Horomonal control of larval diapause and metamorphosis of the southwestern corn borer Diatraea grandiosella

Although we have established that the initiation and maintenance of the facultative mature larval diapause of Diatraea grandiosella is regulated by the juvenile hormone (JH), we have yet to determine the precise functional differences within the endocrine system of diapause and non-diapause larvae (Chippendale & Yin, 1973 ; Yin & Chippendale, 1973). For example, we still need to know how the corpora allata are regulated and whether the circulating JH controls the release of ecdysiotropin or ecdysone in diapause larvae. In the present paper we estimate the activity of the corpora allata in relation to the preapolysis requirements for the cerebral neurosecretory system and the ecdysial glands, and determine the effect of JH mimics on non-diapause larvae. The results provide a necessary framework for comparing the functions of the endocrine system in diapause and non-diapause larvae.

Our colony of D. grandiosella Dyar was originally obtained from the delta region of southeastern Missouri and is presently maintained in a continuous laboratory culture. Larvae were fed an artificial diet and kept in 1 oz clear plastic cups (Chippendale, 1975). Although most larvae were reared under non-diapausing conditions (3O°C 12L:12D), a few groups were subjected to the diapause inducing regime of 23°C 12L:12 Ü. Immaculate diapause larvae are distinct polymorphic variants of the spotted non-diapause ones (Chippendale & Reddy, 1972).

Head capsule measurements of 20 larvae (1 – 6) days or 40 larvae (7 – 19 days) were recorded daily. Beginning at day 6 the sex was determined by dissection. Pupation rates of 90 male and 80 female larvae were observed from 15 days.

Haemolymph samples used in the Galleria wax test were removed from fifth- and sixth-stage larvae and newly ecdysed pupae using graduated microcapillaries. Newly ecdysed fifth-stage larvae were collected at 10 and 11 days and haemolymph samples were obtained at 0 –, 1 –, 2 –, 3 – and 4-day intervals after the ecdysis. Each assay used about 200 μl of haemolymph from 5 – 10 female larvae. Newly ecdysed sixth-stage larvae were collected at 13 and 14 days and haemolymph samples were collected at 0-, 0 · 25 –, 0 · 5 –, 0 · 75 –, 1 –, 2 – and 3-day intervals after the ecdysis. Each assay used about i ml haemolymph collected from 10-20 male and female larvae. Pooled haemolymph samples from males and females were also collected from 25 newly ecdysed white pupae, and 10 newly diapaused immaculate larvae which had been reared at 23 ° C 12L:12D for 50 days.

Galleria wax test

This JH bioassay was slightly modified from that of De Wilde et al. (1968). Haemolymph samples were extracted with chloroform : methanol 2:1 (v/v) immediately after collection (Folch, Lees & Stanley, 1957). The chloroform phase was dried under a stream of nitrogen and the residue was dissolved in peanut oil. This solution was diluted with paraffin wax (m.p. 56 ° C) and applied to a thoracic wound of ten newly ecdysed Galleria pupae. Control pupae treated with a 1:1 mixture of peanut oil and paraffin wax routinely gave a negative response. The Galleria units (GU) per ml of haemolymph were estimated from a serial dilution which gave a positive response in 50 % of the pupae. A single GU corresponds to approximately 5 × 10 ^{− 8}μ g cecropia JH.

Newly ecdysed sixth-stage larvae were collected from 13 to 15 days. Groups of 30 larvae were given a head ligature at 0 · 25 day intervals from 2 · 75 to 4 · 5 days or a thoracic ligature (between the meso- and metathorax) at 0 · 25-day intervals from 3 · 75 ^to 5 days after the ecdysis. The number of larvae and pupae was recorded 5 days after treatment. Pupal abdomens were sufficiently well formed to permit their easy identification.

Groups of 50 early sixth-stage larvae (13 days) were each topically treated on their abdomens with 3 μ g/3 μ 1 acetone of a JH mimic donated by Zoecon Corp. The mimics included ZR 512 (ethyl-3,7,11-trimethyl-2,4-dodecadienoate), ZR 515 (isopropyl-n-methoxy-3,7,11-trimethyl-2,4-dodecadienoate) and ZR 1662 (ethyl-10-methoxy-3,7,n-trimethyl-2,4-dodecadienoate). Untreated larvae and those applied solely with the solvent served as controls. The effect of the mimics on the incidence of ecdyses into the immaculate morph was observed. At 40 days the immaculate larvae were transferred into shell vials containing moist paper strips and their rate of pupation was recorded. Normal diapause larvae reared at 23 ° C for 40 days and then transferred to 30 ° C 12 L: 12 D served as controls (Chippendale & Reddy, 1972).

Mean head capsule widths were plotted against age to determine the number and duration of the instars of the southwestern corn borer (Fig. 1). Beginning in the third instar a sex dimorphism was observed. Males ecdysed into the fourth instar on days 7 and 8 and into the fifth instar on day 10. Most of the males pupated from the fifth instar, with 50% pupation observed at 16 days. The females ecdysed into the fourth instar on days 7 and 8 and into the fifth instar on days 10 and 11. Most of the females, however, entered a sixth instar and therefore pupated later than the males (50% pupation by day 18). For the subsequent timing experiments, then, both male and female newly ecdysed fifth instars were collected at 10 and 11 days, whereas mainly female larvae were collected from 13 to 15 days. These larvae constitute a non-diapause generation and retain their pigmented integumental pinacula in contrast to the diapause larvae which have pigment-free integuments.

Fig. 2 shows the haemolymph JH titres in the 5th and 6th instars and newly ecdysed pupae as estimated by the Galleria wax test. Since the southwestern corn borer has a fused corpus cardiacum – allatum complex it is not possible to monitor directly the effects of allatectomy. Measurements of haemolymph JH concentrations, which provide data on the equilibrium existing between the rates of corpus allatum secretion, tissue uptake, and haemolymph enzymic deactivation, currently provide the only practical method for estimating corpus allatum activity. Two days after the ecdysis the JH titre declined in 5th instars from 600 to 300 GU/ml and then increased to 3000 GU/ml on day 3. The corpora allata probably renewed secretory activity on day 3. On day 4 the titre declined to 700 GU/ml, probably because the rate of uptake by target cells exceeded the rate of secretion. Within 6 h of the ecdysis into the 6th instar the JH titre rapidly declined from 1000 to 140 GU/ml, suggesting a rapid deactivation. Continued deactivation, coupled with inactive corpora allata, led to a JH titre of only 60 GU/ml 3 days after the ecdysis. The subsequent moult was therefore pupal and the newly ecdysed pupae (< 1 h old) still contained ca. 55 GU/ml haemolymph.

Fig. 3 shows the response of larvae to head and thoracic ligatures applied from 2-75 to 5 days after the ecdysis into the 6th instar. A head ligature applied 3 · 5 – 4 days after the ecdysis prevented pupation in about 60 % of the larvae, suggesting that the ecdysis is dependent upon the brain for the first 4 days of the instar. A thoracic ligature applied 4 – 4 · 5 days after the ecdysis prevented pupation in from 45 to 65 % of the larvae, suggesting that the ecdysis is dependent on the ecdysial glands for the first 4 · 5 days after the ecdysis (Yin & Chippendale, 1975). In most larvae the pupal moult occurred independently of the brain at 4 · 25 days and of the ecdysial glands at 4 · 75 days. The results indicate that the requirement for the brain is completed half a day ahead of that for the ecdysial glands.

The formation of dormant immaculate southwestern corn borers from last-stage spotted non-diapause larvae following treatment with a JH mimic has already been described (Yin & Chippendale, 1974). Our present results now support the viewpoint that this ‘artificial’ diapause is induced by tissue responsiveness to the mimics rather than to a reactivation of the recipient’s corpora allata (Fig. 4). The results show the fate of early 6th instars treated with JH mimics. Within 26 days of treatment the mimics yielded 33 %, 45 % and 75 % immaculate larvae, respectively. The larvae responded similarly to JH mimics 512 and 515, but mimic 1662 caused the formation of an earlier and higher incidence of the immaculate morphs. The remaining larvae either became pupae or abnormal larvae with partially developed wing pads. The untreated and solvent control larvae remained spotted, and 50% had pupated by the 19th and 17th day of age, respectively.

The rate of pupation of the immaculate larvae was observed beginning at 40 days. Larvae treated with JH mimic 512 or 1662 pupated at a high rate, but a higher incidence of larval mortality occurred in those treated with 512. A low rate of pupation was observed in larvae treated with mimic 515 and about 70% larval mortality had occurred by 135 days. The normal diapause larvae, which served as controls, reached 50% pupation in 121 days and completed the process in 145 days. These results suggest that the JH mimics have different intrinsic hormonal activities. Additional data to support this interpretation have been obtained from an experiment in which head ligatured diapause larvae, whose endogenous JH had dissipated, received JH mimic and β-ecdysone applications. Under these conditions JH mimic 1662 showed the highest activity (Chippendale & Yin, unpublished).

Fig. 5 summarizes our current information about JH titres in diapause and nondiapause larvae of D. grandiosella. The diagram is compiled from data obtained from JH bioassays performed on haemolymph extracts of non-diapause and newly diapaused immaculate larvae which contained about 1500 GU/ml. Prediapause last instars therefore retain active corpora allata and a haemolymph JH titre which is slightly lower than that necessary to permit a spotted larval ecdysis. An immaculate larval ecdysis marks the onset of diapause and occurs in response to this intermediate JH titre. Since the JH titre falls during diapause development a maintenance titre between 100 and 1600 GU/ml is shown (Chippendale & Yin, unpublished).

The haemolymph JH titre of last-stage non-diapause larvae of Diatraea declines within 12 h of the ecdysis from about 1000 to 110 GU/ml. A low titre is then maintained for the remainder of the instar, suggesting that the corpora allata are inactive. In contrast, the activity of the corpora allata of Hyalophora cecropia gradually declines during the final larval instar, and the corpora allata of Manduca sexta only stop secreting JH when the final-stage larvae weigh about 5 g (Williams, 1961 ; Nijhout & Williams, 1974). In the latter case the circulating JH was deactivated within 24 h, leading to the release of ecdysiotropin and the pupal moult. The presence of a high JH titre during the early phase of the instar was thought to inhibit the release of ecdysiotropin. It is unlikely that a high titre of circulating JH inhibits the release of ecdysiotropin in the corn borer because the titre declines long before the pupal moulting cycle becomes independent of the brain, at 4 · 25 days. The signal for the release of ecdysiotropin in Diatraea is still unknown, but probably involves extero- and proprioception from the central and stomatogastric nervous systems (Chippendale & Yin, 1975).

Treatment of non-diapause corn borers with JH mimics during the early phase of the final instar caused many to moult into the immaculate morph and become dormant (Yin & Chippendale, 1974). The incidence of the polymorphism and the rate of pupation of these ‘diapause’ borers depended upon the chemical nature of the JH mimic, suggesting that the mimics did not activate the recipient’s corpora allata. Within 2 weeks of treatment the mimics elevated the functional JH titre of many of the last instars to the diapause range, thereby permitting an immaculate ecdysis. In a related experiment De Wilde & Lutke Schipholt (1974) examined the effect of JH compounds on diapausing adults of Leptinotarsa decemlineata. They also concluded that the degree of renewed feeding and reproductive activity depended upon the dose and exposure time rather than upon an induction of secretion of the recipient’s corpora allata.

We have shown the involvement of a specific JH titre in the initiation and maintenance of the larval diapause of Diatraea. Much remains to be learned, however, about the diapause relationships within the endocrine system. For example, the mechanism of activation of the corpora allata of prediapause larvae and the diapause function of JH are as yet unknown. The diapause-associated polymorphism of Diatraea probably has no adaptive significance, but may occur because the enzyme system involved in integumental melanization is not activated at an intermediate JH titre. An active role for the corpora allata in larval diapause has now been established, but many intriguing questions about hormonal regulation remain unanswered (Yagi & Fukaya, 1974; Yin & Chippendale, 1974).

We thank Mrs Evelyn Bendbow for her valuable technical assistance. This study was supported in part by NSF grant BMS 74 – 18155 and is a contribution from the Missouri Agricultural Experiment Station, journal series no. 7334.