Limitations in dose-response and surrogate species methodologies for risk assessment of Cry toxins on arthropod natural enemies.

Dose-response assays and surrogate species are standard methods for risk analysis for environmental chemicals. These assume that individuals within a species have unimodal responses and that a surrogate species can predict responses of other related taxa. We exposed immature individuals of closely related aphidophagous coccinellid predators, Cycloneda sanguinea and Harmonia axyridis, to Cry1Ac and Cry1F toxins through uniform and constant artificial tritrophic exposure through Myzus persicae aphids. Both toxins were detected in coccinellid pupae, with individual and interspecific variation. Uptake was significantly higher in H. axyridis than in C. sanguinea, both in the proportion of individuals and the concentrations per individual. We also observed bimodal uptake of the Cry toxins by H. axyridis, which indicated that some individuals had low bioaccumulation and some had high bioaccumulation. This suggests that standard dose-response assays need to be interpreted with caution and future assays should examine the modality of the responses. In addition, the similarity in the biological effects of the Cry toxins in the two predators was due to different biological exposure mechanisms. The majority of H. axyridis were exposed both internally and in the gut, while C. sanguinea was exposed primarily in the gut. Thus, despite their close phylogenetic relatedness, these species would not be good surrogates for each other and the surrogate species methodology should be tested more rigorously.

Artificial tritrophic exposure system for environmental risk analysis on aphidophagous predators.

We evaluated an artificial tritrophic exposure system for use in ecotoxicological evaluations of environmental stressors on aphidophagous predators. It consists of an acrylic tube with a Parafilm M sachet containing liquid aphid diet, into which can be added environmental stressors. Immature Cycloneda sanguinea, Harmonia axyridis and Chrysoperla externa, and adult H. axyridis were reared on Myzus persicae. Larval and pupal development and survival of all species and reproductive parameters of H. axyridis were similar to published results. The system provides a suitable tritrophic exposure route, enables ex-ante evaluation of stressors, and improves the accuracy of the assessment.

Uncovering Trophic Interactions in Arthropod Predators through DNA Shotgun-Sequencing of Gut Contents.

Characterizing trophic networks is fundamental to many questions in ecology, but this typically requires painstaking efforts, especially to identify the diet of small generalist predators. Several attempts have been devoted to develop suitable molecular tools to determine predatory trophic interactions through gut content analysis, and the challenge has been to achieve simultaneously high taxonomic breadth and resolution. General and practical methods are still needed, preferably independent of PCR amplification of barcodes, to recover a broader range of interactions. Here we applied shotgun-sequencing of the DNA from arthropod predator gut contents, extracted from four common coccinellid and dermapteran predators co-occurring in an agroecosystem in Brazil. By matching unassembled reads against six DNA reference databases obtained from public databases and newly assembled mitogenomes, and filtering for high overlap length and identity, we identified prey and other foreign DNA in the predator guts. Good taxonomic breadth and resolution was achieved (93% of prey identified to species or genus), but with low recovery of matching reads. Two to nine trophic interactions were found for these predators, some of which were only inferred by the presence of parasitoids and components of the microbiome known to be associated with aphid prey. Intraguild predation was also found, including among closely related ladybird species. Uncertainty arises from the lack of comprehensive reference databases and reliance on low numbers of matching reads accentuating the risk of false positives. We discuss caveats and some future prospects that could improve the use of direct DNA shotgun-sequencing to characterize arthropod trophic networks.

Detection and decay rates of prey and prey symbionts in the gut of a predator through metagenomics.

DNA methods are useful to identify ingested prey items from the gut of predators, but reliable detection is hampered by low amounts of degraded DNA. PCR-based methods can retrieve minute amounts of starting material but suffer from amplification biases and cross-reactions with the predator and related species genomes. Here, we use PCR-free direct shotgun sequencing of total DNA isolated from the gut of the harlequin ladybird Harmonia axyridis at five time points after feeding on a single pea aphid Acyrthosiphon pisum. Sequence reads were matched to three reference databases: Insecta mitogenomes of 587 species, including H. axyridis sequenced here; A. pisum nuclear genome scaffolds; and scaffolds and complete genomes of 13 potential bacterial symbionts. Immediately after feeding, multicopy mtDNA of A. pisum was detected in tens of reads, while hundreds of matches to nuclear scaffolds were detected. Aphid nuclear DNA and mtDNA decayed at similar rates (0.281 and 0.11 h(-1) respectively), and the detectability periods were 32.7 and 23.1 h. Metagenomic sequencing also revealed thousands of reads of the obligate Buchnera aphidicola and facultative Regiella insecticola aphid symbionts, which showed exponential decay rates significantly faster than aphid DNA (0.694 and 0.80 h(-1) , respectively). However, the facultative aphid symbionts Hamiltonella defensa, Arsenophonus spp. and Serratia symbiotica showed an unexpected temporary increase in population size by 1-2 orders of magnitude in the predator guts before declining. Metagenomics is a powerful tool that can reveal complex relationships and the dynamics of interactions among predators, prey and their symbionts.

Uptake and transfer of a Bt toxin by a Lepidoptera to its eggs and effects on its offspring.

Research on non-target effects of transgenic crop plants has focused primarily on bitrophic, tritrophic and indirect effects of entomotoxins from Bacillus thuringiensis, but little work has considered intergenerational transfer of Cry proteins. This work reports a lepidopteran (Chlosyne lacinia) taking up a Bt entomotoxin when exposed to sublethal or low concentrations, transferring the entomotoxin to eggs, and having adverse effects on the first filial generation (F1) offspring. Two bioassays were conducted using a sublethal concentration of toxin (100.0 ng/µl Cry1Ac) for adults and a concentration equal to the LC10 (2.0 ng/µl Cry1Ac) for larvae. Cry1Ac is the most common entomotoxin expressed in Bt cotton in Brazil. In the adult diet bioassay there was no adverse effect on the parental generation (P0) adults, but the F1 larvae had higher mortality and longer development time compared to F1 larvae of parents that did not ingest Cry1Ac. For the 3rd instar larvae, there was no measurable effect on the P0 larvae, pupae and adults, but the F1 larvae had higher mortality and longer development time. Using chemiluminescent Western Blot, Cry1Ac was detected in F1 eggs laid by P0 butterflies from both bioassays. Our study indicates that, at least for this species and these experimental conditions, a ∼65 kDa insecticidal protein can be taken up and transferred to descendants where it can increase mortality and development time.

Alternative food sources and overwintering feeding behavior of the boll weevil, Anthonomus grandis boheman (Coleoptera: Curculionidae) under the tropical conditions of Central Brazil.

The boll weevil causes serious damage to the cotton crop in South America. Several studies have been published on this pest, but its phenology and behavior under the tropical conditions prevailing in Brazil are not well-known. In this study the feeding behavior and main food sources of adult boll weevils throughout the year in Central Brazil was investigated. The digestive tract contents of insects captured in pheromone traps in two cotton fields and two areas of native vegetation (gallery forest and cerrado sensu stricto) were analyzed. The insect was captured all through the year only in the cerrado. It fed on pollen of 19 different plant families, on Pteridophyta and fungi spores and algae cysts. Simpson Index test showed that the cerrado provided greater diversity of pollen sources. In the beginning of the cotton cycle, the plant families used for pollen feeding were varied: in cotton area 1, the weevil fed on Poaceae(50%), Malvaceae and Smilacaceae (25% each); in cotton area 2 the pollen sources were Malvaceae (50%), Asteraceae (25%) and Fabaceae and Clusiaceae (25% each); in the cerrado they were Chenopodiaceae (67%) and Scheuchzeriaceae (33%). No weevils were collected in the gallery forest in this period. After cotton was harvested, the family Smilacaceae was predominant among the food plants exploited in all the study areas. These results help to explain the survivorship of adult boll weevil during cotton fallow season in Central Brazil and they are discussed in the context of behavioral adaptations to the prevailing tropical environmental conditions.

Sex attractant pheromone from the neotropical red-shouldered stink bug, Thyanta perditor (F.).

Olfactometer bioassays showed that odors from mature Thyanta perditor males attracted females but not males. Furthermore, odors from females did not attract either sex, indicating that like other phytophagous pentatomid bugs, the males produce a sex pheromone. Attraction appeared to peak in late afternoon to evening. The headspace volatiles collected from male and female T. perditor were analyzed by GC-MS and HPLC. A male-specific compound, methyl (2E,4Z,6Z)-decatrienoate (2E,4Z,6Z-10:COOMe), was identified along with a number of other compounds found in extracts from both sexes. Bioassays carried out with 2E,4Z,6Z-10:COOMe showed it was as attractive to females as the crude extract of male volatiles, suggesting that the male-produced sex pheromone consists of 2E,4Z,6Z-10:COOMe as a single component. Consecutive volatiles collections from males showed that 2E,4Z,6Z-10:COOMe began appearing in extracts from males about 9 d after the final molt, as the males became sexually mature.

Alternative food sources and overwintering feeding behavior of the boll weevil, Anthonomus grandis boheman (coleoptera: curculionidae) underthe tropical conditions of central Brazil

The boll weevil causes serious damage to the cotton crop in South America. Several studies have been published on this pest, but its phenology and behavior under the tropical conditions prevailing in Brazil are not well-known. In this study the feeding behavior and main food sources of adult boll weevils throughout the year in Central Brazil was investigated. The digestive tract contents of insects captured in pheromone traps in two cotton fields and two areas of native vegetation (gallery forest and cerrado sensu stricto) were analyzed. The insect was captured all through the year only in the cerrado. It fed on pollen of 19 different plant families, on Pteridophyta and fungi spores and algae cysts. Simpson Index test showed that the cerrado provided greater diversity of pollen sources. In the beginning of the cotton cycle, the plant families used for pollen feeding were varied: in cotton area 1, the weevil fed on Poaceae(50 percent), Malvaceae and Smilacaceae (25 percent each); in cotton area 2 the pollen sources were Malvaceae (50 percent), Asteraceae (25 percent) and Fabaceae and Clusiaceae (25 percent each); in the cerrado they were Chenopodiaceae (67 percent) and Scheuchzeriaceae (33 percent). No weevils were collected in the gallery forest in this period. After cotton was harvested, the family Smilacaceae was predominant among the food plants exploited in all the study areas. These results help to explain the survivorship of adult boll weevil during cotton fallow season in Central Brazil and they are discussed in the context of behavioral adaptations to the prevailing tropical environmental conditions.

Predation as a Mortality Factor in Populations of the Spittlebug, Deois flavopicta Stål (Homoptera: Cercopidae) / A Predação Como Fator de Mortalidade em Populações da Cigarrinha-das-Pastagens, Deois flavopicta Stål (Homoptera: Cercopidae)

A cigarrinha-das-pastagens Deois flavopicta StÕl é a principal praga de pastagens cultivadas nos Cerrados do Brasil Central. O inseto apresenta distribuição sazonal com três gerações anuais discretas durante a estação chuvosa (setembro-abril) e população sincronizada de ovos diapáusicos durante a estação seca (maio-agosto). Experimentos em pastagens cultivadas mostraram que predadores não-específicos foram capazes de alterar significativamente as taxas de mortalidade de ovos diapáusicos e ninfas de D. flavopicta. Predação foi um fator de mortalidade independente da densidade, reduzindo os ovos diapáusicos em aproximadamente 60 por cento e ninfas de 20 por cento a 47 por cento. A taxa de mortalidade das ninfas resultante da predação não variou significativamente entre a primeira e a segunda geração anual. No entanto, a mortalidade de ninfas na terceira geração foi significativamente mais baixa, fato relacionado ao maior grau de agregação das ninfas em refúgios espaciais. Observações diretas de interações predador-presa nas pastagens sugerem que, potencialmente, as formigas representam o grupo que mais contribui para a mortalidade observada, embora esse impacto deva ainda ser verificado experimentalmente. Taxas de mortalidade mais altas em ovos e ninfas de D. flavopicta expostos aos predadores indicam que a predação pode ser um fator importante na determinação da população de adultos. Desta forma, práticas de manejo das pastagens que perturbam a comunidade de predadores podem resultar em densidades mais altas das cigarrinhas.

The spittlebug, Deois flavopicta StÕl, is the main pest in cultivated pastures of the "Cerrados" (savanna) in the central region of Brazil. The insect has three discrete generations during the rainy season (September-April) and a synchronized population of diapausing eggs during the dry season (May-August). Experiments in cultivated pastures showed that nonspecific predators were able to affect significantly the mortality rates of diapausing eggs and nymphs of D. flavopicta. Predation was a density independent mortality factor that reduced diapausing eggs by approximately 60 percent and nymphs by 20 percent to 47 percent. The nymph mortality rate due to predation did not differ during the first and the second generations of the year. For the third generation however, the mortality rate was lower, related to a greater degree of nymphal aggregation in spatial refuges. Our direct observations indicate that among the types of predators in pastures, ants potentially make the greatest contribution to mortality, although their impact needs experimental verification. High rates of mortality of eggs and nymphs of D. flavopicta exposed to predators indicate that predation can be an important factor determining the size of adult populations. Therefore, management practices that disturb the predator community may increase the population densities of the spittlebug.

Cinética da esporulação e viabilidade de conídios de Nomuraea rileyi (Farlow) samson sobre cadáveres da lagarta-da-soja, Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae), em condições de campo / Sporulation kinetics and conidial viability of Nomuraea rileyi (Farlow) samson on cadavers of the velvetbean caterpillar, Anticarsia gemmatalis Hübner (Lep: Noctuidae) in the field

The entomopathogenic fungus Nomuraea rileyi (Farlow) Samson produces epizootics on populations of the velvetbean caterpillar, Anticarsia gemmatalis Hübner, resulting in its natural control. However, sometimes this phenomenon does not occur soon enough to prevent pest population outbreaks and damage to the crop. Observations of N. rileyi conidial production on the surface of A. gemmatalis cadavers in the field showed that high relative humidity and precipitation determine the sporulation dynamics. In fact, initiation of sporulation is delayed and conidia production is reduced when A. gemmatalis cadavers infected with N. rileyi are exposed to water stress (relative humidity 25-30%) in the laboratory. Field observations also demonstrated that N. rileyi conidial viability remain high (>75% germination) until 10 days on A. gemmatalis cadaver surface, acting as an inoculum source for this period.

O fungo entomopatogênico Nomuraea rileyi (Farlow) Samson pode produzir epizootias em populações de lagarta-da-soja, Anticarsia gemmatalis Hübner, controlando essa praga naturalmente. No entanto, o fenômeno nem sempre ocorre a tempo de evitar que populações de A. gemmatalis atinjam o nível de dano econômico na cultura. Observações sobre a produção de conídios em cadáveres de A. gemmatalis em condições de campo mostraram que alta umidade relativa e precipitação são fatores determinantes na cinética da esporulação do fungo in vivo. O início da esporulação foi retardado e a produção de conídios diminuída quando cadáveres de A. gemmatalis infectados com N. rileyi foram submetidos a períodos longos de estresse hídrico (umidade relativa de 25-30%). Observações no campo também demonstraram que a viabilidade de conídios de N. rileyi pode permanecer elevada (>75% germinação) por períodos de até 10 dias na superfície de cadáveres, servindo como fonte de inóculo durante esse período.

The Environmental Effects of Genetically Modified Crops Resistant to Insects: [review] / Efeitos Ambientais de Culturas Geneticamente Modificadas Resistentes a Insetos: revisão]

Cultivares transgênicas de várias culturas estão sendo utilizadas em escala comercial em muitos países. A área dedicada ao cultivo com plantas transgênicas resistentes às pragas em todo o mundo alcançou 13 milhões de hectares em 2001. As cultivares transgênicas proporcionam benefícios, mas também apresentam riscos potenciais. As avaliações do seu impacto no ambiente são conduzidas antes da sua aprovação para uso comercial, como requerido pelas normas de biossegurança. Nesta revisão, serão discutidas as conseqüências ecológicas potenciais do uso comercial na agricultura de cultivares geneticamente modificadas que apresentam resistência aos insetos-pragas. Também serão discutidos os impactos ambientais causados pelas mudanças nas práticas agrícolas, identificando-se falhas e oportunidades de pesquisa, considerando-se essa nova ferramenta tecnológica. Os comentários e análises serão baseados no conhecimento atual que se tem dos riscos e beneficios do uso de cultivares resistentes a insetos, geneticamente modificadas, dentro do contexto dos programas de manejo integrado de pragas tradicionais.

Transgenic crops are currently being cultivated on a commercial scale in many countries. The area devoted to transgenic pest resistant varieties worldwide reached 13 million hectares in 2001. These varieties offer valuable benefits but also pose potential risks. Assessments of their impact on the environment are conducted before they are approved for commercial use, as required by the regulatory biosafety frameworks. In this review, we discuss the potential ecological consequences of the commercial use in agriculture of genetically modified insect resistant crops. We also discuss the impacts caused by the change in agricultural practices, and attempt to identify gaps and possible opportunities for research, considering this new technological tool. We based our analysis and comments on the current knowledge of the risks and benefits of these genetically modified insect resistant crops, within the context of traditional insect management strategies.