JOURNAL
OF INVERTEBRATE
PATHOLOGY
58,
82-87 (1991)
Metacestode-Induced Depression of the Production Response to, Sex Pheromone in the Intermediate Tenebrio molitor
of, and Host
HILARY HURDAND G. PARRY Parasitology
Research Laboratory,
Department of Biological Sciences, University of Keele. Keeie ST5 5BG, United Kingdom
Received June5, 1990; accepted October 3, 1990 Hymenolepis diminuta infection of Tenebrio molitor is associated with an impairment of vitellogenesis and a reduction in host fecundity. In this communication the effect of infection upon an additional aspect of host reproduction, the initiation of mating behavior, has been examined. Copulatory release pheromone, extracted from control virgin females 6-7 days old. was shown to stimulate a positive mating response in 88% of 5- to 6-day-old control males; however, only a 56% response was elicited by pheromone from infected females. In addition, parasitization adversely effected male response to pheromone from control females. A significant (P < 0.001) depression of copulatory response occurred in infected 6- to ‘I-dav-old males (age of peak response) although this ility that an endocrine interaction between effect was not sustained in older beetles. The p metacestodes and host may mediate these effel ts is discussed in the light of our knowledge of the role of host juvenile hormone in controlling bo.h pheromone production and vitellogenesis in T. molitor. 0 1991 Academic Press, Inc. KEY WORDS: Hymenolepis diminuta; Tenebrio molitor: Cestoda; Coleoptera; host fecundity; host sex pheromone; host mating behavior.
An additional role of juvenile hormone is its regulation of sex pheromone production in T. molitor (Menon, 1970, 1976; Menon & Nair, 1970, 1976). This communication describes the use of a biological assay designed to determine whether H. diminuta infection of T. molitor results in disturbance of an additional endocrine-controlled event, that of sex pheromone production by female beetles. A comparison has also been made of the response of male infected and uninfected beetles to female sex pheromone. Any reduction in production of, or response to, sex pheromone may be an additional factor contributing to the reduction of host reproductive output observed in the association.
INTRODUCTION
A reduction in host fecundity is known to occur in associations between metacestodes of Hymenolepis diminuta, the rat tapeworm, and its coleopteran intermediate hosts Tribolium cot&sum (Keymer, 1980, 1981) and Tenebrio molitor (Hurd and Arme, 1986b). This may in part be attributed to a perturbance in events associated with egg production which has been observed in T. molitor 12 days or more postinfection. These include a reduction in fat body synthesis of vitellogenin, the development of patency in the follicular epithelium, and the sequestration of vitellogenin by developing oocytes (see review by Hurd, 1990a). In common with the majority of insects, vitellogenesis in T. molitor is regulated via fluctuations in juvenile hormone titer (Laverdure, 1967) and it has been suggested that metacestodes may interact with the host endocrine system to downregulate the events listed above (Hurd, 1990b).
MATERIALS
Maintenance of Parasite and Host T. molitor pupae were collected weekly from a stock colony, sexed (Bhattacharya et al., 1970), and emergents separated daily. Pupae and adults were segregated ac82
0022-201 l/91 $1.50 Copyright 8 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
AND METHODS
METACESTODES
AND
HOST
83
PHEROMONE
cording to sex and age. All insects used in this study were maintained in plastic, paper-lined Petri dishes in the same incubator at 26” 2 1°C and 60-80% relative humidity. H. diminuta eggs were collected from feces of infected Wistar rats by a salt flotation method, washed, and stored at 4°C for a maximum of 4 weeks. Adult beetles were starved for up to 2 days postemergence and then exposed to a mixture of parasite eggs and apple pulp. Control insects were starved then fed on apple pulp alone. Thereafter, insects were kept in groups of 20-30 and fed on bran. All male T. molitor exposed to H. diminuta eggs were dissected after testing and uninfected individuals removed from the study. No beetles were tested more than once.
and all experiments were performed tween 1400 and 1600.
Bioassay
Investigations
Pheromone extraction was performed by immersing 6- to 7-day-old virgin females in 100% ethanol (1 ml/mg fresh wt) for 2 hr, with initial mixing on a vortex. A pilot study revealed no significant difference (P = 0.05) in male response to pheromone extracts stored for 2 weeks at -20°C. The majority of tests reported in this communication were performed on fresh extracts; however, some extracts were stored at -20°C for a maximum of 2 weeks prior to use. Pieces of glass rod, 4 mm in diameter, were stored in ethanol. Immediately before use they were air dried, immersed in 200 ~1 of pheromone extract for 30 set, air dried, and then anchored in a test arena with a piece of plasticine. Male virgin beetles were placed individually on clean “Benchkot” under an inverted Petri dish base and allowed to acclimatize for 5 min before the introduction of a pheromone-coated glass rod. A positive response was recorded when the beetle attempted to mount the rod, bent the tip of the abdomen downward, and extended the genetalia to touch the glass rod (Fig. I). Any beetles failing to respond after 15 min were recorded as negative. The time intervals of all procedures were kept constant
(1) The effect of infection upon production of pheromone by female beetles was investigated by measurement of the response of 5- to 6-day-old control males to pheromone extracted in I ml of ethanol/mg wet wt female, and to various dilutions of this pheromone extract. The concentration of pheromone eliciting a positive response in 50% of males was calculated. (2) A comparison was made of the response made by control and infected 6- to 7-day-old and 13- to 16day-old males to pheromone collected from control females. In addition, variation in male response with age was investigated to determine whether time of peak response was affected by infection. Data were tested for significance using the G test of independence (Sokal and Rohlf, 1981) with a minimum significance level set at P < 0.05. Male response to pheromone dilution was examined using regression analysis.
FIG. I. Male Tenebrio molitor exhibiting a positive response to a female pheromone-coated glass rod. Genitalia are extended and antennae drum on the rod.
be-
RESULTS
Male beetles exhibited a characteristic behavior pattern in response to a glass rod coated with copulatory release pheromone. Beetles within 10-20 mm of the rod per-
84
HURD AND PARRY
formed a rearing action, raising the anterior legs in the air with the head and antennae thrown backward (Fig. 2). On contact, the rod was mounted and the males beat a “tattoo” (Tschinkel et al., 1967) on the surface of the rod with their antennae while extending the genitalia to touch the rod (Fig. 1). None of these behavior traits were exhibited by males exposed to a rod coated with ethanol alone, or by females. Although 88% of 5- to 6-day-old control males (n = 18) exhibited a positive response to pheromone from control females, only 56% of control beetles responded to pheromone from infected females (n = 23). Application of the G test of significance gave a “G” value of 3.86 and thus demonstrated a significant difference at P < 0.05. A comparison of dose responses obtained by exposing control males to various dilutions of pheromone from control and infected females was made (see Fig. 3). The resulting regressions were found to be significantly different (F = 55.84, critical F value = 3.63 (P = 0.05)). A concentration of 0.59 ml control extract/ml ethanol produced a response in 50% of control males. However, a concentration of 1.Ol ml of infected-extract/ml of ethanol was required to produce the same level of response. Data obtained from a comparison of the responses made by infected and control male beetles at two ages postemergence are
- from infected female *from control female
0 0.1 02 03 0.4 0.5 0.6 0.7 0.8 09 extract
dilution
(ml/ml
FIG. 3. Response of S- to 6-day-old control male Tenebrio molitor to various dilutions of pheromone extracted (1 ml ethanol/ 1 mg wet wt) from control and infected females.
given in Table 1. Infected beetles, 6-7 days old, exhibited a significant reduction in response to pheromone compared with uninfected counterparts; however, at 12-14 days postemergence no such reduction in response was detected. Male response to pheromone was therefore determined daily from days 4 to 10 postemergence and was shown to peak on day 5 in control and infected insects (see Fig. 4). Changes in response to age followed a similar pattern in both groups with the exception of day 8, when the number of control males responding increased from 50 to 65% (n = 20). Although no such increase occurred in infected beetles the response did not vary significantly with infection (P = 0.05). The TABLE
I
COPULATORY RESPONSE OF MALE TO PHEROMONE EXTRACTED T-DAY-OLD
CONTROL
Sample size 6- to 7-day-old males Control
2. Rearing response performed by male beetles 10-20 mm from a female pheromone-coated glass rod. FIG.
1.0
ethanol)
Tenebrio molitor FROM
6- TO
FEMALES
Response
Infected
60 60
91.6% 36.6%
P < 0.001
12- to 14-day-old males Control Infected
40 40
45% 42.5%
NS
METACESTODES
AND
FIG. 4. Change in response to female copulatory release pheromone by control and infected male Tenebrio molitor with age. * Percentage infected males responding is significantly lower than uninfected counterparts (P < 0.05).
percentage of infected insects exhibiting a positive response was significantly lower (P < 0.05) than control beetles on days 5 and 6 postemergence, i.e., 34 days postinfection. DISCUSSION
Several authors have investigated sex pheromone production by T. molitor using methods of bioassay based on either attraction or contact (Tschinkel et al., 1967; Happ, 1970; Happ and Wheeler, 1969; Tschinkel, 1970; August, 1971). Tanaka et al. (1986), employing both methods of bioassay, demonstrated the presence of both a volatile sex attractant, released only by female T. molitor and identified as 4methyl-1-nonanol, and a nonvolatile copulatory release pheromone produced by females and, to a lesser extent, by males. These two pheromones were found to act synergistically. Happ and Wheeler (1969) and Menon and Nair (1976) reported a peak production of female sex pheromone early in adult life (day 4-5 and 7, respectively), a decline in production coinciding with the onset of egg laying. The findings reported in this paper indicate that, at the time of peak output, parasitic infection reduces the production
HOST
PHEROMONE
85
of the nonvolatile copulatory release pheromone by approximately 50%. This reduction occurs at a very early stage in infection, when developing metacestodes are at stages 2-3 (Voge and Heyneman, 1957). Menon (1970) has established that the nutritional state plays no role in the control of pheromone production up to 10 days postemergence and it is unlikely that any element of parasite-induced host-nutrient deprivation occurs at this stage in infection, when parasite biomass is very small. Parasite burden was not assessed in this study but we have been unable to relate intensity of infection to degree of pathophysiology in a previous study (Hurd and Arme, 1984). It has, however, been demonstrated that there is endocrine regulation of pheromone production, the corpora allata being directly involved and juvenile hormone application partially restoring activity in allatectomized females (Menon, 1970). Hurd and Arme, (1984, 1986a. 1987) have previously shown that metacestodes of H. diminuta modulate several juvenile hormoneregulated events associated with female host reproductive physiology (see above). It is possible that the mechanism underlying these effects is similar to that responsible for the reduction in copulatory release pheromone production observed in this study. Topical application of the juvenile hormone analogue methoprene at the time of infection has a rescue effect on the pathophysiological events associated with host vitellogenesis, although hormone synthesis, circulating titers, and rate of degradation are not affected by the presence of metacestodes in the hemolymph (Hurd and Weaver, 1987; Hurd et al., 1990). Thus H. diminuta metacestodes do not appear to reduce the amount of hormone available to play a regulatory role. It is, however, possible that parasitism is associated with some form of competitive inhibition occurring at the level of juvenile hormone receptors and thus affecting all juvenile hormone-mediated events, to varying extents.
86
HURD AND PARRY
De Jong-Brink and co-workers (see review, 1990) have shown that this type of parasite manipulation of host reproduction occurs at the level of hormone receptors in the Trichobilharzia ocellata-Lymnaea stagnalis model. A significant parasite-associated depression in the male copulatory response to female pheromone was observed at the time of peak responsiveness, 5-6 days posteclosion (Happ, 1970). Data indicated that metacestodes may induce a permanent decrease in host sexual vigor rather than a delay in maximum responsiveness. August (1971) suggested that, although tactile stimuli are primarily functional during amplexus in T. molitor, extension of male genitalia in response to copulatory release pheromone is stimulated by contact chemoreception. He observed that copulation can occur when the antennae of the male have been amputated, but sensory receptors on the mouthparts must be functional. Our initial observations support this view. We demonstrated that loo-cl.1 drops of female extract, inserted into the end of a hollow glass tube, did not release copulatory behavior in control males, although the same extract, accessible on the outside of a solid rod, did act as a stimulant. The majority of beetles encountered the pheromone-coated rods during random wandering in the test arena, and the rearing response was usually performed after initial contact with a rod. Both control and infected beetles explored the rod as described earlier, thus we cannot determine whether the infection-associated decrease in response to a nonvolatile female sex pheromone is the result of an impairment of host contact chemoreception or whether the pheromone was detected but did not elicit a response. It is possible that the parasite may inhibit male mating behavior at a stage after the detection of copulatory pheromone. The mechanism underlying this change in male host mating behavior is not understood. In common with the reduction in fe-
male pheromone production, it is manifest 3-4 days postinfection and could be due to one of several factors such as the presence of hatched onchospheres in the host gut, the penetration of onchospheres through the mid-gut, or the effect of secretory/ excretory products produced by the developing metacestodes. Both the reduction in female pheromone production and the decrease in male response to pheromone may be contributary factors responsible for the delay in oviposition and the reduction in fecundity associated with this symbiosis (Hurd, 1990b). In addition, it is conceivable that, in a mixed population of infected and uninfected insects, reduction in pheromone production may indirectly lead to recognition of infected conspecifics and increase the mating chances of uninfected individuals at the expense of the infected ones. Reduced reproductive success has been described in many parasite-invertebrate host associations (see review by Hurd. 1990a). However, to our knowledge, this is the first report of a parasite which may affect host sex pheromone production. ACKNOWLEDGMENTS The senior author acknowledges the financial support of the Leverhulme Trust via a fellowship for women returners to science. We are also grateful to Mr. I. Burns for valuable technical assistance.
REFERENCES AUGUST. C. J. 197 1. The role of male and female pheromones in the mating behaviour of Tenebrio molifor. J. Insect Physiol., 17, 739-751. BHATTACHARYA, A. K., AMEEL, J. J.. AND WALDRAUER. G. P. 1970. A method for sexing living and pupal adult yellow mealworms. Ann. Entomol. Sot. Am.. 63B, 1783. DE JONG-BRINK. M.. HORDIJK. P. L.. SCHALLIG, H. D. F. H.. BERGAMIN-SASSEN, M. J. M., AND OOSTHOEK, P. 1990. Possible mechanisms underlying parasitic castration in trematode infected snails. In “Adv. Invertebr. Reprod.” (M. Hoshi and 0. Yamashita. Eds.). Vol. 5. pp. 141-149. HAPP, G. M. 1970. Maturation of the response of male Tenebrio molitor to the female sex pheromone. Ann. Entomol. Sot. Am., 63, 1782-1783. HAPP. G. M., AND WHEELER, J. 1969. Bioassay, preliminary purification. and effect of age. crowding,
METACESTODES
AND
and mating on the release of sex pheromone by female Tenebrio molitor. Ann. Entomol. Sot. Am., 62, 846-851. HURD, H., AND ARME, C. 1984. Pathophysiology of Hymenolepis diminuta infections in Tenebrio molitor: Effect of parasitism on haemolymph proteins. Parasitology, 89, 253-262. HURD. H.. AND ARME. C., 1986a. Hymenolepis diminuta: Influence of metacestodes upon synthesis and secretion of fat body protein and its ovarian sequestration in the intermediate host, Tenebrio molitor. Parasitology, 93, 11 l-120. HURD, H.. AND ARME, C., 1986b. Hymenolepis diminuta: The effect of metacestodes upon egg production and viability in the intermediate host, Tenebrio molitor. J. Invertebr. Pathol.. 41, 225-23 1. HURD, H., AND ARME, C., 1987. Hymenolepis diminuta: Effect of infection upon the patency of the follicular epithelium of the intermediate host Tenebrio molitor. J. Inrsertebr. Pathol.. 49, 227-234. HURD. H.. AND WEAVER, R. J. 1987. Evidence against the hypothesis that metacestodes of Hymenolepis diminuru inhibit corpora allata functioning in rhe intermediate host, Tenebrio molitor. Parasitology. 95, 93-97. HURD,
H.,
STRAMBI.
C..
AND
BECKAGE,
N.
E. 1990.
Hymeno/epis diminata: An investigation ofjuvenile hormone titre, degradation and supplementation in the intermediate host, Tenebrio molitor. Parusitology.
loo, 445-452.
HURD. H. 199Oa. Physiological and behavioral interactions between parasites and invertebrate hosts. In “Advances in Parasitology” (J. R. Baker and R. Muller. Eds.). Vol 29, pp. 271-317. Academic Press. New York. HURD. H. 1990b. Parasite induced modulation of insect reproduction. In “Adv. lnvertebr. Reprod.” (M. Hoshi and 0. Yamashita, Eds.). Vol. 5, pp. 163169. KEYMER. A. E. 1980. The influence of Hymenolepis diminata on the survival and fecundity of the inter-
HOST
87
PHEROMONE
mediate host, Triboliam
confkum.
Parasitology,
81, 405-421.
KEYMER, A. E. 1981. Population dynamics of Hymenolepis diminuta in the intermediate host. J. Anim. Ecol., 50, 941-950. LAVERDURE, A. M. 1967. Rbles de I’alimentation et des hormones cerkbrales dans la vitellogen&se chez Tenebrio molitor (ColCopt&e). Bull. Sot. Zoo/. Fr.. 92, 629-640. MENON, M. 1970. Hormone-pheromone relationships in the beetle, Tenebrio molitor. J. Insect Physiol.. 16, 1123-1139. M. 1976.
Hormone-pheromone relationships of male Tenebrio molitor. J. Insect Physiol.. 22, 1021-1023. MENON, M., AND NAIR, K. K. 1970. Sex pheromone production and reproductive behaviour in gammairradiated Tenrbrio molitor. J. Insect Physiol.. 18, MENON,
1323-1331.
M.. AND NAIR, K. K. 1976. Age dependent effects of synthetic juvenile hormone on pheromone synthesis in adult females of Tenebrio molitor. Ann. Entomol. Sot. Am., 69, 457-458. SOKAL, R. R.. AND ROHLF, F. R. 1981. “Biometry.” Freeman. San Francisco. MENON.
TANAKA,
Y.,
HONDA.
H.,
OHSAWA,
K.,
AND
YA-
1. 1986. A sex attractant of the yellow mealworm, Tenebrio molitor L., and its role in the mating behaviour. J. Pesticide Sci.. 11, 49-55. TSCHINKEL, W. R. 1970. Chemical studies on the sex pheromone of Tenebrio molitor (Coleoptera: Tenebrionidae). Ann. Entomol. Sor. Am., 63, 626-627. TSCHINKEL, W.. WILLSON, C.. AND BERN, H. A. 1967. Sex pheromone of the mealworm beetle (Tenebrio molitor). J. Exp. Zool.. 164, 81-86. VOGE, M.. AND HEYNEMAN. D. 1957. Development of Hymenolepis nann and Hymenolepis dirninuta (Cestoda: Hymenolepididae) in the intermediate host Tribolium confi~sam. Univ. Culif. Pab. Zool., 59, 549-580. MAMOTO,
OF INVERTEBRATE
PATHOLOGY
58,
82-87 (1991)
Metacestode-Induced Depression of the Production Response to, Sex Pheromone in the Intermediate Tenebrio molitor
of, and Host
HILARY HURDAND G. PARRY Parasitology
Research Laboratory,
Department of Biological Sciences, University of Keele. Keeie ST5 5BG, United Kingdom
Received June5, 1990; accepted October 3, 1990 Hymenolepis diminuta infection of Tenebrio molitor is associated with an impairment of vitellogenesis and a reduction in host fecundity. In this communication the effect of infection upon an additional aspect of host reproduction, the initiation of mating behavior, has been examined. Copulatory release pheromone, extracted from control virgin females 6-7 days old. was shown to stimulate a positive mating response in 88% of 5- to 6-day-old control males; however, only a 56% response was elicited by pheromone from infected females. In addition, parasitization adversely effected male response to pheromone from control females. A significant (P < 0.001) depression of copulatory response occurred in infected 6- to ‘I-dav-old males (age of peak response) although this ility that an endocrine interaction between effect was not sustained in older beetles. The p metacestodes and host may mediate these effel ts is discussed in the light of our knowledge of the role of host juvenile hormone in controlling bo.h pheromone production and vitellogenesis in T. molitor. 0 1991 Academic Press, Inc. KEY WORDS: Hymenolepis diminuta; Tenebrio molitor: Cestoda; Coleoptera; host fecundity; host sex pheromone; host mating behavior.
An additional role of juvenile hormone is its regulation of sex pheromone production in T. molitor (Menon, 1970, 1976; Menon & Nair, 1970, 1976). This communication describes the use of a biological assay designed to determine whether H. diminuta infection of T. molitor results in disturbance of an additional endocrine-controlled event, that of sex pheromone production by female beetles. A comparison has also been made of the response of male infected and uninfected beetles to female sex pheromone. Any reduction in production of, or response to, sex pheromone may be an additional factor contributing to the reduction of host reproductive output observed in the association.
INTRODUCTION
A reduction in host fecundity is known to occur in associations between metacestodes of Hymenolepis diminuta, the rat tapeworm, and its coleopteran intermediate hosts Tribolium cot&sum (Keymer, 1980, 1981) and Tenebrio molitor (Hurd and Arme, 1986b). This may in part be attributed to a perturbance in events associated with egg production which has been observed in T. molitor 12 days or more postinfection. These include a reduction in fat body synthesis of vitellogenin, the development of patency in the follicular epithelium, and the sequestration of vitellogenin by developing oocytes (see review by Hurd, 1990a). In common with the majority of insects, vitellogenesis in T. molitor is regulated via fluctuations in juvenile hormone titer (Laverdure, 1967) and it has been suggested that metacestodes may interact with the host endocrine system to downregulate the events listed above (Hurd, 1990b).
MATERIALS
Maintenance of Parasite and Host T. molitor pupae were collected weekly from a stock colony, sexed (Bhattacharya et al., 1970), and emergents separated daily. Pupae and adults were segregated ac82
0022-201 l/91 $1.50 Copyright 8 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
AND METHODS
METACESTODES
AND
HOST
83
PHEROMONE
cording to sex and age. All insects used in this study were maintained in plastic, paper-lined Petri dishes in the same incubator at 26” 2 1°C and 60-80% relative humidity. H. diminuta eggs were collected from feces of infected Wistar rats by a salt flotation method, washed, and stored at 4°C for a maximum of 4 weeks. Adult beetles were starved for up to 2 days postemergence and then exposed to a mixture of parasite eggs and apple pulp. Control insects were starved then fed on apple pulp alone. Thereafter, insects were kept in groups of 20-30 and fed on bran. All male T. molitor exposed to H. diminuta eggs were dissected after testing and uninfected individuals removed from the study. No beetles were tested more than once.
and all experiments were performed tween 1400 and 1600.
Bioassay
Investigations
Pheromone extraction was performed by immersing 6- to 7-day-old virgin females in 100% ethanol (1 ml/mg fresh wt) for 2 hr, with initial mixing on a vortex. A pilot study revealed no significant difference (P = 0.05) in male response to pheromone extracts stored for 2 weeks at -20°C. The majority of tests reported in this communication were performed on fresh extracts; however, some extracts were stored at -20°C for a maximum of 2 weeks prior to use. Pieces of glass rod, 4 mm in diameter, were stored in ethanol. Immediately before use they were air dried, immersed in 200 ~1 of pheromone extract for 30 set, air dried, and then anchored in a test arena with a piece of plasticine. Male virgin beetles were placed individually on clean “Benchkot” under an inverted Petri dish base and allowed to acclimatize for 5 min before the introduction of a pheromone-coated glass rod. A positive response was recorded when the beetle attempted to mount the rod, bent the tip of the abdomen downward, and extended the genetalia to touch the glass rod (Fig. I). Any beetles failing to respond after 15 min were recorded as negative. The time intervals of all procedures were kept constant
(1) The effect of infection upon production of pheromone by female beetles was investigated by measurement of the response of 5- to 6-day-old control males to pheromone extracted in I ml of ethanol/mg wet wt female, and to various dilutions of this pheromone extract. The concentration of pheromone eliciting a positive response in 50% of males was calculated. (2) A comparison was made of the response made by control and infected 6- to 7-day-old and 13- to 16day-old males to pheromone collected from control females. In addition, variation in male response with age was investigated to determine whether time of peak response was affected by infection. Data were tested for significance using the G test of independence (Sokal and Rohlf, 1981) with a minimum significance level set at P < 0.05. Male response to pheromone dilution was examined using regression analysis.
FIG. I. Male Tenebrio molitor exhibiting a positive response to a female pheromone-coated glass rod. Genitalia are extended and antennae drum on the rod.
be-
RESULTS
Male beetles exhibited a characteristic behavior pattern in response to a glass rod coated with copulatory release pheromone. Beetles within 10-20 mm of the rod per-
84
HURD AND PARRY
formed a rearing action, raising the anterior legs in the air with the head and antennae thrown backward (Fig. 2). On contact, the rod was mounted and the males beat a “tattoo” (Tschinkel et al., 1967) on the surface of the rod with their antennae while extending the genitalia to touch the rod (Fig. 1). None of these behavior traits were exhibited by males exposed to a rod coated with ethanol alone, or by females. Although 88% of 5- to 6-day-old control males (n = 18) exhibited a positive response to pheromone from control females, only 56% of control beetles responded to pheromone from infected females (n = 23). Application of the G test of significance gave a “G” value of 3.86 and thus demonstrated a significant difference at P < 0.05. A comparison of dose responses obtained by exposing control males to various dilutions of pheromone from control and infected females was made (see Fig. 3). The resulting regressions were found to be significantly different (F = 55.84, critical F value = 3.63 (P = 0.05)). A concentration of 0.59 ml control extract/ml ethanol produced a response in 50% of control males. However, a concentration of 1.Ol ml of infected-extract/ml of ethanol was required to produce the same level of response. Data obtained from a comparison of the responses made by infected and control male beetles at two ages postemergence are
- from infected female *from control female
0 0.1 02 03 0.4 0.5 0.6 0.7 0.8 09 extract
dilution
(ml/ml
FIG. 3. Response of S- to 6-day-old control male Tenebrio molitor to various dilutions of pheromone extracted (1 ml ethanol/ 1 mg wet wt) from control and infected females.
given in Table 1. Infected beetles, 6-7 days old, exhibited a significant reduction in response to pheromone compared with uninfected counterparts; however, at 12-14 days postemergence no such reduction in response was detected. Male response to pheromone was therefore determined daily from days 4 to 10 postemergence and was shown to peak on day 5 in control and infected insects (see Fig. 4). Changes in response to age followed a similar pattern in both groups with the exception of day 8, when the number of control males responding increased from 50 to 65% (n = 20). Although no such increase occurred in infected beetles the response did not vary significantly with infection (P = 0.05). The TABLE
I
COPULATORY RESPONSE OF MALE TO PHEROMONE EXTRACTED T-DAY-OLD
CONTROL
Sample size 6- to 7-day-old males Control
2. Rearing response performed by male beetles 10-20 mm from a female pheromone-coated glass rod. FIG.
1.0
ethanol)
Tenebrio molitor FROM
6- TO
FEMALES
Response
Infected
60 60
91.6% 36.6%
P < 0.001
12- to 14-day-old males Control Infected
40 40
45% 42.5%
NS
METACESTODES
AND
FIG. 4. Change in response to female copulatory release pheromone by control and infected male Tenebrio molitor with age. * Percentage infected males responding is significantly lower than uninfected counterparts (P < 0.05).
percentage of infected insects exhibiting a positive response was significantly lower (P < 0.05) than control beetles on days 5 and 6 postemergence, i.e., 34 days postinfection. DISCUSSION
Several authors have investigated sex pheromone production by T. molitor using methods of bioassay based on either attraction or contact (Tschinkel et al., 1967; Happ, 1970; Happ and Wheeler, 1969; Tschinkel, 1970; August, 1971). Tanaka et al. (1986), employing both methods of bioassay, demonstrated the presence of both a volatile sex attractant, released only by female T. molitor and identified as 4methyl-1-nonanol, and a nonvolatile copulatory release pheromone produced by females and, to a lesser extent, by males. These two pheromones were found to act synergistically. Happ and Wheeler (1969) and Menon and Nair (1976) reported a peak production of female sex pheromone early in adult life (day 4-5 and 7, respectively), a decline in production coinciding with the onset of egg laying. The findings reported in this paper indicate that, at the time of peak output, parasitic infection reduces the production
HOST
PHEROMONE
85
of the nonvolatile copulatory release pheromone by approximately 50%. This reduction occurs at a very early stage in infection, when developing metacestodes are at stages 2-3 (Voge and Heyneman, 1957). Menon (1970) has established that the nutritional state plays no role in the control of pheromone production up to 10 days postemergence and it is unlikely that any element of parasite-induced host-nutrient deprivation occurs at this stage in infection, when parasite biomass is very small. Parasite burden was not assessed in this study but we have been unable to relate intensity of infection to degree of pathophysiology in a previous study (Hurd and Arme, 1984). It has, however, been demonstrated that there is endocrine regulation of pheromone production, the corpora allata being directly involved and juvenile hormone application partially restoring activity in allatectomized females (Menon, 1970). Hurd and Arme, (1984, 1986a. 1987) have previously shown that metacestodes of H. diminuta modulate several juvenile hormoneregulated events associated with female host reproductive physiology (see above). It is possible that the mechanism underlying these effects is similar to that responsible for the reduction in copulatory release pheromone production observed in this study. Topical application of the juvenile hormone analogue methoprene at the time of infection has a rescue effect on the pathophysiological events associated with host vitellogenesis, although hormone synthesis, circulating titers, and rate of degradation are not affected by the presence of metacestodes in the hemolymph (Hurd and Weaver, 1987; Hurd et al., 1990). Thus H. diminuta metacestodes do not appear to reduce the amount of hormone available to play a regulatory role. It is, however, possible that parasitism is associated with some form of competitive inhibition occurring at the level of juvenile hormone receptors and thus affecting all juvenile hormone-mediated events, to varying extents.
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De Jong-Brink and co-workers (see review, 1990) have shown that this type of parasite manipulation of host reproduction occurs at the level of hormone receptors in the Trichobilharzia ocellata-Lymnaea stagnalis model. A significant parasite-associated depression in the male copulatory response to female pheromone was observed at the time of peak responsiveness, 5-6 days posteclosion (Happ, 1970). Data indicated that metacestodes may induce a permanent decrease in host sexual vigor rather than a delay in maximum responsiveness. August (1971) suggested that, although tactile stimuli are primarily functional during amplexus in T. molitor, extension of male genitalia in response to copulatory release pheromone is stimulated by contact chemoreception. He observed that copulation can occur when the antennae of the male have been amputated, but sensory receptors on the mouthparts must be functional. Our initial observations support this view. We demonstrated that loo-cl.1 drops of female extract, inserted into the end of a hollow glass tube, did not release copulatory behavior in control males, although the same extract, accessible on the outside of a solid rod, did act as a stimulant. The majority of beetles encountered the pheromone-coated rods during random wandering in the test arena, and the rearing response was usually performed after initial contact with a rod. Both control and infected beetles explored the rod as described earlier, thus we cannot determine whether the infection-associated decrease in response to a nonvolatile female sex pheromone is the result of an impairment of host contact chemoreception or whether the pheromone was detected but did not elicit a response. It is possible that the parasite may inhibit male mating behavior at a stage after the detection of copulatory pheromone. The mechanism underlying this change in male host mating behavior is not understood. In common with the reduction in fe-
male pheromone production, it is manifest 3-4 days postinfection and could be due to one of several factors such as the presence of hatched onchospheres in the host gut, the penetration of onchospheres through the mid-gut, or the effect of secretory/ excretory products produced by the developing metacestodes. Both the reduction in female pheromone production and the decrease in male response to pheromone may be contributary factors responsible for the delay in oviposition and the reduction in fecundity associated with this symbiosis (Hurd, 1990b). In addition, it is conceivable that, in a mixed population of infected and uninfected insects, reduction in pheromone production may indirectly lead to recognition of infected conspecifics and increase the mating chances of uninfected individuals at the expense of the infected ones. Reduced reproductive success has been described in many parasite-invertebrate host associations (see review by Hurd. 1990a). However, to our knowledge, this is the first report of a parasite which may affect host sex pheromone production. ACKNOWLEDGMENTS The senior author acknowledges the financial support of the Leverhulme Trust via a fellowship for women returners to science. We are also grateful to Mr. I. Burns for valuable technical assistance.
REFERENCES AUGUST. C. J. 197 1. The role of male and female pheromones in the mating behaviour of Tenebrio molifor. J. Insect Physiol., 17, 739-751. BHATTACHARYA, A. K., AMEEL, J. J.. AND WALDRAUER. G. P. 1970. A method for sexing living and pupal adult yellow mealworms. Ann. Entomol. Sot. Am.. 63B, 1783. DE JONG-BRINK. M.. HORDIJK. P. L.. SCHALLIG, H. D. F. H.. BERGAMIN-SASSEN, M. J. M., AND OOSTHOEK, P. 1990. Possible mechanisms underlying parasitic castration in trematode infected snails. In “Adv. Invertebr. Reprod.” (M. Hoshi and 0. Yamashita. Eds.). Vol. 5. pp. 141-149. HAPP, G. M. 1970. Maturation of the response of male Tenebrio molitor to the female sex pheromone. Ann. Entomol. Sot. Am., 63, 1782-1783. HAPP. G. M., AND WHEELER, J. 1969. Bioassay, preliminary purification. and effect of age. crowding,
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and mating on the release of sex pheromone by female Tenebrio molitor. Ann. Entomol. Sot. Am., 62, 846-851. HURD, H., AND ARME, C. 1984. Pathophysiology of Hymenolepis diminuta infections in Tenebrio molitor: Effect of parasitism on haemolymph proteins. Parasitology, 89, 253-262. HURD. H.. AND ARME. C., 1986a. Hymenolepis diminuta: Influence of metacestodes upon synthesis and secretion of fat body protein and its ovarian sequestration in the intermediate host, Tenebrio molitor. Parasitology, 93, 11 l-120. HURD, H.. AND ARME, C., 1986b. Hymenolepis diminuta: The effect of metacestodes upon egg production and viability in the intermediate host, Tenebrio molitor. J. Invertebr. Pathol.. 41, 225-23 1. HURD, H., AND ARME, C., 1987. Hymenolepis diminuta: Effect of infection upon the patency of the follicular epithelium of the intermediate host Tenebrio molitor. J. Inrsertebr. Pathol.. 49, 227-234. HURD. H.. AND WEAVER, R. J. 1987. Evidence against the hypothesis that metacestodes of Hymenolepis diminuru inhibit corpora allata functioning in rhe intermediate host, Tenebrio molitor. Parasitology. 95, 93-97. HURD,
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