Growth-blocking peptide titer during larval development of parasitized and cold-stressed armyworm

Growth-blocking peptide (GBP) has been isolated for the first time from the haemolymph of the host armyworm Pseudaletia separata whose development was halted in the last larval instar stage by parasitization with the parasitoid wasp Cotesia kariyai. Recent studies demonstrated that GBP not only exists in the plasma (haemolymph without cells) of parasitized last instar larvae, but also in the plasma of nonparasitized penultimate (5th) instar larvae. Monoclonal antibodies were prepared to measure the titers of GBP in nonparasitized and parasitized larval plasma. One of three monoclonal antibodies raised against GBP, which is the most specific for GBP, was used to quantify the concentration of plasma GBP. As this antibody recognized two plasma peptides other than GBP in crude plasma fractions, each plasma peptide fraction was separated by a reversed phase HPLC, and then plasma GBP level was measured by ELISA. The highest level of plasma GBP detected on Day 0 of the penultimate instar larvae was gradually decreased throughout the larval growth except for the temporary increase on Day 0 of last larval instar. After parasitization on Day 0 of last larval instar, two peaks of plasma GBP titer were detected during the last larval instar, one day and six days after parasitization. This characteristic increase and decrease in plasma GBP level was also observed by transferring last instar larvae of the armyworm from 25 to 10°C, as a result of which larvae delayed pupation by more than 15 days. From these results, it is reasonable to propose that plasma GBP in lepidopteran larvae might control certain upstream steps in a cascade of events leading to pupation; thus, an elevated level of plasma GBP interferes with normal metamorphosis from larvae to pupae.     

C-terminal Elongation of Growth-blocking Peptide Enhances Its Biological Activity and Micelle Binding Affinity

Growth-blocking peptide (GBP) is a hormone-like peptide that suppresses the growth of the host armyworm. Although the 23-amino acid GBP (1–23 GBP) is expressed in nonparasitized armyworm plasma, the parasitization by wasp produces the 28-amino acid GBP (1–28 GBP) through an elongation of the C-terminal amino acid sequence. In this study, we characterized the GBP variants, which consist of various lengths of the C-terminal region, by comparing their biological activities and three-dimensional structures. The results of an injection study indicate that 1–28 GBP most strongly suppresses larval growth. NMR analysis shows that these peptides have basically the same tertiary structures and that the extension of the C-terminal region is disordered. However, the C-terminal region of 1–28 GBP undergoes a conformational transition from a random coiled state to an α-helical state in the presence of dodecylphosphocholine micelles. This suggests that binding of the C-terminal region would affect larval growth activity.Growth-blocking peptide (GBP)² was initially identified from the hemolymph of armyworm Pseudaletia separata as a 25-amino acid peptide (1–25 GBP) that prevents the onset of pupation of the host by parasitization of wasp Cotesia kariyai (1–4). Injection of GBP into nonparasitized armyworm larvae early in the last instar delays larval growth and retards pupation for more than a few days. Our previous studies showed that GBP is a hormone-like biogenic peptide of the host armyworm (5, 6). In nonparasitized larvae, the concentrations of GBP were much higher in the early larval stages than in the latter ones. However, parasitization by wasp induces an elevation of GBP in the last larval stages. This elevation was shown to lead to growth retardation via repression of juvenile hormone esterase activity (7–9). Interestingly, a cDNA analysis indicated that the cDNA encodes a 23-amino acid GBP (1–23 GBP), although GBP purified from parasitized armyworm plasma consists of 25 amino acid residues. GBP was expressed as a 23-residue peptide (1–23 GBP) in nonparasitized armyworm larvae, whereas 1–25 GBP, containing Tyr²⁴ and Gln²⁵, was purified from the parasitized larvae. Moreover, the preliminary peptide sequencing of GBP prepared from parasitized larval hemolymph showed the 26^th and 27^th residues on rare occasions (Leu and Ile, respectively) (6). On the basis of these results, we concluded that the TAG stop codon for the 24^th amino acid was unusually decoded as Tyr upon parasitization by parasitoid wasps (10) and predicted that an intact and mature GBP synthesized in the parasitized armyworm larvae would consist of 28 amino acid residues (1–28 GBP).GBP has multiple functions: adhesion and spreading of a specific class of immune cells (plasmatocytes), proliferation of various cultured cells, and induction of larval paralysis (11–13). More than 10 GBP homologous peptides have been identified in Lepidopteran insects, and based on their N-terminal consensus sequences (Glu¹-Asn²-Phe³), they have been categorized as the ENF peptide family (14). The tertiary structure of 1–25 GBP consists of a disordered N-terminal region (residues Glu¹–Gly⁶), a well ordered core region (residues Cys⁷–Thr²²) stabilized by a disulfide bond and a short antiparallel β-sheet, and a short unstructured C-terminal region (Phe²³–Glu²⁵) (15). Because no GBP receptor or its gene has been isolated yet, the nature of either of them at the cellular and molecular levels is poorly understood at present. In contrast, the relationship between the structure and activity of GBP has been well studied by analyzing the biological activities of several variants of GBP and plasmatocyte-spreading peptide (one of the ENF family peptides). Especially, extensive studies on the N termini (residues 1–6) of GBP and plasmatocyte-spreading peptide demonstrated the importance of Phe³ for exerting their hemocyte stimulating activity, thereby suggesting a possible mechanism for receptor activation that requires binding of the aromatic ring of Phe³ and a closely spaced primary amine with receptor activating properties (16–19).In contrast, the C termini of GBP and other ENF peptides have received less attention, because of the weak secondary structure predictions. Therefore, in this study we focused on the C terminus region of GBP and analyzed its contribution to the expression of some biological activities and to the tertiary structure of this peptide. Especially, we prepared GBP with 28 amino acids and characterized the C-terminal region of 1–28 GBP (residues Phe²³–Thr²⁸), because we knew that GBP is present as a 23-amino acid peptide in nonparasitized healthy larvae and that GBP with 28 amino acids has been found only in parasitized host larvae. Our results suggest that the elongation of the C-terminal region of Phe²³–Thr²⁸ greatly reinforced GBP binding with the membrane. Further, the elongation increased GBP inhibition of larval growth.     

Parasitic castration of Pseudaletia separata by Cotesia kariyai and its association with polydnavirus gene expression

Parasitization by the endoparasitoid Cotesia kariyai caused the inhibition of spermatogenesis of Pseudaletia separata. This phenomenon is called parasitic castration. The degree of castration was dependent on the host stage parasitized. Host parasitized on day 1 of the 4th stadium (the time of primary spermatocyte accumulation), had testicular cells with abnormal chromosomes appearing two days after parasitization, and spermiogenesis was completely inhibited. However, when hosts were parasitized on day 0 of the 6th (final) stadium, the degree of castration was less severe, and elongated cells appeared similar to those found in nonparasitized larvae. Results of this study involving injection of C. kariyai polydnavirus (CkPV) and venom suggested that these wasp components caused the appearance of abnormal chromosomes in specific germ cells, which were in mitotic or meiotic prophases. The amount of CkPV gene expression in host testes increased immediately after parasitization and reached a maximum 12h later. The early-expressed CkPV gene(s) may be related to the parasitic castration phenomenon.     

Inhibition of testicular growth and development in Manduca sexta larvae parasitized by the braconid wasp Cotesia congregata

Tobacco hornworm larvae parasitized by the gregarious larval endoparasitoid Cotesia congregata exhibited an inhibition in testicular growth and development, the extent of which was determined by the age and developmental stage of the host at the time of parasitization. The degree of parasitic castration, as assessed by measurements of testicular volume, was correlated with the stadium in which parasitization occurred. A mathematical formula requiring the measurement of testicular length, width and depth was used to calculate testicular volume. The use of the depth parameter revealed a negative correlation between host weight and testicular volume in parasitized larvae. Testicular volumes of fifth instar hosts, which had been parasitized in the first stadium, were significantly smaller than those originally parasitized as fourth or fifth instar larvae and were not correlated with parasitoid load. Effects of natural parasitism were not duplicated by injections of C. congregata polydnavirus and venom, topical treatment with the juvenile hormone analog methoprene, or starvation of nonparasitized larvae. Larvae receiving virus plus venom or methoprene grew larger due to delayed wandering and had larger testes than controls. Deleterious effects on host testes may be due to the effects of nutrient competition between the developing parasitoid progeny and the gonads, combined with the juvenilizing effects believed to be caused by the polydnavirus.     

A putative new juvenile peptide hormone in lepidopteran insects.

A growth-blocking peptide (GBP) with repressive activity against juvenile hormone (JH) esterase has been isolated from the last (6th) instar larval plasma of the armyworm Pseudaletia separata (Lepidoptera: Noctuidae) parasitized by the parasitoid wasp Apanteles kariyai (Hymenoptera: Braconidae) (1,2). This study demonstrates that GBP not only exists in the plasma of parasitized last instar larvae, but also in the plasma of unparasitized penultimate (5th) instar larvae, while the plasma of last instar larvae does not contain any detectable amount of GBP. The detection of GBP in unparasitized penultimate instar larvae, before the final larval molt, demonstrates that this factor is naturally occurring in the insect larva before the last larval instar and is seemingly coordinating, along with JH, the regulation of juvenile characteristics. This finding suggests the existence of a new type of juvenile peptide hormone in lepidopteran insects.     

The endoparasitoid Cotesia kariyai (Ck) regulates the growth and metabolic efficiency of Pseudaletia separata larvae by venom and Ck polydnavirus

It was previously demonstrated that parasitization by Cotesia kariyai caused a decrease in weight gain and food consumption in host larvae, resulting in a lower final weight for parasitized hosts. It is predicted that C. kariyai regulates the physiological condition of the host to obtain maximum food under restricted nutritional conditions. Approximate digestibility (AD) was higher following parasitization but the efficiency of conversion of digested food (ECD) of the parasitized hosts was lower. This suggests that resources available to the parasitoid larvae are enhanced in the parasitized hosts. We evaluated the physiological changes caused by injection of calyx fluid (polydnavirus) plus venom (C+V) in nonparasitized hosts. Injection of C+V into the nonparasitized hosts duplicated the effects of parasitism, namely it increased the AD and decreased the ECD. Furthermore, C+V injections elevated trehalose concentrations in nonparasitized host 7 to 10 d after injection (2nd stadium of the parasitoid larva). Protein content also increased on days 9 and 10 after C+V injection. These results suggest that the nutrients that parasitoid larvae require for their growth increase in the hemolymph of the host during the 2nd stadium of the parasitoid larva.     

A putative protein translation inhibitory factor encoded by Cotesia plutellae bracovirus suppresses host hemocyte-spreading behavior

An endoparasitoid, Cotesia plutellae (Hymenoptera: Braconidae), possesses a mutualistic bracovirus (CpBV), which plays significant roles in the parasitized host, Plutella xylostella (Lepidoptera: Plutellidae). CpBV15beta, a viral gene encoded by CpBV, is expressed at early and late parasitization periods, suggesting that it functions to manipulate the physiology of the parasitized host. This paper reports a physiological function of CpBV15beta as an immunosuppressive agent. The effect of CpBV15beta on cellular immunity was analyzed by assessing hemocyte-spreading behavior. Parasitization by C. plutellae caused altered behavior of hemocytes of P. xylostella, in which the hemocytes were not able to attach and spread on glass slides. CpBV15beta was expressed in Sf9 cells using a baculovirus expression system and purified from the culture media. When hemocytes of nonparasitized P. xylostella were incubated with purified CpBV15beta protein, spreading behavior was impaired in a dose-dependent manner at low micro-molar range. This inhibitory effect of CpBV15beta could also be demonstrated on hemocytes of a non-natural host, Spodoptera exigua. CpBV15beta protein significantly inhibited F-actin growth of hemocytes in response to an insect cytokine. Similarly, cycloheximide, a eukaryotic translation inhibitor, strongly inhibited the spreading behavior and F-actin growth of P. xylostella hemocytes. Under in vitro condition, hemocytes of nonparasitized P. xylostella released proteins into the surrounding medium. Upon incubation of hemocytes with either CpBV15beta or cycloheximide, their ability to release protein molecules was markedly inhibited. This study suggests that CpBV15beta suppresses hemocyte behavior by inhibiting protein translation.     

Structure of a growth-blocking peptide present in parasitized insect hemolymph

Last instar larvae of the insect armyworm, Pseudaletia separata, parasitized with the parasitoid wasp, Apanteles kariyai, do not initiate metamorphosis and, ultimately, the wasp larvae emerge from the host larvae about 10 days after parasitization (Tanaka, T., Agui, N., and Hiruma, K. (1987) Gen. Comp. Endocrinol. 67, 364-374). It is necessary for the parasitoid wasp to perturb the armyworm's endocrinological processes that control normal metamorphosis from larvae to pupae. This endocrinological perturbation allows the parasitoid to complete its larval growth before emerging from the host larvae. It is obligatory for the parasitoid larvae to emerge while the host is still in a larval stage because the sclerotized pupal cuticle is impenetrable for the parasitoid larvae. A growth-blocking peptide with repressive activity against juvenile hormone esterase has been proven to exist in the parasitized host larval plasma (Hayakawa, Y. (1990) J. Biol. Chem. 265, 10813-10816). Here, I describe the detailed structure of this peptide and also the corresponding synthetic peptide to confirm this structure.     

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.     

Parasitism-induced effects on host growth and metabolic efficiency in Plutella xylostella larvae parasitized by Cotesia vestalis or Diadegma semiclausum

The nutritional physiology of the diamondback moth, Plutella xylostella, larvae was examined after parasitization by the solitary endoparasitoids Cotesia vestalis or Diadegma semiclausum. Examinations were performed in two phases, one was examined at the time point of 24 h post‐parasitization, and the other was done at the end of the 4th instar larvae of host. Rates of growth, food consumption, assimilation, excretion, and respiration were calculated as well as approximate digestibility and the rate ratios ECI (percent efficiency of conversion of ingested food to body substance), and ECD (percent efficiency of conversion of digested food to body substance). Parasitization by C. vestalis resulted in significant decrease in the rates of growth, feeding, excretion, assimilation, and respiration, but the final dry rate of respiration at the end of last larval stadium was elevated. The ECI and ECD were also reduced as the result of parasitization, but digestibility was increased. All these parameters in the larvae parasitized by D. semiclausum at 24 h post‐parasitization were also significantly changed compared to the control; however, these differences were quantitatively, but not qualitatively before pupation, similar to those resulted from parasitization by C. vestalis. In spite of the similarities of the parasitism‐induced effects caused by these endoparasitoids, the final metabolic rate, that is, the rate of intake of nutrients required to compensate for metabolism, was much lower in the larvae parasitized by C. vestalis than that of the larvae parasitized by D. semiclausum. All of the results discussed here will contribute toward explaining the different ways these two wasps regulate the parasitoid‐host relationship.     

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.     

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.     

THE PARASITOID,Cotesia flavipes (CAMERON) (HYMENOPTERA: BRACONIDAE), INFLUENCES FOOD CONSUMPTION AND UTILIZATION BY LARVAL Diatraea saccharalis (F.) (LEPIDOPTERA: CRAMBIDAE)

Parasitoids exploit host insects for food and other resources; they alter host development and physiology to optimize conditions to favor parasitoid development. Parasitoids influence their hosts by injecting eggs, along with a variety of substances, including venoms, polydnaviruses, ovarian fluids, and other maternal factors, into hosts. These factors induce profound changes in hosts, such as behavior, metabolism, endocrine events, and immune defense. Because endoparasitoids develop and consume tissues from within their hosts, it is reasonable to suggest that internal parasitization would also influence host food consumption and metabolism. We report on the effects of parasitism by Cotesia flavipes on the food consumption and utilization of its host, Diatraea saccharalis. Cotesia flavipes reduces the host food consumption, but parasitized larvae considered a unit with their parasitoid's attained the same final weight as the nonparasitized larvae. Nutritional indices, midgut activities of carbohydrases, and trypsin of parasitized and nonparasitized D. saccharalis were assessed. Parasitized larvae had reduced relative food consumption, metabolic and growth rates, coupled with higher efficiency for conversion of the digested, but not ingested, food into body mass. Parasitism also affected food flux through the gut and protein contents in the midgut of parasitized larvae. The activity of α‐amylase and trehalase in parasitized host was enhanced in the first day after parasitism relative to control larvae. Saccharase activity remained unchanged during larval development. Trypsin activity was reduced from the fifth to ninth day after parasitism. We argue on the mechanisms involved in host food processing after parasitism.     

Morphological abnormalities and lethality in silkworm (Bombyx mori) larvae treated with high concentrations of insect growth-blocking peptide

Insect growth-blocking peptides (GBPs) exhibit growth-blocking and paralytic activity. Low concentrations of GBP stimulate larval growth, whereas high concentrations of GBP significantly retard larval growth. Here, we show that morphological abnormalities and lethality were induced in silkworm (Bombyx mori) larvae by high concentrations of GBP. Active B. mori GBP (BmGBP) was produced by treating recombinant proBmGBP (expressed in baculovirus-infected insect cells) with bovine factor Xa. When silkworm larvae on day 1 of the fifth-instar stage were injected between the seventh and eight abdominal segments with BmGBP (100 or 500 ng/larva), the larval–pupal and pupal–adult transformations of these silkworms were delayed in a dose-dependent manner. However, a high concentration (2000 ng/larva) of BmGBP or Spodoptera exigua GBP (SeGBP) acutely induced morphological abnormalities and death in silkworm larvae. In silkworm larvae treated with high concentrations of GBPs, the ingested food excessively accumulated in the foregut, which caused extreme swelling in both the thorax and the foregut and resulted in larval death. Therefore, these results not only provide insight into the effect of insect GBPs on gut physiology but also reveal a novel function of insect GBPs.     

A novel polydnaviral gene family, BEN, and its immunosuppressive function in larvae of Plutella xylostella parasitized by Cotesia plutellae

A full genome sequence of the episomal form of Cotesia plutellae bracovirus (CpBV) suggests 11 BEN family genes. This study analyzed their expression and physiological function in the viral host, Plutella xylostella. All 11 BEN family genes were expressed during entire parasitization period of P. xylostella larvae. In addition, these BEN family genes were expressed in fat body, gut, epidermis, and hemocytes in final larval instar of parasitized P. xylostella. The 11 BEN family genes were transiently expressed in nonparasitized larvae by injection of each viral segment containing its corresponding BEN family gene. The transient expression of BEN family genes significantly suppressed hemocyte nodule formation in response to bacterial challenge. Subsequent injection of double-stranded RNA specific to each BEN family gene suppressed the expression of the BEN family gene and rescued the immunosuppression. These results indicate that 11 BEN family genes are expressed in larvae parasitized by C. plutellae and play crucial role in inducing immunosuppression. Homologous BEN family genes were found in other bracoviral genomes. We propose BEN domain-containing genes as a new functional gene family in polydnaviruses.