 3.66 3.00 2.00 8.66 % 30.90 15.45 7.73 45.92 42.3 34.61 23.07

2. Parasitoids associated with insect pests infesting stored leguminous seeds:

Three hymenopteran parasitoids were detected in the samples collected at the leguminous seed warehouses at El Sahel. Two of them were belonging to family Pteromalidae being Eupelmus vuilleti (Crawford) and Dinarmus basalis (Rond.). The third one Uscana lariophaga Steffan was belonging to Trichogrammatidae. Data presented in Table 3, shows that a total of 104.00 adult parasitoid individuals per 12 kilograms were counted. The most abundant species was E. vuilleti being 44 adult during the year of survey, followed by 36 adult insects of D. basalis and the least abundance was 24 adults by U. lariophaga. These species could be respectively represented by 42.30, 34.61 and 23.07 % of the total number of detected parasitoids. In Table 4, the statistical analysis revealed that there were significantly positive correlations between the population size of each of C.maculatus and C.chinensis, and that of their two parasitoids E.vuilleti and U.lariophaga. The correlation coefficient values were 0.66; 0.60 and 0.51, 0.54 respectively. Meanwhile “r” values were 0.20 and 0.44 for the third parasitoid D.basalis. B.incarnatus population dynamics were also positively correlated with those of their associated parasitoid E.vuilleti (r= 0.62), but not with those of D.basalis and U.lariophaga where “r” values were 0.28 and 0.46, respectively. The “r” value was not significant in regard correlation between population size of A.obtectus and its associated parasitoids where it ranged 0.07-0.20. The combined effects of the three parasitoids showed that these natural enemies were affected as a group with 25%, 39%, 18% and 51% on the population dynamics of C.chinensis, B.incarnatus, A.obtectus, and C.maculatus, respectively.

931 J. Appl. Sci. Res., 9(1): 928-936, 2013

Table 4: Simple correlation and partial regression between changes in numbers of bruchid insect pests and their associated parasitoids. Bruchid host species Parasitoids C. chinensis B. incarnatus A. obtectus C. maculates E. vuilleti r 0.51* 0.62* 0.07 0.66* b 0.94 0.98 0.56 2.64 S.E. 1.7 0.66 0.86 1.80 D. basalis r 0.44 0.28 0.20 0.20 b 1.20 0.07 0.83 1.49 S.E. 1.98 0.77 1.01 2.11 U. lariophaga r 0.54* 0.46 0.17 0.60* b 0.16 0.08 0.91 1.48 S.E. 1.55 0.61 0.79 1.66 a 11.67 2.44 0.25 9.64 E.V.% 25 39 18 51 a = regression constant, r=correlation coefficient, b=regression coefficient, S.E.=Standard Error, E.V.= Explained variance

Molecular Identification:

The four coleopteran insect pests and their three associated parasitoids considered in the present work were subjected to RAPD-PCR with eight random primers; their photographed profiles are shown in Fig.1. The amplified fragments were weighted according to the molecular weights of known marker and classified to band types for insect pest and parasitoid samples (Table 5 A and B). Primer F04 failed to detect any specific positive or negative marker with either the insect pest or parasitoid samples. This primer often gave monomorphic bands. Also, primer B17 failed to detect any positive or negative markers with the three parasitoid samples but gave just one negative marker (480 bp) for only C.maculatus but not with other three insect pests samples. Two other primers showed few unique patterns in the considered samples of which primer F07 gave one positive marker (748 bp) for the beetle C.maculatus and also for the parasitoid U.lariophaga sample (861 bp) and another negative (1250 bp) were observed. In addition, primer B14 detected one positive marker (286 bp) for A.obtectus and two specific markers, a positive (550 bp) and a negative markers (854 bp) with D. basalis and U.lariophaga, respectively. Other specific markers scored for both pest and parasitoid samples were primers A07, B20, F01 and F09:- (i) The first primer A07 exhibited five positive markers, i.e. 1490, 1139, 1002, 764 and 355 bp and two negative ones 711 and 596 bp for the insect pest samples and clearly marked the sample of A.obtectus. Furthermore, this primer gave a negative pattern (854 bp) for U. lariophaga and a positive one (550 bp) for D.basalis. (ii) The second primer B20 scored two positive patterns with B. incarnatus (3572 bp and 2836 bp) and a negative pattern with C. maculatus (1175 bp). Also, a positive pattern (699 bp) was scored with E. vuilleti and three negative patterns (950 bp, 596 bp and 349 bp) for D. basalis. (iii) The third primer F01 detected five positive bands (1873 bp, 1584 bp, 978 bp, 450 bp and 361 bp) four of these bands appeared for A. obtecus and one (1584 bp) for C. chinensis . In addition, two positive bands 562 bp and 511bp were detected with the two parasitoids E. vuilleti and D. basalis, respectively. Meanwhile, two negative bands 399 bp and 277 bp were detected in B. incarnates and A. obtecus, respectively and a third band 361 bp for U. lariophaga. (iv) The fourth primer F09 gave three positive markers 962 bp, 721 bp and 452 bp, the former two were detected for A. obtecus and the latter was in C. chinensis . Another three positive bands 764 bp, 509 bp and 355 bp appeared with E. vuilleti parasitoid. The two negative markers 2125bp and 391 bp of this primer were evident in B. incarnates and one (1002 bp) for D. basalis. As seen in Table 5 A, as many as 25 out of 165 (about 15.16 %) bands total specific markers were useful RAPD markers for Coleoptern species. Of these bands 17 scored for the presence of a unique band i.e. positive markers, meanwhile 8 scored for the absence of a common band i.e. negative markers. Table 5B shows that 17 total specific markers out of 106, i.e. 16.04% were useful RAPD markers for the parasitoid insects. Of these 9 scored positive for the presence of a unique band, meanwhile 8 scored as negative markers. In summation, of the Coleoptern insect samples, A.obtectus scored the highest number of specific markers (14 bands) followed by B. incarnatus (5 bands), C. maculatus (4 bands) then the last C. chinensis (2 bands). Of the three parasitoids, D.basalis scored 7 specific markers meanwhile both of E.vuilleti or U.lariophaga scored 5 specific markers.

932 J. Appl. Sci. Res., 9(1): 928-936, 2013

All of the RAPD primers used in the present study allowed for enough distinction among the four Coleoptern insect species except F04. Also, except for the two primers F04 and B17 the other primers were successful in the distinction of the three parasitoid species, (Fig.1). The comparison among insect species by the eight primers revealed the ability of RAPD-PCR in distinguishing among closely related species. These markers can be used in subsequent experiments to detect molecular markers for polymorphic genes among these species.

Fig.1-a: profile of amplified fragments of Primer Fig.1-b: Profile of amplified fragments of Primer (A07) (B14)

Fig.1-c: profile of amplified fragments of Primer Fig.1-d: Profile of amplified fragments of Primer (B17) (B20)

933 J. Appl. Sci. Res., 9(1): 928-936, 2013

Fig.1- e:profile of amplified fragments of Primer Fig1-f:Profile of amplified fragments of Primer (F01) (f04)

Fig.1 -g:profile of amplified fragments of Primer Fig.1-h:Profile of amplified fragments of Primer (F07) (f09)

Fig. 1: Comparison of the RAPD –PCR banding patterns of four pests and three parasitoids samples amplified with random primers. M: 1kb ladder DNA Molecular marker. Lanes 1 -4 refers to pest samples and 5-7 refers to parasitoid samples.

Table 5A: Number of amplified fragments markers of four Coleoptern species based on RAPD– PCR analysis. RAPD Primers Insects species A07 B14 B17 B20 F01 F04 F07 F09 Total B. incarnatus AF 4 6 4 5 5 3 4 3 34 SM 0 0 0 2 1 0 0 2 5 C.maculatus AF 3 7 3 2 6 4 6 10 41 SM 1 0 1 1 0 0 1 0 4 C. chinensis AF 4 6 4 4 7 4 4 7 40 SM 0 0 0 0 1 0 0 1 2 A.obtectus AF 8 8 4 4 9 3 3 11 50 SM 6 1 0 0 5 0 0 2 14 TSM 7 1 1 3 7 0 1 5 25 TAF 19 27 15 15 27 14 17 31 165 PB 6 2 7 5 6 2 4 16 48 TAF = Total amplified fragment, PB = Polymorphic bands, AF = Amplified fragment, SM = specific marker, including either the presence or absence of a band in pest species, TSM = Total no. of specific markers across pest species.

934 J. Appl. Sci. Res., 9(1): 928-936, 2013

Table 5B: Number of amplified fragments markers of three parasitoid species based on RAPD –PCR analysis. RAPD Primers parasitoid species A07 B14 B17 B20 F01 F04 F07 F09 Total E.vuilleti AF 4 4 3 6 4 9 4 6 40 SM 0 0 0 1 1 0 0 3 5 U.lariophaga AF 3 3 3 5 2 9 4 3 32 SM 1 1 0 0 1 0 2 0 5 D. basalis AF 5 5 3 2 4 9 4 2 34 SM 1 1 0 3 1 0 0 1 7 TSM 2 2 0 4 3 0 2 4 17 TAF 12 12 9 13 10 27 12 11 106 PB 2 2 0 6 2 0 2 2 16 TAF = Total amplified fragment, PB = Polymorphic bands, AF = Amplified fragment, SM = specific marker, including either the presence or absence of a band in parasitoid species, TSM = Total no. of specific markers across parasitoid species.

As exhibited in Table 6, the two bruchids, C. maculatus and C.chinensis were shown to be the most genetically-close species with a similarity index of about 81.50%. B.incarnatus was similar to C.chinensis with 81.10% and to C.maculatus with 77.30% which indicates that B.incarnatus was closer to C.chinensis than to C.maculatus. Meanwhile, A. obtectus was slightly closer to C.chinensis and C.maculatus ( 73.300% and 72.50%, respectively) than to B. incarnatus with 66.70%.

Table 6: Similarity matrix of insect pests as revealed by RAPD data. Insect species B. incarnatus C.maculatus C. chinensis A.obtectus B. incarnatus 1.000 0.773 0.811 0.667 C.maculatus 0.773 1.000 0.815 0.725 C. chinensis 0.811 0.815 1.000 0.733 A.obtectus 0.667 0.725 0.733 1.000

The parasitoid species E. vuilleti and D. basalis were shown to be the most genetically similar species with a similarity index of 81.30%. Meanwhile, the similarity between U. lariophaga and also D. basalis was 81.50 %, while with E. vuilleti was 79.60 (Table7).

Table 7: Similarity matrix of three parasitoids as revealed by RAPD data. Insect species E.vuilleti U. lariophaga D. basalis E.vuilleti 1.000 0.796 0.813 U. lariophaga 0.796 1.000 0.815 D. basalis 0.813 0.815 1.000

Discuusion:

Although leguminous seeds are infested by many insects, bruchids were the most important field and storage insect pests (Abate and Ampofo, 1996; Akkaya, 1998 and Sanon et al., 1998). In Niger, Alebeek (1996) found that eggs of C. maculatus composed 96% of samples after sampling cowpea seeds collected from storage units. In Portugal, Mateus and Carvalho El-Mexia (2003) found that Bruchus rufimanus and B. pisorum were the most abundant insects infesting stored beans and peas. In Brazil, C. analis was a major pest to stored soybean (Costa et al., 2007). In Mexico, Corral et al. (1992) found Acanthoscelides obtectus only in pinto beans (Phaseolus vulgars). Bruchids that infect cowpea seeds are generally associated with several entomophagous species of Hymenoptera. The most widespread ectoparasitoids were reported to be Uscana lariophaga, Eupelmus vuilleti and Dinarmus basalis (Sanon et al., 1998; Zaghloul and Mourad, 1998; Gauthier et al., 1999; Islam, 2001; Schmale et al., 2001; Amevoin et al., 2007; Ndoutoume-Ndong and Rojas-Rousse, 2007 and Velten et al., 2007). The analysis of PCR provides an effective dimension for distinguishing among organism according to the banding patterns of PCR products (Bebeli et. al.1997). By the use of eight primers complete identification was obtained for the four studied Coleopteran insect species, B. incarnatus, C.maculatus, C. chinensis and A. obtectus and their three parasitoid, Eupelmus vuilleti, Dinarmus basalis and Uscana lariophaga. These results confirm the importance of RAPD-PCR technique as an efficient tool for identification of insect species especially when they were closely related. Also, this technique is most important for the identification of species which were not easily identified by morphological characters. These markers can be used in subsequent experiments to identify polymorphic genes and therefore distinguish among these closely related species. Many authors used this method for insect species identification, for example, Gawel and Barro and Driver (1997), Beitia et al. (1997), Guirao et al. (1997); Moreno et al.(2010) and Veijalainen et al.(2011).

935 J. Appl. Sci. Res., 9(1): 928-936, 2013

References

Abate, T. and J.K.O. Ampofo, 1996. Insect pests of beans in Africa, their ecology and management. Annual Rev. Entomol., 41: 45-73. Akkaya, A., 1998. Review of harmful insect fauna associated with lentil in Turkey. Lens Newsletter, 25(1/2): 63-65. Alebeek, F.A.N., 1996. A survey of bruchid pests and their parasitoids in stored cowpea in Niger. Proc. Sec. Experim. Applied Entomol Netherlands Entomol. Soc., 5: 145-150. Amevoin, K., I.A. Glitho, Y. Nuto and J.P. Monge, 2006. Population dynamics of natural bruchids and their larval and pupae parasitoids in experimental cowpea storage in Guinean zone. Tropicultura. AGRI- Overseas, Brussels, Belgium, 24(1): 45-50. Amevoin, K., A. Sanon, M. Apossaba and I.A. Glitho, 2007. Biological control of bruchids infesting cowpea by the introduction of Dinarmus basalis (Rondani) (Hymenoptera: Pteromalidae) adults into Farmers' stores in West Africa. J. Stored Prod. Res., 43(3): 240-247. Antoine,S., D. Clementine, O.A. Patoin and H. Jacques, 2005. Field occurrence of bruchid pests of cowpea and associated parasitoids in a subhumid zone of Burkina Faso: importance on the infestation of two cowpea varieties at harvest. Plant Pathol. J., 4(1): 14-20. Bebeli, P.J., Z. Zhou, D.J. Somers and J.P. Gustafson, 1997. PCR primed with minisatellite core sequences yields DNA fingerprinting probes in wheat. Theor. Appl. Genet., 95: 276-283. Beitia, F., I. Mayo, E.M. Robles-Chillida, P. Guirao, J.L. Cenis, R. Albajes and A. Carnero, 1997. Current status of Bemisia tabaci (Gennadius) in Spain: the presence of biotypes of this species. Bull. OILB SROP, 20: 99- 107. Cenis, J.L., P. Perez and A. Fereres, 1993. Identification of Aphid (Homoptera: Aphididae) species and clones by Random Amplified Polymorphic DNA. Entmol. Soc. Amer., 86: 545-550. Corral, F.J.W., M.O.C. Rocha, J.B. Flores and F.B. Andrade, 1992. Insect species infesting grain stored in rural Communities in the northeast of Sonora, Mexico. Southwestern Entomologist, 17(4): 327-331. Costa,V.A., E.C. Guzzo, A.L. Lourencao, M.A.G.C. Tavares and J.D. Vendramim, 2007. Occurrence of Dinarmus basalis in Callosobruchus analis in stored Soybean in Sao Paulo, Brazil. Scientia Agricola, 64(3): 301-302. Espinal, R., R. Higgins and V. Wright, 2004. Economic losses associated with Zabrotes subfasciatus (Boheman) (Coleoptera: Bruchidae) and Acanthoscelides obtectus (Say) (Coleoptera: Bruchidae) infestations of stored dry red beans (Phaseolus vulgaris L.) in Southeastern Honduras. CEIBA. Zamorano Academic Press, Tegucigalpa, Honduras, 45(2): 107-119. Fox, C.W., W.G. Wallin, M.L. Bush, M.E. Czesak and F.J. Messina, 2012. Effects of seed beetles on the performance of desert legumes depend on host species, plant stage, and beetle density. Journal of Arid Environments, 80: 10-16. Gauthier, N., A. Sanon, J.P. Monge and J. Huignard, 1999. Interspecific relations between two sympatric species of Hymenoptera, Dinarmus basalis (Rond) and Eupelmus vuilleti (Crw), Ectoparasitoids of the bruchid Callosobruchus maculatus (F). J. Insect Behavior, 12(3): 399-413. Graham, M.W.R., de V., 1969. The Pteromalidae of North Western Europe (Hymenoptera, Chalcidoidea). Bull. Br. Mus. Nat. His. Entmol. (Suppl)., 16: 908. Guirao, P., F. Beitia and J.L. Cenis, 1997. Biotype determination of Spanish populations of Bemisia tabaci (Hemiptera: Aleyrodidae). Bull. Entmol. Res., 87: 587-593. Gurney, T. jr, R. Elbel, D. Ratnapradipa and R. Brossard, 2000. Introduction to the molecular phylogeny of insects. Pages 63-79 in Tested studies for laboratory teaching, 21 (S.J. Karcher, Editor). Huignard, J., S. Dugravot, K.G. Ketoh, E. Thibout and A.I. Glitho, 2005. Use of secondary plant products to protect the seeds of a legume, cowpea. Effects on insect pests and their parasitoids. Biopesticides of Plant Origin. Lavoisier Publishing, Paris, France: 123-137. Islam, W., 2001. Effect of adult nutrition on longevity and fecundity of Dinarmus basalis (Rond.) (Hymenoptera: Pteromalidae). J.Control. Soc. for Biocontrol Advancement, Bangalore, India, 15(1): 5-10. Mateus, C. and A. Carvalho, E.L. Mexia, 2003. Bruchidae (Coleoptera) in stored Leguminosae:a survey conducted In Portugal.Advances in stored product protection. Proceedings of the 8th International Working Conference on Stored Product Protection,York,UK, CABI Pub.,Wallingford, UK, 406-409. Moreno, F., C. Menendez, I. Perez-Moreno and V. Marco, 2010. Molecular identification of a local population of Trichogramma cacoeciae Marchal, 1927 (Hym., Trichogrammatidae) collected in La Rioja (Spain). Boletin de Sanidad Vegetal, Plagas, 36(1): 91-99. Ndoutoume-Ndong, A. and D. Rojas-Rousse, 2007. Is there elimination of Eupelmus orientalis Crawford by Eupelmus vuilleti Crawford (Hymenoptera: Eupelmidae) out of the niebe (Vigna unguiculata Walp) stocks? Annal Soc. Entomol. France, 43(2): 139-144.

936 J. Appl. Sci. Res., 9(1): 928-936, 2013

Purves, W.K., G.H. Orians, H.C. Heller and D. Sadava, 1997. Life, the science of biology.Fifth edition. Sinauer Associates, Sunderland, Massachusetts, 1243 pages. Sanon, A., A.P. Ouedraogo, Y. Tricault, P.F. Credland and J. Huignard, 1998. Biological control of bruchids in cowpea stores by release of Dinarmus basalis (Hymenoptera: Pteromalidae) adults. Environ. Entomol., 27(3): 717-725. Sanon, A., C. Dabire, A.P. Ouedraogo and J. Huignard, 2005. Biological control of Callosobruchus maculatus F. (Coleoptera: Bruchidae) populations by two sympatric parasitoid species, Dinarmus basalis Rond. and Eupelmus vuilleti Crw. Belgian J. Entomol.,7(1): 57-69. Schmale, I., F.L. Wackers, C. Cardona and S. Dorn, 2001. Control potential of three hymenopteran parasitoid species against the bean weevil in stored beans: the effect of adult parasitoid nutrition on longevity and progeny production. Biol. Control, 21(2): 134-139. Shomar, N.F.H., 1963. A monographic revision of the Bruchidae of Egypt (U.A.R.). Bull. Soc. Entmol. Egypte, 47: 141-196. Smartt, J., 1976. Tropical pulses.Textbook,Longman Eds. London, pp: 348. Veijalainen, A., G.R. Broad, N. Wahlberg, J.T. Longino and I.E. Saaksjarvi, 2011. DNA barcoding and morphology reveal two common species in one: Pimpla molesta stat. rev. separated from P. croceipes (Hymenoptera, Ichneumonidae). Zookeys, 124: 59-70. Velten, G., A.S. Rott, C. Cardona and S. Dorn, 2007. Effects of a plant resistance protein on parasitism of the common bean bruchid Acanthoscelides obtectus (Coleoptera: Bruchidae) by its natural enemy Dinarmus basalis (Hymenoptera:Pteromalidae).Biol. Control, 43(1): 78-84. Vijay, L.S., B. Suman, K.G. Tajinder, A.B. Adnan, K. Mamtesh, J. Singh Jagmohan and C.S. Ranbir, 2006. Molecular characterization of two species of butterflies through RAPD - PCR Technique, Cytologia, 71: 81-85. Whiting, M.F., J.C. Carpenter, Q.D. Wheeler and W.C. Wheeler, 1997. The tresipera problem: Phylogeny of the homometabolous insect orders inferred from 18S and 28S ribosomal DNA sequences and morphology. Systematic Biol., 46: 1-68. Zaghloul, O.A. and A.K. Mourad, 1998. Studies on the egg-parasitoid, Uscana lariophaga Steffan(Hymenoptera: Trichogrammatidae), with reference to its role in the assessment of cowpea losses due to Callosobruchus maculatus (F.) (Coleoptera: Bruchidae) in Egypt. Mededelingen Faculteit Landbouwkundige en Toegepaste Biologische Wetenschappen, Univer. Gent., 63(2b): 441-453.