Eaptian Journal of Biologico1 Pes! Control 5 (l), 19Yjp25-31

SUSCEPTIBILITY OF PACHNODA SA WGNY G.&P. LARVAE (COLEOPTERA: ) TO THE ENTOMOPATHOGENIC NEMATODE STEINEWEMA GLASERZ (STEINER) (FtHABDIT1DA:STEINERNEMATIDAE).

FI. Abou Bakr*, JX Adel* and M. emillet** * Biological Control Research Lbratory, Fac. Agric., air0 University, Giza, Egypt ** Institut Francais de Reserche Scientifique pour le Eveloppmmtk en Coopkauon (OSTKOM) (Received,August 13, 1995: Accepted August 30,1995).

ABSTR4CI' - - -_ ___- - -. -P *n -r-. ,.-,.-m . . . I - -. ,.L..L- thlrmr: ,.-.-.-+~--L..I---L--- c.^ - - - A laboratory screening of 10 strains of entomopathogenic nematodes belonging to Sternernemu spp. revealed that S. glasen is the most effective steinernematid against the 3rd instar larvae of the peach cockchafèr, Puchnoda savigny (ColeopkmScarabaeidae). Bioassays indicated that both (IJs)2nd and 3rd instar grubs are susc tible to S. glasen using nematode concentrations ranging between 8,000soil and 35,000 infective juveniles per a surface area of 156 cm ??p. Higher mortalities occurred when bioassay was canied out in sandy than in a soil mixture composed of equal volumes of sand, loam and cattle manure. The 3rd instar larvae ofP. savigny killed with S. glasen proved to be a suitable environment for the multiplication of S. gluseri infectivejuveniles. The number of emerging IJs ranged between 30,000 and 125,000 IJdgrub depending on the infective inoculum. preliminary trials indicated that infxtivity of S. gherito P. savigny larvae could be enhanced by the passage of nematode through the larvae of the target host.

Key Words: Entomopathogenicnematodes, Steinemema glasen'',Scarabaeidae, Puchnoda savigny, efficacy, screening, in vivo production, infectivity improvement.

INTRODUCTION laboratory against L2 and L3 of the same . Laboratory experiments included the effect of soil composition and The peach cockchafer, Pachnoda savigny Gory & nematode concentration on nematode-induced larval mor- Percheron (Rigout, 1989), which was previously howin tality. Nematode multiplication inside P. savigny dead lar- Egypt as P. fasciata F. (Alfieri, 1976), is a common scara- vae was also examined. The infectivity of the IJs produced baeid in the country. The adults appear in late June in P. savigny larviie against L3 of the same insectwas and early July and last up to September where they attack compared to that of IJs produced in Galleria mellonella lar- flowers and ripe fits of the summer orchards as well as vae. some ornamental plants (Abou Bakr et al., 1989a). As eggs are laid in the soil, larval development takes place in soil MATERIALS AND METHODS rich in organic matter upon which the grubs feed (Abou Bark ef al., 1989b, Helmi et al., 1989). The populations of Labomtory Maintenance of&?savìgny P. saVigny have been steadily increasing in different parts P. saVigny adults were collected by hand picking and of Egypt during the recent decade. In order to reduce the insect net in the field during the period of June-September, use of chemical insecticides, efforts have been directed to 1992 and 1993. In the laboratoq, were held in biological and agricultural control methods, among which 60~60x75 cm metal wire- mesh, wooden-earned ages the entomopathogenic nematodes of the families Stei- provid.4 with pieces of banana for feeding the , and nernematidae and Heterorhabditidaepossess many qualities plastic boxes containing a moistened autoclaved mixture of that make them excellent biological control agents equal parts of sand, loam and cattle manure as oviposition (Gaugler, 1988). They have a broad host range, kill their media (Abou Bakr et al., 1989a). Food was renewed daily, host rapidly and are environmentally safe. Isolation of S. and soil moisture was continuously kept at a suitable level. gluseri from Japanese beetle larvae in New Jersey in 1930s The boxes were inspected eveq two days where eggs were resulted in the first effort to use nematodes against insect picked up and kept in plastic boxes (14 cm diameter', 4.5 pests (Klein, 1993). Recent field tests with nematode in the high) filled with the previously mentioned autoclaved soil genus Steinemema (=Neoaplectana) have shown that they with suitable moisture. The boxes were covered with @o- can be effective biological control agents against scarabaeid rated plastic lids. Eggs were kept for hatching at28OC insects (Klein, 1990). Steinemema glasen' is known as where larval development took place. Under such condi- particularly efficient against various scarabaeids, e.g. tions, P. savigny grubs reach the 2nd larval instar within Anomala (Manuya, 1989)', Ligvrus (Sosa and Hall, 1989), about 20 days; the 3rdlarval instar may last for up to 256 Phyllophaga (Kard et al., 1988; Forschler and Gardner, days. ,1991a,b), Popillin japonicu (Shetlar et al., 1988), and Mzizotrogtrs niajalis (Villani and Wright, 1988). In the pre- Nematode Production and Storage sent study a comparison was made'between the infectivity Steinemema strains used in this work were provided by O of 1 strains belonging to different Sfeineniemuspecies, Dr. C. Laumond, "Laboratoire d'Entomonhatologie", isolated in different parts of the world, against the 3rd in- INRA, Antibes, France. The nematodes were produced in star larvae of P. smtigny. Then,$. glaseti was tested n the vivo by infecting last instar larvae of the greater wax moth; ..“I..I”. -^____ -- I - _I.. ---. -... .,..-*. - diamñeterplaster disc (modified after Woodring and Kaya, boxes served as coiitrol where the soil was moistened wtli 1988) kept inside an extraction chaniber, a rounded plastic tap water. Boxes were covered with perforated lids and box with a thin layer of 0.1% formalin in distilled water. kept at a temperature of 28°C. Boxes were inspected each The boxes were tightly covered with plastic lids to avoid two days for larval mortality. Dead larvae were removed, the entry of Drosophila flies. After four days of incubation washed with distilled water and individually placed on the at 28°C the new IJs individuals started emerging from the previously described plaster discs to assure that death was Galleria cadavers taking their way down to water around because of the nematode infection. the plaster disc. IJs were collected daily and finally stored in 100 ml d.w. with 0.1% formalin in transparent plastic Nematode Production in Nematode-Killed Grubs boxes 8 cm diruneter, 5 cm high) in a refrigerator (9.011”C) Nematode-killed L3 of P. srivigny were kept individu- at density of lo6 IJs/lOO ml water. Bioassay testes were ally in the extraction chambers at rooin temperature of Carried out using nematodes stored for 34weeks. 28°C until the emergence of the new generation of the IJs which were collected and counted daily throughout the pro- Screening Tests duction period. Then distilled water was added to the daily Ten strains of entoiiiopatliogenicnematodes, all belong- harvest of LTs to reach a standard volume of 1OQml.One nil ing to the genus Steitiemenia, isolated in different parts of of the suspension was pipetted and spread onto the Haw- the world (Table 1) were screened for their virulence skley slide and counted. This was repeated sis tinies and against the 3rd instar larvae of P. savigny in sand. A con- the average of six readings was used to express the number centration of 10,000 IJs of each strain was used for each 5 of IJs/ml. Both daily and total productions were based on grubs kept in a plastic box (1 56 cm2 area) containing 375 the average IJs numbers produced from 5 P. savigtzy 3rd in- cm3 of sand. Four replicates were used for each nematode star larvae for each nematode concentration initially used strain. Similar 4 boxes containing a sum of 20 L3 larvae fçr the infection. The IJs produced in nematode-killed L3 of served as control where sand was moistened with tap water. P. savigny were then tested for their virulence against 3rd Larval mortality was recorded up to 10 days after nematode instar larvae of the same beetle in comparison with IJs pro- exposure at a temperature of 28°C. duced in G. mellonella larvae.

Soil Preparation Statistical Analysis Moderately fme sand, loam taken from autoclaved soil Data were subjected to analysis of variance (ANOVA) in the Experimental Fami at the Faculty ofAgriculture, and means (*SE) were compared at P<0.05 level signifi- Giza and dry cattle manure taken from husbandry cance. section in the same fmn, were used for preparing the soil mixture. Sand was washed and dried; both loam and cattle RESULTS mDISCUSSION manure were separately autoclaved. Two types of soil were experimentally prepared for testing the effect of soil com- Screening of 10 Steiriernertia strains YS 3rdInstar Lar- position on the infectivity of S. glaseri on the 2nd and 3rd vae of€?savigny instar larvae of P. savigny. Type I soil consisted of plain A list of the tested nematode strains and their sources is san& and type 2 was made of equal volumes of sand, loam given in Table (1). As shown in this table, no larval niortal- and cattle manure. Saniples of the two types of soil were ity was recorded 10 days after larval exposure to the nema- subjected to physical and chemical analysis (Table 2). todes of strains: All (S. carpocapsae), E2 (S. kushidai),J26 (Steinemema sp.), K43 (Steinemema sp.), K66 (S. Infectivity Tests with S. &seri kraussei), K91 (S. ayfinis), or K92 (S. feltiae).Mortalities Three-week old LTs of S. glasen were taken out of their of 20% and 13% were recorded among larvae exposed to storage in the refrigerator and lefi at room temperature for strains K35 (Steinemenla sp.) and K27 (S. curpocapsare). about one hour for reactivation. Then they were inicro- On the other hand, a remarkable mortality of 95% were re- scopically examined for viability, and numerically adjusted corded among larvae treated with S. glaseri. to the wanted concentrations as number of IJs/ml water us- These results indicate that S. gluseri is the most eKw- ing Hawskley counting slide. Nematode concentrations tive among the 10 tested steinemematid strains against P. generally ranged between 8,000 and 35,000 IJslbox. saVigny grubs. Tlie lack of infectivity of some entoniopa- The previously described plastic boxes (of 156 cm2 thogenic steinemematids towards scarabs was a point ol‘ area) were filled with fixed volumes (375 cm3) of the concem among many researchers. Klein (1993) concluded tested soils. Accordingly, nematode concentrations were that S. glaseri and H. Dacteriopliorn are the most inkkctinus the calculated as number of IJshox, or IJs/cm2area as iiidi- nematodes for use against scarab larvae. Wang et al. ( 1994) I I c

Table (1): Mortality % 10 days &er treatment, among the 3rd instar larvae of P. savigny treated with 10 Y Steinememu strains in sandy soil (nematode concentration: 10,000 Ud5 larvae). ’

Nematode strain Source Mortality % All strain (S. capocapsue) USA O E2 (S. kushidai) Japan O 526 (Steinememu sp.) Jamaica O EC27 (S. catpocapsae)Breton strain France 13*1.1 K35 (Steinemema sp.) France 2Ml.l K43 (Steinemema sp.) France O K66 (S. kraussei) Czechoslovakia O K91 (S. afziiis) Australia 0. K92 (S. feltiaej T 3 19 strain Australia O S. glaset? USA 95M.57

Table (2): Physical and chemical properties of two soil types used in nematode bioassay.

Soil Coarse Fine Silt Clay Texture pH EC Caco3 Solublecations(mg/lOOgmsoil) Soluble anions (mg1100 gm soil) type sand% sand% % % Ca* Mg* Na+ K+ CO,-- HCO3- Cl- SO4-- - 1* 84.910 10.663 2.848 1.579 Sandy 7.89 2.20 3.280 0.20 0.08 0.09 0.01 0.08 0.05 0.26 2** 32.894 34.201 29.287 3.618 Sandyloam 7.69 2.90 3.108 0.74 0.22 0.14 1.12 - 0.09 0.11 . 0.91

* Sand ** Mixture of equal volumes of sand, loam and cattle manure. c

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S. compared the infectivity of S. carpocapsae, S. glasen, 90% in L2 and 55% in L3. All L2 exposed to a concentra- scapterisci and H. bacteriophoru to P. japonica larvae us- tion of l0,bOO IJs died on the 4th das the same concentra- ing external exposure and haemocoelic injection. They re- tion caused mortality in L3 after the sameperiod. S. 9Yh ported that only H. bacteriophora and glasen caused However, a higher concentration of 15,000 Us induced high mortality &er external exposure to 10,000 Us, while 100% mortality among the two tested larval instars &er 4 S. carpocapsae had a low level of infectivity. days of exposure. S. Since the nematodes and white grubs have co-evolved When glasen was tested vs the 2~1dand 3rd instar in the soil environment, larvae possess a number of defence larvae of P. savigny in a soil mixture composed of equal mechanisms such as low carbon oxide output, sieve-plates volumes of sand, loam and cattle manure (Table 2), higher on their spiracles, frequent defecation, wiping tlíeir mouth nematode concentrations and longer period of time were parts to remove nematodes and possession of peritrophic needed to obtain considerable larval mortalities than those membrane that serves as a barrier once nematode enter the previously obtained in the sandy soil. Theresdtsofthis insect gut. All of these defences prevent scarabs from being bioassay are shown in Figs. (3&4). Nevertheless, according highly susceptible to nematode parasites (Bedding and to these figures, the observed larval mortality was generally Molyneux, 1982; Froschler and Gardner, 1991~;Klein, inconsistent. Nomortality was recorded among L2 up to the 1993). Our results find supports by previous field studies of 6th day of exposure, while mortalities between 5%aiid various researchers indicating that S. gloseri can provide 30% were recorded among L3 during the first 4 days fol- acceptable levels of P. japonica control (Wright et al., lowing the nematode treatment (Fig. 4). Ten days after 1988; Villani and Wright, 1988, Klein, 1990;Kleinand treatment, larval mortality among L2 did not exceed 55% Georgis, 1992; Selvan et al., 1993), whereas other species (conc. 30,000 Uslbox) and this mortality did not change such as S. carpocapsae were less effective (Georgis and during the following days (not illustrated in the histogram). Gaugler, 1991; Smits, 1994). Georgis and Gaugler (1941) On the other hand, the maxi" mortality recorded among L3 was 70% in the nematode concentxation of 30,000 Us attributed differences in nematode efficacy to differences in A levels of host-parasite adaptation. Cui et al. (1993) reported after 3 days. comparison between mortality values in the S. loam that infective juveniles of glaseri are highly mobile two tested soils clearly indicates that the presence of "cruisers" that actively search for hosts whereas S. cur- and organic matter in the soil mixture causes an obvious pocapsae mostly wait for hosts to come to them as decrease in larval mortality among the two tested larval in- "ambushers". Gaugler (1993) attributed the superior per- stars in terms of both nematode concentration and the time formance of S. glasen' against scarabs in large part to their needed to obtain high larval death. cruiser host-finding strategy. On the other hand, lackof Statistical analysis of the obtained results showed no scarab suswptibility to entonlopathogenic nematodes could significant differences in the susceptibility of L2 and L3 be attributed to encapsulation and melanization inside the when they were exposed to the infective juveniles in sand, regardless of the nematode concentration used, although the host. Studies of Wang and his co-workers (1994) on P. ja- initial mortality among L2 was higher than in L3. But when ponica showed that few S. glasen' (2.8%) were encapsu- S. lated and melanized, whereas nearly all infective juveniles the exposure to glasen' took place in the soil mixture of of other tested species became encapsulated and melanized. sand, loam and cattle manure, L3 was significantly more S. glasen in P. japonica can evade the immune response susceptible than L2 regardless of the nematode concentra- because the two organisms, the nematode and the grub, as- tion. Similar findings were recorded by Smits (1994) who sociate under natural conditions. Accorckg to Wang et az. stated that grubs become more sensitive to nematode infec- (1994) the mechanism of this avoidance is unclear and may tion with increasing age. However, he added that the data differ among nematode species. Therefore, screening for may still toll sporadic to allow such a general conclusion. nematode ability to overcome or evade the host defence re- He suggested that the natural openings are usually larger in sponse may provide an important first step in narrowing îhe the older larvae and therefore may provide a better oppor- list of suitable nematode species. tunity for nematodes to enter the body. Physiological de- fence mechanisms may also show differences between the Susceptibility ofR savigny Larvae to S gloseri instars. Nevertheless, since P. savigny grubs do not feed As Steinemema glasen' proved to be the most virulent upon living plant material, and since the 3rd larval ins$ steinemematid amongst the 10 tested strains, susceptibility represents the longest stage in the insect life cycle (Abou of both 2nd and 3rd instar larvae of P. savigny was tested Bakr et al., 1989a), the increased susceptibility of the L3 using nematode concentrations ranged between 8,000 and should be considered as an advantage fkom thepointof 15,000 Us/plastic box. Mortality % induced by the different view of pest control. The results also showed a significant nematode concentrations among L2 and L3 is graplucally decrease in nematode infectivity when bioassays were car- illustrated in Figs. (1&2), respectively. Two days after grub ried out in a soil mixture containing silty soil and organic exposure to the infectivejuveniles, much higher mortalities matter. Glaser et al. (1940) mentioned that parasitism by were recorded amongst L2 than L3 in all the tested nema- insect nematodes varied depending on soil moisture, nema- tode concentrations. For instance, the lowest concentration tode dosage, soil temperature, and host density. Georgis and Poinar (1983) attributed the decreased nematode infec- of 8,000 IJs induced 70% mortality among L2 and about 10% mortality in L3. Four days after exposure, larval mor- tivity in soils containing increased proportion of clay and tality induced by this concentration (8,000 Us) reached silt to the diffxult movement of the juvenile nematodes in 100

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M) 80 70 rp 70 -Ib 60 (D 50 P5' 40 30 30 20 20 10 10 O 000000000000 O 000000000000 0000000000000000 OOOOOOOOOOOO o o o o o o o o o 0-0 o o o o o OInOIn OOOOOOOOOOOOOO0O NNOOs%g%%"ooNOO ~~g~o~gg8~g~g"omNO- ( 6 days )( 8days )( 10 days ) ( 2 daYs )( 4 days )( 6 days )( 8 days ) IJ5 Conoentratlon/5 L2 Fig. (3) : Mortallty % among L2 of Psavlgny Concentration Fig. (4) : Mortality 96. among 213 of P.saVigny treated with S.alaserC-in soll mixture. treated with S.glaseri in soil mixture.

3 1 < 30 these types of soils to migrate and infect the host; and they some stability (as in Fig. 5) or decline gradually or rapidly 6,7&8). observed that the greatest dispersal and infectivity occurred (Figs. This peak was reached on the 2nd day of the in pure silica sand and coarse sandy loam. More explana- initial emergence in all. cases, except in case of using 5,000 tions were given by Molynew and Bedding (1984), they lid5 L3 (1,000 UsL3) as initial infective dose (Fig. 6). reported that nematode movement within the soil requires The total period of active emergence ranged between4 water filled pores or films of sufficient thickness and conti- days (when very high infective dose of 2,400 IJsL3 was 8) nuity to allow migration. They added that the higher clay used, Fig. and 7 days when 1,600 UsL3 was used. How- content of the clay loam is associated with more pores of ever, the initial infective dose seems to have a slight effect small diameter and greater tortuosity which limits nema- on the total active period of nematode production; rather it tode movement, reflected in lower larval mortality. Mean- affects the level of the peak reached in each case: the high- while, the IeveIs of parasitism occurring in the sandy soils est infective dose (2,400 ITsL3) resulted in thehighest at high moisture potentials reflect the amount of aeration in peak (Fig. 8).Moreover, a proportional relationship can be these soils at high moisture contents. detected between the initial infective dose and the total The soil mixture in which the bioassay was canried out number of ITS produced in the dead grub. As shown in Fig. contained a great part of organic matter (cattle manure) to (9) a gradual increase in nematode production is recorded which the decreased nematode infectivity is attributed. The by the increase of the infective dose. Number of emerging ITS effect of manure and increased organic matter in the soil ranged between ca 30,000 IJs and 125,000 IJs using in- was a point of concern among several workers. Mullens et fective doses of 200 and 2,400 IJsL3, respectively. Accord- S. al. (1987) reported that the application of carpocapsae ing to our experiments (unpublished) a 111mature Galle- and H. bacteriophoru to control maggots in poultry houses ria niellonella larva may produce about 43,000 IJs when an produqed conflicting results. The failure was attributed to initial infective dose of 100 IJs of S. glasen was used. It is poor nkmatode survival in manure because of the Sgh am- noteworthy that the weight of a 3rd instar larva of P. savi- bient temperature, toxic ammonia and salts (Georgis et al., gny ranges between 1.8-2.6 gm with an average of 1987), or" possibly predatory mites (Wicht and Rodriguez, 2.22*0.24 gm, while the weight of G. mellonella larva 1970). ranges between 0.112- 0.184 gm with an average of In the present work our data show that grub mortality 0.156*0.02 gm. Although we did not examine the size of does not necessarily increase as the nematode dosage in- individual juveniIes produced in each infective dose, Sel- creases. That was more obvious when nematode was ap- van et al. (1993) reported that the longest infective juve- plied in the soil mixture (Figs. 3&4). No clear explanation niles were produced at the lowest nematode densities, indi- is available so far, and several previous works pointed to cating a trade off between size and numberofprogeny. the inconsistency of the results of nematode applications. They also stated that effects of high density appear to re- al. However Selvan et (1993) observed that although the sult from competition for limited nutrients within the host. number of invading nematodes increased with increasing dose, percentage penetration declined. density-de- This Virulence of S. glaseri IJs Produced in Different Hosts I. pendent penetration was not sufficient to prevent the detri- A comparison was made between mortalities among the mental effects of overcrowding. Although the exposure 3rd instar Iarvae of P. savigny when they were infected method followed by Selvan and his co-workers assured host with Us of S. glasen' produced in two different hosts, i.e. G. than contact, no more half of the nematode population initi- niellonella and P. savigny, using an infective dose of ated host infection. They reported that their results bol- 10,000 Ud5 L3 P. savigny. As shown in Fig. (lo),the IJs stered Fan and Hominick's (1 991) contention that most en- produced in P. savigny larvae were more virulent than tomopathogenic nematode infective juveniles are not infec- those produced in G. mellonella. Four days after nematode tive at a given point in time. In addition, Bohan and exposure 60% mortality was recorded among P. savigny in- Hominick (1994) mentioned that there is a proportion of fected with S. glaseri produced in-G: mellonella, while dauers that are infectious at any given time and that this 100% mortality was recorded after the same period among proportion changes through the time. the P. savigny L3 infected with nematode produced in nematode-killed grubs. These results indicate that virulence Multiplication of S. glmeri in the Cadavers of€? savigzy of S. glasen vs P. smigny larvae could be enhanced by Nematode-killed 3rd instar larvae of P. savigny were S. passing the nematode into the larvae of the target insect. examined for their suitability for the multiplication of The present results are of one-passage nematodes and more glasen, taking into consideration the initial nematode con- passages may result in more virulence. Another trial to im- centration used for larval infection. Results shown in Figs. ITS prove the activity of a nematode strain against grubs was (5-8) indicate that at a temperature of 28°C the began recently made by Smits (1994) who passed a strain of S. to emerge from the cadavers 14- 19 days after death of the gluserì four times through M. melolontha larvae and found infected larvae. The longest period (19 days) was related to a 10- fold increase in the biological activity. the least nematode concentration used for infation, i.e. 1,000 LTs/5 L3 (= 200 IJsL3). The rate of Us mergence Acknowledgement: We are indebted to Dr. C. Laumond seems to follow a general pattern regardless of the initial fassi, ITS and E. Bon Laboratoire d Entonionematologie,Ih?RA. infective dose: a gradual increase of emerging until Antibes, France, for providìng the nematode strains and for reaching a peak, then nematode emergence may show the valuable help they offered. We also thank Dr. S.E. Bakr, 1000

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1O0 O 19 25 27 29 21 23 15 16 17 18 19 21 . days after larval death 14 20 days after larval death Flg (5) : Number of S. giaserl IJs emerged from L3 cadavers of Psavlgny In?ected Fig (6) : Number of S.glaseri IJs emerged with 1000 lJsl5 larvae in sand. from L3 cadavers of P. savlgny Infected with.. 5000, IJsl5 larvae In sahd.

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O 16 18 19 20 21 22 23 14 16 18 20 22 days after larical death days after larval death Fig. (7) : Number of Sglaserl IJs emerged Flg. : Number of S.glaserl IJs emerged from cadavers Psavlgny Infected (e) L3 of from L3 cadavers of P.saVigny treated with ßO00 lJel5 larvae In sand. with 12 008 lJsl 5 latbae In sand.

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Gmellonella P.savigny G.me/lone/la Psavlgny (’ 2 days )( 4 days ) 200 2400 1000 1600 Source of IJs IJs conc. IL3 used for infection Flg.(lO): Mortallty YO among L3 P. savlgny Hg. (9) : Total number of Sglaseri IJs Infected with S.glaser1 (10000 IJsl 5 produced In P. savlgny L3 Infected with larvae) produced In two dlfferent hosts. dlfferent nematode concentratlons.

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