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.

Background: Halyomorpha halys (Stål), the brown marmorated stink bug, is a highly invasive insect species due in part to its exceptionally high levels of polyphagy. This species is also a nuisance due to overwintering in humanmade structures. It has caused significant agricultural losses in recent years along the Atlantic seaboard of North America and in continental Europe. Genomic resources will assist with determining the molecular basis for this species' feeding and habitat traits, defining potential targets for pest management strategies.

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.

The brown marmorated stink bug, Halyomorpha halys, is a devastating invasive species in the USA. Similar to other insects, olfaction plays an important role in its survival and reproduction. As odorant-binding proteins (OBPs) are involved in the initial semiochemical recognition steps, we used RNA-Sequencing (RNA-Seq) to identify OBPs in its antennae, and studied their expression pattern in different body parts under semiochemical stimulation by either aggregation or alarm pheromone or food odorants. Thirty full-length putative HhalOBPs were identified, corresponding to 22 'classic' OBPs and eight 'Plus-C' OBPs. The similarity amongst them ranged from 4.95-70.92%, and with another 325 hemipteran OBPs similarity ranged from 1.94-91.51%, the highest levels being with other stink bug OBPs. Phylogenetic analysis confirmed the monophyly of seven groups of stink bug and other hemipteran OBPs. All 30 HhalOBPs were expressed and about 2/3 were expressed primarily in antennae. The expression of 21 HhalOBPs was higher in the antennae under alarm pheromone stimulus, indicating that multiple OBPs may be responding to this pheromone. Two were highest in antennae under aggregation pheromone stimulus. These findings should provide a basis for understanding the physiological functions of HhalOBPs and the chemosensory perception of this pest, which may help to uncover new control targets for behavioural interference.

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.

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.

The boll weevil (Anthonomus grandis Boheman) (Coleoptera: Curculionidae) is an introduced pest in Brazil, which in 30 yr has successfully expanded to various eco-regions and became the most important pest of cotton (Gossypium hirsutum, Malvaceae). Given the limited knowledge about the adaptive mechanisms that allowed successful establishment of the pest population in a tropical region, in this work we studied the potential of the Midwest population of boll weevils to enter a reproductive dormancy and identified the importance of the feeding source for induction of dormancy. We investigated morphological and physiological characters as indicators of the dormancy. We also investigated the occurrence of reproductive dormancy in boll weevils populations from cotton farms of the Midwestern region of Brazil during the cotton and noncotton seasons of 2009 and 2010. The studies revealed that boll weevils entered facultative reproductive dormancy; however, unlike what has been observed for boll weevils from temperate and subtropical regions, the hypertrophy of fat body and hexamerin levels did not straightly correlated to reproductive dormancy. The food source and field conditions during early adult development were decisive factor for the induction of reproductive dormancy. The incidence of reproductive dormancy increased progressively as the phenology of cotton plant advanced, reaching approximately 90% at the end of the crop season. During the noncotton season, the boll weevil was predominantly found in reproductive dormancy, especially females; however, there is evidence of use of multiple adaptive strategies to colonize the next harvest.

Administration of nitrous oxide with desflurane results in maintenance of normal IOP and prevents a decrease in horizontal PD during anesthesia. Therefore, this may be a suitable protocol in dogs undergoing intraocular surgeries that require mydriasis and maintenance of normal IOP.