RESUMO
The structure of macroinvertebrate communities in agroecosystems has been assumed to be modular and organized around key herbivore pests. We characterized the macroinvertebrate community in the annual organic brassica agroecosystem in tropical central Brazil to determine if the community was a random assemblage of independent populations or was organized into repeatable multi-species components. We sampled 36 macroinvertebrate taxa associated with six organic brassica farms at biweekly intervals during the dry season during two years in the Distrito Federal, Brazil. We used an unconstrained ordination based on latent variable modeling (boral) with negative binomial population counts to analyze community composition independent of variation in sample abundance. We evaluated observed community structure by comparing it with randomized alternatives. We found that the community was not a random assemblage and consistently organized itself into two modules based around the major herbivores; one with lepidoptera and whiteflies and their associated natural enemies which was gradually replaced during the season by one with brassica aphids, aphid parasitoids and coccinellids. This analysis suggests that the historical and present-day focus on pest herbivores and their associated species in agroecosystems may be justified based on community structure.
Assuntos
Afídeos , Brassica , Lepidópteros , Animais , Herbivoria , BrasilRESUMO
The search for effective biological control agents without harmful non-target effects has been constrained by the use of impractical (field direct observation) or imprecise (cage experiments) methods. While advances in the DNA sequencing methods, more specifically the development of high-throughput sequencing (HTS), have been quickly incorporated in biodiversity surveys, they have been slow to be adopted to determine arthropod prey range, predation rate and food web structure, and critical information to evaluate the effectiveness and safety of a biological control agent candidate. The lack of knowledge on how HTS methods could be applied by ecological entomologists constitutes part of the problem, although the lack of expertise and the high cost of the analysis also are important limiting factors. In this review, we describe how the latest HTS methods of metabarcoding and Lazaro, a method to identify prey by mapping unassembled shotgun reads, can serve biological control research, showing both their power and limitations. We explain how they work to determine prey range and also how their data can be used to estimate predation rates and subsequently be translated into food webs of natural enemy and prey populations helping to elucidate their role in the community. We present a brief history of prey detection through molecular gut content analysis and also the attempts to develop a more precise formula to estimate predation rates, a problem that still remains. We focused on arthropods in agricultural ecosystems, but most of what is covered here can be applied to natural systems and non-arthropod biological control candidates as well.
Assuntos
Artrópodes , Animais , Ecossistema , Agentes de Controle Biológico , DNA/análise , Comportamento Predatório , Sequenciamento de Nucleotídeos em Larga EscalaRESUMO
Quantifying species trophic interaction strengths is crucial for understanding community dynamics and has significant implications for pest management and species conservation. DNA-based methods to identify species interactions have revolutionized these efforts, but a significant limitation is the poor ability to quantify the strength of trophic interactions, that is the biomass or number of prey consumed. We present an improved pipeline, called Lazaro, to map unassembled shotgun reads to a comprehensive arthropod mitogenome database and show that the number of prey reads detected is quantitatively predicted from the prey biomass consumed, even for indirect predation. Two feeding bioassays were performed: starved coccinellid larvae consuming different numbers of aphids (Prey Quantity bioassay), and starved coccinellid larvae consuming a chrysopid larvae that had consumed aphids (Direct and Indirect Predation bioassay). Prey taxonomic assignment against a mitochondrial genome database had high accuracy (99.8% positive predictive value) and the number of prey reads was directly related to the number of prey consumed and inversely related to the elapsed time since consumption with high significance (r2 = .932, p = 4.92E-6). Aphids were detected up to 6 h after direct predation plus 3 h after indirect predation (9 h in total) and detection was related to the predator-specific decay rates. Lazaro enabled quantitative predictions of prey consumption across multiple trophic levels with high taxonomic resolution while eliminating all false positives, except for a few confirmed contaminants, and may be valuable for characterizing prey consumed by field-sampled predators. Moreover, Lazaro is readily applicable for species diversity determination from any degraded environmental DNA.
Assuntos
Afídeos , Besouros , Animais , Cadeia Alimentar , Besouros/genética , Comportamento Predatório , Afídeos/genética , DNA/genéticaRESUMO
BACKGROUND: A central challenge of DNA gut content analysis is to identify prey in a highly degraded DNA community. In this study, we evaluated prey detection using metabarcoding and a method of mapping unassembled shotgun reads (Lazaro). RESULTS: In a mock prey community, metabarcoding did not detect any prey, probably owing to primer choice and/or preferential predator DNA amplification, while Lazaro detected prey with accuracy 43-71%. Gut content analysis of field-collected arthropod epigeal predators (3 ants, 1 dermapteran, and 1 carabid) from agricultural habitats in Brazil (27 samples, 46-273 individuals per sample) revealed that 64% of the prey species detections by either method were not confirmed by melting curve analysis and 87% of the true prey were detected in common. We hypothesized that Lazaro would detect fewer true- and false-positive and more false-negative prey with greater taxonomic resolution than metabarcoding but found that the methods were similar in sensitivity, specificity, false discovery rate, false omission rate, and accuracy. There was a positive correlation between the relative prey DNA concentration in the samples and the number of prey reads detected by Lazaro, while this was inconsistent for metabarcoding. CONCLUSIONS: Metabarcoding and Lazaro had similar, but partially complementary, detection of prey in arthropod predator guts. However, while Lazaro was almost 2× more expensive, the number of reads was related to the amount of prey DNA, suggesting that Lazaro may provide quantitative prey information while metabarcoding did not.
Assuntos
Artrópodes , Animais , Artrópodes/genética , Artrópodes/metabolismo , Brasil , DNA/metabolismo , Ecossistema , Humanos , Análise de Sequência de DNARESUMO
The intergenerational transfer of plant defense compounds by aposematic insects is well documented, and since 2006, has been shown for Cry toxins. Cry toxins are proteins naturally produced by the soil bacterium Bacillus thuringiensis (Bt) and its genes have been expressed in plants to confer insect pest resistance. In this work we tested if non-aposematic larvae of a major maize pest, Spodoptera frugiperda, with resistance to Cry1F, could transfer Cry1F from a genetically engineered maize variety to their offspring. Resistant 10-day-old larvae that fed on Cry1F Bt maize until pupation were sexed and pair-mated to produce eggs. Using ELISA we found that Cry1F was transferred to offspring (1.47-4.42 ng Cry1F/10 eggs), a toxin concentration about 28-83 times less than that detected in Cry1F Bt maize leaves. This occurred when only one or both sexes were exposed, and more was transferred when both parents were exposed, with transitory detection in the first five egg masses. This work is an unprecedented demonstration that a non-aposematic, but resistant, species can transfer Cry1F to their offspring when exposed to Bt host plant leaves as immatures.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Endotoxinas/genética , Endotoxinas/metabolismo , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Spodoptera/efeitos dos fármacos , Spodoptera/metabolismo , Zea mays/genética , Zea mays/metabolismo , Animais , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/farmacologia , Endotoxinas/farmacologia , Feminino , Proteínas Hemolisinas/farmacologia , Resistência a Inseticidas/genética , Larva/efeitos dos fármacos , Larva/genética , Óvulo/metabolismo , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , Transporte Proteico , Spodoptera/genéticaRESUMO
Constitutive expression of Odorant-Binding Proteins (OBPs) in antennae and other body parts has been examined mainly to infer their involvement in insect olfaction, while their regulation in response to semiochemical stimuli has remained poorly known. Previous studies of semiochemical response were basically done using electrophysiology, which integrates the response of the set of OBPs present in an antenna or sensillum, without revealing the regulation of OBPs or which ones might be involved. In this study we used boll weevil as a model and mined its OBPs by RNA-Seq to study their simultaneous antennal expression by qPCR under controlled semiochemical stimuli with aggregation pheromone and plant volatiles. In the absence of a semiochemical stimulus, 23 of 24 OBPs were constitutively expressed in the antenna in both sexes. Semiochemicals changed systemically the expression of OBPs in both sexes. There were different patterns of up- and down-regulation in female antennae for each semiochemical stimulus, consistent with female chemical ecology. On the other hand, the only response in males was down-regulation of some OBPs. We suggest that these systemic changes in OBP expression might be related to enhancing detection of the semiochemical stimuli and/or priming the olfactory system to detect other environmental chemicals.
Assuntos
Regulação da Expressão Gênica , Proteínas de Insetos/genética , Feromônios/metabolismo , Receptores Odorantes/genética , Gorgulhos/genética , Sequência de Aminoácidos , Animais , Antenas de Artrópodes/química , Antenas de Artrópodes/metabolismo , Feminino , Proteínas de Insetos/análise , Masculino , Receptores Odorantes/análise , Alinhamento de Sequência , Caracteres Sexuais , Transcriptoma , Gorgulhos/química , Gorgulhos/metabolismoRESUMO
Mitogenome sequences are highly desired because they are used in several biological disciplines. Their elucidation has been facilitated through the development of massive parallel sequencing, accelerating their deposition in public databases. However, sequencing, assembly and annotation methods might induce variability in their quality, raising concerns about the accuracy of the sequences that have been deposited in public databases. In this work we show that different sequencing methods (number of species pooled in a library, insert size and platform) and assembly and annotation methods generated variable completeness and similarity of the resulting mitogenome sequences, using three species of predaceous ladybird beetles as models. The identity of the sequences varied considerably depending on the method used and ranged from 38.19 to 90.1% for Cycloneda sanguinea, 72.85 to 91.06% for Harmonia axyridis and 41.15 to 93.60% for Hippodamia convergens. Dissimilarities were frequently found in the non-coding A+T rich region, but were also common in coding regions, and were not associated with low coverage. Mitogenome completeness and sequence identity were affected by the sequencing and assembly/annotation methods, and high within-species variation was also found for other mitogenome depositions in GenBank. This indicates a need for methods to confirm sequence accuracy, and guidelines for verifying mitogenomes should be discussed and developed by the scientific community.
Assuntos
Genoma Mitocondrial , Sequenciamento de Nucleotídeos em Larga Escala/métodos , AnimaisRESUMO
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.
Assuntos
Besouros/fisiologia , Cadeia Alimentar , Conteúdo Gastrointestinal/química , Insetos/fisiologia , Análise de Sequência de DNA/métodos , AnimaisRESUMO
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.
Assuntos
Afídeos , Besouros/fisiologia , Comportamento Predatório/fisiologia , Estresse Fisiológico , Animais , Meio Ambiente , Larva , ReproduçãoRESUMO
BACKGROUND: The frequency of resistance alleles is a major factor influencing the rate of resistance evolution. Here, we adapted the F2 screen procedure for Spodoptera frugiperda (J. E. Smith) with a discriminating concentration assay, and extended associated statistical methods to estimate the frequency of resistance to Cry1F protein in S. frugiperda in Brazil when resistance was not rare. RESULTS: We show that F2 screen is efficient even when the resistance frequency is 0.250. It was possible to screen 517 isoparental lines from 12 populations sampled in five states of Brazil during the first half of 2012. Western Bahia had the highest allele frequency of Cry1F resistance, 0.192, with a 95% confidence interval (CI) between 0.163 and 0.220. All other states had a similar and lower frequency varying from 0.042 in Paraná to 0.080 in Mato Grosso do Sul. CONCLUSION: The high frequency in western Bahia may be related to year-round availability of maize, the high population density of S. frugiperda, the lack of refuges and the high adoption rate of Cry1F maize. Cry1F resistance alleles were not rare and occurred at frequencies that have already compromised the useful life of TC1507 maize in western Bahia. © 2016 Society of Chemical Industry.
Assuntos
Proteínas de Bactérias/genética , Endotoxinas/genética , Frequência do Gene , Proteínas Hemolisinas/genética , Resistência a Inseticidas/genética , Spodoptera/genética , Animais , Toxinas de Bacillus thuringiensis , Brasil , Plantas Geneticamente Modificadas/genética , Zea mays/genéticaRESUMO
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.
Assuntos
Afídeos/fisiologia , Proteínas de Bactérias/toxicidade , Besouros/fisiologia , Endotoxinas/toxicidade , Proteínas Hemolisinas/toxicidade , Controle Biológico de Vetores/métodos , Animais , Toxinas de Bacillus thuringiensis , Relação Dose-Resposta a Droga , Larva , Medição de RiscoRESUMO
BACKGROUND: Dominance of resistance has been one of the major parameters affecting the rate of evolution of resistance to Bt crops. High dose is the capacity of Bt crops to kill heterozygous insects and has been an essential component of the most successful strategy to manage resistance to these crops. Experiments were conducted to evaluate directly and indirectly whether the TC1507 event is high dose to Spodoptera frugiperda (JE Smith). RESULTS: About 8% of heterozygote neonate larvae were able to survive, complete larval development and emerge as normal adults on TC1507 leaves, while susceptible larvae could not survive for 5 days. The estimated dominance of resistance was 0.15 ± 0.09 and significantly higher than zero; therefore, the resistance to Cry1F expressed in TC1507 was not completely recessive. A 25-fold dilution of TC1507 maize leaf tissue in an artificial diet was able to cause a maximum mortality of only 37%, with growth inhibition of 82% at 7 days after larval infestation. CONCLUSION: Resistance to Cry1F in TC1507 maize is incompletely recessive in S. frugiperda. TC1507 maize is not high dose for S. frugiperda. Additional or alternative resistance management strategies, such as the replacement of single-trait Bt maize with pyramided Bt maize, which produces multiple proteins targeting the same insect pests, should be implemented wherever this technology is in use and S. frugiperda is the major pest.
Assuntos
Proteínas de Bactérias/farmacologia , Endotoxinas/farmacologia , Proteínas Hemolisinas/farmacologia , Resistência a Inseticidas , Inseticidas/farmacologia , Spodoptera/efeitos dos fármacos , Zea mays/genética , Animais , Toxinas de Bacillus thuringiensis , Brasil , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/genética , Spodoptera/crescimento & desenvolvimentoRESUMO
In the past 10 years, sequestration of Cry toxins and transfer to offspring has been indicated in three insect species in laboratory studies. This work directly demonstrates the sequestration and intergenerational transfer of Cry1F by the parents of the aphidophagous coccinellid predator, Harmonia axyridis, to its offspring. Recently emerged adults (10 individual couples/cage/treatment) were exposed during 20 days to aphids (100 Myzus persicae each day) that fed on a holidic diet containing 20 µg/mL Cry1F (and a control-group). Egg batches and neonate larvae were monitored daily, and counted and weighed for immunodetection of Cry1F by ELISA. At the end of the bioassay, the parents were weighed and analyzed by ELISA. Cry1F was detected in the offspring, both eggs and neonate larvae, of exposed H. axyridis adults. On average the neonate larvae had 60% of the Cry1F concentration of the eggs from the same egg batch. The Cry1F concentration in the adults was positively correlated with the concentration in their eggs. These three results provided independent evidence of transfer to offspring. No detrimental effects of Cry1F were observed on the age of first reproduction, total number of eggs laid per female, age-specific fecundity, egg development time, hatching rate, or fertility rate. The occurrence and generality of intergenerational transfer of Cry toxins should be investigated in the field to determine its potential ecological implications.
Assuntos
Besouros/metabolismo , Endotoxinas/metabolismo , Animais , Afídeos/fisiologia , Besouros/crescimento & desenvolvimento , Endotoxinas/análise , Endotoxinas/genética , Ensaio de Imunoadsorção Enzimática , Feminino , Larva/metabolismo , Óvulo/metabolismo , Comportamento PredatórioRESUMO
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.
Assuntos
Afídeos/genética , Besouros/fisiologia , DNA/genética , DNA/isolamento & purificação , Enterobacteriaceae/genética , Trato Gastrointestinal/química , Metagenômica , Animais , Afídeos/classificação , Afídeos/microbiologia , Enterobacteriaceae/classificação , Dados de Sequência Molecular , Comportamento Predatório , Análise de Sequência de DNARESUMO
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 â¼65 kDa insecticidal protein can be taken up and transferred to descendants where it can increase mortality and development time.
Assuntos
Proteínas de Bactérias/metabolismo , Ovos , Endotoxinas/metabolismo , Proteínas Hemolisinas/metabolismo , Lepidópteros/metabolismo , Animais , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/toxicidade , Bioensaio , Endotoxinas/toxicidade , Feminino , Proteínas Hemolisinas/toxicidade , Larva/efeitos dos fármacos , Lepidópteros/crescimento & desenvolvimento , Plantas Geneticamente ModificadasRESUMO
Under the umbrella of the International Organisation of Biological Control (IOBC), an international working group of public sector scientists entitled on "Transgenic Organisms in Integrated Pest Management and Biological Control" has been organized. The group will develop scientific principles and detailed scientific guidelines for biosafety testing of transgenic crops. The key elements of this project are: (1) An international initiative including expert scientists from leading research institutions in developed and developing countries; (2) coordination of the development and implementation of the guidelines as a dynamic process, which will include scientific and technical capacity building and communication among scientists and between scientists and policy makers; (3) rapid serial publication of sections of the guidelines as they are completed; and (4) rapid and timely revision of previously published sections. The guidelines will be constructed on a case-by-case basis and will have no regulatory legitimacy themselves.