Interaction between two entomopathogenic nematode species in the same host

JOURNAL

OF INVERTEBRATE

Interaction

PATHOLOGY

57,

ld

(1991)

between Two Entomopathogenic the Same Host

Nematode

Species in

RAQUELALATORRE-ROSAS'AND HARRYK. KAYA Department

of Nematology,

University

of California,

Davis,

California

95616

Received September 14, 1989; accepted March 26, 1990 When Steinernema carpocapsae and Heterorhabditis bacteriophora (= heliothidis) simultaneously competed for the same insect species in Petri dishes, S. carpocapsae infected significantly more Galleria hosts than H. bacteriophora. When S. carpocapsae was added first followed by H. bacteriophora up to 15 hr, S. carpocapsae was the dominant species. When H. bacteriophora was added first, followed by S. carpocapsae at 0, 10, and 15 hr, S. carpocapsae infected 68% of the insect hosts at 0 hr and 67% at 10 hr, whereas H. bacteriophora infected 71% of the insects at 15 hr. Total percentage mortality by both nematode species in combination was higher than mortality for either species alone. A Steinernema species and H. bacteriophora cannot coexist when both species are inoculated directly into the hemocoel of an insect host. S. carpocapsae, S. feltiae (= bibionis), or S. glaseri, inoculated before H. bacteriophora at 3-hr intervals, was the dominant species. When a steinemematid species was inoculated in the same host after H. bacteriophora, steinemematid dominated for the first 6 hr. Thereafter, H. bacteriophora was the dominant speties . 0 1991 Academic Press, Inc. KEY

WORDS:

sae; S. feltiae

Interspecific

(= bibionis);

comvetition:

Heterorhabditis

bacteriophora;

Steinernema

carpocap-

S. glaseri.

INTRODUCTION

neously in sand in the presence of insect hosts, H. bacteriophora invariably infected and killed more hosts located at a greater distance from the nematode placement site than the Steinernema sp. The Steinernema sp. infected and killed more hosts located closer to the nematode placement site. Usually only one nematode species was found in each host, but in a few hosts, both nematode species were present. In the latter situation, half of the body of the dead insect was either ocher, indicating the presence of Xenorhabdus nematophilus which is the associated bacterium of Steinernema, or red, indicating the presence of X. luminescens which is the associated bacterium of Heterorhabditis. In some hosts, the cadavers were ocher with red spots. Dissection of the hosts revealed dead immature or adult nematodes, indicating that if both nematode species occurred in the same insect, neither species could survive. We report herein further studies on interaction between Heterorhabditis and Steinernema when both species occur in the same insect host.

Little is known about interspecific competition between entomogenous nematode species when they occur in the same insect host. The occurrence of two or more nematode species in the same insect host is not unusual, especially in bark beetles (Kaya, 1984; Choo et al., 1987; Lieutier, 1980, 1982). Lieutier (1980) found two species of Parasitaphelenchus occurring independently of each other in the same bark beetle host. However, incompatibility was observed when Parasitaphelenchus sexdentati and Contortylenchus diplogaster occurred in the same host. The incompatibility was believed to be associated with the production of progeny by Contortylenchus. Recently, Alatorre-Rosas and Kaya (1990) demonstrated that when Heterorhabditis bacteriophora (= heliothidis) and a Steinernema sp. were placed simultar Present address: Department of Entomology and Acarology, Colegio de Postgraduados, Chapingo, Mexico, Edo. 56230. 1

0022-201 l/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

ALATORRE-ROSAS

MATERIALS

AND METHODS

Insect and nematode rearing. A colony of wax moth, Galleria mellonella, was reared on a bran diet (Dutky et al., 1962). In all experiments, last instar larvae were used. S. carpocapsae, S. glaseri, S. feltiae (=bibionis), or H. bacteriophora was cultured in last instar wax moth larvae (Dutky et al., 1964). The infective nematodes were harvested and stored in distilled water (15,000 nematodes/ml) at 10°C and used within 10 days of harvest. Compatibility of nematodes in Petri dishes. Compatibility of Steinernema and Heterorhabditis in G. mellonella larvae was

initially tested in Petri dishes (100 x 15 mm) lined with two 9-cm-diameter (Whatman No. 1) filter papers. In the first test, 10, and increments of 10 up to 100 S. carpocapsae in 0.5 ml of water and an equal number of H. bacteriophora in 0.5 ml of water, were distributed on the filter papers and 12 Galleria larvae were placed in each dish. Controls consisted of equal numbers of S. carpocapsae alone or H. bacteriophora alone (20, 40, 60 . . . etc.). In the second test, 50 S. carpocupsae infective nematodes in 0.5 ml of water were placed first in the dish followed by 12 Galleria larvae. Subsequently, at 5-, lo-, and 15-hr intervals, 50 H. bacteriophora infective nematodes were added to the dish. The reciprocal test was also conducted. Controls for these tests were 100 infective nematodes of S. carpocapsae alone or H. bacteriophora alone per dish. The Petri dishes were held at 25 + 2°C in plastic bags to minimize desiccation. After 72 hr, the insects were examined for mortality and changes in coloration. An ocher color was classified as a S. carpocapsae infection and a red color was classified as a H. bacteriophora infection (Poinar, 1979). Six days later, the dead larvae were dissected and examined. In this and all subsequent tests when nematodes were present, slides containing females, males, and juveniles from the thorax and abdomen

AND

KAYA

of the host were prepared for microscopic observation. Twenty-five nematodes were examined to determine which species was present in the insect. Heterorhabditids and steinernematids were readily separated by several morphological characters, including tail shape, presence or absence of male bursa, and excretory pore location. In all tests, there were two trials with 12 Galleria larvae per trial. Inoculation of nematodes in the same host. Infective nematodes were surface

sterilized according to the method of Lownsbery and Lownsbery (1956) and Hara et al. (1981). Surface sterilization was accomplished by placing several thousand infective nematodes in a separatory funnel containing a solution of 0.013% Aretan (methoxyethyl mercury chloride) and 0.5% dihydrostreptomycin sulfate. After 3 hr the nematodes were delivered to a lower separatory funnel containing 5 ml of sterilized distilled water for 3 additional hours. Finally, the nematodes were delivered to a vial containing 20 ml of sterile water. The nematodes were stored in the vial at 10°C until used. The sterilized infective nematodes were injected in the thoracic region of a Galleria larva as follows. Five infective nematodes of S. carpocapsae and H. bacteriophora were inoculated into the hemocoel using a fine-tipped glass needle (ca. 0.2 mm inside diameter). The needle with a rubber tube for a mouthpiece allowed individual nematodes to be aspirated from the water suspension. The infective nematode of each species was either introduced simultaneously or one species was introduced first followed by the second species at 3-hr intervals up to 27 hr. There were three trials with 15 Galleria per treatment. Controls consisted of larvae inoculated with five infective nematodes of S. carpocapsae or H. bacteriophora to check their viability and infectivity. Two days later, the insects were checked for mortality and changes in coloration, and 6 days later the dead larvae were dissected.

INTERACTION

BETWEEN

Similar experiments were conducted between S. feltiae and H. bacteriophora and S. glaseri and H. bacteriophora. The reciprocal test was conducted where H. bacteriophora was first inoculated in Galleria larvae followed by Steinernema spp. Statistical analysis. Analysis of variance was performed on the infectivity of S. carpocapsae alone, H. bacteriophora alone, and/or S. carpocapsae + H. bacteriophora. Regression analysis was used to test variability on the infectivity by Steinernema spp. (S. carpocapsae, S. feltiae, S. glaseri) when they were alone or in combination with H. bacteriophora in Galleria larvae (Steel and Torrie, 1985). RESULTS Compatibility in Petri dishes. In the first test when S. carpocapsae and H. bacteriophora were placed in equal numbers in Petri dishes, S. carpocapsae infected a higher percentage of Galleria larvae than H. bacteriophora at all concentrations tested (Fig. 1). At concentrations of 20 to 140 infective nematodes per dish, signifi-

0

3

NEMATODES

cantly lower nematode infections were observed between S. carpocapsae alone or H. bacteriophora alone than S. carpocapsae + H. bacteriophora

(F = 4.28, P = 0.05).

At concentrations >160 infective nematodes, the treatments, when the species were alone or combined, were not signifcantly different. In the second test when S. carpocapsae preceded the placement of H. bacteriophora by 5-hr intervals, S. carpocapsae consistently infected a higher percentage of Galleria larvae than H. bacteriophora (Table I). In the reciprocal test, S. carpocapsae infected a high percentage of Galleria up to 10 hr, but at 15 hr H. bacteriophora was the dominant species. Intrahemocoelic inoculation. When S. carpocapsae was inoculated into the hemocoel of Galleria first followed by H. bacteriophora at 3-hr intervals up to 27 hr, S. carpocapsae was dominant at all inoculation times (Fig. 2). Similar results were observed when S. feltiae and S. glaseri were inoculated before H. bacteriophora (Fig. 2).

SC alone Hb alone b SC + Hb

-Pm

.b

J’

b

i 20 Nematode

Concentration

1. Galleria larvae infected with Steinernema carpocapsae (SC) or Heterorhabditis bacteriophora (Hb) alone and in combination in Peti dish tests. Diierent letters above the bars for each nematode concentration indicate significant differences at P < 0.05. FIG.

4

ALATORRE-ROSAS

AND

TABLE INFECTIVITY

Incumbent species S. carpocapsaeC

1

OF Steinernema carpocapsae (SC) AND Heterorhabditis bacteriophora (Hb) TO Galleria IN PETRI DISHES WHEN NEMATODES WERE INTRODUCED AT DIFFERENT TIMES x % Galleria

Lead time (hr) for incumbent species

SC a a 80 a 94 a

0

69 83

5 10 15 H. bacteriophorad

KAYA

0

68 72 67 20

5 10 1.5 0 Mean percentage of two trials with five replicates/trial. letters are-significaky different (P < 0.05). b Dual nematode infection. ’ S. carpocapsae added before H. bacteriophora. d H. bacteriophora added before S. carpocapsae.

In the reciprocal test, S. carpocapsae prevailed between 0 and 6 hr, but at 9 hr and later H. bacteriophora was the dominant species (Fig. 3). With H. bacteriophora and S. feltiae or H. bacteriophora and S. glaseri, both Steinernema species also prevailed from 0 to 6 hr but after this time H. bacteriophora became the dominant species (Fig. 3). Inoculation of the steinernematid species or H. bacteriophora alone into Galleria hosts showed that the infective nematodes were viable. DISCUSSION Our working hypothesis was that Steinernema spp. would be the dominant species over H. bacteriophora whenever Steinernema spp. had the competitive advantage in time over H. bacteriophora. This hypothesis held true in the Petri dish test when Galleria larvae were placed with Heterorhabditis and Steinernema. Steinernema carpocapsae infected the insect host faster than H. bacteriophora and this relationship was true eve? when H. bacteriophora was introduced up to 15 hr before S. carpocapsue. The Petri dish tests confirmed our earlier studies in sand where Steinernema spp. generally outcompeted H. bacteriophora for insect hosts located proximal to nema-

a a a b

Hb

LARVAE

infected” SC and Hbb

b b b 5b

22 12 12

16 b 17 b 23 b 71 a

Mean for SC and Hb in a row followed by different

tode placement (Alatorre-Rosas and Kaya, 1990). Direct inoculation of a steinernematid species and H. bacteriophora simultaneously into the hemocoel of an insect host also demonstrated that the steinernematid was the dominant species. The steinemematid was the dominant species even when H. bacteriophora had a 6-hr time advantage in the hemocoel of the host before inoculation of the steinernematid. At inoculation times ~9 hr, H. bacteriophora dominated over the steinernematid species when it was inoculated first. The dominance of S. carpocapsae over H. bacteriophora in the Petri dish test and of S. carpocapsae, S. feltiae, and S. glaseri over H. bacteriophora during the early phases of intrahemocoelic inoculation was expected. Even if they lagged up to 6 hr behind H. bacteriophora in infection time, the steinemematids still could overcome H. bacteriophora infection. While the steinernematids have a shorter life cycle than H. bacteriophora, a shorter life cycle per se is probably not critical to the competitive edge of the steinernematids over the heterorhabditid. The steinernematids may release their mutualistic bacteria Xenorhabdus nematophilus (for S. carpocapsae), X.

INTERACTION

100

BETWEEN

NEMATODES

5

the nematode (Burman, 1982; Boemare et al., 1982) orXenorhabdus (Paul et al., 1981; Akhurst, 1982) may prevent the development of H. bacteriophora (interference competition). The inoculation of H. bacteriophora before the steinernematids demonstrated that H. bacteriophora required a 6-hr time advantage in the host’s hemocoel before it be-

-

so *

60 -

40 -

20 -

IS

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24

27 IOO-

b 60-

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20o-

0

loo0 w + ” w L

80 -

60 -

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2. Interaction between Steinernema carpocapor S. gfaseri and Hefewhen the steinernematid was inoculated first into the hemocoel of Galleria followed by H. bacteriophora. Steinernema sp., nonshaded; Heterorhabditis bacteriophora, shaded. FIG.

sae, S. feitiae (=bibionis), rorhabditis bacteriophora

bovienii (for S. feftiae), and X. poinarii (for S. glaseri) sooner than H. bacteriophora releases its mutualistic bacterium, X. lumi-

nescens. Once the bacteria are released, the steinernematids begin their development. In contrast, H. bacteriophora may take longer to release its bacterial symbiont, giving the competitive advantage to the steinernematids. Once development is initiated, toxins or other byproducts from

z e w

2

zo-

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,ooSO-

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FIG.

0

3

6

9

12 15 IS Time ( Hr 1

3. Interaction between Heterorhabditis

riophora and Steinernema (= bibionis), or S. glaseri

carpocapsae,

21

24

27

bacteS. feltiae

when the heterorhabditid was inoculated first into the hemocoel of Galleria followed by Steinernema sp. Steinernema sp., nonshaded; Heterorhabditis bacteriophora, shaded. Different letters above the bars for each time period indicate significant differences at P < 0.05.

6

ALATORRE-ROSAS

came the dominant species. These data demonstrate that the steinemematids overcame the time advantage given to H. bacteriophora which is probably related to the rapid release of the mutualistic bacteria by S. carpocapsae and S. feltiae. Another possibility is that bacterial release by the nematode species is not significantly different, but that the growth rate of X. luminescens is slower than Xenorhabdus spp. from steinernematids. Thus, Xenorhabdus spp. from steinernematids can overcome X. luminescens allowing the steinernematid nematodes to develop rather than H. bacteriophora .

The competition between steinemematid and heterorhabditid nematodes is very complex. It involves the host and two nematodes and their associated mutualistic bacteria. However, the competition has been studied only under one set of environmental conditions and with one species of insect host. Further research on interspecific competition is needed to determine whether our discussion adequately explains the results obtained. At present we conelude that internal competition between the entomogenous nematode species appears to be interference rather than exploitative. ACKNOWLEDGMENTS We thank Drs. Lester E. Ehler and Charles L. Judson, Department of Entomology, University of California, Davis, for critically reading the manuscript. This research was supported, in part, by a Jastro Shields Research Grant and CONACYT-Mexico provided to the first author.

REFERENCES AKHURST, R. J. 1982. Antibiotic activity ofxenorhabdus spp., bacteria symbiotically associated with insect pathogenic nematodes of the families Heterorhabditidae and Steinemematidae. J. Gen. Microbiol., 128, 3061-3066. ALATORRE-ROSAS, R., AND KAYA, H. K. 1990. Interspecific competition between entomopathogenic nematodes in the genera Heterorhabditis and Stein-

AND

KAYA

ernema for an insect host in sand. J. Invertebr. BOAST’ “,“r yz -188. AND LUCIANI, J. 1982. 1 7 UMOND,C., Mise en evidencl e d’une toxicogenese provoquee par le nematode axemiaue entomonhaee Neoaolectana carpocapsae Weiser chez l’insecte axenique Galleria mellonellaL. C.R. Acad. Sci. (Paris) Ser. 3, 295, 543-546. BURMAN, M. 1982. Neoaplectana carpocapsae: Toxin production by axenic nematodes. Nematologica, 28, 62-70. CHOO, H. Y., KAYA, H. K., SHEA, P., AND NOFFSINGER, E. M. 1987. Ecological study of nematode parasitism in Zps beetles from California and Idaho. J. Nematol., 19, 495-502. DUTKY, S. R., THOMPSON, J. V., AND CANTWELL, G. E. 1962. A technique for mass rearing the greater wax moth (Lepidoptera: Galleriidae). Proc. Entomol. Sot. Wash., 64, 495-502. DUTKY, S. R., THOMPSON, J. V., AND CANTWELL, G. E. 1964. A technique for mass propagation of the DD-136 nematode. J. Insect Pathof., 6, 417-422. HARA, A. H., LINDEGREN, J. E., AND KAYA, H. K. 1981. “Monoxenic Mass Production of the Entomogenous Nematode, Neoaplectana carpocapsae Weiser, on Dog Food/Agar Medium.” U.S. Department of Agriculture AAT-W-16. KAYA, H. K. 1984. Nematode parasites of bark beetles. Zn “Plant and Insect Nematodes” (W. R. Nickle, Ed.), pp. 727-754. Marcel Dekker, New York. LIEUTIER, F. 1980. Le parasitisme d’lps sexdentatus (Boem) (Coleoptera: Scolytidae) par les nematodes du genere Parasitaphelenchus Fuchs: Relations avec le parasitisme par Contortylenchus diDlopaster (v. Lini.). Rev. Nematol., 3, 271-281. _ LIEUTIER, F. 1982. Action des nematodes endoparasites sur la ponte du Scolytidae Zps sexdentatus Boemer (Insecta: Coleoptera). Acta Ecol. Appl., 3, 191-204. LOWNSBERY, B. F., AND LOWNSBERY, J. W. 1956. A procedure for testing the sterility of large numbers of nematodes after treatment with various sterilants. Plant Dis. Rep., 40, 989-990. PAUL, V. J., FRAUTSCHY, S., FENICAL, W., AND NEALSON, K. H. 1981. Isolation and structure assignment of several new antibacterial compounds from the insect-symbiotic bacteria Xenorhabdas spp. J. Chem. Ecol., 7, 589-597. POINAR, G. O., JR. 1979. “Nematodes for Biological Control of Insects.” CRC Press, Boca Raton, FL. STEEL, R. G. D., AND TORRIE, J. H. 1985. “Bioestadistica: Principios y procesamientos.” McGrawHill, Bogata, Colombia.