Organization of the macroinvertebrate community in a tropical annual agroecosystem into modules.

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.

Quantitative prey species detection in predator guts across multiple trophic levels by mapping unassembled shotgun reads.

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.

Changes in metabolism and antioxidant systems during tropical diapause in the sunflower caterpillar Chlosyne lacinia (Lepidoptera: Nymphalidae).

Insect diapause shares many biochemical features with other states of metabolic depression, including the suppression of global metabolism, reorganization of metabolic pathways and improved stress resistance. However, little is known about the biochemical changes associated with the diapause phenotype in tropical insects. To investigate biochemical adaptations associated with tropical diapause, we measured the activities of metabolic and antioxidant enzymes, as well as glutathione levels, in the sunflower caterpillar Chlosyne lacinia at different times after initiation of diapause (<1, 20, 40, 60, and 120 days) and after arousal from diapause. Biochemical changes occurred early in diapausing animals, between the first 24 h and 20 days of diapause. Diapausing animals had reduced oxidative capacity associated with a decrease in the activities of peroxide-decomposing antioxidant enzymes. There was no sign of redox imbalance either during diapause or after recovery from diapause. Noteworthy, glutathione transferase and isocitrate dehydrogenase-NADP+ activities sharply increased in diapausing animals and stand out as diapause-associated proteins. The upregulation of these two enzymes ultimately indicate the occurrence of Preparation for Oxidative Stress in the tropical diapause of C. lacinia.

Transfer of Cry1F from Bt maize to eggs of resistant Spodoptera frugiperda.

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.

Mitogenome sequence accuracy using different elucidation methods.

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.

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.

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.

Uptake and bioaccumulation of Cry toxins by an aphidophagous predator.

Uptake of Cry toxins by insect natural enemies has rarely been considered and bioaccumulation has not yet been demonstrated. Uptake can be demonstrated by the continued presence of Cry toxin after exposure has stopped and gut contents eliminated. Bioaccumulation can be demonstrated by showing uptake and that the concentration of Cry toxin in the natural enemy exceeds that in its food. We exposed larvae of the aphidophagous predator, Harmonia axyridis, to Cry1Ac and Cry1F through uniform and constant tritrophic exposure via an aphid, Myzus persicae, and looked for toxin presence in the pupae. We repeated the experiment using only Cry1F and tested newly emerged adults. Both Cry toxins were detected in pupae, and Cry1F was detected in recently emerged, unfed adults. Cry1Ac was present 2.05 times and Cry1F 3.09 times higher in predator pupae than in the aphid prey. Uptake and bioaccumulation in the third trophic level might increase the persistence of Cry toxins in the food web and mediate new exposure routes to natural enemies.

Sequestration and Transfer of Cry Entomotoxin to the Eggs of a Predaceous Ladybird Beetle.

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.

Transcriptome-Based Identification of Highly Similar Odorant-Binding Proteins among Neotropical Stink Bugs and Their Egg Parasitoid.

Olfaction plays a fundamental role in insect survival through resource location and intra and interspecific communications. We used RNA-Seq to analyze transcriptomes for odorant-binding proteins (OBPs) from major stink bug pest species in Brazil, Euschistus heros, Chinavia ubica, and Dichelops melacanthus, and from their egg parasitoid, Telenomus podisi. We identified 23 OBPs in E. heros, 25 OBPs in C. ubica, 9 OBPs in D. melacanthus, and 7 OBPs in T. podisi. The deduced amino acid sequences of the full-length OBPs had low intraspecific similarity, but very high similarity between two pairs of OBPs from E. heros and C. ubica (76.4 and 84.0%) and between two pairs of OBPs from the parasitoid and its preferred host E. heros (82.4 and 88.5%), confirmed by a high similarity of their predicted tertiary structures. The similar pairs of OBPs from E. heros and C. ubica may suggest that they have derived from a common ancestor, and retain the same biological function to bind a ligand perceived or produced in both species. The T. podisi OBPs similar to E. heros were not orthologous to any known hymenopteran OBPs, and may have evolved independently and converged to the host OBPs, providing a possible basis for the host location of T. podisi using E. heros semiochemical cues.

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.

Identification and sequence analysis of the Condylorrhiza vestigialis MNPV p74 gene.

The baculovirus Condylorrhiza vestigialis multiple nucleopolyhedrovirus (CoveMNPV), isolated from C. vestigialis infected larvae in Paraná (Brazil), was identified in our laboratory. A full-length clone was obtained from the CoveMNPV genome, of the gene that encodes the homolog to baculoviral p74, essential for oral infectivity which was then sequenced and characterized. The CoveMNPV p74 gene (GenBank accession number EU919397) contains an ORF of 1935 bp that encodes a deduced protein of 73.61 kDa. The phylogenetic affiliations of the CoveMNPV gene were determined by a heuristic search of 40 aligned baculovirus p74 nucleotide sequences using maximum parsimony (PAUP 4.0b4a). The phylogenetic analysis placed CoveMNPV within lepidopteran nucleopolyhedrovirus (NPV) Group I, Clade A, as being the closest to Choristoneura fumiferana defective NPV.

Expression and purification of a small heat shock protein from the plant pathogen Xylella fastidiosa.

The small heat shock proteins (smHSPs) belong to a family of proteins that function as molecular chaperones by preventing protein aggregation and are also known to contain a conserved region termed alpha-crystallin domain. Here, we report the expression, purification, and partial characterization of a novel smHSP (HSP17.9) from the phytopathogen Xylella fastidiosa, causal agent of the citrus variegated chlorosis (CVC). The gene was cloned into a pET32-Xa/LIC vector to over-express the protein coupled with fusion tags in Escherichia coli BL21(DE3). The expressed HSP17.9 was purified by immobilized metal affinity chromatography (IMAC) and had its identity determined by mass spectrometry (MALDI-TOF). The correct folding of the purified recombinant protein was verified by circular dichroism spectroscopy. Finally, the HSP17.9 protein also proved to efficiently prevent induced aggregation of insulin, strongly indicating a chaperone-like activity.