Differential responses of tiger swallowtail subspecies to secondary metabolites from tulip tree and quaking aspen

Summary Two subspecies of the eastern tiger swallowtail butterfly, Papilio glaucus, exhibit reciprocal inabilities to survive and grow on each other's preferred foodplant. P. g. canadensis R. & J. performs well on quaking aspen (Populus tremuloides Michx.) but not on tulip tree (Liriodendron tulipifera L.); P. g. glaucus L. performs well on tulip tree but not on quaking aspen. This study was designed to test the hypothesis that secondary metabolites in tulip tree and quaking aspen are responsible for these differential utilization abilities. We extracted and fractionated leaf constituents into different chemical classes, applied them to a mutually acceptable diet (black cherry, Prunus serotina, leaves), and bioassayed them against neonate larvae (survival) and penultimate instar larvae (survival, growth, digestibility and conversion efficiencies). For each plant species, one fraction in particular showed activity against the unadapted subspecies. One tulip tree fraction dramatically reduced survival of P. g. canadensis neonates, and reduced consumption rates, growth rates, and ECI's of fourth instar larvae. The tulip tree constituents most likely responsible for these effects are sesquiterpene lactones. One quaking aspen fraction greatly lowered survival of P. g. glaucus neonates, and decreased survival, consumption rates, growth rates and ECD's of fourth instar larvae. The compounds responsible for these results are probably simple phenols or phenolic glycosides. Surprisingly, P. g. glaucus and P. g. canadensis showed slightly poorer performance on the active tulip tree and quaking aspen fractions, respectively, indicating that even adapted insects incur a metabolic cost in the processing of their host's phytochemicals.     

Ascobotryozyma americana gen. nov. et sp. nov. and its anamorph Botryozyma americana, an unusual yeast from the surface of nematodes

A new teleomorphic genus Ascobotryozyma, with a single species, A. americana, is proposed. Its anamorph is a Botryozyma that differs from the type species, B. nematodophila, on distributional, physiological, and molecular criteria; it is described as Botryozyma americana, anam. sp. nov. Ascobotryozyma is characterized by globose asci bearing four lunate ascospores. Fusion of thallus cells precedes ascus formation. Ascobotryozyma americana was isolated from the surface of nematodes (Panagrellus dubius) associated with galleries of the poplar borer (Saperda calcarata) in trembling aspen (Populus tremuloides) in eastern Washington, USA. The teleomorph has not been produced in pure culture.     

Ingested prey increase risks of visual predation in transparent Chaoborus larvae

Summary Transparency reduces the chances of detection of large planktonic animals by visual predators. An important constraint on the transparency of planktonic animals may be ingested food which could be seen through the body, thereby increasing the vulnerability of transparent zooplankton to visual predators. To test this hypothesis, we presented fed and un-fed Chaoborus larvae to juvenile coho salmon (Oncorhynchus kisutch). Overall, the presence of prey in the gut of Chaoborus increased their probability of capture by 68%. Predation risks due to the visibility of ingested food increased in proportion to meal size: larvae with nearly full gut were captured about three times faster on the average than larvae which had little food in their gut. Although Chaoborus larvae may be able to reduce this increased predation risk by migrating downward to low light levels, this behavior would reduce feeding opportunities by removing the larvae from surface waters where prey density is generally high. In this way, visual predators may limit the growth and the maximum size that can be achieved by transparent animals.     

Insectivory and social digestion inDrosophila

It has long been believed thatDrosophila larvae feed almost entirely by ingesting yeast and possibly other microorganisms that are associated with fermenting fruits or other vegetable matter. However, we have discovered that the larvae of a number ofDrosophila species can consume such diverse substrates as insect tissues, including the exoskeleton. Experiments reported here, which include raising sterile dechorionated eggs to adulthood on adult carcasses under axenic conditions, show that larvae can consume complex chitinous substrates directly without the assistance of microorganisms. We show thatDrosophila larvae are able externally to digest amylose, cellulose, and chitin, without coming into physical contact with them. We conclude that not only doDrosophila larvae produce enzymes enabling them to digest a wide variety of substrates, but also these enzymes are egested onto the substrates so that at least some digestion, especially of large polymers, takes place externally. Finally, we suggest that the phenomenon of external digestion explains both the previously unexplained massiveness ofDrosophila salivary glands and their chromosomes and the tendency of larvae to cluster, which may also be true of other dipterans.This investigation was supported by NIH (AREA) Grant IR 15AG05897.     

Location of ecdysteroid 22-O-acyltransferase in the larvae ofHeliothis virescens

Larvae of the tobacco budworm,Heliothis virescens, are resistant to high levels of ingested 20-hydroxyecdysone which could cause potential inhibition to the development of many other lepidopteran species. This resistance is attributed to the ability of the larvae to metabolize this molting hormone to its 22-acyl ester forms. When tobacco budworm larvae were fed large quantities of 20-hydroxyecdyone, the hormonal metabolites were found in gut and fat body tissues. When incubated with 20-hydroxyecdysone gut tissue converted 20-hydroxyecdysone into its 22-acyl ester metabolites. Lumen site of the midgut was found to be the major location of this bio-transformation. In contrast, fat body tissue failed to convert 20-hydroxyecdysone to 22-acyl ester metabolitesin vitro. After the oral injection of³H-ecdysone, the major metabolites formed were ecdysone 22-acyl esters whereas the majority of³H-ecdysone was transformed to polar metabolites after it was injected into the hemocoel of the larvae. Similar distributions of ecdysteroid 22-O-acyltransferase and alkaline phosphatase activity in subcellular fractions demonstrates the co-localization of these enzymes in plasma membrane of the gut epithelial cells. These results suggest that gut brush border membrane is the major site of ecdysteroid 22-acyl ester formation inH. virescens larvae.     

Effect of nitrogen fertilization upon the secondary chemistry and nutritional value of quaking aspen (Populus tremuloides Michx.) leaves for the large aspen tortrix (Choristoneura conflictana (Walker))

Summary We investigated the effects of nitrogen fertilization upon the concentrations of nitrogen, condensed tannin and phenolic glycosides of young quaking aspen (Populus tremuloides) leaves and the quality of these leaves as food for larvae of the large aspen tortrix (Choristoneura conflictana), a Lepidopteran that periodically defoliates quaking aspen growing in North America. Nitrogen fertilization resulted in decreased concentrations of condensed tannin and phenolic glycosides in aspen leaves and an increase in their nitrogen concentration and value as food for the large aspen tortrix. These results indicate that plant carbon/nutrient balance influences the quality of aspen leaves as food for the large aspen tortrix in two ways, by increasing the concentrations of positive factors (e.g. nitrogen) and decreasing the concentrations of negative factors (eg. carbon-based secondary metabolites) in leaves. Addition of purified aspen leaf condensed tannin and a methanol extract of young aspen leaves that contained condensed tannin and phenolic glycosides to artificial diets at high and low levels of dietary nitrogen supported this hypothesis. Increasing dietary nitrogen increased larval growth whereas increasing the concentrations of condensed tannin and phenolic glycosides decreased growth. Additionally, the methanol extract prevented pupation. These results indicate that future studies of woody plant/insect defoliator interactions must consider plant carbon/nutrient balance as a potentially important control over the nutritional value of foliage for insect herbivores.     

The effect of parasitism byHyposoter didymator [Hym.: Ichneumonidae] on food consumption and utilization bySpodoptera litura [Lep.: Noctuidae]

The effect of parasitism byHyposoter didymator (Thunb.) [Hym.: Ichneumonidae] on food consumption and utilization bySpodoptera litura (Fb.) [Lep.: Noctuidae] was studied for seven days, during which the parasitoid completed its larval development. Food consumption, weight gained and faeces produced were significantly less in parasitized larvae than in unparasitized larvae after the 4^th day following parasitization. Approximate digestibility was higher in parasitized larvae after the 2^nd day following parasitization. Efficiency of conversion of ingested and digested food into body weight was greater in unparasitized larvae after the 2^nd day of parasitization. There seems to be a definite immediate advantage to the crop on releasing the parasitoid due to the reduced consumption of food. Contribution No. 46004 of Biological Control Centre (NCIPM), Bangalore 560 024.     

Ectomycorrhizae formed in vitro by quaking aspen: including Inocybe lacera and Amanita pantherina

Ectomycorrhizae formed in synthesis tubes by aspen (Populus tremuloides) seedlings and each of seven fungal isolates are described. Isolates of Amanita muscaria v. formosa, A. pantherina, Inocybe lacera, and Paxillus vernalis, from sporocarps collected in aspen stands in southwestern Montana, developed mantles and Hartig nets on aspen roots, as did the broad-hostrange fungi Cenococcum geophilum and Pisolithus tinctorius from the VPI culture collection. Chalciporus piperatus failed to form mycorrhizae, and Piloderma croceum formed a mantle, but no Hartig net. The first syntheses of I. lacera and A. pantherina with aspen are reported.     

Indirect effects of the fairy shrimp,Branchipus schaefferi and two ostracod species on Bacillus thuringiensis var Israelensis-induced mortality in mosquito larvae

Bacillus thuringiensis var israelensis (BTI), when ingested by mosquito larvae, is highly toxic to them. Many other aquatic invertebrates feed on bacteria but, in general, BTI is not toxic to them. We tested in the laboratory the hypothesis that certain crustaceans indirectly benefit mosquito larvae by reducing mortality caused by BTI. We presumed the mechanism to be ingestion of the bacteria by the crustaceans resulting in a lower concentration available to the mosquito larvae.Mortality of Aedes aegypti larvae exposed to BTI was reduced in the presence of the fairy shrimp, Branchipus schaefferi and the ostracod, Cypridopsis vidua (only during summer trials and not autumn trials for the latter species) but was not reduced in the presence of the ostracod, Heterocypris incongruens. By contrast, H. incongruens preyed upon infected, though still-moving, larvae. Feeding on the bacteria by the crustaceans may not be an important mechanism; our data indicates that the crustaceans did not reduce BTI in the water. Moreover, B. schaefferi, introduced into water and then removed prior to the introduction of BTI and mosquitoes, also reduced mosquito mortality.The mechanism for the protective effect of B. schaefferi and C. vidua is unknown. However, these results suggest that the abundance of certain organisms co-occurring with mosquito larvae may partially explain why the effective concentration of BTI varies among habitats.     

Cuticular and non-cuticular substrate influence on expression of cuticle-degrading enzymes from conidia of an entomopathogenic fungus,Nomuraea rileyi

Larval cuticle fromTrichoplusia ni, Helicoverpa (=Heliothis)zea, andHeliothis virescens and a cellulose substrate were used to quantify release of proteolytic, chitinolytic, and lipolytic enzymes by germinating conidia of the entomopathogenic fungus,Nomuraea rileyi. There was no significant difference in conidial viability incubated withT. ni, H. zea or cellulose substrates. Conidial viability onH. virescens cuticle, however, was significantly lower (ca. 19–25%) than the other three substrates. The presence of cuticle substrates, especially cuticle ofT. ni, stimulated germination. The nature of the substrate influenced both the time and quantity of the enzymes expressed. Specific proteases (aminopeptidase, chymoelastase, trypsin) generally were expressed earlier and/or in greater quantities on cuticular than on the cellulose substrate. Although both chitinolytic enzymes (endochitinase, N-acetylglucosaminidase) were detected on all three cuticular substrates, their activity was substantially lower than that of the proteolytic enzymes. Lipase activity was only minimally present. Early concurrent release of both proteases and chitinases suggested that both may be important in the penetration of the larval integument by germinating conidia ofN. rileyi. Expression of proteases and chitinases, especially aminopeptidase and endochitinase was probably a specific response to cuticle, because little or no activity was expressed on the non-host, cellulose substrate.This article reports the results of research only. Mention of a proprietary product in this paper does not constitute a recommendation for use by the US Department of Agriculture.     

Xylem-specific and tension stress-responsive coexpression of KORRIGAN endoglucanase and three secondary wall-associated cellulose synthase genes in aspen trees

In nature, angiosperm trees develop tension wood on the upper side of their leaning trunks and drooping branches. Development of tension wood is one of the straightening mechanisms by which trees counteract leaning or bending of stem and resume upward growth. Tension wood is characterized by the development of a highly crystalline cellulose-enriched gelatinous layer next to the lumen of the tension wood fibers. Thus experimental induction of tension wood provides a system to understand the process of cellulose biosynthesis in trees. Since KORRIGAN endoglucanases (KOR) appear to play an important role in cellulose biosynthesis in Arabidopsis, we cloned PtrKOR, a full-length KOR cDNA from aspen xylem. Using RT-PCR, in situ hybridization, and tissue-print assays, we show that PtrKOR gene expression is significantly elevated on the upper side of the bent aspen stem in response to tension stress while KOR expression is significantly suppressed on the opposite side experiencing compression stress. Moreover, three previously reported aspen cellulose synthase genes, namely, PtrCesA1, PtrCesA2, and PtrCesA3 that are closely associated with secondary cell wall development in the xylem cells exhibited similar tension stress-responsive behavior. Our results suggest that coexpression of these four proteins is important for the biosynthesis of highly crystalline cellulose typically present in tension wood fibers. Their simultaneous genetic manipulation may lead to industrially relevant improvement of cellulose in transgenic crops and trees.Suchita Bhandari and Takeshi Fujino contributed equally to this research.     

A genetically engineered mosquitocidal cyanobacterium

Larvae of the mosquitoAedes aegypti ingested, and developed into adults, on a diet of 1O of 14 different species of cyanobacteria includingAgmenellum quadruplicatum PR-6 (=Synechococcus PCC7002). Mosquito larvae ingested and grew on cells of PR-6 adapted to growth in the absence of NaCl. ThecryIVD gene ofBacillus thuringiensis var.israelensis was cloned into a PR-6 expression vector to form pAQRM56, which was transformed into PR-6. Expression of the CryIVD protein in PR-6 was demonstrated by immunocytochemistry and larvicidal activity. Immunogold labelling indicated production of an electron-dense material among the thylakoid membranes of PR-6. Cells of PR-6 carrying pAQRM56 were toxic to the larvae ofA. aeqypti whereas control cells were not. Growth of PR-6 cells carrying pAQRM56 was slower than the growth of control cells and these cells were also larger.     

Comparison of allozyme,RFLP, and RAPD markers for revealing genetic variation within and between trembling aspen and bigtooth aspen

We examined genetic variation in allozyme loci, nuclear DNA restriction fragment length polymorphisms (RFLPs), and random amplified polymorphic DNAs (RAPDs) in 130 trembling aspen (Populus tremuloides) and 105 bigtooth aspen (P. grandidentata) trees. In trembling aspen 10 out of 13 allozyme loci assayed (77%) were polymorphic (P), with 2.8 alleles per locus (A) and an expected heterozygosity (H_e) of 0.25. In contrast, bigtooth aspen had a much lower allozyme genetic variability (P=29%; A=1.4; H_e=0.08). The two species could be distinguished by mutually exclusive alleles at Idh-1, and bigtooth aspen has what appears to be a duplicate 6PG locus not present in trembling aspen. We used 138 random aspen genomic probes to reveal RFLPs in HindIII digests of aspen DNA. The majority of the probes were from sequences of low copy number. RFLP results were consistent with those of the allozyme analyses, with trembling aspen displaying higher genetic variation than bigtooth aspen (P=71%, A=2.7, and H_e=0.25 for trembling aspen; P=65%, A=1.8, and H_e=0.13 for bigtooth aspen). The two species could be distinguished by RFLPs revealed by 21 probes (15% of total probes assayed). RAPD patterns in both species were studied using four arbitrary decamer primers that revealed a total of 61 different amplified DNA fragments in trembling aspen and 56 in bigtooth aspen. Assuming a Hardy-Weinberg equilibrium, estimates of P=100%, A=2, and H_e=0.30 in trembling aspen and P=88%, A=1.9, and H_e=0.31 in bigtooth aspen were obtained from the RAPD data. Five amplified DNA fragments were species diagnostic. All individuals within both species, except for 2 that likely belong to the same clone, could be distinguished by comparing their RAPD patterns. These results indicate that (1) RFLPs and allozymes reveal comparable patterns of genetic variation in the two species, (2) trembling aspen is more genetically variable than bigtooth aspen at both the allozyme and DNA levels, (3) one can generate more polymorphic and species-specific loci with DNA markers than with allozymes in aspen, and (4) RAPDs provide a very powerful tool for fingerprinting aspen individuals.     

Chitinolytic enzymes from Streptomyces albidoflavus expressed in tomato plants: effects on Trichoplusia ni

Tomato (Lycopersicon esculentum ) cultivars were transformed with genes that encode bacterial chitinolytic enzymes (i.e., endochitinase and chitobiosidase) from Streptomyces albidoflavus. Transgenic tomato plants producing these enzymes were found to have enhanced resistance to cabbage looper, Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae), consistently reducing the growth rates of larvae. Mortality was significantly increased in two of three feeding trials. Ingestion of endochitinase and chitobiosidase not only affected development of larval T. ni from neonate to ultimate instar, but they also caused mortality and decreased insect weight when exposure began during the third instar. The results of this study provide some insight into the mode of action of the chitinolytic enzymes, by supporting the hypothesis that ingested chitinolytic enzymes damage the chitin component of the peritrophic envelope, leading to increased permeability. The size of marker molecules (FITC-dextrans) that permeated the peritrophic envelopes of T. ni feeding on transgenic plants were 50% larger than those permeating the peritrophic envelopes of T. ni feeding on the control plants. Further research is needed to more clearly identify the sites and modes of action of these chitinolytic enzymes, and the potential for synergy between these enzymes and pathogens, allelochemicals, and other environmental factors.     

Feeding habits of larval Mirogrex terraesanctae (Steinitz, 1952) in Lake Kinneret (Israel) I. Field study

Analysis of the food composition, distribution, lengths and dry weights of larval bleak, Mirogrex terraesanctae (Steinitz, 1952) in Lake Kinneret, Israel was conducted. Indices of electivity of ingested prey organisms were also determined. Larvae were surveyed throughout the 1989–90 reproductive seasons (Dec–Mar). Small larvae (<10.28 mm SL) preferentially select prey items smaller than 180 µ. Dominant zooplankters in the larval guts included: Bosmina longirostris, Ceriodaphnia reticulata and C. rigaudi, Asplanchna priodonita, Synchaeta oblonga, S. pectinata and juvenile cyclopoid copepods. Diaphanosoma brachiurum was rarely found (instars only) and no adult copepods were observed in larval guts. Some larvae specimens were found with up to 500 undigested (intact) accidentally ingested cells of Peridinium spp. per gut.     

Microfungi on the Pandanaceae:Polytretophora macrospora sp. nov.

Polytretophora macrospora is introduced based on specimens from two species ofPandanus in Seychelles. The new species is compared with currently accepted species. A key and a comparative synopsis toPolytetophora species are provided. Numerous specimens ofP. calcarata, collected on members of the Pandanaceae from Australia, Fiji, Hong Kong, Malaysia, New Caledonia, Palau, Philippines, Seychelles and Solomon Islands are also reported, along withP. dendroidea onPandanus sp. from Malaysia.     

Laboratory and field studies supporting the development of Heringia calcarata as a candidate biological control agent for Eriosoma lanigerum in New Zealand

Studies supporting a project seeking to introduce Heringia calcarata (Loew) (Diptera: Syrphidae) to New Zealand (NZ) to supplement biological control of woolly apple aphid (WAA), Eriosoma lanigerum (Hausmann) (Hemiptera: Aphididae) are reported. Annual surveys of H. calcarata presence and abundance in a Virginia, USA apple orchard revealed a bimodal distribution, with peaks in mid-June and mid-September. In the field, female H. calcarata oviposited on sentinel apple shoots infested with WAA, providing an efficient method for egg collection and larval production. Similarly, most field-collected females readily deposited viable eggs on WAA colonies in laboratory cages, demonstrating that mated females will oviposit in captivity. Survivorship of eggs and larvae transported to NZ was good, yielding adult flies in containment in Auckland. Adult, virgin female H. calcarata reared from eggs in captivity developed mature oocytes, providing an important step toward future mating studies in containment. Oviposition and larval feeding studies examined aspects of the intraguild interactions between H. calcarata and Aphelinus mali (Haldeman) (Hymenoptera: Aphelinidae), the sole biological control agent of WAA in NZ. Field tests using paired sentinel apple shoots with a non-parasitized or parasitized WAA colony revealed that although H. calcarata deposited eggs on both parasitized and non-parasitized colonies, fewest eggs were deposited on heavily parasitized colonies. Feeding studies showed that larval H. calcarata consumed fewer mummified aphids or aphids in an earlier stage of parasitization than non-parasitized aphids.     

Molecular cloning of cDNAs encoding a range of digestive enzymes from a phytophagous beetle, Phaedon cochleariae

To gain better knowledge of the variety of digestive enzymes in phytophagous coleopteran pests, a sequencing screen of 76 random cDNAs from a gut library from Phaedon cochleariae larvae was performed. The screen yielded 21 cDNAs encoding amino-acid sequences homologous to known digestive enzymes, most of them were cell wall-hydrolysing enzymes. The deduced protein sequences of 7 cDNAs encoding putative α-amylase, cysteine proteinase, trypsin, chymotrypsin, cellulase, pectinase and xylanase display all the structural features that characterize these enzymes in other eukaryotic organisms. Except the α-amylase and chymotrypsin cDNAs, the other cDNAs probably derive from multigene families. The distribution of the corresponding enzymatic activities at various developmental stages of P. cochleariae was examined. α-amylase activity is present in guts of larvae and adults, proteinases are abundant in guts of larvae and adults, but scarce in eggs and larval carcasses, xylanases are present in the guts of larvae and adults, as well as in carcasses of larvae, whereas cellulase and pectinase activities are distributed in larval and adult guts, larval carcasses, and eggs. Only a minor fraction of the cellulases is secreted by microorganisms, suggesting that P. cochleariae synthesizes most of its own cell-wall hydrolysing enzymes. The physiological role of the enzymes is discussed, as well as the significance of these results for pest management strategies involving transgenic plants expressing enzyme inhibitors.     

Host plant exploitation within a population of a generalist herbivore, Choristoneura rosaceana

Feeding performance of larvae of the obliquebanded leafroller, Choristoneura rosaceana (Harris), was estimated on apple and three alternative hosts in the Okanagan Valley. The obliquebanded leafroller is native to the study area and apple was introduced in the valley at the beginning of the century. Components of insect fitness were measured to help formulate predictions about the evolution of host preference in this generalist herbivore. In the summer, females attained a higher pupal weight on apple than on trembling aspen, wild rose and snowberry. Males performed best on apple and trembling aspen. In the field, females arising from caterpillars fed on apple leaves had a 26% greater mean fecundity than if fed on alternative hosts. The diet also affected development time; females initiated pupation later on trembling aspen than on the other hosts. Finally, no differences were found in larval survival nor in the proportion of larvae diapausing when fed on different hosts. These components of fitness indicate apple as the most suitable host in the summer.In the fall, diet influenced larval propensity to initiate diapause in the second or third instar, the time elapsed from hatching to diapause initiation and the weight of the instars initiating diapause. However, no difference in overwintering survival among larvae fed on different hosts was detected. The rank order of water and nitrogen contents of leaves of the host plants did not match the rank order of larval feeding performance in either season. A high suitability of apple and its abundance in the Okanagan valley are factors that would favor the evolution of an increased utilization of this host by the obliquebanded leafroller.