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Comparison of ecdysteroid concentration in different morphs of aphids
ISSN 0742~8413/96/$15.00 PII SO742-8413(96)00104-l
Come. B&hem. Physiol. Vol. 11X, No. 2, pp. 179-183, 1996 Copynght 0 1996 Elsevier Science Inc.
ELSEVIER
Comparison of Ecdysteroid Concentration in Different Morphs of Aphids L. A.
Polgcir,I* B . Duwus,’ W. V&l,* P. Purcher~n,~A. SQ~&S,’ and S. Szehger’
OF ZOOLOGY, PLANTPROTECTION INSTITUTE,HUNGARIANACADEMYOF SCIENCES,P.O. Box 102, H-1525 BUDAPEST,HUNGARY;‘DEPARTMENT OF ANIMALECOLOGY, UNIVERSITY OF BAYRJKJTH, POSTFACH10 1251, D-95770 BAYREUTH,GERMANY;AND 3B~~~~~~~~ ET PHYSIOL~~XE DU ~~VELOPPEMENT, ECOLENORMALESUP~RIEURE, CNRS-URA 686, 46 RUE D’ ULM, 75230 PARISCEDEX05, FRANCE IDEPARTMENT
ABSTRACT. The amounts of ecdysteroids were determined in different morphs of holocyclic monoecious aphids (Acyrrhosiphon pisum Harris, Dysaphis d.evecta Walk., Lachnus roburis L., Schizoluchnus pin& F.) and holocyclic heteroecious aphids (Aphis sumbuci L., Rhop&sighum padi L.) by means enzyme immunoassay. Among the parthenogenetic morphs (fundatrices, virginoparae, and oviparae), the fundatrices have consistently higher amounts of ecdysteroids than those of other morphs of the same species. Alate and apterous virginoparae showed slight differences in their ecdysteroid titer both in heteroecious and monoecious aphids. The migrant morphs (i.e., alate fundatrices and gynoparae) have the lowest amounts of ecdysteroids within a species. With the exception of D. deuecta, the oviparae of both heteroecious and monoecious aphids have the second greatest amounts of ecdysteroids among the morphs living on same host plant. Polyphenism, dispersal behavior, and fecundity in connection with weight, ovariole number, and ecdysteroid concentration of different morphs of aphids are discussed. I(’ ‘. Coprright 0 1996 Elsevier Science Inc. COMPBIOCHEM PHYSIOL115C;2:179-183, 1996. KEY WORDS. Aphid morphs, ecdysteroids, immunoassay, Acyrthosiphon pisum, Aphis sambud, IDysaphisdeuecta, Lachnw roboris, Rho@osiphum padi, Schizoluchnus pineti
INTRODUCTION
life cycle of holocyclic species of aphids includes several parthenogenetic morphs that alternate with a heterosexual generation (6,25). Holocyclic, non-host-alternating (monoecious) aphids live on the same host plants throughout the season. The parthenogenetic morphs of a monoecious aphid species, in sequence of appearance, are fundatrices (i.e., first parthenogenetic generation emerging from overwintered eggs), followed by virginoparae (i.e., summer parthenogenetic generations), which later give birth to oviparae (i.e., heterosexual females) and males, generally in autumn. A holocyclic, host-alternating (heteroecious) aphid species shows wider polyphenic variability. Oviparae develop on woody host plants (i.e., winter host) only, while the summer generations of virginoparae generally reject them. For this reason, these aphids have at least two further morphs (i.e., emigrants and immigrants) that differ reproductively, usually morphologically (15), and mainly in their behaviour (polyethism) from virginoparae. The alate fundatrigeniae (i.e., last parthenogenetic generation on winter The
Address rejxint requests to: L. A. PolgPr, Department of Zoology, Plant Protection Institute, Hungarian Academy of Sciences, P.O. Box 102, H-1525 Budapest, Hungary. Tel 36-11-769555; Fax 36-11~769729; E-mail [email protected]. Received 15 February 1996; accepted 22 May 1996.
host) seek different, usually annual, hosts. The gynoparae (i.e., last parthenogenetic generation on summer hosts) are also alate and return to the winter host. These polyphenisms and polyethisms (4) have evolved largely in response to predictable seasonal changes in habitat quality (6). The environmental signals such as population density (crowding), photoperiod, temperature, and nutrition (i.e., host plant effect) are transformed to chemical cues by neurosecretory cells and are mediated by insect hormones (10). Among the insect hormones, the role of juvenile hormone (JH) was studied intensively in relation to polyphenism and polyethism of aphids (10,14,23) because of the effect of JH on their reproduction (11) and host plant preference (13). In contrast, there is currently no information about differences in ecdysteroid titers among the various aphid morphs (12). Ecdysteroids occur in most adult insects also, and may play an important role in reproduction (5,9,21). There is a report for the aphid Schizaphis gmminum (Rondani) of huge amounts of phytosterols ,(stigmasterol, sitosterol, and campesterol) besides cholesterol, which originated from the host plant, Sorghum bicolor (L.) (3). Additionally, some researchers (2) measured ecdysone in Hyalopterus pruni Geofi. alongside vertebrate-type (estradiol and progesterone) steroids, but they did not consider aphid morph and age. However, very small amounts of ma,
180
L. A. PolgPr et al.
kisterone A besides ecdysone and 20-hydroxyecdysone have been reported in adults of apterous Megoura viciae Buck. (20). Recently, amounts of ecdysteroids were determined from parasitized and nonparasitized instars and adults of A. pisum( 28). In the present study, we measured the ecdysteroid concentration in adults of six aphid species. We compared different morphs of two host-alternating and four non-hostalternating aphids to obtain basic information about ecdysteroid titers among aphid morphs. This study thus represents the first attempt to obtain comparative data in aphids, with a special interest in polyphenism, dispersal behavior, and fecundity.
sone-specific antibodies and an ecdysone-acetylcholinesterase enzyme conjugate as a tracer. The cross-sensitivity of the antibody provided by this kit is 100% to ecdysone and 20-hydroxyecdysone, and 88%, 70%, 63%, and 43% to 2deoxy-20+hydroxyecdysone, polypodine B, 2-deoxyecdysone, and ponasterone A, respectively. The binding rate of other ecdysteroids such as makisterone A and 26-hydroxyecdysone did not exceed 5%. Samples were analyzed in at least two duplicates and incubated in a 96-well microplate. The amount of ecdysteroids in each sample was compared to the 20-hydroxyecdysone standard curve and expressed as pg/mg 20-hydroxyecdysone equivalents. The calorimetric reaction was measured at 415 nm.
MATERIALS Insects
RESULTS
AND METHODS
We tested parthenogenetic and heterosexual morphs (i.e., males and oviparae) of the heteroecious species Rhopalosiphum @di L. (fundatrices, alate and apterous fundatrigeniae, gynoparae, and oviparae from the winter host, Prunus pudus L., and alate and apterous virginoparae from the summer host, Hordeurn vulgare L.) and Aphis sambuci L. (fundatrices, alate and apterous virginoparae, gynoparae, and oviparae, all morphs from the winter host, Sambucus nigra L.), and different morphs of the monoecious species Dysaphis dewecta Walk. (fundatrices, apterous virginoparae, and oviparae from M&s domestica Bork.), Acyrthosiphon pisum Harris (apterous and alate virginoparae from Vicia faba L.), Luchnus roboris L. (fundatrices, alate and apterous virginoparae, and oviparae from Quercus roboris L.), and Schizoolachnus pineti F. (fundatrices, apterous virginoparae, oviparae, and males from Pinus silvestris L.). With the exception of S. pineti, where different instars were mixed, we collected only adult aphids. Most of the collected adults were in prereproductive stage. Parthenogenetic females gave birth to few (zero or one) offspring. Wet weights of aphids were measured by groups of 10 individuals. The mean value of aphid weight was calculated from five measurements. In the case of L. roboris, different morphs were measured individually. Numbers of ovarioles were determined by means of light microscope after dissection of 10 individuals from each morph.
Ecdysteroid
Analysis
For the ecdysteroid measurements, aphid groups with a fresh weight of at least 0.2 g were used. The samples of whole bodies of aphids were prepared according to the method of Porcheron et al. (29). After purification of the samples, they were equalized to 0.1 g fresh weight and dried under Nz. The dried samples were stored at -50°C until analysis by enzyme immunoassay (EI A). Ecdysteroid measurements were carried out with 20-hydroxyecdysone EIA kits from Cayman Co. The competitive immunoassay system utilizes immobilized 20-hydroxyecdy-
Amounts of ecdysteroids differed both between aphid species and morphs and within a species. With the exception of D. devecta, fundatrices have the largest amounts of ecdysteroids in all tested aphid species. This difference between fundatrices and other morphs of the same species in their ecdysteroid concentration is more pronounced in the hostalternating R. padi and A. sambuci (Tables 1, 2) than in the non-host-alternating L. roboris and S. pineti (Table 3; Fig. 1). Generally, fundatrices have the largest weight within a species and, with the exception of R. padi, they have more ovarioles than other tested morphs. Wingless (apterous) females are heavier and have more ovarioles than winged (alate) morphs, and alate morphs have lower amount of ecdysteroids than their apterous counterparts within a species. However, these differences were not significant in all cases. Alate virginoparae of S. pineti and D. dtwectu were not tested, but a similar tendency was observed in two other non-host-alternating aphid species, L. roboris (Table 3) and A. pisum (Fig. 1). The migrant morphs of R. pudi (i.e., alate fundatrigeniae and gynoparae) have the lowest weights and amounts of ecdysteroids among the alate morphs of this species (Table 1). Similarly, the gynoparae have the lowest ecdysteroids, in the case of A. sumbuci (Table 2). Ecdysteroid concentration was measured in males of S. pineti only (Fig 1). Males were found to have the lowest ecdysteroid concentration ( 1.44 t 0.2 pg/mg) and the lowest weight of insects (2.5 5 0.3 mg/lO aphids) in comparison to other morphs of the species. Different morphs of non-host-alternating L. roboris have very similar weights (Table 3). Oviparae of this species have same number of ovarioles as apterous virginoparae, but they have significantly more ecdysteroids than virginoparous morphs. Similarly, in the host-alternating R. pad and A. sambuci, the oviparae are lighter and have similar numbers or fewer ovarioles but have more ecdysteroids than virginoparae from the same host plant. However, this difference is not significant in the case of A. sambuci. Contrary to all other species investigated, in the case of D. devecta the fun-
181
Ecdysteroids and Aphids
TABLE 1. 20.Hydroxyecdysone
equivalents (pglmg, * *SE) in different morphs of Rbopalosiphum 20E equivalents (pg/mg fresh weight)
Fresh weight (mglAphid)
Aphid morph
Fundatrices Fundatrigeniae, apterous Fundatrigeniae, alate Virginoparae, apterous Virginoparae, alate
2.60 1.48 0.69 1.14 0.96
I 0.45 2 0.32 + 0.28 2 0.12 +- 0.11
Gynoparae Oviparae
0.38 0.15
2 0.02 -+ 0.03
A B BC CD
6.85 0.77 1.06 3.75
? 2 ? 2
DE E E
3.19 5 0.42 0.47 t- 0.15 1.53 I 0.23
Within each column, means sharing the same capital letter do not differ at p < 0.05 (Mann-Whitney *Ecdysteroids other than ecdysone and 20.hydroxyecdysone have high probability. ‘rn = 2. data from Wellings et al. (38).
TABLE
2. 20.Hydroxyecdysone
Fresh weight (mg/aphid)
Fundatrices Virginoparae, apterous Virginoparate, alate Gynoparae Oviparae
TABLE
3.18 1.58 0.92 0.65 0.56
A B B c
10 12$ 103 6
C D E
4 6 6
U-test).
? 2 2 I? ”
0.20 0.12 0.07 0.12 0.05
20E equivalents (pglmg fresh weight)
A B C D D
6.88 0.53 0.75 0.68 0.95
2 2 2 ? -e
means sharing the same capital letrer do not differ at p < 0.05 (Mann-Whitney
3. 20+Hydroxyecdysone
0.85*t 0.04 0.08 0.54
Number of ovarioles
equivalents (pg/mg; *SE) in different morphs of Aphis sambuci (n = 4)
Aphid morph
Within each column, *n = 2.
pad; (n = 4)
0.76 A 0.24 B 0.16 B 0.06” B 0.04% B
Number of ovarioies
12 8 6 8 6
U-test).
equivalents (pg/mg ; *SE) in different morphs of Lachnus roboris (II = 4)
Aphid morph
Fresh weight (mg/aphid)
Fundatrices Virginoparae, apterous Virginoparate, alate Oviparae
12.4 11.6 11.2 12.9
-+ 2.2 2 0.8 2 1.7 2 1.5
A A A A
20E equivalents (pglmg fresh weight)
4.86 1.93 1.71 2.24
+- 0.61 -c 0.22 I? 0.64 5 0.19
Within each column, means sharing the same capital letter do not differ at p < 0.05 (Mann-Whitney
A B B C
Number of ovarioles
16 14 12 14
U-test).
datrices have the lowest ecdysteroid concentration and the oviparae have the highest among the morphs tested. The standard 20-hydroxyecdysone curve and the curves from the samples of fundatrices of R. padi and oviparae of A. pisum showed a different shape, which is a sign of ecdysteroids with different cross-reactivity than 20shydroxyecdysone.
DISCUSSION S. pineti
D. dew&a
A. pim
Aphid species and morphs FIG. I. Ecdysteroid concentration in different morphs of aphids. Data on males are included only for S. pineti. Alate virginoparae of S. pinetiand D. devecta and fundatrices of A. pisum were not tested.
The main sources of ecdysteroids in adult insects are the follicle cells (females) and testes (males) in general (reviews in 16,17), but other sources such as epidermis and oenocytes have also been demonstrated (30,37) in the adult cricket, Gqllus bimacuIatus. The role of ecdysteroids in both parthenogenetic and heterosexual morphs of adult aphids can be
L. A. Polgir et al.
similar, namely to promote the early phase of reproduction (i.e., gametogenesis and embryogenesis). There have been studies on fecundity in relation to ovariale number and body size of subsequent generations in the case of some monoecious and heteroecious aphids (22,38). It was stated that fecundity depends on both ovariole number and size of an aphid within a generation. While the ovariole number is stable within a generation, the size of aphids depends on the food quality. Therefore, large aphids are more fecund than small aphids in the same generation because they have more embryos per ovariole at maturity. The number of ovarioles varies in summer generations of heteroecious Aphis f&e (17), R. padi, and Metopolophium dirhodum (38), but the monoecious Drepanosiphum p&unoidis and Eucallipterus tiliae have a constant ovariole number within each generation through the season, with only the first generation (fundatrices) having two more ovarioles than subsequent generations (38). Our finding that fundatrices have generally higher ecdysteroid levels within a species complements the observation that they have higher reproductive rates, and adults contain more embryos than virginoparae of the same species. Similarly, less fecund gynoparae of R. padi (36) had the lowest amounts of ecdysteroids. However, apterous fundatrigeniae of R. padi have two more ovarioles than fundatrices (38), but their weight differs markedly (Table 1). All these observations suggest that different titers of ecdysteroids may be related to fecundity and movement behavior (i.e., dispersal and migration) for an aphid species. Adult aphids have small amounts of ecdysteroids in comparison to related heteropteran species (2). One experiment showed 50 times more (ca. 3 pg/mg) 20-hydroxyecdysone (20E) than makisterone A (ca. 0.06 pg/mg) and 10 times more 20E than ecdysone (ca. 0.3 pg/mg) in M. viciae (20). It seems to be that in aphids, contrary to many heteropteran species, the main ecdysteroids are 20-hydroxyecdysone and ecdysone instead of makisterone A. Recently, some authors reported approximately 4 pg/mg ecdysteroids (expressed as ecdysone equivalents) from whole-body extracts of A. pisum 2 days after the onset of reproductive activity of adults (28). All these investigations were carried out by radioimmunoassay. However, we used single enzyme immunoassay, and our results (3.43 -+ 0.37 pg/mg 20E equivalents) in apterous virginoparae of A. piston met with these observations. We found that fundatrices and oviparae of all tested species, with the exception of D. deuecta, have higher amounts of ecdysteroids than other morphs from same host plant. This trend is more evident in the case of L. roboris, where the different morphs have similar weights (Table 3). The reason for this finding may be sought in potential differences in the ecdysteroid production of ovarioles producing embryos or yolky eggs. Ecdysteroids may also occur in male insects, but in much lower titers than found in females. In males, ecdysteroids
may play a role in spermatogenesis (24). Our results are consistent with the relative size of the gonads in this species. While oviparae have very large ovarioles, the testes are comparably small. It has also been suspected that differences in the hormonal state of the various morphs and in the balance between JH III and ecdysteroids could be involved in diapause induction in aphid parasitoids (26,27). The present results indicate that such differences may actually occur. Besides crowding, photoperiod, and temperature, the “host plant effect” is known to play a significant role in aphid polyphenism. Senescent host plants usually induce alate virginoparae and/or oviparae morphs of holocyclic aphids (18,3 1,34). During plant life, phytoecdysteroids alter in quality and quantity as well (35), according to measurements in Ajuga reptans L. These results raise the question of whether phytoecdysteroids are the secondary plant substances involved in at least one part of the “host plant effect” in aphid polyphenism. We did not find considerable differences in ecdysteroid concentration between alate and apterous virginoparae of R. Dadi from their summer host, H. uulgure, nor between alate and apterous fundatrigeniae collected from their winter host, I’. J&US. These four morphs have similar weight, but they differ markedly in number of ovarioles. AIate and apterous fundatrigeniae reared on their winter host have more ovarioles but lower ecdysteroids levels than the virginoparous generation grown on their summer host, H. vulgare. The fact that fundatrigeniae have more ovarioles but lower ecdysteroids than virginoparae may also indicate some “host plant effect” too. In addition, fundatrices of all tested species have higher concentrations of ecdysteroids than would be expected from their weight and number of ovarioles in comparison with other morphs within the species. In plant samples, besides common phytosteroids such as sitosterol, stigmasterol, and campesterol, the most frequent phytoecdysteroids are 20-hydroxyecdysone, ponasterone A, polypodine 8, ecdysone, and pterosterone (1). Insects cannot synthesize sterols de nouo, and they have a dietary sterol requirement that is met by phytosterols in phytophagous species or by cholesterol in carnivorous insects (19). In the aphid My~us persicae Sulz., the mycetocyte-symbionts synthesize sterols (8). It was also demonstrated that the aphid S. graminum is able to dealkylate phytosterols (i.e., sitosterol) via their symbionts (3). There are many “orphan” ecdysteroid receptors for which no ligands have been identified, suggesting that there may be many undiscovered steroid-like insect hormones (32,33). Consequently, the final question may seem strange but is significant: can secondary plant substances, like phytoecdysteroids, act as “exogenous ” insect hormones (i.e., ligands of “orphan” ecdysteroid receptors) in cases of aphid polyphenism and dormancy of their parasitoids? Further research in this area is ongoing.
Ecdysteroids
183
and Aphids
We thank J. Hardie, K. H. Hoffmann, and J. Lurenz for their helpful comments on earlier drafts of manuscript. The Hungarian Research Council (OTKA 1526) pouided financial support for this research.
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Come. B&hem. Physiol. Vol. 11X, No. 2, pp. 179-183, 1996 Copynght 0 1996 Elsevier Science Inc.
ELSEVIER
Comparison of Ecdysteroid Concentration in Different Morphs of Aphids L. A.
Polgcir,I* B . Duwus,’ W. V&l,* P. Purcher~n,~A. SQ~&S,’ and S. Szehger’
OF ZOOLOGY, PLANTPROTECTION INSTITUTE,HUNGARIANACADEMYOF SCIENCES,P.O. Box 102, H-1525 BUDAPEST,HUNGARY;‘DEPARTMENT OF ANIMALECOLOGY, UNIVERSITY OF BAYRJKJTH, POSTFACH10 1251, D-95770 BAYREUTH,GERMANY;AND 3B~~~~~~~~ ET PHYSIOL~~XE DU ~~VELOPPEMENT, ECOLENORMALESUP~RIEURE, CNRS-URA 686, 46 RUE D’ ULM, 75230 PARISCEDEX05, FRANCE IDEPARTMENT
ABSTRACT. The amounts of ecdysteroids were determined in different morphs of holocyclic monoecious aphids (Acyrrhosiphon pisum Harris, Dysaphis d.evecta Walk., Lachnus roburis L., Schizoluchnus pin& F.) and holocyclic heteroecious aphids (Aphis sumbuci L., Rhop&sighum padi L.) by means enzyme immunoassay. Among the parthenogenetic morphs (fundatrices, virginoparae, and oviparae), the fundatrices have consistently higher amounts of ecdysteroids than those of other morphs of the same species. Alate and apterous virginoparae showed slight differences in their ecdysteroid titer both in heteroecious and monoecious aphids. The migrant morphs (i.e., alate fundatrices and gynoparae) have the lowest amounts of ecdysteroids within a species. With the exception of D. deuecta, the oviparae of both heteroecious and monoecious aphids have the second greatest amounts of ecdysteroids among the morphs living on same host plant. Polyphenism, dispersal behavior, and fecundity in connection with weight, ovariole number, and ecdysteroid concentration of different morphs of aphids are discussed. I(’ ‘. Coprright 0 1996 Elsevier Science Inc. COMPBIOCHEM PHYSIOL115C;2:179-183, 1996. KEY WORDS. Aphid morphs, ecdysteroids, immunoassay, Acyrthosiphon pisum, Aphis sambud, IDysaphisdeuecta, Lachnw roboris, Rho@osiphum padi, Schizoluchnus pineti
INTRODUCTION
life cycle of holocyclic species of aphids includes several parthenogenetic morphs that alternate with a heterosexual generation (6,25). Holocyclic, non-host-alternating (monoecious) aphids live on the same host plants throughout the season. The parthenogenetic morphs of a monoecious aphid species, in sequence of appearance, are fundatrices (i.e., first parthenogenetic generation emerging from overwintered eggs), followed by virginoparae (i.e., summer parthenogenetic generations), which later give birth to oviparae (i.e., heterosexual females) and males, generally in autumn. A holocyclic, host-alternating (heteroecious) aphid species shows wider polyphenic variability. Oviparae develop on woody host plants (i.e., winter host) only, while the summer generations of virginoparae generally reject them. For this reason, these aphids have at least two further morphs (i.e., emigrants and immigrants) that differ reproductively, usually morphologically (15), and mainly in their behaviour (polyethism) from virginoparae. The alate fundatrigeniae (i.e., last parthenogenetic generation on winter The
Address rejxint requests to: L. A. PolgPr, Department of Zoology, Plant Protection Institute, Hungarian Academy of Sciences, P.O. Box 102, H-1525 Budapest, Hungary. Tel 36-11-769555; Fax 36-11~769729; E-mail [email protected]. Received 15 February 1996; accepted 22 May 1996.
host) seek different, usually annual, hosts. The gynoparae (i.e., last parthenogenetic generation on summer hosts) are also alate and return to the winter host. These polyphenisms and polyethisms (4) have evolved largely in response to predictable seasonal changes in habitat quality (6). The environmental signals such as population density (crowding), photoperiod, temperature, and nutrition (i.e., host plant effect) are transformed to chemical cues by neurosecretory cells and are mediated by insect hormones (10). Among the insect hormones, the role of juvenile hormone (JH) was studied intensively in relation to polyphenism and polyethism of aphids (10,14,23) because of the effect of JH on their reproduction (11) and host plant preference (13). In contrast, there is currently no information about differences in ecdysteroid titers among the various aphid morphs (12). Ecdysteroids occur in most adult insects also, and may play an important role in reproduction (5,9,21). There is a report for the aphid Schizaphis gmminum (Rondani) of huge amounts of phytosterols ,(stigmasterol, sitosterol, and campesterol) besides cholesterol, which originated from the host plant, Sorghum bicolor (L.) (3). Additionally, some researchers (2) measured ecdysone in Hyalopterus pruni Geofi. alongside vertebrate-type (estradiol and progesterone) steroids, but they did not consider aphid morph and age. However, very small amounts of ma,
180
L. A. PolgPr et al.
kisterone A besides ecdysone and 20-hydroxyecdysone have been reported in adults of apterous Megoura viciae Buck. (20). Recently, amounts of ecdysteroids were determined from parasitized and nonparasitized instars and adults of A. pisum( 28). In the present study, we measured the ecdysteroid concentration in adults of six aphid species. We compared different morphs of two host-alternating and four non-hostalternating aphids to obtain basic information about ecdysteroid titers among aphid morphs. This study thus represents the first attempt to obtain comparative data in aphids, with a special interest in polyphenism, dispersal behavior, and fecundity.
sone-specific antibodies and an ecdysone-acetylcholinesterase enzyme conjugate as a tracer. The cross-sensitivity of the antibody provided by this kit is 100% to ecdysone and 20-hydroxyecdysone, and 88%, 70%, 63%, and 43% to 2deoxy-20+hydroxyecdysone, polypodine B, 2-deoxyecdysone, and ponasterone A, respectively. The binding rate of other ecdysteroids such as makisterone A and 26-hydroxyecdysone did not exceed 5%. Samples were analyzed in at least two duplicates and incubated in a 96-well microplate. The amount of ecdysteroids in each sample was compared to the 20-hydroxyecdysone standard curve and expressed as pg/mg 20-hydroxyecdysone equivalents. The calorimetric reaction was measured at 415 nm.
MATERIALS Insects
RESULTS
AND METHODS
We tested parthenogenetic and heterosexual morphs (i.e., males and oviparae) of the heteroecious species Rhopalosiphum @di L. (fundatrices, alate and apterous fundatrigeniae, gynoparae, and oviparae from the winter host, Prunus pudus L., and alate and apterous virginoparae from the summer host, Hordeurn vulgare L.) and Aphis sambuci L. (fundatrices, alate and apterous virginoparae, gynoparae, and oviparae, all morphs from the winter host, Sambucus nigra L.), and different morphs of the monoecious species Dysaphis dewecta Walk. (fundatrices, apterous virginoparae, and oviparae from M&s domestica Bork.), Acyrthosiphon pisum Harris (apterous and alate virginoparae from Vicia faba L.), Luchnus roboris L. (fundatrices, alate and apterous virginoparae, and oviparae from Quercus roboris L.), and Schizoolachnus pineti F. (fundatrices, apterous virginoparae, oviparae, and males from Pinus silvestris L.). With the exception of S. pineti, where different instars were mixed, we collected only adult aphids. Most of the collected adults were in prereproductive stage. Parthenogenetic females gave birth to few (zero or one) offspring. Wet weights of aphids were measured by groups of 10 individuals. The mean value of aphid weight was calculated from five measurements. In the case of L. roboris, different morphs were measured individually. Numbers of ovarioles were determined by means of light microscope after dissection of 10 individuals from each morph.
Ecdysteroid
Analysis
For the ecdysteroid measurements, aphid groups with a fresh weight of at least 0.2 g were used. The samples of whole bodies of aphids were prepared according to the method of Porcheron et al. (29). After purification of the samples, they were equalized to 0.1 g fresh weight and dried under Nz. The dried samples were stored at -50°C until analysis by enzyme immunoassay (EI A). Ecdysteroid measurements were carried out with 20-hydroxyecdysone EIA kits from Cayman Co. The competitive immunoassay system utilizes immobilized 20-hydroxyecdy-
Amounts of ecdysteroids differed both between aphid species and morphs and within a species. With the exception of D. devecta, fundatrices have the largest amounts of ecdysteroids in all tested aphid species. This difference between fundatrices and other morphs of the same species in their ecdysteroid concentration is more pronounced in the hostalternating R. padi and A. sambuci (Tables 1, 2) than in the non-host-alternating L. roboris and S. pineti (Table 3; Fig. 1). Generally, fundatrices have the largest weight within a species and, with the exception of R. padi, they have more ovarioles than other tested morphs. Wingless (apterous) females are heavier and have more ovarioles than winged (alate) morphs, and alate morphs have lower amount of ecdysteroids than their apterous counterparts within a species. However, these differences were not significant in all cases. Alate virginoparae of S. pineti and D. dtwectu were not tested, but a similar tendency was observed in two other non-host-alternating aphid species, L. roboris (Table 3) and A. pisum (Fig. 1). The migrant morphs of R. pudi (i.e., alate fundatrigeniae and gynoparae) have the lowest weights and amounts of ecdysteroids among the alate morphs of this species (Table 1). Similarly, the gynoparae have the lowest ecdysteroids, in the case of A. sumbuci (Table 2). Ecdysteroid concentration was measured in males of S. pineti only (Fig 1). Males were found to have the lowest ecdysteroid concentration ( 1.44 t 0.2 pg/mg) and the lowest weight of insects (2.5 5 0.3 mg/lO aphids) in comparison to other morphs of the species. Different morphs of non-host-alternating L. roboris have very similar weights (Table 3). Oviparae of this species have same number of ovarioles as apterous virginoparae, but they have significantly more ecdysteroids than virginoparous morphs. Similarly, in the host-alternating R. pad and A. sambuci, the oviparae are lighter and have similar numbers or fewer ovarioles but have more ecdysteroids than virginoparae from the same host plant. However, this difference is not significant in the case of A. sambuci. Contrary to all other species investigated, in the case of D. devecta the fun-
181
Ecdysteroids and Aphids
TABLE 1. 20.Hydroxyecdysone
equivalents (pglmg, * *SE) in different morphs of Rbopalosiphum 20E equivalents (pg/mg fresh weight)
Fresh weight (mglAphid)
Aphid morph
Fundatrices Fundatrigeniae, apterous Fundatrigeniae, alate Virginoparae, apterous Virginoparae, alate
2.60 1.48 0.69 1.14 0.96
I 0.45 2 0.32 + 0.28 2 0.12 +- 0.11
Gynoparae Oviparae
0.38 0.15
2 0.02 -+ 0.03
A B BC CD
6.85 0.77 1.06 3.75
? 2 ? 2
DE E E
3.19 5 0.42 0.47 t- 0.15 1.53 I 0.23
Within each column, means sharing the same capital letter do not differ at p < 0.05 (Mann-Whitney *Ecdysteroids other than ecdysone and 20.hydroxyecdysone have high probability. ‘rn = 2. data from Wellings et al. (38).
TABLE
2. 20.Hydroxyecdysone
Fresh weight (mg/aphid)
Fundatrices Virginoparae, apterous Virginoparate, alate Gynoparae Oviparae
TABLE
3.18 1.58 0.92 0.65 0.56
A B B c
10 12$ 103 6
C D E
4 6 6
U-test).
? 2 2 I? ”
0.20 0.12 0.07 0.12 0.05
20E equivalents (pglmg fresh weight)
A B C D D
6.88 0.53 0.75 0.68 0.95
2 2 2 ? -e
means sharing the same capital letrer do not differ at p < 0.05 (Mann-Whitney
3. 20+Hydroxyecdysone
0.85*t 0.04 0.08 0.54
Number of ovarioles
equivalents (pg/mg; *SE) in different morphs of Aphis sambuci (n = 4)
Aphid morph
Within each column, *n = 2.
pad; (n = 4)
0.76 A 0.24 B 0.16 B 0.06” B 0.04% B
Number of ovarioies
12 8 6 8 6
U-test).
equivalents (pg/mg ; *SE) in different morphs of Lachnus roboris (II = 4)
Aphid morph
Fresh weight (mg/aphid)
Fundatrices Virginoparae, apterous Virginoparate, alate Oviparae
12.4 11.6 11.2 12.9
-+ 2.2 2 0.8 2 1.7 2 1.5
A A A A
20E equivalents (pglmg fresh weight)
4.86 1.93 1.71 2.24
+- 0.61 -c 0.22 I? 0.64 5 0.19
Within each column, means sharing the same capital letter do not differ at p < 0.05 (Mann-Whitney
A B B C
Number of ovarioles
16 14 12 14
U-test).
datrices have the lowest ecdysteroid concentration and the oviparae have the highest among the morphs tested. The standard 20-hydroxyecdysone curve and the curves from the samples of fundatrices of R. padi and oviparae of A. pisum showed a different shape, which is a sign of ecdysteroids with different cross-reactivity than 20shydroxyecdysone.
DISCUSSION S. pineti
D. dew&a
A. pim
Aphid species and morphs FIG. I. Ecdysteroid concentration in different morphs of aphids. Data on males are included only for S. pineti. Alate virginoparae of S. pinetiand D. devecta and fundatrices of A. pisum were not tested.
The main sources of ecdysteroids in adult insects are the follicle cells (females) and testes (males) in general (reviews in 16,17), but other sources such as epidermis and oenocytes have also been demonstrated (30,37) in the adult cricket, Gqllus bimacuIatus. The role of ecdysteroids in both parthenogenetic and heterosexual morphs of adult aphids can be
L. A. Polgir et al.
similar, namely to promote the early phase of reproduction (i.e., gametogenesis and embryogenesis). There have been studies on fecundity in relation to ovariale number and body size of subsequent generations in the case of some monoecious and heteroecious aphids (22,38). It was stated that fecundity depends on both ovariole number and size of an aphid within a generation. While the ovariole number is stable within a generation, the size of aphids depends on the food quality. Therefore, large aphids are more fecund than small aphids in the same generation because they have more embryos per ovariole at maturity. The number of ovarioles varies in summer generations of heteroecious Aphis f&e (17), R. padi, and Metopolophium dirhodum (38), but the monoecious Drepanosiphum p&unoidis and Eucallipterus tiliae have a constant ovariole number within each generation through the season, with only the first generation (fundatrices) having two more ovarioles than subsequent generations (38). Our finding that fundatrices have generally higher ecdysteroid levels within a species complements the observation that they have higher reproductive rates, and adults contain more embryos than virginoparae of the same species. Similarly, less fecund gynoparae of R. padi (36) had the lowest amounts of ecdysteroids. However, apterous fundatrigeniae of R. padi have two more ovarioles than fundatrices (38), but their weight differs markedly (Table 1). All these observations suggest that different titers of ecdysteroids may be related to fecundity and movement behavior (i.e., dispersal and migration) for an aphid species. Adult aphids have small amounts of ecdysteroids in comparison to related heteropteran species (2). One experiment showed 50 times more (ca. 3 pg/mg) 20-hydroxyecdysone (20E) than makisterone A (ca. 0.06 pg/mg) and 10 times more 20E than ecdysone (ca. 0.3 pg/mg) in M. viciae (20). It seems to be that in aphids, contrary to many heteropteran species, the main ecdysteroids are 20-hydroxyecdysone and ecdysone instead of makisterone A. Recently, some authors reported approximately 4 pg/mg ecdysteroids (expressed as ecdysone equivalents) from whole-body extracts of A. pisum 2 days after the onset of reproductive activity of adults (28). All these investigations were carried out by radioimmunoassay. However, we used single enzyme immunoassay, and our results (3.43 -+ 0.37 pg/mg 20E equivalents) in apterous virginoparae of A. piston met with these observations. We found that fundatrices and oviparae of all tested species, with the exception of D. deuecta, have higher amounts of ecdysteroids than other morphs from same host plant. This trend is more evident in the case of L. roboris, where the different morphs have similar weights (Table 3). The reason for this finding may be sought in potential differences in the ecdysteroid production of ovarioles producing embryos or yolky eggs. Ecdysteroids may also occur in male insects, but in much lower titers than found in females. In males, ecdysteroids
may play a role in spermatogenesis (24). Our results are consistent with the relative size of the gonads in this species. While oviparae have very large ovarioles, the testes are comparably small. It has also been suspected that differences in the hormonal state of the various morphs and in the balance between JH III and ecdysteroids could be involved in diapause induction in aphid parasitoids (26,27). The present results indicate that such differences may actually occur. Besides crowding, photoperiod, and temperature, the “host plant effect” is known to play a significant role in aphid polyphenism. Senescent host plants usually induce alate virginoparae and/or oviparae morphs of holocyclic aphids (18,3 1,34). During plant life, phytoecdysteroids alter in quality and quantity as well (35), according to measurements in Ajuga reptans L. These results raise the question of whether phytoecdysteroids are the secondary plant substances involved in at least one part of the “host plant effect” in aphid polyphenism. We did not find considerable differences in ecdysteroid concentration between alate and apterous virginoparae of R. Dadi from their summer host, H. uulgure, nor between alate and apterous fundatrigeniae collected from their winter host, I’. J&US. These four morphs have similar weight, but they differ markedly in number of ovarioles. AIate and apterous fundatrigeniae reared on their winter host have more ovarioles but lower ecdysteroids levels than the virginoparous generation grown on their summer host, H. vulgare. The fact that fundatrigeniae have more ovarioles but lower ecdysteroids than virginoparae may also indicate some “host plant effect” too. In addition, fundatrices of all tested species have higher concentrations of ecdysteroids than would be expected from their weight and number of ovarioles in comparison with other morphs within the species. In plant samples, besides common phytosteroids such as sitosterol, stigmasterol, and campesterol, the most frequent phytoecdysteroids are 20-hydroxyecdysone, ponasterone A, polypodine 8, ecdysone, and pterosterone (1). Insects cannot synthesize sterols de nouo, and they have a dietary sterol requirement that is met by phytosterols in phytophagous species or by cholesterol in carnivorous insects (19). In the aphid My~us persicae Sulz., the mycetocyte-symbionts synthesize sterols (8). It was also demonstrated that the aphid S. graminum is able to dealkylate phytosterols (i.e., sitosterol) via their symbionts (3). There are many “orphan” ecdysteroid receptors for which no ligands have been identified, suggesting that there may be many undiscovered steroid-like insect hormones (32,33). Consequently, the final question may seem strange but is significant: can secondary plant substances, like phytoecdysteroids, act as “exogenous ” insect hormones (i.e., ligands of “orphan” ecdysteroid receptors) in cases of aphid polyphenism and dormancy of their parasitoids? Further research in this area is ongoing.
Ecdysteroids
183
and Aphids
We thank J. Hardie, K. H. Hoffmann, and J. Lurenz for their helpful comments on earlier drafts of manuscript. The Hungarian Research Council (OTKA 1526) pouided financial support for this research.
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