Comparative Genomics of Pesticide-Degrading Enterococcus Symbionts of Spodoptera frugiperda (Lepidoptera: Noctuidae) Leads to the Identification of Two New Species and the Reappraisal of Insect-Associated Enterococcus Species.

Enterococcus species have been described as core members of the microbial community of Spodoptera frugiperda (Lepidoptera: Noctuidae) and have been previously reported as insecticide degrading agents. This study aimed to investigate the molecular composition of these microbial symbionts of S. frugiperda to better understand their association with the host and their potential for insecticide metabolization. Through phenotypic assays and comparative genomic analyses of several pesticide-degrading Enterococcus isolated from the gut of S. frugiperda larvae, we identified two new species: Enterococcus entomosocium n. sp. and Enterococcus spodopteracolus n. sp. Their identities as new species were confirmed by whole genome alignment, utilizing cut-offs of 95-96% for the average nucleotide identity (ANI) and 70% for the digital DNA: DNA hybridization (dDDH) values. The systematic positioning of these new species within the genus Enterococcus was resolved using genome-based analysis, revealing Enterococcus casseliflavus as a sister group of E. entomosocium n. sp., and Enterococcus mundtii as a sister group of E. spodopteracolus n. sp. Comparative genomic analyses of several isolates of E. entomosocium n. sp. and E. spodopteracolus n. sp. provided a better assessment of the interactions established in the symbiotic association with S. frugiperda and led to the discovery of misidentified new species of Enterococcus associated with insects. Our analyses indicated that the potential of E. entomosocium n. sp. and E. spodopteracolus n. sp. to metabolize different pesticides arises from molecular mechanisms that result in rapid evolution of new phenotypes in response to environmental stressors, in this case, the pesticides their host insect is exposed to.

The molecular interplay of the establishment of an infection - gene expression of Diaphorina citri gut and Candidatus Liberibacter asiaticus.

BACKGROUND: Candidatus Liberibacter asiaticus (CLas) is one the causative agents of greening disease in citrus, an unccurable, devastating disease of citrus worldwide. CLas is vectored by Diaphorina citri, and the understanding of the molecular interplay between vector and pathogen will provide additional basis for the development and implementation of successful management strategies. We focused in the molecular interplay occurring in the gut of the vector, a major barrier for CLas invasion and colonization. RESULTS: We investigated the differential expression of vector and CLas genes by analyzing a de novo reference metatranscriptome of the gut of adult psyllids fed of CLas-infected and healthy citrus plants for 1-2, 3-4 and 5-6 days. CLas regulates the immune response of the vector affecting the production of reactive species of oxygen and nitrogen, and the production of antimicrobial peptides. Moreover, CLas overexpressed peroxiredoxin, probably in a protective manner. The major transcript involved in immune expression was related to melanization, a CLIP-domain serine protease we believe participates in the wounding of epithelial cells damaged during infection, which is supported by the down-regulation of pangolin. We also detected that CLas modulates the gut peristalsis of psyllids through the down-regulation of titin, reducing the elimination of CLas with faeces. The up-regulation of the neuromodulator arylalkylamine N-acetyltransferase implies CLas also interferes with the double brain-gut communication circuitry of the vector. CLas colonizes the gut by expressing two Type IVb pilin flp genes and several chaperones that can also function as adhesins. We hypothesized biofilm formation occurs by the expression of the cold shock protein of CLas. CONCLUSIONS: The thorough detailed analysis of the transcritome of Ca. L. asiaticus and of D. citri at different time points of their interaction in the gut tissues of the host led to the identification of several host genes targeted for regulation by L. asiaticus, but also bacterial genes coding for potential effector proteins. The identified targets and effector proteins are potential targets for the development of new management strategies directed to interfere with the successful utilization of the psyllid vector by this pathogen.

Microbial Diversity and Chemical Multiplicity of Culturable, Taxonomically Similar Bacterial Symbionts of the Leaf-Cutting Ant Acromyrmex coronatus.

Insects are a highly diverse group, exploit a wide range of habitats, and harbor bacterial symbionts of largely unknown diversity. Insect-associated bacterial symbionts are underexplored but promising sources of bioactive compounds. The community of culturable bacteria associated with the leaf-cutting ant Acromyrmex coronatus (Fabricius) and the diversity of their metabolites produced were investigated. Forty-six phylotypes belonging to Actinobacteria, Firmicutes, and Proteobacteria were identified. The chemical profiles of 65 isolates were further analyzed by LC-MS/MS, and principal components analysis (PCA) was used to group the isolates according to their chemical profiles. Historically, selection of bacterial strains for drug discovery has been based on phenotypic and/or genotypic traits. Use of such traits may well impede the discovery of new compounds; in this study, several indistinguishable phylotypes cultured in identical nutritional and environmental conditions produced completely different chemical profiles. Our data also demonstrated the wide chemical diversity to be explored in insect-associated symbionts.

Culturable symbionts associated with the reproductive and digestive tissues of the Neotropical brown stinkbug Euschistus heros.

Symbionts are widely distributed in eukaryotes, and potentially affect the physiology, ecology and evolution of their host. Most insects harbour free-living bacteria in their haemocoel and gut lumen, intracellular-living bacteria in a range of tissues or bacteria in host-derived specialized cells. Stinkbugs, as do many arthropods, harbour extracellular bacteria in the gut that may affect the fitness of their host. This study identified the culturable symbionts associated with the ovaries, spermatheca, seminal vesicle and posterior midgut region (V4) of males and females of Euschistus heros (F.) (Hemiptera: Pentatomidae). Several culture media were used to isolate the bacteria associated with these structures. The selected colonies (morphotypes) were cultured in liquid medium, subjected to genomic DNA extraction, 16S rRNA gene amplification, and restriction fragment length polymorphism (RFLP) analyses. Morphotypes with distinct RFLP patterns were purified and sequenced, and the sequences obtained were used for putative identification and phylogenetic analysis. Comparison of the sequences with those available in the EzTaxon-e database and the use of a matrix of paired distances grouped the isolates in phylotypes belonging to the Phylum Proteobacteria. Proteobacteria was represented by γ-Proteobacteria phylotypes belonging to Enterobacteriaceae, while Firmicutes had Bacilli phylotypes distributed in Enterococcaceae and Staphylococcaceae. Some of the phylotypes identified were associated exclusively with single structures, such as ovaries, spermatheca and the V4 midgut region of males and females. All culturable bacteria associated with the seminal vesicle were also associated with other tissues.

Tapping the biotechnological potential of insect microbial symbionts: new insecticidal porphyrins.

BACKGROUND: The demand for sustainable agricultural practices and the limited progress toward newer and safer chemicals for use in pest control maintain the impetus for research and identification of new natural molecules. Natural molecules are preferable to synthetic organic molecules because they are biodegradable, have low toxicity, are often selective and can be applied at low concentrations. Microbes are one source of natural insecticides, and microbial insect symbionts have attracted attention as a source of new bioactive molecules because these microbes are exposed to various selection pressures in their association with insects. Analytical techniques must be used to isolate and characterize new compounds, and sensitive analytical tools such as mass spectrometry and high-resolution chromatography are required to identify the least-abundant molecules. RESULTS: We used classical fermentation techniques combined with tandem mass spectrometry to prospect for insecticidal substances produced by the ant symbiont Streptomyces caniferus. Crude extracts from this bacterium showed low biological activity (less than 10% mortality) against the larval stage of the fall armyworm Spodoptera frugiperda. Because of the complexity of the crude extract, we used fractionation-guided bioassays to investigate if the low toxicity was related to the relative abundance of the active molecule, leading to the isolation of porphyrins as active molecules. Porphyrins are a class of photoactive molecules with a broad range of bioactivity, including insecticidal. The active fraction, containing a mixture of porphyrins, induced up to 100% larval mortality (LD50 = 37.7 µg.cm-2). Tandem mass-spectrometry analyses provided structural information for two new porphyrin structures. Data on the availability of porphyrins in 67 other crude extracts of ant ectosymbionts were also obtained with ion-monitoring experiments. CONCLUSIONS: Insect-associated bacterial symbionts are a rich source of bioactive compounds. Exploring microbial diversity through mass-spectrometry analyses is a useful approach for isolating and identifying new compounds. Our results showed high insecticidal activity of porphyrin compounds. Applications of different experiments in mass spectrometry allowed the characterization of two new porphyrins.

Symbiont Diversity of Aphis (Toxoptera) citricidus (Hemiptera: Aphididae) as Influenced by Host Plants.

Aphids are well known for their association with endosymbiont bacteria. Almost all aphids harbor Buchnera aphidicola as an obligate symbiont and several other bacteria as facultative symbionts. Associations of facultative symbionts and aphids are quite variable in terms of diversity and prevalence across aphid species. Facultative symbionts can have a major impact on aphid bioecological traits. A number of factors shape the outcome of the facultative symbiont-aphid association, including aphid clone, bacterial genotype, geography, and host plant association. The effects of host plant on aphid-facultative symbiont associations are the least understood. We performed deep sequencing of the bacterial community associated with field populations of the oligophagous aphid Aphis (Toxoptera) citricidus collected from different host plants. We demonstrate that (i) A. citricidus has low symbiont diversity, (ii) symbiont diversity is affected by host plant, and (iii) host plants affect the relative abundance of the obligate symbiont Buchnera and an unknown genus of Enterobacteriaceae.

Comparative transcriptome analysis of lufenuron-resistant and susceptible strains of Spodoptera frugiperda (Lepidoptera: Noctuidae).

BACKGROUND: The evolution of insecticide resistance in Spodoptera frugiperda (Lepidoptera: Noctuidae) has resulted in large economic losses and disturbances to the environment and agroecosystems. Resistance to lufenuron, a chitin biosynthesis inhibitor insecticide, was recently documented in Brazilian populations of S. frugiperda. Thus, we utilized large-scale cDNA sequencing (RNA-Seq analysis) to compare the pattern of gene expression between lufenuron-resistant (LUF-R) and susceptible (LUF-S) S. larvae in an attempt to identify the molecular basis behind the resistance mechanism(s) of S. frugiperda to this insecticide. RESULTS: A transcriptome was assembled using approximately 19.6 million 100 bp-long single-end reads, which generated 18,506 transcripts with a N50 of 996 bp. A search against the NCBI non-redundant database generated 51.1% (9,457) functionally annotated transcripts. A large portion of the alignments were homologous to insects, with the majority (45%) being similar to sequences of Bombyx mori (Lepidoptera: Bombycidae). Moreover, 10% of the alignments were similar to sequences of various species of Spodoptera (Lepidoptera: Noctuidae), with 3% of them being similar to sequences of S. frugiperda. A comparative analysis of the gene expression between LUF-R and LUF-S S. frugiperda larvae identified 940 differentially expressed transcripts (p ≤ 0.05, t-test; fold change ≥ 4). Six of them were associated with cuticle metabolism. Of those, four were overexpressed in LUF-R larvae. The machinery involved with the detoxification process was represented by 35 differentially expressed transcripts; 24 of them belonging to P450 monooxygenases, four to glutathione-S-transferases, six to carboxylases and one to sulfotransferases. RNA-Seq analysis was validated for a number of selected candidate transcripts by using quantitative real time PCR (qPCR). CONCLUSIONS: The gene expression profile of LUF-R larvae of S. frugiperda differs from LUF-S larvae. In general, gene expression is much higher in resistant larvae when compared to the susceptible ones, particularly for those genes involved with pathways for xenobiotic detoxification, mainly represented by P450 monooxygenases transcripts. Our data indicate that enzymes involved with the detoxification process, and mostly the P450, are one of the resistance mechanisms employed by the LUF-R S. frugiperda larvae against lufenuron.

Population dynamics and growth rates of endosymbionts during Diaphorina citri (Hemiptera, Liviidae) ontogeny.

The infection density of symbionts is among the major parameters to understand their biological effects in host-endosymbionts interactions. Diaphorina citri harbors two bacteriome-associated bacterial endosymbionts (Candidatus Carsonella ruddii and Candidatus Profftella armatura), besides the intracellular reproductive parasite Wolbachia. In this study, the density dynamics of the three endosymbionts associated with the psyllid D. citri was investigated by real-time quantitative PCR (qPCR) at different developmental stages. Bacterial density was estimated by assessing the copy number of the 16S rRNA gene for Carsonella and Profftella, and of the ftsZ gene for Wolbachia. Analysis revealed a continuous growth of the symbionts during host development. Symbiont growth and rate curves were estimated by the Gompertz equation, which indicated a negative correlation between the degree of symbiont-host specialization and the time to achieve the maximum growth rate (t*). Carsonella densities were significantly lower than those of Profftella at all host developmental stages analyzed, even though they both displayed a similar trend. The growth rates of Wolbachia were similar to those of Carsonella, but Wolbachia was not as abundant. Adult males displayed higher symbiont densities than females. However, females showed a much more pronounced increase in symbiont density as they aged if compared to males, regardless of the incorporation of symbionts into female oocytes and egg laying. The increased density of endosymbionts in aged adults differs from the usual decrease observed during host aging in other insect-symbiont systems.

The parasitoid, Cotesia flavipes (Cameron) (Hymenoptera: Braconidae), influences food consumption and utilization by larval Diatraea saccharalis (F.) (Lepidoptera: Crambidae).

Parasitoids exploit host insects for food and other resources; they alter host development and physiology to optimize conditions to favor parasitoid development. Parasitoids influence their hosts by injecting eggs, along with a variety of substances, including venoms, polydnaviruses, ovarian fluids, and other maternal factors, into hosts. These factors induce profound changes in hosts, such as behavior, metabolism, endocrine events, and immune defense. Because endoparasitoids develop and consume tissues from within their hosts, it is reasonable to suggest that internal parasitization would also influence host food consumption and metabolism. We report on the effects of parasitism by Cotesia flavipes on the food consumption and utilization of its host, Diatraea saccharalis. Cotesia flavipes reduces the host food consumption, but parasitized larvae considered a unit with their parasitoid's attained the same final weight as the nonparasitized larvae. Nutritional indices, midgut activities of carbohydrases, and trypsin of parasitized and nonparasitized D. saccharalis were assessed. Parasitized larvae had reduced relative food consumption, metabolic and growth rates, coupled with higher efficiency for conversion of the digested, but not ingested, food into body mass. Parasitism also affected food flux through the gut and protein contents in the midgut of parasitized larvae. The activity of α-amylase and trehalase in parasitized host was enhanced in the first day after parasitism relative to control larvae. Saccharase activity remained unchanged during larval development. Trypsin activity was reduced from the fifth to ninth day after parasitism. We argue on the mechanisms involved in host food processing after parasitism.

Evolutionary and phylogenetic insights from the mitochondrial genomic analysis of Diceraeus melacanthus and D. furcatus (Hemiptera: Pentatomidae).

The mitochondrial genomes of D. melacanthus and D. furcatus were sequenced and used to investigate the phylogenetic relationships with 54 species of Pentatomidae. Their mitogenomes are 17,197 and 15,444 bp-long, respectively, including 13 protein-coding genes (PCGs), 2 ribosomal RNA genes, and 22/21 transfer RNA genes, with conserved gene arrangement. Leu, Lys, and Ser were the most common amino acids in their PCGs. PCGs evolutionary analysis indicated their mitogenomes are under purifying selection, and the most conserved genes are from the cytochrome complex, reinforcing their suitability as markers for molecular taxonomy. We identified 490 mtSSRs in 56 Pentatomidae species, with large variation and a positive correlation between mtSSR number and genome size. Three mtSSRs were identified in each Diceraeus species. Only the mtSSR in the nad6 (D. melacanthus) and nad4 (D. furcatus) appear to have application as molecular markers for species characterization. Phylogenetic analysis confirmed the monophyly of Pentatomidae. However, our analysis challenged the monophyly of Pentatominae and Podopinae. We also detected unexpected relationships among some tribes and genera, highlighting the complexity of the internal taxonomic structure of Pentatomidae. Both Diceraeus species were grouped in the same clade with the remaining Carpocorini analyzed.

Susceptibility monitoring and comparative gene expression of susceptible and resistant strains of Spodoptera frugiperda to lambda-cyhalothrin and chlorpyrifos.

BACKGROUND: Spodoptera frugiperda (J. E. Smith) is a widespread agricultural pest with several records of resistance to different insecticides and Bt proteins, including the neurotoxic insecticides chlorpyrifos (organophosphate) and lambda-cyhalothrin (pyrethroid). Here, we (i) characterized and monitored the susceptibility of field populations of S. frugiperda to chlorpyrifos (194 populations) and lambda-cyhalothrin (197 populations) collected from major maize-growing regions of Brazil from 2003 to 2016, and (ii) compared gene expression levels of laboratory-selected, chlorpyrifos- and lambda-cyhalothrin-resistant strains to a susceptible reference strain (Sf-ss) of S. frugiperda. RESULTS: The susceptibility monitoring detected average survival ranging from 29.3% to 36.0% for chlorpyrifos, and 23.1% to 68.0% for lambda-cyhalothrin. The resistance ratio of the chlorpyrifos-resistant strain (Clo-rr) was 25.4-fold and of the lambda-cyhalothrin-resistant strain (Lam-rr) was 21.5-fold. We identified 1098 differentially expressed genes (DEGs) between Clo-rr and Sf-ss, and 303 DEGs between Lam-rr and Sf-ss. Functional analyses of the DEGs revealed the up-regulation of several detoxification enzymes, mainly cytochrome P450 belonging to CYP3 and CYP6 clans. Genes associated with regulatory processes, such as the forkhead box class O (FoxO) transcription factor were also up-regulated. Variant analysis of target-site mutations for both pesticides identified the A201S and F290V mutations in acetylcholinesterase-1, both occurring in heterozigosis in the Clo-rr S. frugiperda strain. CONCLUSION: Our data show that the overexpression of the enzymatic detoxification machinery is the main difference to explain the resistance of Clo-rr and Lam-rr strains of S. frugiperda to chlorpyrifos and lambda-cyhalothrin, although a target-site mutation also contributes to the Clo-rr resistance to chlorpyrifos. © 2023 Society of Chemical Industry.

The genome sequence of the Neotropical brown stink bug, Euschistus heros provides insights into population structure, demographic history and signatures of adaptation.

The Neotropical brown stink bug, Euschistus heros, is a major pest of soybean in South America. The importance of E. heros as a pest has grown significantly in recent times due to increases in its abundance and range, and the evolution of insecticide resistance. Recent work has begun to examine the genetic diversity, population structure, and genetic mechanisms of insecticide resistance in E. heros. However, to date, investigation of these topics has been hampered by a lack of genomic resources for this species. Here we address this need by assembling a high-quality draft genome for E. heros. We used a combination of short and long read sequencing to assemble an E. heros genome of 1.4 Gb comprising 906 contigs with a contig N50 of 3.5 MB. We leveraged this new genomic resource, in combination with genotyping by sequencing, to explore genetic diversity in populations of this species in Brazil and identify genetic loci in the genome which are under selection. Our genome-wide analyses, confirm that there are two populations of E. heros co-occurring in different geographical regions in Brazil, and that, in certain regions of the country these populations are hybridizing. We identify several regions of the genome as under selection, including markers associated with putative insecticide resistance genes. Taken together, the new genomic resources generated in this study will accelerate research into fundamental aspects of stinkbug biology and applied aspects relating to the sustainable control of a highly damaging crop pest.

The Genome of "Candidatus Liberibacter asiaticus" Is Highly Transcribed When Infecting the Gut of Diaphorina citri.

The Asian citrus psyllid, Diaphorina citri, is the vector of the bacterium "Candidatus Liberibacter asiaticus" (Las), associated with the devastating, worldwide citrus disease huanglongbing. In order to explore the molecular interactions of this bacterium with D. citri during the vector acquisition process, cDNA libraries were sequenced on an Illumina platform, obtained from the gut of adult psyllids confined in healthy (H) and in Las-infected young shoots (Las) for different periods of times (I = 1/2 days, II = 3/4 days, and III = 5/6 days). In each sampling time, three biological replicates were collected, containing 100 guts each, totaling 18 libraries depleted in ribosomal RNA. Reads were quality-filtered and mapped against the Chinese JXGC Las strain and the Floridian strain UF506 for the analysis of the activity of Las genome and SC1, SC2, and type 3 (P-JXGC-3) prophages of the studied Las strain. Gene activity was considered only if reads of at least two replicates for each acquisition access period mapped against the selected genomes, which resulted in coverages of 44.4, 79.9, and 94.5% of the JXGC predicted coding sequences in Las I, Las II, and Las III, respectively. These genes indicate an active metabolism and increased expression according to the feeding time in the following functional categories: energy production, amino acid metabolism, signal translation, cell wall, and replication and repair of genetic material. Pilins were among the most highly expressed genes regardless of the acquisition time, while only a few genes from cluster I of flagella were not expressed. Furthermore, the prophage region had a greater coverage of reads for SC1 and P-JXGC-3 prophages and low coverage in SC2 and no indication of activity for the lysis cycle. This research presents the first descriptive analysis of Las transcriptome in the initial steps of the D. citri gut colonization, where 95% of Las genes were active.

Chromosomal scale assembly of parasitic wasp genome reveals symbiotic virus colonization.

Endogenous viruses form an important proportion of eukaryote genomes and a source of novel functions. How large DNA viruses integrated into a genome evolve when they confer a benefit to their host, however, remains unknown. Bracoviruses are essential for the parasitism success of parasitoid wasps, into whose genomes they integrated ~103 million years ago. Here we show, from the assembly of a parasitoid wasp genome at a chromosomal scale, that bracovirus genes colonized all ten chromosomes of Cotesia congregata. Most form clusters of genes involved in particle production or parasitism success. Genomic comparison with another wasp, Microplitis demolitor, revealed that these clusters were already established ~53 mya and thus belong to remarkably stable genomic structures, the architectures of which are evolutionary constrained. Transcriptomic analyses highlight temporal synchronization of viral gene expression without resulting in immune gene induction, suggesting that no conflicts remain between ancient symbiotic partners when benefits to them converge.

Beyond host regulation: Changes in gut microbiome of permissive and non-permissive hosts following parasitization by the wasp Cotesia flavipes.

Koinobiont parasitoids regulate the physiology of their hosts, possibly interfering with the host gut microbiota and ultimately impacting parasitoid development. We used the parasitoid Cotesia flavipes to investigate if the regulation of the host would also affect the host gut microbiota. We also wondered if the effects of parasitization on the gut microbiota would depend on the host-parasitoid association by testing the permissive Diatraea saccharalis and the non-permissive Spodoptera frugiperda hosts. We determined the structure and potential functional contribution of the gut microbiota of the fore-midgut and hindgut of the hosts at different stages of development of the immature parasitoid. The abundance and diversity of operational taxonomic units of the anteromedial (fore-midgut) gut and posterior (hindgut) region from larvae of the analyzed hosts were affected by parasitization. Changes in the gut microbiota induced by parasitization altered the potential functional contribution of the gut microbiota associated with both hosts. Our data also indicated that the mechanism by which C. flavipes interferes with the gut microbiota of the host does not require a host-parasitoid coevolutionary history. Changes observed in the potential contribution of the gut microbiota of parasitized hosts impact the host's nutritional quality, and could favor host exploitation by C. flavipes.

Regulation of the Larval Transcriptome of Diatraea saccharalis (Lepidoptera: Crambidae) by Maternal and Other Factors of the Parasitoid Cotesia flavipes (Hymenoptera: Braconidae).

Koinobiont endoparasitoid wasps regulate the host's physiology to their own benefit during their growth and development, using maternal, immature and/or derived-tissue weaponry. The tools used to subdue the wasps' hosts interfere directly with host transcription activity. The broad range of host tissues and pathways affected impedes our overall understanding of the host-regulation process during parasitoid development. Next-generation sequencing and de novo transcriptomes are helpful approaches to broad questions, including in non-model organisms. In the present study, we used Illumina sequencing to assemble a de novo reference transcriptome of the sugarcane borer Diatraea saccharalis, to investigate the regulation of host gene expression by the larval endoparasitoid Cotesia flavipes. We obtained 174,809,358 reads and assembled 144,116 transcripts, of which 44,325 were putatively identified as lepidopteran genes and represented a substantial number of pathways that are well described in other lepidopteran species. Comparative transcriptome analyses of unparasitized versus parasitized larvae identified 1,432 transcripts of D. saccharalis that were up-regulated under parasitization by C. flavipes, while 1,027 transcripts were down-regulated. Comparison of the transcriptomes of unparasitized and pseudoparasitized D. saccharalis larvae led to the identification of 1,253 up-regulated transcripts and 972 down-regulated transcripts in the pseudoparasitized larvae. Analysis of the differentially expressed transcripts showed that C. flavipes regulated several pathways, including the Ca+2 transduction signaling pathway, glycolysis/gluconeogenesis, chitin metabolism, and hormone biosynthesis and degradation, as well as the immune system, allowing us to identify key target genes involved in the metabolism and development of D. saccharalis.

Shifting the Balance: Heat Stress Challenges the Symbiotic Interactions of the Asian Citrus Psyllid, Diaphorina citri (Hemiptera, Liviidae).

Global warming may impact biodiversity by disrupting biological interactions, including long-term insect-microbe mutualistic associations. Symbiont-mediated insect tolerance to high temperatures is an ecologically important trait that significantly influences an insect's life history. Disruption of microbial symbionts that are required by insects would substantially impact their pest status. Diaphorina citri, a worldwide citrus pest, is associated with the mutualistic symbionts Candidatus Carsonella ruddii and Candidatus Profftella armatura. Wolbachia is also associated with D. citri, but its contribution to the host is unknown. Symbiont density is dependent on a range of factors, including the thermosensitivity of the host and/or symbiont to heat stress. Here, we predicted that short-term heat stress of D. citri would disrupt the host-symbiont phenological synchrony and differentially affect the growth and density of symbionts. We investigated the effects of exposing D. citri eggs to different temperatures for different periods of time on the growth dynamics of symbionts during the nymphal development of D. citri (first instar to fifth instar) by real-time polymerase chain reaction. Symbiont densities were assessed as the number of gene copies, using specific molecular markers: 16S rRNA for Carsonella and Profftella and ftsZ for Wolbachia. Statistical modeling of the copy numbers of symbionts revealed differences in their growth patterns, particularly in the early instars of heat-shocked insects. Wolbachia was the only symbiont to benefit from heat-shock treatment. Although the symbionts responded differently to heat stress, the lack of differences in symbiont densities between treated and control late nymphs suggests the existence of an adaptive genetic process to restore phenological synchrony during the development of immatures in preparation for adult life. Our findings contribute to the understanding of the potential deleterious effects of high temperatures on host-symbiont interactions. Our data also suggest that the effects of host exposure to high temperatures in symbiont growth are highly variable and dependent on the interactions among members of the community of symbionts harbored by a host. Such dependence points to unpredictable consequences for agroecosystems worldwide due to climate change-related effects on the ecological traits of symbiont-dependent insect pests.

Spiroplasma affects host aphid proteomics feeding on two nutritional resources.

Bacterial symbionts are broadly distributed among insects, influencing their bioecology to different degrees. Aphids carry a number of secondary symbionts that can influence aphid physiology and fitness attributes. Spiroplasma is seldom reported as an aphid symbiont, but a high level of infection has been observed in one population of the tropical aphid Aphis citricidus. We used sister isolines of Spiroplasma-infected (Ac-BS) and Spiroplasma-free (Ac-B) aphids reared on sweet orange (optimum host) and orange jasmine (suboptimum host) to demonstrate the effects of Spiroplasma infection in the aphid proteome profile. A higher number of proteins were differently abundant in aphids feeding on orange jasmine, indicating an impact of host plant quality. In both host plants, the majority of proteins affected by Spiroplasma infection were heat shock proteins, proteins linked to cell function and structure, and energy metabolism. Spiroplasma also induced changes in proteins involved in antimicrobial activity, carbohydrate processing and metabolism, amino acid synthesis and metabolism in aphids feeding on orange jasmine. We discuss on how the aphid host proteome is differentially affected by Spiroplasma infection when the host is exploiting host plants with different nutritional values.

Loci under selection and markers associated with host plant and host-related strains shape the genetic structure of Brazilian populations of Spodoptera frugiperda (Lepidoptera, Noctuidae).

We applied the ddRAD genotyping-by-sequencing technique to investigate the genetic distinctiveness of Brazilian populations of the noctuid moth Spodoptera frugiperda, the fall armyworm (FAW), and the role of host-plant association as a source of genetic diversification. By strain-genotyping all field-collected individuals we found that populations collected from corn were composed primarily of corn-strain individuals, while the population collected from rice was composed almost entirely of rice-strain individuals. Outlier analyses indicated 1,184 loci putatively under selection (ca. 15% of the total) related to 194 different Gene Ontologies (GOs); the most numerous GOs were nucleotide binding, ATP binding, metal-ion binding and nucleic-acid binding. The association analyses indicated 326 loci associated with the host plant, and 216 loci associated with the individual strain, including functions related to Bacillus thuringiensis and insecticide resistance. The genetic-structure analyses indicated a moderate level of differentiation among all populations, and lower genetic structure among populations collected exclusively from corn, which suggests that the population collected from rice has a strong influence on the overall genetic structure. Populations of S. frugiperda are structured partially due to the host plant, and pairs of populations using the same host plant are more genetically similar than pairs using different hosts. Loci putatively under selection are the main factors responsible for the genetic structure of these populations, which indicates that adaptive selection on important traits, including the response to control tactics, is acting in the genetic differentiation of FAW populations in Brazil.

The gut microbiota of insecticide-resistant insects houses insecticide-degrading bacteria: A potential source for biotechnological exploitation.

The exploration of new niches for microorganisms capable of degrading recalcitrant molecules is still required. We hypothesized the gut microbiota associated with insect-resistant lines carry pesticide degrading bacteria, and predicted they carry bacteria selected to degrade pesticides they were resistant to. We isolated and accessed the pesticide-degrading capacity of gut bacteria from the gut of fifth instars of Spodoptera frugiperda strains resistant to lambda-cyhalothrin, deltamethrin, chlorpyrifos ethyl, spinosad and lufenuron, using insecticide-selective media. Sixteen isolates belonging to 10 phylotypes were obtained, from which four were also associated with the susceptible strain. However, growth of gut bacteria associated with larvae from the susceptible strain was not obtained in any of the insecticide-based selective media tested. Growth of isolates was affected by the concentration of insecticides in the media, and all grew well up to 40 µg/ml. The insecticide-degrading capacity of selected isolates was assessed by GC or LC-MS/MS analyses. In conclusion, resistant strains of S. frugiperda are an excellent reservoir of insecticide-degrading bacteria with bioremediation potential. Moreover, gut-associated bacteria are subjected to the selection pressure imposed by insecticides on their hosts and may influence the metabolization of pesticides in insects.