Developmental synchrony between Spodoptera littoralis (Boisd.) and its parasite Microplitis rufiventris Kok.

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J. Insecl Phykd. vol. 34, No. 8, PP. 773-778, 1988 Printedin Great Britain. All rights reserved

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1988 Pergamon Press

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DEVELOPMENTAL SYNCHRONY BETWEEN SZ’ODOPTERA LZTTORALZS (BOISD.) AND ITS PARASITE MZCROPLZTZS RUFZVENTRZS KOK. E. M.

HEGAZI*,

A.

SCHOPF~, E. FuHRERt

and S. H.

FOUAD~

*Faculty of Agriculture, Alexandria University, Egypt, TForest Entomology Institute, Agricultural University of Vienna, Austria and IFaculty of Agriculture, Cairo University, Egypt (Received 27 July 1987; revised 30 November 1987) developmental interaction between Spodoptera littoralis(Boisd.) and its endoparasite, MicroplitisrujiventrisKok. were studied under controlled conditions of short (6 h light-18 h dark) and long (18 h light-6 h dark) photoperiods, 20 + 1°C and 60* 5% r.h. The developmental times of both species were reduced under short-day photoperiod conditions. Photoperiod as well as parasitism markedly affected the number of moults of the host larvae and the developmental time of the parasite was correlated with its sex, the host’s age and photoperiod. Juvenile hormone titres in the haemolymph of parasitized and unparasitized larvae were determined using the Galleria bioassay. The haemolymph juvenile hormone titres of parasitized larvae were significantly higher than those of unparasitized larvae. The high levels of juvenile hormone were recorded prior and subsequent to parasite emergence. Importantly, parasitism interferes with the normal development of the host larvae. Abstract-The

Key Word Index: Photoperiod, parasitism, instars, Microplitisrufiventris, Spodoptera littoralis, juvenile hormone INTRODUCTION

Microplitis rujiventris Kok. (Hymenoptera: Braconidae) is a solitary endoparasite. It oviposits and develops in many noctuid caterpillars including the cotton leafworm, Spodoptera littoralis [Boisd.] (ElMinshawy, 1963; Shalaby, 1968; Gerling, 1969; Hegazi et al., 1973; Ibrahim and Tawfik, 1975). S. littoralis is one of the most important cotton pests in Egypt (Hosny and Isshak, 1967). It is a polyphagous insect and its activity continues almost all the year under different climatic conditions. S. littoralis has seven generations per year: four on clover in winter and spring; two to three on cotton in summer and a possible generation on maize in late summer and autumn (Hafez, 1972). Studies on the combined effect of photoperiod and parasitism revealed that photoperiod, especially in conjunction with low temperatures, affects the developmental periods of the immature stages of M. rufiventris within S. littoralis larvae (Hegazi and Fiihrer, 1985). Consequently, the present study was carried out to investigate the developmental interactions between S. littoralis and M. rufiverttris under two different photoperiod conditions. In addition, the juvenile hormone titres in the haemolymph of parasitized and parasite-free larvae were measured. MATERIALS AND METHODS

Cultures of Spodoptera littoralis and Microplitis rujiventris were obtained from the Institute of Forest

Zoology, Gottingen, West Germany. Mass rearing of both species was achieved in ways similar to those described by Hegazi and El-Minshawy (1979). S. littoralis larvae were divided into two groups; one was exposed to short-day photoperiod (6 h light per day)

and the other to long-day (18 h light per day) photoperiod. At the same time both groups were maintained at 20 f 1°C and 60 f 5% r.h. Parasitism was accomplished by introducing a single female parasite to a single S. littoralis larva in a small glass vial. All vials remained under observation until parasitism occurred, usually within a few minutes. The host instars subjected to parasitization were late first, early and late second and early third. Parasitized and unparasitized larvae of the same age were transferred individually to small Petri-dishes (3.5 cm dia), fed on meridic diet (Hegazi et al., 1977a) and remained under the same environmental conditions as those outlined above. Daily examinations were done to record the occurrence of moulting of parasitized and parasite-free larvae and the timing of parasite emergence. In order to study the internal developmental time of the parasite, a sample of twenty-five to thirty host larvae that were parasitized on the first day of their third instar and reared under the two photoperiodic conditions, was dissected at daily intervals in Ringer’s solution and the duration of the egg and larval stages of the parasite was carefully checked. The juvenile hormone titres of the haemolymph of parasitized larvae were measured, prior and subsequent to parasite emergence and compared to unparasitized larvae of the same age, in days, using the Galleriu test (De Wilde et al., 1968). The parasitized larvae (weighing 29.12 f 0.9 mg prior parasite emergence and 24.06 + 0.8 one day later to parasite emergence) were fifth instar while the unparasitized host larvae (weighing 353.6 f 2.69 mg) were in the active sixth (last) instar. Therefore, fifteen and fifty unparasitized and parasitized larvae were needed respectively, to get 25&450~1 haemolymph for a 773

E. M.

HEGAZI

et al.

RESULTS

ti

4th 5th 61 Pm-pupa Instar Fig. 1. Effect ofshortm(6L:18D)andlonglJ(18L:6D) photoperiod on the developmental durations of unparasitized S. litloralislarvae. 1st

2nd

3rd

3 times and used for the bioassay within a few weeks. In this study, the juvenile hormone titres were expressed in Galleria units; (GU)/pl haemolymph. One (GU)/pl was equal to juvenile activity of 25 pg/pl juvenile hormone I (cis-10,l I-Epoxy-7-ethyl-3,l I-dimethyl-trans, trans2,6-tridecadienoic acid methyl ester) [Fa. Sigma]. sample. Each sample was replicated

Furasitized

instar

Age ( days )

The photoperiod affected the total developmental time of unparasitized and parasitized larvae of Spodoptera littoralis. The developmental time of each instar of the unparasitized larvae (Fig. 1) was significantly longer under the long-day regime as shown by a t-test comparison of the means (P < 0.05). The development of Spodoptera larvae was faster under the short-day than under the longday regimen (Fig. 1). The developmental time of the larval stage required an average of 39.56 + 0.81 days the long-day regime, and 24.42 f 0.42 days under the short day. The same effect was also observed in the parasitized larvae (Fig. 2). The developmental time of parasitized was significantly shorter under the shortday regime than those larvae reared under the longday photoperiod condition. To clarify the effect of parasitism on the duration of host instar, a middle-instar such as the third host’s instar in Fig. 2 systems a-c could be compared with the corresponding unparasitized ones (Fig. 1). The effect was significant in hosts raised under the shortday regime. The combined effect of parasitism and photoperiod, in some cases, caused an increasing in the overall total required time for development of Spadoptera larvae. Under the long-day regime, parasitization of the “older” second-instar larvae as com-

: 1st :2

Parasitized

instar

Age (days)

n

: 3rc :1

d

7

5

3

-::

1

2

0i

1 s1 2nd 3rd 4 th

I

-

Parasitized Age (days)

I

b

0 2nd 3rd

mstar

3rd

: 2nd

4th

5th

6th

instar : 2nc

Parasitized

Age (days)

:2

n

I

1;th

2nd 3rd Host larval instar

4th

5th

Fig. 2. Effect of short n (16L: 18D) and long 0 (18L:6D) photoperiods and instar and age of S. litforulislarvae parasitized by 44. rufiuenrris on the developmental durations of host instars.

Host-parasite developmental synchrony Table 1. Effect of short (6 h) and long (18 h) photoperiods and age and instar of Spodopteralittorah larvae parasitized by Microplifis rujiioentris on the number and percentages of the host’s moults and the timing of parasite emergence at 20 f 1°C Host instar at onset of parasite emergence

Host moults Host instar parasitized 1st

Host age of instar (day) 2

2nd

Photoperiod regime Short (30)’ Long (31) Short (30) Long (33)

I

2

Short (35)

Long (29) 3rd

1

Short (31) Long (29)

*Number of observations.

No.

% Of total larvae

3.0 4.0 3.0 2.0 2.0 3.0 4.0 I.0 2.0 3.0 2.0 3.0 1.0 2.0 1.0 2.0 3.0

93.30 6.66 100.00 100.00 81.80 9.90 9.90 7.14 35.71 57.14 62.50 37.50 6.66 93.33 7.69 84.90 7.10

Long and short photoperiods

pared to “younger” larvae lengthens the time required for host larvae (Fig. 2, systems b and c). However, the growth difference of parasitized larvae raised under the short-day regime was not significant. In general, parasitized larvae of S. littoralis passed through fewer moults than the parasite-free larvae. No larval moults to the sixth (last) instar were reported under short-day photoperiod conditions, while, under the long-day photoperiod regime, only 9.9 and 7.1% of the parasitized larvae were able to moult to the sixth instar when parasitization was initiated in the host’s early second and early third instars, respectively (Table 1). By contrast, the unparasitized larvae always moulted to the sixth instar prior to pupation. Furthermore, the two variable regimes induced a supernumerary larval instar as 25.0 and 10.0% of unparasized larvae moulted to seventh instar under the short- and long-day photoperiods, respectively. Micropfitis rujioentris parasites emerged, in a descending order of frequency, from the host’s fourth, fifth, sixth and third instars. The highest and lowest percentages of parasite emergence, under short-day conditions, were 100 and 6.66 and they took place in the host’s fourth and fifth instars, respectively. By contrast, these percentages under long-day conditions were 100 and 7.69 and they occurred in the fourth instar (Table 1). The relationship between the timing of parasite emergence and the host’s age at the onset of parasitism was obvious (Table 1). When parasitism was accomplished during the host’s late first and early second instar, most of M. rujiuentris larvae emerged from the fourth-instar of S. littorolis larvae. Thus, when parasite eggs were deposited in late secondinstar of the hosts, most of the wasps emerged from the fourth and fifth instars. When parasitization was initiated in the early third instar of the host, more than 80% of the wasps from host’s fifth instar. The developmental times of M. rujiventris and S. littoralis larvae and their relationship to photoperiod, are illustrated in Fig. 3. Dissections of host larvae in which parasitization was initiated during their early

3rd

4th

5th

6th

x x x x x x x x x x x x x x x x x

were 18 h and 6 h light per day respectively.

third instar indicated that parasite eggs hatched to the first instar on the second or on the fourth day after oviposition under the short-day and long-day respectively. regimes, Without exception, M. rz@entris passes through three instars but moults 3 times within the host larvae. In the present case, parasite eggs hatched in the host’s third instar, then Microplitis larvae ecdysed to the second instar during host’s ecdysis to the fifth instar. A few hours later, parasite larvae ecdysed to the third instar. Thus, the parasite larvae ecdyse twice during the host’s late fourth and early fifth instars and emerge during host’s late fifth instar. When the parasite larva becomes full grown and be ready to emerge and metamorphose (about 4.7 mm length and 0.9 mm width), it tears a hole on the host cuticle and ecdyses for the third time leaving its mandibles and cast skin blocking the exit hole. Moreover, emergence of the parasite never coincided with moulting of the host epidermis. Host locomotion and feeding cease before parasite emergence, and the host rests quietly while emerging parasites spin their cocoons and metamorphose. Following emergence, the developmental arrest of host larvae continued for 2-l 1 days before their eventual death. The described mode of parasite ecdysis was the same under the two photoperiodic regimes, although there was conspicuous prolongation in the duration of development of host larvae reared under long-day conditions. The prolongation in the host’s development was correlated with a prolongation in the parasite’s development. Additionally, parasites developing in hosts raised under the above regimes of photoperiod did not enter diapause following emergence. Dissections revealed that the parasite’s second-instar larvae develop along the dorsal posterior part of the host’s mid-gut and as the development of the parasite proceeds and the larva increases in size, it puts a pressure on the host’s mid-gut, minimizing the host’s internal volume relative to the parasite. The relationship between the two variables of

E. M. HEGAZI et

al.

Egg I

Ll

J

I

L2

,

L3

I

Ll

I

Duration

I

1

(days)

Fig. 3. Effect of short and long photoperiods on the moulting times of S. littoralisn and M. rufuentris 0 larvae. L: larval instar.

photoperiod and the host’s instar, and the age within host’s instar and the rate of development of h4. rujiuentris parasite is illustrated in Fig. 4. The duration of internal and external development following emergence of the parasite varied according to the following: (1) The parasite’s sex; as the duration of the larval and pupal stages of male parasites were shorter by 1 or 2 days than the corresponding developmental periods of females. (2) The host’s instar; as the developmental period of the parasite within the host’s first instar was significantly longer than the period within the host’s third instar, though. the duration of the pupal stage of the parasite emerging from both of these type of hosts was almost the same. (3) The age within host’s instar; the development of the egg-larval stage of the parasite was clearly accelerated versus delayed when parasitization was initiated in host’s “young” and “old” second instars, respectively. However, this phenomenon reversed in the pupal stage of the parasite. (4) Photoperiod; the two photoperiodic regimes caused significant differences in the developmental duration of the immature stages of the parasite (larval and/or pupal stages). The parasite’s development was much more rapid under short-day conditions to synchronize the rapid development of the host. Under the long-day conditions, 3-S% of the hosts had unemerged parasites, and dissections of these hosts revealed the presence of third-instar parasite larvae which were alive and unencapsulated by host haemocytes. These parasite larvae remained alive until the host larvae died, when they were about 17-25 days old. The skin of these parasite larvae was “melanized” and still surrounded the larval body indicating that the larva had suffered from a moulting

failure. In some cases the mandibles of the parasite larvae were embedded in the host body wall, and the host tissues around the mouth parts were “melanized”, suggesting that the parasite larva had some problems during the laceration of host cuticle at emergence time. The larva was also “melanized” and had not completed its third (last) larval ecdysis. It was easy to discover such cases without dissecting the host larvae as a black spot was always found on one of the sides of the posterior part of host’s abdomen. This black spot indicates the “melanized” host’s tissues around the parasite mandibles as described above. The juvenile hormone titres in the haemolymph of parasitized larvae were extremely high with respect to unparasitized larvae of the same age, in days (Table 2). There was a significant elevation of juvenile hormone titre prior to parasite emergence. Following emergence, the juvenile hormone titre decreased to a low level, but was still significantly higher than the juvenile hormone titre of parasite-free larvae of the same age. DISCUSSION

The shortened developmental times of unparasitized larvae of Spodoptera littoralis under short-day photoperiod conditions (Fig. 1) were due to a direct stimulatory effect of photoperiod (Beck, 1968). The effect of the two photoperiods on the number of moults of parasitized larvae is likely to be explained by the presence of a metabolic reaction and endocrinological changes. The timing of parasite emergence was irregular under short-day conditions and it was controlled by host’s age (Table 1). This is probably due to the response of the internal-

Host-parasite krasitized

developmental synchrony

instar

1 st

2

Age (days)

777

Parasitized instar : 3r :1 Age (days)

14 1c

6

Parasitized instar Age (days)

2nd 1

Parasitized instar Age (days)

: 2r 12

6

Egg-Larvae

Pupae Parasite

Egg -Larvae

Pupae

stages

Fig. 4. Effect of short and long photoperiods and instar and age of S. lirroralis larvae on the durations of immature stages of male q and female n adults of M. rufiuenrris. L. long day (18 h light-6 h dark), S. short day (6 h light-18 h dark).

parasite development; older host larvae (third instar) are able to provide the parasite with more food than the younger larvae (first or second instars). Similarly, It was obvious that the parasite established an other studies suggest that the development of endointimate physiological association with its host to parasites is regulated by host’s hormones, as well as ensure its own survival by synchronizing its develnutritional factors (Cals-Usciati, 1969 and 1975; opment with that of its host though the indirect Baronio and Sennal, 1980; Jones, 1986). Of interest sensitivity of the parasite to the daylengths used (Fig. is the early emergence of male adults of M. rujiventris, 3). This apparent synchrony may be caused by the and perhaps this enables them to feed on nectars for parasite cueing in on the host’s hormones for growth 1 or 2 days, then become ready to fertilize the female and development. adults, as soon as they emerge from their pupae. The lengthened developmental times of the larval Beckage et al., 1987, reported that larvae of the stage of female parasites compared to males may gregarious endoparasite wasps Cotesia congregata result from the increased nutritional requirements of frequently failed to emerge from host Manduca sexta the female parasite during its endoparasitic devellarvae treated with high doses of benzyle-1,3 benzoopment since females live longer (Fig. 4). Also, the dioxole derivative J-2710, particularly when the hosts rapid endoparasitic development in older host larvae fail to feed normally. This was attributed to nutrimay emphasize the role of nutritional factors in tional inadequacy caused by the inability of the host to feed and not to the direct action of J-2710. In the present study, some hosts raised under long-day Table 2. Juvenile hormone titres (in Gall&a units, GU/rI) in the never had emerged parasites but were haemolympb of S. MroraliF larvae parasitized by M. rufwnfris in conditions comparison with unparasitized larvae found to contain unencapsulated pharate Microplitis larvae when they died. It seems that these parasites Average f SD Host missed a developmental “cue”. 340.3 ?r. 107.7 a Unparasitized larvae: Table 2 shows the elevation of juvenile hormone Parasitized larvae: 24499.1 + 6318.6 b Prior to parasite emergence titres in the haemolymph of parasitized larvae prior 18350.5 + 403 I .6 c Subsequent to parasite emergence to parasite emergence. This elevation is possibly due development of the parasite to endocrinological changes occurring in the host larvae (Vinson and Iwantsch, 1980).

E. M. HEGAZIef al

778

to any or all of the following factors: (1) Decreased

haemolymph volume of parasitized larvae. The parasitized larvae (29.12 f 0.9 mg) were significantly smaller than the unparasitized larvae (353.6 f 2.69 mg). Therefore,250450 PI haemolymph were obtained from 50 parasitized larvae, while the same amount was obtained from only 15 unparasitized larvae. Thus, parasitism causes a dramatic decrease in the volume of haemolymph of host larvae which may cause an increase in juvenile hormone titre. However, Beckage and Riddiford (1982) mentioned that the hormonal changes seen during parasitism of Manduca sexta by Apanteles congregatus reflect a direct effect on the juvenile hormone titre rather than changes in the haemolymph volume. (2) Increased juvenile hormone production by the parasitized host’s corpora allata. (3) Decreased juvenile hormone-specific esterase activity in the haemolymph. Additionally, Beckage and Riddiford (1982) reported that parasitism by Apanteles congregatus in Manduca sexta caused an elevation in the juvenile hormone titres of fifth-instar larvae, then the titres declined to their initial level prior to parasite emergence. Contrary to their findings the haemolymph juvenile hormone titres of S. littoralis larvae parasitized by M. rujventris elevated prior to parasite emergence. High levels of juvenile hormone titres are presented in the parasitized larvae to suppress their metamorphoses and arrest their development. Similar to many parasitized insects, parasitized larvae of S. littoralis cease feeding prior to parasite emergence until their death (Smilowitz et al., 1976; Hegazi et al., 1977b; Beckage and Riddiford, 1978). Possibly, the developmental stress of the parasite has an effect on the muscles of the host’s gut and its muscles lose the ability to process food. Indeed, some food remained inside the alimentary canals of these parasitized hosts following cessation of food consumption. It appears likely that the physiological stage of the hosts (early or late second instars), the nutrient available for the parasite larvae (host’s size) and the hormone titres in the host are of the important factors for the development of M. rufventris parasite. Acknowledgement-Professor Dr Hegazi wishes to thank the Alexander-van-Humboldt-Foundation grant.

for the research

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