Movement of Heterorhabditis amazonensis and Steinernema arenarium in search of corn fall armyworm larvae in artificial conditions

Spodoptera frugiperda (Smith, 1797) (Lepidoptera: Noctuidae) is considered to be the main pest of maize crops in Brazil. Entomopathogenic nematodes (EPN) may be used to control this pest and exhibit different, unique abilities to search for their hosts. The movement of EPN in relation to S. frugiperda was evaluated. To test for horizontal movement, a styrofoam enclosure filled with sand was divided into segments, nematodes were placed at the entrance to the enclosure and a larva was placed at the end of each division. The same approach was used to evaluate vertical movement; however, PVC pipes were used in this case. In general, the mortality was inversely proportional to the initial distance between host and nematodes. In the vertical displacement test, both nematodes were able to kill the larvae up to a distance of 25 cm. Therefore, the infective juveniles of H. amazonensis and S. arenarium can search out, infect and kill larvae of S. frugiperda at distances of up to 60 cm and 25 cm of horizontal and vertical displacement, respectively.

Three in one: fixing marine nematodes for ecological, molecular, and morphological studies

Multidisciplinary benthic studies are still hindered by the lack of a unique fixative that satisfactorily preserves morphology and DNA, and that is simultaneously adequate for ecological surveys. The objective of this study is to test the performance of five fixatives: formalin, ethanol, dimethylsulfoxide with EDTA and NaCl salts (DESS), methanol with acetic acid (METHAC), and ethanol with acetic acid (ETHAC), for the preservation of estuarine and exclusively marine nematode assemblages for morphological, molecular, and ecological studies. The presence of the stain rose bengal in each fixative was also evaluated in the yield of PCR reactions. For molecular analyses, one species of each habitat was considered. Results revealed that fixative performance for morphological studies is habitat-and species-dependent. For studies of estuarine sediment nematodes, we recommend the use of pure ethanol, because it caused little morphological distortion (<10% of the assemblage), preserved all the species for ecological studies, and yielded high quality DNA sequences. For studies of exclusively marine environments, METHAC or DESS are the most adequate. The first performed better for morphological and ecological surveys, whereas the second was more appropriate for molecular research. For ecological studies, DESS should be used in comparison with formalin, in order to cross check the results. Finally, staining of samples with rose bengal is not recommended, because it hindered DNA amplification regardless of the fixative used.

Underground leaves of Philcoxia trap and digest nematodes

The recently described genus Philcoxia comprises three species restricted to well lit and low-nutrient soils in the Brazilian Cerrado. The morphological and habitat similarities of Philcoxia to those of some carnivorous plants, along with recent observations of nematodes over its subterranean leaves, prompted the suggestion that the genus is carnivorous. Here we report compelling evidence of carnivory in Philcoxia of the Plantaginaceae, a family in which no carnivorous members are otherwise known. We also document both a unique capturing strategy for carnivorous plants and a case of a plant that traps and digests nematodes with underground adhesive leaves. Our findings illustrate how much can still be discovered about the origin, distribution, and frequency of the carnivorous syndrome in angiosperms and, more generally, about the diversity of nutrient-acquisition mechanisms that have evolved in plants growing in severely nutrient-impoverished environments such as the Brazilian Cerrado, one of the world's 34 biodiversity hotspots.

The importance of vertical and horizontal dimensions of the sediment matrix in structuring nematodes across spatial scales

Intensive surveys have been conducted to unravel spatial patterns of benthic infauna communities. Although it has been recognized that benthic organisms are spatially structured along the horizontal and vertical dimensions of the sediment, little is known on how these two dimensions interact with each other. In this study we investigated the interdependence between the vertical and horizontal dimensions in structuring marine nematodes assemblages. We tested whether the similarity in nematode species composition along the horizontal dimension was dependent on the vertical layer of the sediment. To test this hypothesis, three-cm interval sediment samples (15 cm depth) were taken independently from two bedforms in three estuaries. Results indicated that assemblages living in the top layers are more abundant, species rich and less variable, in terms of species presence/absence and relative abundances, than assemblages living in the deeper layers. Results showed that redox potential explained the greatest amount (12%) of variability in species composition, more than depth or particle size. The fauna inhabiting the more oxygenated layers were more homogeneous across the horizontal scales than those from the reduced layers. In contrast to previous studies, which suggested that reduced layers are characterized by a specific set of tolerant species, the present study showed that species assemblages in the deeper layers are more causal (characterized mainly by vagrant species). The proposed mechanism is that at the superficial oxygenated layers, species have higher chances of being resuspended and displaced over longer distances by passive transport, while at the deeper anoxic layers they are restricted to active dispersal from the above and nearby sediments. Such restriction in the dispersal potential together with the unfavorable environmental conditions leads to randomness in the presence of species resulting in the high variability between assemblages along the horizontal dimension.

Genetic Variability of Beauveria bassiana and a DNA Marker for Environmental Monitoring of a Highly Virulent Isolate Against Cosmopolites sordidus

The banana weevil Cosmopolites sordidus (Germar) is one of a number of pests that attack banana crops. The use of the entomopathogenic fungus Beauveria bassiana as a biological control agent for this pest may contribute towards reducing the application of chemical insecticides on banana crops. In this study, the genetic variability of a collection of Brazilian isolates of B. bassiana was evaluated. Samples were obtained from various geographic regions of Brazil, and from different hosts of the Curculionidae family. Based on the DNA fingerprints generated by RAPD and AFLP, we found that 92 and 88 % of the loci were polymorphic, respectively. The B. bassiana isolates were attributed to two genotypic clusters based on the RAPD data, and to three genotypic clusters, when analyzed with AFLP. The nucleotide sequences of nuclear ribosomal DNA intergenic spacers confirmed that all isolates are in fact B. bassiana. Analysis of molecular variance showed that variability among the isolates was not correlated with geographic origin or hosts. A RAPD-specific marker for isolate CG 1024, which is highly virulent to C. sordidus, was cloned and sequenced. Based on the sequences obtained, specific PCR primers BbasCG1024F (5'-TGC GGC TGA GGA GGA CT-3') and BbasCG1024R (5'-TGC GGC TGA GTG TAG AAC-3') were designed for detecting and monitoring this isolate in the field.

Biological and behavioral parameters of the parasitoid Cotesia flavipes (Hymenoptera: Braconidae) are altered by the pathogen Nosema sp (Microsporidia: Nosematidae)

A major issue for mass rearing of insects concerns sanitary conditions and disease. Microsporidian infection (Nosema sp.) in laboratory colonies of Diatraea saccharalis (Fabr.) (Lepidoptera: Crambidae), used in producing the parasitoid. Cotesia flavipes Cameron (Hymenoptera: Braconidae), is representative of the problems faced by growers and industry. Although C. flavipes has been produced for several years in Brazil for biological control of D. saccharalis, we have only recently observed that the parasitoid becomes infected when developing inside hosts infected with Nosema sp. We assessed the effects of Nosema sp. on C. flavipes, including the ability to locate and select hosts, and evaluated pathogen transmission. Third instar larvae of D. saccharalis were inoculated with Nosema sp. spores at different concentrations and were parasitized when larvae reached fifth instar. Heavily infected D. saccharalis larvae did not support parasitism. Parasitoids that developed in infected D. saccharalis larvae exhibited increased duration of larval and pupal stages, decreased adult longevity and number of offspring, and reduced tibia size compared to parasitoids developing in uninfected D. saccharalis larvae. Infection by Nosema sp. reduced the ability of the C. flavipes parasitoid to distinguish between volatiles released by the sugarcane infested by healthy larvae and pure air. Uninfected parasitoids preferred plants infested with uninfected hosts. But infected C. flavipes did not differentiate between uninfected hosts and those infected with Nosema sp. The pathogen is transmitted from host to parasitoids and parasitoids to hosts. Pathogenic effects of the microsporidium in C. flavipes are sufficiently severe to justify disease management efforts, particularly considering the importance of C. flavipes as a biological control agent in sugarcane. (C) 2012 Elsevier Inc. All rights reserved.

LOW GENETIC DIVERSITY IN Wolbachia-INFECTED Culex quinquefasciatus (DIPTERA: CULICIDAE) FROM BRAZIL AND ARGENTINA

Culex quinquefasciatus is a vector of human pathogens, including filarial nematodes and several viruses. Although its epidemiological relevance is known to vary across geographical regions, an understanding of its population genetic structure is still incipient. In light of this, we evaluated the genetic diversity of Cx. quinquefasciatus and Cx. pipiens x Cx. quinquefasciatus hybrids collected from nine localities in Brazil and one site in Argentina. We used mitochondrial genes cox1 and nd4, along with the coxA and wsp genes of the maternally-inherited Wolbachia endosymbiont. The nd4 fragment was invariant between samples, whilst cox1 exhibited four haplotypes that separated two types of Cx. quinquefasciatus, one clustered in southern Brazil. Low sequence diversity was generally observed, being discussed. Both Brazilian and Argentinian mosquitoes were infected with a single Wolbachia strain. As reported in previous studies with these populations, cox1 and nd4 diversity is not congruent with the population structure revealed by nuclear markers or alar morphology. Future Cx. quinquefasciatus research should, if possible, evaluate mtDNA diversity in light of other markers.

Compatibility of Beauveria bassiana commercial isolate with botanical insecticides utilized in organic crops in southern Brazil

Products based on botanical insecticides and entomopathogenic fungi have been widely used in organic farming, especially in southern Brazil. Thus, this study investigated, in vitro, the effect of aqueous extracts and commercial formulations of plants with insecticidal activity on Beauveria bassiana. The treatments comprised the botanical insecticides Neempro (azadiractin +3-tigloylazadirachtol), at the concentrations of 0.25, 0.5, 0.75, and 1.0% (v/v), and DalNeem (neem oil emulsifiable), at 0.5, 1.0, 1.5, and 2.0% (v/v) (both commercial formulations of Azadirachta indica (neem)), and the aqueous extracts, at the concentrations of 2.5, 5.0, 7.5, and 10.0% (w/v), of neem seeds, tobacco powder (Nicotiana tabacum), and catigua leaves (Trichilia clausenii). In potato, dextrose, and agar culture medium, the effects of each product on the mycelial growth and the production and viability of conidia of B. bassiana were estimated. According to the adopted compatibility index, the aqueous extracts of neem seeds and leaves catigua, depending on the concentration used, and the botanical insecticide Neempro, were classified as compatible with the entomopathogen, becoming important alternatives to integrate programmes of integrated pest management, especially in organic farming systems.

POPULATION BIOLOGY AND DIET OF THE SOUTHERN KINGCROAKER Menticirrhus americanus (LINNAEUS, 1758) (PERCIFORMES: SCIAENIDAE) IN CARAGUATATUBA BAY, SOUTHEASTERN BRAZIL

This study evaluated the spatio-temporal distribution, population biology and diet of Menticirrhus americanus in Caraguatatuba Bay. Samples were taken monthly between August 2003 and October 2004, by trawling in two previously selected areas. The northern area is more exposed to wave activity and is influenced by a river, functioning as a small estuary. In contrast, the southern area is relatively sheltered from wave energy and influenced to a lesser degree by smaller rivers. The fishes' length was measured, and the sex and gonadal stage macroscopically identified. The abundance of this species was compared between areas and among months. The diet was identified and quantified. M. americanus occurred in equal proportions in the two study areas, being most abundant in April 2004, followed by December 2003 and January 2004. The population was dominated by small immature individuals. The few individuals in maturation or mature that were captured showed no seasonal pattern of distribution. This species had a varied diet, feeding on worms (nemerteans, sipunculans and echiurans), mollusks (bivalves and cephalopods), polychaetes, crustaceans and fish. The presence of intact nematodes in the intestine suggests that these are parasites. The results demonstrated that M. americanus has a homogeneous spatial and temporal distribution in Caraguatatuba Bay, being uniformly distributed between the south and north areas as well as across the months. This species can be considered a carnivorous predator, showing a preference for consuming benthic sandy-beach species such as glycerids and other polychaetes, crustaceans, and bivalve siphons.

Parasitism by Oestrus ovis: Influence of sheep breed and nematode infections

Previous studies showed that Santa Ines (SI) hair sheep were more resistant to gastrointestinal nematode infections (GIN) than Ile de France (IF) sheep. The present experiment aimed to evaluate if that reported resistance difference against GIN also occurred against Oestrus ovis infestation and also to evaluate the influence of O. ovis infestation on the gastrointestinal nematodes (GIN) infections. SI (n = 12) and IF (n = 12) young male lambs were weaned at 2 months of age and moved to a paddock (0.3 ha) with Brachiaria decumbens grass, where they also received concentrate ration. The animals were kept together during the experimental period (September to early December 2009). Fecal and blood samples were taken from all animals every 2 weeks and body weight and nasal discharge score (oestrosis clinic signs) were recorded on the same occasion. In early December 2009, all lambs were sacrificed and O. ovis larvae and GIN were recovered, counted and identified according to the larval stage. All animals were infested by different larval instars of O. ovis without any statistical difference between breeds (P > 0.05). The SI lambs had an average of 24.8 larvae, and the intensity of infection ranged between 14 and 39 larvae, while the IF lambs showed an average of 23.5 larvae with the minimum and maximum from 11 to 36 larvae, respectively. SI lambs presented the lowest nematode fecal egg counts (FECs) and the lowest mean numbers of Haemonchus contort us, Trichostrongylus colubriformis and Strongyloides papillosus, however, there was no significant differences between group means (P > 0.05). Inverse relationship between numbers of O. ovis larvae and gastrointestinal nematodes was observed in both breeds. SI sheep showed a significant increase in blood eosinophils and total IgE serum levels and these variables were negatively correlated with nematode FEC. A negative correlation was observed between total IgE serum level and H. contortus burden in both breeds. In conclusion, there was no breed difference regarding O. ovis infestation and in each breed, animals with more nasal bot fly larvae tended to display smaller worm burden. (C) 2011 Elsevier B.V. All rights reserved.

Pathogenicity of Metarhizium anisopliae towards Rhipicephalus (Boophilus) microplus under laboratory and field conditions

Metarhizium anisopliae is one of the most studied agents of biological control of several arthropod plagues, including the cattle tick Rhipicephalus (Boophilus) microplus. Studies have been conducted to assess the fungal complex infection process towards its hosts. To accomplish that, mutant strains overexpressing or lacking assumed determinant genes for the process were constructed over the years. A fundamental experiment to demonstrate a particular gene or set of genes participation is the bioassay. The comparison of bioassays using wild and engineered strains is an essential tool to affirm a given gene is crucial in the process. Therefore, the in vitro bioassays should mimic the results obtained in tests under field conditions. In this study, tests under laboratory and filed conditions were done and a correlation analysis was performed in order to statistically validate in vitro bioassays. Tick egg laying, larvae hatching and host mortality were recorded in each experiment through 21 days, both under laboratory and field conditions. In all cases, M. anisopliae treatments were statistically different from the control treatments. A linear regression analysis was performed between the cases. Laboratory results showed a statistically significant correlation with the field conditions using the Pearson's Correlation Test (P < 0.01 host mortality - 0.969, tick egg laying - 0.977 and larvae hatching - 0.956). These results legitimize the in vitro bioassays and, therefore, constitute them as a valid tool for studying this fungus behavior, so they can be used to infer M. anisopliae response towards R. (Boophilus) microplus.

Acaricidal action of destruxins produced by a marine-derived Beauveria felina on the bovine tick Rhipicephalus (Boophilus) microplus

The increasing resistance of Rhipicephalus (Boophilus) microplus tick to commercial insecticides requires alternative methods for the control of this cattle plague. The enthomopathogenic fungus Beauveria feline produces destruxins in culture media, cyclic depsipeptides which display an array of biological activities. The present investigation aimed to evaluate the acaricide action of destruxins isolated from B. felina culture media on R. (B.) microplus engorged females. B. felina was grown in MF medium under 19 different growth conditions. HPLC-PDA analysis of chromatographic fractions obtained from the 19 different growth media extracts indicated the presence of destruxins in all lipophylic fractions. Such fractions were combined and subjected to separation by HPLC. Fractions containing distinct destruxins composition were tested against R. (B.) micro plus. Two fractions, composed of destruxin Ed(1) and pseudodestruxin B and/or pseudodestruxin C (fraction P1) as well as by hydroxyhomodestruxin B and/or destruxin D and/or roseotoxin C (fraction P7), displayed 30% and 28.7% acaricidal efficacy, respectively. This activity profile in such low concentration is adequate to consider destruxins as potential leading compounds to be developed for tick biological control. (C) 2012 Elsevier Inc. All rights reserved.

HELMINTH PARASITES OF HYPSIBOAS PRASINUS (ANURA: HYLIDAE) FROM TWO ATLANTIC FOREST FRAGMENTS, SAO PAULO STATE, BRAZIL

Seventy-seven males of Hypsiboas prasinus from 2 Atlantic forest fragments in the municipalities of Botucatu and Jundiai, sao Paulo State, Brazil, were examined for endoparasites. The frogs were captured in summer (January until March) and winter (July/August) of 2008 and 2009. Thirty-three males (75%) from Botucatu were infected with Rhabdias cf. fuelleborni, cosmocerciid nematodes, and Cylindrotaenia americana. Twenty-five tree frogs (78.5%) from Jundiai were infected by Rhabdias cf. fuelleborni, Physaloptera sp., and cosmocerciid nematodes. Only cosmocerciid nematodes presented a statistically significance difference in prevalence (z = 4.345; P < 0.001) and mean abundance (t = 562.0; P < 0.001) between Botucatu and Jundiai during the winter. Also, the cosmocerciids exhibited higher mean abundance (t = 196.0; P = 0.034) in winter when compared with summer at the Jundiai site. Moreover, to our knowledge, this is the first report of C. americana in the Brazilian Hylidae. This study presents 4 new records of nematodes in H. prasinus.

Habitat alteration and community-level effects of an invasive ecosystem engineer: a case study along the coast of NSW, Australia

We investigated the effects of the habitat-modifying green algae Caulerpa taxifolia on meiobenthic communities along the coast of New South Wales, Australia. Samples were taken from unvegetated sediments, sediments underneath the native seagrass Zostera capricorni, and sediments invaded by C. taxifolia at 3 sites along the coast. Meiofaunal responses to invasion varied in type and magnitude depending on the site, ranging from a slight increase to a substantial reduction in meiofauna and nematode abundances and diversity. The multivariate structure of meiofauna communities and nematode assemblages, in particular, differed significantly in sediments invaded by C. taxifolia when compared to native habitats, but the magnitude of this dissimilarity differed between the sites. These differential responses of meiofauna to C. taxifolia were explained by different sediment redox potentials. Sediments with low redox potential showed significantly lower fauna abundances, lower numbers of meiofaunal taxa and nematode species and more distinct assemblages. The response of meiofauna to C. taxifolia also depended on spatial scale. Whereas significant loss of benthic biodiversity was observed locally at one of the sites, at the larger scale C. taxifolia promoted an overall increase in nematode species richness by favouring species that were absent from the native environments. Finally, we suggest there might be some time-lags associated with the impacts of C. taxifolia and point to the importance of considering the time since invasion when evaluating the impact of invasive species.

First assessment of the endoparasitic nematode fauna of four psammophilous species of Tropiduridae (Squamata: Iguania) endemic to north-eastern Brazil

Tropiduridae (Squamata: Iguania) is a lizard taxon widely distributed in the neotropics. Among its representatives, some species are classified as generalists regarding habitat usage. Others exhibit a very restricted and probably relict distribution, and are strongly associated with predominantly sandy and dry habitats. Within this rather ecologically similar than phylogenetically closely related group we examined specimens of Eurolophosaurus amathites, E. divaricatus, Tropidurus hygomi, T. psammonastes for endoparasites. In all four species examined we recorded parasitic nematodes (Nemathelminthes: Nematoda). At least three nematode species were recovered: Parapharyngodon sp., Physaloptera lutzi and Strongyluris oscari, with Ph. lutzi being the most abundant parasite encountered in all lizard species examined. In spite of the hosts' habitat specialization, these parasites are also found frequently in non-psammophilous tropidurid species as well as in other squamates. Individual species richness per lizard was low, with usually just one species parasitizing at a time. These are the first parasites registered for these tropidurids and constitute a total of six new host records.