Relative pathogenicity of nuclear polyhedrosis viruses from Heliothis punctigera and Heliothis zea for larvae of Heliothis armigera and Heliothis punctigera

Laboratory bioassays indicated that the potency of a nuclear polyhedrosis virus from Heliothis zea derived from a commercial American formulation was similar to that of a naturally occurring nuclear polyhedrosis virus from H. punctigera in Australia. Both viruses exhibited high virulence for neonate larvae of H. armigera and H. punctigera, the major pest species in this genus in Australia. Hence evaluation of the virus in Australia can proceed employing virus from either H. punctigera or H. zea.     

Temperature-sensitive mechanism regulating diapause in Heliothis zea

Pupal diapause in Heliothis zea is regulated by a temperature-sensitive mechanism which prevents ecdysone production despite the release of prothoracicotropic hormone. To determine how this mechanism functioned, donor prothoracic glands were implanted into prothoracic gland-ablated hosts to test their ability to produce ecdysone in a diapause-sustaining temperature of 19°C. Results of these experiments ruled out the possibility that ecdysis production was regulated by the nervous system or by a mechanism intrinsic to the prothoracic glands, and suggested that a humoral factor was required for diapause termination.Haemolymph injection experiments supported this humoral factor hypothesis, i.e. haemolymph from non-diapausing donor pupae terminated diapause in hosts maintained at 19°C, whereas haemolymph from diapausing donor pupae had no such effect. These findings indicate that the temperature-sensitive mechanism regulating H. zea diapause functions by controlling the availability of a humoral factor necessary for ecdysone production by the prothoracic glands.     

Utilizable surface nutrients on Heliothis zea available for growth of Beauveria bassiana

Amino acids and glucosamine are present on the surface of Heliothis zea larvae. The amino acid compositions vary among instars and with time following completion of the gross molting process. Larvae collected from ears of corn have many surface amino acids. All amino acid combinations found on larval surfaces are sufficient for initiation of germination and growth by Beauveria bassiana. Amines and peptides are also present; these do not inhibit germination of B. bassiana or Aspergillus niger.     

Hemolymph responses in Heliothis zea to inoculation with Bacillus thuringiensis or Micrococcus lysodeikticus

Direct injection into the hemolymph of Heliothis zea of either an entomopathogen (Bacillus thuringiensis subsp. kurstaki) or a nonpathogen (Micrococcus lysodeikticus) is followed by a rapid phagocytosis and extensive removal of the organisms within 2 hr. The bacteria that survive this initial clearance initiate a new round of growth that is clearly evident 6–8 hr after injection. When the infecting organism is M. lysodeikticus, a second period of clearance occurs 8–12 hr after injection and nearly complete removal (many by lysis) is evident by the 12th hr. Larvae usually survive infection with this organism. When B. thuringiensis is the infecting organism, 60–80% of the phagocytized bacteria are lysed, however, the second wave of clearance seen with M. lysodeikticus does not occur; instead, the bacteria multiply extensively and death of the larvae results 12–16 hr after injection. This death does not appear to be caused either by crystalline protein or by the β-exotoxin. Analysis of hemolymph proteins using one-dimensional polyacrylamide gel electrophoresis indicated that although some quantitative changes were observed in some experiments, in the faster moving proteins when the infecting agent was B. thuringiensis, they were not consistent enough to support the idea that hemolymph proteins were either synthesized or used up during the time larvae were responding to the infectious agent. Dramatic changes were evident when the larvae were near death. No changes were ever observed when M. lysodeikticus was used as the infecting organism. A rapid response to infection using free spores of B. thuringiensis (sickness within 2–4 hr followed by death at 6–8 hr) may indicate that the spore germinating process is accompanied by release of a highly toxic material.     

Toxic components on the larval surface of the corn earworm (Heliothis zea) and their effects on germination and growth of Beauveria bassiana

Caprylic acid is present on the surface of corn earworm, Heliothis zea, and fall armyworm, Spodoptera fragiterda, larvae. Because caprylic acid inhibits germination of Beauveria bassiana, presence of this compound will be determined to the establishment of an infection of larvae by this fungus. Other free fatty acids present on the surface of the H. zea and S. frugiterda are tentatively identified as valeric and nonanoic acids; these also possess mycostatic activity toward B. bassiana. Depending on concentration, caprylic acid inhibits germination of conidia for different amounts of time (R. J. Smith and E. A. Grula, 1981, J. Invertebr. Pathol., 37, 222–230). We now further report that inhibition and/or growth is also related to the source of carbon, nitrogen, and energy present in the growth medium. This observation of selective toxicity in the presence of different nutrients was also observed using nonanoic acid. Our data therefore make it necessary to interpret the effects of certain fatty acids on germination and growth of B. bassiana (and probably other fungi as well) in terms of nutrients for the germination process.     

The effects of the structure of sterols on the development of Heliothis zea

Heliothis zea was reared on artificial diets which lacked supplementation with plant materials but were supplemented with different sterols in order to determine how certain structural features of a sterol molecule affect the development of this insect. We found that sitosterol and cholesterol supported a more rapid rate of growth than did campesterol. Larvae did not moult when they ingested 5-pregnen-3β-ol. Larvae utilized spinasterol more efficiently than lathosterol. Such a pronounced effect was not observed in the Δ⁵-series. The substitution of a Δ⁷-bond (spinasterol, dihydrospinasterol, lathosterol) for the Δ⁵-bond (stigmasterol, sitosterol, cholesterol) in the 24-ethyl- and desalkylsterols reduced the rate of growth of the larvae. Although larvae developed normally on cholesterol, the addition of a Δ⁷-bond to give the Δ^5,7-diene system apparently altered the functionality of the molecule because 7-dehydrocholesterol did not support larval development. The growth of larvae was also inhibited, although not prevented, when they consumed diets which contained ergosterol. In contrast, the larvae completed their development more rapidly on brassicasterol which lacked the Δ⁷-bond. Cholestanol supported the complete development of the insect. H. zea is unusual among investigated insects because it develops both on cholestanol and lathosterol but does not utilize ergosterol efficiently and fails to grow on 7-dehydrocholesterol.     

Detection of Autographa californica and Heliothis zea baculovirus proteins by enzyme-linked immunosorbent assay (ELISA)

The structural proteins of Autographa californica (AcMNPV) and Heliothis zea (HzSNPV) nuclear polyhedrosis viruses were detected by indirect enzyme-linked immunosorbent assay (ELISA). The immunoassay detected less than 1 ng of AcMNPV protein. The extent of immunological relatedness between AcMNPV-occluded virus and AcMNPV polyhedral protein, AcMNPV-nonoccluded virus, Estigmene acrea granulosis virus, Amsacta moorei entomopoxvirus Heliothis zea NPV, and Lymantria dispar NPV was determined. No immunological relatedless was detected between HzSNPV, AcMNPV, and a persistent rod-shaped virus isolated from the Heliothis zea cell line (IMC-Hz-1). The polyhedral proteins of HzSNPV and AcMNPV were found to be immunologically identical.     

Comparison of the time-mortality response of Heliothis zea to 14 isolates of Heliothis nuclear polyhedrosis virus

Twelve singly embedded isolates (SEV) and two multiply embedded isolates (MEV) of nuclear polyhedrosis viruses from Heliothis larvae were compared by time-mortality assays in neonate H. zea larvae. The isolates could be separated into six groups based on differences in the 50% survival time (ST₅₀) values. Isolates with identical restriction endonuclease (REN) profiles did not differ significantly in their ST₅₀ values, whereas isolates with several different REN cleavage sites also had significantly different ST₅₀ values. With the exception of one isolate from India, the singly embedded isolates acted faster than the multiply embedded isolates.     

Interactions of the microsporidium Vairimorpha necatrix with a bacterium,virus, and fungus in Heliothis zea

Interactions between Vairimorpha necatrix and three other pathogens of Heliothis zea were evaluated with dose-mortality studies in order to find a synergistic combination which could be tested for field control of H. zea. The effect of the microsporidium combined with the bacterium Bacillus thuringiensis was at least additive, with indications of synergism. The interaction between V. necatrix and Heliothis nuclear-polyhedrosis virus was antagonistic except that the highest microsporidian dose overcame the antagonism with a resultant independent action. The interaction between V. necatrix and the fungus Nomuraea rileyi was additive, though response varied some-what with different proportions of the two pathogens. Even though none of these interactions is likely to be valuable in microbial control, V. necatrix has the potential to synergize or antagonize any biological or chemical agent that acts on the midgut epithelium of host insects.     

In vivo events associated with entomopathology of Beauveria bassiana for the corn earworm (Heliothis zea)

When conidia of Beauveria bassiana are injected into the hemocoel of corn earworm larvae, it appears that a positive correlation exists between exocellular proteolytic activity of the fungus and entomopathological manifestations. Once inside the hemolymph, defense mechanisms (including phagocytosis) are incapable of overcoming the fungus and one important event in a terminal mycocidal cascade involves preferential invasion of the gut wall. Such invasion helps explain the observed inhibition of feeding by infected larvae. Although histopathological changes seen in gut tissues suggest that a gut toxin is produced, evidence for such a toxin could not be obtained in preliminary tests. Biochemical changes are seen in hemolymph components; however, these are viewed as being due to general starvation rather than to specific activities of the fungus, at least up to the time that a general mycosis is established. With the host larva under physiological stress (starvation, nutrient depletion, and, possibly, toxin production in gut tissues) and failure of defense mechanisms, the infection spreads quickly and a terminal mycosis results.     

产绿原酸内生菌的分离及其绿原酸合成途径关键基因的克隆和功能研究

【目的】分离产绿原酸的内生菌并对其绿原酸合成途径的一种关键酶基因进行克隆和功能研究。【方法】采用表面消毒法从金银花中分离内生菌,以高效液相色谱(HPLC)和液相色谱-质谱联用(LC-MS)筛选确定产绿原酸的内生菌,克隆、表达该内生菌的组氨酸解氨基酶(HAL)并进行酶活测定。【结果】从金银花根中分离到一株产绿原酸的内生细菌RP1,同时在该内生菌发酵液中检测到了绿原酸代谢的中间物肉桂酸。对RP1的分子鉴定表明该内生菌为枯草芽孢杆菌(Bacillus subtilis)。对RP1的HAL基因进行克隆和原核表达,酶活测定表明该酶具有HAL和PAL (苯丙氨酸解氨基酶)双功能,其PAL活性产生的肉桂酸与LC-MS检测的结果一致。【结论】推测该内生菌可能利用其HAL的苯丙氨酸解氨基活性将苯丙氨酸的催化产物肉桂酸导入苯丙烷途径,从而产生绿原酸。     

Effects of cotton condensed tannin on feeding and digestion in the cotton pest, Heliothis zea

Condensed tannin of cotton incorporated into an artificial diet caused growth inhibition in H. zea larvae whose susceptibility was dependent upon the concentration of the dietary tannin, and also upon the size of the feeding larvae.Larvae feeding on tannin-treated diet exhibited both decreased protease and invertase activities in the midgut caecal wall, as well as lowered total protein and sugar levels in the haemolymph as compared to controls. These differences, however, had no effect on assilimation and efficiency of conversion of digested matter into animal biomass and therefore may be secondary effects. Ultimately, inhibition of growth is attributed to a reduction in food consumption.     

Serial passage of Heliothis zea singly embedded nuclear polyhedrosis virus in a homologous cell line

This paper describes the replication and serial passage of Heliothis zea nuclear polyhedrosis virus (NPV) in a H. zea cell line. It was demonstrated that long-term serial passages of the H. zea NPV in homologous host cell culture decreased both the total number of polyhedral inclusion bodies (PIBs) produced and the infectivity of the supernatant as measured by TCID₅₀. The growth curve indicated that infectious material was released from cells 24 hr postinfection (p.i.) and approached a maximal titer 3 days p.i. The kinetics of H. zea NPV decay at 4°, 27°, and 37°C were determined. Infectivity was not detected after 3 weeks at 37°C, but approximately 10^3.5 TCID₅₀/ml activity was still present after 3 and 8 weeks storage at 27° and 4°C, respectively. Electron microscopy confirmed the presence of single embedded virions in the inoculated cells.     

Release of prothoracicotropic hormone and potentiation of developmental ability during diapause in the bollworm, Heliothis zea

In Heliothis zea, pupal diapause is not due to a deficiency of the prothoracicotropic hormone (PTTH), as it is in many other insects. However, PTTH is essential for diapause termination and adult development. Removal of the pupal brain 4 hr after larval-pupal ecdysis blocks the insect's ability to initiate adult development. Transplantation of brain neurosecretory cells restores this ability, whereas other tissues such as corpora allata have no effect. In the diapausing pupa, PTTH is released from the brain within 24 hr after larval-pupal ecdysis. Subsequent removal of the brain fails to block the ability for diapause termination, because PTTH potentiates the ability for adult development. Since diapause termination is suppressed in a temperature of 21°C, the bollworm retains the ability to initiate development in 27°C whereas it remains in diapause in 21°C. Diapause continues even though pupae are supplied with additional PTTH via neurosecretory cell transplantation.Ecdysone injection and prothoracic gland-ablation experiments indicate that the prothoracic glands are the source of the prohormone α-ecdysone, and that diapause is maintained by an α-ecdysone deficiency. This evidence, in conjunction with the above results, suggests that PTTH release potentiates prothoracic gland function in the diapausing pupa which is then regulated by a temperature dependent process.     

Interaction of calcineurin with its activator,chlorogenic acid revealed by spectroscopic methods

Chlorogenic acid (CHA) has been proved to be an activator of calcineurin (CN) in our previous research. In this study, the activation of single chain calcineurin (BA) by CHA, their interaction and concomitant changes in protein conformation were studied using fluorescence and Fourier transform infrared spectroscopy. Evidence is present that binding of CHA to CN is responsible for the stimulation of enzyme and results in structural changes. Aromatic residues reorient into new environments upon binding of CHA, the binding constant for the reaction was (2.76 ± 0.64) × 10⁴ M⁻¹ by one binding site, which indicated that CHA bound to BA statically and the change of secondary structure was mainly due to reduced α-helical content and increased β-turns. The results obtained in this study should be useful for understanding the molecular mechanisms underlying the interactions between CN and its activators.     

Selection for resistance to a nucleopolyhedrosis virus in laboratory populations of the cotton bollworm, Heliothis zea

Resistance to a nucleopolyhedrosis virus (Baculovirus heliothis) did not develop in laboratory populations of the cotton bollworm, Heliothis zea. A selection pressure of LD₅₀ to 70 was maintained throughout 20 to 25 generations of selection. No significant changes in LD₅₀, slope, or intercept of dose-mortality lines were detected. Laboratory populations under selection were as susceptible to the virus as nonselected or wild populations of H. zea. The resistance ratio (LD₅₀ of selected generation/initial generation) ranged from 0.5 to 1.2.     

Infection of the corn earworm, Heliothis zea, with Nosema acridophagus and Nosema cuneatum from grasshoppers: Relative virulence and production of spores

Per os inoculations of 4- to 6-day-old larvae of the corn earworm, Heliothis zea, with suspensions containing 10⁶ spores of Nosema acridophagus or 10⁴, 10⁵, and 10⁶ spores of Nosema cuneatum retarded the growth and development of the larvae. Migratory grasshoppers, Melanoplus sanguinipes, inoculated with N. acridophagus produced fewer spores than similarly inoculated corn earworms, but spore production was similar in these insects when they were inoculated with N. cuneatum. Standard bioassay procedures showed that spores of both microsporidians were some-what more virulent when they were produced in corn earworms than when they were produced in grasshoppers. Spores of these microsporidians might be produced more efficiently in corn earworm larvae than in grasshoppers.     

Mode of infection of the corn earworm (Heliothis zea) by Beauveria bassiana as revealed by scanning electron microscopy

Conidia from highly pathogenic mutants of Beauveria bassiana germinate quickly (within 18 hr) on the surface of corn earworm larvae (Heliothis zea) and immediately begin penetration of the cuticle. Enzymes produced by the penetrating hyphae degrade the cuticle since holes are formed at the point of entry. Clustering of conidia around nodules of larvae is often seen, but penetration is not restricted to such areas. Direct evidence is presented to show that conidia can also germinate inside of spiracle openings and could invade larvae by this route. Once inside the hemocoel, the fungus multiplies extensively; however, larval death occurs with only minimal breakdown of internal tissues. During mummification, outgrowths of fungal hyphae occur first and most extensively in the intersegmental regions of larvae. Conidia from mutants exhibiting low levels of pathogenicity are either significantly delayed or do not germinate on larval surfaces. When germination does occur, hyphae from such mutants do not penetrate immediately; instead, extensive surface hyphal growth, with or without eventual penetration of the integument, is evident.     

Occurrence of rosmarinic acid,chlorogenic acid and rutin in Marantaceae species

In a survey of the higher plants for families with rosmarinic acid-accumulating species we could show for the first time, that some species of the family Marantaceae of the order Zingiberales accumulate rosmarinic acid. Other compounds detected in Marantaceae are chlorogenic acid and rutin (quercetin 3-O-rutinoside). Out of 35 species coming from 9 different genera extracted and analysed, two species of Maranta (Maranta leuconeura, Maranta depressa) and one Thalia species (Thalia geniculata) showed the presence of rosmarinic acid. The two Maranta species additionally contained chlorogenic acid, which was also present in Stromanthe amabilis. Rutin was detected in the genera Calathea, Ctenanthe, Maranta, Pleiostachya and Thalia. For a comparison, species from six other families of the Zingiberales were analysed as well.     

The biosynthesis of flavonoid pigments: On the incorporation of phloroglucinol and phloroglucinyl cinnamate into rutin in Fagopyrum esculentum

The problem of whether phloroglucinol is a direct biosynthetic precursor of flavonoids was reinvestigated. Phloroglucinol-2,4,6-¹⁴C was found to be incorporated into rutin in Buckwheat (Fagopyrum esculentum) but most of the activity was found in the sugar moiety, the remainder being approximately equally distributed among the A- and B-rings of the aglycone, quercetin. This indicates extensive degradation of the added phloroglucinol prior to its utilization in the biosynthesis of the flavonoid. The hypothesis of a bio-Fries rearrangement of phloroglucinyl cinnamate to a chalcone, and hence to flavonoids, was also eliminated by comparing the efficiency of incorporation of ¹⁴C-labelled phloroglucinyl cinnamate and those of labelled phloroglucinol and cinnamic acid.