Effect of parasitism by Cardiochiles nigriceps on food consumption and utilization by Heliothis virescens

Parasitism of Heliothis virescens larvae by the endoparasitoid Cardiochiles nigriceps resulted in a reduction in the amount of food consumed by parasitized larvae. This effect was attributed in part to inoculation of material from the accessory reproductive glands of the female at the time of oviposition. Injection of solutions of either the calyx fluid or the poison gland (0·04 gl/larva) into non-parasitized larvae resulted in a reduction in the amount of food consumed by these larvae. A 1 : 1 mixture of these glands (total of 0·04 gl/larva) appeared to be more active than either of the two glands alone. Both of these glands were essential for total activity since larvae parasitized by females lacking the poison gland (poi gl^? female) continued to eat and consumed more food than did those parasitized by a normal female.Parasitism resulted in a slower rate of crop-emptying. This effect was, however, shown to be a result of the quantity of food consumed. Inhibition of gut movement was therefore not considered the cause for the reduction in the amount of food consumed by parasitized larvae.The effect of parasitism on the ability of H. virescens larvae to utilize ingested food was partially reduced by parasitism. Larvae parasitized by a normal female were less efficient than non-parasitized larvae in digesting food. Those larvae parasitized by a poi gl^? female did not convert as much of their food to body substance as did non-parasitized larvae. Injection of solutions of accessory glands into non-parasitized larvae did not cause these effects.     

Regulation of host larval development by the egg-larval endoparasitoidChelonus insularis [Hym.: Braconidae]

In laboratory studies the effect of parasitism by the egg-larval endoparasitoidChelonus insularis Cresson on the resulting larvae of 2 host species,Heliothis virescens (F.) andSpodoptera ornithogalli Guénée) were determined by comparing daily measurements of larval weights. Growth of parasitized larvae of both host species was slower than growth of unparasitized larvae. Injections of fluids from the female parasitoid's calyx or poison gland intoH. virescens eggs retarded subsequent larval growth. However, a combination of fluids from these 2 organs produced the most significant reduction in the host larval growth rate. The growth reducing factor(s) was also effective when injected into 5-day-old host larvae.     

Effect of a virus associated with the reproductive system of the parasitoid wasp,Campoletis sonorensis,on host weight gain

The particulate fraction of the calyx fluid of the endoparasitoid, Campoletis sonorensis, reduces host weight gain when manually injected into healthy Heliothis virescens larvae. Reduced weight gain of the host, H. virescens, is normally associated with parasitism by C. sonorensis. Electron microscopy has confirmed that the particulate fraction of the calyx fluid is composed of virus particles and it appears that this virus, injected with the egg at oviposition, actually reduces host weight gain. The effect of the virus is negated when the calyx fluid is exposed to ultraviolet light prior to injection. Furthermore, the calyx fluid is effective only if injected into hosts; there is no effect on host weight gain when hosts are fed or topically treated with the virus-containing calyx fluid.     

Ultrastructure of host tissues exposed to the calyx fluid of the parasitoid,Campoletis sonorensis (Cameron) (Hymenoptera: Ichneumonidae)

Campoletis sonorensis (Cameron) (Hymenoptera: Ichneumonidae) is a solitary endoparasitoid of Heliothis virescens. The lateral oviducts of the female parasitoid contain a particulate suspension called calyx fluid. The particles in calyx fluid are a polydnavirus (CsV) which, when injected into last-instar H. virescens, stimulates degeneration of the host's prothoracic glands. In order to determine if CsV-induced degeneration is specific to prothoracic glands, last-instar H. virescens larvae were injected with C. sonorensis calyx fluid. After 4 days, a variety of host tissues were dissected from both calyx fluid-injected and uninjected control larvae and fixed for transmission electron microscopy. Prothoracic glands from injected larvae were ultrastructurally degenerated by 4 days post-injection, whereas control glands remained intact. Other tissues from calyx fluid-injected larvae (tracheal epithelia, corpora allata, Malpighian tubules, fat body, skeletal muscle, and the brain) showed no signs of ultrastructural degeneration or gross abnormalities as compared with control tissues. These observations suggested that CsV-induced degeneration is specific to the host's prothoracic glands.     

Changes in differential haemocyte count and in vitro behaviour of plasmatocytes from host Heliothis virescens caused by Campoletis sonorensis polydnavirus

Campoletis sonorensis is a habitual parasitoid of 3rd-instar larvae of Heliothis virescens. C. sonorensis eggs and small glass rods were encapsulated in 5th-instar host larvae implanted in the absence of wasp calyx fluid; prior injection of calyx fluid into larvae suppressed the encapsulation response. Within 8 h of calyx fluid injection there was a removal of approx. 75% of the circulating capsule-forming haemocytes (plasmatocytes). The remaining subpopulation of plasmatocytes, in addition to being incapable of encapsulating targets in vivo, spread at a significantly reduced rate in vitro. Identical changes in plasmatocyte count and behaviour were observed after injection of virus purified from calyx fluid. Additionally, the activity of calyx fluid was abolished after ultraviolet irradiation. The onset of haemocytic abnormalities occurred more rapidly after natural parasitism of 3rd-instar host larvae. The cell-free haemolymph of calyx fluid-injected 5th-instar larvae also retarded the spreading of plasmatocytes from non-injected control larvae in vitro. We conclude that the abnormalities induced in H. virescens plasmatocytes by C. sonorensis virus contribute to the suppression of encapsulation.     

Ecdysteroid-titre reduction and developmental arrest of last-instar Heliothis virescens larvae by calyx fluid from the parasitoid Campoletis sonorensis

Fifth-instar Heliothis virescens larvae did not pupate after injections of Campoletis sonorensis calyx fluid in or before the burrow-digging stage of development. Arrested development occurred in 40% of larvae injected at the cell-formation stage. Further experiments showed that the particles in calyx fluid were responsible for developmental arrest. Arrested development due to calyx fluid could be reversed by injecting 10 μg of either ecdysone or 20-hydroxyecdysone, although a second injection of 20-hydroxyecdysone was needed for some larvae 3 days after the first treatment. Ecdysteroid production ceased for up to 10 days in 5th-instar H. virescens after calyx-fluid injection. After 10 days, some experimental larvae began to produce ecdysteroids again but remained developmentally arrested. The head, thorax, or abdomen of larvae were isolated by ligations and calyx fluid injected into the isolated body region. After 24 h, ligatures were released and the larvae observed for developmental arrest. Only injections into the isolated thorax stopped development. This, along with ecdysteroid data, indicated that C. sonorensis calyx fluid may directly affect the prothoracic glands of 5th-instar H. virescens.     

The infectivity of a nuclear polyhedrosis virus of the velvetbean caterpillar for eight noctuid hosts

Eight species of noctuid larvae were tested for susceptibility to a nuclear polyhedrosis virus of the velvetbean caterpillar, Anticarsia gemmatalis. Velvetbean caterpillar larvae were highly susceptible to crude preparations of polyhedral inclusion bodies (PIBs; LD₅₀ = 4.7 PIBs/larva), but preparations of purified polyhedra were much less effective against these larvae (LD₅₀ = 319.7 PIBs/larva). Of seven other noctuid species tested, only Heliothis virescens was as susceptible to the virus as A. gemmatalis. High dosages were required to kill Heliothis zea, Trichoplusia ni, Pseudoplusia includens, and Spodoptera ornithogalli. Plathypena scabra and Spodoptera frugiperda were not susceptible.     

Dose-dependent influence of Campoletis sonorensis polydnavirus on the development and ecdysteroid titers of last-instar Heliothis virescens larvae

Campoletis sonorensis calyx fluid arrests the development of last-instar Heliothis virescens larvae and is associated with the gross degeneration of the host's prothoracic glands. Through manipulations of ovary supernatant, Campoletis sonorensis polydnavirus (CsV) was found to be the only component of calyx fluid responsible for causing host developmental arrest. Venom from C. sonorensis had no effect on host development. Suspensions of CsV were quantified, and various doses were injected into last-instar hosts. The percentage of larvae developmentally arrested was dose dependent. In addition, larvae not arrested by injection with CsV suspensions were developmentally delayed in a dose-dependent manner. Hosts were delayed in the stage in which they were injected and, after recovery, developed at normal rates. Measurements by radioimmunoassay indicated that developmental delay was due to a suppression of ecdysteroid titers. After a dose-dependent period of suppression, hemolymph ecdysteroid titers recovered and reached titers comparable to those observed in saline-injected controls. Examination of prothoracic glands from developmentally delayed larvae revealed that partial degeneration occurred. Comparisons of the number and mean size of surviving gland cells and the length of developmental delay suggested that surviving gland cells may compensate for degenerated cells by increasing their ecdysone production.     

Consumption and utilization of a soyflour-wheat germ diet by larvae of the tobacco budworm parasitized by the tachinidEucelatoria sp

The number ( \(\bar X\) =2.4) ofEucelatoria sp. maggots that completed development in 4th- or 5th-instar larvae of the tobacco budworm (TBW),Heliothis virescens (F.), was significantly greater (P<0.05) than the number ( \(\bar X\) =1.2) that completed development in 3rd-instar larvae. Maggot development time decreased with increasing number of maggots per host larva. It also decreased with advancing larval instars. The range was 6.9±1.1 days in early 3rd-instar TBW larvae and 5.0±0.8 days in early 5th-instar TBW larvae. Unparasitized 3rd- or 4th-instar TBW larvae consumed significantly more food than did similar aged larvae parasitized byEucelatoria sp., but larvae parasitized during the early 5th-instar consumed more food than did similar aged unparasitized larvae. Consumption by 4th- or 5th-instar larvae increased significantly as maggot densities increased from 1 to 3 per host larva, but decreased at a density of 4 or more maggots per host larva. Although body weight gain and consumption were both significantly reduced 48 and 120 h after parasitization of late 3rd-instar larvae (6 days old), the approximate digestibility (AD) value was significantly greater for parasitized than for unparasitized larvae. Unparasitized larvae were more efficient in converting digested food to body substance (ECD) than parasitized larvae, but the efficiency in conversion of ingested food to body substance (ECI) was similar for both parasitized and unparasitized larvae.     

Polydnavirus of Microplitis croceipes prolongs the larval period and changes hemolymph protein content of the host,Heliothis virescens

Polydnaviruses from certain parasitoid Hymenoptera have been reported to interfere with both host immunity and host development. Heliothis virescens larvae injected with either calyx fluid or sucrose gradient-purified polydnavirus from Microplitis croceipes (McPDV) gained less weight than saline-injected larvae. The active feeding portion of the fifth stadium larva (time to reach the burrowing-digging stage) was doubled (7.0 vs. 3.4 days) when a 0.25 wasp equivalent (WE) of sucrose gradient-purified McPDV was injected into a newly ecdysed fifth stadium host. Many of the treated larvae were unable to pupate, successfully and died at a point of incomplete larval-pupal ecdysis. Pupae that did result from the treated larvae weighed significantly less than controls, even at 0.025 WE. The rate of weight gain and extent of delay of development were dose-dependent; as little as 0.1 WE extended the time of active feeding by 1.5 days and yielded only 25% adults. A 0.05 WE dose yielded 78% adults compared to 95% for controls. The total protein content of hemolymph from individuals injected with McPDV was significantly less than that of controls at any McPDV dose equal to or greater than 0.1 WE. SDS-PAGE profiles of hemolymph proteins from control and McPDV-injected larvae revealed a marked inhibition of the normal accumulation of storage proteins during the fifth stadium and a lesser reduction of serine protease inhibitor protein. Thus, McPDV-injected larvae exhibited some symptoms (less total hemolymph protein and reduced amounts of storage protein) similar to those shown by both parasitized larvae and by larvae injected with M. croceipes teratocytes. However, McPDV affected development during the active feeding stage of the larva, while teratocytes primarily impacted larvae at the time when larval-pupal transformation processes are initiated. © 1995 Wiley-Liss, Inc.     

Regulation of Heliothis virescens prothoracic glands by Cardiochiles nigriceps polydnavirus

Heliothis virescens (F.) Larvae parasitized by the endophagous braconid Cardiochiles nigriceps Viereck fail to attain the pupal stage. This developmental alteration is caused by both an inactivation of prothoracic glands of last-instar larvae and an altered ecdysone metabolism. Decrease in ecdysteroidogenesis in vitro was already evident in glands explanted from larvae that have attained the early cell formation stage (day 4 of fifth instar), 6 h after parasitoid oviposition. Ecdysteroidogenesis nearly ceased by 24 h after parasitoid oviposition. The degree of this biosynthetic depression increased as the time between parasitization and gland dissection increased. A time-course study allowed us to determine if both the degree of phosphorylation of regulatory target proteins, the rate of general protein synthesis and ecdysteroidogenesis decreased in concert over time. The results provide further evidence in support of the hypothesis that these cellular activities in prothoracic gland cells are functionally correlated in steroidogenic responses. Treatment with calyx fluid and venom of C. nigriceps duplicates the parasitism-induced inactivation of host prothoracic glands. A 6-h conditioning in vitro of pupally committed host prothoracic glands with these parasitoid female reproductive secretions resulted in a significant depression of their ecdysteroid production. However, glands lost their sensitivity to calyx fluid and venom treatment when explanted from hosts that had already attained the cell formation stage. This was further supported by the fact that nearly all the host larvae parasitized on day 4 of fifth instar (cell formation stage) pupated, while parasitization on day 3 resulted in only 11% pupation. The coupled trioxsalen/UV irradiation treatment of C. nigriceps calyx fluid and venom eliminated their negative effect on biosynthetic activity in vitro by host prothoracic glands. This result indirectly demonstrates that C. nigriceps polydnavirus is the major regulating factor involved in the host prothoracic gland inactivation. Arch. Insect Biochem. Physiol. 38:1–10, 1998. © 1998 Wiley-Liss, Inc.     

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.     

Interference with function of plasmatocytes of Heliothis virescens in vivo by calyx fluid of the parasitoid Campoletis sonorensis

Summary Immature stages of the ichneumonid parasitoid, Campoletis sonorensis, develop within the haemocoel of its noctuid host, Heliothis virescens. The host cannot encapsulate the parasitoid egg owing to the suppressive effect of the polydnavirus-laden calyx fluid injected by the female parasitoid during oviposition. We have examined the effects of injection of calyx fluid on the following haemocytic manifestations of the immune system of 5th-instar larvae of H. virescens: encapsulation, nodulation, phagocytosis, erythrocyte rosetting and coagulation. Of these phenomena, only those requiring the formation of a multicellular sheath of plasmatocytes were affected. In general, encapsulation was fully suppressed; all of the C. sonorensis eggs and most of the glass rods implanted as targets were devoid of attached haemocytes 3 days after implantation although a few of the latter were coated by a sparsely distributed layer of granulocytes. Plasmatocytes also appeared to be present in thicker depositions of haemocytes. In nodulation, only the second, encapsulation-like phase was inhibited. The resistant first stage, involving the entrapment of particles by haemocytes, only resulted in the formation of amorphous, disorganized nodules. Granulocyte-dependent aspects of the immune system (phagocytosis, rosetting and possibly coagulation and the first stage of encapsulation and nodulation) occurred normally. The data suggest that in 5th-instar hosts injection of calyx fluid acts specifically on plasmatocyte function.     

Microplitis croceipes teratocytes cause developmental arrest of Heliothis virescens larvae

Microplitis croceipes teratocytes placed into nonparasitized Heliothis virescens larvae survived in the absence of a parasitoid larva and caused developmental changes in the host. Expressions of these changes included delayed larval mortality, incomplete larval-pupal ecdysis, or delayed pupation. Two day old 4th stadium H. virescens larvae were more sensitive to injected teratocytes than were 5th stadium larvae. Three day old teratocytes were more effective than were 6 day old teratocytes. The degree of response was related to the number of injected teratocytes. For example, 750 three day old teratocytes (the approximate number from a single parasitoid egg) caused delayed larval mortality in 96% of the treated larvae whereas 175 three day old teratocytes caused delayed larval mortality in only 33% of the treated larvae. Even a dose of 80 teratocytes resulted in 15% incomplete larval-pupal ecdysis compared to 0% for controls. Treatment with hemocyte-and teratocyte-free hemolymph from parasitized larvae, hemocytes from nonparasitized H. virescens, unfertilized M. croceipes eggs, Cotesia congregata teratocytes, or Micrococcus lysodeikticus cells all had very little effect either on larval growth or development time.     

Interspecific competition among endoparasitoids of tobacco budworm larvae [Lep.: Noctuidae]

The ability of female larvae endoparasitoids [Microplitis croceipes Cresson:Cardiochiles nigriceps Viereck andCumpoletis sonorensis (Carlson)] to distinguish between unparasitized tobacco budworm,Heliothis virescens (F.), larvae andH. virescens larvae parasitized by the egg-larval parasitoidChelonus insularis,Cresson, was determined in laboratory studies. The 3 species of larval endoparasitoid females did not appear capable of distinguishing between unparasitized andC. insularis parasitized larvae resulting in multiple parasitoidism. The results of the ensuing competition between the 3 species for possession of the host demonstrated that bothC. sonorensis andM. croceipes were intrinsically superior toC. insularis. Both larva endoparasitoids destroyed the olderC. insularis larvae by physically attacking the latter. The presence ofC. insularis larvae in the host was found to prevent the hatch of compeatingC. nigriceps eggs through physiological suppression. The results show that the early attack of a host, as in the egg-larval parasitoid habit, is not necessarily advantageous.     

Potential detoxification of gossypol by UDP-glycosyltransferases in the two Heliothine moth species Helicoverpa armigera and Heliothis virescens

The cotton bollworm Helicoverpa armigera and the tobacco budworm Heliothis virescens are closely related generalist insect herbivores and serious pest species on a number of economically important crop plants including cotton. Even though cotton is well defended by its major defensive compound gossypol, a toxic sesquiterpene dimer, larvae of both species are capable of developing on cotton plants. In spite of severe damage larvae cause on cotton plants, little is known about gossypol detoxification mechanisms in cotton-feeding insects. Here, we detected three monoglycosylated and up to five diglycosylated gossypol isomers in the feces of H. armigera and H. virescens larvae fed on gossypol-supplemented diet. Candidate UDP-glycosyltransferase (UGT) genes of H. armigera were selected by microarray studies and in silico analyses and were functionally expressed in insect cells. In enzymatic assays, we show that UGT41B3 and UGT40D1 are capable of glycosylating gossypol mainly to the diglycosylated gossypol isomer 5 that is characteristic for H. armigera and is absent in H. virescens feces. In conclusion, our results demonstrate that gossypol is partially metabolized by UGTs via glycosylation, which might be a crucial step in gossypol detoxification in generalist herbivores utilizing cotton as host plant.     

Role of the exoprotease InA in the pathogenicity of Bacillus thuringiensis in pupae of Hyalophora cecropia

Two geographical biotypes of Nomuraea rileyi (from Ecuador and the United States) were topically bioassayed against seven lepidopteran species, i.e., Anticarsia gemmatalis, Heliothis zea, Heliothis virescens, Heliothis subflexa, Pseudoplusia includens, Spodoptera exigua, and Trichoplusia ni. There was an average difference of 1.7-fold in mortality in how cultures of the same insect species from different sources responded to topical applications of either biotype of N. rileyi. Regression equations and LC₅₀ values were obtained for each insect species and fungal biotype combination. Larvae of S. exigua were equally susceptible to both biotypes of N. rileyi. Although larvae of A. gemmatalis were moderately susceptible to the Ecuadoran biotype, they were relatively nonsusceptible to the Mississippian biotype. Species of Heliothis (H. zea, H. virescens, and H. subflexa) were about equally susceptible to the Mississippian biotype. Larvae of H. subflexa and H. virescens, however, were significantly less susceptible than H. zea to the Ecuadoran biotype. When the integumental barrier was breached via intrahemocoelic injections, larvae of H. virescens were as susceptible as H. zea larvae to blastospores of either biotype of N. rileyi.     

Juvenile hormone esterase activity and ecdysteroid titer in Heliothis virescens larvae injected with Microplitis croceipes teratocytes

Juvenile hormone esterase (JHE) activity in the hemolymph of 5th-instar Heliothis virescens larvae injected with Microplitis croceipes teratocytes was inversely related to the number of teratocytes injected. JHE activity in the hemolymph of larvae injected with 750 3-day-old teratocytes (the approximate number from one parasitoid embryo) was depressed to less than 5% of those levels found in control larvae. During the latter portion of the digging stage and in the burrowing-digging (BD) stage JHE activity in larvae treated with 350 teratocytes was approximately 40% of control values. However, injection of 180 teratocytes did not significantly affect JHE titers. Two-day-old teratocytes caused the greatest reduction in JHE titer with decreasing effects observed with injections of 3- to 6-day-old teratocytes. Nevertheless, because 2-day-old teratocytes were difficult to separate from host hemocytes, 3-day-old teratocytes were used in most of these studies. Injections of nonparasitized H. virescens hemolymph plasma, Micrococcus luteus bacterial cell walls, washed M. croceipes eggs, or teratocytes from Cotesia congregata did not depress JHE titers. Teratocyte injections also significantly reduced growth of host fat body. Ecdysteroid titers in cell formation, day 2 (CF2) larvae injected as new 5th instars with 350 3-day-old teratocytes failed to increase, as compared to noninjected and saline-injected controls. An injection of 1 μg/larva of 20-hydroxyecdysone at the BD stage permitted normal pupation in 50% of the teratocyte-treated larvae as compared to 0% pupation for teratocyte-treated control larvae not treated with 20-hydroxyecdysone. Teratocytes seem to be responsible for the inhibition of JHE release and thus indirectly impact on ecdysteroid titers. © 1992 Wiley-Liss, Inc.