Repercussion of a deficiency in mitochondrial ß-oxidation on the carbon flux of short-chain fatty acids to the peroxisomal ß-oxidation cycle in Aspergillus nidulans.

The fungus Aspergillus nidulans contains both a mitochondrial and peroxisomal ß-oxidation pathway. This work was aimed at studying the influence of mutations in the foxA gene, encoding a peroxisomal multifunctional protein, or in the scdA/echA genes, encoding a mitochondrial short-chain dehydrogenase and an enoyl-CoA hydratase, respectively, on the carbon flux to the peroxisomal ß-oxidation pathway. A. nidulans transformed with a peroxisomal polyhydroxyalkanoate (PHA) synthase produced PHA from the polymerization of 3-hydroxyacyl-CoA intermediates derived from the peroxisomal ß-oxidation of external fatty acids. PHA produced from erucic acid or heptadecanoic acid contained a broad spectrum of monomers, ranging from 5 to 14 carbons, revealing that the peroxisomal ß-oxidation cycle can handle both long and short-chain intermediates. While the ∆foxA mutant grown on erucic acid or oleic acid synthesized 10-fold less PHA compared to wild type, the same mutant grown on octanoic acid or heptanoic acid produced 3- to 6-fold more PHA. Thus, while FoxA has an important contribution to the degradation of long-chain fatty acids, the flux of short-chain fatty acids to peroxisomal ß-oxidation is actually enhanced in its absence. While no change in PHA was observed in the ∆scdA∆echA mutant grown on erucic acid or oleic acid compared to wild type, there was a 2- to 4-fold increased synthesis of PHA in ∆scdA∆echA cells grown in octanoic acid or heptanoic acid. These results reveal that a compensatory mechanism exists in A. nidulans that increases the flux of short-chain fatty acids towards the peroxisomal ß-oxidation cycle when the mitochondrial ß-oxidation pathway is defective.

Oral Terbinafine and Itraconazole Treatments against Dermatophytes Appear Not to Favor the Establishment of Fusarium spp. in Nail.

Background: Fusarium onychomycoses are weakly responsive or unresponsive to standard onychomycosis treatments with oral terbinafine and itraconazole. Objective: To examine whether the use of terbinafine and itraconazole, which are highly effective in fighting Trichophyton onychomycoses, could be a cause of the high incidence of Fusarium nail infections. Methods: Polymerase chain reaction methods were used to detect both Fusarium spp. and Trichophyton spp. in nails of patients who had either received treatment previously or not. Results: No significant microbiological differences were found between treated and untreated patients. In 24 of 79 cases (30%), Fusarium spp. was detected in samples of patients having had no previous antifungal therapy and when Trichophyton spp. grew in culture. Conclusion: Oral terbinafine and itraconazole treatments do not appear to favor the establishment of Fusarium spp. in onychomycosis. © 2014 S. Karger AG, Basel.

A novel family of dehydrin-like proteins is involved in stress response in the human fungal pathogen Aspergillus fumigatus.

 During a search for genes controlling conidial dormancy in Aspergillus fumigatus, two dehydrin-like genes, DprA and DprB, were identified. The deduced proteins had repeated stretches of 23 amino acids that contained a conserved dehydrin-like protein (DPR) motif. Disrupted DprAΔ mutants were hypersensitive to oxidative stress and to phagocytic killing, whereas DprBΔ mutants were impaired in osmotic and pH stress responses. However, no effect was observed on their pathogenicity in our experimental models of invasive aspergillosis. Molecular dissection of the signaling pathways acting upstream showed that expression of DprA was dependent on the stress-activated kinase SakA and the cyclic AMP-protein kinase A (cAMP-PKA) pathways, which activate the bZIP transcription factor AtfA, while expression of DprB was dependent on the SakA mitogen-activated protein kinase (MAPK) pathway, and the zinc finger transcription factor PacC. Fluorescent protein fusions showed that both proteins were associated with peroxisomes and the cytosol. Accordingly, DprA and DprB were important for peroxisome function. Our findings reveal a novel family of stress-protective proteins in A. fumigatus and, potentially, in filamentous ascomycetes.

Optimização da Produção de Frutooligossacáridos por Aspergillus

Este trabalho foi proposto no âmbito do Projecto BIOLIFE – Produção de ingredientes para alimentos funcionais, em curso na BIOTEMPO, Lda desde 2004. Com este trabalho, pretendeu-se avaliar o potencial da produção de frutooligossacáridos (FOS) por Aspergillus sp, recorrendo a um sistema de fermentação descontínuo. Frutooligossacáridos (FOS) são oligossacáridos que ocorrem naturalmente em produtos de origem vegetal, tais como o tomate, cevada, cebola, banana, centeio, espargos, alcachofras, entre outros. Para além de existirem naturalmente, os FOS podem ser produzidos a partir da sacarose usando enzimas ou recorrendo a microrganismos produtores. A tecnologia de fermentação tem como vantagem face à tecnologia enzimática, actualmente a mais utilizada no mercado, o facto de evitar o passo de produção e separação das enzimas, simplificando o processo e reduzindo os custos de operação. Os FOS são prebióticos que, para além de serem compostos indigeríveis, possuem um bom poder adoçante que, apesar de inferior ao dos açúcares comuns é bastante significativo. Esta característica permite a sua utilização como adoçantes não calóricos. Adicionalmente, os prebióticos apresentam outras propriedades benéficas tais como o aumento da absorção de cálcio e magnésio, o aumento da velocidade de metabolismo dos lípidos e efeitos anticancerígenos. A produção de FOS por Aspergillus sp foi estudada recorrendo a um desenho experimental para restringir o número de experiencias requeridas num processo de optimização. Inicialmente foram efectuadas sete fermentações com o intuito de encontrar o ponto óptimo de produção de FOS. Os parâmetros de optimização utilizados foram a temperatura e a agitação. Posteriormente, foram incluídas mais quatro fermentações no referido desenho. O tratamento estatístico dos resultados conduziu a um modelo cúbico que foi posteriormente validado comprovando o seu ajuste. Concluindo, o desenho experimental permitiu obter um modelo que descreve a produção de FOS por Aspergillus sp, bem como determinar as condições para os quais o rendimento de produção de FOS é máximo.

Identification of a key lysine residue in heat shock protein 90 required for azole and echinocandin resistance in Aspergillus fumigatus.

Heat shock protein 90 (Hsp90) is an essential chaperone involved in the fungal stress response that can be harnessed as a novel antifungal target for the treatment of invasive aspergillosis. We previously showed that genetic repression of Hsp90 reduced Aspergillus fumigatus virulence and potentiated the effect of the echinocandin caspofungin. In this study, we sought to identify sites of posttranslational modifications (phosphorylation or acetylation) that are important for Hsp90 function in A. fumigatus. Phosphopeptide enrichment and tandem mass spectrometry revealed phosphorylation of three residues in Hsp90 (S49, S288, and T681), but their mutation did not compromise Hsp90 function. Acetylation of lysine residues of Hsp90 was recovered after treatment with deacetylase inhibitors, and acetylation-mimetic mutations (K27A and K271A) resulted in reduced virulence in a murine model of invasive aspergillosis, supporting their role in Hsp90 function. A single deletion of lysine K27 or an acetylation-mimetic mutation (K27A) resulted in increased susceptibility to voriconazole and caspofungin. This effect was attenuated following a deacetylation-mimetic mutation (K27R), suggesting that this site is crucial and should be deacetylated for proper Hsp90 function in antifungal resistance pathways. In contrast to previous reports in Candida albicans, the lysine deacetylase inhibitor trichostatin A (TSA) was active alone against A. fumigatus and potentiated the effect of caspofungin against both the wild type and an echinocandin-resistant strain. Our results indicate that the Hsp90 K27 residue is required for azole and echinocandin resistance in A. fumigatus and that deacetylase inhibition may represent an adjunctive anti-Aspergillus strategy.

Comparative biology of Cycloneda sanguinea (Linnaeus, 1763) and Hippodamia convergens Guérin-Méneville, 1842 (Coleoptera, Coccinellidae) focusing on the control of Cinara spp. (Hemiptera, Aphididae)

The giant conifer aphids Cinara pinivora (Wilson, 1919) and Cinara atlantica (Wilson, 1919) (Hemiptera, Aphididae) have been observed attacking Pinus spp. in Southern and Southeastern Brazil. The coccinellids, on the other hand, were found feeding on these aphids in the field, which can be regarded as potential biological control agents. The biological cycle and mortality rate of larvae of Cycloneda sanguinea (Linnaeus, 1763) and Hippodamia convergens Guérin-Méneville, 1842 (Coleoptera, Coccinellidae) were evaluated using twenty larvae of each predator species fed with nymphs of Cinara. The vials with the insects were kept under 15 ºC, 20 ºC and 25 ºC, with 12h photophase and 70 ± 10% relative humidity. The consumption was evaluated every 24 hours and the nymphs replaced. For C. sanguinea, the egg incubation time was 10.5, 5.0 and 4.0 days; the average larval development period was 33.3, 15.8 and 8.6 days and the larval mortality rate 20%,0% and 15%, respectively at 15 ºC, 20 ºC and 25 ºC. For H. convergens, the larval development time was 41.9, 19.3 and 10.9 days at 15 ºC, 20 ºC and 25 ºC, respectively. The larval mortality rate was 35%, 15% and 0% under the three temperatures. Both species developed adequately when fed nymphs of Cinara, however, C. sanguinea performed better than H. convergens, even at 15 ºC, at which temperature the biological cycles of the coccinellids are prolonged, but the temperature is favorable for the development of Cinara populations in the field.

Consumption of Cinara spp. (Hemiptera, Aphididae) by Cycloneda sanguinea (Linnaeus, 1763) and Hippodamia convergens Guérin-Méneville, 1842 (Coleoptera, Coccinellidae)

Adults and larvae of coccinellids were observed feeding on populations of the giant conifer aphids Cinara spp. on Pinus spp., in Southern Brazil. The objective of this research is to evaluate the consumption capacity of Cycloneda sanguinea (Linnaeus, 1763) and Hippodamia convergens Guérin-Méneville, 1842 (Coleoptera, Coccinellidae) on these aphid species, in order to obtain information for biological control programs. Ten larvae of each predator species were fed with aphids of small size (nymphs of 1st and 2nd instars), and 10 with aphids of medium size (nymphs of 3rd and 4th instars), maintained under 15ºC, 20ºC and 25ºC, 12 h photophase and 70 ± 10% relative humidity. The aphids were counted every 24 hours, replacing those that were consumed. The total consumption of Cinara by the larvae of C. sanguinea was not statistically different at the three temperatures: 325.5; 322.2 and 324.8 of small aphids and 121.3; 140.4 and 109.9 of medium ones, respectively at 15ºC, 20ºC and 25ºC. The consumption by H. convergens was higher than by C. sanguinea and increased noticeably with temperature: 444 aphids at 15ºC; 491.3 at 20ºC and 513.3 at 25ºC, considering the small aphids, and 187.1; 205.1 and 216.6 of medium aphids at the three temperatures. The small aphids weigh about half as much as medium ones and were preferred by all larval instars probably because they are easier to manipulate than the large ones. Both predators, especially the 4th instar larvae, showed high consumption capacity on the Cinara nymphs at all temperatures and can be regarded as promising biological control agents.

Riqueza de formigas (Hymenoptera, Formicidae) epigéicas em povoamentos de Eucalyptus spp. (Myrtaceae) de diferentes idades no Rio Grande do Sul, Brasil

Este estudo descreve as comunidades de formigas de solo em povoamentos de eucalipto implantados em ecossistema de restinga no Rio Grande do Sul. As coletas de formigas foram feitas em seis povoamentos de Eucalyptus grandis Hill ex Maiden e de Eucalyptus saligna Smith com idades de 31, 19, sete e cinco anos. Para as coletas de formigas, foram selecionados ao acaso 24 talhões, quatro por povoamento. Em cada talhão, foram traçados três transectos com 100 m de comprimento, afastados entre si 12 m. Ao longo dos transectos, foram enterradas 30 armadilhas, tipo pitfall, com iscas de sardinha, afastadas entre si 10 m e mantidas por 24 horas. Foi coletado um total de 21.033 formigas pertencentes a cinco subfamílias, 12 tribos, 19 gêneros e 49 espécies. De acordo com o estimador de riqueza jackknife de primeira ordem, não houve diferenças significativas entre as riquezas das comunidades de formigas considerando as espécies de eucalipto (U = 81,500; g.l.=1; P=0,582) e as idades dos povoamentos (U=2,504; g.l.=3; P=0,547). Os resultados indicam que a riqueza de espécies de formigas não está relacionada à espécie de eucalipto e/ou à idade do povoamento implantado na restinga.

Occurrence of Cinara spp. (Hemiptera, Aphididae) on Pinus spp. (Pinaceae), in the county of Lages-SC, Brazil

A population survey of the giant conifer aphid Cinara spp. was carried out in two areas of Pinus elliottii using yellow pan traps, during two years, from July 1997 to June 1999. During the collection period, the predominant species was Cinara pinivora (Wilson, 1919), representing 99% of the collected species. A few specimens of Cinara maritimae (Dufour, 1833) and only one winged female of Cinara fresai Blanchard, 1939 were collected. The population of C. pinivora was statistically higher in the area with 4.5 year-old trees than in the 1.5 year-old ones. The highest population peaks were registered in July 1997 and in August 1998. An unexpected high number of winged parthenogenetic females was collected in December 1998. C. fresai Blanchard, 1939 is recorded for the first time for Brazil.

Natural parasitism in eggs of Anticarsia gemmatalis Hübner (Lepidoptera, Noctuidae) by Trichogramma spp. (Hymenoptera, Trichogrammatidae) in Brazil

Field surveys were carried during four soybean seasons in Southern Brazil to evaluate the occurrence of parasitoids in eggs of Anticarsia gemmatalis Hübner, 1818 and their incidence along the crop season. Eggs were collected by visual search on soybean leaves and from plants kept inside cages where A. gemmatalis moths were allowed to lay eggs. Trichogramma acacioi Brun, Moraes & Soares, 1984 was recorded for the first time in eggs of A. gemmatalis and the citations in the literature of Trichogramma lasallei Pinto, 1998 in Brazil where based on the material collected in this survey. Apart from these species, Trichogramma pretiosum Riley, 1879, Trichogramma rojasi Nagaraja & Nagarkatti, 1973 and Trichogramma atopovirilia Oatman & Platner, 1983 were also collected, all of which have been previously recorded in this host. Parasitized eggs were collected all over the period of occurrence of A. gemmatalis, from January to April each year. Total parasitism ranged from 4.8% in 2000 and 2002, 23.3% in 2001 and 28.9% in 2003. T. pretiosum and T. acacioi accounted for more than 80% of the parasitoids emerged each year, followed by T. atopovirilia, T. rojasi and T. lasallei, with less than 20% of incidence. Both the sex ratio and the mean number of parasitoids/egg did not differ among the species. Searching for A. gemmatalis eggs proved to be time consuming in comparison to the collection of eggs laid by moths inside the cages, which showed to be a useful method to provide qualitative estimates of parasitism in eggs of A. gemmatalis.

Seleção de Trichogramma spp., para o controle de Ecdytolopha aurantiana, com base na biologia e exigências térmicas

O objetivo deste trabalho foi avaliar o potencial de utilização de Trichogramma atopovirilia Oatman & Platner, 1983 e T. pretiosum Riley, 1879 como agentes de controle de Ecdytolopha aurantiana (Lima, 1927) (Lepidoptera: Tortricidae), importante praga da citricultura paulista. Foram realizados estudos de biologia em diferentes temperaturas, exigências térmicas e capacidade de parasitismo, visando fornecer subsídios a programas de controle biológico com estes parasitóides. A razão sexual não foi afetada pelas temperaturas estudadas (18, 20, 22, 25, 28, 30 e 32ºC), porém, a longevidade de fêmeas das duas espécies foi maior a 22 e 25ºC. Para a porcentagem de emergência e longevidade de fêmeas, a temperatura que tendeu a ser mais adequada foi a de 25ºC. A duração do período ovo-adulto para as duas espécies foi inversamente relacionada com o aumento da temperatura, sendo as exigências térmicas dessas espécies muito próximas, em torno de 108 GD. O hospedeiro natural, E. aurantiana, ou o alternativo, Anagasta kuehniella (Zeller, 1879) (Lepidoptera, Pyralidae), não afetaram o número de ovos parasitados por fêmea de Trichogramma. Porém, a porcentagem de parasitismo e o número de adultos emergidos por ovo, quando provenientes de ovos de E. aurantiana, foram maiores do que os criados sobre ovos de A. kuehniella. A porcentagem de emergência foi, no entanto, maior quando os parasitóides foram provenientes de ovos de A. kuehniella. As duas espécies de Trichogramma estudadas poderão ser testadas em campo para controle do bicho-furão-dos-citros, havendo uma indicação de que as exigências térmicas também podem servir como um bom parâmetro para seleção de espécies e/ou linhagens de Trichogramma, aliadas ao estudo de capacidade de parasitismo.

Breeding sites of Neotropical Drosophilidae (Diptera): IV. living and fallen flowers of Sessea brasiliensis and Cestrum spp. (Solanaceae)

The flowering phenologies of Sessea brasiliensis and three species of Cestrum (C. amictum, C. schlechtendalii and C. sendtnerianum), all belonging to the Solanaceae, were monitored in a fragment of the montane Atlantic rain forest within São Paulo city from July 1993 through June 1995. A total of 11,021 flies belonging to five species of the flavopilosa group of Drosophila (D. cestri, D. cordeiroi, D. hollisae, D. incompta and D. mariaehelenae), plus 1,244 flies belonging to four species of Zygothrica (Z. dispar, Z. vittimaculosa, Z. vittinubila and Z. aff. vittipunctata) emerged from 33,955 sampled flowers. Cestrum amictum, C. sendtnerianum and Sessea brasiliensis are recorded for the first time as host plants for the species of the Neotropical flavopilosa group ofDrosophila. Additionally, at least ten less abundant taxa belonging also to the Drosophilidae, plus 1,073 microhymenopteran parasitoids (Braconidae, Eucoilidae, and Pteromalidae), emerged from the sampled flowers of S. brasiliensis e Cestrum spp. The present study raised from nine to eleven the number of species of Cestrum previously recorded as breeding sites for these flies. A comprehensive list of species of Solanaceae known to host six species of Drosophila and four species of Zygothrica in southeastern and southern Brazil is included.

Secreted glutamic protease rescues aspartic protease Pep deficiency in Aspergillus fumigatus during growth in acidic protein medium.

In an acidic protein medium Aspergillus fumigatus secretes an aspartic endoprotease (Pep) as well as tripeptidyl-peptidases, a prolyl-peptidase and carboxypeptidases. In addition, LC-MS/MS revealed a novel glutamic protease, AfuGprA, homologous to Aspergillus niger aspergillopepsin II. The importance of AfuGprA in protein digestion was evaluated by deletion of its encoding gene in A. fumigatus wild-type D141 and in a pepΔ mutant. Either A. fumigatus Pep or AfuGprA was shown to be necessary for fungal growth in protein medium at low pH. Exoproteolytic activity is therefore not sufficient for complete protein hydrolysis and fungal growth in a medium containing proteins as the sole nitrogen source. Pep and AfuGprA constitute a pair of endoproteases active at low pH, in analogy to A. fumigatus alkaline protease (Alp) and metalloprotease I (Mep), where at least one of these enzymes is necessary for fungal growth in protein medium at neutral pH. Heterologous expression of AfuGprA in Pichia pastoris showed that the enzyme is synthesized as a preproprotein and that the propeptide is removed through an autoproteolytic reaction at low pH to generate the mature protease. In contrast to A. niger aspergillopepsin II, AfuGprA is a single-chain protein and is structurally more similar to G1 proteases characterized in other non-Aspergillus fungi.

Parasitismo por Neodohrniphora spp. Malloch (Diptera, Phoridae) em operárias de Atta sexdens rubropilosa Forel (Hymenoptera, Formicidae)

Parasitóides do gênero Neodohrniphora Malloch, 1914 geralmente atacam operárias de Atta sexdens rubropilosa Forel, 1908 em baixa freqüência, mas os ataques alteram o ritmo de forrageamento das colônias, reduzindo o número e tamanho das operárias e aumentando o abandono de fragmentos vegetais ao longo das trilhas. O forrageamento de A. sexdens rubropilosa pode ser diurno ou noturno, mas existem dúvidas se Neodohrniphora spp. atacam as operárias dessa formiga também durante a noite. Os objetivos deste estudo foram verificar a ocorrência de ataques e avaliar o grau de parasitismo de forídeos do gênero Neodohrniphora em operárias de A. sexdens rubropilosa sob três níveis de luminosidade em laboratório, incluindo a ausência de luz. Os níveis de luminosidade foram: alta (0,65 µmol/m³/s); baixa (0,05 µmol/m³/s) e ausência de luz (0,0 µmol/m³/s). Fêmeas de Neodohrniphora tonhascai Brown, 2001 e Neodohrniphora elongata Brown, 2001 coletadas no campo e liberadas em laboratório somente efetuaram ataques e foram obtidas operárias parasitadas sob alta luminosidade, sugerindo que essas espécies não são ativas no campo durante o período noturno (ausência de luz). Os resultados sugerem também que o estímulo visual em Neodohrniphora spp. pode ser um componente essencial para a localização e reconhecimento do hospedeiro.