Endogenous levels of insect juvenile hormones in larval, pupal and adult stages of the tomato moth, Lacanobia oleracea

Pergamon

0022-1910(95)00031-3

J. Insect Physiol. Vol. 41, No. 8, pp. 641-651, 1995 Copyright 0 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0022-1910/95 $9.50 + 0.00

Endogenous Levels of Insect Juvenile Hormones in Larval, Pupal and Adult Stages of the Tomato Moth, Lacanobia oleracea J. P. EDWARDS,*?

T. S. CORBITT,*

H. F. McARDLE,*

J. E. SHORT,*

R. J. WEAVER*

Received 7 November 1994; revised 21 February 1995

Endogenous levels of juvenile hormones 0, I, II and III have been measured each day during the 4th, 5th and 6th larval stadia of the Tomato moth, Lacanobiu oZeracea, using physico-chemical methods. Levels of the same hormones have also been measured at selected times during pupal development, and over the first 5 days of adult life in males, and in mated and virgin females. JH I and JH II were the predominant homologues in this insect in all developmental stages, although JH III was also present in some samples. Hormone levels were relatively high in 4th and 5th instar larvae, hut were much reduced in 6th (final) instar larvae. Hormone levels increased in larvae that were about to moult to the subsequent stage. Juvenile hormones were absent in pupae during the first half of pupal development, but increased in pharate adults. Adult males and females contained substantially more hormone than pre-adult stages, although levels in virgin moths were lower than in mated adult females. JH 0 was detected in some extracts of mated adult female moths. These results are discussed in relation to the likely role of juvenile hormones in modulating various aspects of the physiology of this insect. Juvenile hormone

Lepidoptera

Lacanobia oleracea

INTRODUCTION

Reproduction

III appears to be present) lepidopterans are known to produce at least 5 JHs (Bergot et al., 1980, 1981a; Schooley et al., 1984). In addition, radioimmunoassay methods may be unable to differentiate between JHs and some of their metabolites and precursors (e.g. JH acids). The development of the in vitro radiochemical method for measuring the biosynthetic activity of isolated corpora allata (Pratt and Tobe, 1974; Tobe and Pratt, 1974) provided a fast and reliable technique for estimating JH levels extrapolated from the rate of hormone biosynthesis by corpora allata in vitro and, at least in cockroaches, in vitro rates of JH biosynthesis have indeed been shown to correlate closely with directly-measured endogenous hormone titres (Tobe et al., 1985, Edwards et al., 1990). Notwithstanding the introduction of new bioassay methods (e.g. Munduca black larval assay (Truman et al., 1973) the refinement of radioimmunoassay techniques (Goodman et al., 1990) and the development of several micro-chemical techniques (Mauchamp et al., 1979; Rembold and Lackner, 1985) it appears that the most accurate and reliable method for measurement of in vivo JH levels is the physico-chemical method developed by Bergot et al. (1981b). This latter technique employs chromatography/mass spectroscopy gas (GC/MS) to identify and quantify methoxyhydrin de-

In insects, juvenile hormones (JHs) control several important physiological processes including metamorphosis, reproduction, diapause, polymorphism and pheromone production. However, despite the importance of these molecules as regulators of insect physiology, in only very few species has there been a thorough investigation of fluctuations in levels of juvenile hormones over a substantial part of post-embryonic development. In part, this has been due to the difficulties inherent in measuring sub-nanogramme quantities of JHs in insect tissues or haemolymph. Early methods for estimating JH levels using bioassays (e.g. the Galleriu wax test, Gilbert and Schneiderman, 1960) were relatively crude and inaccurate. Similar problems are inherent in the use of indirect methods (e.g. measurement of corpus allatum volume or cell size), and more recently developed techniques (e.g. radioimmunoassay; Baehr et al., 1976; Strambi et al., 1981) have been prone to lack of specificity and reproducibility. Specificity (i.e. the ability to differentiate between JH homologues) is particularly important in studies of lepidopterans since, unlike the majority of other insect orders (where only JH *Central Science Laboratory, Ministry of Agriculture, Fisheries Food, London Road, Slough, Berks SL3 7HJ, U.K. tTo whom all correspondence should be addressed.

Metamorphosis

and

641

642

J. P. EDWARDS

rivatives of the JHs, following their extraction from insect tissues and subsequent purification. This method has been used to produce the most detailed studies of JH titres during the last stage of larval development in the lepidopterans Munduca sexta (Baker et al., 1987) and Trichoplusia ni (Jones et al., 1990). Lepidopteran species in which studies of JH identity or levels have been made in various developmental stages include, in the Saturnidae, Hyalophora cecropiu (Roller et al., 1967; Meyer et al., 1968), Hyalophora gloveri (Dahm and Roller, 1970), Samia Cynthia (Roller and Dahm 1974), Bombyx mori (Kimura et al., 1989) and Attacus arias (Baker et al., 1985); in the Pyrallidae, Galleria mellonellu (Hsiao and Hsiao, 1977; Plantevin et al., 1984; Rembold and Sehnal, 1987); in the Sphingidae, M. sexta (Judy et al., 1973; Fain and Riddiford, 1975; Peter et al., 1976; Bergot et al., 1980; Baker et al., 1986; Edwards, 1987); and in the Noctuidae, Mamestra brassicae (Vargas et al., 1976), Spodoptera littoralis (Zimowska et al., 1989), Mamestra oleracea = Lacanobia oleracea (Yagi, 1976; Vargas et al., 1992) T. ni (Jones et al., 1990), Pseudaletia unipuncta (Cusson et al., 1993) and Heliothis zea (Satyanarayana et al., 1991). However, only in those studies with M. sexla, A. atlas and T. ni has the physico-chemical method of Bergot et al. (198 1b) been employed to determine JH levels. Moreover, despite these studies, there has, to date, been no detailed study of the quantitative or qualitative variations in JH levels over a substantial part of the post embryonic development of any lepidopterous insect. The Tomato moth, Lacanobia oleracea (L.) is a member of the economically important noctuid group of lepidopterous pests, and is an occasional pest of glasshouse tomatoes in the U.K. (Lloyd, 1920; Burgess and Jarrett, 1976). In the past, infestations of L. oleracea were easily controlled with chemical insecticides, and this species was not considered to be a major pest. Recently, the extensive use of biological control agents for the control of other pests affecting tomatoes (e.g. whitefly and red spider mite) has led to a concomitant reduction in the use of conventional (neurotoxic) insecticides on glasshouse-grown crops. In turn, this has opened the way for a resurgence of Tomato moth infestations in greenhouse tomatoes and other crops. Currently, we are investigating alternative ways of controlling the Tomato moth and other lepidopterous pests, that are compatible with the safety of the environment and of the consumer, and with the use of biological control agents. For example, we are evaluating the use of juvenile hormone analogues, anti juvenile hormone agents and allatostatic neuropeptides as potential control agents for L. oleracea, because such molecules have considerable potential as environmentally acceptable insecticides (Staal, 1977; Edwards and Menn, 1980; Menn et al., 1989). Since molecules of this type are intended to disrupt the insect’s endocrine system (particularly that controlled by JHs), we decided to investigate the natural endogenous variations in JH levels in L. oleracea, in order to conduct bioassays of novel molecules at the most appropriate

et al.

times during larval, pupal or adult development. In addition, we wished to further explore the ways in which changing levels of JHs might modulate or alter a variety of physiological and behavioural phenomena in this insect. With these aims in mind, we have measured the levels of juvenile hormones 0, I, II and III during the 4th, 5th and 6th larval stadia of L. oleracea. In addition, levels of these hormones have been measured at selected times during the pupal stage, in adult male, and in virgin and mated adult female moths. These results are discussed in relation to their possible significance in terms of the regulation of physiological processes in this species, and in relation to results previously obtained in other lepidopteran species.

MATERIALS

AND METHODS

The insects used in these experiments were from laboratory cultures of L. oleracea, maintained inside Gallenkamp F-400 environmental cabinets (Sankyo Ltd). These moths originated from a wild population collected from tomato glasshouses on Jersey in 1990. Larval moths were reared at 20°C and 65% r.h. in polythene sandwich boxes (25 x 25 x 5 cm) with muslin tops, and were fed on a maize-flour based noctuid artificial diet (Koran0 Ltd, Montalieu, France). Larvae were kept under a 16 h light : 8 h dark (16L : SD) photoperiod (long-day) which was non-diapause inducing. Pupae were kept in constant darkness at 20°C and 70% r.h. Adult moths were kept under the same conditions as larvae, and were fed with cotton wool pads moistened with an aqueous solution containing 10% sucrose, 2 g/l (w/v) methyl-4-hydroxybenzoate and 2.0% (v/v) of a mixed vitamin solution. All larval insects were selected within 24 h windows from each of the last three larval stadia using standard physiological stage indicators such as head capsule slippage, ecdysis, head capsule width etc. (T. S. Corbitt, unpublished results). These larvae were equivalent to the gate 2 L. oleracea larvae in which Vargas et al. (1992) studied JH levels. We made no attempt to distinguish between the sexes of any larvae used in the experiments. Thus, JH levels were measured on each day of development during the 4th, 5th and 6th larval stadia. In the 4th and 5th larval stadia, day 5 insects were examined and only those exhibiting head capsule slippage (clear head capsule stage) were used in the JH titre determinations. The 4th and 5th larval stadia both have a mean duration of 5.0 days at 20°C (T. S. Corbitt, unpublished results) and 5 day old clear head capsule larvae normally moult within 24 h of selection. In the final larval stadium, 9 and 10 day old larvae were also examined and classified either as wandering larvae (day 9) or pre pupae (day lo), and only these individuals were used in the JH determinations for the respective days. In L. oleracea, the pupal stage lasts for about 25 days at 20°C (T. S. Corbitt, unpublished results). Juvenile hormone levels were measured in pupae aged 1,2, 5, 10,

JUVENILE

HORMONE

LEVELS

15, 20 and 25 days after pupal eclosion. Day 25 pupae were examined prior to selection, and only those showing characteristics of the pharate adult stage were used in the experiments. Furthermore, these pharate adults (day 25 “pupae”) were examined and the sexes segregated on the basis of external morphological characteristics (T. S. Corbitt, unpublished results) so that JH titres could be measured separately in male and female pharate adults. Adult moths were staged at daily intervals from the time of adult eclosion. Adult females were either kept isolated with food (virgin females), or were kept in the presence of both males and food (subsequently referred to as “mated” females). Virgin adults were obtained by segregating batches of male or female pupae before adult eclosion, and allowing adult emergence to take place in isolation. Batches of larvae, pupae, or adults weighing between 3 and 5 g (wet wt) were accurately counted and weighed, and quick-frozen by immersion in liquid nitrogen, before being stored in a low temperature freezer ( < - 20°C) prior to being processed for JH titre determination. Although preliminary experiments had shown no detectable effect of this procedure on JH levels when samples were stored for several months (J. P. Edwards, unpublished results) these frozen samples were usually processed within 1 month of collection. Juvenile hormone titre determination was carried out using the method of Bergot et al. (1981 b) which utilizes a variety of selective extraction/partition processes, chemical conversion of the hormones to stable methoxyhydrin derivatives, further purification by high performance liquid chromatography (HPLC) and identification and quantification of JHs by gas chromatography-mass spectroscopy (GC/MS) using selective ion monitoring (SIM). Thus, ions at m/z = 76 were used to quantify JH III and internal standard (3H iso-JH II ethyl ester) methoxyhydrins, and ions at m/z = 90 were used for quantification of JH 0, JH I and JH II methoxyhydrins. Calibration of the mass spectrometer was accomplished with an external standard solution containing known quantities of JH and internal standard methoxyhydrins (-0.080 pgglpl). In addition, some samples (especially those where the detection of JH III was inconsistent) were re-analysed by the “accurate mass” method described by Short and Edwards (1992) whereby selective ion monitoring was performed at m/z = 76.084. This procedure can enable accurate measurement of the intensity of ions due exclusively to the JH III methoxyhydrin fragment, without interference from ions deriving from potential contaminants (e.g. lipids and phthalates). For each day of larval development from the moult to the 4th larval stadium to the formation of the pupa, a minimum of three separate determinations were made with different batches of insects at different times. For pupae, where JHs were generally absent, only one determination was made unless there was evidence that JHs were present, in which case, the analyses were repeated. In adult moths, only one determination was made unless unexpectedly high or low JH levels were detected, where-

IN THE

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643

upon determinations were repeated. JH levels were measured at daily intervals for the first 5 days of adult life in males, “mated” females and virgin females. Juvenile hormone levels were calculated as means of the number of determinations per day or stage (replicates). However, as with all determinations, each individual replicate was based on the average JH levels present in several insects. Juvenile hormone levels are expressed as rig/g wet weight of live insect tissue.

RESULTS

Table 1 shows the daily changes in average individual wet weight of the L. oleracea larvae used in these experiments, along with the total number of individuals used in each JH determination. During the 4th and 5th stadia, there was a daily increase in the mean wet weight of larvae as each stadium proceeded, with the 5th instar larvae gaining weight more rapidly than the 4th instar larvae. In the first 5 days of the final (6th) stadium, mean daily weight gain was extremely rapid (Table 1). Subsequently, as the final instar larave entered the wandering phase (days 6, 7, 8 and 9) mean weight decreased each day, presumably due to increased locomotory activity, cessation of feeding and gut purge. During the pre-pupal stage (day 10) further slight weight loss occurred (Table 1). The mean levels of the 3 juvenile hormone homologues (JH I, II and III) detected in larval and pupal stages of L. oleracea are presented in Table 2, along with the number of replicates upon which each JH titre determination was based. For clarity and ease of comparison, JH levels present in these stages are diagrammatically presented in Fig. 1. TABLE of 4th,

1. Total number of insects, and average wet weights 5th and 6th stadium L. oleracea larvae used in juvenile hormone titre determinations

Larval stage, age (days) and description 4th 4th 4th 4th 4th 5th 5th 5th 5th 5th 6th 6th 6th 6th 6th 6th 6th 6th 6th 6th

stadium stadium stadium stadium stadium stadium stadium stadium stadium stadium stadium stadium stadium stadium stadium stadium stadium stadium stadium stadium

day day day day day day day day day day day day day day day day day day day day

No. of insects

I

722 342 306 218 238 206 132 93 82 72 44 39 32 23 24 27 29 33 56 52

2 3 4 5 (CHC)

1 2 3 4 5 (CHC) 1 feeding 2 feeding 3 feeding 4 feeding 5 feeding 6 wandering 7 wandering 8 wandering 9 wandering 10 (prepupa)

“CHC = clear head capsule

stage.

Mean

weight (g)

0.0185 0.0362 0.0432 0.0575 0.0572 0.0748 0.1114 0.1726 0.1975 0.2079 0.2837 0.3850 0.4896 0.6991 0.6975 0.6486 0.5598 0.5576 0.3441 0.3039

644

J. P. EDWARDS

et al.

TABLE 2. Levels of juvenile hormones I, II and III (rig/g wet weight + SEM”) during the 4th, 5th and 6th larval stadia, and at selected times during pupal development of the Tomato moth, L. oleracea Total JH I and II

Replicates Larval 4th L 4th L 4th L 4th L 4th L 5th L 5th L 5th L 5th L 5th L 6th L 6th L 6th L 6th L 6th L 6th L 6th L 6th L 6th L 6th L Pupa Pupa Pupa Pupa Pupa Pupa Pupa

stage/day

(n =)

Dl D2 D3 D4 D5 (CHC)b Dl D2 D3 D4 D5 (CHC)b Dl D2 D3 D4 D5 D6 D7 D8 D9 DlO prepupab Dl D5 DlO D15 D20 D25P D25mC

3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 4 3 1

1 1 1 1 2 2

“SEM values co.01 are given as 0.00. bCHC = clear head capsule stage. ‘Female (f) and male (m) pharate adult

JH II + SEM

JH III & SEM 0.000 0.008 0.000 0.002 0.065 0.016 0.018 0.000 0.017 0.006 0.000 0.015 0.027 0.000 0.010 0.002 0.000 0.008 0.003 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000

f. 0.00 & 0.00 k 0.04 + 0.02 + 0.02 + 0.01 f 0.00 ~ & 0.02 f 0.01 f 0.00 k 0.00 * 0.00 ~ ~ -

JH I&SEM

0.463 + 0.16 0.969 f 0.23 0.995 f 0.41 0.778 & 0.18 1.078 & 0.12 0.398 & 0.19 0.412 + 0.07 0.313 f 0.13 0.340 + 0.05 1.071 + 0.21 0.000 0.001 * 0.00 0.000 0.000 0.000 0.008 + 0.00 0.009 & 0.00 0.018 f 0.02 0.046 + 0.01 0.043 * 0.02 0.000 0.000 0.000 0.000 0.025 1.706f0.11 1.315 f 0.19

0.109 0.181 0.194 0.212 0.406 0.097 0.041 0.053 0.095 0.355 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.013 0.011 0.000 0.000 0.000 0.000 0.005 0.792 0.301

f f f + + + f f * *

f +

f +

0.03 0.04 0.06 0.01 0.12 0.05 0.00 0.00 0.02 0.02 ~ ~ 0.00 0.00 0.33 0.02

0.572 1.150 1.189 0.990 1.484 0.495 0.453 0.366 0.435 1.426 0.000 0.001 0.000 0.000 0.000 0.008 0.009 0.018 0.062 0.054 0.000 0.000 0.000 0.000 0.030 2.498 1.616

stage.

In the 4th and 5th stadia JH II was the predominant homologue, with lesser quantities of JH I also being present. JH III was detected inconsistently (and at very low levels) in some samples of 4th, 5th and 6th instar larvae. It is possible that the inconsistent presence of such very low levels of JH III could have been

artefactual. No JH 0 was detected in any of the larval stadia examined, or in pupae. The ratio of JH II to JH I during the 4th and 5th larval stadia remained remarkably constant (approx. 4: 1) irrespective of fluctuations in total juvenile hormone levels. Total JH levels were generally higher in 4th instar larvae than in 5th instars

Juvenile

4th stadium

hormone

__

PuPa

6th (fmel) stadium Feeding

Wandering

I,, 123451*3451

2

1, 3

4

5

6

II&&_ 7

8

9

/ 1s

,

5

10

h. ,5

10 15F25M

Age (days) of larval and pupal development. FIGURE

1. Average

levels (k SEM) of JH I and II found each day in 4th, 5th and 6th stadium selected times during pupal development.

L. oleracea larvae,

and at

JUVENILE

HORMONE

LEVELS

(mean total JH I + JH II = 1.07 and 0.63 rig/g,, respectively). However, despite the quantitative differences in JH levels in these two stadia, the changing pattern of JH levels was similar in both. Thus, during the first 4 days of each stadium, combined JH I and II levels remained relatively constant, before increasing in 5 day old (clear head capsule) larvae just prior to the moult to the next stadium (Fig. 1). By contrast with JH levels detected in the antepenultimate and penultimate larval stadia (4 and 5) JH levels were extremely low during the major part of the final (6th) larval stadium. Neither JH I nor JH II were detected in day 1, 3, 4 and 5 day old 6th instar larvae, and only a trace of JH II was present in day 2 6th instars (Table 2). In addition, there was some evidence that low levels (ca 0.02 rig/g)) of JH III were present in some 6th instar larvae (Table 2). Once again, the presence of JH III in these insects was not detected consistently in all samples, and could have been spurious. During the second half of the 6th larval stadium (on days 6, 7 and 8) there was consistent evidence of the presence of low levels of JH II (Table 2, Fig. 1). Finally, in larvae at the end of the wandering phase (day 9) and in day 10 an increase in pre-pupae (i.e. -24 h prior to pupation), JH II levels (to ca 0.04 rig/g)) was detected, as were pro-rata increases in JH I (Table 2, Fig. 1). In L. oleracea pupae, we were unable to detect the presence of any JH homologue at any of the times examined during the first 15 days of pupal development (Table 2). We therefore conclude that, for the first half of pupal development, this insect is essentially devoid of JH. Later however, in 20 day old pupae, both JH I and JH II were present in small quantities (ca 0.03 rig/g total JH), and in pharate adults (25 day old “pupae”) we found a marked increase in JH I and II levels (Table 2, Fig. 1). Interestingly, we detected slightly higher JH I and II levels in female pharate adults than in males

IN THE

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645

TABLE 3. Levels of juvenile hormones I, II, III, and 0 in fed adult females in the presence of males (FEM) adult males (MAL) and fed virgin adult females (FVIR) of L. oleracea. JH levels given as rig/g wet weight

Stage/day FEM FEM FEM FEM FEM MAL MAL MAL MAL MAL FVIR FVIR FVIR FVIR FVIR

n =

JH III

JH II

JH I

JH 0

Total JH I and II

2 1 2 2 1 1 1 1 1 1 2 2 2 2 2

0.438 0.515 0.356 0.335 0.600 0.427 0.000 0.341 0.345 0.483 0.148 0.136 0.093 0.176 0.258

15.267 15.740 18.530 16.171 29.803 25.509 27.274 33.932 34.515 39.794 20.406 10.864 2.854 2.464 4.893

11.904 31.531 30.990 26.324 34.480 5.900 16.962 26.515 30.385 47.611 17.724 12.953 4.022 2.143 2.919

0.000 0.000 0.085 0.081 0.180 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000

27.171 47.271 41.389 42.495 64.283 3 1.409 44.236 60.447 64.900 87.405 38.130 23.817 6.876 4.607 7.812

Dl D2 D3 D4 D5 Dl D2 D3 D4 D5 Dl D2 D3 D4 D5

2.5 and - 1.6 rig/g respectively). We did not detect the presence of either JH III or JH 0 in any of the pupae examined. In adult L. oleracea, JH levels were much higher than those found in pre-adult stages (Table 3, Fig. 2). In females kept in the presence of males and in adult males, combined JH I and II levels appeared to increase slightly over the first 5 days of adult life. JH III was also detected in l-5 day-old “mated” females and in males, but at much lower levels (-0.4 rig/g)) than the predominant homologues JH I and JH II. In addition, in extacts of 3, 4 and 5 day old “mated” females, low levels (-0.2 rig/g)) of JH 0 were detected (Table 3). By contrast with the relative abundance of the two primary homologues (JH I and JH II) found in larval L. oleracea (where JH II was always the predominant homologue), the ratio of JHI : JH II in adult females and (-

50

45 40

---Jq-

35 30 25

VIRGIN FEMALE

20 15 10 5 0 FDI

FD2

FD3

FD4

FDS

MD1 MD2 MD3 MD4 MD5

Age (days) FIGURE

2. Levels of JH I and II found

during

VDI

vD2

vD3

vD4

MS

of adult life.

the first 5 days of adult L. oleracea.

life in males, and in mated

and virgin

females

of

J. P. EDWARDS et al.

646

in adult males was much closer to 1: 1 and, in some samples, JH I was present in greater quantity than JH II (Table 3, Fig. 2). Combined JH I and II levels were generally much higher in adult males (reaching 87 rig/g on day 5) than in “mated” adult females, and we were unable to detect JH 0 in any of the male samples (Table 3). In virgin female moths, we found approximately the same quantities of JH I and II in 1 day old virgins as we found in 1 day old females kept in the presence of males (Table 3). However, by contrast with the results obtained with “mated” females, combined JH levels in virgins showed an apparent decline during the first 5 days of adult life, from - 38 on day 1 to - 8.0 rig/g on day 5. JH 0 was not detected in any of the virgin female samples (Table 3). DISCUSSION

Since the pioneering studies on insect juvenile hormones by Wigglesworth (1934, 1935) almost 50 years ago, investigation of the precise roles of these molecules in regulating various aspects of developmental, behavioural and reproductive physiology in insects has been the subject of a plethora of scientific papers, too numerous to cite. From these studies it is generally accepted that the juvenile hormone(s) are present, at relatively high levels, throughout all but the final larval stadium. The presence of JH in larval insects serves to maintain the larval state, and prevent metamorphosis. In the last larval stadium JH levels drop, and this allows the insect to proceed with the moult to the adult stage. In hemimetabolous insects, adult features develop gradually throughout nymphal development, and the moult to adult proceeds directly from the final nymphal stage without the intervention of a pupal stage. By contrast, holometabolous insects, including the Lepidoptera, exhibit a more complex metamorphosis wherein an additional developmental stage (the pupa) intervenes between the final larval stadium and the adult stage. It is generally believed that JH is absent during pupal development in order that the metamorphosis may continue, and adult structures may differentiate and develop. Subsequently, in the adult insect, it is believed that JH is produced again to act as a gonadotropin, modulating various aspects of reproductive physiology including vitellogenin synthesis and uptake (Englemann, 1970; Koeppe et al., 1985). From the present experiments, we have obtained confirmation that, in general, these supposed changes in the levels and functions of JH do indeed occur in viva in a representative lepidopterous insect. JH levels were clearly higher in the antepenultimate and penultimate larval stadia than in the final (pre-metamorphosis) larval stadium. JHs were also detected in pre-pupae and in pharate adults, although the majority of the pupal stage was devoid of these hormones. Finally, high levels of JHs were present in adult insects of both sexes, but were much reduced in virgin female moths.

On considering our results, we see that the predominant homologues in this species are JH I and JH II. Although it appears from our data that JH III was occasionally present, we suggest either that the presence of this homologue was artefactual [since levels detected were very close to the limit of accurate detection (-0.01 rig/g)) and the presence of JH III was sporadic], or that JH III is indeed present, at very low levels, throughout much of post-embryonic development. Unfortunately, the very low levels of JH III made unequivocal confirmation of its presence very difficult, even using the accurate mass method of Short and Edwards (1992). This is because the increased precision with which the JH III methoxyhydrin fragmentation ion can be detected and quantified, is counterbalanced by a reduction in overall sensitivity. Interestingly, other researchers have also found inconsistent evidence of the presence of JH III in other moth species (Baker et al., 1987; Jones et al., 1990). JH II was the predominant homologue in larval L. oleracea, and this result was consistent with the results obtained by direct physico-chemical methods in two other noctuid larvae: T. ni and S. littoralis. In T. ni (Jones et al., 1990) JH II levels ranged from 0.001 to 0.04 rig/g (values somewhat lower than those found in 4th and 5th stadium L. oleracea). In S. littoralis (Zimoska et al., 1989) measured 1.75 pmole JH II/ml of haemolymph from newly-moulted last instar larvae. Although direct comparison of haemolymph and whole body JH titres is difficult, this value approximates to about 0.5 rig/g--again levels comparable to those found in L. oleracea. In addition, in newly-moulted 4th (penultimate) stadium larvae of M. sexta, JH II was also found to be the predominant homologue, and levels of 0.5-0.9 rig/g were reported by Baker et al. (1986) and by Edwards (1987). As well as the predominant homologue JH II, we also found significant levels of JH I in larval L. oleracea. In fact, the ratio of the relative quantities of the two homologues JH I and JH II remained remarkably constant (- 1:4) irrespective of overall fluctuations in JH levels. Thus, in larval L. oleracea, the presence of JH II was always associated with a lesser quantity of JH I. A similar association between these two homologues also occurs in 4th stadium M. sexta larvae (Baker et al., 1986; Edwards, 1987; Edwards et al., 1983). However, in T. ni, Jones et al. (1990) found only very low levels of JH I and concluded that the presence of this homologue was probably artefactual. In Pieris brassicae, Mauchamp et al. (1979) found only JH I in last stadium larvae (at levels of approx. 7.0 rig/g)) and did not find significant amounts of JH II or III. Similarly, Vargas et al. (1992) found only JH I in newly-moulted 6th stadium L. oleracea larvae ( - 1.O rig/g)) and was unable to detect the presence of either JH II or III in this species. M. sexta and T. ni are the only other moth species in which changing JH levels have been monitored [using the method of Bergot et al. (1981b)] at regular intervals over a period of larval development. Unfortunately, in

JUVENILE

HORMONE

LEVELS

M. sex&, only the final larval stadium was used (Baker et al., 1987) and, in T. ni, JH levels were measured throughout the final stadium, and in larvae that were at the very end of the penultimate larval stadium (Jones et al., 1990). For this reason, we have little information about detailed fluctuations of the JH levels in antepenultimate and penultimate instar larvae of lepidopterans. In our experiments, we found that there was a pronounced peak in total JH levels in clear head capsule 4th and 5th instar larvae (i.e. those approaching the moult to the subsequent stadium). Interestingly, Jones et al. (1990) also found a high titre (ca 0.42 rig/g)) of JH II just prior to the moult to the final stadium in T. ni. By contrast, in M. sexta, high levels of JH I and II are found immediately following the moult to the penultimate (4th) larval instar (Edwards et al., 1983; Baker et al., 1987; Edwards, 1987). We are unable to say whether it is the continued presence of JH throughout the 4th and 5th stadia, or the pre-ecdysial peak of JH that is primarily responsible for maintaining the larval state during the subsequent stadium. In L. oleracea, the levels of JHs were markedly lower in the final larval stadium by comparison with the two previous stadia. It is quite likely that the paucity of JHs during this last larval stage is responsible for the initiation of metamorphosis. There have been several previous studies in which JH levels have been measured in last instar lepidopteran larvae. For example, in T. ni, the pattern of changing JH (II) levels (Jones et al., 1990) is remarkably similar to that found in L. oleraceu. Thus, following relatively high JH levels prior to the moult to the last stadium, JH levels fell to very low levels during the majority of the final stadium before increasing again approx. 24 h before pupal ecdysis. A similar pattern, involving both JH II and JH I, also occurs in final instar M. sextu larvae (Baker et al., 1987). In last instar Pieris brassicae larvae, the situation was, again, very similar although JH I was detected in larvae prior to the wandering stage (Mauchamp et al., 1979). By contrast, in L. oleruceu, we detected no JH I and only a trace of JH II during the feeding phase in last instar larvae, and a slight increase in JH I and II levels coincident with the wandering phase of these larvae. A similar pattern of JH fluctuation was reported in M. brussicue where increases in JH levels were associated with the wandering stage (Vargas et al., 1976) although Zimowska et al. (1989) were unable to detect JH during the wandering stage of S. littoralis. From these examples, it can be seen that there has been considerable variation in the types and levels of juvenile hormones reported in a range of lepidopterous larvae. No doubt, the main reason for this is the difficulty of accurately identifying and precisely measuring the very small quantities of these closely related molecules in insect tissues. None the less, the results of many of these studies concur in general terms about the overall pattern of hormone levels in last instar larvae and, in particular, that there are increases in JH levels associated with the pre-pupal stage in several lepidopteran species. The appearance of increased JH

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levels immediately prior to the moult in antepenultimate and penultimate instar larvae, coupled with similar increased hormone levels in pre-pupae and in pharate adults, leads us to suggest that these pre-ecdysial increases may be intimately associated with the induction of the moulting process, and possibly with the stimulation of ecdysteroid production by the prothoracic glands. However, the precise significance of the more subtle changes in JH titre during larval development remains a subject for speculation. On the basis of our results, we suggest that the almost complete absence of JHs during the first half of the final larval stadium of L. oleraceu enables the process of metamorphosis. Furthermore, it is possible that the reappearance of JHs in 6, 7, 8 and 9 day old final instar larvae may be associated with the change in behaviour from the relatively sedentary feeding stage to the wandering stage and the subsequent formation of the pupal cell (burrowing). However, this interpretation would be contrary to the situation that obtains in T. ni where it appears that the absence or reduction in JH activity initiates the cessation of feeding and the initiation of wandering (Jones, 1985). At the end of larval development, in pre-pupae, the presence of still more JH may serve to initiate ecdysis. In the closely-related species M. brussicue, previous studies have shown that JH mimics can indeed activate the prothoracic glands (Hiruma et al., 1978; Hiruma, 1980). Similarly, in T. ni the presence of JH during the pre-pupal stage has been shown to be critical for the success of the larval/pupal ecdysis (Jones and Hammock, 1985). Another possible role for the pre-pupal rise in JH levels might be to prevent too rapid a metamorphosis to the adult stage. In this respect, it would be interesting to see if similar increases in JHs occur in hemimetabolous insects (which moult directly from larva to adult). In addition to their role in the regulation and co-ordination of behaviour prior to metamorphosis as well as the process of metamorphosis itself, JHs have been implicated in the modulation of several other aspects of the physiology of larval insects; notably, diapause determination and polymorphism [for reviews see Denlinger (1985) Hardie and Lees (1985)]. Several authors have linked increased JH levels in larval noctuids (including L. oleruceu) with the initiation of diapause development. In M. brussicue, Yagi (1976) Vargas et al. (1976) and Vargas and Mauchamp (1988) reported higher levels of JH in those larvae that had been exposed to short-day (diapause inducing) conditions than in larvae that were reared under long-day conditions. In L. olerucea, Vargas et al. (1992) recorded increased levels of JH I (2-6-fold) in last instar larvae reared under short-day (12L : 12D) conditions compared to larvae reared in a 16L : 8D photoperiod. In a different study, Vargas and Mauchamp (1988) reported 2.1 ng JH I/ml in haemolymph of newly-moulted 6th instar M. brassicae larvae reared under long-day conditions, and as much as 27.0 ng/ml in haemolymph from diapause-determined larvae (i.e. those reared in a 12L: 12D photoperiod). In

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the present experiments, L. oleracea larvae were reared continuously in long-day (16L : 8D) conditions, under which the majority ( > SOO/,) do not enter pupal diapause (T. S. Corbitt, pers. commun.). In these larvae, we were unable to detect the presence of any JH homologue in newly-moulted (day 1) final instar larvae, despite the fact that Vargas et al. (1992) reported haemolymph JH I levels of 1.0 ng/ml (i.e. approx. 1.O rig/g)) in exactly comparable larvae of this species. We have no explanation for this discrepancy. In the closely-related species M. brassicae, application of a juvenile hormone analogue to late last instar larvae prevents pupal diapause (Hiruma, 1979), so it is quite possible that pupal diapause in L. oleracea is influenced by the relative quantity of JH present in the latter stages of development of the final instar larvae. There is very little published information on JH levels in lepidopterous pupae. The accepted view is that JH is largely absent in the pupa in order that development of adult structures may proceed. The absence of JHs during the major part of pupal development in L. oleracea is consistent with this accepted role of JH as acting to maintain the status quo. In late (day 20) pupae and pharate adults (day 25 “pupae”) of L. oleracea we detected significant amounts of both JH I and JH II. In pharate adults total JH levels were similar (- 1.5 rig/g)) to those found in 4th and 5th stadium larvae at the clear head capsule stage (i.e. just prior to ecdysis). Moreover, we found JH levels to be slightly higher in female pharate adults than in males. Sexual dimorphism in relation to JH levels or the biosynthetic capabilities of the corpora allata has been reported previously in pharate adult moths (Gilbert and Schneiderman, 1961) and may be a characteristic of the Lepidoptera (Bhaskaran et al., 1988). We suggest that the increased levels of JH in pharate adult L. oleracea may be required to initiate the imaginal ecdysis, and in females may serve to prime either the organs of reproduction or the pheromone production system. However, we do not believe that the increase in JH in pharate adult males is linked directly with reproductive physiology in males since spermatogenesis is initiated (and often completed) in male lepidopterans during late larval or early pupal development. Thus, the newly-emerged male moth will be reproductively functional at the time of adult eclosion. In L. oleracea this is certainly the case, and adult males are competent to mate during the first 24 h after emergence if receptive females are present. It is possible that high levels of JH in adult male noctuids may be important in determining receptivity to sex pheromones (Cusson et al., 1994a) or in modulating aspects of male courtship behaviour. Nevertheless, the function, if any, of the very high JH levels in adult male moths remains unexplained. In newly-emerged female moths, copulation and egg production are preceded by pheromone production, although duration of the calling period may vary considerably between species (Barth, 1965). Adult female L. oleracea commence pheromone production shortly after

et al.

emergence and pheromone levels increase during the first 9 days of adult life if mating does not occur (T. S. Corbitt, pers. commun.). We suggest that the increase in JH in pharate adult females might, in addition to inducing ecdysis, be responsible for priming pheromone production. The initiation of pheromone production by female moths has been associated with the secretion of pheromone biosynthesis activating neuropeptide (PBAN; Raina et al., 1989) and this, in turn, may be modulated (at least in P. unipuncta) by JH (Cusson and McNeil, 1989; Cusson et al., 1994a). The association between PBAN and pheromone production has also been confirmed in the related noctuid M. bra&cue (Jacquin et al., 1994). Thus, it is quite possible that in L. oleracea, the presence of JH in pharate adult females is responsible for stimulating the production of PBAN and, consequently, the production of pheromone, by newly-emerged female moths. However, the pheromonotropic activity of JH is not universal in lepidopterans (Barth, 1965) especially in species like L. oleracea that do not have a significant pre-reproductive period, and further experiments are needed in order to establish unequivocally any direct relationship between JH in pharate adults and stimulation of the pheromone production system. In the present experiments we recorded a continuous decline in combined JH levels during the first 5 days of virgin adulthood, yet levels of pheromone extractable from pheromone glands of virgin L. oleracea show a general increase from N 5.0 ng at adult eclosion, to - 110 ng on day 9 (T. S. Corbitt unpublished results). It therefore seems most unlikely that JHs are associated with the modulation of pheromone production in L. oleracea, after the time of adult eclosion. The presence of high levels of JHs in adult moths has long been recognized. Indeed, it was as a result of the existence of very high levels of JH in adult males of several saturnid moths, especially Hyalophora cecropia, that researchers were able originally to identify the chemical structure of JH I (Roller et al., 1967). Thus, it was not surprising to find levels of JH in adult Lacanobia that were substantially higher than those found in pre-adult stages. The presence of both JH I and JH II has been reported in several other adult lepidopterans. In Attacus atlas, Baker et al. (1985) found more JH II than JH I in adults and (as is apparent in L. oleracea) higher levels of JH in males than in females. In addition, these authors reported low levels (- 0.2 rig/g)) of JH 0 in adult female moths aged between 2 and 5 days old. In other noctuid moths, in vitro studies of JH production by isolated corpora allata have provided evidence that both JH I and II are produced by virgin adult female Heliothis zea (Satyanarayama et al., 1991) and P. unipuncta (Cusson et al., 1993). In both species, in vitro JH production increased with age, although our in vivo data suggest that JH levels actually decrease in virgin female L. oZeracea if mating is prevented. These apparently contradictory results might be explained if the surgical removal of corpora allata for in vitro studies of

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JH biosynthesis, removes the inhibitory effects of nerves or neurohormones present in the intact animal. It is likely that the high JH levels found in adult females are responsible for the stimulation of vitellogenesis and the initiation of egg production, as has been clearly demonstrated in a number of other moth species (e.g. P. unipuncta, Cusson et al., 1994b). In our laboratory cultures of L. oleracea, where newly-emerged males and females are normally allowed unrestricted access to each other, mating usually takes place during the scotophase of the first 24 h period following eclosion, and females do not lay eggs during the first day of adult life (T. S. Corbitt, unpublished results). Following mating on day 1, egg laying begins slowly on days 2 and 3 ( - 10 eggs/female/day) before increasing sharply on day 4 to more than 120 eggs/female/day, and then declining slowly over the 25 days of adult life (T. S. Corbitt, unpublished results). In this respect, it may be significant that JH levels appear to increase substantially in 2 day old females, and we believe that this increase is intimately linked with the onset of vitellogenesis. Furthermore, we suggest that this increase may be stimulated by the act of copulation, as appears to be the case in M. sexta (Sasaki and Riddiford, 1984). The maintenance of high JH levels in mated females on days 3, 4 and 5 of adult life is consistent with the gonadotropic role of JH, and is compatible with the maintenance of the maximum rates of oviposition that occur during this period (T. S. Corbitt, unpublished results). In addition to the presence of JH I and II in adult females, we consistently detected the presence of JH 0 in samples from 3, 4 and 5 day old mated females. This homologue had hitherto been absent from any of our samples. Since this homologue has previously been identified only in eggs of M. sexta and Heliothis virescens (Bergot et al., 1980, 198la) it is possible that the JH 0 in our samples may have been present in eggs inside the gravid females. The significance of high JH levels in adult male moths, which also apparently showed a general increase over the first 5 days of adult life, remains intriguing since, in the majority of cases, mating will have occurred on day 1. However, unlike females which generally mate only once, males are capable of mating up to 7 times if suitable females are available (T. S. Corbitt, unpublished results). It is therefore possible that JHs do play a significant role in the reproductive physiology of adult male moths. However, since in some male moths (e.g. H. cecropia, Williams, 1959) allatectomy does not appear to reduce reproductive performance, and since there is no conclusive evidence that the high levels of JH found in several adult male moths have any direct effect on reproduction, we suggest that the effects of JH on male reproductive capacity (if present) may be mediated through less obvious pathways (e.g. behaviour) than through direct effects on the reproductive glands. In some male lepidopterans previous studies have reported an absence of JHs which (due to the absence of JH acid methyl transferase) are replaced by JH acids (Bhaskaran et al., 1988; Hebda et al., 1994). In the present exper-

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iments we have obtained evidence that both JH I and JH II do occur in L. oleracea adult males, although we are unable to say whether or not the respective JH acids occur in this species. Ultimately, the levels of juvenile hormone(s) present in an insect will reflect a balance between the rates at which JH is biosynthesized and the rate at which it is metabolized or excreted. In the Lepidoptera JH metabolism appears to be predominantly mediated by the enzymic action of juvenile hormone esterases (JHE)-the activity or production of which may be synchronized with times when hormone levels are declining (Hammock et al., 1984, Samburg et al., 1975; Hammock, 1985). JH biosynthesis in Lepidoptera occurs predominantly in the corpora allata where these molecules are built up from simple precursors like acetate, propionate and mevalonate (Schooley et al., 1973). In M. sexta, the corpora allata are themselves regulated by neuropeptides that stimulate (Mas allatotropin, Kataoka et al., 1989) or inhibit (Mas allatostatin, Kramer et al., 1991) the biosynthetic activity of the glands. It seems likely that neuropeptides with similar actions exist in other Lepidoptera including L. oleracea. On the basis of the present study, we would expect to find maximum aflatotropic activity at times when JH levels are increasing (i.e. in adults, pharate adults and in larvae immediately prior to ecdysis). Conversely, we anticipate allatostatins and JH esterases to act at times when JH levels are reduced or absent (i.e. during much of the final larval stadium and the majority of pupal development). Finally, the discovery that, unlike the majority of other insect orders (which appear to make and utilize only JH III) lepidopterans are unique in that they appear to use several JH homologues (Schooley et al., 1984) prompted speculation that each homologue might perform a different role. For example, the higher homologues (JH I and II) might predominantly have a morphogenetic role and be responsible for regulating development and metamorphosis, while the lower homologue (JH III) might have a purely gonadotropic role. The results of the present experiments lend no support to these earlier speculations and, at least in L. oferacea, it appears that JH I and JH II are present (or absent) at critical physiological points, and that the changing levels of these two homologues appear to be largely responsible for all physiological activities of JH throughout larval, pupal and adult life in this species.

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Acknowledgements-The authors wish to thank F. C. Baker (Sandoz Crop Protection) for the generous gift of several chemicals, J. P. Wilkins and G. Keenan (CSL) for mass spectroscopy, and S. Olieff (CSL) for technical assistance. This study was supported by the Pesticides Safety Directorate of the Ministry of Agriculture, Fisheries and Food.