Influence of larval dietary supply on the food consumption, food utilization efficiency, growth and development of the lacewingChrysoperla carnea

In order to derive quantitative estimates of predation rate from serological gut analysis data, one must have an estimate of the interval during which a meal can be detected after feeding. In practice this has turned out to be ‘D_max,’ defined as ‘...the time from finishing a meal until that meal could just no longer be detected in any individuals.’ However D_max substitutes an absolute limit for what is really a continuous variable with significant variation. We examined this problem in a study of the detectability ofHelicoverpa zea Boddie (Lepidoptera: Noctuidae) fifth instar remains in the guts ofPolistes metricus Say (Hymenoptera: Vespidae). Wasps were maintained onTrichoplusia ni (Hübner) (Lepidoptera: Noctuidae) fifth instars before being fed a singleH. zea fifth instar. They were killed and frozen at 0, 24, 48 and 96 h intervals, with those held for more than 24 h fed a singleT. ni fifth instar at 24 h intervals in order to simulate continued feeding. Wasp abdomens were assayed by immunodot, using a monoclonal antibody toH. zea arylphorin. There was a logarithmic decay in the proportion ofP. metricus positive over time, a singleH. zea fifth instar meal having a detectability half-life of 19.4 h at field temperatures. If prey antigen detectability decays exponentially, then a detectability half-life is a more appropriate unit of detectability than an absolute detectability period.     

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.     

The humoral immune response of sheep to antigens from larvae of the sheep blowfly (Lucilia cuprina)

The sheep blowfly, Lucilia cuprina, is a myiasis-causing insect whose larvae evoke an immune response in sheep. By means of an immuno-dot blot and Western immuno-blot assays it has been demonstrated that sheep experimentally infected with larvae produce antibodies against a wide array of components from all three larval instars, with each instar displaying a differing set of antigens. The electrophoretic profiles of the proteins in various larval extracts and the patterns of antibody reactivity were very different. Of the extracts tested (1st, 2nd and 3rd instar larval excretions/secretions and visceral homogenates, extracts of 3rd instar salivary glands, mid guts, haemolymph and cuticle) the most intense antibody reaction was detected against the salivary gland extract: preparations of larval excretions/secretions and from the larval mid gut also reacted strongly. In contrast a cuticle extract reacted minimally with infected sheep sera.     

Regulation ofHelicoverpa zea larval behavior by the parasitoidEucelatoria bryani

The parasitoidEucelatoria bryani Sabrosky regulates the larval behavior of its hostHelicoverpa zea (Boddie). Parasitized third, fourth and fifth instars burrow into the soil 0.7–3.4 days earlier than unparasitized larvae that normally enter the soil to pupate at the end of the fifth and final larval instar. Parasitized third instars molt once then burrow as fourth instars, one instar earlier than normal. WhenE. bryani pupariated on the soil surface in the field, none survived to the adult stage. However,E. bryani adults emerged from 49.2% of hosts that had burrowed into the soil. By accelerating the timing ofH. zea burrowing behavior and causing host larvae to enter the soil before death,E. bryani ensures its pupariation in an environment with improved protection against natural enemies and lethal temperatures.     

Regulation of expression of arylphorin and female-specific protein mRNAs in the tobacco hornworm, Manduca sexta

Two non-cross-hybridizing cDNA clones were isolated from a lambda gt11 cDNA library prepared from Day 2 fifth instar female fat body of Manduca sexta and shown by hybrid selection to code respectively for the two storage proteins arylphorin and female-specific protein (FSP). Analysis of the developmental expression of arylphorin showed its presence during the feeding phases of the penultimate (fourth) and final (fifth) larval instars and its absence during the molt. Abdominal ligation of larvae followed by infusion of Grace's medium showed that this amino acid-rich medium was able to maintain arylphorin expression in fourth instar larvae, but not continued high expression in fifth instar larvae. This nutrient medium however was sufficient to allow initiation of expression in newly ecdysed fifth larval abdomens. Infusion of 5 micrograms 20-hydroxyecdysone (20HE) caused a significant reduction of arylphorin RNA in ligated fourth larval abdomens, whereas 50 micrograms was required in Day 2 fifth larval abdomens to suppress this RNA. Thus, both the lack of incoming nutrients and the rising titer of ecdysteroid contribute to the loss of arylphorin mRNA at the molts and at wandering. By contrast, FSP mRNA was first detected in females on Day 2 of the fifth instar, but not in males until wandering, and then was present throughout the prepupal period. In females allatectomy caused the precocious appearance of FSP mRNA which was prevented by application of 10 micrograms methoprene, a juvenile hormone analog. Expression of FSP mRNA in males however appeared to be independent of hormonal milieu.     

Molecular differentiation in pea powdery-mildew haustoria

Monoclonal antibodies have been raised against haustorial complexes isolated from pea (Pisum sativum L.) leaves infected by the biotrophic powdery mildew fungus Erysiphe pisi D.C. Immuno-localisation studies, using isolated haustorial complexes and infected pea leaf material, have shown that one of the antibodies, designated UB7, binds to fungal wall and plasma membranes present in both haustoria and mycelia. However, a second antibody, UB8, binds specifically to the haustorial plasma membrane, and does not label fungal plasma membranes in mycelia. Western blotting and antigen-modification techniques have shown that UB8 recognises a protein epitope of a 62-kDa antigen. A reduction in molecular weight of this component after endo-F treatment indicates that the antigen is an N-linked glycoprotein. UB7 also recognises a 62-kDa glycoprotein, which is susceptible to endo-F treatment, and the antibody binds to a carbohydrate epitope. Differences in molecular weights of the products after endo-F treatment of antigens show that the 62-kDa glycoproteins recognised by the antibodies are distinct molecules, in accordance with the localisation results. Overall, the results provide evidence for molecular differentiation associated with the development of haustoria in a biotrophic infection.Abbreviations ehm extrahaustorial membrane - ELISA enzyme-linked immunosorbent assay - HC haustorial complex - hpm haustorial plasma membrane - IIF indirect immunofluorescence - MAb monoclonal antibody - M_r apparent molecular weight - PMSF phenylmethylsulfonyl fluoride - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis We thank Mr. D. Mills and Mr. P. Stanley for help with the EM immunogold techniques. This work was supported by an Agricultural and Food Research Council grant and a studentship from the Science and Engineering Research Council.     

Monoclonal antibodies against chromosomal proteins of Drosophila melanogaster

Total nuclear protein from the embryonic D. melanogaster cell line Kc and crude hydroxyapatite fractions thereof were used for immunization of mice. From the spleen cells of these mice we established 755 permanent lymphoid cell lines using the hybridoma technique originally developed by Köhler and Milstein (1975). Radioimmunoassay showed 455 of these cell lines secreted antibodies which bound to component(s) contained in the antigen mixtures used for immunization. Screening of 311 cell lines using indirect immunofluorescence revealed 58 lines whose antibodies showed a highly selective staining pattern on polytene chromosomes from the salivary glands of D. melanogaster third instar larvae. Eight of these cell lines were cloned and further characterized. We were able to order the staining patterns into three distinct classes based on the staining behaviour of the monoclonal antibodies: staining of active regions, staining of phase dark bands or staining of most interbands. The molecular weight of those antigens against which the monoclonal antibodies were directed was determined in SDS polyacrylamide gels.     

Developmental profiles of the mRNAs for Manduca arylphorin and two other storage proteins during the final larval instar of Manduca sexta

When fat body mRNA from the tobacco hornworm larva, Manduca sexta, was translated in a rabbit reticulocyte lysate system, three major polypeptides were found, each having a different developmental profile. One mRNA coded for a 74 kilodalton (K) polypeptide doublet precipitated by an antibody to the arylphorin (manducin). This mRNA was present only during the intermolt feeding phase of the penultimate and the final larval instars. Its appearance 16–24 hr after larval ecdysis was dependent upon the incoming nutrient supply and independent of the juvenile hormone (JH) level. Immunoblots of proteins of the fat body, epidermis, and cuticle revealed the presence of arylphorin in all three tissues. Additionally, several small polypeptides that cross-reacted with the arylphorin antibody were found in the fat body during and up to 24 hr after the last larval molt and in the tanning pupal cuticle. The larval epidermis was also found to contain a small amount of arylphorin mRNA. At the time of the JH decline prior to the onset of metamorphosis, a female-specific mRNA coding for a 79 K translation product appeared. In allatectomized larvae this mRNA was detectable earlier, and its appearance in intact larvae was prevented by application of methoprene, indicating that JH regulates its appearance. At wandering a new mRNA that also codes for a 79 K polypeptide appeared in both sexes and was the major messenger present during the prepupal stage. Neither it nor the female-specific mRNA were translatable after pupal ecdysis.     

Quaternary and subunit structure of Calliphora arylphorin as deduced from electron microscopy,electrophoresis, and sequence similarities with arthropod hemocyanin

Summary Arylphorin was purified from larvae of the blowfly Calliphora vicina and studied in its oligomeric form and after dissociation at pH 9.6 into native subunits. In accordance with earlier literature, it was electrophoretically shown to be a 500 kDa hexamer (1×6) consisting of 78 kDa polypeptides (= subunits). Electron micrographs of negatively stained hexamers show a characteristic curvilinear, equilateral triangle of 12 nm in diameter (top view) and a rectangle measuring 10×12 nm (side view). Alternatively, particles in the top view orientation exhibit a roughly circular shape 12 nm in diameter. Crossed immunoelectrophoresis revealed the presence of a major subunit type; the nature of a very minor and a third immunologically separated component remains unclear. A novel 2×6 arylphorin particle was detected and isolated. It comprises less than 10% of the total arylphorin material and shows a long, narrow interhexamer bridge in the electron microscope. An arylphorin dissociation intermediate identified as a trimer (1/2×6) was isolated; its possible quaternary structure is discussed on the basis of electron micrographs. The epitope of monoclonal antibody Ec-7 directed against tarantula (Eurypelma californicum) hemocyanin subunit d and also reactive to Calliphora arylphorin was traced to a highly conserved peptide of 27 amino acids localized in the center of the protein. The primary structure of Calliphora arylphorin as published in our preceding paper (Naumann and Scheller 1991) is compared in detail to the sequences of spider and spiny lobster hemocyanin. This revealed a basic framework of 103 strictly conserved amino acids. Isofunctional exchanges are proposed for another 76 positions. On the basis of these similarities, and the published three-dimensional model of spiny lobster hemocyanin, a detailed model of the quaternary structure of Calliphora arylphorin is presented. A second larval storage protein previously termed protein II was purified from Calliphora hemolymph. It was demonstrated to be a 500 kDa hexamer of 83 kDa subunits. In the electron microscope it shows a cubic view 9 nm in length with a large central hole and a rectangular view (9×10 nm) with a large central cavity. A morphologically very similar hemolymph protein was detected in Drosophila melanogaster larvae. From its structural appearance it is uncertain whether protein II belongs to the hemocyanin superfamily or not.Abbreviations FPLC fast performance liquid chromatography - HPLC high performance liquid chromatography - LSP Larval serum protein - PAGE Polyacrylamide gel electrophoresis - SDS Sodium dodecyl sulphate - Tris Tris-(hydroxymethyl)-aminomethane     

Major hemolymph protein of Aedes aegypti larvae

Aedes aegypti larval hemolymph proteins were analyzed, and the major protein was characterized. The major protein, designated P1, is hexameric and is composed of subunits with molecular weights estimated to be 83,000. P1 is dissociated into its subunits when the pH is elevated from 7 to 9. This protein accumulates during the last larval instar and is not detected in adult mosquitoes. These characteristics, together with the high content of aromatic amino acids, include P1 in the arylphorin group of the insect storage proteins. Arch. Insect Biochem. Physiol. 34:191–201, 1997. © 1997 Wiley-Liss, Inc.     

Utilization of Δ5,7 - and Δ8-sterols by larvae of Heliothis zea

Larvae from two populations of Heliothis zea were reared on artificial diets containing various sterols, which supported suboptimal growth, and their tissue sterols were characterized in order to determine how these dietary sterols are utilized by this insect. The sterols studied included Δ^5,7-sterols (7-dehydrocholesterol or ergosterol), Δ⁸-sterols (lanosterol and/or 24-dihydrolanosterol), and a Δ⁵-sterol (4,4-dimethylcholesterol). Although larvae did not develop on 4,4-dimethylcholesterol, those fed primarily Δ⁸-4,4,14-trimethylsterols developed to the third instar. When the latter sterols were spared with cholesterol, the larvae reached the sixth instar and contained 4,4,14-trimethylsterols as well as cholesterol in their tissues. When larvae were fed 7-dehydrocholesterol, <1% of the larvae from one population developed to the sixth instar and these larvae contained 7-dehydrocholesterol as their principal sterol. The other larvae successfully completed their larval stage when they were transferred from the diet containing 7-dehydrocholesterol (or no sterol) to a diet containing cholesterol within at least 9 days. The sterol composition of larvae transferred from a diet containing cholesterol to a diet containing 7-dehydrocholesterol, after they had reached 60% of their final weight, was 54% cholesterol and 46% 7-dehydrocholesterol. The major sterol isolated from the tissues of the larvae fed ergosterol was also 7-dehydrocholesterol. Therefore, although the larva of H. zea can dealkylate and saturate the side chain of the Δ^5,7,22-24β-methylsterol, it carries out little metabolism of the B ring of the nucleus. These studies demonstrate that, when Δ^5,7- or Δ⁸-sterols are the principal sterols in the diet of H. zea, they are absorbed and incorporated into its tissues, although they slow the rate of growth and may prevent complete development of the larva.     

Expression of carbohydrate epitopes L2/HNK-1 and L3 in the larva and imago of Drosophila melanogaster and Calliphora vicina

Summary The carbohydrate epitopes L2/HNK-1 and L3 belong to two overlapping families of adhesion molecules in the vertebrate, and probably the invertebrate nervous systems. To investigate their pattern of expression during the development of insects, cryosections of late third instar larvae and imagoes of Drosophila melanogaster and Calliphora vicina were studied by indirect immunofluorescence using several monoclonal antibodies to the L2/HNK-1 and one monoclonal antibody to the L3 epitope. Each monoclonal antibody to the L2/HNK-1 epitope showed a different immunohistological staining pattern, which differed from that of the L3 monoclonal antibody. In both insect species the immunohistological staining patterns for the two carbohydrate epitopes were similar at the two developmental stages, with immunoreactivity not confined to the nervous system. In larvae, immunoreactivities of the monoclonal antibodies L2.334 and L3.492 were predominantly associated with the extracellular matrix as indicated by co-localization with laminin, particularly in the imaginal discs, while L2.349 revealed a more cell surface-associated distribution. In imagoes, immunoreactivities were detectable in most organs studied.     

Drosophila hemolymph proteins: Purification,characterization, and genetic mapping of larval serum protein 2 in D. melanogaster

Three of the major protein species present in the hemolymph of Drosophila melanogaster larvae just prior to pupation are absent from second instar larvae but accumulate rapidly during the third instar. This article describes the purification and characterization of one of these, larval serum protein (LSP) 2, using an immunological assay. It is a homohexamer of molecular weight about 450,000, with a polypeptide molecular weight of 78,000–83,000. Fast and slow electrophoretic variants of this protein map between the markers vin and gs, at 36–37 on chromosome 3.This work was partially supported by M.R.C. Research Studentships to J.W. and M.E.A.     

Effects of prococene II on the nutritional physiology of last instar Heliothis zea larvae

When precocene II was fed to last instar larvae of Heliothis zea, it caused significant reductions in the calculated rate of growth, rate of nutrient assimilation, and conversion of ingested and digested food to body mass. No change in the rate of food consumption occurred but respiration was significantly higher. Transport of the nutrient [1-¹⁴C] linoleic acid across midgut tissue was hindered when larvae were fed precocence II. There was also a precocene-induced change in the apical (luminal) morphology of midgut cells, including absence of the glycocalyx and loss of the microvillar absorptive surface. The influence of precocene II on the midgut physiology and metabolic processes in last instar larvae of H. zea may account for the observed reduced growth and delayed development. © 1992 wiley-Liss, Inc.     

Binding of insecticides to lipophorin and arylphorin,two hemolymph proteins of Heliothis zea

The binding of different insecticides to hemolymph proteins of the corn earworm, Heliothis zea, was studied by an analytical isoelectric focusing technique. Two proteins capable of binding hydrophobic substrates were discovered and identified as lipophorin and arylphorin. Lipophorin, the major lipoprotein of Heliothis zea, demonstrated rather unspecific binding of xenobiotics of different hydrophobicities. Arylphorin, a putative storage protein, revealed strong affinity for compounds of medium polarity, binding only weakly to insecticides of higher or lower polarity.     

Behavioral changes and growth inhibition in last instar larvae of Heliothis zea induced by oral and topical application of precocene II

In diet choice tests, newly-ecdysed fifth instar Heliothis zea larvae randomly chose diet containing 4 mM precocene II or diet without precocene II but initially preferred to feed on diet without precocene II. After two days, however, they were found more frequently on diet containing precocene II. Nevertheless, larval growth and development were inhibited when forced to eat diet containing precocene II. Removal of the maxillae, the site of the primary gustatory receptors, did not prevent the growth inhibition caused by larval consumption of diet with precocene II. Topical treatments with precocene II also caused larval growth inhibition, and daily treatments were more detrimental to the larvae than a large single dose. Addition of methylenedioxyprecocene (6,7-methylenedioxy-2,2-dimethylchromene) to the diet did not reverse the observed larval growth inhibition and this compound was itself significantly toxic. These results indicate that larval growth and development are disrupted after precocene II ingestion, but not because of preingestive discrimination by olfaction and gustation.     

Importance of predation by Coleomegilla maculata larvae in the natural control of the corn earworm in sweet corn

The corn earworm, Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) is a serious pest of many crops in North America, particularly sweet corn. The larvae of Coleomegilla maculata (DeGeer) (Coleoptera: Coccinellidae) are the most frequently observed predators of H. zea eggs in sweet corn. Companion cropping strategies have been developed to augment densities of C. maculata in sweet corn plantings. In this study, the importance of predation by C. maculata larvae was evaluated in experiments when larval abundance was manipulated using hand removal and physical exclusion. In 2003 and 2004, sentinel H. zea eggs were exposed on corn for 48 h. Egg mortality was 13.8% greater in the treatment where C. maculata larvae were present. In 2004 and 2005 eggs were exposed on corn under the same hand removal and physical exclusion techniques and monitored at 3-h intervals for 24 h to directly observe predation on sentinel eggs. Coleomegilla maculata larvae were the most frequently observed predators of the eggs, accounting for 45.9% of 85 total observed predation events in the control. Whereas in the treatment where C. maculata larvae were reduced, egg survival was 26.13% greater and only 37 total predation events were observed. All pest eggs were eliminated from individual ears 22.1% more often when C. maculata larvae were present at natural densities indicating that predation by this predator stage lessens crop damage by H. zea. The results of this study suggest that C. maculata larvae are an irreplaceable source of natural mortality for H. zea eggs on sweet corn.     

Dynamic changes of gut bacterial communities present in larvae of Anoplophora glabripennies collected at different developmental stages

The Asian long-horned beetle, Anoplophora glabripennies (Motschulsky), is a destructive wood-boring pest that is capable of killing healthy trees. Gut bacteria in the larvae of the wood-boring pest is essential for the fitness of hosts. However, little is known about the structure of the intestinal microbiome of A. glabripennies during larval development. Here, we used Illumina MiSeq high-throughput sequencing technology to analyze the larval intestinal bacterial communities of A. glabripennies at the stages of newly hatched larvae, 1st instar larvae and 4th instar larvae. Significant differences were found in larval gut microbial community structure at different larvae developmental stages. Different dominant genus was detected during larval development. Acinetobacter were dominant in the newly hatched larvae, Enterobacter and Raoultella in the 1st instar larvae, and Enterococcus and Gibbsiella in the 4th instar larvae. The microbial richness in the newly hatched larvae was higher than those in the 1st and 4th instar larvae. Many important functions of the intestinal microbiome were predicted, for example, fermentation and chemoheterotrophy functions that may play an important role in insect growth and development was detected in the bacteria at all tested stages. However, some specific functions are found to be associated with different development stages. Our study provides a theoretical basis for investigating the function of the intestinal symbiosis bacteria of A. glabripennies.     

Major hemolymph proteins from larvae of the black swallowtail butterfly,Papilio polyxenes

Three major hemolymph proteins of Papilio polyxenes larvae were isolated and characterized. Density gradient ultracentrifugation of hemolymph resulted in flotation of the major lipoprotein, lipophorin. P. polyxenes larval lipophorin is composed of two apoproteins, apolipophorin-I and apolipophorin-II, plus a mixture of lipids, to give a density of 1.13 g/ml. Immunoblotting experiments using antisera directed against Manduca sexta apolipophorin-I and apolipophorin-II, respectively, revealed cross-reactivity of apoLp-I with Manduca sexta apoLp-I, and apoLp-II with M. sexta apoLp-II. Gel permeation chromatography of the subnatant obtained following density gradient ultracentrifugation revealed the presence of a major protein peak which was shown to contain three major serum proteins, two of which were isolated and characterized. One of these proteins was purified by lectin affinity chromatography. Both proteins have native molecular weights in the range of 450,000 and appear to be hexamers of a single subunit type. Major serum protein-1 is nonglycosylated and has a subunit molecular weight of 75,000. Major serum protein-2 is glycosylated and has a subunit molecular weight of 74,000. Amino acid analysis of this protein revealed a tyrosine plus phenylalanine content of 20 mole percent, characteristic of the arylphorin class of insect storage proteins. Using antibodies against M. sexta larval hemolymph proteins, both the P. polyxenes major serum proteins were shown to be immunologically related to serum proteins of other lepidopteran species.