Metabolic discrimination of sucrose starvation from<Emphasis Type="Italic">Arabidopsis</Emphasis> cell suspension by<Superscript>1</Superscript>H NMR based metabolomics

Whole cell extracts ofArabidopsis cell cultures maintained on various sucrose concentrations (0,3, and 6%) were analyzed by¹H NMR spectroscopy to determine the comprehensive metabolic change in these cultures during sucrose starvation. The amount of sucrose, glucose, and fructose in the cells decreased to almost nothing after 12 h of culture in medium without sucrose. In contrast, the total free amino acid content of the cells increased as the culture proceeded. Among the free amino acids, phenylalanine and malic acid increased the most, followed by asparagine and alanine, whereas glutamic acid did not change significantly. These results are in agreement with previous studies using HPLC.¹H NMR spectroscopy enabled measurement of changes in the sugar and free amino acid content of whole cell extracts without fractionation and complicated sample preparation. These results indicate that comprehensive metabolic changes in the cells can be determined by a simple, rapid method using whole cell extracts and¹H NMR spectroscopy.     

Intraspecific larval competition in two solitary parasitoids, Apoanagyrus (Epidinocarsis) lopezi and Leptomastix dactylopii

Analysis of total aromatic amino acid (free and bound) in some cucumber accessions selected previously for resistance to western flower thrips, Frankliniella occidentalis (Pergande) [Thysanoptera: Thripidae], indicated that low concentrations of these essential nutrients, relative to total leaf protein, were correlated with a reduction in damage by the insect. Further analysis of samples of four important horticultural crops (lettuce, tomato, pepper and cucumber) with unknown levels of resistance to thrips showed a significant genotypic variation in the concentrations of total aromatic amino acids relative to the total leaf protein. Accessions from each crop with low or high concentrations of aromatic amino acids in proteins were exposed to thrips larvae. Regression analysis showed a highly significant positive correlation between aromatic amino acid concentration in leaf protein and thrips damage, regardless of crop species. It is concluded that higher concentrations of aromatic amino acids in plant proteins are important for successful thrips development. These results provide plant breeders with a promising tool for indirect selection without using undesirable insect bioassays.     

Deadly venom of Asobara japonica parasitoid needs ovarian antidote to regulate host physiology

Asobara japonica (Braconidae) is an endophagous parasitoid developing in Drosophila larvae. The present study shows that A. japonica was never encapsulated in Drosophila melanogaster, and that it caused an overall inhibition of the host encapsulation reaction since injected foreign bodies were never encapsulated in parasitized hosts. Both the number of circulating hemocytes and the phenoloxidase activity decreased in parasitized larvae, and the hematopoietic organ appeared highly disrupted. We also found that A. japonica venom secretions had atypical effects on hosts compared to other braconid wasps. A. japonica venom secretions induced permanent paralysis followed by death of D. melanogaster larvae, whether injected by the female wasp during an interrupted oviposition, or manually injected into unparasitized larvae. More remarkably, these effects could be reversed by injection of ovarian extracts from female wasps. This is the first report that the venom of an endophagous braconid parasitoid can have a deadly effect on hosts, and moreover, that ovarian extracts can act as an antidote to reverse the effects of the wasp's venom. These results also demonstrate that A. japonica secretions from both venom gland and ovary are required to regulate synergistically the host physiology for the success of the parasitoid.     

Suppression of Boll Weevil Infestations (Coleoptera: Curculionidae) Occurring on Fallow-Season Cotton in Southern Texas by Augmentative Releases ofCatolaccus grandis(Hymenoptera: Pteromalidae)

Augmentative releases ofCatolaccus grandis(Burks) were conducted in the Lower Rio Grande Valley of Texas in an attempt to suppress infestations of boll weevil,Anthonomus grandisBoheman, occurring in stands of “fallow-season” cotton (i.e., fields in violation of the 1 September stalk destruction deadline mandated by Texas law). In each of five release sites monitored during the study period (October, 1994–March 1995), augmentative releases ofC. grandiswere accompanied by an appreciable increase in the incidence of parasitized boll weevils (primarily third-stage larvae and pupae infesting abcised cotton squares) within a relatively brief time period. The relatively high incidence of host mortality caused byC. grandisin each release site was largely indispensible (i.e., would not have occurred in the absence of the parasite) and served to destroy significant numbers of immature boll weevils that appear to have been predisposed to successfully overwinter. The potential role of parasite augmentation in the management of the overwintering boll weevil population in southern Texas is discussed.     

Prey selectivity inEuseius tularensis [Acari: Phytoseiidae]

Differences in responsiveness to potential prey and pollen were tested in the facultative predator,Euseius tularensis Congdon. Following isolation without food for 16 h, adult female mites were observed with one of the following potential foods: pollen ofMalephora crocea (Jacquin) Schwantes,Scirtothrips citri (Moulton) 2nd instar larvae,Tetranychus pacificus McGregor nymphs and ♂,Panonychus citri (McGregor) nymphs and ♂,E. hibisci (Chant) larvae, andE. tularensis larvae. Responses were compared using the proportion of encounters that resulted in consumption. This proportion was highest when pollen was encountered, followed byS. citri andT. pacificus. Consumption was much lower in response toP. citri and congeneric larvae. WhenS. citri larvae were encountered a 2nd time, 1 to 2 h after the initial capture and consumption, responsiveness increased to a level equal to the response observed with pollen. These results suggest thatE. tularensis is a more effective biological control agent of citrus thrips (S. citri) than of citrus red mite (P. citri).     

Photoautotrophic growth of soybean cells in suspension culture IV. Free amino acid pools and the effect of nitrogen on chlorophyll levels

A photoautotrophic soybean suspension culture was used to study free amino acid pools during a subculture cycle. Free amino acid analysis showed that the intracellular concentrations of asparagine, serine, glutamine, and alanine reached peaks of 200, 10, 9 and 7 mM, respectively, at specific times in the 14-day subculture cycle. Asparagine and serine levels peaked at day 14 but glutamine level rose quickly after subculture, peaking at day three and then declined gradually. Roughly similar patterns were found in the conditioned culture medium although the levels were 1000-fold lower than those found in cells. Photoautotrophic (SB-P) and photomixotrophic (SB-M) cultures were quantitatively similar with regard to free asparagine and serine but not glutamine or free ammonia. Heterotrophic (SB-H) cells had 81–85% less free asparagine on day seven than did SB-M or SB-P cells. Hence, similar to the phloem sap of a soybean plant, asparagine, glutamine, alanine and serine were the predominant amino acids in photoautotrophic soybean cell cultures. Varying the amount of total nitrogen in culture medium for two subcultures at 10, 25, 50, and 100% Of normal levels showed that growth was inhibited only at the 10 and 25% levels but that growth on medium containing 50% of the normal nitrogen was as good as that on 100% nitrogen. Moreover, cellular chlorophyll content correlated exceptionally well with initial nitrogen content of the medium. Thus, the photosynthesis of SB-P cells was not limited by chlorophyll content. SB-P cells grown for two subcultures on 10% nitrogen contained very low free amino acid levels and only 1% of the free ammonia levels found in cells growing on a full nitrogen complement.Abbreviations SB-P photoautotrophic soybean cells (no sucrose, high CO₂, high light) - SB-M photomixotrophic soybean cells (1% w/v sucrose, high light) - SB-H heterotrophic soybean cells (3% sucrose, dark)     

Evaluation of herbicide-coated larvae ofBactra verutana [Lep.: Tortricidae] to control nutsedges [Cyperus rotundus L. andC. Esculentus L.]

Feeding by 10-day old mid-instar larvae of the native moth,Bactra verutana Zeller, coated with solutions of bentazon [3-isopropyl-1 H-3-benzothiadiazin-4(3H)-one 2,2-dioxide] or glyphosate [N-(phosphonomethyl) glycine], reduced the dry weight of purple (Cyperus rotundus L.) or yellow nutsedge (C. esculentus L.) an average 25% more than did uncoated larvae. The herbicide-coated larvae reduced dry weight by 71% for 4-week yellow nutsedge and by 80% for 2-week purple nutsedge plants within 15 days after release. This integrated approach was apparently not pratical, however, because larvae randomly damaged the non-target crops, cotton (Gossypium hirsutum L.) and turnip (Brassica rapa L.). The more mature larvae killed about 3% of the cotton and 14% of the turnip plants. Newly-emerged larvae were highly sensitive to the herbicides. Mature larvae are less host-specific than are newly-emerged larvae. Therefore, the use of repeated broadcasts of newly-emergedB. verutana larvae is still the best method known for using insects to control nutsedge.     

Isolation and Characterization of a Serine Protease,Ba III-4, from Peruvian <Emphasis Type="Italic">Bothrops atrox</Emphasis> venom

A serine protease from Bothrops atrox (Peruvian specimen’s venom) was isolated in two chromatographic steps in LC molecular exclusion and reverse phase-HPLC. This protein was denominated Ba III-4 (33,080.265 Da determinated by MALDI-TOF mass spectrometry) and showed pI of 5.06, Km 0.2 × 10⁻¹ M and the V _máx 4.1 × 10⁻¹ nmoles p-NA/lt/min on the synthetic substrate BapNA. Ba III-4 also showed ability to coagulate bovine fibrinogen. The serine protease was inhibited by soyben trypsin inhibitor and DA2II, which is an anti-hemorrhagic factor isolated from the opossum specie Didelphis albiventris. The primary structure of Ba III-4 showed the presence of His(44), Asp(94) and Ser(193) residues in the corresponding positions to the catalytic triad established in the serine proteases and Ser(193) are inhibited by phenylmethylsulfonylfluoride (PMSF). Amino acid analysis showed a high content of Asp, Glu, Gly, Ser, Ala and Pro, as well as 12 half-cysteine residues. Ba III-4 contained 293 amino acid residues and the primary structure of VIGGDECDIN EHPFLAFMYY SPRYFCGMTL INQEWVLTAA HCRYFCGMTL IHLGVHRESE KANYDEVRRF PKEKYFIFCD NNFTDDEVDK DIMLIRLDKP VSNSEHIAPL SLPSNPPSVG SVCRIMGWGQ TTTSPIDVLS PDEPHCANIN LFDNTVCHTA HPQVANTRTS TDTLCAGDLQ GGRDTCNGDS GGPLICNEQL HGILSWGGDP CAQPNKPAFY TKVYYFDHPW IKSIIAGNKK TVNFTCPPLR SDAKDDSTTY INQEWDWVLT AEHCDRTHMR NSFYDYSSIN SDS. Titration experiments did not show the presence of free sulfhydryl groups after 4 h incubation, nor were differences found in relation to titration kinetics in the presence of nondenaturating buffer. The isolation of this protein, Ba III-4, is of potential interest for the understanding of the pathomechanism of the snake venom action and for the identification of new blood coagulation enzymes of natural sources.     

Proteomic analysis of parasitized Plutella xylostella larvae plasma

Insects use their innate immunity to defend themselves against foreign invaders, such as microorganisms, nematodes and parasites. Cotesia plutellae, an endoparasitoid wasp that parasitizes the diamondback moth Plutella xylostella, uses several strategies to attack the host immune system, such as injection of viruses, venom, and serosal membrane-derived cells denoted teratocytes. However, the proteome profiles related to these immune deficiency systems have yet to be clearly defined. In this study, we investigate differences in protein expression patterns in parasitized P. xylostella larvae, with a view to identifying parasitism-specific factors. Using 2D polyacrylamide gel electrophoresis, proteins in the host plasma were assessed every 48 h after parasitism by C. plutellae. A large number of protein spots (350 in total) were detected, and approximately 50 spots were differentially expressed in the parasitized P. xylostella larvae every 48 h. In total, 26 potential candidates, including P. xylostella Serpin 2 (pxSerpin 2), translationally controlled tumor protein, signal transduction histidine kinase, apolipophorin-III, and fatty-acid binding protein were identified through quadrupole time-of-flight tandem mass spectrometry and sequence homology analysis. These proteins were classified into the following functional groups: immunity, signaling, lipid metabolism, energy metabolism, amino acid/nucleotide metabolism, and others. The pxSerpin 2 gene was cloned, and its expression profile investigated during the course of parasitism. Real-time PCR analysis of pxSerpin 2 revealed a poor correlation between the mRNA level and protein abundance. Our results clearly suggest that parasitism-specific proteins participate in suppression of the host immune response.     

Biochemical analysis of four species ofCassia L. pollen

The present paper describes the comparative biochemical studies in terms of quantitative analyses of carbohydrates, lipids, proteins, free amino acids, nucleic acids, minerals, ash and moisture as well as the identification of free amino acids of pollen of four species ofCassia L. (C. alata L,C. fistula L,C. occidentalis L andC. siamea Lam.). A significant variation in the chemical constituents was observed among the four species.C. occidentalis showed the highest levels of carbohydrate (15.15%) and protein (22.45%), andC. siamea had the lowest levels of carbohydrate (7.15%), lipid (6.2%) and protein (13.85%).C. alata andC. fistula showed intermediate results. However,C. alata showed the highest amount of free amino acids (3.8%) and the least of 1.42% was found inC. fistula. Thin layer chromatography (TLC) of free amino acids of the four species showed some homology in their amino acid content, of which proline, glutamic acid, methionine and phenyl-alanine were the most dominant. The level of nucleic acids and minerals was found to be comparatively low.C. siamea andC. alata showed an exceptionally high level of ash content (8.6 and 8.8%, respectively) while moisture content varied from 8 to 11%.     

Simulated acid rain reduces the susceptibility of the European pine sawfly (Neodiprion sertifer) to its nuclear polyhedrosis virus

Summary The study dealt with the effect of simulated acid rain (both H₂SO₄ and HNO₃; acidities of pH 4 and pH 3) on the susceptibility of the larvae of Neodiprion sertifer to its nuclear polyhedrosis virus. Scots pines growing in a subarctic area with low ambient pollution levels were irrigated with simulated acid rain during two summers. Neodiprion larvae fed with foliage from the experimental trees were infected with a dilute virus suspension. The acid treatment of host trees had a significant effect on the proportion of virus-treated larvae alive 16 days after the virus application: there were almost no differences between the controls and the pH 4 irrigation group, but on the needles of pH 3-treated trees larval survival was twice as high as with other treatments. The direct spraying of acid water on the needles before they were fed to the larvae did not significantly affect the survival of virus infected larvae. Our results suggest that acid rain may reduce the susceptibility of Neodiprion larvae to virus disease via changes in the quality of pine foliage.     

Distribution of isoasparagine among different characean species

Thirteen species of Characeae were analyzed for their free amino acid contents. Large amounts of isoasparagine, accounting for 10 to 50% of the total free amino acids, were found in extracts fromChara (5 species including one unidentified),Nitellopsis (1 species), andLamprothamnium (1 species). In contrast, no isoasparagine was detected inNitella (5 species) andTolypella (1 species), except forN. flexilis in which as much as 40% of the free amino acids was isoasparagine. Other major amino acids found in the tested materials were Ala, Asp, Glu and Gln.     

Astacin-like metalloproteases are a gene family of toxins present in the venom of different species of the brown spider (genus Loxosceles)

Brown spiders have a worldwide distribution, and their venom has a complex composition containing many different molecules. Herein, we report the existence of a family of astacin-like metalloprotease toxins in Loxosceles intermedia venom, as well as in the venom of different species of Loxosceles. Using a cDNA library from the L. intermedia venom gland, we cloned two novel cDNAs encoding astacin-like metalloprotease toxins, LALP2 and LALP3. Using an anti-serum against the previously described astacin-like toxin in L. intermedia venom (LALP1), we detected the presence of immunologically-related toxins in the venoms of L. intermedia, Loxosceles laeta, and Loxosceles gaucho. Zymographic experiments showed gelatinolytic activity of crude venoms of L. intermedia, L. laeta, and L. gaucho (which could be inhibited by the divalent metal chelator 1,10-phenanthroline) at electrophoretic mobilities identical to those reported for immunological cross-reactivity. Moreover, mRNAs extracted from L. laeta and L. gaucho venom glands were screened for astacin-like metalloproteases, and cDNAs obtained using LALP1-specific primers were sequenced, and their deduced amino acid sequences confirmed they were members of the astacin family with the family signatures (HEXXHXXGXXHE and MXY), LALP4 and LALP5, respectively. Sequence comparison of deduced amino acid sequences revealed that LALP2, LALP3, LALP4, and LALP5 are related to the astacin family. This study identified the existence of gene family of astacin-like toxins in the venoms of brown spiders and raises the possibility that these molecules are involved in the deleterious effects triggered by the venom.     

Olfactory host location and learning in the granary weevil parasitoidLariophagus distinguendus (Hymenoptera: Pteromalidae)

The sources of volatiles that attract the parasitoid Lariophagus distinguendus(Först.) to its hosts, the larvae of the granary weevil Sitophilus granarius(L.) infesting grains of Triticum aestivumL., were investigated in bioassays. The influence of oviposition experience on the responsiveness of the parasitoids to the sources of volatiles was considered. The potential sources of attractants are the host larvae, their feces, and the grain infested by the host. Weevil larvae were not attractive. Feces were attractive to females without and females with oviposition experience. Infested grains from which the larvae and feces were removed were slightly attractive to inexperienced, but very attractive to experienced females. Healthy and artificially damaged grains were attractive to experienced parasitoids only. Refering to the “variable response model,” we discuss the effect of experience on the response of the parasitoids to the several stimuli. Stimuli from different trophic levels are used by the foraging parasitoids: feces of the host larvae is an important source of attractants, but also the grains, particularly after experience.     

Transmission of a nuclear polyhedrosis virus inHeliothis zea [Lep.: Noctuidae] larval populations on soybean

Transmission of a nuclear polyhedrosis virus (NPV) inHeliothis zea (Boddie) on soybean was examinated. Artificial infestations ofH. zea were established at densities of 6.5, 19.5 and 58.5 larvae/row-m. Additional larvae infected to die from NPV in the 2nd stage were released into subplots to simulate 5 and 25% mortality levels. Virus transmission from infected to noninfected larvae was correlated with the initial incidence of infection in the population but not the density of larvae/row-m. Deposition of virus on plants from cadavers of larvae that died of virus infection was correlated with the initial incidence of infection in the populations and the density of larvae/row-m. After pupation of larvae in the 1st population, noninfected larvae only were again released to examine transmission of viral inoculum remaining on plants and soil. The percent mortality of larvae collected from the 2nd release was low and did not differ significantly between treatments. The concentration of virus on foliage and in soil after the 2nd release was directly correlated with density of larvae/row-m but not the incidence of infection within the population in the 1st release. This material is based upon work supported in part by the U.S. Department of Agriculture under Agreement N^o 82 CRSR-2-1000.     

Host behaviour and its influence on foraging and acceptance by the solitary parasitoid wasp,Venturia canescens (Hym: Ichneumonidae)

Behavioural interactions between the solitary koinobiont parasitoid,Venturia canescens, and two of its hosts,Plodia interpunctella andCorcyra cephalonica, were investigated. The response of both hosts to simulated antennation using a two-haired brush was examined over instars 3 (L3) to 5 (L5). YoungP. interpunctella larvae predominantly adopted escape tactics (writhe, trash) whereas L5P. interpunctella usually froze after the stimulus was applied. L3C. cephalonica larvae were more aggressive (headrear, flick) thanP. interpunctella in response to the application of the stimulus, but olderC. cephalonica responded less aggressively than in earlier instars. AlthoughV. canescens readily jabbed its ovipositor at both hosts after antennation,P. interpunctella was considerably more susceptible to parasitoid attack thanC. cephalonica, irrespective of size in the final (L5) instar.C. cephalonica, the larger, more aggressive host, actively resisted parasitism whereasP. interpunctella responded much more passively after parasitoid contact. Parasitoids examined and jabbed their ovipositors at dead hosts, but this behaviour was not sustained, implying that host movement stimulates parasitoid attack. On patches containingV. canescens, L5C. cephalonica andP. interpunctella, mostP. interpunctella larvae responded by freezing after parasitoid contact.P. interpunctella that froze usually avoided parasitism, whereas larvae that attempted to escape by crawling were pursued with vigour byV. canescens and usually parasitized. Irrespective of behaviour after parasitoid contact,C. cephalonia displayed more aggressive behaviour and had much greater success in warding off parasitoid attack. Host acceptance byV. canescens is clearly affected by the size and species of the host it attacks. The influence of host defensive behaviour is discussed in relation to the evolution of parasitoid counter-defences and oviposition strategies.     

Host selection and larval interactions among three primary parasitoids ofPlathypena scabra (Lep.: noctuidae)

Three hymenopteran parasitoids that attackPlathypena scabra (F.) larvae often oviposit into consecutive instars of the host. We investigated host discrimination by adults and competitive interactions among larvae of these three parasitoid species. Avoidance of superparasitism byCotesia marginiventris (Cresson) andDiolcogaster facetosa Ashmead was tested. Females of each species were presented either withP. scabra parasitized by a different female of the same species 6 h earlier or unparasitizedP. scabra. Under these conditions,C. marginiventris attacked similar numbers of parasitized and unparasitized hosts.D. facetosa attacked 31% fewer parasitized than unparasitizedP. scabra. The avoidance of multiple parasitism byD. facetosa was studied in a similar bioassay. AlthoughD. facetosa females parasitized fewerP. scabra that had been attacked byC. marginiventris 6 h previously, the reduction in parasitism was only about 23%. In competition studies, immatureD. facetosa were better competitors than immatureC. marginiventris. Aleiodes (=Rogas) nolophanae (Ashmead) was an inferior competitor against bothC. marginiventris andD. facetosa when the duration between parasitism events was 1 h, but their competitive ability increased when they multiply parasitized hosts at least 32 h after the initial oviposition.