Expansion of cytochrome P450 and cathepsin genes in the generalist herbivore brown marmorated stink bug.

BACKGROUND: The brown marmorated stink bug (Halyomorpha halys) is an invasive pest in North America which causes severe economic losses on tree fruits, ornamentals, vegetables, and field crops. The H. halys is an extreme generalist and this feeding behaviour may have been a major contributor behind its establishment and successful adaptation in invasive habitats of North America. To develop an understanding into the mechanism of H. halys' generalist herbivory, here we specifically focused on genes putatively facilitating its adaptation on diverse host plants. RESULTS: We generated over 142 million reads via sequencing eight RNA-Seq libraries, each representing an individual H. halys adult. The de novo assembly contained 79,855 high quality transcripts, totalling 39,600,178 bases. Following a comprehensive transcriptome analysis, H. halys had an expanded suite of cytochrome P450 and cathepsin-L genes compared to other insects. Detailed characterization of P450 genes from the CYP6 family, known for herbivore adaptation on host plants, strongly hinted towards H. halys-specific expansions involving gene duplications. In subsequent RT-PCR experiments, both P450 and cathepsin genes exhibited tissue-specific or distinct expression patterns which supported their principal roles of detoxification and/or digestion in a particular tissue. CONCLUSIONS: Our analysis into P450 and cathepsin genes in H. halys offers new insights into potential mechanisms for understanding generalist herbivory and adaptation success in invasive habitats. Additionally, the large-scale transcriptomic resource developed here provides highly useful data for gene discovery; functional, population and comparative genomics as well as efforts to assemble and annotate the H. halys genome.

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.

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.

The use of refuge in host plant resistance systems for the control of virulent biotype adaptation in the soybean aphid (Hemiptera: Aphididae.

Host plant resistant (HPR) crop varieties offer control of many insect pest species. However, the evolution of virulent biotypes capable of overcoming plant resistance poses challenges for the implementation of HPR. Widespread planting of HPR crops further reduces HPR efficacy by increasing selection pressure on pests, favoring the rapid proliferation of virulence. An analogous situation occurs in managing insect resistance to transgenic Bt crops, where planting of susceptible refuges effectively delays the evolution and spread of Bt resistance. We investigated the applicability of susceptible refuges in HPR as a tactic to manage virulent biotypes, using the soybean aphid (Aphis glycines Matsumura) as a model system. The virulent biotype 3 and avirulent biotype 1 were reared in greenhouse microcosms using a variety of refuge size, HPR gene, and biotype mixture treatments, allowing us to discern how the presence of a refuge alters the relative fitness and movement of biotypes both by themselves and in competition. The virulent biotype had greater relative fitness in 10 of 12 tested microcosms, with the greatest advantage observed in refuge-free microcosms. In microcosms with a refuge, avirulent fitness increased significantly as these biotypes moved to and used refuge plants. When the two biotypes were reared in the same microcosm, biotype 3's fitness increased significantly relative to when reared in isolation, while biotype 1's fitness was slightly, but not significantly, increased. Our findings suggested that while susceptible refuges would be incapable of reversing the proliferation of virulent biotypes, they could slow the spread of virulence by maintaining avirulence.

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.

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.

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.

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.

Characterizing Resistance to Soybean Aphid (Hemiptera: Aphididae): Antibiosis and Antixenosis Assessment.

Host-plant resistance (HPR) remains a vital tool to manage soybean aphid (Aphis glycines Matsumura), a major pest of soybean in Midwestern United States and southern Canada. HPR can be overcome by virulent biotypes of A. glycines; thus, in order to increase the durability of resistant cultivars, HPR needs to be deployed strategically. To improve the strategic deployment, a complete understanding of HPR in existing resistant germplasm will help ensure HPR success. In this study, we characterized HPR soybean to determine antibiosis and antixenosis categories of resistance to different biotypes of A. glycines. No-choice and free-choice tests were performed on 11 previously reported plant introductions (PIs) possessing resistance to at least one A. glycines biotype (1, 2, and 3). Overall, we found that the PIs manifested differences of a particular resistance category in response to infestation by different biotypes. Our data from no-choice tests indicate that all tested PIs possess antibiosis-based resistance to three biotypes. However, the strength of antibiosis was variable as some PIs showed stronger antibiosis toward a given biotype than others. All tested PIs manifested antixenosis, in addition to antibiosis. Furthermore, detached leaf assays revealed that resistance to A. glycines was not retained in excised soybean leaves. Characterization of resistance in this study can contribute to develop strategies for future deployment of resistant cultivars developed from these PIs.

Aphid resistance is the future for soybean production, and has been since 2004: efforts towards a wider use of host plant resistance in soybean.

The soybean aphid (Aphis glycines) is an important pest of soybeans in the Midwestern US. The first aphid resistance genes were identified in the early 21st century and resistant varieties have been commercially available for 10 years, but have been very underutilized. Major seed companies have avoided commercializing aphid resistant soybean varieties for conventional farmers (i.e., not organic), in part because of the discovery of virulent biotypes in North America. The emergence of soybean aphid populations resistant to insecticides creates a greater incentive for the use of host plant resistance. New research on aphid genetics and markers, plant gene expression and in-plant refuges, suggest important avenues for insect resistance management (IRM) which may encourage more widescale commercialization of this valuable pest management tool.

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.

Jasmonic Acid-Isoleucine (JA-Ile) Is Involved in the Host-Plant Resistance Mechanism Against the Soybean Aphid (Hemiptera: Aphididae).

Host-plant resistance (HPR) is an important tool for pest management, affording both economic and environmental benefits. The mechanisms of aphid resistance in soybean are not well understood, but likely involve the induction of the jasmonic acid (JA) pathway, and possibly other phytohormone signals involved in plant defense responses. Despite the efficacy of aphid resistance in soybean, virulent aphids have overcome this resistance through mostly unknown mechanisms. Here, we have used metabolomic tools to define the role of plant phytohormones, especially the JA pathway, in regulating interactions between aphid-resistant soybean and virulent aphids. We hypothesized that virulent aphids avoid or suppress the JA pathway to overcome aphid resistance. Our results suggested that aphid-resistant soybean increased accumulation of JA-isoleucine (JA-Ile) only when infested with avirulent aphids; virulent aphids did not cause induction of JA-Ile. Further, applying JA-Ile to aphid-resistant soybean reduced subsequent virulent aphid populations. The concentrations of other phytohormones remained unchanged due to aphid feeding, highlighting the importance of JA-Ile in this interaction. These results increase our knowledge of soybean resistance mechanisms against soybean aphids and contribute to our understanding of aphid virulence mechanisms, which will in turn promote the durability of HPR.

Soybean aphids adapted to host-plant resistance by down regulating putative effectors and up regulating transposable elements.

In agricultural systems, crops equipped with host-plant resistance (HPR) have enhanced protection against pests, and are used as a safe and sustainable tool in pest management. In soybean, HPR can control the soybean aphid (Aphis glycines), but certain aphid populations have overcome this resistance (i.e., virulence). The molecular mechanisms underlying aphid virulence to HPR are unknown, but likely involve effector proteins that are secreted by aphids to modulate plant defenses. Another mechanism to facilitate adaptation is through the activity of transposable elements, which can become activated by stress. In this study, we performed RNA sequencing of virulent and avirulent soybean aphids fed susceptible or resistant (Rag1 + Rag2) soybean. Our goal was to better understand the molecular mechanisms underlying soybean aphid virulence. Our data showed that virulent aphids mostly down regulate putative effector genes relative to avirulent aphids, especially when aphids were fed susceptible soybean. Decreased expression of effectors may help evade HPR plant defenses. Virulent aphids also transcriptionally up regulate a diverse set of transposable elements and nearby genes, which is consistent with stress adaptation. Our work demonstrates two mechanisms of pest adaptation to resistance, and identifies effector gene targets for future functional testing.

Whole genome sequence of the soybean aphid, Aphis glycines.

Aphids are emerging as model organisms for both basic and applied research. Of the 5,000 estimated species, only three aphids have published whole genome sequences: the pea aphid Acyrthosiphon pisum, the Russian wheat aphid, Diuraphis noxia, and the green peach aphid, Myzus persicae. We present the whole genome sequence of a fourth aphid, the soybean aphid (Aphis glycines), which is an extreme specialist and an important invasive pest of soybean (Glycine max). The availability of genomic resources is important to establish effective and sustainable pest control, as well as to expand our understanding of aphid evolution. We generated a 302.9 Mbp draft genome assembly for Ap. glycines using a hybrid sequencing approach. This assembly shows high completeness with 19,182 predicted genes, 92% of known Ap. glycines transcripts mapping to contigs, and substantial continuity with a scaffold N50 of 174,505 bp. The assembly represents 95.5% of the predicted genome size of 317.1 Mbp based on flow cytometry. Ap. glycines contains the smallest known aphid genome to date, based on updated genome sizes for 19 aphid species. The repetitive DNA content of the Ap. glycines genome assembly (81.6 Mbp or 26.94% of the 302.9 Mbp assembly) shows a reduction in the number of classified transposable elements compared to Ac. pisum, and likely contributes to the small estimated genome size. We include comparative analyses of gene families related to host-specificity (cytochrome P450's and effectors), which may be important in Ap. glycines evolution. This Ap. glycines draft genome sequence will provide a resource for the study of aphid genome evolution, their interaction with host plants, and candidate genes for novel insect control methods.

Differential Expression of Cytochrome P450 CYP6 Genes in the Brown Marmorated Stink Bug, Halyomorpha halys (Hemiptera: Pentatomidae).

Cytochrome (CYP) P450s are a superfamily of enzymes that detoxify xenobiotics and regulate numerous physiological processes in insects. The genomes of phytophagous insects usually contain large numbers of P450s, especially within the CYP3 clan. Within this clan, CYP6 subfamily members help detoxify plant host secondary metabolites. In this study, we analyzed three CYP6 genes in the highly polyphagous invasive pest, Halyomorpha halys (Stål), commonly known as brown marmorated stink bug. We characterized and validated the expression of HhCYP6BQ27, HhCYP6BK13, and HhCYP6BK24 among sexes, tissues (gut, fat body, and Malpighian tubules) and hosts (apple, corn, soybean). Sequence characterization by amino acid alignments confirmed the presence of conserved motifs typical of the P450 superfamily. No significant differences existed in gene expression among sexes or when fed different hosts, suggesting that these transcripts might have broad substrate specificities. However, significant differences in gene expression were observed among the tissues studied and were gene-dependent. Collectively, the results show that H. halys differentially expressed CYP6 genes among tissues, which may be related to important and specific physiological functions. This study has increased our understanding of H. halys biology that can be useful for functional studies and can potentially be exploited in developing sustainable pest management strategies.

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.

Genetic Structure of Liriomyza trifolii (Diptera: Agromyzidae) Associated With Host Plants From Southeastern Mexico.

Host-associated differentiation (HAD) has played a major role in insect diversification at both macroevolutionary and microevolutionary scales. This evolutionary process has been reported in insects associated with wild and domesticated plant species. In particular, domesticated species harbor large genetic and phenotypic diversity associated with traits of human interest, including variation in nutrition, phenology, fruit, and leaf shape. This diversity may alter selection regimes affecting insect evolution and host specialization. The genus Liriomyza includes highly polyphagous species that are characterized for living and feeding inside plant leaves. Ecological and genetic data suggest the presence of cryptic species within this genus. Moreover, there is evidence of HAD in a group of populations of Liriomyza trifolii (Burgess) associated with Capsicum annum L. (Solanaceae). In this work, we explored HAD in L. trifolii populations from southeastern Mexico, and inquire into differentiation specific to peppers based on cytochrome oxidase I. We also evaluated the relationship between the genetic structure of leafminers and the different types of C. annuum. Our main results did not support previous findings of specialization of L. trifolli on C. annuum. Nevertheless, we found a divergent group of haplotypes associated to Allium cepa (Aspargales: Amaryllidaceae) in sympatric condition to Physalis philadelphica Lam. (Solanales: Solanaceae) and C. annum, suggesting the presence of HAD, as well as significant genetic differentiation of L. trifolii associated to peppers from Oaxaca and Yucatán.

Mechanisms of aphid adaptation to host plant resistance.

Host-plant resistant (HPR) crops can play a major role in preventing insect damage, but their durability is limited due to insect adaptation. Research in basal plant resistance provides a framework to investigate adaptation against HPR. Resistance and adaptation are predicted to follow the gene-for-gene and zigzag models of plant defense. These models also highlight the importance of insect effectors, which are small molecules that modulate host plant defense signaling. We highlight research in insect adaptation to plant resistance, and then draw parallels to virulence adaptation. We focus on virulent biotype evolution within the Aphididae, since this group has the highest number of described virulent biotypes. Understanding how virulence occurs will lead to more durable insect management strategies and enhance food production and security.

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.

Establishment of in vitro soybean aphids, Aphis glycines (Hemiptera: Aphididae): a tool to facilitate studies of aphid symbionts, plant-insect interactions and insecticide efficacy.

BACKGROUND: Studies on plant-insect interactions of the soybean aphid, Aphis glycines (Matsumura), can be influenced by environmental fluctuations, status of the host plant and variability in microbial populations. Maintenance of aphids on in vitro-grown plants minimizes environmental fluctuations, provides uniform host materials and permits the selective elimination of aphid-associated microbes for more standardized controls in aphid research. RESULTS: Aphids were reared on sterile, in vitro-grown soybean seedlings germinated on plant tissue culture media amended with a mixture of antimicrobials. For initiation and maintenance of in vitro aphid colonies, single aphids were inoculated onto single in vitro seedlings. After three rounds of transfer of 'clean' aphids to fresh in vitro seedlings, contamination was no longer observed, and aphids performed equally well when compared with those reared on detached leaves. The addition of the insecticides thiamethoxam and chlorantraniliprole to the culture medium confirmed uptake and caused significant mortality to the in vitro aphids. The use of the antimicrobial mixture removed the associated bacteria Arsenophonus but retained Buchnera and Wolbachia within the in vitro aphids. CONCLUSION: The in vitro aphid system is a novel and highly useful tool to understand insecticidal efficacy and expand our knowledge of tritrophic interactions among plants, insects and symbionts. © 2016 Society of Chemical Industry.