Chelonus sp.: suppression of host ecdysteroids and developmentally stationary pseudoparasitized prepupae

When eggs of the caterpillar Trichoplusia ni are stung by Chelonus sp. (near C. curvimaculatus) (Braconidae), the developing host larvae precociously spin a cocoon but then remain developmentally stationary in the prepupal stage. The latter event happens even in hosts which were stung and precociously spin cocoons but which, upon dissection, contain no obvious parasite. Injection of radiolabeled ecdysone into either pseudoparasitized or allatectomized larvae demonstrates suppressed rates of conversion of ecdysone to 20-hydroxyecdysone when compared with controls. The data indicate that the occurrence of developmentally stationary pseudoparasitized prepupae is due to less production of ecdysteroid and less conversion of ecdysone to 20-hydroxyecdysone, both probably as a result of suppressed juvenile hormone titer.     

Protein expression during parasite redirection of host (Trichoplusia ni) biochemistry

Expression of proteins during normal egg and larval development of Trichoplusia ni was compared with that occurring in hosts stung as eggs by the parasitic wasp Chelonus sp. near curvimaculatus. Those stung hosts which produced a parasite (truly parasitized), precociously expressed proteins associated with larval-pupal metamorphosis, as did those stung hosts which did not contain a developing endoparasite (pseudoparasitized). No highly abundant, low-intermediate molecular weight hemolymph proteins were observed in truly or pseudoparasitized larvae which did not also occur at some point in the development of normal larvae. A low abundance, high molecular mass (160,000 Da) protein was observed in the hemolymph of truly parasitized larvae, but not of normal or pseudoparasitized larvae. The protein is glycosylated and very acidic (pI near 4.5). The data show that any parasitization proteins injected or induced by the ovipositing female parasite are in low abundance, in contrast to situations reported for parasitic wasps which sting hosts as larvae.     

Endocrine interrelationship between the parasitoid Chelonus sp. and its host Trichoplusia ni

The egg-larval parasitoid Chelonus sp. induces the precocious onset of metamorphosis in the 4th (penultimate) stadium of its host Trichoplusia ni, emerges from the prepupa, and then feeds on it. Qualitative and quantitative changes in ecdysteroids and juvenile hormone were measured. Hemolymph of 3rd-to 4th-instar host larvae and the parasitoids they contained, as well as nonparasitized and parasitized eggs, were analyzed. In the host hemolymph a broad peak of ecdysteroids during molting into the 4th stadium and a continuous increase from day 2 (onset of precocious wandering) until day 4 (emergence of parasitoid) were observed; 20-hydroxyecdysone and 20,26-dihydroxyecdysone were predominant. The juvenile hormone titer fluctuated in the 3rd and early 4th stadium and fell to undetectable levels shortly before the precocious onset of wandering. The parasitoid's ecdysteroids started to increase on the molt to the 2nd instar (= early 4th instar of the host) and thereafter fluctuated on a high level, 20-hydroxyecdysone, 20,26-dihydroxy-ecdysone, and ecdysone being predominant. The juvenile hormone titer was high in late 1st-instar parasitoids, decreased to low levels at ecdysis into the 2nd instar, and increased again to high levels in the 2nd-instar larvae at the time when their shape changed from flat to cylindrical. After ecdysis to the 3rd instar the juvenile hormone titer fell. A comparison revealed that both ecdysteroids and juvenile hormone fluctuate independently in parasitoid and host at most stages, suggesting that the parasitoid produces its own hormones. The first data on ecdysteroids and juvenile hormones in the egg stage of a parasitoid/host system are reported. At the stage of eye pigmentation parasitized eggs contained more immunoreactive midpolar ecdysteroids than non-parasitized ones. 20-Hydroxyecdysone and 20,26-dihydroxyecdysone were the predominant ecdysteroids in both nonparasitized and parasitized eggs, but the latter contained several additional ecdysteroids which were not seen in nonparasitized eggs. The titer of juvenile hormone was similar in both. Shortly before hatching the ecdysteroids were low in parasitized and nonparasitized eggs, but the content of juvenile hormone was much higher in the former. At this stage the majority of parasitoids have already eclosed and teratocytes are released. The results of HPLC analysis indicated the presence of juvenile hormone III together with juvenile hormones I and II in parasitized eggs, but only juvenile hormones I and II in nonparasitized eggs.     

Manipulation of fifth-instar host (Manduca sexta) ecdysteroid levels by the parasitoid wasp Cotesia congregata

Although 5th (last) instar parasitized Manduca sexta larvae undergo developmental arrest and do not wander, they exhibit a small hemolymph ecdysteroid peak (300-400pg/&mgr;l) which begins one day prior to the parasitoid's molt to the 3rd (last) instar and concomitant emergence from the host. Ecdysteroids present in this peak were 20-hydroxyecdysone, 20,26-dihydroxyecdysone and one or more very polar ecdysteroids, as well as small amounts of 26-hydroxyecdysone and ecdysone. In parasitized day-1 and -2 5th instars ligated just behind the 1st abdominal proleg, hemolymph ecdysteroid levels increased in both anterior and posterior portions (100-500pg/&mgr;l), while in unparasitized larvae, hormone levels only increased in the anterior portion (100-350pg/&mgr;l). Thus, the ecdysteroid peak observed in host 5th instars was probably produced, at least in part, by the parasitoids. It may serve to promote Cotesia congregata's molt from the second to the third instar and/or to facilitate parasitoid emergence from the host. In parasitized day-1 and -2 5th instars ligated between the last thoracic and 1st abdominal segments, hemolymph ecdysteroid titers reached much higher levels (500-3500pg/&mgr;l) in the anterior portion (no parasitoids present) than in the posterior portion (150-450pg/&mgr;l). Therefore, it appears that the parasitoid's regulation of hemolymph ecdysteroid titers occurs at two levels. First, parasitization neutralizes the host's ability to maintain its normal hemolymph ecdysteroid levels. Second, in a separate action, the parasitoid manipulates the ecdysteroid-producing machinery so that hemolymph levels are maintained at the 200-400pg/&mgr;l characteristic of day 3-4 hosts. This is the first report of a parasitoid's ability to interfere with the normal inhibitory mechanisms which prevent prothoracic gland production of ecdysteroid at inappropriate periods of insect growth and development.     

Parasite regulation of host insect metamorphosis: a new form of regulation in pseudoparasitized larvae ofTrichoplusia ni

Summary When eggs ofTrichoplusia ni (Lepidoptera) are stung by a parasitic wasp,Chelonus sp., the developing host larvae precociously initiate metamorphosis ten days later. Precocious initiation of metamorphosis occurs even in ‘pseudoparasitized’ stung hosts which contain no living parasites at the time of symptoms of host regulation by the parasite. In feeding, penultimate instar, pseudoparasitized hosts, the corpora allata activity, hemolymph juvenile hormone esterase activity, in vivo rates of juvenile hormone metabolism and changes in hemolymph protein composition all follow the pattern of the normal last instar. This and other evidence suggests the entire developmental pattern of the last larval instar is precociously expressed in penultimate instar, pseudoparasitized hosts. The cause of precocious expression of the developmental program leading to metamorphosis is a significant decrease in the critical size parameter that, in normal larvae, signals attainment of the last instar. The induction, in preultimate instar larvae, of the entire feeding stage developmental program leading to metamorphic commitment, using either biochemical, surgical or parasitic experimental probes, has not been previously reported. The results have important implications for the study of host-parasite endocrine interaction, of normal insect metamorphosis and even of human puberty.     

Physiological and endocrine changes associated with polydnavirus/venom in the parasitoid-host system Chelonus inanitus-Spodoptera littoralis

As shown earlier, parasitization by the egg-larval parasitoid C. inanitus causes in its host the precocious onset of metamorphosis in the 5th instar followed by developmental arrest in the prepupal stage. Polydnavirus/venom were shown to be responsible for the developmental arrest. We investigated how polydnavirus/venom affect growth of the host larvae and found that head capsule widths were smaller from the 4th to 6th stadium and weights were lower in the 6th stadium in polydnavirus/venom-containing larvae than in non-parasitized larvae. In an attempt to identify endocrine parameters that are modified by polydnavirus/venom and might be responsible for the developmental arrest in the prepupa, we compared juvenile hormones, juvenile hormone esterase and ecdysteroids between non-parasitized and polydnavirus/venom-containing larvae from the 4th instar until pupation or developmental arrest, respectively. Obvious differences became manifest only in the 6th instar at the pupal cell formation stage, i.e. 12 days after entry of polydnavirus/venom into the host egg. Then, prothoracic glands of polydnavirus/venom-containing larvae released less ecdysteroids and ecdysteroid titres were lower than in non-parasitized larvae; this was followed by a delayed, reduced and desynchronized increase in prepupal juvenile hormones and juvenile hormone esterase and a slightly modified metabolism of ecdysone. This indicates that polydnavirus/venom affects the endocrine system of the host only after pupal commitment and that inhibition of prothoracic gland activity is the first detectable effect.     

Stage dependent influences of polydnaviruses and the parasitoid larva on host ecdysteroids

In the solitary egg-larval parasitoid Chelonus inanitus (Braconidae) both polydnavirus and the parasitoid larva manipulate host development. Parasitization leads to a premature drop in juvenile hormone titre and a precocious onset of metamorphosis in the 5th larval instar. The C. inanitus bracovirus (CiBV) alone causes a reduction in host ecdysteroid titres at the pupal cell formation stage and prevents pupation. Here we report three new findings. (1) We show that parasitization causes a reduction in haemolymph ecdysteroid titre immediately after the moult to the 5th instar; similarly low values were seen in nonparasitized larvae after the moult to the 6th instar. These data along with parasitoid removal experiments indicate that the low ecdysteroid titre after the moult is a very early sign of the upcoming metamorphosis. (2) In vitro experiments with prothoracic glands and brain extracts showed that CiBV affects both prothoracic glands and prothoracicotropic hormone after the stage of pupal cell formation. (3) In the haemolymph of parasitized larvae the ecdysteroid titre increased in the late cell formation stage, i.e. immediately before egression of the parasitoid. In vitro experiments showed that late 2nd instar parasitoids release ecdysteroids and are thus very likely responsible for the rise in host ecdysteroids.     

Growth and development of Cardiochiles nigriceps viereck (hymenoptera,braconidae) larvae and their synchronization with some changes of the hemolymph composition of their host,Heliothis virescens (F.) (Lepidoptera,Noctuidae)

Larval development of the parasitoid Cardiochiles nigriceps Viereck occurs in the last instar larva of its host, Heliothis virescens (F.). This allows the parasitoid to exploit the nutritional increase in the biosynthetic activity occurring in the host in preparation for metamorphosis. To understand the biochemical basis of this host parasitoid developmental synchrony, we undertook host ligation studies and analyzed host hemolymph for proteins and glycerol esters. Parasitization affected the biochemical profile of the host. The hemolymph protein concentration of parasitized last instar H. virescens larvae increased through time, whereas unparasitized (control) larvae were characterized by a decrease in the protein titer when they reached the prepupal stage. The effect of parasitism on glyceride titers of host hemolymph was not as pronounced as the effect on proteins. Ligation conducted on 5th instar hosts, which were parasitized as 4th instars, affected parasitoid development in a time-dependent way. The percentage of successfully developing C. nigriceps larvae increased with the increase of the time interval between parasitization and ligation. Ligation performed before day 2 of the 5th larval instar of H. virescens completely inhibited parasitoid development. Ligations that disrupted parasitoid developmentwere associated with a low host hernolymph protein concentration. Parasitoid development was successful when hernolymph protein titer was high, as occurred when ligations were performed after day 3 of the 5th host instar in both control and parasitized larvae. Ligations in both situations resulted in a slight increase in glyceride titers. The results suggest that host proteins and/or some factor(s) associated with them may play a role in parasitoid growth and development. © 1993 Wiley-Liss, Inc.     

Endocrine alteration and precocious premetamorphic behaviors in the greater wax moth larvae,Galleria mellonella,parasitized by an endoparasitoid,Apanteles galleriae

Parasitization of Galleria mellonella (Lepidoptera: Pyralididae) larvae by a larval endoparasitoid Apanteles galleriae (Hymenoptera: Braconidae) leads to the precocious expression of premetamorphic behavior in the sixth (normally penultimate) instar host larvae prior to the parasitoid's emergence. We investigated the role of parasitization with A. galleriae on the alteration of development and/or behavior of its host. The ecdysteroid titer in the hemolymph of parasitized sixth instar larvae (the last instar of parasitized larvae) was higher than that of unparasitized ones, and the high ecdysteroid concentrations induced premetamorphic behaviors such as wandering and cocoon spinning. However, the epidermis of the parasitized larvae was not pupally committed through this stage. The activity of JH esterase in the parasitized larvae remained low, and application of a JH analogue to these larvae caused the production of a larval-type cocoon. These facts suggest that the parasitization by A. galleriae induces precocious premetamorphic behaviors of G. mellonella larvae by changing host endocrine conditions without causing the typical larval-pupal metamorphosis. Arch. Insect Biochem. Physiol. 34:257–273, 1997. © 1997 Wiley-Liss, Inc.     

Entomogenous fungus Nomuraea rileyi inhibits host insect molting by C22-oxidizing inactivation of hemolymph ecdysteroids

The entomogenous fungus Nomuraea rileyi reportedly secretes a proteinaceous substance inhibiting larval molt and metamorphosis in the silkworm Bombyx mori. We studied the possibility that N. rileyi controls B. mori development by inactivating hemolymph molting hormone, ecdysteroids. Incubation of ecdysone (E) and 20-hydroxyecdysone (20E) in fungal-conditioned medium resulted in their rapid modification into products with longer retention times in reverse-phase HPLC. Each modified product from E and 20E was purified by HPLC, and identified by NMR as 22-dehydroecdysone and 22-dehydro-20-hydroxyecdysone. Some other ecdysteroids with a hydroxyl group at position C22 were also modified. Injection of the fungal-conditioned medium into Bombyx mori larvae in the mid-4th instar inhibited larval molt but induced precocious pupal metamorphosis, and its injection into 5th instar larvae just after gut purge blocked pupal metamorphosis. In hemolymph of injected larvae, E and 20E disappeared and, in turn, 22-dehydroecdysone and 22-dehydro-20-hydroxyecdysone accumulated. These results indicate that N. rileyi secretes a specific enzyme that oxidizes the hydroxyl group at position C22 of hemolymph ecdysteroids and prevents molting in B. mori larvae.     

Positive feedback regulation of prothoracicotropic hormone secretion by ecdysteroid – A mechanism that determines the timing of metamorphosis

When insect larvae have fully grown, prothoracicotropic hormone (PTTH) is released from the brain, triggering the initiation of metamorphic development through stimulation of ecdysteroid secretion by the prothoracic glands. The present study analyzes the mechanism that regulates the occurrence of this PTTH surge. In the silkworm Bombyx mori, the PTTH surge occurs on day 6 of the fifth instar and is preceded by a small rise in hemolymph ecdysteroid titer, which occurs late on day 5. We therefore hypothesized that this rise of ecdysteroid titer is involved in the induction of the PTTH surge. To test this hypothesis, two experiments were conducted. First, a small amount of 20-hydroxyecdysone was injected on day 4, two days before the expected day of the PTTH surge, to simulate the small rise in hemolymph ecdysteroid titer on day 5. This injection led to a precocious surge of PTTH the next day. Next, the hemolymph ecdysteroid titer on day 5 was artificially lowered by injecting ecdysteroid-22-oxidase, which inactivates 20-hydroxyecdysone. After this treatment, the PTTH surge did not occur on day 6 in 80% of the animals. These results indicate that a small rise of the hemolymph ecdysteroid titer plays a critical role in the induction of the PTTH surge. Since basal ecdysteroidogenic activity of the prothoracic glands increases with larval growth, a circulating level of ecdysteroids may convey information about larval maturity to the brain, to coordinate larval growth and metamorphosis. This is the first report in invertebrates to demonstrate positive feedback regulation of the surge of a tropic hormone by a downstream steroid hormone.     

Host regulation effects on Heliothis virescens (F.) larvae inducd by teratocytes of Cardiochiles nigriceps Viereck (Lepidoptera,Noctuidae-Hymenoptera,Braconidae)

Teratocytes deriving from the serosal membrane of Cardiochiles nigriceps Viereck, obtained “in vitro” from embryos hatched on a semidefined medium, were injected at different numbers and in different developmental stages of nonparasitized Heliothis virescens (F.) last instar larvae. Host development was affected by teratocyte injections and the responses registered ranged from normal to complete inhibition of pupation, according to the number of teratocytes injected and the developmental stage of the larva at time of injection. Complete pupation failure was observed when teratocytes derived from 4C nigriceps embryos were injected into 1st day 5th instar (new-slender stage) host larvae. Complete pupation occurred when teratocytes from 2 embryos were injected into 3rd or 4th day 5th instars (burrow-digging or day 1 cell formation stage). Intermediate responses, such as the formation of pupal cuticle without ecdysis or with only partial ecdysis, were obtained with intermediate teratocyte numbers, or host developmental stages. All pupae derived from teratocyte injected larvae failed to develop into adults normally obtained from control injected larvae. The larval weight just before pupation was negatively affected only when teratocyte injections were performed on 1st day 5th instar H. virescens larvae. Teratocyte injections altered the hemolymph protein titer to a level similar to that occurring in parasitized larvae. At the same time the ecdysteroid titer was characterized by a late significant increase, which reached values almost 3 times greater than found in normally parasitized larvae, and also surpassed the highest values registered for nonparasitized larvae. Ligation of parasitized larvae between the meso- and metathorax demonstrated that when the prothoracic glands were excluded, there was almost no ecdysteroid production posterior to the ligation. Ligations performed on parasitized larvae to isolate parasitoid eggs before hatching in the last abdominal segments, demonstrated that only virus and venom determined a reduction of the ecdysteroid titer. On the basis of these results the possible role of teratocytes in affecting the biological activity of ecdysteroids is postulated and discussed in a wider context of host-parasitoid physiological interactions.     

Expression of a parasitism-specific protein in lepidopteran hosts of Chelonus sp.

The hemolymph of each noctuid species successfully parasitized by Chelonus near curvimaculatus possessed a parasitism-specific protein (PSP) previously identified in host T. ni (Insect Biochem. 19:445; 21:845). Expression of PSP occurred in a stage-specific manner in the stadium during which the host undergoes precocious metamorphosis. The appearance of the protein was not due to nutritional stress associated with parasitism of hosts, since starved nonparasitized larvae did not produce the protein, or to low juvenile hormone titers occurring in precociously metamorphosing hosts, but rather was dependent on the presence of the endoparasite larva. Results of in vivo incorporation experiments with [³⁵S]-methionine showed that synthesis and subsequent appearance of the protein in the hemolymph of parasitized hosts was abrogated by prior surgical removal of endoparasite. Immunoprecipitation analysis of proteins from C. near curvimaculatus larvae cultured in vitro using antibodies specific to PSP indicated that the source of the protein was the endoparasite. Synthesis of PSP by the endoparasitic larvae with its subsequent secretion into the hemocoel of hosts was specific to the advanced stages of parasite development prior to its egression from the host. © 1993 Wiley-Liss, Inc.     

Mode of action of the venom of the ectoparasitic wasp,Euplectrus comstockii, in causing developmental arrest in the European corn borer,Ostrinia nubilalis

Euplectrus comstockii Howard (Hymenoptera: Eulophidae), is an ectoparasitic, gregarious wasp which parasitizes the larval stage of several important lepidopteran pests. Parasitization of both natural and unnatural hosts prevents molting in the parasitized instar. Here we report the effect of wasp venom on the European corn borer (unnatural host), an important pest of corn and other vegetables. Venom collected from venom glands of adultE. comstockii, when injected intoO. nubilalis 5th instars, inhibited the growth rate, development and molting of the injected larvae. The observed effect on molting was dose and age dependent. When 3rd, 4th and 5th instarO. nubilalis were envenomated by adult wasps, the larvae also were developmentally arrested and failed to undergo a molt. However, 3rd and 4th instars underwent apolysis (separation of the epidermis from the old cuticle) and produced new cuticle. Fifth instars did not. A premolt hemolymph ecdysteroid peak was not observed in these experimental 5th instars, but injections of 20-hydroxy-ecdysone induced apolysis and new cuticle formation. Envenomated 4th instars (on becoming pharate 5th instars) exhibited a premolt hemolymph ecdysteroid peak. HPLC/RIA revealed that 20-hydroxyecdysone was present in the hemolymph of these pharate 5th instars. Thus, in the European corn borer, the mode of action of the venom depended upon the instar parasitized. Our results support the presence of a venom component(s) that, in 4th instar hosts, inhibited ecdysis, but did not prevent hemolymph ecdysteroid levels from increasing sufficiently to stimulate apolysis. In 5th instars, the same, or perhaps, a different component(s) ofE. comstockii venom prevented the synthesis/release of ecdysteroid by inhibiting a previously unknown molt-regulating physiological event that occurs between days 3 and 4 of the instar. Deceased     

Immunoanalysis of unique protein in Trichoplusia ni larvae parasitized by the braconid wasp Chelonus near curvimaculatus

Parasitization in insects brings about profound biochemical and physiological effects in the host which may include complete overriding of the normal endocrinological program, resulting in precocious metamorphosis and in blockage of pupal development. The subtle effects of parasitization include changes in the expression of hemolymph proteins and the appearance of proteins which are unique to parasitized hosts. One such protein has been identified in the hemolymph of Trichoplusia ni larvae parasitized by the braconid wasp Chelonus near curvimaculatus. In this study, purified preparations of the parasitism-specific protein were used to generate polyconal antibodies against the protein. Results from the immunocharacterization indicate the antibodies obtained are highly specific for the protein and are present in a high titer (1:8000 antiserum dilution yielded strong signals in analysis of the protein in 0.25 μl hemolymph). Subsequently, the expression of the parasitism-specific protein in the hemolymph and tissues was analyzed by immunoblotting during the entire course of development in normal and parasitized insects. The parasitism-specific protein was not detected in normal, unparasitized larvae. In parasitized insects, expression of the parasitism-specific protein appears to be stage-specific in that it is only detected during the last larval stadium of precociously metamorphosing larvae, but is absent from all earlier stages of development.     

Ecdysteroid synthesis and molting by the tobacco hornworm, Manduca sexta, in the absence of prothoracic glands

When a pair of prothoracic glands (PGs) were removed from Manduca sexta pupae on the day of pupation, the hemolymph ecdysteroid titer remained at a low level. When a portion of the gland pair was extirpated from pupae after the critical period for prothoracicotropic hormone release, the maximum hemolymph ecdysteroid titer was reduced in proportion to the mass of the PGs removed. These findings clearly showed that the PGs in intact pupae are responsible for the elevated ecdysteroid titer required to elicit adult development on schedule. When brains were removed on the day of pupation, the initiation of adult development was delayed for weeks or months. In contrast, pupae whose PGs were removed on the day of pupation initiated development only 7 days late, indicating the existence of an additional source of pupal ecdysteroids. Further, abdomens of male M. sexta that were isolated on the day of pupation initiated adult development spontaneously within 70 days. The implantation of day 0 pupal brains into these isolated abdomens accelerated the initiation of adult development and elicited synchronous adult development. The hemolymph ecdysteroid titer of those isolated abdomens receiving implants of brains increased within 5 days and reached a maximum level of 1.5 micrograms/ml. The analysis of hemolymph ecdysteroids by reverse-phase HPLC revealed that ecdysone was the major moiety and that the ecdysteroid composition was similar to that of normal, intact pupae that had just initiated adult development. These results demonstrate that the PGs are not requisite for adult development. An increased hemolymph ecdysteroid titer was also observed in isolated abdomens from which the testes were removed and in abdomens devoid of their digestive tract. Indeed, in the latter case, the ecdysteroid titer attained much higher levels than those observed for abdomens with intact guts. Despite numerous attempts to identify the tissue(s) in the isolated abdomens responsible for the increase in ecdysteroid titer, its identity remains unknown.     

Hemolymph ecdysteroid titer and ecdysteroid-dependent developmental events in the last-larval stadium of the silkworm,Bombyx mori: role of low ecdysteroid titer in larval-pupal metamorphosis and a reappraisal of the head critical period

The endocrine regulation of larval-pupal metamorphosis was studied in the silkworm, Bombyx mori, by measuring the following changes: hemolymph ecdysteroid titer, the secretory activity of prothoracic glands and the responsiveness of larvae to ecdysteroids and prothoracicotropic hormone (PTTH), with regard to developmental events such as the occurrence of spinneret pigmentation, initiation of cocoon spinning and onset of wandering stage as indicated by gut purge. These measurements were concentrated especially on the time before and after the head critical period (HCP) which falls 3-4 days before the gut purge ([Sakurai, 1984]). A small increase in the hemolymph ecdysteroid titer was first found during the HCP, and then the titer increased with daily fluctuations. Small but significant titer peaks were found prior to the occurrence of both spinneret pigmentation and gut purge, indicating that an individual titer peak could possess a specific role in development. Responsiveness of larvae to exogenous 20-hydroxyecdysone (20E) after the HCP was markedly higher than that before the HCP. The sensitivity of the prothoracic gland to PTTH also changed during the HCP. The results thus showed that the HCP is not the period after which an additional PTTH release is not required for the developmental events occurring on schedule, but rather it is the period during which complex events occur not only in the endocrine glands but also in the peripheral tissues. In addition, various developmental phenomena before gut purge are brought about by the hemolymph ecdysteroid whose concentration gradually increased with daily fluctuations, and these precise changes in the titer appeared to be important for the sequential occurrence of developmental events in the larval-pupal metamorphosis.     

Developmental interaction between suboptimal instars of Spodoptera littoralis (Lepidoptera:Noctuidae) and its parasitoid Microplitis rufiventris (Hymenoptera:Braconidae)

Relative effects of parasitism by Microplitis rufiventris on the development of the third instar Spodoptera littoralis (preferable, optimal host) with the development of penultimate (5th) and last (6th) instars (suboptimal hosts) were investigated. Newly molted 6th instar hosts were more acceptable for parasitization by the wasp female than older hosts. In singly parasitized 3rd instar hosts, 82.0 +/- 3.9% of the parasitoid eggs developed to full-grown instar wasp larvae. However, parasitoid eggs deposited singly in 73.9 +/- 3.3% of 5th and 100% of 6th instar hosts failed to develop. Superparasitization in the 3rd instar hosts reduced the production of pseudoparasitized larvae and, conversely, all parasitized hosts yielded viable parasitoid offspring. In suboptimal hosts, the development interaction between the parasitoid and its host larvae was highly influenced by the age of hosts at parasitism, load of deposited eggs, and other parasitoid factors. The latter factors, e.g., mainly calyx fluid particles, might be involved in establishing parasitoid eggs in the suboptimal hosts. In the last two host instars, superparasitization significantly increased the number of parasitoid larvae successfully reaching their final instar. Variation in host quality, e.g., physiological status, might be attributed, in part, to the partial breakdown of the solitary habit observed in the earlier instars. More parasitoid eggs developed to mature parasitoid larvae in hosts superparasitized as 6th instar than parasitoid eggs laid in 5th instar hosts. Superparasitization significantly lengthened the developmental period of 5th and 6th host instars and inhibited their development to the pupal stage. Studying parasitoid development in suboptimal instars of its habitual host provided physiological insight, as shown here. The results may have implication for biological control and in vitro mass rearing programs with solitary parasitoids.     

Decreased JH biosynthesis is related to precocious metamorphosis in recessive trimolter (rt) mutants of the silkworm, Bombyx mori

In recessive trimolter (rt) mutants of the silkworm, Bombyx mori, that have four larval instars rather than five larval instars of normal B. mori, a decrease after a small increase in the hemolymph ecdysteroid titer during the early stages of the last (fourth) larval instar appeared to be a prerequisite for larvae to undergo precocious metamorphosis. The present study was carried out to investigate the possible mechanism underlying this decrease in the ecdysteroid titer. It was found that juvenile hormone (JH) biosynthetic activity of the corpora allata (CA) increased during the first day of the last larval instar, but its absolute JH biosynthesis activity was relatively lower compared to that of normal fourth-instar larvae in tetramolters. This lowered JH biosynthetic activity appeared to be related to a decrease in prothoracic gland ecdysteroidogenesis during the second day of the last instar, because hydroprene application prevented this decrease in prothoracic gland ecdysteroidogenesis, leading to the induction of a supernumerary larval molt. The in vitro incubation of prothoracic glands with hydroprene showed that hydroprene did not directly exert its action on prothoracicotropic hormone (PTTH) release. Further study showed that the application of hydroprene enhanced the competency of the glands to respond to PTTH. From these results, it was supposed that the lowered JH biosynthesis of the CA during the first day of last instar in rt mutants was related to decreased ecdysteroidogenesis in the prothoracic glands during the second day, thus playing a role in leading to precocious metamorphosis.