Characterization and field aDDlication of Heterorhabditis bacteriophori ;train HP88 (Heterorhabditidae : ) George O. POINAR,Jr. and Ramon GEORGIS Departenlent of Entomology and Parasitology, University of California, Berkeley, California 94720, and Biosys, Inc., 1057 E. Meadow Circle, Pal0 Alto, California 94303, USA.

SUMMARY

Strain HP88of Heterorhabditis bacteriophorafrom western United States is described and compared to the type strain (HB1) from Australia. Using a dorsal and two smaller subventral terminal cuticular processes, the infective stagesof HP88 were able to bore through the external cuticleof worker termites. The HP88 strainof H. bacteriophora could equal or surpass the control provided by chemical insecticides in field trials against larvae of the Japanese beetle (Popillia japonica).

%SUME

Caractérisation et application au champ de la souche HP88 de Heterorhabditis bacteriophora (Heterorhabditidae :Rhabditida)

La souche HP88 de Heterorhabditis bacteriophora provenant de l’ouest des États-Unis est décriteet comparée à la souche type (HB1) originaire d’Australie. Grâce à des procès cuticulaires terminaux- un dorsal, deux subventraux - les stades infestants de la souche HP88 peuvent forer la cuticule externe des termites ouvriers. Lors d’essais au champ pour lutter contre les larves de Popillia japonica, la souche HP88 deH. bacteriophora a montré une action égaleou supérieure à celle des insecticides chimiques.

The insect-parasitic ,Heterorhabditis bacter- were held at 22 OC and dissected on days 4 and 5 to iophora was first described from a noctuid moth larva recover the first generation hermaphroditic females,and (Heliothis punctigeraHal 1) at Brecon, South Australia on days 7 and 8 to obtain the males and second-gener- (Poinar, 1976). It was subsequently designatedas strain ationamphimictic females. Infective stage juveniles HBl, therebyseparating it from strain CS406 from were examined after being heldfor 3 months in water at Tainan, China and strain V16 from Geelong, Victoria, 12 OC. Australia (Akhurst,1987). We now describe anew strain, Al1 were killed in hot (55 OC) Ringer’s, HP88, which originated from the United States, thus fiied in TM and processed to glycerin for measure- showing the wide distribution of this species. This strain ments. has been mass produced and used successfully in field For studies on host penetration, smallblocks of 1.0 O/o trialsagainst several insectpests in North America water agar were used. In each of ten agar blocks (1 cm (Georgis & Poinar, 1989). long by0.5 cm wide by 0.5 cm deep) a smallgroove was made which could containthe body of a worker western subterraneantermite, Reticulitemzeshesperus Banks. Materials and methods About a hundred infective stage nematodeswere added (in a smalldrop of water) to each groove andthe termite The HP88 strain was isolated from infected June was covered with liquid agar. Observations and photo- beetle larvae (Phyllophaga sp.; Scarabaeidae : Coleop- graphs were made with a Nikon Optiphot microscope tera)which had been sent tothe seniorauthor on fitted for differential interference contrast. September 20th, 1982 from thevicinity of Logan, Utah. Host preference studies were performed in the labora- The nematodes were then maintained on larvae of the tory using 9 cm Petri dishes lined withtwo filter papers. wax moth, L. For greenhouse and To each dishwere added 50 infective stageHP88 in2 ml field trials, nematodes were supplied by Biosys (Palo of water. Ten individuals of each of thirteeninsect Alto, CA) where they were produced in vitro. species were added to each dish (withfive replications). For the descriptive portion of the paper, the nema- Mortality was recorded 72 h after the nematodes and todes were grown in Galleria larvae. Infected insects hosts were placed together and maintained at 22 OC.

Revue Nérnatol. 13 (4) :387-393 (1990) 387 G. O. Poinar, Jr Br R. Georgis

In thegreenhouse tests, potting soil (moisture content Heterorhabditis bacteriophora about 26 %,based on volumetric measurements) was Poinar, strain HP88 added to 3.78 liters (= 1 gallon) sized pots (without plants). In thefirst trial, ten, thirdand fourthstage black MEASUREMENTSAND DESCRIPTION vine weevil larvae (Otiorhynchus sulcatus) were placed 10 cm below the soil surface. In the second trial, the Adults (general) : Head truncate toslightly rounded; same number of larvae were placed 20 cm below the soil six distinct lips surroundingthe mouthopening; each lip surface. In both trials, the larvae were enclosed in a mesh bears an inner labial papilla; at the base of each sub- screen cage (8 cm indiameter and 1 cm high) to restrict dorsaland sublateral lips are two additional papillae their movements.To the surface of each potwere added representing anouter labialpapilla and a cephalic 25 O00 infectivestage HP88in 200 ml water.For papilla; base of each lateral lip contains an elliptical comparative purposes, the samenumber of Steinernema amphidial opening and an outer labial papilla; stoma carpocapsue. Al1 strain and S. bibionis SN strain were mostly collapsed, cheilorhabdionslining the non-col- addedto separate pots with weevil larvae. Controls lapsed portion;two additional segments in thecollapsed contained only insect larvae in pottedsoil. Al1 trials were portion may represent separate pro and mesorhabdions replicated four times andthe evaluation was made or fused pro and mesorhabdions and separate metar- 10 days after the application. habdions; additional characters similarto those of HB1 Field effectiveness of HP88 was compared with two described earlier (Poinar, 1976). insecticides (Bendiocarb and Trichlorfon) in an exper- iment to control larvae of the Japanese beetle, Popillia Hemzaphroditicfemale (n = 15). (Fig. 1 F) : juponica Newman, in a golf course at Sunbury, Ohio on Length = 3.9 (3.3-4.4) mm; greatest diameter = 185 August 20, 1987. The fainvay was planted with Ken- (144-216) pm;length of portion of stoma = 9.5 tuckybluegrass (Pou prutensis) (30 Yo), bentgrass (6.0-15.0) pm;width of stomaopening = 16 (Agrostis sp.) (25 "O) and annual bluegrass (Pou annuu) (12-21) ym; distance from anterior end to nerve ring = 132 (114-159) ym; distance from anteriorend to (45 "0). The experimentaldesign was a randomized complete block with five replicates; individual plots were excretorypore = 205 (174-240) Pm;distance from 3 meters square. The nematodes were applied to the anterior end to base of pharynx = 184 (171-207) pm, surface of the turfat therate of 2.5 and 5 billiodha ( = 1 length of tail = 81 (69-90) ym; body diameter at anus and 2 billiodacre) in568 liters(= 150 gallons) of water. = 49(36-60) pm; V = 43 (35-48). Vulva functiond They were applied usinga Coz sprayer withfour 800 pm and protruding slightly (6-9) ym from body contour; Tee jet nozzles mounted on a 2 meter boom operating anus located on protrusion. at 32psi. Immediately after treatment,the experimental Amphimictic female (n = 15). (Figs. 1 A,B, D) : area was irrigated 1.3 cm of water. Thereafter the with Length = 1.4(1.0-1.7) mm; greatestdiameter = 91 area was irrigated with0.7-1.3 cm of water each evening (63-113) Pm; length of noncollapsed pokion of stoma unless rain had fallen during the day. This amount of = 3.8(3.2-4.5) p; width of stomaopening = 7.6 irrigation was a regular managerial practice for the golf (6.4-9.6) pm; distance from anterior end to nerve ring course. = 83 (72-88) ym; distance from anterior end to excre- Insecticides were applied in granular form with a tory pore = 130 (177-149) pm; distance anterior end precalibrated Gandy 2.5 spreader. The Japanese beetle head to base of pharynx = 123 (112-136) p;length of grubs were mainly in the top 3 cm of soil with 80 O/o in tail = 71 (66-80) ym; bodydiameter at anus = 24 the third instar and 20 O/O in the second instar. (19-26)Fm; V = 48 (43-50). Vulva nonfunctional, Evaluations were made on October 2 (42 days after notprotruding from body contour and often sur- treatment) by taking four 930 cm2 (= 1 ft') samples of rounded by a mating deposit; anus not located on anal turf fromeach plotand counting the numberof healthy protrusion but usually just anterior to anal protrusion. grubs. At the time of application, soil temperature at 3 cm depthwas 22 OC(73 OF) and soil moisture 26 %.At Mule (n = 15). (Figs 1 E, G) : Length = 687 the timeof sampling, soil temperature at 3 cm depthwas (567-762) pm;greatest diameter = 43 (36-48) ym; 18 OC (64 OF) and soil moisture was 28 O/O. The soil was length of noncollapsed portionof stoma = 2.7 (2-3) pm; a silty loam with 9 O/o organic matter and a pH of 4.3. width of stoma opening = 4.6 (4-5) Pm; distance from Data was analyzed by ANOVA for a randomized anterior end to nerve ring = 70 (66-77) pm; distance complete block design(Ryan, Joiner & Ryan, 1985) and fromanterior endto excretorypore = 113 mean separations were based on Duncan's (1955) (105-120) pm, distance from anterior end to base of multiple range test (P = 0.05). pharynx = 95 (91-105) ym; reflexion of testis = 78

388 Revue Nématol. 13 (4) :387-393 (1990) Heterorhabditis bacteriophora strain HP88

(63-86) Pm; length of tail = 28 (22-30) Pm; body width of gubernaculum = 0.12 (0.10-0.18) Pm; distance diameterat cloacal opening = 18 (12-23) p;spic- from the firstgenital papilla to thecloacal opening = 43 ules = 39(38-42) Pm; greatest width of spicules 0.6 (37-50) Pm;ratio length of gubernaculum/length of (Q.5-0.7) Pm; gubernaculum = 19 (13-21) Pm; greatest spicules = 0.48 (0.33-0.54) Pm.

Fig. 1. Heterorhabditis bacteriophoru strain HP88.A : Amphimictic female, lateral view ofmouth region; B : Amphimictic female, en faceview; C :Third stage infective juvenile, en face view;D : Amphimictic female, tail, lateral view;E : Male, tail, ventral view; F : Hermaphroditic female, tail, lateral view;G : Male, tail, lateral view. (Bar equivalent :A,C, 0, E, F, G = 10 pz; B = 5 pz.) G. O. Poinar, Jr di R. Georgis

Fig. 2. Heterorhabditis bacteriophoru strain HP88. A :Third stage infective juvenile(n = nerx ring, e = excretory pore, p= basal pharyngeal bdb, b = bacteria in the intestinal lumen); B : Infective juvenile, head (ventral view) (d = dorsal tooth; arrows = subventral teeth); C : Infective juvenile, head (lateral view) (d = dorsal tooth; arrow = subventral tooth); D : Infective juvenile, exploratory movements prior to penetration (arrow shows head pressed against inter-segmented membrane ofE :termite); Infective juvenile, striations on second stage cuticle;F : Infective juvenile, double paired lateral lines the on cuticle; G :Infective stage juvenile passing through the external body cuticle of a termite worker; arrow shows border of insect cuticle.(Bar eqzrivalent :A = 22 Pm; B, C = 2 Pm; 0, G = 44 Pm; E, F = II prn). Heterorhabditis bacteriophora main HP88

Infective 3rd stage juvenile (n = 15). (Figs 1 C, 2) : preference. Table 1 shows that Coleoptera and Lepidop- The following measurements were made on exsheathed tera appear to be the preferred hosts for this strain. third stage juveniles. Length = 538 (480-570) pm; greatestdiameter = 20 (18-24) p;distance from Table 1 anterior end to nerve ring = 85 (72-93) pm, distance Host preference of Heterorhabditis bacteriophora strain HP88. from anterior end to excretory pore = 101 (87-110) p; distance from anterior end to base of pharynx = 116 Order Insect host Insect Morrality (100-125) pm; length of taiI = 56 (46-65) Pm. Head level* stage bearing a dorsal tooth that is elevated 0.5-1.0 pm from the topof a cushion whichis roughly 2-3 wide at the p Orthoptera Blatella gennanica males C base; subventral sectors each bearing a smaller tooth (German cockroach) only about one-third the lengthof the dorsal tooth (Figs 2 B, C); leftsubventral sector may only support a Orthoptera Scapteriscus vicinus males C sclerotized plate; cuticle of third stage infective bears (Mole cricket) two distinct double lateral lines (Fig.2 F) whereas cuticle Orthoptera Periplaneta atnericana males D of second stage juvenile bears numerous longitudinal (American cockroach) striations (Fig. 2 E). The intestinal lumen of the infective Orthoptera Schistocerca nitens females B stages contain bacterialcells of Xenorhabdus luminescens (Vargant grasshopper) (Fig. 2 A) which are released whenthe nematode enters Diptera Musca domestica second stage B the hemocoel of a host. (House fly) larvae Coleoptera Diabrotica sp. third stage A VOUCHER SPECIMENS (Southern corn rootworm) larvae ,’ Division of Nematology,University of California, Coleoptera Cyclocephalla borealis third stage A Davis, California, USA. (Northern masked chafer) larvae Coleoptera Rhizotrogus tnialis third stage A BIOLOGICALDATA (European chafer) larvae Coleoptera Phyllophaga sp. third stage B Host penetration :Penetration of the thirdstage infec- (May beetles) larvae tives of HP88 was observed with worker termites. The infectives were attracted to the insect and first crawled Coleoptera Otiorhynchus sulcatus PuPae A over its surface, stopping now and then to make more (Black vine weevil) intricate contact withthe body surface. When a suitable Lepidoptera Galleria tnellonella last stage A area was found, thejuvenile wouldflatten itself against the (Wax moth) larvae host, then startmoving its head backand forthover the Lepidoptera Manduca quinqoemaculata fourth stage A termite’s cuticle. Preferredareas were intersegmental (Tomato hornworm) larvae membranesand smooth areas on the segments. The Lepidoptera Trichoplusia ni fouth stage A head would be pushedso hard against the cuticle that it (Cabbage looper) lawae would make animpression. While maintaining this pressure, the infective juvenile would then continuously * A = ove1 80 % monality; B = 60-80% mortality, C = 40-60 % monality; D = 20-40 % mortality, rasp back and forth untila tear was made. Immediately the nematode would enter the wound andcrawl into the EXPERIMENTALRESULTS body cavity. Soft areas on the body surface (especially around theanus) were most frequently selected as areas Greenhouse tests :The results of the greenhouse tests to initiatepenetration attempts. Even though entry of placing nematodesin soil with black vine weevil larvae through thecuticle occurred, the majority of nematodes showed that HP88was effective in locating insectlarvae, entered the anus and mouth of the termite and pen- even when the insects had been placed at a depth of etrated through the alimentary tract. After entering the 20 cm (Table 2). These results suggest that HP88 has host, the infectives continued to migrate through the the ability to reach insect larvae in the soil more rapidly host’s body cavity, stopping to rasp at interna1 tissues. than the othertwo nematode species. This could be relat- Many foundtheir way into thelegs, antennae and mouth ed to their small size and host seeking ability. palps of the host (Fig. 2 D). Field trials :The results obtained by comparing two Host selectivity :AIthough, once inside the hemocoel different dosagesof HP88 with standard insecticides for of an insect, most Heterorhabditis spp. will be able to the control of Japanese beetle larvae (Table 3) show mature and reproduce, not al1 insects are equally sus- that at both dosages (2.5 / 109/ha and 7.5 x 109/ha)the ceptible to thesenematodes. There may be various nematodes canequal the effect of Bendiocarb and physical and chemical factors which influence the host surpass the control provided by Trichlorfon.

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Table 2 overlap (Poinar, 1976). This indicates that adultsize and Numbers of surviving black vine weevil larvae (Otiorhynchus certain quantitative values cannot be used for specific sukatus) placed at two depths in soil 10 days after an applica- characters. Nuclear DNA analyses of the genome of tion of 25 O00 nematodes per pot. HB1 and HP88 using majorthe sperm proteinas a probe showed that both nematodestrains were almost identical Nematodespecies numberMean of surviving larvae (in contrast to other species of Heterorhabditis) (Jim White, pers. comm.). Insects at 10 cm depth Insects at 20 cm depth Values associated with the infective stages are more stable. Although the values for the total length of the (n = 10) (n = 10) nematode and the length of the tail presented here are shorter than those for HB1, the measurements of HP88 h: buctmophoru HP88 2.8 It 0.6 (0.8-6.2)* [72]** 0.5 t 0.1 (0.1-1.9) [95] infectives were made without their enclosing cuticles S. cuqocapsaeALL 1.0 f 0.1 (0.6-3.4) [90] 3.8 t l.O(l.7-5.5)[60] (thecuticles were shed while thenematodes were in S. bibionis SN 4.8 f 1.3(1.6-7.2) [51] 5.3 t 1.5 (4.7-6.1)[44] storage), whereas the measurements of HB1 infectives Control 9.8 It 1.3 (7.3-10.3 9.5 t 1.6 (7.0-10.5) included the2nd stageenclosing cuticle. Since the enclosing cuticle adds roughly40 pm to the total length * 95 %confidence level; ** % control. and tail length of each individual, there are actually no quantitative(or qualitative) differences between the Table 3 infectives of the two strains. Thus, it is important to indicate whether the 2nd stage cuticle was present or Comparison of H. bacteriophoru HP88 with chemical insecti- absent when measurementsof infective stages are taken. cides against the Japanese beetle,Popillia japonica, on a golf The presence of a large dorsal and two smaller sub- course in Sunbury, Ohio (August-October 1987). ventral terminal teeth on the infective stage is interest- ing. The presence and arrangement of the subventral Treatment NumberTreatment of living teeth may be characteristicof this species since in H. me- fwbs 42days Control gadis, only a dorsal toothwas mentioned (Poinar, 1987) after treatment* and Wouts (1979) described a " dorsal tooth on a star shaped base, opposite a sclerotized ventral plate" for a HP88(2.5 109/ha) 3.5 a 79 New Zealand Heterorhabditis population, now consid- HP88 (7.5 x 109/ha) 2.2 a 87 ered a separate species. The presence of teeth were not Bendiocarb (4.70 kg [aillha) 3.2 a 81 mentioned in the original description of HB1 (Poinar, Trichlorfon (8.96 kg [ai]/ha) 62 6.5 b 1976), probably because theywere masked by the enclos- ing 2nd stage cuticle. Control 16.9 c - The role of these terminal teeth in HP88is undoubt- edly for entry into an insect host as shown here and * Mean of five replicates. Means followed by the same letter are not earlier by Bedding and Molyneux (1982). Although it is significantly different (p < 0.05) according to Duncan's multiple possible for heterorhabditids to enter thebody cavity of range test. hosts with thin, intersegmental membranes by penetrat- ing through the cuticle, penetration into the hemocoel through natural openings certainly does occur as em- Discussion phasized by Mracek, Hanzaland Kodrik (1988). In such cases, the terminal hooks most certainly assist the The descriptive studies of HP88 add to OUT knowl- nematode in penetrating the alimentary tract and tra- edge of the variability foundamong the species of cheal walls. Heterorhabditis. Although this strain is qualitatively Host preference is an important consideration in HP88 similar morphologically to the HB1 (type) strain, quanti- andother entomogenous nematodes. Although these tative differences occur betweenthe strains. The size of nematodes have a wide host range, they have generally the females can Vary considerably and they grow after adapted to specific hosts in specific soil habitats. Since becoming sexually mature. The amphimictic females of HP88 was originally recoveredfrom a coleopterous HP88 were smaller than those of HB1 (Poinar, 1976), larva, it is not surprising that field trialsshowed a high with a length rangingfrom 1 050-1 740 pm in compari- mortality level with coleopterous hosts. son to 3 180-3 850 pm in HB1. Similarly, the distance Hostfinding behavior is another variable among from the head to the excretory pore, nerve ring and strains of insectparasitic rhabditoids. In the present pharynx were significantly less in HP88 than in HBl in study, theHP88 strain was cornpetitive in locating the amphimictic females.In addition, the males of HP88 insects placed 20 cm below the surface of the soil. This are significantly smallerthan those of HB1 (567-762 for rapid host finding ability andthe preference for coleop- HP88 us 780-960 for HB1) yet al1 other measurements terous larvae weretwo important factors which con-

392 Revue Neinatol. 13 (4) :387-393 (1990) Heterorhabditis bacteriophora strain HP88 tributedto the successful field application of strain GEORGIS, R.& POINAR, Jr.,G.O. (1989). Field effectivenessof HP88 againstJapanese beetle larvae. Other factors entomophilic nematodes Neoaplectana and Heterorhabditis. which contributed to the field success were adequate In : Leslie, A. & Metcalf, R. (Eds) Integrated Pest manage- levels of temperature and moisture to allow movement ment in turf and ornamentals. Washington,D.C. Govt and infection to occur. Printing Office. In summary, H. bacteriophora strain HP88 has proven MRACEK,Z., HANZAL, R. & KODNK,D. (1988). sites of to be a highly successful biological control agent. Not penetration of juvenile steinernematids and heterorhabditids only has it shown promise against the Japanese beetle, (Nematoda) into the larvae of Galleria ?neIlonella(Lepidop- but it has equaled or bettered the control obtained by tera). J. Invert. Pathol., 52 : 477-478. insecticides in fieldtests with the black vine weevil (Otiorhynchus sulcatus), Northern masked chafer (C’clo- POINAR, Jr., G. O. 1976. DescripGon and biology of a new cephala borealis), European chafer (Rhizotrogus majalis) parasitic rhabditoid,Heterorhabditis bacteriophoran. gen., n. and bluegrass billbug (Sphenophorus parvaZus) (Georgis sp. (Rhabditida; Heterorhabditidae n. fam.). Nematologica, & Poinar, 1989). 21 : 463-470. POINAR,Jr., G. O., JACKSON, T. & KLEIN, M. (1987). Hetero- REFERENCES rhabditis megidis sp.n. (Heterorhabditidae : Rhabditida) parasitic in the Japanese beetle, Popillia japonica (Scara- AKHURST, R. J. (1987). Use of starch gel electrophoresis in the baeidae : Coleoptera) in Ohio. Proc. hehninth. Soc. Wash., of the genusHeterorhabditis (Nematoda :Hetero- 51 : rhabditidae). Nematologica, 33 : 1-9. 53-59.

BEDDING, R.A. & MOLYNEUX,A. S. (1982). Penetration of RYAN,B. F., JOINER, B. L. & RYAN,Jr., T. A. (1985). Minilab insect cuticle by infective juveniles of Heterorhabditis spp. handbook, 2nd Ed. Duxbury, Mass. 254 p. (Heterorhabditidae : Nematoda). Nematologica, 28 : 354- 359. WOUTS, W. M. (1979). The biology and life cycle of a New DUNCAN,D. B. (1955). Multiple range and multiple F test. Zealand population of Heterorhabditis heliothidis (Heteror- Biometrics, 11 : 1-42. habditidae). Nematologica, 25 : 191-202.

Accepté pour publication le 13 novembre 1989.

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