World List Abbreviation (Egypt. J. Bid. Pest Control) P 5 (liez) 1995 Published by Egyptian Society for Biological Control of pests (E§BCP) CAIRO Fonds Documentaire ORSTOM Cote: B%S?%Y EY A ~ EGYPTIAN JOURNAL OF BIOLOGICAL PEST CONTROL (Egypt. J. Biol. Pest Control) 0 A scientificjownal published twice a year by ESBCP. I 0 The Egyptian Society for Biological Control of Pests was founded in February 1989 in view to 'encowage and promote all aspects of biological and integrated pest control. 0 Annual subscription fee is 15 Egyptian pounds for .the Egyptian members due in January. Annual subscription fee is 15 US $ for members and institutions of foreign countsies. '0 Volume price (two issues) is L.E.' 20 for Egyptians and US $20 foidnon Egyptians (volumes #areavailable). i All coiyespondence should be addressed to: Secreta; General of society PO. Box, 154 Code 12211 - Giza, Egypt. (2), Egyptian Journal of Biological Pest Control, 5 1995, 127-130 HOST RANGE OF THE AUTO6;RAPHA CAIFORNKA MULTICAPSID NUCLEAR POLYHEDROSIS VIJ3US (ACMNPV) PN RELATION TO GWNULOSIS VIRUSES SUSCEPTIBLE LEPIDOPTERAN SPECIES IN EGYPT A. Taha", J. Giannotti*, X. LCry", M. Ravàlec** and S. Abo17ELA* * Entomovirology Laboratoly ORSTOM, Faculty of Agriculture. B.O. 26, Giza Code 1221 1, Cairo, Egypt ** Station de Recherche de Pathologie Compar&, 30380, St Christol-lè-Alès, France (Received,November 4,1995:Accepted, December 25, I995) ABSTRACT The host range OEAutographa calgomica multicapsid nuclear polyhedrosis virus (AcMNPV) was studied in Egypt on 3 pest species susceptible to granulosis viruses. Among the cotton leafworm, Spodoptera littoralis, the maize com borer, Sesamia cretica and the potato tuber moth Phfhorimaeaoperculella, only S. CretiCQand P. operculella were susceptible to the AcMNPV. Ultrastructural studies and virus detection by nucleic probe showed that S. cretica was highly susceptible, while only 5% infection ,was obtained in P. operadella. -. Key Words: AcMNPV, host range, GV, Spodoptera littoralis. Sesamia cretica, Phthorimaea operculella. small potato tubers were used as natural feeding source, while S. cretica was reared on a new semiartificial diet The Abtographa califmica multicapsid polyhedrosis (unpublished data). virus (AcMNPV) became a reference model for the viral ge- netic researches, biotechnology and biological control of in- Land Infection sects. Its wide range of interest was due to several reasons. AcMNPV produced on P. gosT'ella larvae was puri- A large number of lepidopteran species belonging to twelve fied and concentrated up to 60x10 PlBs per milliliter. families, often representing important pests with high geo- AcMNPV produced on the SF9 cell line waspurifiedas graphical distribution, were susceptible to AcMNPV follows: infected cells were scraped from the flask walls-and (Albrecht, 1986). The AcMNPV could be mass produced centrifuged at 13000 g for 15 min. The pellets were ;&us- ml and several of its hosts could be reared industrially on arti- pended in 1 tris (10 mMpH7.8).Thepolyhedrawere I =i ficial diets (Vail et al., 1972). The AcMNPV could be mul- mixed with Triton-X at the final concentration of O. 1%, then tiplied in vitro on a certain number of insect cell lines with used as infectious suspension on the artificial diet or potato large scale of origin (Granados and Hashimoro, 1989). The tubers. AcMNPV could be used as expression vector for broad Newly hatched larvae of S. littoralis and P. operculella range of genes with different origins (Eldridge et al., 1992) were exposed to contaminated diets while 2nd and 3rd in- and its great capacity of recombination could beusedto stars of S. cretica and P, gossypiella were used for experi- modify its pathogenicity or host range by introducing for- mental infections. eign genes (Miller et al., 1983). As in our laboratory, the use of granulosis viruses (GVs) Extraction and Analysis of Viral DNA as biological control agents and as models formolecular Infected larvae were homogenized in 50 mM Tris, 2 mM studies represented a great importance, AcMNPV affrnty of SDS pH 7.8, then filtered through cheesecloth. The suspen- certain hosts which are susceptible to GVs in Egypt was ex- sion was purified by centrifugation on sucrose gradient 30- amined. (Abol-Ela et al., 1994;, Matthiessen et al., 1978;' 70% (W/W). Virions were liberated by alkaliie treatment Fidière et al., 1992). meobjective of this study is to obtain with Na2CO3 (60 mM final, PH 11). DNA was extracted by models which will permit the analysis of viruses produced addition of 2% sodium lauroyl sarcosine and 200 pg/ml of from genetic recombinations between GVs and NPVs. proteinase K for 2 hrs at 50"C,followed by two extractions with phenol-chloroform3soamyl alcohol (25/24/1), two MATERIALS AND METHODS phenol and two chloroform isoamyl (24/1) extractions. Fi- nely, the DNA was precipitated with 2 volumes of glacial vim5 ethanol in presence of 1/10 volume of sodium acetate (3M) . AcMNPV strain was obtained from Dr. Croizier (INRA, at -20°C ovemight. Electrophoretic mobilities of different France). For consekgthe initial viral properties, the virus DNAs, digested with Eco R and Hind II restriction endonu- strain was multiplied in §F9 cell line grovin in Grace's cleases and the migrations through 1% agarose gel were modified medium (L.éry and Fédière, 1990). The AcMNPV realized according to Maniatis et al. (1989). was also multiplied on Pecti?iophoragossypiella (Vail et al., 1972) and used as a source of infectious haemolymph. Ultrastructural Studies. Larval tissues were prepared for electron microscope hectRearing observations as follows: small pieces of fat body were'fixed Both P. pectinophora and S. littoralis were reared under in 2.5% glutaraldehyde prepared in 0.01 M sodium cacody- laboratory conditions on semiartificial diets (Shorey and late, then post-fixed in osmium tetroxide l% indistilled Hale, 1965; Couilloud and Giret, 1980). For P. operculella, water and embedded in Epon Araldite. Finally, ultrathin 128 sections were stained with uranylacetate saturated in 50% type and ScGV in the cytoplasm (Fig. 3). ethanol, then by lead . citrate (Reynolds, 1963). Grids were The different results obtained on the 3 tested insects, re- examined through transmission electron microscope ZEISS garding their susceptibility ta AcMNPV could be considered EMlO. according to two points of view. Firstly, if the tested host is not susceptible to AcMNPV (S. littoralis), it can play the Preparatiun of Non Radioactive Nucleic Probe and Hy- important role of filter for the selection of genetically modi- bridization fied AcMNPV recombined with GV, in order to enlarge its Synthesis of non radioactive and dot blot hybridization host range. Secondly, if the insect is susceptible to AcMN- for both Ach4NPV qd GVs were performed according to PV- (S. cretica). the tested insects will be added to the host the supplier's .protocol (Boehringer Mannheim). list of AcMNPV, which could represent a possible altema- tive baculovirus for biological control after the achievement RESULTS AND DISCUSSION of necessary studies of the viral pathogenicity ad its persis- tence, particularly at the Delta and Nile valley. Four trials with AcMNPV (10x103 PIBs/ml) were car- In case of P. opercrdella, the fact that a little response to ried out to infect P. opercu1ella. No viral infection was ob- AcMNPV was observed. could permit us to consider the served. No late disease symptoms could be seen. Oh the concept of filter. other hand, infection of P. operculella with AcMNPV poly- In conclusion, using these different models, the poten- hedra, purified from infected cells of the SF9 cell line, tialities of AeMNPV could be enhanced to play a new role caused 4% death and 13% abnormalities in larvae. Exami- in biological control programs, where the GV was the only nation through optical microscope demonstrated the exis- possibility. tence of inclusion bodies with cubic-shape and a significant number of granules (PTMGV) distributed in tested tissues REFERENCES were observed as well. Nucleic probes originated from both AcMNPV and PTMGV. separately applied, revealed that Abol-Ela, S.; G. Fédière; A. Nour El-Din; O. Khamis and only 5% of larvae mixed infected with AcMNPV and M. Salah. 1994. Restriction endonuclease analysis and PTMGV, while 75% of total insects were only infected with diagnosis of the granulosis virus isolated fiPm GV (results not shown). No AcMNPV infection alone was. Spodoptera Iittoralis Boisd. 'in West fica and found. The presence of both GVs and AcMNPV could lead multiplied in Egypt. Bull. Fac. Agric., Univ. of Cairo, to the role, not yet precised, ofPTMGV as an enhancing 45: 919-932. factor like other GVs previously studied (Yafeng eí al.. Albrecht, G. 1986. Specificity and sdety of baculoviruses. 1989; Hashimoto et al., 1991). ,In: The Biology of Baculoviruses, R.R. Granados and B. Conceming the trials for AcTV niultiplication in Federtci eds., CRC Press. Inc.. 276. pp. PTM healthy larvae. only 3 larvae died at the 2nd instar 9 Couilloud, R. and M. Giret. 1980. Elevage du Cryptophle- days post-infection showing high infection by AcMNPV bia (= Argyroploce) Ieiicotreta Meyr. (Lep., (Fig. l), while 40 larvae seemed to be normal and active at Tortricidae). Cot Fib. Trop., 35: 225-227. 4th to 5th instar and completed their life cycle. These low Eldridge, R.; D.R. OReilly and L.K. Miller. 1992. Efficacy levels of AcMNPV multiplication in P. opercrrlellaper os of a baculovirus pesticide expressing an eclosion was not correlated with the successful multiplication in vitro hormone gene. Biol. Cont., 2: 104-110. of the same virus on P. OperculeIIa cell lines (Léry et al.. Fédière, G.; A. Taha; S. Abol-Ela, X. Léry; J.L. Zeddam; 1995, in press). This contradiction could be due to one or J.C. Veyrunes and J. Giannotti. 1993. Mise en évidence more of the following reasons. Different levels of suscepti- d'un virus de granulose chez Sesamia cretica Led.