Eicosanoids mediate microaggregation and nodulation responses to bacterial infections in black cutworms,Agrotis ipsilon,and true armyworms,Pseudaletia unipuncta

Nodulation is the first, and quantitatively predominant, cellular defense reaction to bacterial infection in insects and other invertebrates. Inhibition of eicosanoid biosynthesis in true armyworms, Pseudaletia unipuncta, and black cutworms, Agrotis ipsilon, immediately prior to intrahemocoelic injections with heat-killed preparations of the bacterium, Serratia marcescens, severely impaired the nodulation response. Five eicosanoid biosynthesis inhibitors, including dexamethasone (a phospholipase A(2) inhibitor), indomethacin, ibuprofen (cyclooxygenase inhibitors), phenidone (dual lipoxygenase/cyclooxygenase inhibitor) and eicosatetraynoic acid (an arachidonic acid analog that inhibits all arachidonic acid metabolism) severely reduced nodulation in infected insects. The dexamethasone effects were reversed by treating true armyworms with arachidonic acid immediately after infection. In addition to these pharmacological findings, we demonstrate that an eicosanoid biosynthesis system is present in these insects. Arachidonic acid is present in fat body phospholipids at about 0.4% of total phospholipid fatty acids. Fat body expressed a phospholipase A(2) that can hydrolyze arachidonic acid from the sn-2 position of cellular phospholipids. Fat body preparations were competent to biosynthesize prostaglandins, of which PGE(2) was the major product. These findings support the hypothesis that eicosanoids mediate cellular immune reactions in insects.     

Prostaglandin biosynthesis by fat body from larvae of the beetle Zophobas atratus

We describe prostaglandin (PG) biosynthesis by microsomal-enriched fractions of fat body prepared from larvae of the tenebrionid beetle, Zophobas atratus. PG biosynthesis was sensitive to incubation time, temperature, pH, substrate and protein concentration. Optimal PG biosynthesis conditions of those we examined included 2 mg of microsomal-enriched protein, incubated at 22 degrees C for 2 min at pH 6. These preparations yielded four major PGs: PGA(2), PGE(2), PGD(2) and PGF(2 alpha). PGA(2) and PGF(2 alpha) were the predominant eicosanoids produced under these conditions. Two non-steroidal anti-inflammatory drugs, indomethacin and naproxen, effectively inhibited PG biosynthesis in low concentrations. In vitro PG biosynthetic reaction conditions, using vertebrate or invertebrate enzyme sources, usually include a cocktail of reaction co-factors. The Z. atratus preparation similarly performs better in the presence of co-factors. Arch.     

Cardioacceleratory effects of Manduca sexta allatotropin in the true armyworm moth, Pseudaletia unipuncta

Manduca sexta allatotropin (Manse-AT), a peptide originally isolated on the basis of its ability to stimulate juvenile hormone (JH) biosynthesis in the tobacco hornworm, is a potent in vitro stimulator of the corpora allata (CA) in Pseudaletia unipuncta (Lepidoptera: Noctuidae). At 10(-6)M, Manse-AT stimulated in vitro rates of JH biosynthesis by CA of day 0 and 6 adult females 15- and 10-fold respectively. Both Manse-AT and serotonin were also shown to be dose-dependent stimulators of heart rate in day 0, 3 and 6 adult males and females. Furthermore, analysis suggests that there are differences in both resting and Manse-AT-stimulated heart rates depending on age and rearing conditions.     

Characterization and baculovirus-directed expression of a myosuppressin encoding cDNA from the true armyworm, Pseudaletia unipuncta

Insect myosuppressins are a highly conserved sub-family of peptides which are primarily characterized by the ability to suppress contraction of visceral muscles in a variety of insect species. We have isolated a cDNA from the true armyworm, Pseudaletia unipuncta, that encodes a prohormone containing a peptide identical to ManducaFLRFamide. We have shown that this myosuppressin gene appears to be expressed in late larval and adult insects. In Manduca sexta, a number of extended-FLRFamide peptides have previously been purified including ManducaFLRFamide, F7D (DPSFLRFamide), F7G (GNSFLRFamide) and two larger peptides F24 and F39 that contain the shorter ManducaFLRFamide sequence at their C-terminus. Comparison with the true armyworm prepropeptide characterized here identifies F24 and F39 as partially processed products from the same precursor. Expression in the true armyworm was shown by in situ hybridization to occur in over 150 cells throughout the adult brain and nerve cord, and also to occur in both open and closed endocrine type cells of the gut. Overexpression of the P. unipuncta FLRFamide cDNA from a baculovirus vector in cabbage looper caterpillars was used to assess the potential for myosuppressin expression as a means of enhancing virus efficacy. Viral expression of the armyworm prohormone cDNA resulted in raised levels of RFamide-like products in the hemolymph of infected insects, but the products were found to be chemically distinguishable from authentic mature peptide and probably represent partially processed hormone.     

Adipokinetic hormone of the true armyworm, Pseudaletia unipuncta: immunohistochemistry, amino acid analysis, quantification and bioassay

Abstract A cluster of five to seven AKH-like immunoreactive cells lie in each lobe of the paired corpora cardiaca of the true armyworm, Pseudaletia unipuncta. These cells form a mesh work of immunoreactive processes within the corpora cardiaca, and immunoreactive tracts projecting posteriorly over the aorta.
Reversed-phase high performance liquid chromatography of extracts of the corpora cardiaca of P.unipuncta revealed a single large U.V. absorbent peak with a retention time identical to synthetic Manduca- AKH. Amino acid analysis of the contents of this peak yielded a composition identical to that of synthetic Manduca-AKH which was analysed in a parallel manner. Furthermore the material within the peak possessed adipokinetic activity when bioassayed in day 2 adult male P. unipuncta. The corpora cardiaca of similar individuals were found to contain approximately 17.6ng (17.6pmol) of Manduca-AKH equivalents per pair.
Injection of Manduca-AKH into 2-day-old adult male P.unipuncta resulted in a dose-dependent elevation in haemolymph lipid levels with a maximum level of 80–90μmg/μl obtained with 5–10 ng of Manduca-AKH. Continuous flight also elevated haemolymph lipid levels in day 4 adult males with a significant elevation evident in the first samples taken after 15 min of flight and lipid levels plateauing at approximately 100 μg/μl by about 60 min of flight.     

The combined effect of photoperiod and temperature on the calling behaviour of the true army worm, Pseudaletia unipuncta

ABSTRACT. Pseudaletia unipuncta (Haw.) (Lepidoptera: Noctuidae) virgin females, maintained at either 10 or 25d?C under LD 12:12 or 16:8 h, started calling at different ages. For a given photoperiod, calling was initiated 11 days later at 10d?C than at 25d?C, while for a given temperature, calling at LD 12:12 h was 3–4 days later than at LD 16:8 h. At 10d?C 50.8% of females did not call within 35 days at LD 12:12 h compared with 30.8% at LD 16:8 h. Calling started earlier in the scotophase at 10d?C than at 25d?C and at LD 16:8 h than at LD 12:12 h. Under all treatments calling generally advanced on successive nights. The time elapsed between the mean onset time of calling and the mid-scotophase was relatively constant under both photoperiod conditions at 25d?C, but at 10d?C was more variable. The mean time spent calling increased significantly with calling age but did not differ significantly between the four experimental conditions tested. Older (15 days) females transferred from 10d?C, LD 16:8 h to 25d?C at either LD 163 or 12:12 h, required less time to initiate calling than younger (5 days) ones. Those transferred from 10d?C, LD 12:12 h took the same time, regardless of their age at the time of the transfer. Females experiencing either a decrease or increase in daylength as well as a temperature increase, required respectively more or less time to initiate calling, compared with individuals that only experienced an increase in temperature. If temperature was the only parameter changed females that initiated calling soon after the transfer immediately adjusted their calling periodicity to prevailing conditions. When both temperature and photoperiod were altered, it took several days before calling periodicity adjusted to the new regime. The ecological implications of temperature and photoperiodic conditions on the possible autumn migration of P. unipuncta are discussed.     

Primary and continuous midgut cell cultures from Pseudaletia unipuncta (Lepidoptera: Noctuidae)

Midgut epithelial cells were isolated from fifth-instar Pseudaletia unipuncta larvae by collagenase treatment of midgut tissue, and cultured in TNM-FH medium. Long-term continuous culture and maintenance of midgut cells were achieved with P. unipuncta armyworm intestinal cells. Several cells lines were obtained from these P. unipuncta primary cultures, and they have been subcultured and maintained for over 24 mo. The three major midgut cell types were present in the cultures, including stem (regenerative), columnar, and goblet cells. In vitro morphogenesis and differentiation of columnar and goblet cells from stem cells were observed. There appeared to be a cycle of cell death of goblet and columnar cells followed by their replacement from stem cells every 7-8 wk. After approximately six passages, the cell density in T-flasks appeared to be somewhat constant, reaching 10(3)-10(4) cells per milliliter of medium. The columnar cells are round to rectangular in shape and possess a brush border, while the goblet cells have a classic flask-like shape with a central cavity. Peritrophic membrane-like secretions were observed in all the culture flasks. Infection of these cells with multiply embedded nucleopolyhedrovirus was confirmed, and we conclude that these midgut cells can be used as an in vitro model system to study early events in baculovirus infection.     

The establishment of new cell lines from Pseudaletia unipuncta with differential responses to baculovirus infection

Summary Six insect cell lines from Pseudaletia unipuncta embryos were established and characterized, and their susceptibility to Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) infection was investigated. These embryonic P. unipuncta cell lines had characteristics distinct from each other in morphology and growth, and showed differential responses to AcMNPV infection. Among the six cell lines, two were highly susceptible to virus infection. One of these two cell lines, BTI-Pu-A7S, produced over 100 AcMNPV occlusion bodies per cell, on average. Three cell lines showed an apoptotic response following AcMNPV infection. One cell line did not support complete virus replication through the late phase of virus growth and did not exhibit apoptosis. The P. unipuncta cell lines could be distinguished from SF21 and BTI-Tn-5B1-4 cells by their isozyme markers.     

The effect of photoperiod on the calling behaviour of virgin females of the true armyworm,Pseudaletia unipuncta (Haw.) (Lepidoptera: Noctuidae)

The effect of photoperiod on the calling behaviour of Pseudaletia unipuncta virgin females was examined under five different photoperiodic regimes at 25°C, 65% r.h. The age at which females called for the first time following emergence varied with photoperiod; generally calling was later under long-scotophase conditions. However under a 6 h scotophase there was also a delay in calling and >63% of the females tested never called. There was a considerable variability in the daily calling patterns between the different photoperiods, and the mean onset time of calling was not constant with either “lights on” or “light off”. There was however a constancy of the mean onset time of calling relative to the mid-point of both the photo- and scotophase at all photoperiods tested, indicating that females could measure the absolute duration of either the photo- or scotophase. Transferring females from one photoperiodic condition to another once calling had been initiated, determined that it was the “lights off” signal that P. unipuncta females used to phase set the clock governing circadian calling behaviour.Females subjected to a decrease of 4 or 6 h in the length of the scotophase following the initiation of calling required several days to adjust to the new photoperiodic regime and a high proportion of females did not call during the night following the transfer. A 4 or 6 h increase in the scotophase did not inhibit calling on the night following the transfer but females still required several days to adjust completely. However, females experiencing a 2 h increase or decrease in the duration of the scotophase were able to maintain normal calling behaviour. The results of these experiments are discussed in relation to the seasonal biology of the true armyworm and the hypothesis that this is a migrant species.     

Biological activity of essential oils from seven Azorean plants against Pseudaletia unipuncta (Lepidoptera: Noctuidae)

Ten essential oils from seven Azorean plant species were evaluated for their insecticidal, ovicidal, feeding‐deterrence and growth inhibition activities against Pseudaletia unipuncta. The oils of Laurus azorica (leaves), and Juniperus brevifolia (leaves) showed strong moderate insecticidal effect on fourth‐instar larvae causing 93.3% and 46.7% mortality, respectively. Juniperus brevifolia (leaves), L. azorica (leaves), Persea indica (leaves), Hedychium gardnerianum (leaves) and Pittosporum undulatum (fruits and leaves) significantly affected the hatching of P. unipuncta eggs (<8% eclosions). Five oils showed significant feeding deterrent activities (L. azorica, 92.4%, J. brevifolia, 93.6%, P. undulatum leaves, 95.5% and fruits, 83.8% and H. gardnerianum, 88.2%). All of the essential oils tested, significantly inhibited the larval growth after 5 days of feeding on the treated diet. Essential oils from L. azorica and J. brevifolia were the most potent growth inhibitors among the oils tested, producing a decrease in the initial larva weight (?14.8 and ?14.5 mg, respectively). Our results indicate that L. azorica (leaves), J. brevifolia (leaves), P. indica (leaves), H. gardnerianum (leaves), and P. undulatum (leaves and fruits) can be exploited for the development of bioactive compounds as a new source of agrochemicals. Further emphasis on isolation and identification of active constituents can be useful to develop new environment‐friendly insect control agents.     

Manduca sexta allatotropin and the in vitro biosynthesis of juvenile hormone by moth corpora allata: a comparison of Pseudaletia unipuncta females from two natural populations and two selected lines

We compared the effects of Manduca sexta allatotropin (Manse-AT) on the rate of in vitro juvenile hormone (JH) biosynthesis by the corpora allata (CA) of different-aged virgin females from migrant (Quebec) and non-migrant (Azores) populations of the armyworm, Pseudaletia unipuncta, as well as from early- and late-calling lines selected from the Quebec population. There was a significant age x strain interaction, with the observed rates of JH biosynthesis in early adult life closely reflecting strain-specific differences in the age at onset of calling. In considering data for all ages combined, treatment of CA with Manse-AT resulted in a significant increase in the rate of JH biosynthesis for all but the Late strain, although significant differences for this strain were detected at certain ages. The CA of females from the Azores strain showed the strongest stimulation, with those of 0- and 1-day-old individuals displaying a singularly high degree of sensitivity. Selection for early- and late-calling lines resulted in significant differences in the temporal patterns of JH biosynthesis but did not markedly affect the sensitivity of the CA to Manse-AT. These findings are discussed within the context of the age-related differences observed in the rates of in vitro JH biosynthesis and JH haemolymph titers previously reported in comparisons of the Quebec and Azorean strains of the true armyworm.     

Phospholipid,an enhancing component in the synergistic factor of a granulosis virus of the armyworm,Pseudaletia unipuncta

The synergistic factor (SF) in the capsule of a granulosis virus (Hawaiian strain) of the armyworm, Pseudaletia unipuncta, contained polypeptides and phospholipids. Its molecular weight estimated by SDS-polyacrylamide gel electrophoresis was 126,000 ± 8,700. The capsule proteins were digested by a proteinase released from the capsule under alkaline conditions, and by trypsin added to the proteinase-free capsules. Neither enzyme affected the synergistic factor or its activity. The synergistic factor was slowly depolymerized by 2% sodium dodecyl sulfate and was more rapidly depolymerized when phospholipase C (phosphatidylcholine cholinephosphohydrolase) was also added. Phospholipase C alone did not decompose the synergistic factor, but it did destroy the capacity of the synergistic factor to enhance the nuclear polyhedrosis virus. In contrast, phospholipase A₂ (phosphatidyl 2-acylhydrolase) had no effect on the synergistic factor. The different reactions of the two phospholipases on the synergistic factor suggested that the hydrophilic group of the phospholipid was exposed to the action of phospholipase C and was associated with the synergistic activity. This interpretation was supported by the detection of a phospholipid in the SF by thin-layer chromatography.     

Interference between two nuclear polyhedrosis viruses of the armyworm,Pseudaletia unipuncta [Lep.: Noctuidae]

The larva ofPseudaletia unipuncta (Haworth) is susceptible to 2 nuclear polyhedrosis viruses (NPV), the typical (TNPV) and the hypertrophy (HNPV) strains. The interference between the 2 viruses was studied primarily in the tracheal and fat tissues. When both viruses were microfed simultaneously, no larva had mixed virus infections and most were infected with TNPV. In order to obtain 50% infection by each virus the inoculum of HNPV had to be increased by 1000 polyhedra/ml at the LD₅₀ value of TNPV. Mixed infection resulted when TNPV was inoculated 1 and especially 2 days after HNPV inoculation. In mixed infection, each virus attacked certain areas of the tracheae instead of random cells. These results indicated an interference or antagonism between the 2 viruses. The possible cause for the interference is discussed.

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Résumé La larve dePseudaletia unipuncta (Haworth) est sensible à 2 virus à polyèdres nucléaires (NPV), la souche typique (TNPV) et la souche causant une hypertrophie (HNPV). L'interférence entre ces 2 virus a été étudiée essentiellement dans les trachées et le corps adipeux. Si les deux virus sont inoculés simultanément par micro ingestion aucune larve ne présente d'infection mixte et la plupart d'entre elles est infectée par le TNPV. Pour obtenir 50% d'infection par chacun des virus, l'inoculum de HNPV doit être accru de 1000 polyèdres par ml par rapport à l'inoculum provoquant la DL 50 du TNPV. Une infection mixte est enregistrée quand le TNPV est inoculé 1 jour et de préférence 2 jours après l'introduction du HNPV. Dans ce cas d'infection mixte chaque virus attaque certaines régions des trachées et non pas des cellules réparties au hasard. Ces résultats prouvent une interférence ou un antagonisme entre les 2 virus, dont la cause est discutée.

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Portion of a dissertation submitted by the senior author for the degree of doctor of philosophy from the University of California, Berkeley. Part of the study was supported by a NSF grant provided to the junior author.     

Prostaglandin biosynthesis by midgut tissue isolated from the tobacco hornworm, Manduca sexta

We describe prostaglandin (PG) biosynthesis by isolated midgut preparations from tobacco hornworms, Manduca sexta. Microsomal-enriched midgut preparations yielded four PGs, PGA/B(2), PGD(2), PGE(2) and PGF(2alpha), all of which were confirmed by analysis on gas chromatography--mass spectrometry (GC--MS). PGA and PGB are double bond isomers which do not resolve on TLC but do resolve by GC; for convenience, we use the single term PGA(2) for this product. PGA(2) was the major product under most conditions. The midgut preparations were sensitive to reaction conditions, including radioactive substrate, protein concentration (optimal at 1mg/reaction), reaction time (optimal at 0.5 min), temperature (optimal at 22 degrees C), buffer pH (highest at pH 6), and the presence of a co-factor cocktail composed of reduced glutathione, hydroquinine and hemoglobin. In vitro PG biosynthesis was inhibited by two cyclooxygenase inhibitors, indomethacin and naproxen. Subcellular localization of PG biosynthetic activity in midgut preparations, determined by ultracentrifugation, revealed the presence of PG biosynthetic activity in the cytosolic and microsomal fractions, although most activity was found in the cytosolic fractions. This is similar to other invertebrates, and different from mammalian preparations, in which the activity is exclusively associated with the microsomal fractions. Midgut preparations from M. sexta pupae, adult cockroach, Periplaneta americana, and corn ear worms, Helicoverpa zea, also produced the same four major PG products. We infer that insect midguts are competent to biosynthesize PGs, and speculate they exert important, albeit unrevealed, actions in midgut physiology.     

Location of a synergistic factor in the capsule of a granulosis virus of the armyworm, Pseudaletia unipuncta

A synergistic factor (SyF), which enhanced the infection of nuclear polyhedrosis viruses, was purified from capsules of a Pseudaletia unipuncta granulosis virus (Hawaiian strain) by immune affinity chromatography. The isolated SyF consisted primarily of a protein with molecular mass 98 kDa. The recovery rate depended on the alkali used to dissolve the capsules: the highest rate occurred with 0.05 M Na2CO3-0.05 M NaCl, followed in turn with 0.02-0.05 M NaOH and 0.04 M NaOH-0.05 M glycine. The solubilized components from untreated capsules contained 98- and 100-kDa proteins in addition to the matrix protein (29 kDa) and its decomposed products, while those from heat-treated capsules contained only the 100-kDa protein. Virons liberated from the capsules with the glycine buffer contained three proteins (33, 98, and 100 kDa) serologically related to the SyF. Immunoelectron microscopy of infected tissue and purified virions revealed the localization of the SyF antigens on the viral envelope.