Evaluating the role of insecticidal seed treatment and refuge for managing soybean aphid virulence.

BACKGROUND: Managing insect virulence can extend the durability of host-plant resistant crops. Genetically modified resistant crops continue to be successful because of insect-resistant management strategies that delay resistance such as multiple toxins and a susceptible refuge. These strategies may also be useful for host-plant resistant crops, but more research is needed on their applicability. We investigated the interaction between a susceptible refuge and an insecticidal seed treatment to manage virulence in the soybean aphid. We tested four scenarios of an insecticidal seed treatment (plus an untreated control) in a microcosm containing 25% aphid-susceptible (refuge) and 75% aphid-resistant soybeans. Independent cohorts of plants were infested every week with avirulent and virulent aphids at equal frequencies. We used a molecular marker to estimate the change in virulence frequency across different plant maturities (from 7 to 42 days after planting). RESULTS: The presence of an insecticidal seed treatment on either the susceptible or resistant soybean decreased the overall population size of the soybean aphid. However, the insecticidal seed treatment impacted both virulent and avirulent aphids similarly, and only altered frequencies in favor of virulence when the sole susceptible plant (i.e., refuge) was treated. CONCLUSION: Under our experimental conditions, the frequency of avirulent aphids persisted with the use of a refuge. Although an insecticidal seed treatment decreased the overall aphid population size, it did not appear to benefit virulence management. © 2021 Society of Chemical Industry.

Using host-associated differentiation to track source population and dispersal distance among insect vectors of plant pathogens.

Small, mobile insects are notoriously challenging to track across landscapes and manage in agricultural fields. However, genetic differentiation among insect populations and host plants acquired through host-associated differentiation could be exploited to infer movement within crop systems and damage potential. Although many insects exhibit host-associated differentiation, management strategies for insect vectors of plant pathogens assume a homogenous population. Nevertheless, phenotypic changes derived from host-associated differentiation could manifest in altered behavior or physiology affecting the likelihood of vector-pathogen-plant interactions, or the subsequent efficiency of pathogen transmission. We used SNPs to assess genotypic structure and host-associated differentiation in the cowpea aphid, Aphis craccivora Koch (Hemiptera: Aphididae). To do so, we sampled A. craccivora across the Midwestern United States. from two host plants, alfalfa (Medicago sativa) and black locust (Robinia pseudoacacia)-putative source populations for winged migrants. Simultaneously, we sampled winged A. craccivora landing in pumpkin fields where they transmit viruses. Structure analyses supported host-associated differentiation by identifying two major genotypic groups: an alfalfa group containing a single multilocus genotype and a locust group containing all others. Winged locust-group aphids landed at a much greater magnitude within focal fields during year 2 than year 1, while those in the alfalfa group remained fairly consistent. Spatial autocorrelation analyses indicated locust-group aphid movement was characterized by small-scale dispersal during year 2, likely originating from populations within 10 km. We also detected strong temporal differences in colonization from the two host plants. Early in the summer, most winged aphids (79.4%) derived from the locust group, whereas late in the summer more (58.3%) were from the alfalfa group. Because early crop growth stages are more susceptible to damage from aphid-vectored viruses, these data implicate locust as the more important source and illustrate how host-associated differentiation can be used to track dispersal and inform management of heterogeneous pest populations.

Genome-Wide Association Mapping of Host-Plant Resistance to Soybean Aphid.

Soybean aphid [ Matsumura (Hemiptera: Aphididae)] is the most damaging insect pest of soybean [ (L.) Merr.] in the Upper Midwest of the United States and is primarily controlled by insecticides. Soybean aphid resistance (i.e., genes) has been documented in some soybean accessions but more sources of resistance are needed. Incorporation of the resistance into marketed varieties has also been slow. Genome-wide association mapping can aid in identifying resistant accessions by correlating phenotypic data with single nucleotide polymorphisms (SNPs) across a genome. Aphid population measures from 2366 soybean accessions were collected from published studies screening cultivated soybean () and wild soybean ( Siebold & Zucc.) with aphids exhibiting Biotype 1, 2, or 3 characteristics. Genotypic data were obtained from the SoySNP50K high-density genotyping array previously used to genotype the USDA Soybean Germplasm Collection. Significant associations between SNPs and soybean aphid counts were found on 18 of the 20 soybean chromosomes. Significant SNPs were found on chromosomes 7, 8, 13, and 16 with known genes. SNPs were also significant on chromosomes 1, 2, 4 to 6, 9 to 12, 14, and 17 to 20 where genes have not yet been mapped, suggesting that many genes remain to be discovered. These SNPs can be used to determine accessions that are likely to have novel aphid resistance traits of value for breeding programs.

RNA-Seq reveals a xenobiotic stress response in the soybean aphid, Aphis glycines, when fed aphid-resistant soybean.

BACKGROUND: While much recent research has expanded our understanding of the molecular interactions between aphids and their host plants, it is lacking for the soybean aphid, Aphis glycines. Since its North American invasion, A. glycines has become one of the most damaging insect pests on this important crop. Five soybean genes for host plant resistance to A. glycines have been identified, but populations of A. glycines have already adapted to overcome these resistance genes. Understanding the molecular interactions between resistant soybean and A. glycines can provide clues to its adaptation mechanisms. Here, we used RNA-Sequencing to compare and contrast A. glycines gene expression when fed resistant (Rag1) and susceptible soybean. RESULTS: Combining results from a previous A. glycines transcriptome, we generated 64,860 high quality transcripts, totaling 41,151,086 bases. Statistical analysis revealed 914 genes with significant differential expression. Most genes with higher expression in A. glycines on resistant plants (N = 352) were related to stress and detoxification such as cytochrome P450s, glutathione-S-transferases, carboxyesterases, and ABC transporters. A total of 562 genes showed lower transcript abundance in A. glycines on resistant plants. From our extensive transcriptome data, we also identified genes encoding for putative salivary effector proteins (N = 73). Among these, 6 effector genes have lower transcript abundance in A. glycines feeding on resistant soybean. CONCLUSIONS: Overall, A. glycines exhibited a pattern typical of xenobiotic challenge, thereby validating antibiosis in Rag1, presumably mediated through toxic secondary metabolites. Additionally, this study identified many A. glycines genes and gene families at the forefront of its molecular interaction with soybean. Further investigation of these genes in other biotypes may reveal adaptation mechanisms to resistant plants.

Microbiome diversity of Aphis glycines with extensive superinfection in native and invasive populations.

Associations among insects and microbes can lead to beneficial or parasitic interactions. Using 454 sequencing of 16S RNA genes, we compared microbiome diversity and abundance among field-collected (F) and laboratory-reared (L) populations of the invasive soybean aphid (Aphis glycines), a pest of soybean. Additionally, we screened A. glycines populations from native (Japan, South Korea and China) and invasive regions (North America) to broadly determine the microbiome diversity. Our results suggested that Arsenophonus (relative abundance of 54.6%), Buchnera (38.7%) and Wolbachia (3.7%) were the major bacteria associated with A. glycines. Arsenophonus was the most abundant in F populations but was significantly reduced in L populations; additional bacteria species also had lower relative abundances in L populations. Native and invasive populations were largely similar in bacteria communities and revealed substantial superinfection of Arsenophonus and Wolbachia. The lone exception was a lack of Arsenophonus in A. glycines from Japan. Divergent selection pressures among natural and laboratory populations were inferred as factors driving the differential bacterial communities observed. Our results will allow for improved comparative aphid-symbiont research and broaden our understanding of the interactions among insects, endosymbionts and their environments.

Western corn rootworm (Diabrotica virgifera virgifera) transcriptome assembly and genomic analysis of population structure.

BACKGROUND: Western corn rootworm (WCR) is one of the most significant insect pests of maize in North America. WCR has dramatically increased its range in the last century, invading key maize production areas in the US and abroad. In addition, this species has a history of evolving traits that allow it to escape various control options. Improved genetic and genomic resources are crucial tools for understanding population history and the genetic basis of trait evolution. Here we produce and analyze a transcriptome assembly for WCR. We also perform whole genome population resequencing, and combine these resources to better understand the evolutionary history of WCR. RESULTS: The WCR transcriptome assembly presented here contains approximately 16,000 unigenes, many of which have high similarity to other insect species. Among these unigenes we found several gene families that have been implicated in insecticide resistance in other species. We also identified over 500,000 unigene based SNPs among 26 WCR populations. We used these SNPs to scan for outliers among the candidate genes, and to understand how population processes have shaped genetic variation in this species. CONCLUSIONS: This study highlights the utility of transcriptomic and genomic resources as foundational tools for dealing with highly adaptive pest species. Using these tools we identified candidate gene families for insecticide resistance and reveal aspects of WCR population history in light of the species' recent range expansion.

Implementing an evolutionary framework for understanding genetic relationships of phenotypically defined insect biotypes in the invasive soybean aphid (Aphis glycines).

Adaptive evolution of pest insects in response to the introduction of resistant cultivars is well documented and commonly results in virulent (i.e., capable of feeding upon resistant cultivars) insect populations being labeled as distinct biotypes. Phenotypically defined, biotypes frequently remain evolutionarily indistinct, resulting in ineffective application of virulence control measures and shorter durability of resistant cultivars. Here, we utilize an evolutionary framework to discern the genetic relationship between biotypes of the soybean aphid (Aphis glycines, Matsumura). The soybean aphid is invasive in North America and is among the most destructive pests of commercial soybean on the continent. Attempts to breed host-plant-resistant soybean have been hampered by the emergence of virulent aphid biotypes that are unaffected by the plant's resistance mechanism(s). Comparative population genetic analysis of virulent and avirulent (i.e., unable to feed on resistant cultivars) biotypes found populations to be genetically indistinguishable across biotype and geographic distance, with high rates of interpopulation immigration and admixture. The lack of genetic distinction between biotypes coupled with elevated genotypic diversity within all populations suggested virulence has a nongenetic-based or includes a gene complex that is widely distributed throughout soybean aphid populations, which undergo regular dispersal and unimpeded sexual recombination.

Identification of novel sources of host plant resistance to known soybean aphid biotypes.

While soybean cultivars with resistance to the soybean aphid (Aphis glycines Matsumura) have been commercially released, the presence of virulent biotypes capable of overcoming plant resistance threatens the durability of host plant resistance as a stable management tactic. Novel sources of host plant resistance are needed to combat rapid biotype evolution. In this study, we screened 1,061 soybean plant introductions (PIs) for resistance to three known biotypes of A. glycines. Based on a series of growth chamber and field screenings, we identified 11 PIs that showed resistance to biotype 1 of A. glycines. Among these 11 PIs, 7 PIs were resistant to biotype 2 and 5 PIs were resistant to biotype 3. Further, two PIs (PI 606390A from Vietnam and PI 340034 from South Korea) showed resistance to all three biotypes of A. glycines. We also identified 11 PIs that were potentially tolerant to A. glycines, illustrated by no adverse impact on plant quality because of A. glycines infestation. As resistant PIs identified in this study belong to maturity group II-IV, they can be readily crossed to early maturing cultivars adapted to north-central states of the United States, where A. glycines is a major pest. The genetic characterization of resistance in these PIs and incorporation of novel resistant genes into elite soybean cultivars will broaden the defense against multiple biotypes of A. glycines.

Molecular characterization and expression analysis of soluble trehalase gene in Aphis glycines, a migratory pest of soybean.

In insects, the enzyme trehalase plays a crucial role in energy metabolism, chitin synthesis and possibly during plant-insect interactions. We have characterized a soluble trehalase gene (Tre-1) from cDNA of Aphis glycines, a serious migratory pest of soybean. The full-length cDNA of Tre-1 in A. glycines (AyTre-1) was 2550 bp long with an open reading frame of 1770 bp that encoded for a 589 amino acid residues protein. Sequence assessment and phylogenetic analysis of the putative protein suggested that the selected cDNA belongs to soluble trehalase group. Quantitative PCR (qPCR) analysis in different tissues and developmental stages revealed peak mRNA levels of AyTre-1 in the gut (compared with other tissues assayed) and highest expression in the second instar compared with the other developmental stages assayed. Interestingly, a significantly increased expression of AyTre-1 (1.9-fold, P < 0.05) was observed in the alate morphs compared with that in apterate morphs. However, there was no significant difference in AyTre-1 expression in A. glycines-nymphs fed with resistant and susceptible plants. Expression patterns identified in this study provide a platform to investigate the role of AyTre-1 in physiological activities such as flight and feeding in A. glycines. The characterization of soluble trehalase gene may help to develop novel strategies to manage A. glycines using trehalase inhibitors and using RNA interference for knock-down of AyTre-1 expression.

Core RNAi Machinery and Sid1, a Component for Systemic RNAi, in the Hemipteran Insect, Aphis glycines.

RNA interference (RNAi) offers a novel tool to manage hemipteran pests. For the success of RNAi based pest control in the field, a robust and systemic RNAi response is a prerequisite. We identified and characterized major genes of the RNAi machinery, Dicer2 (Dcr2), Argonaute2 (Ago2), and R2d2 in Aphis glycines, a serious pest of soybean. The A. glycines genome encodes for at least one copy of Dcr2, R2d2 and Ago2. Comparative and molecular evolution analyses (dN/dS) showed that domain regions of encoded proteins are highly conserved, whereas linker (non-domain) regions are diversified. Sequence homology and phylogenetic analyses suggested that the RNAi machinery of A. glycines is more similar to that of Tribolium casteneum as compared to that of Drosophila melanogaster. We also characterized Sid1, a major gene implicated in the systemic response for RNAi-mediated gene knockdown. Through qPCR, Dcr2, R2d2, Ago2, and Sid1 were found to be expressed at similar levels in various tissues, but higher expression of Dcr2, R2d2, and Ago2 was seen in first and second instars. Characterization of RNAi pathway and Sid1 in A. glycines will provide the foundation of future work for controlling one of the most important insect pests of soybean in North America.

Characterization of a chitin synthase encoding gene and effect of diflubenzuron in soybean aphid, Aphis glycines.

Chitin synthases are critical enzymes for synthesis of chitin and thus for subsequent growth and development in insects. We identified the cDNA of chitin synthase gene (CHS) in Aphis glycines, the soybean aphid, which is a serious pest of soybean. The full-length cDNA of CHS in A. glycines (AyCHS) was 5802 bp long with an open reading frame of 4704 bp that encoded for a 1567 amino acid residues protein. The predicted AyCHS protein had a molecular mass of 180.05 kDa and its amino acid sequence contained all the signature motifs (EDR, QRRRW and TWGTR) of chitin synthases. The quantitative real-time PCR (qPCR) analysis revealed that AyCHS was expressed in all major tissues (gut, fat body and integument); however, it had the highest expression in integument (~3.5 fold compared to gut). Interestingly, the expression of AyCHS in developing embryos was nearly 7 fold higher compared to adult integument, which probably is a reflection of embryonic molts in hemimetabolus insects. Expression analysis in different developmental stages of A. glycines revealed a consistent AyCHS expression in all stages. Further, through leaf dip bioassay, we tested the effect of diflubenzuron (DFB, Dimilin ®), a chitin-synthesis inhibitor, on A. glycines' survival, fecundity and body weight. When fed with soybean leaves previously dipped in 50 ppm DFB solution, A. glycines nymphs suffered significantly higher mortality compared to control. A. glycines nymphs feeding on diflubenzuron treated leaves showed a slightly enhanced expression (1.67 fold) of AyCHS compared to nymphs on untreated leaves. We discussed the potential applications of the current study to develop novel management strategies using chitin-synthesis inhibitors and using RNAi by knocking down AyCHS expression.

Validation of reference genes for gene expression studies in Aphis glycines (Hemiptera: Aphididae).

Quantitative real-time polymerase chain reaction (qRT-PCR) is a common and robust tool for accurate quantification of mRNA transcripts. To normalize results, a housekeeping gene ([HKG], reference gene or endogenous control gene) is mandatory. Soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), is a significant soybean, Glycine max (L.) Merr., pest, yet gene expression and functional genomics studies are hindered by a lack of stable HKGs. We evaluated seven potential HKGs (SDFS, succinate dehydrogenase flavoprotein subunit; EF1a, elongation factor-la; HEL, helicase; GAPDH, glyceraldehyde-3 phosphate dehydrogenase; RPS9, ribosomal protein S9; TBP, TATA-box binding protein; and UBQ, ubiquitin-conjugating protein) to determine the most efficient HKGs that have stable expression among tissues, developmental stages, and aphids fed on susceptible and host plant-resistant soybean. HKG stability was determined using GeNorm and NormFinder. Results from three different experimental conditions revealed high stability of TBP compared with the other HKGs profiled across the samples assayed. RPS9 showed stable expression among aphids on susceptible and resistant plants, whereas EF1a showed stable expression in tissues and developmental stages. Therefore, we recommend the TBP as a suitable HKG for efficient normalization among treatments, tissues, and developmental stages of A. glycines. In addition, RPS9 may be used for host-plant resistance experiments and EF1a could be considered for testing differential expression across tissues or developmental stages. These results will enable a more accurate and reliable normalization of qRT-PCR data in A. glycines.