Bulk segregant mapping and transcriptome analyses reveal the molecular mechanisms of spinetoram resistance in Spodoptera frugiperda.

The evolution of resistance to insecticides poses a significant threat to pest management programs. Understanding the molecular mechanisms underlying insecticide resistance is essential to design sustainable pest control and resistance management programs. The fall armyworm, Spodoptera frugiperda, is an important insect pest of many crops and has a remarkable ability to evolve resistance to insecticides. In this study, we employed bulk segregant analysis (BSA) combined with DNA and RNA sequencing to characterize the molecular basis of spinetoram resistance in S. frugiperda. Analysis of genomic data derived from spinetoram selected and unselected bulks and the spinetoram-resistant and susceptible parental strains led to the identification of a three-nucleotide deletion in the gene encoding the nicotinic acetylcholine receptor α6 subunit (nAChR α6). Transcriptome profiling identified the upregulation of few genes encoding detoxification enzymes associated with spinetoram resistance. Thus, spinetoram resistance in S. frugiperda appears to be mediated mainly by target site insensitivity with a minor role of detoxification enzymes. Our findings provide insight into the mechanisms underpinning resistance to spinetoram in S. frugiperda and will inform the development of strategies to control this highly damaging, globally distributed crop pest.

Transcriptomic investigation of the molecular mechanisms underlying resistance to the neonicotinoid thiamethoxam and the pyrethroid lambda-cyhalothrin in Euschistus heros (Hemiptera: Pentatomidae).

BACKGROUND: Laboratory-selected resistant strains of Euschistus heros to thiamethoxam (NEO) and lambda-cyhalothrin (PYR) were recently reported in Brazil. However, the mechanisms conferring resistance to these insecticides in E. heros remain unresolved. We utilized comparative transcriptome profiling and single nucleotide polymorphism (SNP) calling of susceptible and resistant strains of E. heros to investigate the molecular mechanism(s) underlying resistance. RESULTS: The E. heros transcriptome was assembled, generating 91 673 transcripts with a mean length of 720 bp and N50 of 1795 bp. Comparative gene expression analysis between the susceptible (SUS) and NEO strains identified 215 significantly differentially expressed (DE) transcripts. DE transcripts associated with the xenobiotic metabolism were all up-regulated in the NEO strain. The comparative analysis of the SUS and PYR strains identified 204 DE transcripts, including an esterase (esterase FE4), a glutathione-S-transferase, an ABC transporter (ABCC1) and aquaporins that were up-regulated in the PYR strain. We identified 9588 and 15 043 nonsynonymous SNPs in the PYR and NEO strains. One of the SNPs (D70N) detected in the NEO strain occurs in a subunit (α5) of the nAChRs, the target site of neonicotinoid insecticides. Nevertheless, this residue position in α5 is not conserved among insects. CONCLUSIONS: Neonicotinoid and pyrethroid resistance in laboratory-selected E. heros is associated with a potential metabolic resistance mechanism by the overexpression of proteins commonly involved in the three phases of xenobiotic metabolism. Together these findings provide insight into the potential basis of resistance in E. heros and will inform the development and implementation of resistance management strategies against this important pest. © 2023 Society of Chemical Industry.

Host-Adapted Strains of Spodoptera frugiperda Hold and Share a Core Microbial Community Across the Western Hemisphere.

The fall armyworm Spodoptera frugiperda is an important polyphagous agricultural pest in the Western Hemisphere and currently invasive to countries of the Eastern Hemisphere. This species has two host-adapted strains named "rice" and "corn" strains. Our goal was to identify the occurrence of core members in the gut bacterial community of fall armyworm larvae from distinct geographical distribution and/or host strain. We used next-generation sequencing to identify the microbial communities of S. frugiperda from corn fields in Brazil, Colombia, Mexico, Panama, Paraguay, and Peru, and rice fields from Panama. The larval gut microbiota of S. frugiperda larvae did not differ between the host strains nor was it affected by the geographical distribution of the populations investigated. Our findings provide additional support for Enterococcus and Pseudomonas as core members of the bacterial community associated with the larval gut of S. frugiperda, regardless of the site of collection or strain. Further investigations are required for a deeper understanding of the nature of this relationship.

The Microbiota of a Mite Prey-Predator System on Different Host Plants Are Characterized by Dysbiosis and Potential Functional Redundancy.

Microbiota has diverse roles in the life cycles of their hosts, affecting their growth, development, behavior, and reproduction. Changes in physiological conditions of the host can also impact the assemblage of host-associated microorganisms. However, little is known of the effects of host plant-prey-predatory mite interactions on mite microbiota. We compared the microbial communities of eggs and adult females of the two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae), and of adult females of the predatory mite Neoseiulus californicus (McGregor) (Acari: Phytoseiidae) on four different host plants (cotton, maize, pinto bean, and tomato) by metabarcoding sequencing of the V3-V4 region of the 16S ribosomal RNA gene (16S rRNA), using the Illumina MiSeq platform. Only the egg microbiota of T. urticae was affected by the host plant. The microbiota of the predatory mite N. californicus was very different from that of its prey, and the predator microbiota was unaffected by the different host plant-prey systems tested. Only the microbiota of the eggs of T. urticae carried Serratia as a high fidelity-biomarker, but their low abundance in T. urticae adult females suggests that the association between Serratia and T. urticae is accidental. Biomarker bacteria were also detected in the microbiota of adult females of T. urticae and N. californicus, with different biomarkers in each host plant species. The microbiota associated with eggs and adult females of T. urticae and adult females of N. californicus differed in their functional potential contributions to the host mite.

Non-targeted metabolomics reveals differences in the gut metabolic profile of the fall armyworm strains when feeding different food sources.

Spodoptera frugiperda (fall armyworm - FAW) is an important polyphagous agricultural pest feeding on nearly 350 host plants. FAW is undergoing incipient speciation with two well-characterized host-adapted strains, the "corn" (CS) and "rice" (RS) strains, which are morphologically identical but carry several genes under positive selection for host adaptation. We used non-targeted metabolomics based on gas chromatography/mass spectrometry to identify differences in metabolite profiles of the larval gut of CS and RS feeding on different host plants. Larvae were fed on artificial diet, maize, rice, or cotton leaves from eclosion to the sixth instar, when they had their midgut dissected for analysis. This study revealed that the midgut metabolome of FAW varied due to larval diet and differed between the FAW host-adapted strains. Additionally, we identified several candidate metabolites that may be involved in the adaptation of CS and RS to their host plants. Our findings provide clues toward the gut metabolic activities of the FAW strains.

Modelling movement and stage-specific habitat preferences of a polyphagous insect pest.

BACKGROUND: The feeding preferences of Diabrotica speciosa (Coleoptera: Chrysomelidae) cause a parent-offspring conflict, as providing the best host for the offspring development is detrimental to adult survival and fecundity. Understanding the implications of this conflict could help entomologists to implement pest-management programs. With this in mind, the foraging behaviour of D. speciosa was investigated using an individual-based model in two distinct scenarios. METHODS: In an intercropping scenario, parent-offspring conflict was simulated when adult insects exploit two crops (corn and soybean) that provide different nutritional advantages for each insect stage. First, we compared three hypothetical types of adult dispersal, considering a continuous oviposition over time: diffusion, attracted to a fixed host and alternating the preference between hosts with frequency 1 τ , where τ is the time in days spent foraging for each host. We also simulated two principles: "mother knows best" (adult females foraging for corn during the oviposition period) and "optimal bad motherhood" (adult females remain foraging for soybean to maximise their own fitness during the oviposition period), but considering the existence of a pre-oviposition period. In a landscape scenario, we investigated the population dynamics in an area composed by 4 crop plots that change over time. RESULTS: Among dispersal types considering continuous oviposition, the crop-alternating movement a-3 performed best, when close to an optimal τ. Additionally, τ was predicted to be influenced mainly by the width of crop rows. We also verified that the "mother knows best" strategy is better for the population growth than the "optimal bad motherhood". In the landscape scenario, we observed that including fallow periods in the crop calendar and adopting a more-heterogeneous arrangement of crop plots reduced the density of this insect. CONCLUSION: Both the continuous and sequential oviposition simulations indicate that foraging involving switching of target crop benefits population fitness. In the landscape scenario, arranging crop plots more heterogeneously and avoiding vast areas of soybean can help farmers to control this insect pest. Additionally, fallow periods can also reduce significantly D. speciosa populations.

Two genomes of highly polyphagous lepidopteran pests (Spodoptera frugiperda, Noctuidae) with different host-plant ranges.

Emergence of polyphagous herbivorous insects entails significant adaptation to recognize, detoxify and digest a variety of host-plants. Despite of its biological and practical importance - since insects eat 20% of crops - no exhaustive analysis of gene repertoires required for adaptations in generalist insect herbivores has previously been performed. The noctuid moth Spodoptera frugiperda ranks as one of the world's worst agricultural pests. This insect is polyphagous while the majority of other lepidopteran herbivores are specialist. It consists of two morphologically indistinguishable strains ("C" and "R") that have different host plant ranges. To describe the evolutionary mechanisms that both enable the emergence of polyphagous herbivory and lead to the shift in the host preference, we analyzed whole genome sequences from laboratory and natural populations of both strains. We observed huge expansions of genes associated with chemosensation and detoxification compared with specialist Lepidoptera. These expansions are largely due to tandem duplication, a possible adaptation mechanism enabling polyphagy. Individuals from natural C and R populations show significant genomic differentiation. We found signatures of positive selection in genes involved in chemoreception, detoxification and digestion, and copy number variation in the two latter gene families, suggesting an adaptive role for structural variation.

Genetic Diversity and Structure of Brazilian Populations of Diatraea saccharalis (Lepidoptera: Crambidae): Implications for Pest Management.

The sugarcane borer, Diatraea saccharalis (F.), is the main pest of sugarcane in Brazil. Genetic variability and gene flow among 13 Brazilian populations of the species were evaluated based on mitochondrial DNA sequences to estimate the exchange of genetic information within and among populations. We found high genetic structure among sampled localities (ΦST=0.50923), and pairwise genetic distances were significantly correlated to geographic distances. Demographic analysis and genealogical network of mitochondrial sequences indicate population growth and admixture of D. saccharalis populations, events likely related to the sequential expansion of the corn and sugarcane crops in Brazil. The implications of these findings for pest management are discussed.

The genetic structure of an invasive pest, the Asian citrus psyllid Diaphorina citri (Hemiptera: Liviidae).

The Asian citrus psyllid Diaphorina citri is currently the major threat to the citrus industry as it is the vector of Candidatus Liberibacter, the causal agent of huanglongbing disease (HLB). D. citri is native to Asia and now colonizes the Americas. Although it has been known in some countries for a long time, invasion routes remain undetermined. There are no efficient control methods for the HLB despite the intensive management tools currently in use. We investigated the genetic variability and structure of populations of D. citri to aid in the decision making processes toward sustainable management of this species/disease. We employed different methods to quantify and compare the genetic diversity and structure of D. citri populations among 36 localities in Brazil, using an almost complete sequence of the cytochrome oxidase I (COI) gene. Our analyses led to the identification of two geographically and genetically structured groups. The indices of molecular diversity pointed to a recent population expansion, and we discuss the role of multiple invasion events in this scenario. We also argue that such genetic diversity and population structure may have implications for the best management strategies to be adopted for controlling this psyllid and/or the disease it vectors in Brazil.

Landscape diversity influences dispersal and establishment of pest with complex nutritional ecology.

We studied the effects of landscape structure on species with resource nutritional partition between the immature and adult stages by investigating how food quality and spatial structure of a landscape may affect the invasion and colonization of the insect pest, Diabrotica speciosa. To this end, we formulated two bidimensional stochastic cellular automata, one for the insect immature stage and the other for the adult stage. The automata are coupled by adult oviposition and emergence. Further, each automata site has a specific culture type, which can affect differently the fitness attributes of immatures and adults, such as mortality, development and oviposition rates. We derived the mean-field approximation for these automata model, from which we obtained conditions for insect invasion. We ran numerical simulations using entomological parameters obtained from laboratory experiments (using bean, soybean, potato, and corn crops), and we compared the results of the automata with the ones given by the mean-field approximation. Finally, using artificially generated landscapes, we discussed how the structured heterogeneous landscape can affect dispersal and establishment of insect populations.

Geometric morphometry and molecular analysis clarified the identity of Opius sp. aff. bellus (Hymenoptera, Braconidae), a fruit fly parasitoid in Brazil.

The aim of this study was to clarify the identity of specimens designated as Opius sp. aff. bellus using geometric morphometry and analysis of the ITS2 region of the ribosomal DNA (rDNA) and region D2 of the 28S rDNA. Opius bellus Gahan is a koinobiont endoparasitoid of fruit fly larvae (Diptera: Tephritidae) exclusively found in the Neotropical region, but widely distributed from Mexico to Argentina. Opius sp. aff. bellus is morphologically similar to Opius bellus but exhibits yellowish, instead of dark-brown, posterior tibiae. Twenty anatomical landmarks on the wings from these two taxa sampled from three Brazilian states were analyzed. Multivariate morphometric analysis showed a large amount of morphological similarity between the specimens, indicating they are the same species. Sequence analysis of the ITS2 showed that intrapopulation variability was similar to interpopulation variability and that the morphotypes were also highly similar. In addition, the D2 region of the 28S rDNA displayed high similarity between sequences. Therefore, based on morphometric and molecular analyses, specimens thought to be Opius sp. aff. bellus actually belong to O. bellus. 

The gastric caeca of pentatomids as a house for actinomycetes.

BACKGROUND: Microbes are extensively associated with insects, playing key roles in insect defense, nutrition and reproduction. Most of the associations reported involve Proteobacteria. Despite the fact that Actinobacteria associated with insects were shown to produce antibiotic barriers against pathogens to the hosts or to their food and nutrients, there are few studies focusing on their association with insects. Thus, we surveyed the Actinobacteria diversity on a specific region of the midgut of seven species of stinkbugs (Hemiptera: Pentatomidae) known to carry a diversity of symbiotically-associated Proteobacteria. RESULTS: A total of 34 phylotypes were placed in 11 different Actinobacteria families. Dichelops melacanthus held the highest diversity with six actinobacteria families represented by nine phylotypes. Thyanta perditor (n = 7), Edessa meditabunda (n = 5), Loxa deducta (n = 4) and Pellaea stictica (n = 3) were all associated with three families. Piezodorus guildini (n = 3) and Nezara viridula (n = 3) had the lowest diversity, being associated with two (Propionibacteriaceae and Mycobacteriaceae) and one (Streptomyceataceae) families, respectively. Corynebacteriaceae and Mycobacteriaceae were the most common families with phylotypes from three different insect species each one. CONCLUSIONS: Many phylotypes shared a low 16S rRNA gene similarity with their closest type strains and formed new phyletic lines on the periphery of several genera. This is a strong indicative that stinkbug caeca can harbor new species of actinobacteria, which might be derived from specific associations with the species of stinkbugs studied. Although the well-known role of actinobacteria as a source of biomolecules, the ecological features of these symbionts on the stinkbugs biology remain unknown.

Genetic structure and gene flow among Brazilian populations of Heliothis virescens (Lepidoptera: Noctuidae).

Population genetic studies are essential to the better application of pest management strategies, including the monitoring of the evolution of resistance to insecticides and genetically modified plants. Bacillus thuringiensis Berliner (Bt) crops have been instrumental in controlling tobacco budworm, Heliothis virescens (F.) (Lepidoptera: Noctuidae), a pest that has developed resistance to many common insecticides once used for its management. In our study, microsatellite markers were applied to investigate the genetic structure and patterns of gene flow among Brazilian populations of H. virescens from cotton, Gossypium hirsutum L., and soybean, Glycine max (L.) Merr., fields, aiming to propose means to improve its management in the field. In total, 127 alleles were found across nine microsatellites loci for 205 individuals from 12 localities. Low levels of gene flow and moderate to great genetic structure were found for these populations. Host plant association, crop growing season, and geographic origin were not responsible for the genetic structuring among Brazilian populations of H. virescens. Other factors, such as demographic history and seasonal variability of intrapopulation genetic variation, were suggested to be molding the current pattern of genetic variability distribution.

Isolation and characterization of actinobacteria ectosymbionts from Acromyrmex subterraneus brunneus (Hymenoptera, Formicidae).

The ectosymbiont actinobacterium Pseudonocardia was isolated from the integument of Acromyrmex leaf-cutter ants and seems to play a crucial role in maintaining asepsis of the nest. Currently, there has been an intensive search for Pseudonocardia associated with several attine species, but few studies have indicated that other actinobacteria may be associated with these ants as well. We therefore characterized the culturable actinobacteria community associated with the integument of the fungus-growing ant Acromyrmex subterraneus brunneus Forel, 1893 (Hymenoptera: Formicidae). Ectosymbionts were isolated using four different media and characterized by morphological and molecular (16S rDNA) methods. A total of 20 strains were isolated, of which 17 were characterized as Streptomyces spp., and one isolate each as Pseudonocardia, Kitassatospora and Propionicimonas. Unlike other Acromyrmex species, A. subterraneus brunneus is associated with a diversity of actinobacteria. Even though Pseudonocardia is present on this leaf-cutting ant's integument, the number and diversity of Streptomyces spp. found differs from those of previous studies with other attine ants and suggest that different culturing approaches are needed to characterize the true diversity of microbes colonizing the integument of attine ants. Moreover, understanding the diversity of the culturable actinobacteria associated with A. subterraneus brunneus should increase our knowledge of the evolutionary relationship of this intricate symbiotic association.

Exploitation of mitochondrial nad6 as a complementary marker for studying population variability in Lepidoptera.

The applicability of mitochondrial nad6 sequences to studies of DNA and population variability in Lepidoptera was tested in four species of economically important moths and one of wild butterflies. The genetic information so obtained was compared to that of cox1 sequences for two species of Lepidoptera. nad6 primers appropriately amplified all the tested DNA targets, the generated data proving to be as informative and suitable in recovering population structures as that of cox1. The proposal is that, to obtain more robust results, this mitochondrial region can be complementarily used with other molecular sequences in studies of low level phylogeny and population genetics in Lepidoptera.

Exploitation of mitochondrial nad6 as a complementary marker for studying population variability in Lepidoptera

The applicability of mitochondrial nad6 sequences to studies of DNA and population variability in Lepidoptera was tested in four species of economically important moths and one of wild butterflies. The genetic information so obtained was compared to that of cox1 sequences for two species of Lepidoptera. nad6 primers appropriately amplified all the tested DNA targets, the generated data proving to be as informative and suitable in recovering population structures as that of cox1. The proposal is that, to obtain more robust results, this mitochondrial region can be complementarily used with other molecular sequences in studies of low level phylogeny and population genetics in Lepidoptera.

[Ovarian development and analysis of mating effects on ovary maturation of Diaphorina citri Kuwayama (Hemiptera: Psyllidae)]. / Desenvolvimento ovariano e influência da cópula na maturação dos ovários de Diaphorina citri Kuwayama (Hemiptera: Psyllidae).

As many other insects with a restricted diet, the citrus psyllid Diaphorina citri Kuwayama, which vector the causing agent of the Huanglongbing, the bacteria Candidatus Liberibacter spp., is intimately associated with symbiotic microorganisms. These mutualist symbionts play a key role on their host nutritional ecology, and are vertically transmitted to the progeny. However, despite the role symbionts play on host development and reproduction, and the growing opportunities of exploitation of the association insect-symbiont to control insect vectored-pathogens, there are very few studies on the host reproductive biology and on the symbiont transovarial transmission. Therefore, we aimed at analyzing the ovary development during D. citri adulthood, and at verifying for the mating requirement as a trigger to initiate ovary development. Newly-emerged D. citri females were grouped as virgin or mated and ovary development was observed during adulthood. Newly-emerged females have immature ovaries, and ovaries remain without any mature eggs until females mate. Once female mates, the vitellogenesis synthesis and uptake are estimulated, and oocytes are quickly developed. Oocytes maturation in ovarioles is metachronic, with only one oocyte developing at a time in each oogenic cycle. Morphological observations of the reproductive system including the ovaries and spermatheca, after the first cluster of eggs is laid, indicated D. citri is polyandrous, and may require multiple mating to develop additional oogenic maturation cycles.

Geographic variation of sex pheromone and mitochondrial DNA in Diatraea saccharalis (Fab., 1794) (Lepidoptera: Crambidae).

(9Z,11E)-hexadecadienal and (Z11)-hexadecenal, the main sex pheromone components of the sugarcane borer, Diatraea saccharalis, were identified and quantified from four Brazilian and one Colombian populations using GC-EAD, GC-MS and GC analyses. Three different ratios were observed, 9:1, 6:1, and 3:1. The pheromone concentration for the major component, (9Z,11E)-hexadecadienal, varied from 6.8 ng/gland to 21.9 ng/gland and from 1.7 ng/gland to 6.5 to the minor component, (Z11)-hexadecenal. The 25 D. saccharalis cytochrome oxidase II sequences that were analyzed showed low intra-specific variation and represented only 11 haplotypes, with the most frequent being the one represented by specimens from São Paulo, Paraná, and Pernambuco states. Specimens from Colombia showed the highest genetic divergence from the others haplotypes studied. Data on the genetic variability among specimens, more than their geographic proximity, were in agreement with data obtained from analyses of the pheromone extracts. Our data demonstrate a variation in pheromone composition and a covariation in haplotypes of the D. saccharalis populations studied.

Desenvolvimento ovariano e influência da cópula na maturação dos ovários de Diaphorina citri Kuwayama (Hemiptera: Psyllidae) / Ovarian development and analysis of mating effects on ovary maturation of Diaphorina citri Kuwayama (Hemiptera: Psyllidae)

As many other insects with a restricted diet, the citrus psyllid Diaphorina citri Kuwayama, which vector the causing agent of the Huanglongbing, the bacteria Candidatus Liberibacter spp., is intimately associated with symbiotic microorganisms. These mutualist symbionts play a key role on their host nutritional ecology, and are vertically transmitted to the progeny. However, despite the role symbionts play on host development and reproduction, and the growing opportunities of exploitation of the association insect-symbiont to control insect vectored-pathogens, there are very few studies on the host reproductive biology and on the symbiont transovarial transmission. Therefore, we aimed at analyzing the ovary development during D. citri adulthood, and at verifying for the mating requirement as a trigger to initiate ovary development. Newly-emerged D. citri females were grouped as virgin or mated and ovary development was observed during adulthood. Newly-emerged females have immature ovaries, and ovaries remain without any mature eggs until females mate. Once female mates, the vitellogenesis synthesis and uptake are estimulated, and oocytes are quickly developed. Oocytes maturation in ovarioles is metachronic, with only one oocyte developing at a time in each oogenic cycle. Morphological observations of the reproductive system including the ovaries and spermatheca, after the first cluster of eggs is laid, indicated D. citri is polyandrous, and may require multiple mating to develop additional oogenic maturation cycles.