The transformation of Saperda calcarata (Coleoptera: Cerambycidae) into a cellulose digester through the inclusion of fungal enzymes in its diet

Summary The larvae of the aspen borer, Saperda calcarata, which feed on the inner bark and sapwood of living aspen stems, are unable to digest cellulose. However, they can be transformed into cellulose digesters by adding the active cellulase complex of the fungus, Penicillium funiculosum to their diet. S. calcarata larvae are preadapted to exploit the digestive potential of ingested microbial enzymes. We argue that ingested fungal enzymes may be responsible for cellulose digestion in many, perhaps most or even all, cellulose digesting cerambycid beetles.     

Variation in diapause and sex ratio in the parasitoidAsobara tabida

Asobara tabida Nees (Hymenoptera: Braconidae) is a widespread parasitoid, attacking larvae ofDrosophila (Diptera: Drosophilidae) species in fermenting substrates. In this species, geographic variation is found in the percentage of parasitoids entering diapause and in the sex ratio of emerging parasitoids. Percentage diapause appears to be influenced by host species (more parasitoids enter diapause inD. melanogaster Meigen than inD. subobscura Collin) and temperature. It is not correlated with any of the abiotic factors investigated, but is correlated with survival probability inD. melanogaster larvae and with the time of year in which the experiment was conducted (even though none of the parasitoids experienced natural day light). Sex ratio was only found to correlate with percentage diapause, suggesting that males enter diapause more frequently than females. It is concluded thatA. tabida uses diapause to survive both unfavourable abiotic and biotic circumstances.     

Mutations in tumour suppressor genes,l(2)gl andl(2)gd,alter the expression ofwingless inDrosophila

To understand the roles of two well known tumour suppressor genes.l(2)gl andl(2)gd in normal imaginal disc development inDrosophila, we have initiated a study to examine effect of mulations of these genes on the expression of genes involved in the patterning of the imaginal discs. In this study we show that the expression ofwingless, theDrosophila orthologue of the mammalian oncogeneWnt, is affected in the imaginal discs ofl(2)gl ⁴ andl(2)gd ¹ mutant individuals. In the tumourous wing imaginal discs froml(2)gl mutant larvae, the pattern ofwingless expression was progressively disrupted with an increase in the area of expression, Tumourous wing imaginal discs froml(2)gd homozygous individuals exhibited progressive broadening and extension of the wingless expressing domains. We suggest thatl(2)gl andl(2)gd might be involved in regulating post embryonic expression ofWingless.     

Effects of the cytolytic seed protein enterolobin on coleopteran and lepidopteran insect larvae

Enterolobin, a novel 55 KDa cytolytic and inflammatory protein fromEnterolobium contortisiliquum seeds, was tested for its toxic effects on larvae of the coleopteranCallosobruchus maculatus and the lepidopteranSpodoptera littoralis. Bioassays performed with enterolobin incorporated into artificial seeds showed that the phytocytolysin was toxic to larvae ofC. maculatus, causing 70% mortality at a concentration of 0.01% (w/w) and 100% mortality at 0.025%. The protein proved to be innocuous to larvae ofS. littoralis.In vitro proteolysis studies using larval gut enzymes, analysed on SDS-PAGE, showed that onlyS. littoralis proteases could digest enterolobin, suggesting that the insect's digestive proteases were able to inactivate the cytolysin before it could exert any toxic effect;C. maculatus proteases, on the other hand, were unable to hydrolyse enterolobin. The mechanism of toxicity of enterolobin did not appear to involve any damage to the microvilli of the epithelial gut cells ofC. maculatus as shown by electron microscopy. Some tentative hypotheses are considered in order to explain the toxic mechanism of action of enterolobin towardsC. maculatus.     

Host habitat finding and host selection of theDrosophila parasitoidLeptopilina australis (Hymenoptera,Eucoilidae), with a comparison of the niches of EuropeanLeptopilina species

Summary Decaying petioles of giant hogweed,Heracleum mantegazzianum Sommier & Levier, are used as a breeding site by six species ofDrosophila and the drosophilidScaptomyza pallida. The most numerous parasitoid species associated with this community isLeptopilina australis. BecauseL. australis was previously unknown in western Europe, we present the characters to distinguish it form its close relativeL. clavipes. Experiments on host species selection and survival ofL. australis showed that this parasitoid mainly usesD. limbata as host. Olfactometer experiments showed thatL. australis is attracted by the odour of decaying hogweed stalks, especially when these contain larvae ofD. limbata. L. australis is also strongly attracted by the odour of stinkhorns, a habitat in which it has never been found in nature.D. phalerata is the dominant fly species in stinkhorns, and is not a host ofL. australis. We offer a possible functional explanation for this unexpected habitat choice, by showing thatD. transversa andD. kuntzei, both species found to breed in fungi, are also suitable hosts forL. australis. We also discuss habitat choice with regard to a proposed phylogeny of theLeptopilina species in temperate Europe. Finally, we discuss niche overlap ofL. australis with the otherLeptopilina species.     

Gut fungi

Herbivores consume large quantities of cellulose and other plant cell wall (fibre) carbohydrates yet generally lack the enzymes to digest them. This has led to the evolution of specialized portions of the gut, such as the rumen and caecum, which contain large populations of digestive anaerobic microorganisms. Diverse bacteria and protists from this environment have been studied for over a hundred years but it is only recently that a significant population of highly specialized flagellate fungi have been identified. These fungi are important in fibre digestion. Their diversity, properties, activities, phylogeny and possible economic significance are the subjects of this review.     

Restriction enzyme digestion of heterochromatin inDrosophila nasuta

In situ digestion of metaphase and polytene chromosomes and of interphase nuclei in different cell types ofDrosophila nasuta with restriction enzymes revealed that enzymes like AluI, EcoRI, HaeIII, Sau3a and SinI did not affect Giemsa-stainability of heterochromatin while that of euchromatin was significantly reduced; TaqI and SalI digested both heterochromatin and euchromatin in mitotic chromosomes. Digestion of genomic DNA with AluI, EcoRI, HaeIII, Sau3a and KpnI left a 23 kb DNA band undigested in agarose gels while withTaqI, no such undigested band was seen. TheAluI resistant 23 kb DNA hybridized insitu specifically with the heterochromatic chromocentre. It appears that the digestibility of heterochromatin region in genome ofDrosophila nasuta with the tested restriction enzymes is dependent on the availability of their recognition sites.     

Mutations in somePolycomb group genes ofDrosophila interfere with regulation of segmentation genes

Mutations in severalPolycomb (Pc) group genes cause maternal-effect or zygotic segmentation defects, suggesting thatPc group genes may regulate the segmentation genes ofDrosophila. We show that individuals doubly heterozygous for mutations inpolyhomeotic and six otherPc group genes show gap, pair rule, and segment polarity segmentation defects. We examined double heterozygous combinations ofPc group and segmentation mutations for enhancement of adult and embryonic segmentation defects.Posterior sex combs andpolyhomeotic interact withKrüppel ² and enhance embryonic phenotypes ofhunchback andknirps, andpolyhomeotic enhanceseven-skipped. Surprisingly, flies carrying duplications ofextra sex combs (esc), that were heterozygous for mutations ofeven-skipped (eve), were extremely subvital. Embryos and surviving adults of this genotype showed strong segmentation defects in even-numbered segments. Antibody studies confirm that expression ofeve is suppressed by duplications ofesc. However,esc duplications have no effect on other gap or pair rule genes tested. To our knowledge, this is only the second triplo-abnormal phenotype associated withPc group genes. Duplications of nine otherPc group genes have no detectable effect oneve. Expression ofengrailed (en) was abnormal in the central nervous systems of mostPc group mutants. These results support a role forPc genes in regulation of some segmentation genes, and suggest thatesc may act differently from otherPc group genes.     

Comparisons of yeast florae from natural substrates and larval guts of southwestern Drosophila

Summary The yeast florae in the natural substrates of four desert and three non-desert Drosophila species were compared both qualitatively and quantatively to the yeast present in the guts of Drosophila larvae living in those substrates. The desert species breed in rotting cacti and the other Drosophila were found breeding in necrotic oranges. Larvae of one cactophilic species, D. mojavensis, and larvae of all of the species utilizing oranges (D. melanogaster, D. pseudoobscura, and D. arizonensis) were found to contain non-random samples of the yeasts available in their respective substrates. Larval preference behavior is most likely responsible for these differences. The other cactophilic Drosophila (D. nigrospiracula, D. mettleri, and D. pachea) did not exhibit significant differences when the yeast florae of their larvae and substrates were compared. Selective feeding by larvae appears to be related to the degree of polyphagy in that only larvae of polyphagous species are selective. Trade-off between generalism and specialism at two biological levels is discussed.     

Dissociation of photoreceptors from whole heads of the fruit fly,Drosophila melanogaster

Photoreceptor cells that were mostly free of extracellular material and suitable for most electrophysiological study procedures were dissociated from whole heads of the fruit fly, Drosophila melanogaster, by a simple smash technique employing gentle chopping by a razor blade through Parafilm sheets. A variety of commonly available proteolytic and glycolytic digestion enzymes were tested as additions to the basic dissociation procedure described. With the aid of Nomarski interference contrast optics, periodic acid-Schiff staining, and fluorescent labeling and microscopy methods, it was determined that proteolytic enzymatic digestion does little to enhance the dissociation procedure, and instead, often damages the cells that one is attempting to recover. Unexpectedly, certain glycolytic enzymes, when added to the basic procedure, appear to enhance the recovery of intact viable Drosophila photoreceptors that are stripped of most extracellular material. Based on these results, a hypothesis concerning the biochemical nature of the extracellular matrix of the Drosophila retina is proposed. Drosophila photoreceptors are an interesting model system for the study of invertebrate phototransduction and photoreceptor cell biology because of their many well-characterized mutant strains. The technique described here should produce clean viable photoreceptors or ommatidia that respond to light, and that are suitable for patch clamping or cell culture.     

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.     

Carbohydrate digestion in Lutzomyia longipalpis' larvae (Diptera - Psychodidae)

Lutzomyia longipalpis is the principal species of phlebotomine incriminated as vector of Leishmania infantum, the etiological agent of visceral leishmaniasis in the Americas. Despite its importance as vector, almost nothing related to the larval biology, especially about its digestive system has been published. The objective of the present study was to obtain an overview of carbohydrate digestion by the larvae. Taking in account that phlebotomine larvae live in the soil rich in decaying materials and microorganisms we searched principally for enzymes capable to hydrolyze carbohydrates present in this kind of substrate. The principal carbohydrases encountered in the midgut were partially characterized. One of them is a α-amylase present in the anterior midgut. It is probably involved with the digestion of glycogen, the reserve carbohydrate of fungi. Two other especially active enzymes were present in the posterior midgut, a membrane bound α-glucosidase and a membrane bound trehalase. The first, complete the digestion of glycogen and the other probably acts in the digestion of trehalose, a carbohydrate usually encountered in microorganisms undergoing hydric stress. In a screening done with the use of p-nitrophenyl-derived substrates other less active enzymes were also observed in the midgut. A general view of carbohydrate digestion in L. longipalpis was presented. Our results indicate that soil microorganisms appear to be the main source of nutrients for the larvae.     

Electrophoretic evidence for the origin ofDryopteris yakusilvicola (Dryopteridaceae)

To investigate the origin of the triploid agamosporous speciesD. yakusilvicola, an electrophoretic analysis was made for five enzymes of theD. sparsa complex.Dryopteris yakusilvicola showed a monomorphic banding pattern for the five enzymes and was heterozygous in all six gene loci coding them. Comparison of enzyme banding patterns suggests that the genome ofD. yakusilvicola was derived through hybridization betweenD. sabaei and either a sexual tetraploid or an agamosporous triploid ofD. sparsa. Cytological evidence (Darnaediet al., 1989) supports the idea that of the two types ofD. sparsa the sexual tetraploid is a parent. The monomorphic pattern implies thatD. yakusilvicola originated from a single hybrid between the parental species, and that it is a neo-endemic of Yakushima Island.     

Biosynthesis of methionine-derived glucosinolates in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>: recombinant expression and characterization of methylthioalkylmalate synthase,the condensing enzyme of the chain-elongation cycle

The major class of glucosinolates in Arabidopsis thaliana (L.) Heynh. are biosynthesized from methionine involving a three-step chain-elongation cycle. Each passage through the cycle results in the net addition of a single methylene group, with up to six cycles of elongation occurring in A. thaliana. The first reaction of the cycle is catalyzed by a methylthioalkylmalate synthase (MAMS), which condenses a -methylthio-2-oxoalkanoic acid with acetyl-CoA. Here we have demonstrated that MAM1, one of two similar genes in the A. thaliana ecotype Columbia, encodes a MAMS catalyzing the condensing reactions of the first two elongation cycles but not those of further cycles. The Columbia ecotype is dominated by compounds that have undergone only two elongation cycles. The A. thaliana MAM1 protein exhibits basic sequence similarity to other previously described enzymes catalyzing the condensation of 2-oxo acids and acetyl-CoA, such as isopropylmalate synthase (EC 2.3.3.13), an enzyme of leucine biosynthesis, and homocitrate synthase (EC 2.3.3.14). It also shares similar properties with them, including the catalytic requirements for a divalent metal ion and an adenine nucleotide. However, the MAM1 protein does not show activity with the substrates of any of these other enzymes, and was chromatographically separable from isopropylmalate synthase in extracts of A. thaliana. Thus, MAM1 is exclusively an enzyme of secondary metabolism, distinct from primary metabolic enzymes catalyzing similar reactions.Abbreviations IPMS Isopropylmalate synthase - MAM Methylthioalkylmalate - MAMS Methylthioalkylmalate synthase     

Genetic and epigenetic variation inDrosophila larvae suitability to a hymenopterous endoparasitoid

Suitability ofDrosophila melanogaster larvae to their endoparasiteLeptopilina boulardi (Nordlander, 1980) shows wide genetic and epigenetic variation. Experiments were done onDrosophila isofemale lines. When reared in optimal laboratory conditions only 50 % of parasitizedDrosophila larvae gave rise to an adult wasp, and significant variation was observed between lines. In crowded conditions, this average value increased up to 90 % and no more significant variability could be detected between lines. Genetic and demographic consequences of these results are discussed. Associé au C.N.R.S. n^o 243     

Differential characterization of two leucine aminopeptidases in Drosophila melanogaster

Two leucine aminopeptidases from Drosophila melanogaster larvae have been partially purified. The LAP A and D enzymes have similar biochemical characteristics including molecular weights of 280,000 daltons, Michaelis-Menten constants of 0.05 mM, associations with metal cofactors, and specificities toward natural and chromogenic substrates. They differ in their pH optima and spatial distributions. If the closely linked genes that code for these enzymes have resulted from a tandem gene duplication event, it is suggested that there has been subsequent evolutionary divergence. This would provide Drosophila larvae with two related, but functionally distinct enzymes.Virginia K. Walker was supported by an NRC Predoctoral Scholarship and a Killam Merit Scholarship.     

Drosophila acetylcholinesterase: Characterization of different mutants resistant to insecticides

Selection of field populations originating from several countries allowed us to isolate 13 strains ofDrosophila melanogaster resistant to parathion.In vitro studies of acetylcholinesterase inhibition by paraoxon have been carried out on purified enzymes: most of the resistant strains harbor an altered acetylcholinesterase. Enzymes with higher resistance levels have been characterized with respect to their cross-resistance toward several insecticides. The patterns obtained have permitted us to group them and to delineate four categories. The existence of four distinct types of protein suggests that several mutations of acetylcholinesterase are responsible for insecticide resistance inDrosophila.     

Cholera toxin and pertussis toxin substrates and endogenous ADP-ribosyltransferase activity in Drosophila melanogaster

Cholera toxin- and pertussis toxin-catalyzed ADP-ribosylation were used to identify and localize G protein substrates in Drosophila melanogaster and in Manduca sexta. Cholera toxin catalyzes ADP-ribosylation of 37 kDa and 50 kDa polypeptides, but these polypeptides are also substrates for an ADP-ribosyltransferase (EC 2.4.2.30) activity endogenous to the Drosophila extracts. Pertussis toxin modifies 37 kDa and 39 kDa polypeptides in Drosophila homogenates. The pattern of proteolysis of the 39 kDa pertussis toxin substrate is similar to that of mammalian G_o and is influenced by guanyl nucleotide binding. The 39 kDa G_o-like Drosophila and Manduca pertussis toxin substrates are found primarily in neural tissues. These studies provide further evidence that G proteins are present in Drosophila and that this organism can therefore be used to investigate the physiological roles of these enzymes using advanced genetic manipulations.     

The cytotaxonomy and origin ofRanunculus yaegatakensis,an endemic taxon of Yakushima Island

Ranunculus yaegatakensis, a close relative of the widespreadR. silerifolius, is endemic to the alpine region (ca. 1,600 m alt.) of Yakushima Island. This species differs morphologically fromRanunculus silerifolius mainly due to its dwarfism and leaf shape, but has an identical karyotype with one (Karatsu-type) of the four cytotype ofR. silerifolius. Ranunculus yaegatakensis is highly cross-compatible with the Karatsu-type plants ofR. silerifolius. Their F₁ hybrids show regular meiotic behaviors and set seeds successfully. In addition, the Karatsu-type ofRanunculus silerifolius is found in individuals that are collected not only from the lowland of Yakushima Island but from its neighboring islands. It is supposed to be more specialized than other cytotypes. This evidence suggests thatRanunculus yaegatakensis has been derived from the Karatsu-type plants ofR. silerifolius. We suggest thatRanunculus yaegatakensis be reduced to a variety ofR. silerifolius.     

Negative regulation ofUltrabithorax expression byengrailed is required for proper specification of wing development inDrosophila melanogaster

In both vertebrates and invertebrates, homeotic selector genes confer morphological differences along the antero-posterior axis. However, insect wing development is independent of all homeotic gene functions, reflecting the ground plan of an ancestral pterygote, which bore wings on all segments. Dipteran insects such asDrosophila are characterized by a pair of wings in the mesothoracic segment. In all other segments, wing development is essentially repressed by different homeotic genes, although in the metathorax they are modified into a pair of halteres. This necessitates that during development all homeotic genes are to be maintained in a repressed state in wing imaginal discs. In this report we show that (i) the function of the segment polarity geneengrailed (en) is critical to keep the homeotic selector geneUltrabithorax (Ubx) repressed in wing imaginal discs, (ii) normal levels of En in the posterior compartment of haltere discs, however, are not enough to completely repressUbx, and (iii) the repression ofUbx byen is independent of Hedgehog signalling through which the long-range signalling ofen is mediated during wing development. Finally we provide evidence for a possible mechanism by whichen repressesUbx. On the basis of these results we propose thaten has acquired two independent functions during the evolution of dorsal appendages. In addition to its well-known function of conferring posterior fate and inducing long-range signalling to pattern the developing appendages, it maintains wing fate by keepingUbx repressed.