BEHAVIOR Reproductive Behavior of pallidovirens (Heteroptera: )

QIAO WANG1 AND JOCELYN G. MILLAR2 Department of Entomology, University of California, Riverside, CA 92521

Ann. Entomol. Soc. Am. 90(3): 380-388 (1997) ABSTRACT Reproductive behavior of the stink bug (Stal) was studied in the laboratory, including the determination of the sexual maturation period for both sexes, Downloaded from https://academic.oup.com/aesa/article/90/3/380/106886 by guest on 04 October 2021 the effects of the number of copulations on fecundity, and the function of prolonged copu- lation. Mean premating periods for males and females were 3.6 ± 0.5 and 4.1 ± 0.6 d, respectively. Preoviposition period averaged 6.6 ± 0.9 d. Females preferred sheltered, rough- textured oviposition sites. Paired adults mated =13 times on average during their lifespan under laboratory conditions. The mean duration of copulations involving virgin females was >17 h, significantly longer than that of subsequent copulations, which lasted between 6.9 and 8.3 h. Multiple matings significantly increased female fecundity. Virgin males donated =17% of their body mass to a virgin female during mating. Duration of mating may be regulated by space available in the spermatheca for ejaculatory material. There was strong evidence of male mate choice, with males terminating copulation with small females before sperm was trans- ferred. There was no indication of male mate-guarding behavior after copulation. Females almost always approached males from distances of >30 cm, suggesting that males attract females from a distance. Within 15 cm, males discern and approach females. Courtship be- haviors of males included antennation of female antennae and abdomens, stroking the abdo- mens with the hind legs, and periodic male abdominal vibrations. Possible functions of these behaviors are discussed.

KEY WORDS Pentatomidae, Thyanta pallidovirens, reproductive behavior, nuptial gift

THE NATIVE STINK bug Thyanta pallidovirens or more under optimal conditions (Schotzko and (Stal) is distributed through the far western states O'Keeffe 1990a). of the United States and into southern British Co- Aspects of the reproductive behavior of the Pen- lumbia (Schotzko and O'Keeffe 1990a). It was tatomidae have been studied by various authors identified as a major pest of nut crops in California (Borges et al. 1987; Kon et al. 1988; Todd 1989; in the mid-1980s. Damage to nuts caused by this McLain 1989, 1992; Drickamer and McPherson includes epicarp lesions, nut abortion and 1992). Reproductive behavior is often complex, kernel necrosis (Rice et al. 1985, 1988; Michailides with individuals of both sexes commonly mating et al. 1987). This insect is also a serious pest of many times (e.g. Kawada and Kitamura 1983, various legume and brassica crops (Schotzko and McLain et al. 1990), and large numbers of egg O'Keeffe 1990a, b). masses being oviposited over periods spanning sev- Adult bugs overwinter in orchard floor debris, eral months (e.g., Schotzko and O'Keeffe 1990a). under rocks, in soil cracks around the bases of host Mate location can involve chemical (Aldrich 1988) plants, or in other shielded places in and around and acoustic cues (Ota and Cold 1991), and there orchards (Rice et al. 1988). In California pistachio is evidence for some degree of mate choice by orchards, overwintering adults first appear in April both sexes (McLain 1980, 1989). or May, with adults of the 1st new generation In one of the few previous studies of T. palli- emerging in June or July. One or 2 additional over- dovirens reproductive behavior, Schotzko and lapping generations may develop during summer O'Keeffe (1990a) investigated ovipositional and and autumn. In the laboratory, this species will re- mating rhythms, and the variability in oviposition produce year-round under long-day conditions, S6s in response to different legume hosts. Because T. with the period from egg to adult averaging 25 pallidovirens is a serious pest of a variety of crops, d (vide infra), and adult longevity reaching 2 mo a better knowledge of its life history is needed. We report here the results of detailed investigations of the reproductive behavior in this species. In par- 'Current address: Department of Plant Science, Massey Uni- versity, Private Bag 11222, Palmerston North, New Zealand. ticular, our objectives were to observe and describe 2To whom correspondence should be addressed. T. pallidovirens reproductive behavior and behav-

0013-8746/97/0380-0388$02.00/0 © 1997 Entomological Society of America May 1997 WANG AND MILLAR: REPRODUCTIVE BEHAVIOR OF T. pallidovirens 381 ioral sequences; to measure parameters associated To determine the premating period for each sex with reproduction in this species, including sexual separately, 2 combinations of pairings were per- maturation periods, effects of single versus multi- formed: (1) newly emerged males were paired with ple matings on fecundity and egg hatch rates, the 10-d-old virgin females (n = 21), (2) newly duration and number of copulations, the number emerged females were paired with 10-d-old virgin of egg masses oviposited and the period between males (n = 19). sequential oviposition bouts; and to investigate the To determine preoviposition periods, 31 pairs of extent and effects of male investment in reproduc- freshly emerged were set up in individual tion in this species. containers. The time between emergence and the 1st copulation was recorded as the premating pe- riod, and that between emergence and 1st ovipo- Materials and Methods sition as the preoviposition period. Daily Mating Rhythms. Forty-one 10-d-old Downloaded from https://academic.oup.com/aesa/article/90/3/380/106886 by guest on 04 October 2021 Insects. In early August 1994, a laboratory col- pairs (1 pair per tube) were set up in 150-ml open- ony of T. pallidovirens was started from adults (31 ended plastic tubes with each end covered with males and 50 females) and nymphs (22 individuals) window screen, and observed hourly for 3 d, start- collected from alfalfa fields near Davis, CA, and ing 4 h into the photophase of the 1st observation from specimens obtained from a laboratory colony day. A piece of filter paper (6 by 2 cm), several (5 males, 7 females, and 30 nymphs) maintained sunflower seeds and half a green bean were placed at the Department of Entomology, University of in each half of the chamber. The number of cop- California, Davis. Insects were reared at 27 ± 1°C, ulating pairs was recorded hourly. 60 ± 10% RH, and a photoperiod of 16:8 (L:D) Effect of Single Versus Multiple Matings on h, with lighting provided by fluorescent lights. Fecundity. To determine whether the number of Food for nymphs and adults consisted of thor- matings influenced egg hatch rates and total num- oughly washed green beans from a supermarket, ber of eggs oviposited, bugs were set up in the organically grown sunflower seeds, and alfalfa cut- following 4 treatments, using 150-ml plastic tubes tings from an alfalfa plot in University of California as described above: (1) paired treatment. Thirty- Riverside Agricultural Operations. Green beans one pairs were kept in individual tubes for their and alfalfa were replaced every other day. All in- entire lifespans. The total number of matings per sects used for experiments were obtained from off- pair was determined by observing pairs thrice daily spring of the initial colony. (=1200 hours, 1700 hours, and just before lights The breeding colony was maintained in 2 wood- out each day). Because copulations are initiated en cages (76 by 43 by 43 cm), each containing 70- during the photophase, and because copulations 80 adults of both sexes. New adults were added to last for a number of hours (see Results), copula- the breeding cages every other day. Egg masses tions could be accurately tallied from these 3 ob- were collected from breeding cages every other servations. (2) Mated once treatment. Twenty- day, and kept in plastic petri dishes (10 by 1.5 cm), three pairs were placed in individual tubes and with 4 egg batches per dish. Upon hatching, males were removed immediately after the 1st nymphs were provided with fresh green beans and mating. (3) Mated 3 times treatment. Twenty-three sunflower seeds every other day. When nymphs females were allowed to mate 3 times with the reached the 2nd instar, they were transferred to same male at 5-d intervals in individual tubes, fol- cylindrical paper ice cream cartons (15 by 12.5 cm) lowed by permanent removal of the males. (4) Un- with window screen lids (^lOO nymphs per car- mated treatment. Twenty virgin females were ton). After the final molt, the adults were segre- maintained in tubes individually. gated by sex for use in experiments or added back For all treatments, egg masses were collected 3 to the breeding colony. times daily for the duration of the experiment and Sexual Maturation and Preoviposition Peri- incubated until hatching in individual petri dishes ods. Freshly emerged pairs (1 male, 1 female) of (10 by 1.5 cm diameter). The total number of eggs insects were set up in individual mating containers per mass and the number of eggs per mass that constructed from two 150-ml plastic tubes. One did not hatch were counted to determine hatch end of each tube was covered with window screen rates. and the open ends were fastened together with Male Investment in Reproduction. To examine transparent adhesive tape, and a piece of paper the relationship between the duration of copula- was used to separate the 2 halves of the chamber. tion and the amount of semen transferred by the A piece of filter paper (6 by 2 cm), several sun- male to the female during mating, body weights of flower seeds and half a green bean were placed in both sexes were measured before and after pro- each half of the chamber. A male was placed in longed 1st mating (>10 h; n = 16) or before and one end of each chamber, and a female in the oth- after short 1st mating (<10 h; n = 11. If pairs did er. The separating papers were removed daily for not separate naturally at 10 h after mating, the 10 h during the scotophase. The separating papers pairs were artificially separated. Body weights of were removed permanently after the 1st mating control groups of virgin bugs of each sex (n = 25), occurred for each pair. with each bug held separately, were measured be- 382 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 90, no. 3 fore and after the experiment. Sunflower seeds were provided for all insects in treatments and —•—* d mating pilra controls. -•—ffolmaUnss The relationship between the duration of mating Mttatedatlkmx and the amount of semen transferred by males also was determined by measurement of the dilation of the spermatheca after mating. Females were dis- sected and the length and width of spermathecae were measured with a stereo microscope. Mea- surements were recorded for sexually mature vir- gins (n = 14), individuals immediately after pro- longed first mating (>10 h, n = 14), individuals 16

10 Downloaded from https://academic.oup.com/aesa/article/90/3/380/106886 by guest on 04 October 2021 immediately after short 1st mating (<10 h, n = Time since lights on 11), and individuals immediately after starting 2nd mating but before 1st opposition (having mated Fig. 1. Orcadian mating rhythms of 41 pairs of sex- >10 h in the 1st mating, n = 9). ually mature T. pallidovirens recorded at hourly intervals To determine whether previous experience af- for 3 d. Lights were on from 0000 to 1600 hours and off fected mating duration, the following 2 additional from 1600 to 2400 hours. treatments were set up: (1) 15 experienced males (3 d after previous mating) and 15 virgin females 20 green beans, 2 bouquets of fresh alfalfa cut- were maintained as individual pairs; (2) 15 expe- tings, each in a plastic bottle with fresh water, 10 rienced females (3 d after previous mating) and 15 pieces of crumpled filter paper (12.5 cm diameter) virgin males were maintained as individual pairs. and 10 pieces of crumpled paper towel (15 by 16 Duration of the 1st mating between each pair was cm) provided. Eggs were collected daily and recorded. counted for 15 d, and green beans and alfalfa cut- All virgin insects used in these experiments were tings were replaced every other day. Observations 10 d after the final molt, and all experienced in- of oviposition behavior were made during the sco- sects were 13 d old. Eggs were collected 3 times tophase for 3 d. Female behaviors, such as walking, daily, and the number of eggs per mass and the sampling the substrate with appendages, and ovi- hatch rates were determined as described above. positing were recorded. The times taken to ovi- Observations of Mating Behavior. Four co- posit and the resting times after oviposition also horts were used for observations of mating behav- were recorded. ior: (1) Ten-day-old adults were obtained from a mixed-sex colony reared under the standard Results conditions described above, and were presumed to have mated, and females to have oviposited. Three Mating and Fecundity. Both sexes required a treatments were set up: 5 females + 2 males, 5 sexual maturation period after the final molt to males + 2 females, and 5 males + 5 females. (2) adult. Premating periods were significantly longer Three groups with the same combinations of adults for females (Mann-Whitney rank sum test, P = as treatment 1 were set up, but with all adult, 10-d- 0.04), ranging from 3.2 to 5.4 d (mean ± 1 SD = old virgins. (3) Six virgin males and 6 virgin fe- 4.1 ± 0.6 d; n = 19) for females and 2.5 to 4.2 d males, all 10 d old, were mixed and observed. (4) (3.6 ± 0.5 d; n = 21) for males. The total preovi- Ten females immediately after prolonged 1st mat- position period ranged from 4.1 to 7.6 d (mean = ing (>10 h) and 10 virgin males at 10 d of age 6.6 ± 0.9 d; n = 31). Paired adults mated an av- were paired. erage of 13.1 ± 4.6 times (range, 3-22 times; n = Each group in treatments 1 and 2 was main- 31) over their life spans, and females mated a tained in a transparent plastic box (18 by 7.5 cm, mean of 1.6 ± 0.7 times (range, 1-3; n = 31) be- Fisher, Pittsburgh, PA). Insects in treatment 3 fore the 1st eggs were oviposited. were held in a wooden cage (76 by 43 by 43 cm) Mating pairs were observed during both the fitted with a glass top. Each pair of insects in treat- photophase and scotophase because of the long ment 4 was maintained in a 150-ml plastic tube duration of each copulation. Copulation was initi- with screened ends. Treatment 1 was observed for ated only during the photophase (Fig. 1), but pairs 10 h/d for 6 d, making observations between 0800 that began mating during the photophase re- and 1800 hours. Treatments 2 and 3 were observed mained coupled into, and sometimes through, sco- for 5 and 6 d, respectively, for 10 h/d. Treatment tophase. Initiation of copulation followed a bimod- 4 was observed for a total of 30 h. Fresh virgin al cycle, with the numbers of pairs initiating insects were used for each day of observation in copulation peaking 4 and 10 h into photophase treatments 2 and 3. (Fig. 1). Observations of Oviposition Behavior. Ovi- Periods between consecutive copulations for the position behaviors were studied with 20 sexually first 10 copulations are shown in Fig. 2. A clear mature pairs held in a wooden cage (79 by 43 by trend was not observed for the first 8 copulations, 43 cm) fitted with a glass top, with sunflower seeds, with the period between matings varying from 34.8 May 1997 WANG AND MILLAR: REPRODUCTIVE BEHAVIOR OF T. pallidovirens 383

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Periods between consecutive mating events Periods between consecutive oviposition events Fig. 2. Periods between successive matings for pairs Fig. 3. Periods between successive ovipositions by fe- of T! pallidovirens, for the first 10 matings. The experi- male T. pallidovirens in caged pairs. The experiment ment started with 31 pairs and ended with 23 because of started with 31 pairs and ended with 23 because of the the deaths of one or both insects of 8 pairs during the deaths of one or both insects of 8 pairs during the ex- experiment. Bars surmounted by different letters are sig- periment. Bars surmounted by different letters are sig- nificantly different (Analysis of variance, (ANOVA) fol- nificantly different (Kruskal-Wallis ANOVA on ranks, fol- lowed by Student-Neuman-Kuels test for separation of lowed by Dunn's all pairwise multiple comparison means; P < 0.05). procedure for separation of means, P < 0.05 [Jandel 1992]). to 57.3 h. From the 8th to the 10th matings, the interval between copulations increased to 85.6 h. Fecundity and the total hatch rate of eggs were Intervals between mating and opposition averaged affected by the number of copulations (Table 1). 23.0 ± 12.1 h (n = 22) and 28.7 ± 15.6 h (n = A significant difference was not detected between 22) for the 1st and 2nd copulations, respectively. continuously paired females and females mated 3 Intervals between consecutive ovipositions are times when comparing mean number of eggs ovi- shown in Fig. 3. The periods were significantly lon- posited per female and mean egg hatch rate. How- ger (45.5-58.6 h) at the beginning and end of the ever, both the number and the hatch rate of eggs experiment than the 5 middle periods between the oviposited by females mated only once were sig- 3rd and the 8th ovipositions (29.1-35.9 h). Inter- nificantly less than those for continuously paired vals between the 3rd and 8th ovipositions were not females (and females mated 3 times). Females that significantly different from each other. mated only once did not produce significantly The duration of the first 10 copulations ranged more eggs than virgin females (t = 1.20, df = 41, from 3 to 28 h (mean = 8.77 ± 4.44 h; n = 295; P > 0.1). A more detailed analysis of data from Fig. 4). Copulation duration was strongly affected females mated only once showed that hatch rate by previous experience, with the first copulation between virgin insects being significantly longer (18.61 ± 3.27 h; n = 31) than that of the next 9 copulations. These varied between 6.86 and 8.32 h in duration, and were not significantly different from each other (Fig. 4). To investigate the significance of the duration of mating in more detail, experiments were conduct- ed by pairing virgin and experienced individuals. The mean duration of the 1st copulation between experienced males and virgin females (mean = 17.07 ± 2.05 h; n = 15) was significantly longer than that between experienced females and virgin males (mean = 7.13 ± 1.77 h; n = 15) (f-test, t = 111111111 14.21; df = 28; P < 0.0001). Furthermore, the du- 1st 2nd 3rd 4th 5th 6th 7th 8th 9th 10th ration of mating between experienced males and Copulation number virgin females was the same as that recorded for Fig. 4. Duration of the first 10 copulations for caged the previous experiment with virgin males and fe- pairs of T. pallidovirens. The experiment started with 31 males (mean = 18.61 ± 3.27 h; n = 31; t = 1.67, pairs and ended with 23 because of the deaths of one or df = 44, P > 0.05). Also, the duration of mating both insects of 8 pairs during the experiment. Bars sur- between experienced females and virgin males was mounted by different letters are significantly different the same as that found in the previous experiment (Kruskal-Wallis ANOVA on ranks, followed by Dunn's all between multiply mated adults (6.86-8.32 h, Fig. pairwise multiple comparison procedure for separation of 4). means; P < 0.05 [Jandel 1992]). 384 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 90, no. 3

Table 1. Effect of the number of copulations on T. pallidovirens lifetime fecundity

Mean ± SD Mean hatch Treatment eggs/female rate, % Paired 31 500.13 ± 192.95a 93.6 ± 5.37a Mated 3 times 23 462.52 ± 139.50a 91.4 ± 5.89a Mated 1 time 23 335.30 ± 148.38b 50.8 ± 22.86b Virgin 20 283.35 ± 134.07b 0

Means followed by the same letter are not significantly different (Student— Neuman—Keuls all pairwise multiple comparisons pro- cedure, P < 0.05).

>10 hr <10 hr virgin controls Downloaded from https://academic.oup.com/aesa/article/90/3/380/106886 by guest on 04 October 2021

Mating duration decreased approximately exponentially in succes- sive egg masses (Fig. 5). Fig. 6. Changes in body weights of virgin male and Body weights of virgin males and females par- female T. pallidovirens after long (>10 h) or short (<10 ticipating in long 1st copulations changed dramat- h) 1st copulations. Different letters indicate significant differences between cohorts (ANOVA, followed by Stu- ically during mating (Fig. 6), indicating that sperm dent-Neuman-Kuels test for separation of means; P < and associated materials transferred during copu- 0.05). lation constituted a significant proportion of male body weight. This outcome also demonstrated a substantial investment in reproduction by males. hatch rates greater than 90% for multiply mated Mean body weights of these sexually mature virgin females (Table 1). males and females before mating were 61.8 ± 8.0 Spermathecae from females in different states and 75.1 ± 13.8 mg, respectively. Immediately af- of mating are illustrated in Fig. 7. The spermathe- ter the prolonged 1st copulation (range, 14.4-26 h; ca of a virgin female was a long narrow ovoid, with mean = 18.9 h), mean male weight decreased by a sclerotized medial rod (Fig. 7A). A partially di- 10.79 mg (17.5% of virgin male mass) whereas lated spermatheca taken from a female mated for mean female weight increased by 8.94 mg (11.9% <10 h is shown in Fig. 7B. A spermatheca taken of virgin female mass). The mean body weights of from a female immediately after prolonged 1st control virgin insects of both sexes held for 20 h mating was widely dilated (Fig. 7C), and unlike the changed only slightly (<2 mg). spermathecae from virgin females or females mat- The mean initial body weights (46.2 ± 6.4 mg; ed for <10 h, the end of the sclerotized rod blocks n = 8) of females involved in short 1st copulations the spermathecal opening. Spermathecae taken (3.4-10 h; mean = 6.6 h) were significantly less from females >24 h after the 1st mating were in- than those of the long-mating females described termediate between those of recently mated and above (75.1 ± 13.8 mg; t-test, P < 7 X 106). The virgin females. mean weights of both sexes immediately after short Oviposition Behavior. Before ovipositing, fe- 1st copulations changed only slightly (<2 mg). males actively searched for an appropriate ovipo- Furthermore, eggs from females with a short 1st sition site by walking on a substrate and frequently copulation did not hatch, in comparison to the sampling the substrate with their antennae, pro- 50.8% mean lifetime hatch rate of eggs from fe- boscis, and tarsi. When a satisfactory site was lo- males with a single prolonged 1st copulation and cated, females commenced ovipositing, laying eggs

7 8 9 10 11 12 13 14 15 16 17 18 egg mass number Fig. 5. Change in the egg hatch rate for successive egg masses oviposited by female T. pallidovirens mated only once. May 1997 WANG AND MILLAR: REPRODUCTIVE BEHAVIOR OF T. pallidovirens 385

DSP DSP DSP

SD Downloaded from https://academic.oup.com/aesa/article/90/3/380/106886 by guest on 04 October 2021

ABC Fig. 7. Spermathecae from female T. pallidovirens. (A) Spermatheca of virgin female. (B) Partially dilated sper- matheca from a female mated for <10 h. (C) Fully dilated spermatheca from a female immediately after prolonged 1st mating. DSP, dilation of spermatheca; SD, spermathecal duct; SP, spermathecal pump; SR, sclerotized rod (terms after Rider and Chapin 1991). transversely from left and right in rows to form a oviposited in the 2nd and 3rd egg masses. Egg regularly ranked egg mass. Oviposition duration numbers were not significantly different for the 1st was 50-182 min (mean = 95.8 ± 34.6 min; n = and 4th-8th egg masses, but began to decline for 23), depending on the number of eggs in an egg the 9th and 10th egg masses. mass. After ovipositing, the female rested beside Mating Behavior. The period between 1st con- the egg mass for 10-66 min (mean = 34.7 ± 14.5 tact and initiation of copulation for virgin sexually min; n = 23). mature insects varied between 6 and 45 min (mean During the 15 d that oviposition behavior of 20 = 16.5 ± 9.5 min, n = 22). For experienced adults, females was observed, more egg masses were ovi- this period was even more variable. Of 39 matings posited on alfalfa leaves (109) than on other sub- observed between experienced individuals, only 5 strates (container wall, 6; filter paper, 42; paper copulations were initiated in <36 min (mean = towel, 49). Of the 109 egg masses on alfalfa, 71 17.4 ± 12.7 min); in most cases (n = 23), the were on the undersides of leaves and 38 on the courtship period ranged from 150 min to 20 h upper side. No eggs were oviposited on either (mean = 404.8 ± 261.7 min). During these ex- green beans or sunflower seeds. tended periods, the male remained in proximity to Numbers of eggs in the first 10 egg masses are the female, periodically contacting her. When shown in Fig. 8. The largest numbers of eggs were freshly mated females were caged with sexually mature virgin males, only 1 out of 10 pairs initiated copulation within 8 h, and no mating occurred in the other 9 pairs within 30 h, indicating that freshly mated females were refractory to further mating attempts. Courtship and mating behavior of T. pallidovi- rens involved a sequence of courtship steps, with- out which females would not submit to copulation. Mating behavior was initiated by a female ap- proaching a stationary male. Antennation often ac- companied the directed movement toward the male. At an intermediate distance from the male (5-15 cm, n = 102), the responses to the opposite 1st 2nd 3rd 4th Sth 6th 7th 8th 9th 10th gender followed 1 of 2 patterns. Usually the ap- Oviposition bout number proaching female (91/102 cases) slowed or stopped Fig. 8. Number of eggs in the first 10 egg masses moving toward the male, her antennae waved oviposited by female T. pallidovirens continuously paired slowly, and the male approached her, periodically with a male. The experiment started with 31 pairs and vibrating his abdomen up and down while also ended with 23 because of the deaths of one or both in- slowly antennating. The male abdominal vibration sects of 8 pairs during the experiment. Bars surmounted period ranged from 2.34 to 3.23 s (mean = 2.95 by different letters are significantly different (Kruskal- Wallis ANOVA on ranks, followed by Bonferroni's meth- ± 0.27 s; n = 20), with intervals between vibration od for separation of means; P < 0.05 [Jandel 1992]). periods averaging 2.62 ± 0.62 s (range, 1.74-3.42 386 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 90, no. 3 s; n = 20). In a few cases (n = 11), the female by a 2nd male never resulted in a breakup of the approached the male quite rapidly. mating pair. Upon 1st contact, the sexes responded in 1 of 3 During mating, even when not disturbed, the ways. In a few cases (n = 6), both sexes moved female occasionally walked a short distance while away from each other with no attempt at further coupled with her mate. While coupled, the male courtship. More frequently (n = 107), the female rapidly stroked the female's ventral abdomen at in- continued walking slowly with no apparent re- tervals with his hind legs. After mating, both in- sponse to the male, and the male followed her, sects rested nearby for a period before moving antennating the abdomen. He also continued the away to forage. Females did not begin ovipositing periodic abdominal vibrations. In a minority of until at least 1 hour after mating (delay after 1st cases (24/107 females), the female held her abdo- mating, 23.0 ± 12.1 h, range 10-54 h, n = 22; delay after 2nd mating, 28.7 ± 15.6 h, range 1-50 h, n men low, thus denying the male access to her gen- Downloaded from https://academic.oup.com/aesa/article/90/3/380/106886 by guest on 04 October 2021 italia. The remainder of the mating encounters (83/ = 22). 107) eventually resulted in successful copulation. Female-female encounters (n = 48) were non- In these cases, both sexes antennated the others' aggressive, with both insects retreating with no antennae, followed by the male moving around the specific response after brief antennal contact. female while continuing to vibrate his abdomen at Male-male encounters (n = 31) had 4 outcomes. intervals and antennating her abdomen. After sev- In about half the cases (n = 16), both retreated eral minutes of courtship, the male commenced without specific response upon touching. In 3 stroking the ventral surface of the female's abdo- cases, males fought briefly by lashing with the an- men until she raised her abdomen, at which point tennae and kicking with the hind legs. Fighting the male quickly turned and attempted to couple lasted <10 s. In 9 cases, 1 male exhibited behavior his genitalia with hers. Successful coupling in lin- typically associated with courtship toward the oth- ear, end-to-end fashion resulted in copulation. If er, including antennation of the antennae and the the male was not successful in coupling with the abdomen, abdominal vibration, and extrusion of female after several attempts, the abdominal strok- the genitalia in an attempt to couple with the 2nd ing sequence was repeated and was followed by male. The latter male usually submitted passively further coupling attempts. If the female persisted to the attentions of the courting male, and even- in maintaining her abdomen in a lowered unrecep- tually moved away. These behaviors lasted 4-13 tive position, the male eventually abandoned min (mean = 6.8 ± 2.5 min). Finally, in 3 cases, 2 courtship attempts. males vigorously displayed courtship behaviors to each other for considerable periods (8.5, 14.2, and Thus, female rejection of male courtship con- 19 min). This homosexual behavior occurred even sists of ignoring male courtship attempts and mov- though females were present. ing away, or of refusing to adopt a receptive stance. Females were never receptive to male courtship during or immediately after opposition, although Discussion males often loitered near ovipositing females and attempted courtship once opposition was com- Mating Rhythms. We found the circadian mat- plete. ing pattern of T. pallidovirens to be similar to that reported by Schotzko and O'Keeffe (1990a). Our In 13 observed cases, a 2nd male exhibited ab- results indicated that the number of pairs in copulo dominal vibrations characteristic of courtship and peaked «5 h into the photophase, then gradually apparently attempted to compete with a male al- decreased to a minimum at the end of the scoto- ready courting a female. In 1 case, this led to a phase. In our study, initiation of copulation was brief skirmish between the males. In other cases, bimodal, with a large peak 4 h into the photophase, 1 of the 2 males either retreated (n = 5) while the and a smaller peak in midafternoon, ^lO h into other continued courting or both males courted the photophase. the female simultaneously (n = 8) until she ac- Investment in Reproduction. Prolonged mat- cepted one. Once the female coupled with 1 of the ing in the Heteroptera has usually been explained 2 males, the superfluous male briefly continued his as male mate-guarding to reduce sperm displace- courtship behavior before breaking off the encoun- ment upon reinsemination of the female (e.g., ter. McLain 1980, 1989; Sill6n-Tullberg 1981; Carroll Because a pair can remain coupled for many and Loye 1990; Carroll 1991; Alcock 1994). Our hours, one or more single males frequently ap- experiments with T. pallidovirens, while supporting proached the mating pair. These males would ex- this general interpretation, revealed some intrigu- hibit typical courting behavior, including abdomi- ing details. Copulation in T. pallidovirens is always nal vibration and antennation of the female, or protracted, but minimal sperm or associated nu- attempted to grasp the females genitalia. If the trients appear to be transferred during the first courting male accidently touched the mating male, hours of a copulation. Furthermore, females par- the latter responded by kicking with his hind legs. ticipating in unsuccessful first copulations (<10 h) When disturbed, a mating female sometimes were smaller than those which remained in copulo moved away, pulling her mate. Courtship attempts for >10 h, and eggs of these small females did not May 1997 WANG AND MILLAR: REPRODUCTIVE BEHAVIOR OF T. pallidovirens 387 hatch. These observations suggest male mate into eggs for the 1st egg mass. Although females choice, with males preferentially investing sperm receive a large volume of sperm and nutrients from and nutrients in large virgin females. There are the 1st copulation, the decreased lifetime egg pro- several possible explanations for the prolonged 1st duction and declining hatch rate for females mated copulation. From an evolutionary perspective, be- only once indicate that female reproductive suc- cause investment in reproduction is substantial for cess is optimized by multiple copulations. How- both sexes, the 1st copulation may include a period ever, because less semen in transferred to experi- of prolonged mutual assessment, with consumma- enced females, mate choice by females should be tion only after partners are deemed mutually ac- most pronounced for virgin females. Furthermore, ceptable. Subsequent matings are shorter and although females continuously paired with males equal in duration, indicating that males distinguish mated on average >13 times, their lifetime egg experienced from virgin females, but not the num- production and egg hatch rates were equal to those ber of times that females have mated. Thus, male of females mated only 3 times. Because females Downloaded from https://academic.oup.com/aesa/article/90/3/380/106886 by guest on 04 October 2021 reproductive success is optimized by transferring feed while copulating, the costs of the 10 extra, sperm and nutrients to virgins because the nutri- apparently superfluous copulations may be out- ents help the female to develop large clutches of weighed by the genetic diversity in offspring eggs, most of which probably will be fertilized by gained by multiple mating. sperm of the 1st male. Conversely, males allocate Courtship and Mating Behavior. T. pallidovi- primarily sperm to experienced females where rens mating behaviors were similar to those re- their confidence of paternity is decreased. ported for other pentatomid species (Gamboa and Furthermore, the large quantity of semen trans- Alcock 1973, Lanigan and Barrows 1977, Kawada ferred to a virgin female may fill the spermatheca, and Kitamura 1983, Borges et al. 1987, Todd reducing the amount that can be transferred in 1989). Observations suggested that T. pallidovirens subsequent copulations. This strategy has been re- males attract females from a distance, but it is not ported in other stinkbugs (N. viridula, McLain yet clear whether attraction is caused by one or a 1985) in which the percentage of eggs fertilized by combination of visual, chemical, acoustic, or other sperm of the 1st male is correlated with male size types of signals. At close range, roles were re- (i.e., the size of the ejaculate). Female preference versed, with males following and antennating fe- for large males also occurs in the stinkbug Acro- males while periodically vibrating their abdomens. sternum hilare Say (Capone 1995). Male body vibration is common in bug courting From a nonevolutionary perspective, there may behavior, and it may serve to transmit vibratory or be physiological reasons for prolonged 1st mating. acoustic signals through the substrate (Fish and Al- For example, females may require tactile or hor- cock 1973, Kon et al. 1988, Ota and Cokl 1991), monal cues to complete copulation or there may or aid in the dispersal of male-produced phero- be a physical barrier to insemination. For the male, mones towards the female. the large volume of semen may need to be mobi- Males often approached and courted a copulat- lized from some storage form. An analogous situ- ing female (despite it never resulting in the break- ation occurs with male katydids, which require up of the mating pair) while ignoring single fe- considerable time to form and pass a large sper- males nearby. Male courtship of other males and matophore (Gwynne 1986). even mutual courtship displays between 2 males Recently mated T. pallidovirens females were also occurred. This suggests that short-range court- refractory to further mating attempts, and addi- ship cues may be similar between males and fe- tional sperm transfer may be largely prevented by males; males and females are certainly similar in the sclerotdzed rod blocking the spermathecal color and morphology. This male-male courtship opening (Fig. 8C). Thus, there is no benefit to may occur as a result of males being easily stimu- males to mate-guard after copulation, and a poten- lated to sexual arousal, whereby the cost of the tial cost in terms of missed mating opportunities occasional mistaken courtship of a male is out- and lost feeding time, particularly for males that weighed by the benefit accruing from aggressive have transferred a large fraction of their body mass courtship of all possible mates. to virgin females. Females of several Heteropteran species obtain Acknowledgments nutrients as well as sperm from males during cop- ulation (Mitchell and Mau 1969, Mau and Mitchell We thank J. Stephen McElfresh for technical assis- 1978, Kon et al. 1993), often in the form of large, tance. We also thank John Alcock, John Pinto, Larry Hanks, and Rick Redak for reviewing drafts of the manu- nonfertilizing sperm (Schrader 1960, Kasule 1986). script. We are grateful to the California Pistachio Com- For female T. pallidovirens, shrinking of the sper- mission for financialsuppor t of this work. matheca during the 1st preovipositdon period in- dicated absorbtion of semen nutrients. The benefit from these nutrients was manifested in the 2nd References Cited and 3rd rather than the 1st egg batch because the Alcock, J. 1994. Postinsemination associations be- short interval between copulation and oviposition SB tween males and females in insects: the male-guard- ( 24 h) precluded the conversion of the nutrients ing hypothesis. Annu. Rev. Entomol. 39: 1-21. 388 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 90, no. 3

Aldrich, J. R. 1988. Chemical ecology of the Heter- McLain, D. K. 1980. Female choice and the adaptive optera. Annu. Rev. Entomol. 33: 211-238. significance of prolonged copulation in Nezara viri- Borges, M., P. C. Jepson, and P. E. Howse. 1987. dula (: Pentatomidae). Psyche 87: 325-326. Long-range mate location and close-range courtship 1985. Male size, sperm competition, and the intensity behaviour of the green stink bug, Nezara viridula and of sexual selection in the southern green stink bug, its mediation by sex pheromones. Entomol. Exp. Nezara viridula (Hemiptera: Pentatomidae). Ann. En- Appl. 44: 205-212. tomol. Soc. Am. 78: 86-89. Capone, T. A. 1995. Mutual preference for large 1989. Prolonged copulation as a post-insemination mates in green stink bugs, Acrosternum hilare (He- guarding tactic in a natural population of the ragwort miptera: Pentatomidae). Anim. Behav. 49: 1335-1344. seed bug. Anim. Behav. 38: 659-664. Carroll, S. P. 1991. The adaptive significance of mate 1992. 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