Neoaplectana carpocapsae: Movements of nematode populations on a thermal gradient

EXPERIMENTAL

PARASITOLOGY

Neoaplectana

49,258-265(1980)

carpocapsae: Movements of Nematode on a Thermal Gradient MARTIN

(Accepted

BURMAN

AND

for publication

E.

ALBERT

27 March

Populations

PYE’

1979)

BURMAN, M., AND PYE, A. E. 1980. Neouplectcrnct crrrpocxpse: Movements of nematode populations on a thermal gradient. Experimental Parasitolo,qy 49, 258-265. To gain information on factors which could affect the nematode Neocrplec~tunrr curpcwcrp.snr’s dispersion and infection of insect larvae in the field, nematode populations were tested on a thermal gradient (0.5 C/cm). Infective juveniles grown at 15, 20, and 25 C migrated on the gradient toward their respective growth temperatures when tested immediately after harvesting from their medium. This migration by juveniles, grown at 20 or 25 C, was altered within 12 hr by shifting incubation temperature. The nematodes’ tendency to migrate toward warmer temperatures, when placed below incubation temperature, decreased during 6-7 days incubation at 20 or 25 C and the nematodes reversed direction: incubation at 2-5 C inhibited this reversal. Nematodes incubated at 20 C, then applied to a gradient zone at 12-13 C, had a greater tendency (P < 0.05) to remain aggregated in that zone within a 3-hr period than those applied at 17- 18,22-23, or 27-28 C. Their tendency to migrate from the l2- 13 C zone was significantly (P i 0.05) increased by shifting them to I5 C for 14- 138 hr. INDEX DESCRIPTORS: Neouplectuna curpocupscrr: Nematode, parasitic; Behavior: Biological control: Insect host; Hdohius crhiefk.

carpocapsae, and temperature

may affect infectivity by reducing nematode activity. Therefore, we studied the movement of populations of N. carpocapsae infective juveniles (dauer third-stage larvae) upon a temperature gradient. Factors were identified which can alter the nematodes’ dispersion. Such factors might be used to manipulate the nematode and increase field infection rates in target insects.

Under laboratory conditions, at 25 C and high humidity, the nematode Neoaplectana carpocapsae parasitizes and kills a wide variety of insect larvae (Niklas 1969; Poinar 1975). The nematode is of interest as a potential biological control agent (Benham and Poinar 1973) especially against susceptible, soil-dwelling, pest insects such as the large pine weevil, Hylobius abietis. N. carpocapsae actively invades H. abietis larvae (Pye and Burman 1977, 1978, 1980) and the nematode can reduce larval populations of the insect in the field (Burman et al. 1979). However, low temperatures (about 13 C), as found below the ground surface during the summer, and insect location (below the ground surface) can reduce nematode infections of H. abietis (Pye and Burman 1978). Natural temperature gradients, with ground depth, may affect the behavior of N.

MATERIALS

METHODS

Nematodes. Neoaplectanu carpocupsue

Leningrad strain was isolated from cadavers of Agriotes lineatus larvae, a soildwelling coleopteron (Poinar and Veremtshuk 1970). Nematodes were obtained from G. 0. Poinar, Jr., University of California, Berkeley. They were cultured on liver-kidney medium (Glaser et al. 1942) with M9 salts (Brenner 1974) substituted for NaCl alone. To increase uniformity in cultures, about 8000 infective (dauer) juveniles, surface sterilized with

’ For correspondence. 258 0014-4894/80/020258-08$02.00/O Copyright @ 1980 by Academic Press, Inc. Au rights of reproduction in any faorm reserved.

AND

Neocrplectancr

THERMAL

carpocapsae

0.2% Benzalkon (ACO, Solna, Sweden), were placed on about 25 cm3 of medium to initiate a culture. After 6 days at 25 C, about 4 ml of water was added to the culture and it was then further incubated at either 15, 20, or 25 C. All culturing was in constant darkness. Nematodes cultured at 20 or 25 C were harvested 13- 17 days after initiation (7- 11 days, or more than one generation, after temperature transfer), usually 17 days after initiation. Harvesting within this interval did not affect results. For harvesting, the medium with nematodes, mostly infective juveniles, was mixed l/5 (v/v) with water. The liquid was filtered through cotton cloth and discarded. The cloth was then wrapped to form a pouch and suspended at the surface of a NaCl gradient (O-O.2 M). With this Baerman apparatus, modified from MacInnis and Voge (1970), nematodes began to swim through the cloth and fell to the bottom of the brine. Harvesting took 6 hr and was conducted at the nematodes’ final culturing temperature. During harvesting, the cloth

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259

was periodically washed with distilled water and every 2 hr transferred to fresh brine. Harvested nematodes were washed on a 8-pm Millipore (Molsheim, France) filter, twice with water and once with M9 buffer (Brenner 1974). Nematodes were then suspended in M9 buffer. Temperature shifts. In certain experiments, suspensions of freshly harvested nematodes were divided into subpopulations and transferred, in buffer, to different temperatures. All culturing and incubation temperatures were 20.5 C. Temperature gradient experiments. In order to quickly (1-3 hr) measure the nematodes’ behavior on a temperature gradient, we used the methods of Hedgecock and Russell (1975). Briefly, a 0.5 C/cm gradient from lo-30 C was created by immersing the opposite “legs” of an aluminum “table” in warm and cold water baths. Petri dishes (diameter 8 cm) with M9 buffered 1.7% agar were then centered at 12.5, 17.5, 22.5, and 27.5 C along the top of the table (with a water film under them to increase conductivity) (see Fig. 1). Tem-

Grown

at 20C

Temperature

gradient

-

0.5ClCrn Center pOSItIOn Test

twne

12 5c 3h

17.5 c 2h

22.5 lh

c

2750 lh

Thermal preference

0

l 14

-79

-94

FIG. 1. Location of Nronplrcfana ccrrpoccrpsoe infective juveniles, tested immediately after harvesting, on a temperature gradient. Circles indicate petri dishes; dots the positions of individual nematodes which migrated from the center application zones. Thermal preference = 100 x (H - C)I(H + C): where H = number of nematodes migrating to the warmer half of a dish, C = number migrating to the colder half.

260

BURMANANDPYE

peratures at various positions along the gradient were checked with a temperature probe and were stable kO.1 C. Nematode suspensions (5-20 ~1) were applied to a center zone (r = 5.5 mm) on the agar with the aid of a plastic ring. Test time started when the suspensions were dry enough for the nematodes to migrate from the center. At the end of a test, nematodes were killed with CHCl, vapor. A test population was then divided into three groups: (1) those remaining at the application zone, (2) those migrating toward the hot (H) half of the dish, and (3) those migrating toward the cold (C) half. Test suspensions usually contained 300 to 500 nematodes. The behavior of nematode populations migrating on the gradient was quantified by calculating thermal preference (TP):

(?O. 1 C). About 2500-5000 infective juveniles were applied to the centers and allowed to disperse as before. The experiments were stopped after sufficient time (2.9 hr) for the fastest dispersing nematodes to just reach the edge of the dish at the most favorable temperature (27.5 C). Populations within different radii were then counted. RESULTS

Behavior

Populations of Ncoaplectana carpocapsue infective juveniles (dauer third-stage larvae, “survivalarvae”) applied to the center of agar in petri dishes on a temperature gradient migrated to form predictable patterns. Figure 1 shows a typical pattern produced by nematodes grown and freshly harvested at 20 C. In this test, infective juveniles applied to a zone centered on the hot side of 20 C tended to migrate toward TP = 100 x (H - C)I(H + C). the cold side of their application zone, When the TPs of different experimental negative thermal preference (TP), while populations were averaged, SEM was cal- juveniles applied to a zone centered below culated and used together with a t value to 20 C tended to migrate toward the hot side of their zone, positive TP. Those applied at calculate confidence intervals. For gradient positions 17.5, 22.5, and 27.5 C, (H + C) 12.5 C migrated randomly, TP zero. With was greater than 200. the test times used, migrating nematodes Nematodes migrated more slowly at po- gave patterns on the agar resembling sitions 12.5 and 17.5 C than at 22.5 or 27.5 triangles, with the application zone as an C. In order that (H + C) would be suffi- apex. Individual infective juveniles tended to migrate away from positions 22.5 and ciently large for calculations, test times for migration on the gradient were 3, 2, 1, and 1 27.5 C aligned head to tail with the temperhr, respectively. In controls it was shown ature gradient. Noninfective juveniles and that extending the time at all positions to 3 adult nematodes remained at the applicahr or reducing it to 1 hr did not affect TP, if tion zones, as aggregations, in all experithe test population was large enough. ments. To give constant humidity, the tests were Figure 2 shows the average TP profile conducted with lids on the petri dishes. As from experiments using freshly harvested the nematodes moved on the agar surface infective juveniles grown at 20 C. At posithey were surrounded by a thin film of tion 17.5 C, TP showed more variation than water. Therefore, they could not sense dif- at positions 12.5, 22.5, or 27.5 C. It varied ferences in water vapor concentration over from about zero to strongly positive TP in the agar surface. some selected populations (LJ - -0). Dispersion rates-No gradient. To mea- Populations with high TP at position 17.5 C sure dispersion rates (area/time), petri dish- and low TP at position 22.5 C gave a line es (diameter 13.6 cm) with 1.7% agar M9 bisecting TP-zero near position 20 C. were placed at the surface of water baths Holding such a selected population in

Neoaplecrana

THERMAL

cwrpocapsae:

Grown

Temperature

gradlent,

GRADIENT

261

at ZOC

C

FIG. 2. Thermal preference (TP) profiles from experiments (n = 8) using populations of Neoaplectana carpocapsae infective juveniles tested immediately after harvesting. Experimental details were as in Fig. 1. Bars show 95% confidence intervals. (n--O) One population, with high TP at position 17.5 C, was used for further experimentation (Fig. 3).

buffer at 20 C, after harvesting, gave a deThe TP profiles of N. carpocapsae subcrease and a reversal in TP at position 17.5 populations grown at 20 C, then shifted to C with time (Fig. 3), but there was no lower temperatures, are shown in Fig. 4. change with time in the population percentThe TP decrease at position 17.5 C caused age (about 10%) remaining at the applicaby a 144-hr incubation at 20 C was inhibited tion zone. During the first few hours, the in subpopulations shifted to 5 C, but shiftdecrease in TP was rapid. Afterwards, TP ing to 15 C caused a larger decrease in TP. in this population stabilized slightly below Shifts to the lower temperatures, 5 and 15 zero. In a subpopulation shifted to 15 C, TP C, caused a TP increase, toward zero, at at position 17.5 C remained at about +80 position 27.5 C. during the first 5 hr after the shift, but after Figure 5 shows the TP profile of infective 138 hr (144 hr after harvest start) TP was juveniles grown at 25 C immediately after reversed, to about -75. At position 22.5 C, harvesting. Their profile bisected TP-zero controls, held at 20 C, maintained a near position 25 C on the gradient, with strongly negative TP, about -90, but a sub- strongly positive TP at position 22.5 C and population shifted to 25 C showed a rapid strongly negative TP at position 27.5 C. At increase in TP, with TP reversal after 4 hr positions 17.5 and 12.5 C TP was slightly (10 hr after harvest start). positive. 0 20c .

20-25

Posman I 225c

A 20C 1ms,,,on

Time, h

FIG. 3. Neoaplecrana carpocapsae infective juveniles’ thermal preference at positions 17.5 and 22.5 C on the temperature gradient with time after harvest start and with shifts of incubation temperature. The test population was n--O from Fig. 2.

262

BURMAN

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PYE

0 20C. 144h.

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0 zo-

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n=2

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15C. 144h.

n=4

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-1ooTemperature

gradent.

C

FIG. 4. Thermal preference profiles of Neo~iplrc~tcltm c~lp~c’~p.~e infective juveniles after shifts in incubation temperature. After harvesting (6 hr), subpopulations were incubated in M9 buffer at different temperatures (138 hr additional). n = number of experiments, with setup as in Fig. I; bars show 95% confidence intervals.

The TP, measured at position 22.5 C, of nematodes grown and harvested at 25 C fluctuated during incubation at 25 C; Figure 6 shows the changes in one such population’s TP. Incubation at 25 C gave first a decrease in TP to about zero, followed by a rebound to positive values, followed by a gradual decrease to negative TP. Such a pattern was observed in other populations grown at and harvested at 25 C. However, neither the periodicity of the rebound nor its amplitude were predictable. In some populations there appeared to be several rebounds, but in no case did the percentage of juveniles migrating from the center application zone vary significantly during the TP fluctuations. By shifting a subpopulation to 2 C the fluctuations and decreases in

TP were inhibited (Fig. 6). However, a shift to 20 C caused a rapid decrease in TP to constant low values, -90 to - 100. In still other experiments using infective juveniles grown (26 days) at 15 C, TP profiles were obtained similar to those with nematodes shifted to 15 C (Fig. 4, A), with the exception that TP at position 12.5 C was slightly higher, + 18. Activity

The activity of Neoaplectana carpocapsue infective juveniles, expressed as the percentage remaining at the application zone, varied in populations grown and harvested at 20 C. It depended upon the location of the application zone along the temperature gradient and upon nematode incu-

+ 100 Grow

at 25C

Temperature

FIG. 5. Thermal preference profile of populations grown at 25 C, tested immediately after harvesting. (n 3 6); bars indicate 95% confidence intervals.

gradlent

C

of Neoclplectrrrrtr ~‘N)POUIJI.SOC infective juveniles The setup was as in Fig. 1. The points are averages

Neoaplectana

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carpocapsar:

THERMAL

GRADIENT

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.

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. .._...__..___,___ “-‘~-~~~.. . . . . . ..__.._..__.__.~~.~~,~

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200 -0

Time. h

FIG. 6. Thermal

preference in populations of Neooplectana curpocapsrrr infective juveniles, grown at 25 C, placed at position 22.5 C on the temperature gradient, plotted against time after harvest start. Subpopulations were temperature shifted 6 hr after harvest start. Experimental setup was as in Fig. 1. Points are averages (n = 3); bars indicate 95% confidence intervals.

bation temperature after harvesting (Fig. 7). The majority of infective juveniles grown, harvested, and held at 20 C in buffer remained on the application zone when placed at position 12.5 C (12- 13 C). At position 12.5 C, about 65% of the populations remained aggregated at the application zone, whereas at positions 17.5, 22.5, and 27.5 C (17-18, 22-23, 27-28 C, respectively) only about 10% remained. The results at position 12.5 C were significantly different (P < 0.05) despite that test time was l-2 hr longer than at the other positions. . Subpopulations shifted from 20 C to incubation at 25 C had similar low activity at position 12.5 C. However, subpopulations

12.X.

3h

17.5C. 2h Center

position

shifted to 15 C for 14-138 hr showed significantly greater (P < 0.05) activity at position 12.5 C, fewer remaining at the application zone (12- I3 C) than those held at 20 C or shifted to 25 C (Fig. 7). In other experiments the activity of nematodes shifted to 5 C was similar to those held at 20 C, when tested at position 12.5 C. Likewise, the activity of nematodes grown at 25 C was similar at all positions to those shifted from 20 to 25 C. In still other experiments, the rates of circular dispersion (area/time) were measured at constant temperature (no gradient) on agar using infective juveniles grown at 25 C. After 2.9 hr on the agar at 27.5 C, 85.5% of a population (n > 2500) was within

22.X.

Ih

27.5C, lh

and Test time

7. Percentage of N~wctplectrtnrr ccrrpoccrpsrre infective juveniles remaining at the center application zones (kO.5 C) on the temperature gradient after temperature shifts. Results are averages from control (20 C) and shifted populations tested 14 to 138 hr after the shifts; n = number of experiments. Bars indicate 95% confidence intervals. FIG.

264

BURMAN

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PYE

an area of 121 ? 7 cmZ around the center. temperature, but rather away from it, even The limits for 85.5% of the population at toward warmer temperatures. 29.0, 25.0, 22.5, 17.5, and 12.5 C were, reIn addition to N. carpocapsae’s changed spectively, 121, 92, 95, 92, and 32 cm”. behavior with temperature shifts, the Since individual nematodes had quiescent nematode also shows changes in dispersion periods and periods with irregular moveactivity with temperature shifts. ments, direct observations could only be Nematodes shifted to 15 C, 14-138 hr, besubjective, but tended to agree with the came more apt to migrate from ther 12- 13 other results, much less activity at 13 C. C application zone than those which reIn the course of the experiments at 12- 13 mained at 20 C (Fig. 7). In Croll’s (1967) experiments with Ditylenchus dipsaci, that C, large crystal-like nematode aggregations were observed, with the nematodes lining nematode showed its greatest activity at up side by side. Similar aggregations could about lo- 15 C after 1 month’s storage at 10 also be produced by using heat-killed infec- C. This peak activity at lo- 15 C was due to tive juveniles. However, the size of the activity decreases at the higher tempera“crystals” was reduced by mixing the tures (20-40 C) tested. Absolute activity of nematodes with suspensions of polydextran D. dipsaci was slightly less when measured at lo-15 C in nematodes stored at 10 C, beads (Sephadex). compared with those stored at 20 or 30 C. DISCUSSION With our methods, no decrease in N. carNeoaplectana carpocapsae infective pocapsae’s activity at higher temperatures (dauer) juveniles show thermal preference was detected (Fig. 7). (TP) on a 0.5 C/cm temperature gradient. Our experiments with heat-killed The TP they show is conditioned by their nematodes indicate that the aggregations growth and incubation temperature. Immeobserved at position 12.5 C were due to diately after harvesting from growth surface tension combined with low medium, nematode populations tend to minematode activity. Nematodes with higher grate toward their growth temperature activity would break free from the aggre(Figs. 1, 2, 5). In the temperature range gates. 20-25 C, a temperature shift of newly harIn our experiments, N. carpocapsae vested nematodes results in a rapid shift in grown at 20 or 25 C and tested at positions behavior, toward the new temperature 22.5 and 27.5 C, made tracks in the agar (Figs. 3,6). With time, TP tends to decrease aligned head to tail toward their growth and nematodes migrate toward colder temtemperatures. Such tracks conform with peratures (Figs. 3, 4, 6). These decreases the definition of a thermotaxis (Croll 1970), can be inhibited by holding the nematodes but our efforts to photograph these narrow at 2-5 C (Figs. 4, 6). tracks were unsuccessful. Random moveCroll (1967) and Hedgecock and Russell ments were not responsible for N. car(1975) have also reported thermal acclimapocapsae’s response at 22.5 and 27.5 C. tion in other species of nematodes. Rapid For example, nematodes leaving the center shifts (within 6 hr) in the behavior of at position 27.5 C did not migrate randomly, Caenorhabditis elegans also result from but rather within 45” of directly toward the temperature shifts (Hedgecock and Russell cold side (Fig. 1). 1975). With C. elegans, adult nematodes Hedgecock and Russell (1975) have progave the responses, whereas in our experiposed that C. elegans thermotaxis is conments with N. carpocapsae only the infectrolled by two “drives”: a constant drive tive juveniles responded to the temperature toward colder temperatures and a plastic gradient. With C. elegans, dauer juveniles drive toward warmer. At the eccritic (predid not migrate toward their incubation ferred) temperature, the two drives are bal-

Neoaplecrono

carpucapsae:

anced. Such a model would explain the decreasing TPs with time in N. carpocapsae, decreases inhibited by chilling to 2-5 C (Figs. 4, 6). That is, a metabolizable component would exist in the plastic upward drive. Therefore, it should be possible to select cryophilic strains of N. carpocapsae as Hedgecock and Russell (1975) have done with C. elegans. In nature, N. carpocapsae reproduces in the cadavers of insects it has killed (Poinar 1975). Often these insects are located in the ground. In disintegrating cadavers, a process similar to our harvesting technique would occur. As the nematodes dispersed they would be subjected to temperature gradients present in the ground. Their conditioned TP might help them to locate new hosts present in the same soil stratum as the cadaver they left. If N. carpocapsae is to be used against an insect inhabiting a particular soil stratum, then our results should be considered in designing field experiments. For example, if the insect has a habitat with 20 C, then the nematode’s incubation and culturing should be adjusted to 20 C. In our experiments, N. carpocapsae acclimated to 25 C migrated toward that temperature even if it meant climbing up a petri dish and dying through desiccation. The same sort of behavior could occur in the field and reduce insect mortality. In other cases, field experiments should wait until cryophilic strains of N. carpocapsas have been selected, strains which may be able to deeply penetrate soil. ACKNOWLEDGMENTS The work was supported by grants from Sweden’s National Environmental Protection Board, 7-9/77; and from The Swedish Council for Forestry and Agricultural Research, S 467/P 328, to A.P. L.-E. Sandstrom provided technical assistance. REFERENCES G. S., AND POINAR, G. 0. 1973. Tabulation and evaluation of recent field experiments using the

BENHAM,

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265

DD-136 strain of Neonplectnna cnrpocupse Weiser: A review. Experimental parusitology 33, 248-252. BRENNER, S. 1974. The genetics of Cuenorhabditis elegans. Genetics 77, 71-94. BURMAN, M., PYE, A. E., AND N&ID, N. 0. 1979. Preliminary field trial of the nematode Neouplectunu carpocupsrre against larvae of the large pine weeveil, Hylobius ubietis (Coleoptera, Cur-

culionidae). Annales

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88.

CROLL, N. A. 1967. Acclimatization in the eccritic thermal response of Ditylenchus dipsrrci. Nematologica 13, 385-389. CROLL, N. A. 1970. “The Behaviour of Nematodes Their Activity, Senses and Responses.” Arnold, London. GLASER, R. W., MCCOY, E. E., AND GIRTH, H. B. 1942. The biology and culture of Neoaplectrrnrr chresimu, a new nematode parasitic in insects. Journal oj’Prrrusito/ogy 28, l23- 126. HEDGECOCK, E. M., AND RUSSELL, R. L. 1975. Normal and mutant thermotaxis in the nematode Cuenorhubditis tional Acudemy

elegcrns. Proceedings Sciences, USA

of

of

the

Nn-

4061-4065. MACINNIS, A. J., and VOGE, M. 1970. “Experiments and Techniques in Parasitology.” Freeman, San Francisco. 72,

NIKLAS, bericht

0. F. 1969. Ergtinzungen zum Literaturtiber die Nematode* DD-136 (Neouplectunrr curpocapsrre Weiser, “strain DD- 136,” Rhabditida). Nachrichtenblcrtt

Deutchen

(Brrrrrnsch~~~eig)

21, 71-78.

Pjlrrn;ens~hlrt~dier2st~,.s

POINAR, G. 0. 1975. “Entomogenous Nematodes. A Manual and Host List of Insect-Nematode Associations.” Brill, Leiden, Netherlands. POINAR, G. O., AND VEREMTSHUK, G. V. 1970. A new strain of entomopathogenic nematodes and geographical distribution of Neorrplectrrnrr urpocrrpstre Weiser (Rhabditida, Steinernematidae). Zoo/ogi?e.skij iurnal 49, %6-%9. [in Russian] A. E., AND BURMAN, M. 1977. Pathogenicity of the nematode Neoaplectuncr crrrpoc’crpscre (Rhabditida, Steinernematidae) and certain microorganisms towards the large pine weevil, Hdobirrs rrbietis (Coleoptera, Curculionidae). Annoles Entomolgici Fennici 43, 1 l5- 119.

PYE,

PYE,

A. E., cc~rpocupscre:

pine

weevil

Parctsito/og.v

PYE, A. E., crrrpocrrpscre:

and bacterial in press.

BURMAN, M. 1978. Neocrplectrrnrr Infection and reproduction in large larvae, Hvlobius trbietis. Experimenttrl 46, I - 1 I.

AND

AND

BURMAN,

Nematode gradients.

M. 1980. accumulations Experimentrrl

Neotrplectctncl

on chemical Prrrcrsitology.