Sound production in Scolytidae: Rivalry and premating stridulation of male Douglas-fir beetle

J. Insect Physiol., 1976, Vol. 22, pp. 997 to 1003. Pergamn Press. Printed in Great Britain.

SOUND PRODUCI’ION IN SCOLYTIDAE: RIVALRY AND PREMATING STRIDULATION MALE DOUGLAS-FIR BEETLE

OF

J. A. RU~INSKYand LEE C. RYKER Department of Entomology, Oregon State University, Corvallis, Oregon 97331, U.S.A. (Received 17 December 197% Abatraet-The previously reported attractant chirp of male Dendroctonus pseudorsugae changed to a distinctive tong interrupted chirp after several minutes in the female gailery, and the same chirp occurred during fighting with another male. It was experimentally evoked by synthetic 3-methylcyciohex-2-en-lone and frontalin, both known pheromone components of the male. A different, short, uninterrupted male chirp emitted shortly before copulation, was not evoked by these pheromones. It is suggested that chemostimulus altows or main~ins dual contexts for the interrupted chirp. Observation of premating and mating behaviour showed some initial male aggressiveness with the female that subsided as gallery cooperation was established before mating and as the uninterrupted chirp replaced the interrupted chirp.

INTRODUCIION

MATERIALS AND METHODS

PREVIO~‘Sstudy of ~mmuni~tion of ~e~rocto~~s beetles has concentrated upon signals directly or indirectly affecting aggregation by flying beetles. The work reported here extends the behaviour of the Douglas-fir beetle, Dentroctonus pseudotsugae Hopkins, within its galleries under the bark. E).ps~do~s~ue male stridulation in response to the chemostimulus of the female attractant has been characterised as a simple (not interrupted), multi-impulse chirp (RUDIN~KYand MICHAEL,1972; MIC~-~AJZL and RUKBNSKY, 1972). In other Dendra~tonus, male ‘rivalry’ stridulation was evoked by fighting with another male and/or by synthetic male pheromones; analysis showed its s~gn~cant difference from attractant stridulation in the same species (RUDINSKY, 1973b; RUDINSKYet ab, 1974b; RUDMSKY and MICHAEL, 1974). In the study of male I). pseudo~s~ue pheromones, three substances have been identified: the antiaggregative pheromone 3-methylcyclohex-2-en-lone (3,2-MCH, formerly called MCH) (RUDINSKYet al., 1974a), which is also released by females (KINZER et al., 1971; RUDINSKYet al., 1973); its isomer 3-methylcydohex-3-en-l-one (3,3-MCH) (LIBBEYet af., 1976); and frontalin (RUDINSKYel al., 1976), which is also released by females (KINZER et at., 1971). These substances were released in situations analogous to the rivalry situations studied in pine-infesting Dendroctonus.

We here report results of the study of male D, pseuin response to two of these substances released by males, and further character&e two male premating chirps, one emitted after a few minutes in the gallery and the other longer exposure to the female and preceding mating.

dotsugae stridulation

997

Test beetles were from naturally infested Douglasfir, Pseudotsuga menziesii (Mirb.) France, from western Oregon. The logs were stored outside or at 4°C and brought to beetle emergence temperature as needed. Chemoucoustic

bioassay and recording

~~#uir~ &e~v~o~r. Previous methods for study of

male chemoacoustic response to attractive females in naturai galleries in logs (RUDINSKYand MICHAEL, 1972, 1974) were slightly modified to include a second, rival male near or in the gallery of the beetle pair. Three females were introduced into logs and allowed to feed and become attractive for 2 days; then a male was pfaced in each gallery and allowed to establish himself for one hour before other males were allowed to walk singly to the entry. St~dulation was monitored with the oscilloscope and earphones. Three tests of 60 males each were run on separate days. To record rivalry stridulation, a male placed in the entry of an attractive female gallery was joined after three minutes by a second male and the entry was screened to prevent departure of either male. Three such sets of beetles were recorded for 15 min each, and 25 chirps were taken at random with eight from each beetle set. To observe male-male behavioural interaction without the effect of female attractants and female presence, we placed two males in a hand-bored trough, the diameter of a typical gallery, in a log that had been cut to expose the trough and give direct access for recording each male when the microphone was moved slightly from one to the other. For sampling, 25 chirps were taken from three sets of rival beetles.

998

J. A. RUDINSKY AND LEE C’.RYKER

Premating behaviour. New recordings were made showing the whole sequence of male stridulation with the female, for study of the previously observed change from a full, uninterrupted attractant chirp, which occurs initially, to a double or multi-interrupted chirp (RUDINSKYand MICHAEL,1973, p. 690). Five attractive females in logs were each joined by a male; recording began as the male approached the gallery and continued for 90 min with each beetle pair. Eight male chirps were sampled at six time intervals from each of three pairs, with one chirp at random added to make 25 chirps from each time interval. Walkway tests. Tests of chemostimulus of male chirps were made in the olfactory walkway used previously and equipped with electronic amplifying and recording equipment as described (RUDINSKYand MICHAEL, 1972). Sixty newly emerged, unfed male beetles were tested individually for each treatment. Synthetic pheromones used were: 3,2-MC 99% Aldrich Chemical Co., Milwaukie, Wise., and frontalin 98.5% Chemical Samples Co., Columbus, Ohio. Host tree resin was drawn from Douglas-fir. Required low concentrations were obtained by dilution in absolute ethanol. Acoustic equipment. The recording system used a Hewlett-Packard model 15119A condenser microphone, a Princeton Applied Research model 113 low noise preamplifier, and an Ampex FR 1300 instrumentation tape recorder. A Nagra 4.2 L recorder was used in 1973-1974 instead of the Ampex. The recording system components all showed an essentially flat response from about 0.02 to 22 kHz at 152 cm/set tape speed on the Ampex and 38 cm/set on the Nagra. Frequencies below 0.3 kHz were attenuated by the preamplifier settings to minimise stray noise. Signal parameters were measured on a Tektronix model 5103N storage oscilloscope, and oscillograms were recorded by Polaroid photographs either of triggered single sweeps or of stored tracings. Motions of male.beetles making both simple and interrupted chirps were studied by confining males in a plexiglass chamber just large enough for two beetles end to end. A thin glass side window of the chamber faces a prism, which directs the light path up through a dissecting microscope for observation. Sound production was simultaneously monitored through earphones from the microphone, which was placed next to a screened end of the chamber. Observation of courtship and mating behaviour

To observe the behaviour of beetle pairs in galleries before, during, and after mating, sheets of fresh bark about 20 x 30cm were sandwiched between two plates of l-cm-thick Plexiglas, secured with c-clamps and sealed with electrical tape around the edges (similar to REID, 1958, and others). Females were introduced into a 2-cm pre-cut-gallery through an entrance tunnel cut through Plexiglas and bark on the back side and allowed to feed for 1 to 2 days. Acoustic signals of 14 pairs were recorded with the microphone

covering the entry hole during observations of the inner side. Because only two of 14 males were successful in mating during observation, the parameters of the male courtship chirp were estimated (n = 30) but not compared statistically to other chirps. RESULTS Distinctive chirp in two contexts Rivalry context. As shown previously (RUDINSKY and MICHAEL.1972) males arriving at the screened entry of an attractive female gallery were arrested there, performed digging motions, and gave attractant chirps (Fig. la). Several minutes after a male had been allowed to join the female, other males were no longer arrested and typical passing behaviour occurred, indicating that the antiaggregative pheromone had been released. However, when the gallery entry was left unscreened so that pedestrian males were able to enter the gallery with both male and female inside, a new behaviour appeared which may be called ‘rivalry’. About 88od of the tested males (n = 180) entered the hole. Most of these came back out within 15 to 30 sec. Some males, however, were kicked and pushed out of the gallery by the resident male, which continuously gave strong interrupted chirps, long chirps broken by two or more interruptions (Fig. Id) (Table 1A). Stridulations of some of the invading males gradually changed during this aggressive interaction from attractant chirps to interrupted chirps as the two males engaged in an ‘acoustic duel’. Long uninterrupted chirps were also produced intermittently (Fig. lc). Tests performed with two males confined in an artificial gallery left open above for microphone access showed that female presence was not necessary for the male to produce the interrupted rivalry chirp (Table 1B). Placed head to head, the males emitted long strings of interrupted chirps (Fig. lh) with one male predominantly chirping and the other silent. Premating context. When a male was allowed to enter the gallery of an attactive female, he produced attractant chirps (Fig. la) as he was digging in and through the frass for the first several minutes. This response was the same as the ‘announcing’ attractant chirps at the chemostimulus of the female reported in walkway and field tests (RUDINSKY, 1969; RUDINSKYand MICHAEL,1972). However, after about 3 min inside the gallery, the male changed to interrupted chirps (Fig. lb) and occasional long chirps. The change from the attractant chirp to the interrupted chirp is shown by comparison of acoustic parameters of chirps sampled within the first minute of male entry and at later intervals (Table lc). Chirps after 3 min are more frequently interrupted, have a slightly slower toothstrike rate, and are longer in duration, having more toothstrike per chirp. Chemostimulus of interrupted chirp. Finding the same male chirp in both premating and rivalry con-

Fig. 1. Oscillograms of male Douglas-fir beetle chirps. In female gallery: (a) attractant chirp after 30 xc, (b) interrupted chirp after 3 min. With another male (rivalry situation) above gallery of attractive female: (c) long chirp, (d) interrupted chirp and more intense click of female (single pulse), Stimulated by synthetic 3,2-MCH and frontalin: (e) long chirp, (f) interrupted chirp. Before copulation: (g) courtship chirp. With another male and no female stimulus: (h) interrupted chirp. Each tracing = 0.2 sec. All at 26” to 28°C.

1001

Sound production in scolytidae Table I. Acoustic

parameters of male Douglas-fir beetle chirps Correctadc;c;~ duration

Sample Stimulus

situation

A.

2 bb

6.

2 dd

with %

C.

g+dlNLOG

without %

o*

NO. ts par chjr# Mea&

Range

Mean

No. interruptions/chirp

Mean

Melo

Range

Range

%#O

47.6b**

34-67

8g.5bcf*

66-122.5

538ab*

429-658

1*88bc**

o-4

84

25

41.4bW

26-58

79.?bc**

50-110

526abw

364-724

1.6ab**

1-2

100

O-l MINUTES

25

19.6'

26

38.gb**

4-5.5 NINUTES

25

48.9b**

16-74

12-16 MINUTES

25

51.7bH

32-67

MINUTES

25

45.2b"*

30-60

60-70 MINUTES

25

47.2b**

17-75

25

50.1b**

19-84

with MCH

Range

ts rate (ts/sec)

25

2.5-3 MINUTES

30

0.

size

9-42 17-61

33.ga

14-77.0

643b

383-833

0.08a

o-1

8

64.?b**

34-112.5

614ab

358-911

0.96abX*

O-3

58

E&1bc**

555ab**

353-729

1.6ab**

o-5

72

60-152.5

518"**

361-667

3.0SC**

O-4

96

89.4bCf*

61-127.5

516a**

347-709

l.ROb**

O-5

72

80 gbc**

31-122.5

57rJab*

426-985

l.76b**

O-6

68

87:3bC**

32.5-157.5

57aab*

336-773

1.88bc**

1-3

100

101.4c*

36-140

11 ts = toothstrikes 21 Letters indicate groupings of means by the Scheffd test fPcO.05); a is smallestgroup, c is largest. l

Mean is different from O-l MINUTE mean by Least Significant Difference Test (PdO.05).

l*

Mean Is different from O-l MINUTE man

by LSD test (P~0.01).

texts occasioned the hypothesis that this chirp is evoked by a chemostimulus common to both contexts. This hy~thesis was tested in the olfactory walkway. In the premating situation, female frass contains both frontalin and 3,2-MCH; more of each substance is released after the male arrives (RUDINSKY et al., 1973, 1974a, 1976). The timing of the male change from attractant chirp to interrupted, i.e. after several minutes near or in the female gallery (Table lC), corresponds to this release of more 3,2-MCH and frontalin. In the rivalry situation, occurrence of the interrupted chirp corresponds to the demonstrated release of 3,2-MCH and frontalin when two males are together (RUDINSKYet al., 1974a, 1976). Thus substantial quantities of these substances are present in both situations where the interrupted chirp naturally occurs. Results of the walkway tests (Table 2) support this hypothesis. The attractant chirp occurred with the control of host tree resin and frontalin but occurred significantly (P < 0.05) more often with 0.0002% and 0.002% concentrations of 3,2-MCH, confirming similar previous tests (RUDINSKYand MICHAEL, 1972; RUDINSKY et al., 1974a). The interrupted chirp occurred only when the concentration of 3,2-MCH reached 0.02% and 0.2%. We do not exclude the possibility that changes in the concentration of frontalin affect male stridulation, as this compound is also released by the male as well as the female. However, we have not seen any effects of concentration difference in our tests with frontalin, once the threshold quantity for attraction is reached, and varied amounts were used by several workers without reported differences. Not shown are preliminary tests of MCH without frontalin and host terpenes; they were not pursued because the compound does not exist in nature

without the other substances. Similar tests were given by RUDINSKYand MICHAEL(1972). Statistical analysis showed that the interrupted chirps produced in the four tested situations (i.e. the male after several minutes in a female gallery, fighting another male in a gallery with a female present, fighting another male in a non-attractive artificial gallery, and responding to a medium concentration of MCH) did not differ sign~cantly in the measured acoustic parameters (Table 1). Observed

courtship

and mating

Interrupted chirp. Observation of male and female behaviour in Plexiglas-bark sandwiches supported previous evidence that the interrupted chirp is used in both male rivairy and premating contexts. A complex sequence of behaviour was recorded from 11 pairs within the first 3 hr, and mating was recorded from two pairs. Attraction chirps were produced as the male burrowed through the frass. Males giving attraction chirps changed to interrupted chirps 1 to 14 min after con~cting the femaie. While continuously emitting interrupted chirps, each male jostled the female, striking her with his forelegs and biting her elytra. Scraping of the male’s mandibles across the tubercles on the female’s elytral declivity produced loud grating noises. Each female responded to a male biting and jostling by orienting her heavily sclerotised elytral declivity toward the male, and tilting so that the lower margin of her elytra protected her legs from the male’s mandibles. Females also repeatedly thrust strongly backwards, wedging in the tunnel against the pushing male between thrusts. Three females that had fed only 3 hr in the sandwich actually ejected the males. During the first hour of

1002

J. A. RUDINSKY ANDLEEC. RYKER

the other tests, periods of quiet occurred and slowly increased in frequency and duration between bouts of reciprocal pushing and male biting and emitting interrupted chirps. Gallery cooperation in the form of female boring and male removal of frass appeared within 10 to 85 min, during the quiet periods. After the male and female had initiated gallery cooperation, reoccurrences of biting or nudging and interrupted or courtship chirping (see below) usually appeared when the male returned to the female after a trip pushing frass out the entry hole. This pattern of behaviour can be explained if one assumes that the male undergoes olfactory disadaptation at the entrance and then exhibits renewed response to the pheromone concentration around the female. Uninterrupted ‘courtship’ chirp. Within 20 min before copulation (or after about 1.5 hr if mating was not observed), a new stridulation appeared, replacing the interrupted chirp from that time on in the gallery. The new chirp may be called a ‘courtship chirp’ as this type of chirp directly preceded copulation. Courtship chirps were simple (uninterrupted) chirps given with gentle nudging and biting of the female’s elytra (Fig. lg). To mate, the male turned around and gave simple chirps while backing up the gallery, bumping against the female, and revolving to the mating position (venter to venter). He became silent when the genitalia were engaged. Copulation lasted 4 to 6 min. Courtship chirps are quite distinct from the long chirps and interrupted chirps that precede them. They are similar to attractant chirps in duration (0.037 set), but have more toothstrikes (30.3) and therefore a faster toothstrike rate (792 ts/sec) (compare to Table 1A above and MICHAELand RUDINSKY,1972, Table 3). Abdominal motion during male stridulation. Observation and acoustic monitoring of males confined in the Plexiglas chamber showed that both the simple chirps and the interrupted chirps are produced by a single downward movement of the abdomen. The upper portion of the elytral file is contacted in simple chirps, and the lower portion in the interrupted chirp.

The mechanism for interrupting the abdominal movement is not known. Similar interrupted chirps are found in other species of Dendroctonus (RUDINSKY and MICHAEL,1974). DISCUSSION These data indicate that male communicative behaviour is very complex. Although we initially considered the jostling behaviour and interrupted chirps of the male in the gallery to be courtship behaviour, it became clear that the essence of this premating interaction is aggressive. In fact, the striking, biting, and chirping of the newly arrived male appeared identical to rivalry behaviour between two males. The initial response of the female was also aggressive as she pushed and crowded the male backwards toward the entry. The slow lessening of aggressive behaviour, the initiation of cooperation behaviour, and the change in male stridulation are indications of mutual habituation, which is necessary before mating can occur. This is a familiar pattern in the behaviour of nesting or territorial vertebrates (BASTOCK,1967, p. 6). Interrupted chirps emitted in the premating context may function to overcome the defense reflexes of the female and to release her mating behaviour, as suggested for grasshoppers by DUMORTIER (1963. p. 597). However, the existence of a distinctive courtship chirp that is not evoked by 3,2-MCH and frontalin, the commofi chemostimulus for all observed instances of interrupted chirp response, and the association between interrupted chirps and aggressive behaviour, also support the idea that interrupted chirps are part of the D. pseudotsugae male aggressive behaviour pattern, which appears in several contexts. Data on the chemostimulus of the interrupted chirp support previous reports on the multifunctionality of 3,2-MCH (RUDINSKY,1973a; RUDINSKYet al., 1974a). In reporting the presence of this substance in hindguts of male D. pseudotsugae, PITMAN and V~ti (1974) assumed that its only function was the well-estab-

Table 2. Walkway test response of Douglas-fir beetle male to various concentrations of 3.2-MCH Beetle response: mean 595% Chewstimulus

ArrestWY

confidence interval per 20 test males1/

Arrested with attraction chirp

Passed

Passed with interrupted chirp

Contro1Y a.7 + 1.4b

4.3 + 3.81

11.3 + 1.4b

0.0 + o.oa

Control + MCH (0.0002%)

18.3 + 1.4'

14.0 + 6.6b

1.7 21.4"

0.0 + 0.0"

Control + MCH (0.002%)

17.3 + 5.2'

12.0 + B.6b

2.7 + 5.2'

0.0 + 0.0"

Control + MCH (0.02%)

1.7 ?: 1.4"

0.0 + o.oa

18.3 + 1.4c

10.0 + 5.0b

Control + MCH (0.2%)

1.0 + 2.5'

0.0 + 0.0'

19.0 + 2.5'

15.7 + 8.0C

y

Three replications; n - 60.

2/ Letters indicate grouping of means by Scheff; test at PcO.05; a is smallest group, b is largest 3/ Control contains 0.01% resin, 0.01% frontalin in absolute ethanol.

Sound production in valytidae lished antiaggregative effect. PITMANet nl. (1975) questioned the concept of multifunctionality and suggested that reported increases in response were due to synergistic effects of the solvent ethanol, not 3,2-MCH. However, such a possibility is ruled out by the range of dilutions used, as was pointed out in the first report of multifunctionality (RUDINSKY, 1973a). In Tabie 2, synergism may occur with the attractants in the control, but we do not consider it possible that increases in the solvent by fractions of 1% would make a second synergism when the concentration is already 99+%. If a second synergism occurs with ethanol present, it must be due to changes in 3,2-MCH. Total pheromone concentrations from 0.01% to 0.001% can be differentially synergistic, because they represent ten-, hundred-, and thousandfold changes in substances released by the insects themselves. Moreover, the male beetles produced basically different types of stridulation when treated by very slightly ~n~trations of 3,2-MCH with frontalin (Table 2). Ethanol may be a host attractant with a synergistic effect like terpenes on the attractants, as PITMAN et al. (1975) suggest, but this possibility does not affect the acoustic data summarized in Tables 1 and 2. With Dfrontalis similar sonic changes in the parameters of the male chirp occurred with increases in verbenone and/or myrtenol (RUDINSKY, 1973b; RUDMSKY ef al., 1974b; RUDINSKY and MICHAEL,1974). With both species there appears to be genuine pheromonal multifunctionality as indicated by male sonic responses to varible chemostimulus by other males as well as females.

1003

KINZER G. W., FENTIMANA. F. JR., FOLTZ R. L., and RUDINSKYJ. A. (1971) Bark beetle attractants: 3-methyl-2-cyclohexen-l-one isolated from Dendroctonus pseudotsugae. J. econ. Ent. 64, 970-971. LIBBEY L. M., MORGAN M. E., PUTNAM T. B., and RUD~SKY J. A. (1976) Isomer of antia~re~t~ve pheromone identified from male Douglas-fir beetle: 3-methylcyclohex-Een-l-one. J. insect Physiol. In press. MICHAELR. R. and RUDINSKYJ. A. (1972) Sound production in Scolytidae: specificity in male Dendroctonus beetles. J. Insect Physiol. 18, 2189-2201. PITMANG. B., HEDDENR. L., and GARA R. I. (1975) Synergistic effects of ethyl alcohol on the aggregation of Dendroctonus pseud~t~gae (Cot., Scotytidae) in response to pheromones. Z. anqew. Ent. 78. 203-208. PI&AN G. B. and VI& J. P. (1974) Biosynthesis of methylcyclohexenone by male Douglas-fir beetle. Environ. Ent. 3, 886887. REID R. W. (1958) The behavior of the mountain pine beetle, Dendroctonus monticolae Hopk., during mating, egg laying, and gallery construction. Can. Ent. 90, 505-509.

RUDINSKYJ. A. (1969) Masking of the aggregation pheromone in Dendroctonus pseudotsugae Hopk. Science, Wash. 166, 884-885. RUDINSKYJ. A. (1973a) Multiple functions of the Douglasfir beetle pheromone 3-methyl-2-cyclohexen-l-one. Environ. Enf. 2, 579-585. RUDINSKYJ. A. (1973b) Muit~ple functions of the southern pine beetle pheromone verbenone. E~~~~on. Ent. 2, 511-514. RUDINSKYJ. A. and MICHAELR. R. (1972) Sound production in Scolytidae: chemostimulus of sonic signal by the Douglas-fir beetle. Science, Wash. 175, 1386-1390. RUDINSKYJ. A. and MICHAELR. R. (1973) Sound production in Scolytidae: stridulation by female Dendractonus beetles. J. Insect Physioi. 19, 689-705. ~~k~uwle~ement~We thank R. R. MICHAEL, Oregon RUDINSKYJ. A. and MICHAELR. R. (1974) Sound producState University, for assistance with electronic equipment tion in Scolytidae: ‘rivalry’ behaviour of male iendrocand for construction of the Plexiglas chamber; SUELLEN tanus beetles. J. Insect Phvsiol. 20, 1219-1230. VERNOFFfor statistical analyses; and P. T. OESTERfor techRUDINSKY3. A., MORGANMI, LIBBE~L. M., and MICHAEL nical help. This research was suooorted in Dart bv NSF R. R. (1973) Sound production in Scolytidae: 3-methyl-2grant No. BMS 72-02522 and tdd O.S.U. Sihool of Forcyclohexen-l-one released by female Douglas-fir beetle estry. This is O.S.U. Agricultural Experiment Station Techin response to male sonic signal. Environ. Ent. 2, nical Paper N. 4157. 505-509.

REFERENCES BAST~CKM. (1967) Courtship: an ~~~ologieal s&y. Aldine Publ. Co., Chicaga. DUMORTIER B. (1963) Ethological and physiological study of sound emissions in Arthropoda. In Acoustic Behavior of Animals (Ed. by BUSNEL,R. G.), pp. 583-654. Elsevier, New York.

RUDINSKYJ. A., MORGANM. E., LIBBEYL. M., and PUTNAMT. B. (1974a) Additional components of the Douglas-fir beetle (Cal.. Scolytidae) aggregative pheromone and their possible utility in pest control. Z. angew. Enc. 76, 65-77. RUDINSKYJ. A., MORGANM. E., LIBBEYL. M.. and PUTNAMT. B. (1974b) Antia~regative-rivalry pheromone of the mountain pine beetle and a new arrestant uf the southern pine beetle. Environ. Ent. 3, 90-98. RUDINSKYJ. A., MORGANM. E., LIBBEYL. M., and PUTNAM T. B. (1976) Release of frontalin by Douglas-fir beetle male. Z. anger. Ent. In press.