Identification of an aromatic aldehyde synthase involved in indole-3-acetic acid biosynthesis in the galling sawfly (Pontania sp.) and screening of an inhibitor.

Indole-3-acetic acid (IAA), a phytohormone auxin, may be involved in insect gall induction. We previously proposed that the IAA biosynthetic pathway is Trp → indole-3-acetaldoxime → indole-3-acetaldehyde (IAAld) → IAA or Trp → IAAld → IAA. In this study, we surveyed galling sawfly enzymes responsible for the rate-limiting steps using a heterologous protein expression system and identified PonAAS2, an aromatic aldehyde synthase, that catalyzed the conversion of Trp to IAAld. The PonAAS2 gene was highly expressed in early- and mid-stage larvae that contained high concentrations of IAA, but the expression level was almost negligible in larvae that had escaped from their gall in autumn and contained very low concentrations of IAA. An inhibitor of PonAAS2, obtained by screening a chemical library, inhibited IAA production in sawfly enzyme solution by 80%, suggesting the important role of this enzyme in IAA biosynthesis in sawfly.

Cloning and characterization of a novel DNase gene from Trichogramma pretiosum.

DNase is a powerful tool for a series of molecular biology applications. Developing a strategy for large-scale production of DNase with high purity and activity is critical for scientific research. In this study, a previously uncharacterized gene with nuclease activity was found in Trichogramma pretiosum genome. Pichia pastoris GS115 was preferred as the host to overcome the issues related to prokaryotic expression. Under the optimal conditions, the activity of T. pretiosum DNase (Tp-DNase) reached 1940 U/mL of culture supernatant in fed-batch fermentation. Using ion-exchange chromatography and adsorption chromatography, Tp-DNase was produced with a purity of >99% and molecular weight of 45 kDa. In vitro DNA degradation experiments showed that Tp-DNase could effectively degrade dsDNA, and its activity was slightly higher than that of bovine pancreas DNase I under the same conditions. Moreover, Tp-DNase can be used to eliminate nucleic acid contamination and improve the accuracy of nucleic acid detection.

Insights into the biotransformation of 2,4,6-trinitrotoluene by the old yellow enzyme family of flavoproteins. A computational study.

Biodegradation is a cost-effective and environmentally friendly alternative to removing 2,4,6-trinitrotoluene (TNT) pollution. However, mechanisms of TNT biodegradation have been elusive. To enhance the understanding of TNT biotransformation by the Old Yellow Enzyme (OYE) family, we investigated the crucial first-step hydrogen-transfer reaction by molecular dynamics simulations, docking technologies and empirical valence bond calculations. We revealed the significance of the π-π stacking conformation between the substrate TNT and the reduced flavin mononucleotide (FMNH2) cofactor, which is a prerequisite for the aromatic ring reduction of TNT. Under the π-π stacking conformation, the barrier of the hydrogen-transfer reaction in the aromatic ring reduction is about 16 kcal mol-1 lower than that of nitro group reduction. Then, we confirmed the mechanism of controlling the π-π stacking, that is, the π-π interaction competition mechanism. It indicates that the π-π stacking of TNT and FMNH2 occurs only when the π-π interaction between FMNH2 and TNT is stronger than that between TNT and several key residues with aromatic rings. Finally, based on the competition mechanism, the formation of π-π stacking of TNT and FMNH2 can be successfully enabled by removing the aromatic ring of those key residues in enzymes that originally only transform TNT through the nitro group reduction. This testified the validity of the π-π interaction competition mechanism. This work theoretically clarifies the molecular mechanism of the first-step hydrogen-transfer reaction for the biotransformation of TNT by the OYE family. It is helpful to obtain the enzymes that can biodegrade TNT through the aromatic ring reduction.

Expansion of the fatty acyl reductase gene family shaped pheromone communication in Hymenoptera.

Fatty acyl reductases (FARs) are involved in the biosynthesis of fatty alcohols that serve a range of biological roles. Insects typically harbor numerous FAR gene family members. While some FARs are involved in pheromone biosynthesis, the biological significance of the large number of FARs in insect genomes remains unclear.Using bumble bee (Bombini) FAR expression analysis and functional characterization, hymenopteran FAR gene tree reconstruction, and inspection of transposable elements (TEs) in the genomic environment of FARs, we uncovered a massive expansion of the FAR gene family in Hymenoptera, presumably facilitated by TEs. The expansion occurred in the common ancestor of bumble bees and stingless bees (Meliponini). We found that bumble bee FARs from the expanded FAR-A ortholog group contribute to the species-specific pheromone composition. Our results indicate that expansion and functional diversification of the FAR gene family played a key role in the evolution of pheromone communication in Hymenoptera.

Insect venom phospholipases A1 and A2: Roles in the envenoming process and allergy.

Insect venom phospholipases have been identified in nearly all clinically relevant social Hymenoptera, including bees, wasps and ants. Among other biological roles, during the envenoming process these enzymes cause the disruption of cellular membranes and induce hypersensitive reactions, including life threatening anaphylaxis. While phospholipase A2 (PLA2) is a predominant component of bee venoms, phospholipase A1 (PLA1) is highly abundant in wasps and ants. The pronounced prevalence of IgE-mediated reactivity to these allergens in sensitized patients emphasizes their important role as major elicitors of Hymenoptera venom allergy (HVA). PLA1 and -A2 represent valuable marker allergens for differentiation of genuine sensitizations to bee and/or wasp venoms from cross-reactivity. Moreover, in massive attacks, insect venom phospholipases often cause several pathologies that can lead to fatalities. This review summarizes the available data related to structure, model of enzymatic activity and pathophysiological roles during envenoming process of insect venom phospholipases A1 and -A2.

Evolution of the insect desaturase gene family with an emphasis on social Hymenoptera.

Desaturase genes are essential for biological processes, including lipid metabolism, cell signaling, and membrane fluidity regulation. Insect desaturases are particularly interesting for their role in chemical communication, and potential contribution to speciation, symbioses, and sociality. Here, we describe the acyl-CoA desaturase gene families of 15 insects, with a focus on social Hymenoptera. Phylogenetic reconstruction revealed that the insect desaturases represent an ancient gene family characterized by eight subfamilies that differ strongly in their degree of conservation and frequency of gene gain and loss. Analyses of genomic organization showed that five of these subfamilies are represented in a highly microsyntenic region conserved across holometabolous insect taxa, indicating an ancestral expansion during early insect evolution. In three subfamilies, ants exhibit particularly large expansions of genes. Despite these expansions, however, selection analyses showed that desaturase genes in all insect lineages are predominantly undergoing strong purifying selection. Finally, for three expanded subfamilies, we show that ants exhibit variation in gene expression between species, and more importantly, between sexes and castes within species. This suggests functional differentiation of these genes and a role in the regulation of reproductive division of labor in ants. The dynamic pattern of gene gain and loss of acyl-CoA desaturases in ants may reflect changes in response to ecological diversification and an increased demand for chemical signal variability. This may provide an example of how gene family expansions can contribute to lineage-specific adaptations through structural and regulatory changes acting in concert to produce new adaptive phenotypes.

Evolutionary history of eukaryotic α-glucosidases from the α-amylase family.

Although some α-glucosidases from the α-amylase family (glycoside hydrolase family GH13) have been studied extensively, their exact number, organization on the chromosome, and orthology/paralogy relationship were unknown. This was true even for important disease vectors where gut α-glucosidase is known to be receptor for the Bin toxin used to control the population of some mosquito species. In some cases orthologs from related species were studied intensively, while potentially important paralogs were omitted. We have, therefore, used a bioinformatics approach to identify all family GH13 α-glucosidases from the selected species from Metazoa (including three mosquito species: Aedes aegypti, Anopheles gambiae, and Culex quinquefasciatus) as well as from Fungi in an effort to characterize their arrangement on the chromosome and evolutionary relationships among orthologs and among paralogs. We also searched for pseudogenes and genes coding for enzymatically inactive proteins with a possible new function. We have found GH13 α-glucosidases mostly in Arthropoda and Fungi where they form gene families, as a result of multiple lineage-specific gene duplications. In mosquito species we have identified 14 α-glucosidase (Aglu) genes of which only five have been biochemically characterized so far, two are putative pseudogenes and the rest remains uncharacterized. We also revealed quite a complex evolutionary history of the eukaryotic α-glucosidases probably involving multiple losses of genes or horizontal gene transfer from bacteria.

Inhibition of melanization by a Nasonia defensin-like peptide: implications for host immune suppression.

The parasitic wasp Nasonia vitripennis suppresses host immune mechanisms that include melanization reactions. Melanization is an important immune response of hosts induced by wasp infection and thus its inhibition represents a successful strategy for parasitism. However, the molecular basis associated with such inhibition is largely unknown in N. vitripennis. Here, we report recombinant expression, structural and functional characterization of a Nasonia-derived defensin-like peptide (called nasonin-3) whose recombinant product exerts inhibitory effect on host melanization. The possible role of nasonin-3 in immune suppression is also discussed.

Insect-specific irreversible inhibitors of acetylcholinesterase in pests including the bed bug, the eastern yellowjacket, German and American cockroaches, and the confused flour beetle.

Insecticides directed against acetylcholinesterase (AChE) are facing increased resistance among target species as well as increasing concerns for human toxicity. The result has been a resurgence of disease vectors, insects destructive to agriculture, and residential pests. We previously reported a free cysteine (Cys) residue at the entrance to the AChE active site in some insects but not higher vertebrates. We also reported Cys-targeting methanethiosulfonate molecules (AMTSn), which, under conditions that spared human AChE, caused total irreversible inhibition of aphid AChE, 95% inhibition of AChE from the malaria vector mosquito (Anopheles gambia), and >80% inhibition of activity from the yellow fever mosquito (Aedes aegypti) and northern house mosquito (Culex pipiens). We now find the same compounds inhibit AChE from cockroaches (Blattella germanica and Periplaneta americana), the flour beetle (Tribolium confusum), the multi-colored Asian ladybird beetle (Harmonia axyridis), the bed bug (Cimex lectularius), and a wasp (Vespula maculifrons), with IC(50) values of approximately 1-11muM. Our results support further study of Cys-targeting inhibitors as conceptually novel insecticides that may be free of resistance in a range of insect pests and disease vectors and, compared with current compounds, should demonstrate much lower toxicity to mammals, birds, and fish.

Edge effects on the orchid-bee fauna (Hymenoptera: Apidae) at a large remnant of Atlantic Rain Forest in southeastern Brazil

Male orchid bees were sampled with chemical baits monthly from July 1999 to April 2000 at six sites situated at different distances from the forest edge (0 to 4,000 m) at Parque Estadual do Rio Doce, the largest remnant of Atlantic Forest in the state of Minas Gerais, southeastern Brazil. The main goal of this study was to assess the abundance, richness, and the community structure of the orchid-bee fauna at different distances from the edge. In all, 1,183 males from 20 species were collected. Only minor and insignificant variation in richness and abundance were observed among the sites. Two dominant species, Euglossa analis Westwood (Hymenoptera: Apidae) and Eulaema cingulata (Fabricius) (Hymenoptera: Apidae), showed strongly opposing habitat associations: while males of the former were most collected in the interior of the forest, the latter was most found at or close to the edge. The responses of individual species, such as these, and not composite measures of richness and abundance, are more informative regarding forest integrity and edge effects.

Machos de abelhas euglossinas foram atraídos mensalmente com o auxílio de iscas aromáticas e coletados no período de julho de 1999 a abril de 2000 em seis pontos amostrais situados a diferentes distâncias da borda da mata (0 a 4.000 m) no Parque Estadual do Rio Doce, o maior remanescente florestal de Mata Atlântica de Minas Gerais, sudeste do Brasil. O objetivo deste estudo foi avaliar possíveis diferenças na abundância, riqueza e estrutura das comunidades de euglossinas a diferentes distâncias da borda. No total, foram coletados 1.183 machos de 20 espécies. Apenas variações pequenas e insignificantes na riqueza e abundância foram observadas entre os pontos amostrais. Duas espécies dominantes, Euglossa analis Westwood e Eulaema cingulata (Fabricius), apresentaram distribuições contrastantes com relação ao hábitat: enquanto os machos da primeira espécie foram coletados em maior número no interior da mata, machos de E. cingulata foram mais comuns na borda ou próximo a esta. As respostas de espécies como essas, e não índices de riqueza e abundância, são mais informativas com relação à integridade da floresta e efeitos de borda.

Ocorrência e características biológicas de forídeos parasitóides (Diptera: Phoridae) da saúva Atta laevigata (Smith) (Hymenoptera: Formicidae) em Porto Nacional, TO / Occurrence and biological characteristics of parasitoid phorids (Diptera: Phoridae) of the leaf-cutting ant Atta laevigata (Smith) (Hymenoptera: Formicidae) in Porto Nacional, TO, Brazil

Este estudo foi conduzido para verificar a ocorrência de forídeos parasitóides da saúva Atta laevigata (Smith) na região de Porto Nacional, TO, e para avaliar algumas características biológicas dessas moscas. Para tanto, entre outubro/2002 e setembro/2003, em intervalos de aproximadamente 15 dias, foram realizadas 22 coletas, cada uma contendo em média 149,5 ± 82,9 operárias, totalizando 3.704 formigas. No laboratório, as formigas eram distribuídas em grupos de cinco indivíduos por placa de Petri e mantidas a 25 ± 1°C e 80 ± 5 por cento UR, sendo alimentadas diariamente, até sua morte, com solução de mel a 50 por cento. Foram coletadas 106 operárias (2,8 por cento), com ou sem carga (fragmento vegetal), parasitadas por forídeos: 72 operárias (68 por cento) estavam parasitadas por Apocephalus vicosae Disney; 28 (26,4 por cento) por Neodohrniphora erthali Brown; e seis (5,6 por cento) por Apocephalus attophilus Borgmeier. As maiores taxas de parasitismo por A. vicosae (3,9-5,4 por cento) foram registradas nos meses secos de junho a agosto, enquanto que o parasitismo por N. erthali manteve-se abaixo de 2,3 por cento ao longo do ano. As operárias maiores estavam parasitadas pela espécie de maior tamanho N. erthali ou pela espécie que produz o maior número de larvas A. attophilus. O registro das três espécies de forídeos no Cerrado do Tocantins amplia a distribuição desses parasitóides no Brasil. Ao mesmo tempo, o desenvolvimento da larva de A. vicosae no tórax do hospedeiro e a ruptura entre o pronoto e a propleura da formiga para a emergência desta mosca são características inéditas do parasitismo de saúvas por forídeos.

The purpose of this study was to verify the occurrence of parasitism in Atta laevigata (Smith) by phorids in Porto Nacional, Tocantins, Brazil and to evaluate some biological characteristics of those flies. From October/2002 to September/2003, at 15-day intervals, 22 samplings were carried out (149.5 ± 82.9 workers each) comprising 3,704 ants. In the laboratory, the ants were distributed in groups of five individuals per petri dish and maintained at temperature of 25 ± 1°C and 80 ± 5 percent of RH. They were fed daily with a 50 percent honey solution until death. The total of 106 workers (2.8 percent), collected with or without load (plant fragment), were parasitized by phorids: 72 workers (68 percent) were parasitized by Apocephalus vicosae Disney, 28 individuals (26.4 percent) by Neodohrniphora erthali Brown, and six (5.6 percent) by Apocephalus attophilus Borgmeier. The higher parasitism rates by A. vicosae (3.9-5.4 percent) were observed during the dry season (June-August), while the parasitism by N. erthali was lower than 2.3 percent throughout the year. The bigger workers were parasitized by bigger parasitoid N. erthali or by parasitoid with larger number of larvae A. attophilus. The occurrence of three phorid species in Tocantins broadens the distribution of these flies in Brazil. The development of A. vicosae larva within the thorax of the host as well as the rupture between the ant pronotum and propleuron so that the emergence of the fly can take place are characteristics not yet described on parasitism of leaf-cutting ants by phorids.

The N-glycans of yellow jacket venom hyaluronidases and the protein sequence of its major isoform in Vespula vulgaris.

Hyaluronidase (E.C. 3.2.1.35), one of the three major allergens of yellow jacket venom, is a glycoprotein of 45 kDa that is largely responsible for the cross-reactivity of wasp and bee venoms with sera of allergic patients. The asparagine-linked carbohydrate often appears to constitute the common IgE-binding determinant. Using a combination of MALDI MS and HPLC of 2-aminopyridine-labelled glycans, we found core-difucosylated paucimannosidic glycans to be the major species in the 43-45 kDa band of Vespula vulgaris and also in the corresponding bands of venoms from five other wasp species (V. germanica, V. maculifrons, V. pensylvanica, V. flavopilosa and V. squamosa). Concomitant peptide mapping of the V. vulgaris 43 kDa band identified the known hyaluronidase, Ves v 2 (SwissProt P49370), but only as a minor component. De novo sequencing by tandem MS revealed the predominating peptides to resemble a different, yet homologous, sequence. cDNA cloning retrieved a sequence with 58 and 59% homology to the previously known isoform and to the Dolichovespula maculata and Polistes annularis hyaluronidases. Close homologues of this new, putative hyaluronidase b (Ves v 2b) were also the major isoform in the other wasp venoms.

Foraging patterns in a nocturnal swarm-founding wasp, Apoica flavissima van der Vecht (Hymenoptera: Vespidae)

Foraging behaviour has a remarkable significance to colonies of social insects since it is directly related to the supply of food, water, and pulp. Social wasps of the genus Apoica Lepeletier are primarily nocturnal, but studies concerning the general aspects related to this habit are still scarce. This study showed that the rates of foraging flights were higher during the full moon and last quarter moon phases. Frequencies of arrival and departure were correlated with time and temperature at night. The results suggest that the moonlight is an important component to general activities in this genus.

O comportamento de forragear é fundamental para o desenvolvimento das colônias de insetos sociais, pois permite que as colônias sejam supridas de alimento, água e materiais utilizados para a construção dos ninhos. Vespas sociais do gênero Apoica Lepeletier (Hymenoptera: Vespidae) têm comportamento de forrageamento noturno como uma de suas principais características, porém estudos sobre aspectos abióticos que influenciam esse comportamento são ainda escassos. Este estudo mostra que a freqüência de forrageamento foi maior em noites de luas cheia e crescente. Além disso, as saídas e retornos ao ninho correlacionaram-se positivamente com o período da noite e a temperatura. Os resultados sugerem que a luminosidade é um componente importante nas atividades gerais das colônias do gênero.

Ocorrência, morfologia e ultra-estrutura da glândula de Dufour de Scaptotrigona postica Latreille (Hymenoptera: Apidae) / Dufour gland occurence, morphology and ultrastructure in Scaptogrigona postica Latreille (Hymenoptera: Apidae)

A glândula de Dufour é uma glândula acessória do aparelho reprodutivo feminino das abelhas. Nas abelhas neotropicais sem ferrão, tem sido pouca estudada sob todos os aspectos: morfológico, ontogenético e bioquímico. Na tentativa de colaborar com o conhecimento dessa glândula em abelhas sem ferrão, foi realizado um estudo da sua ocorrência, morfologia e desenvolvimento em Scaptotrigona postica Latreille. Os resultados mostraram que ela se encontra ausente nas operárias, como ocorre em muitas outras espécies desse grupo. Nas rainhas, as células glandulares parecem mais ativas nas virgens, possuindo uma desenvolvida rede de retículo endoplasmático liso tubular, grânulos de secreção e polirribossomos dispersos no citoplasma, além de apresentarem núcleos maiores do que os das células glandulares das fisogástricas. Nas rainhas fisogástricas há dois tipos de células glandulares, ambas aparentemente inativas sinteticamente. As glândulas das rainhas fisogástricas são claramente capazes de captar substâncias da hemolinfa, provavelmente lipídios, que não penetram nas células, mas passam pelos espaços intercelulares e, através da cutícula, chegam diretamente à luz da glândula. A bem desenvolvida dupla camada de lâmina basal ao redor da glândula pode atuar no processo de captação de substâncias da hemolinfa. A secreção, e conseqüentemente sua função, pode ser diferente nas duas classes de rainhas.

The Dufour gland is an accessory gland of the bee reproductive female apparatus. In neotropical stingless bees, this gland has been poorly investigated, concerning to developmental, morphological or biochemical characteristics. In order to collaborate with the knowledge of the Dufour gland in stingless bees, a study of its occurrence, morphology and development was performed in Scapatotrigona postica Latreille. The results showed that this gland is absent in workers, although it occurs in workers of many stingless bee species. The glandular cells seem to be very active in virgin queens, presenting a well developed tubular network of smooth endoplasmic reticulum, secretion granules and some polyribosomes in the glandular cell cytoplasm, besides larger nuclei than in physogastric queens. In physogastric queen glands there are two types of cells, both of them apparently not synthetically active. Nevertheless, the physogastric queen glands are clearly able to absorb substances from the haemolymph, probably lipids that do not cross the cells, but pass through the intercellular cell spaces and cuticle, and are released directly to the gland lumen. The very developed double-layered gland basal lamina may actuate in the process of substances uptaking. The secretion, and consequently its function, can be different in these two queen types.

Seasonal changes of methamidophos susceptibility and biochemical properties in Plutella xylostella (Lepidoptera: Yponomeutidae) and its parasitoid Cotesia plutellae (Hymenoptera: Braconidae).

Methamidophos resistance and acetylcholinesterase (AChE) insensitivity to methamidophos, dichlorvos, and carbofuran were determined in the field populations of Plutella xylostella (L.) (Lepidoptera: Yponomeutidae) and its parasitoid Cotesia plutellae Kurdjumov (Hymenoptera: Braconidae) collected from the corresponding hosts between October 1998 and December 2003 in Fuzhou and Minhou, Fijian, China. Resistance levels to methamidophos and AChE insensitivity to the three insecticides in the two species of insects were high during autumn and spring and low during summer. Resistance to methamidophos was 15.3- and 12.6-fold higher in resistant F0 parents of P. xylostella and C. plutellae than in their susceptible F11 progeny, respectively. The bimolecular rate constant (k(i)) values of AChE to methamidophos, dichlorvos, and carbofuran were 4.6-, 6.3-, and 7.7-fold higher in F11 progeny of P. xylostella, and 3.7-, 4.5-, and 3.7-fold higher in F11 progeny of C. plutellae than those in their F0 parents, respectively. Compared with susceptible F11 progeny, the resistance ratios for methamidophos were 4.2-29.8 and 3.8-13.1 in 21 field populations of P. xylostella and C. plutellae, respectively. The k(i) values of AChE to methamidophos, dichlorvos, and carbofuran were 2.0-21.6-, 3.6-9.5-, and 2.6-9.2-fold higher in F11 progeny of P. xylostella, and 1.8-7.6-, 1.9-4.6-, and 2.2-7.6-fold higher in F11 progeny of C. plutellae than those in 21 field populations, respectively. Significant correlative variations of methamidophos resistance as well as significant correlative variations of k(i) values of AChE to insecticides between the two species of insects also were found in space and time. The k(i) values of AChE to insecticides in C. plutellae were far higher than those in P. xylostella. There were no obvious differences in the Km and Vmax of AChE between F0 parents and F11 progeny of P. xylostella and C. plutellae, respectively. But carboxylesterase activity was 1.6-fold higher in F0 parents of C. plutellae than in F11 progeny, and glutathione S-transferase activity was 1.5-fold higher in F0 parents of P. xylostella than in F11 progeny. The results suggested that the AChE insensitivity to insecticides might play the most important role in methamidophos resistance in the two species of insects. From these results, a spatial and temporal correlative evolution of methamidophos resistance and insensitive AChE was found to exist between P. xylostella and C. plutellae.

Differential gene expression during wing morph differentiation of the ectoparasitoid Melittobia digitata (Hym., Eulophidae).

Melittobia digitata is an ectoparasitoid of solitary bees and wasps that displays a trade-off between reproduction and dispersion through the development of two wing morphs (long and short wing morphs (LWM and SWM)). The morph differentiation of this species is an exceptional adaptation to maximize host exploitation and habitat colonization, and an understanding of the mechanisms underlying this developmental process will shed light on how nutrients or environmental elicitors alter regulatory pathways leading to physiological and metabolic changes resulting in such drastic developmental rearrangements. Here we describe the differential gene expression between SWM and LWM larvae of M. digitata in order to unravel the molecular mechanisms controlling the morph differentiation in this minute parasitoid and pinpoint the pathways involved in the regulation of this developmental process. The suppression subtractive hybridization (SSH) methodology was used to isolate differentially expressed genes using mRNA populations collected soon after morph development commitment. Dot blot analysis of 384 clones from a forward SSH library identified approximately 200 differentially expressed clones, including those transcripts present in very low abundance. Further DNA sequence analysis of a sub-population of 42 clones revealed 31 putatively unique transcripts, from which 5 were further analyzed by Northern blot analysis and semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). The complete cDNA of one of these transcripts, a putative metalloprotease, was fully sequenced and is described. The role of the putative differentially expressed genes during the wing morph differentiation of M. digitata is discussed.

Allozyme markers to help define the South American origins of Microctonus hyperodae (Hymenoptera: Braconidae) established in New Zealand for biological control of Argentine stem weevil.

The thelytokous parasitoid, Microctonus hyperodae Loan, was collected from eight South American locations and introduced to New Zealand in 1991 for biological control of Argentine stem weevil, Listronotus bonariensis (Kuschel) (Coleoptera: Curculionidae). Parasitoids from each population were released in equal numbers at each New Zealand site to give them the same opportunities to establish. Population markers have been sought to identify the South American geographic populations that have become most successful in New Zealand. These would assist in determining the importance of concepts such as climate matching and host-parasitoid coevolution to the establishment of natural enemies in new regions for biological control. Vertical polyacrylamide electrophoresis was used to survey 16 enzymes and ten calcium binding proteins, and this paper reports variation at three putative loci. Malate dehydrogenase, a dihydrolipoamide dehydrogenase isozyme and a calcium binding protein exhibited clear genetic variation, each with two alleles. All M. hyperodae isofemale lines from east of the Andes mountains shared one genotype, all but one from west of the Andes shared another, while a population from within the Andes contained both genotypes. This variation was highly congruent with previously described morphometric variation. At two loci, the maintenance of heterozygotes, and the absence of homozygotes, within isofemale lines suggested M. hyperodae thelytoky is apomictic.

Antibacterial and proteolytic activity in venom from the endoparasitic wasp Pimpla hypochondriaca (Hymenoptera: Ichneumonidae).

Venom from the endoparasitic wasp, Pimpla hypochondriaca, is composed of a mixture of high and low molecular weight proteins, possesses phenoloxidase activity, has immunosuppressive properties, and induces paralysis in several insect species. In the present study we demonstrate that P. hypochondriaca venom also contains antibacterial and proteolytic activity. Antibacterial activity was detected against the Gram-negative bacteria Escherichia coli and Xanthamonas campestris but not against Pseudomonas syringae nor against two Gram-positive bacteria, Bacillus cereus and Bacillus subtilis. Endopeptidase and aminopeptidase activity in venom was detected using the synthetic fluorogenic substrates N-t-BOC-Phe-Ser-Arg-AMC, Arg-AMC and Leu-Arg. The aminopeptidase activity towards Arg-AMC was sensitive to amastatin (70% inhibition), an aminopeptidase inhibitor. Angiotensin-converting enzyme (ACE)-like enzyme activity was detected, by reverse-phase HPLC using the synthetic tripeptide Hip-His-Leu as a substrate. This activity was sensitive to captopril, an ACE inhibitor (IC(50) 3.8 x 10(-8) M). Using an antiserum raised against recombinant Drosophila melanogaster ACE-like enzyme, (rAnce), Western blot analysis revealed an immunoreactive protein, with a molecular weight estimate of 74 kDa, in P. hypochondriaca venom. The possibility that the endopeptidase, aminopeptidase and ACE are involved in the processing of peptide precursors in the venom sac is discussed.

Possible function of two insect phospholipid-hydroperoxide glutathione peroxidases.

We compared the functional properties of two insect members of the phospholipid hydroperoxide glutathione peroxidases (PHGPx) family, VLP1, a major component of virus-like particles from the hymenopteran endoparasitoid Venturia canescens and its closest Drosophila relative, one of the putative PHGPx-proteins predicted from the Berkeley Drosophila genome sequence project. Recombinant Drosophila PHGPx shows enzymatic activity towards a number of PHGPx substrates, while the recombinant PHGPx-like domain of VLP1 lacks a functionally relevant cysteine and enzyme activity. A possible function of a non-enzymatic extracellular PHGPx-like protein is discussed.

Molecular evolutionary analyses of mariners and other transposable elements in fire ants (Hymenoptera: Formicidae).

Screens of a library of genomic DNA made during a recent study of the fire ant Solenopsis invicta revealed the presence of three distinct types of transposable elements (TEs). Two of the recovered sequences showed a high similarity to long-terminal repeat (LTR) retrotransposons, while the third showed a high homology to mariner elements. To investigate the distribution and relationships of mariners in related ants, we PCR-amplified these elements from additional Solenopsis species. Phylogenetic analyses showed that they form a single group within the mauritiana subfamily that is part of a larger clade derived from hymenopteran species. We also present partial sequence data for the two LTR-retrotransposons and describe their phylogenetic affinities.