Gypsy moths (Lymantria dispar) in the Niagara Region : population density variation, introduction of an entomopathogenic fungus (Entomophaga maimaiga) and occurrence of nuclear polyhedrosis virus

The gypsy moth, Lymantria dispar, a major defoliator of broad leaf trees, was accidentally introduced into North America in 1869. Much interest has been generated regarding the potential of using natural pathogens for biological control of this insect. One of these pathogens, a highly specific fungus, Entomophaga maimaiga, was accredited with causing major epizootics in populations of gypsy moth across the north-eastern United States in 1989 and 1990 and is thought to be spreading northwards into Canada. This study examined gypsy moth population densities in the Niagara Region. The fungus, .E.. maimaiga, was artificially introduced into one site and the resulting mortality in host populations was noted over two years. The relationship between fungal mortality, host population density and occurrence of another pathogen, the nuclear polyhedrosis virus (NPV), was assessed. Gypsy moth population density was assessed by counting egg masses in 0.01 hectare (ha) study plots in six areas, namely Louth, Queenston, Niagara-on-the-Lake, Shorthills Provincial Park, Chippawa Creek and Willoughby Marsh. High variability in density was seen among sites. Willoughby Marsh and Chippawa Creek, the sites with the greatest variability, were selected for more intensive study. The pathogenicity of E. maimaiga was established in laboratory trials. Fungal-infected gypsy moth larvae were then released into experimental plots of varying host density in Willoughby Marsh in 1992. These larvae served as the inoculum to infect field larvae. Other larvae were injected with culture medium only and released into control plots also of varying host density. Later, field larvae were collected and assessed for the presence of .E.. maimaiga and NPV. A greater proportion of larvae were infected from experimental plots than from control plots indicating that the experimental augmentation had been successful. There was no relationship between host density and the proportion of infected larvae in either experimental or control plots. In 1992, 86% of larvae were positive for NPV. Presence and intensity of NPV infection was independent of fungal presence, plot type or interaction of these two factors. Sampling was carried out in the summer of 1993, the year after the introduction, to evaluate the persistence of the pathogen in the environment. Almost 50% of all larvae were infected with the fungus. There was no difference between control and experimental plots. Data collected from Willoughby Marsh indicated that there was no correlation between the proportion of larvae infected with the fungus and host population density in either experimental or control plots. About 10% of larvae collected from a nearby site, Chippawa Creek, were also positive for .E.. maimaiga suggesting that low levels of .E.. maimaiga probably occurred naturally in the area. In 1993, 9.6% of larvae were positive for NPV. Again, presence or absence of NPV infection was independent of fungal presence plot type or interaction of these two factors. In conclusion, gypsy moth population densities were highly variable between and within sites in the Niagara Region. The introduction of the pathogenic fungus, .E.. maimaiga, into Willoughby Marsh in 1992 was successful and the fungus was again evident in 1993. There was no evidence for existence of a relationship between fungal mortality and gypsy moth density or occurrence of NPV. The results from this study are discussed with respect to the use of .E.. maimaiga in gypsy moth management programs.

Starvation Induces Expression of the Plant-Adhesin Gene, Mad2, of the Entomopathogenic Fungus Metarhizium robertsii.

Metarhizium robertsii is an entomopathogenic fungus that is additionally plant rhizosphere competent. Two adhesin-encoding gens, Mad1 and Mad2, are involved in insect pathogenesis or plant root colonization, respectively. This study examined differential expression of the Mad genes for M robertsii grown on a variety of insectand plant-related substrates. Mad1 was up regulated in response to insect cuticles and up regulation of Mad2 resulted from root exudates, tomato stems and non-preferred carbohydrates. A time course analysis that compared water, minimal media, and nutrient rich broth revealed Mad2 gene expression increased as nutrient availability decreased. The regulation of Mad2 compared to known stress-related genes (Hsp30, Hsp70 and ssgA) under various stresses (nutrient, pH, osmotic, oxidative, temperature) revealed Mad2 to be generally up regulated by nutrient starvation only. Examination of the Mad2 promoter region revealed two copies of a stress-response element (S TRE) known to be regulated under the general stress response pathway.

Production technology for entomopathogenic fungus using a biphasic culture system

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Susceptibility of adult and larval stages of the horn fly, Haematobia irritans, to the entomopathogenic fungus Metarhizium anisopliae under field conditions

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Characterization of a new toxin from the entomopathogenic fungus Metarhizium anisopliae: the ribotoxin anisoplin

Metarhizium anisopliae is an entomopathogenic fungus relevant in biotechnology with applications like malaria vector control. Studies of its virulence factors are therefore of great interest. Fungal ribotoxins are toxic ribonucleases with extraordinary efficiency against target ribosomes and suggested as potential insecticides. Here, we describe this ribotoxin characteristic activity in M. anisopliae cultures. Anisoplin has been obtained as a recombinant protein and further characterized. It is structurally similar to hirsutellin A, the ribotoxin from the entomopathogen Hirsutella thompsonii. Moreover, anisoplin shows the ribonucleolytic activity typical of ribotoxins and cytotoxicity against insect cells. How Metarhizium uses this toxin and possible applications are on perspective.

Characterization of a new toxin from the entomopathogenic fungus Metarhizium anisopliae: the ribotoxin anisoplin

Metarhizium anisopliae is an entomopathogenic fungus relevant in biotechnology with applications like malaria vector control. Studies of its virulence factors are therefore of great interest. Fungal ribotoxins are toxic ribonucleases with extraordinary efficiency against target ribosomes and suggested as potential insecticides. Here, we describe this ribotoxin characteristic activity in M. anisopliae cultures. Anisoplin has been obtained as a recombinant protein and further characterized. It is structurally similar to hirsutellin A, the ribotoxin from the entomopathogen Hirsutella thompsonii. Moreover, anisoplin shows the ribonucleolytic activity typical of ribotoxins and cytotoxicity against insect cells. How Metarhizium uses this toxin and possible applications are on perspective.

Effect of Metarhizium anisopliae fungus on off-host Rhipicephalus (Boophilus) microplus from tick-infested pasture under cattle grazing in Brazil

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Interactions Between the Entomopathogenic Fungi Beauveria bassiana (Ascomycota: Hypocreales) and the Aphid Parasitoid Diaeretiella rapae (Hymenoptera: Braconidae) on Myzus persicae (Hemiptera: Aphididae)

The interactions between the entomopathogenic fungus Beauveria bassiana (Balsamo-Crivelli) Vuillemin (Ascomycota: Hypocreales) and the aphid parasitoid Diaeretiella rapae McIntoch (Hymenoptera: Braconidae) were evaluated under laboratory conditions. Nymphs of Myzus persicae Sulzer (Hemiptera: Aphididae) were first exposed to parasitoid females for 24 h and then 0, 24, and 48 h afterwards sprayed with a solution of B. bassiana. Likewise, aphids were also sprayed with B. bassiana and then exposed to parasitoids at 0, 24, and 48 h afterwards. Parasitism rate varied from 13 to 66.5%, and were signi_cantly lower in treatments where the two agents were exposed within a 0-24 h time interval compared with the control (without B. bassiana). Parasitoid emergence was negatively affected in treatments with B. bassiana spraying and subsequent exposure to D. rapae. Decreases in longevity of adult females of the D. rapae F1 generation were observed in treatments with B. bassiana spraying. The application of these two biological control agents can be used in combination on the control of M. persicae, wherein this use requires effective time management to avoid antagonistic interactions.

Melanism and disease resistance in insects

There is growing evidence that insects in high-density populations invest relatively more in pathogen resistance than those in low-density populations (i.e. density-dependent prophylaxis). Such increases in resistance are often accompanied by cuticular melanism, which is characteristic of the high-density form of many phase polyphenic insects. Both melanism and pathogen resistance involve the prophenoloxidase enzyme system. In this paper the link between resistance, melanism and phenoloxidase activity is examined in Spodoptera lanae. In S. exempta, cuticular melanism was positively correlated with phenoloxidase activity in the cuticle, haemolymph and midgut. Melanic S. exempta larvae were found to melanize a greater proportion of eggs of the ectoparasitoid Euplectrus laphygmae than non-melanic larvae, and melanic S. littoralis were more resistant to the entomopathogenic fungus Beauveria bassiana (in S. exempta the association between melanism and fungal resistance was non-signficant). These results strengthen the link between melanism and disease resistance and implicate the involvement of phenoloxidase.

Pupal cocoons affect sanitary brood care and limit fungal infections in ant colonies.

BACKGROUND: The brood of ants and other social insects is highly susceptible to pathogens, particularly those that penetrate the soft larval and pupal cuticle. We here test whether the presence of a pupal cocoon, which occurs in some ant species but not in others, affects the sanitary brood care and fungal infection patterns after exposure to the entomopathogenic fungus Metarhizium brunneum. We use a) a comparative approach analysing four species with either naked or cocooned pupae and b) a within-species analysis of a single ant species, in which both pupal types co-exist in the same colony. RESULTS: We found that the presence of a cocoon did not compromise fungal pathogen detection by the ants and that species with cocooned pupae increased brood grooming after pathogen exposure. All tested ant species further removed brood from their nests, which was predominantly expressed towards larvae and naked pupae treated with the live fungal pathogen. In contrast, cocooned pupae exposed to live fungus were not removed at higher rates than cocooned pupae exposed to dead fungus or a sham control. Consistent with this, exposure to the live fungus caused high numbers of infections and fungal outgrowth in larvae and naked pupae, but not in cocooned pupae. Moreover, the ants consistently removed the brood prior to fungal outgrowth, ensuring a clean brood chamber. CONCLUSION: Our study suggests that the pupal cocoon has a protective effect against fungal infection, causing an adaptive change in sanitary behaviours by the ants. It further demonstrates that brood removal-originally described for honeybees as "hygienic behaviour"-is a widespread sanitary behaviour in ants, which likely has important implications on disease dynamics in social insect colonies.

Expressão de uma quitinase de Metarhizium anisopliae em Nicotiana tabacum : obtenção de plantas transgênicas resistentes a doenças fúngicas

A resistência a doenças em plantas transgênicas tem sido obtida por meio da expressão de genes isolados de bactérias, fungos micoparasitas e plantas. Neste trabalho, relatamos a utilização de um gene do fungo entomopatogênico Metarhizium anisopliae como modo de gerar resistência a doenças fúngicas em plantas. O gene chit1 codifica a quitinase CHIT42 (EC 3.2.1.14), pertencente a uma classe de glicosil-hidrolases capazes de converter quitina em oligômeros de N-acetil-glicosamina (NAcGlc). Quando presentes em tecidos vegetais, supõese que as quitinases ataquem especificamente a parede celular de fungos invasores, provocando danos às hifas e causando a morte por lise das células fúngicas. Deste modo, dois diferentes grupos de plantas transgênicas de Nicotiana tabacum foram produzidos: no primeiro deles, denominado chitplus, os indivíduos possuem o gene chit1 sob o controle do promotor CaMV 35S. O segundo grupo, demoninado chitless, consiste de plantas transformadas com um T-DNA não contendo o gene do fungo. Trinta e quatro plantas transgênicas resistentes à canamicina (17 de cada grupo) foram regeneradas a partir de discos de folhas infectados por Agrobacterium tumefaciens. A produção da quitinase em extratos protéicos de folhas foi analisada por zimogramas em SDS-PAGE contendo glicol-quitina e corados por calcoflúor branco, na forma de um screening dos transgênicos primários. As plantas transgênicas foram testadas, ainda, por meio de ensaios colorimétricos empregando oligômeros sintéticos de NAcGlc como substratos específicos, além de immunoblot e Western blot com soro anti-quitinase. A quantidade de enzima recombinante nas plantas chitplus variou desde nenhuma atividade detectável a elevados níveis de expressão da enzima. A hibridização de Southern blot demonstrou que o número de cópias do gene chit1 integradas no genoma vegetal foi estimado entre uma e quatro. A primeira geração de plantas transgênicas geradas por autofecundação de parentais portadores de duas cópias do transgene foi testada com relação à estabilidade da herança do transgene e em 43 de um total de 67 descendentes, originados de quatro cruzamentos independentes, o padrão de segregação não diferiu das proporções Mendelianas esperadas. Ensaios de resistência, desafiando as plantas transgênicas com o basidiomiceto Rhizoctonia solani foram realizados e uma evidente diminuição da área foliar contendo lesões fúngicas foi observada entre as linhagens transgênicas, embora variações na atividade quitinolítica tenham influenciado o nível de resistência. Nossos resultados sugerem uma relação direta entre a atividade específica de quitinase e ao aumento nos níveis de resistência às lesões causadas pela infecção por R. solani.

Sensibilidade de Metarhizium anisopliae à temperatura e umidade em três tipos de solos

Este trabalho teve como objetivo investigar o efeito da temperatura e do teor de umidade do solo na sobrevivência de Metarhizium anisopliae (Metsch.) Sorok. em três tipos de solos. Foram utilizados o Latossolo Vermelho textura argilosa, Latossolo Vermelho textura média e Argissolo Vermelho Amarelo textura arenosa média. As temperaturas empregadas foram 21,5; 26,8 e 31,5°C, e os teores de umidade foram 35, 65 e 100% de saturação. A sobrevivência do fungo foi avaliada após zero, 20, 40, 60, 80, 100 e 120 dias de incubação em cada temperatura estudada. Na análise do efeito do teor de umidade, a sobrevivência foi avaliada após zero, 14, 28, 42, 56, 70, 84, 98 e 112 dias de incubação à temperatura de 27,0±1,0°C. em ambos os ensaios, foi determinado o número de unidades formadores de colônias (UFC) em placa de Petri. Houve influência significativa da temperatura e do teor de umidade na sobrevivência do fungo. O maior crescimento e a maior sobrevivência ocorreram nas temperaturas de 21,5 e 26,8°C, enquanto que, no solo incubado a 31,5°C, o fungo cresceu pouco, e a população declinou rapidamente. No teor de 65% de umidade, houve rápido crescimento do fungo, mas no 112° dia foi observado um declínio da população nos três tipos de solos. Nos teores de 35 e 100% de umidade, o crescimento foi menor, mas obteve-se maior sobrevivência do fungo no solo.

Pathogenicity of Metarhizium anisopliae for Ceratitis capitata (Wied.) (Diptera: Tephritidae) in soil with different pesticides

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Associação do óleo de mamona com Beauveria bassiana no controle da traça-das-crucíferas

O objetivo deste trabalho foi avaliar a eficiência, a estabilidade, a forma de aplicação e a compatibilidade do óleo de mamona com o fungo entomopatogênico Beauveria bassiana no controle da traça-das-crucíferas, Plutella xylostella. No primeiro experimento, foram avaliadas a eficiência, a estabilidade após 70 dias de armazenamento e a forma de aplicação do óleo de mamona. No segundo experimento, foi avaliada a compatibilidade do óleo de mamona com B. bassiana. A atividade inseticida do óleo de mamona é instável ao longo do tempo. O óleo de mamona é compatível com B. bassiana no controle de P. xylostella.

Seleção de isolados de Beauveria bassiana potenciais para o controle da traça-das-crucíferas

A traça-das-crucíferas, Plutella xylostella (Lepidoptera: Plutellidae), é considerada a principal praga das brássicas no mundo, sendo o uso de inseticidas o método mais usado para o seu controle. Assim, o objetivo desta pesquisa foi selecionar isolados de Beauveria bassiana com viabilidade para utilização no controle da traça-das-crucíferas. Dezessete isolados e um produto comercial de B. bassiana foram testados. Lagartas de segundo ínstar da traça-das-crucíferas foram pulverizadas com suspensão de conídios na concentração de 10(7) conídios mL-1. Para o bioensaio de concentração letal (CL) sete concentrações espaçadas em escala logarítmica foram testadas. Os isolados CCA/UFES-4, 18, 31 e 35 foram selecionados para o bioensaio de CL por causarem mortalidade confirmada superior a 90%. O isolado padrão ESALQ-447 e o produto comercial tiveram resultados semelhantes e também foram selecionados para o bioensaio de CL. Com base nas estimativas da CL50, os isolados CCA/UFES-4, 18, 31, ESALQ-447 e o produto comercial podem ser selecionados para utilização no controle da traça-das-crucíferas.