Host regulation and the embryonic development of the endoparasitoid Toxoneuron nigriceps (Hymenoptera: Braconidae)

Insect endoparasitoids modulate the host physiology through the injection of maternal-derived substances into the host, inducing physiological and hormonal changes in the host's internal environment to benefit parasitoid development. These changes are direct to control host development and regulate nutrient availability to the developing parasitoid, and they are synchronized with parasitoid development. Eggs of some of these parasitoids have low yolk content and require nutrients from the host hemolymph to initiate and complete embryogenesis. We report changes in the amino acid composition and protein profile of the host hemolymph of the endoparasitoid Toxoneuron nigriceps, and improved the in vitro culture of pre-germ band stage eggs. The protein profile of parasitized larvae was similar to controls throughout the embryonic development, but total amino acid concentration decreased in the first 2 h after parasitization, significantly increasing in the following hours up to 8 h. Amino acid levels were higher in parasitized larvae from 16 to 28 h after parasitization. Comparison of single amino acids indicated amino acids involved in energy metabolism (Krebs cycle) followed a trend during parasitoid embryogenesis, and their changes were correlated with embryonic development. Improvement in the in vitro development of 6 h-old eggs of T. nigriceps was obtained by adding factors released by the host fat body to the artificial medium, while a cell lysate stimulated embryogenesis and allowed the full development of newly laid eggs in vitro.     

Juvenile hormone synthesis, metabolism, and resulting haemolymph titre in Heliothis virescens larvae parasitized by Toxoneuron nigriceps

Last instar larvae of the tobacco budworm, Heliothis virescens F., fail to pupate and have little 20-hydroxyecdysone when parasitized by Toxoneuron nigriceps (Viereck). In this paper, we extend these observations to juvenile hormone (JH) to determine if parasitism by this wasp affects other endocrine systems. To this end, we compared the production of JH by corpora cardiaca-corpora allata complexes (CC-CA), the metabolism of JH by haemolymph enzymes, and the haemolymph titre of JH in parasitized and non-parasitized control larvae of H. virescens during the last larval instar. CC-CA from parasitized and control larvae had similar peaks of JH synthesis on day 1 of the fifth instar, with JH II accounting for more than 90% of total JH in both groups. On subsequent days, JH synthesis dropped to undetectable levels more quickly in non-parasitized controls than in parasitized larvae. JH metabolism by haemolymph of parasitized and control animals increased from low levels on day 1 of the fifth instar to high levels on days 2 and 3 of the instar. JH metabolism was significantly higher in control larvae than in parasitized larvae. After day 3, JH metabolism decreased in both groups, but was significantly higher in parasitized larvae. The major metabolite of JH in both groups was JH acid, though traces of JH diol and JH acid diol were also detected. The haemolymph titre of JH in both groups peaked on day 1 of the fifth instar and, similar to the synthesis of JH by CC-CA, decreased more rapidly in control larvae. As a result, non-parasitized animals had significantly lower JH titres on day 2. The higher JH titres observed in parasitized larvae during the early fifth instar may contribute to their developmental arrest. The possible role of these JH alterations in the host developmental and metabolic redirection is discussed and a more comprehensive physiological model accounting for host-parasitoid interactions is proposed.     

Movement of proteins across the digestive system of the tobacco budworm, Heliothis virescens

Bovine serum albumin (BSA) and anti‐BSA polyclonal antibody were used as model polypeptides to examine the movement of foreign proteins across the insect digestive system and their accumulation in hemolymph of fourth stadium tobacco budworms, Heliothis virescens (Fabricius) (Lepidoptera: Noctuidae). Hydrateable meal pads were developed in these studies as a method for easily introducing compounds into the insect digestive system. When insects were allowed to feed continuously on hydrated meal pads containing 0.8 mg of anti‐BSA per gram diet, the level of antibody found in hemolymph was 2.4 ± 0.1 and 3.4 ± 0.1 µg ml^?1 (average  1 SEM) after 8 and 16 h, respectively, as determined by enzyme‐linked immunosorbant assay (ELISA). Continuous feeding on hydrated meal pads containing the same concentration of BSA produced hemolymph concentrations of 1.5 ± 0.1 and 1.6 ± 0.1 µg ml^?1 hemolymph at 8 and 16 h, respectively. Western blot analyses demonstrated that BSA and anti‐BSA both retained their primary and multimeric structure and that anti‐BSA maintained its antigenic activity in the meal pads and after movement from meal pads into the hemolymph. When 1 µg of anti‐BSA or BSA was injected into the hemocoel of fourth instars, the concentrations decreased with time and 120 min after injection were 20% and 0.6% of the original concentration, respectively. When added at the same concentration to plasma in vitro, the decrease was 81.5% and 57.5%, respectively, at 2 h. The accumulation of native anti‐BSA and BSA protein in insect hemolymph is the result of their rate of movement across the gut and their rate of turnover in hemolymph. Movement of anti‐BSA and BSA across the digestive system was also noted in Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae), Acheta domesticus (L.) (Orthoptera: Gryllidae), and Gromphadorhina portentosa (Schaum) (Blattaria: Blattellidae). Anti‐BSA and BSA were not detected in the hemolymph of Manduca sexta (L.) (Lepidoptera: Sphingidae) after feeding.     

Host plant specialization in the generalist moth Heliothis virescens and the role of egg imprinting

Even though generalist insects are able to feed on many different host plants, local specialization may occur, which could lead to genetic differentiation. In this paper we assessed the level and extent of host plant specialization in the generalist herbivore Heliothis virescens Fabricius (Lepidoptera, Noctuidae). This generalist can grow and survive on many different plant species, belonging to more than 37 families. Previously, two laboratory strains were described that differ in their performance on cotton and chickpea. In this study we explored this phenomenon further. Specifically, we asked the following questions: (1) Do the two strains still differ in their performance on cotton and chickpea? Since we found that the most pronounced difference between the two strains was in their growth on fresh chickpea leaves, we then asked: (2) Does this variation in performance have a genetic basis? In our genetic analysis, we found that growth rates changed over time and that two linkage groups significantly affected the ability to grow on chickpea. One QTL was homologous to Bombyx mori chromosome 15, onto which genes for insecticide resistance and detoxicative enzymes have previously been mapped. (3) Is there a difference in oviposition preference between the two strains? Oviposition experiments revealed no preference in either strain when females were reared on the same artificial diet. However, we did find a maternal inheritance of oviposition preference: daughters collected as eggs from cotton oviposited significantly more eggs on cotton, and daughters collected as eggs from chickpea likewise laid more eggs on chickpea. Thus, Hopkins’ host selection principle seems to holds in this species, although imprinting seems to happen not at the larval but at the egg stage, which is a new finding. This study shows how genetic and nongenetic factors can interact to shape the patterns of local specialization in a generalist herbivore.     

Synthesis and transport of storage proteins by testes in Heliothis virescens.

The synthesis of two storage protein subunits, 76,000-Mr and 82,000-Mr polypeptides, by the testes sheath has been studied in Heliothis virescens. Like fat body, which is the primary site of synthesis for the large extratesticular pool, cells of the testes sheath secrete glycosylated storage proteins assembled into hexamers. The testis sheath differed from fat body in several important respects, including the failure to synthesize an abundant (in the hemolymph) 74,000-Mr storage protein, its relatively reduced expression of the 76,000-Mr polypeptide, and the absence of resorption of storage proteins from the lumen of the testis during pupal development. Cyst cells were also shown to import actively the 82,000-Mr storage protein by pinocytosis of testicular fluid and transfer it to the developing spermatids. Unlike other cell types that sequester storage proteins in the form of cytoplasmic granules, their localization within spermatids was exclusively mitochondrial. These observations suggest that expression of the storage protein genes is regulated tissue specifically and reveal novel pathways for their transport and, perhaps, utilization and function during development.     

Host factors involved in retroviral budding and release

The plasma membrane is the final barrier that enveloped viruses must cross during their egress from the infected cell. Here, we review recent insights into the cell biology of retroviral assembly and release; these insights have driven a new understanding of the host proteins, such as the ESCRT machinery, that are used by retroviruses to promote their final separation from the host cell. We also review antiviral host factors such as tetherin, which can directly inhibit the release of retroviral particles. These studies have illuminated the role of the lipid bilayer as the unexpected target for virus restriction by the innate immune response.     

Spermatophore formation and regulation of egg maturation and oviposition in female Heliothis virescens by the male

This study was undertaken, using various surgical manipulations, to examine the role and functions of accessory sex glands and testes in Heliothis virescens in spermatophore formation and stimulation of egg maturation in mated females. Normal females mated to accessory sex glandectomized (-ASG) males produced fewer eggs and retained most of their eggs in ovarioles compared with females mated to sham-operated (+ASG) or normal males. The ASG are the source of the components for spermatophore formation in H. virescens. Females mated to castrated (-Testes) males showed similar pattern of egg production as did females mated to -ASG males. Females mated to -Testes males and those mated to sham-operated (+Testes) or normal males were found to have spermatophore in the bursa after uncoupling. Normal females mated to allatectomized (-CA) males developed similar numbers of total eggs as did females mated to sham-operated (+CA) males and normal males.     

Evidence and bioassay for diuretic factors in the nervous system of larvalHeliothis virescens

Summary Diuretic factors were studied in the central nervous system of larvae of the tobacco budworm,Heliothis virescens, using [¹⁴C]urea as a sensitive indicator for water movement through isolated Malpighian tubules. The assay required Na⁺ and a pH of 6.0–6.2 for maximum activity. Malpighian tubules had high secretory activity in feeding larvae of the fifth instar, but the activity declined during the burrowing-digging stage that preceded pupation. Malpighian tubules from starved larvae showed a greater response to extracts of nervous tissues than did tubules from feeding larvae, and extracts showed a dose-response relationship with fluid secretion. Diuretic activity was distributed throughout all parts of the central nervous system with the brain having the most activity. Brain extracts increased fluid secretion by in vitro Malpighian tubules by more than 3-fold and doubled the rate of dye clearance from the hemolymph in vivo. Diuretic activity in nervous tissue extracts was unaffected by boiling but sensitive to proteases. Fluid secretion by in vitro tubules was increased by cAMP, dbcAMP, theophylline, octopamine and dopa. These studies provide evidence for the presence of diuretic factors in the central nervous system ofH. virescens larvae and describe a sensitive bioassay for these factors.Abbreviations AR activation ratio - cAMP cyclic AMP - dbcAMP dibutyryl cyclic AMP - dbcGMP dibutyryl cyclic GMP - Dopa dihydroxyphenylalanine - 5-HT 5-hydroxytryptamine - L1 larval instar - VCNS ventral central nervous system     

Developmental and hormonal regulation of midgut remodeling in a lepidopteran insect, Heliothis virescens

Midgut tissue undergoes remodeling during metamorphosis in insects belonging to orders Lepidoptera and Diptera. We investigated the developmental and hormonal regulation of these remodeling events in lepidopteran insect, Heliothis virescens. In H. virescens, programmed cell death (PCD) of larval midgut cells as well as proliferation and differentiation of imaginal cells began at 108 h after ecdysis to the final larval instar (AEFL) and proceeded through the pupal stages. Expression patterns of pro- cell death factors (caspase-1 and ICE) and anti-cell death factor, Inhibitor of Apoptosis (IAP) were studied in midguts during last larval and pupal stages. IAP, Caspase-1 and ICE mRNAs showed peaks at 48 h AEFL, 96 h AEFL and in newly formed pupae, respectively. Immunohistochemical analysis substantiated high caspase-3 activity in midgut at 108 h AEFL. Application of methoprene, a juvenile hormone analog (JHA) blocked PCD by maintaining high levels of IAP, downregulating the expression of caspase-1, ICE and inhibiting an increase in caspase-3 protein levels in midgut tissue. Also, the differentiation of imaginal cells was impaired by methoprene treatment. These studies demonstrate that presence of JHA during final instar larvae affects both midgut remodeling and larval-pupal metamorphosis leading to larval/pupal deformities in lepidopteran insects, a mechanism that is different from that in mosquito, Ae. aegypti where JHA uncouples midgut remodeling from metamorphosis.     

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.     

Hagen's glands of the parasitic wasp Diachasmimorpha longicaudata (Hymenoptera: Braconidae): ultrastructure and the detection of entomopoxvirus and parasitism-specific proteins

Hagen's glands of males of the parasitic wasp Diachasmimorpha longicaudata (Hymenoptera: Braconidae) secrete compounds that are involved in courtship and defense. Like the poison glands of female wasps, the Hagen's glands are secretory, membranous, and of ectodermal origin. The poison glands contain the symbiotic entomopoxvirus, DlEPV and the parasitism-specific protein, PSP 24. DlEPV proteins were detected in homogenates of male wasps. Our goal was to describe the ultrastructure of the Hagen's glands and determine whether they contain DlEPV virions and/or proteins as well as PSP 24. The Hagen's glands are bilateral and each consists of 12-16 tubules arranged in two clusters. In cross-section, a tubule has three zones that enclose a central cuticle-lined lumen. The outermost zone consists of aggregates ('islands') of small vesicles, interconnected by narrow ductules that lead to large cuticle-lined ducts, which transport electron-dense material to the lumen prior to its release from the gland. Large vesicles in Zone 2 and a thick layer of ribosomes and rough endoplasmic reticula in Zone 3 are the likely sites of storage and protein synthesis, respectively. While DlEPV virions were not seen in the Hagen's gland, DlEPV and PSP 24 proteins were present.     

Identity and expression pattern of chemosensory proteins in Heliothis virescens (Lepidoptera, Noctuidae)

Analyzing the chemosensory organs of the moth Heliothis virescens, three proteins belonging to the family of insect chemosensory proteins (CSPs) have been cloned; they are called HvirCSP1, HvirCSP2 and HvirCSP3. The HvirCSPs show about 50% identity between each other and 30–76% identity to CSPs from other species. Overall, they are rather hydrophilic proteins but include a conserved hydrophobic motif. Tissue distribution and temporal expression pattern during the last pupal stages were assessed by Northern blots. HvirCSP mRNAs were detected in various parts of the adult body with a particular high expression level in legs. The expression of HvirCSP1 in legs started early during adult development, in parallel with the appearance of the cuticle. HvirCSP1 mRNA was detectable five days before eclosion (day E-5), increased dramatically on day E-3 and remained at high level into adult life. The tissue distribution and the time course of appearance of HvirCSPs are in agreement with a possible role in contact chemosensation.     

Evidence of circadian rhythm of electric discharge in Eigenmannia virescens system

The gymnotid electric fish, Eigenmannia virescens, exhibits electric discharge rhythmicity both in alternate light-dark (LD; 12h light, 12h dark [LD 12:12]) and in constant dark (DD) conditions. It suggests that the electric discharge rhythm is under control of the circadian clock. The free-running periods (FRPs) of electric discharge rhythms at 21°C in DD are greater than, but close to, 24h. The maximum of the electric discharge in the Eigenmannia system peaks approximately at circadian time 6 (CT6) in the middle of the subjective day. The circadian oscillator in the system is temperature compensated. This original report reveals the relationship between electric discharge activity and the circadian pacemaker in Eigenmannia and provides an alternative system to investigate circadian rhythms in vertebrates. (Chronobiology International, 17(1), 43-48, 2000)