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The effects of two entomopathogenic fungal endophytes, Beauveria bassiana and Purpureocillium lilacinum (formerly Paecilomyces lilacinus), were assessed on the reproduction of cotton aphid, Aphis gossypii Glover (Homoptera:Aphididae), through in planta feeding trials. In replicate greenhouse and field trials, cotton plants (Gossypium hirsutum) were inoculated as seed treatments with two concentrations of B. bassiana or P. lilacinum conidia. Positive colonization of cotton by the endophytes was confirmed through potato dextrose agar (PDA) media plating and PCR analysis. Inoculation and colonization of cotton by either B. bassiana or P. lilacinum negatively affected aphid reproduction over periods of seven and 14 days in a series of greenhouse trials. Field trials were conducted in the summers of 2012 and 2013 in which cotton plants inoculated as seed treatments with B. bassiana and P. lilacinum were exposed to cotton aphids for 14 days. There was a significant overall effect of endophyte treatment on the number of cotton aphids per plant. Plants inoculated with B. bassiana had significantly lower numbers of aphids across both years. The number of aphids on plants inoculated with P. lilacinum exhibited a similar, but non-significant, reduction in numbers relative to control plants. We also tested the pathogenicity of both P. lilacinum and B. bassiana strains used in the experiments against cotton aphids in a survival experiment where 60% and 57% of treated aphids, respectively, died from infection over seven days versus 10% mortality among control insects. Our results demonstrate (i) the successful establishment of P. lilacinum and B. bassiana as endophytes in cotton via seed inoculation, (ii) subsequent negative effects of the presence of both target endophytes on cotton aphid reproduction using whole plant assays, and (iii) that the P. lilacinum strain used is both endophytic and pathogenic to cotton aphids. Our results illustrate the potential of using these endophytes for the biological control of aphids and other herbivores under greenhouse and field conditions.

Virus-induced gene silencing (VIGS) has been a crucial tool for elucidating gene function in Upland cotton ( Gossypium hirsutum ) due to its complex allotetraploid genome. Reverse-transcription quantitative PCR is routine for measuring gene expression in VIGS studies, yet reference gene stability has not been adequately evaluated when using VIGS especially under biotic stress in cotton. Here, we employed several statistical methods (∆Ct, geNorm, BestKeeper, NormFinder, and weighted rank aggregation) to evaluate the stability of six candidate reference genes ( GhACT7 , GhPP2A1 , GhUBQ7 , GhUBQ14 , GhTMN5 , and GhTBL6 ) in wild-type and VIGS-infiltrated plants under cotton aphid herbivory stress over time in a fully factorial experiment. Ranked stability analyses overwhelmingly indicated that the frequently used reference genes GhUBQ7 and GhUBQ14 were the least stable whereas GhACT7 and GhPP2A1 were the most stable under VIGS and cotton aphid herbivory stress. Results were validated by comparing normalization methods of the phytosterol biosynthesis gene GhHYDRA1 in response to aphid herbivory. Normalization using GhACT7 / GhPP2A1 revealed significant upregulation of GhHYDRA1 in aphid-infested plants. Conversely, normalization using GhUBQ7 reduced sensitivity to detect expression changes, highlighting the importance of stable reference gene selection for accurate expression normalization and interpretation. This study will facilitate future investigations of the genetics underpinning cotton-herbivore interactions necessary for novel pest management biotechnology discovery.

Insect immune responses to multiple pathogen groups including viruses, bacteria, fungi, and entomopathogenic nematodes have traditionally been documented in model insects such as Drosophila melanogaster , or medically important insects such as Aedes aegypti . Despite their potential importance in understanding the efficacy of pathogens as biological control agents, these responses are infrequently studied in agriculturally important pests. Additionally, studies that investigate responses of a host species to different pathogen groups are uncommon, and typically focus on only a single time point during infection. As such, a robust understanding of immune system responses over the time of infection is often lacking in many pest species. This study was conducted to understand how 3 rd instar larvae of the major insect pest Helicoverpa zea responded through the course of an infection by four different pathogenic groups: viruses, bacteria, fungi, and entomopathogenic nematodes; by sampling at three different times post-inoculation. Physiological immune responses were assessed at 4-, 24-, and 48-hours post-infection by measuring hemolymph phenoloxidase concentrations, hemolymph prophenoloxidase concentrations, hemocyte counts, and encapsulation ability. Transcriptional immune responses were measured at 24-, 48-, and 72-hours post-infection by quantifying the expression of PPO2 , Argonaute-2 , JNK , Dorsal , and Relish . This gene set covers the major known immune pathways: phenoloxidase cascade, siRNA, JNK pathway, Toll pathway, and IMD pathway. Our results indicate H . zea has an extreme immune response to Bacillus thuringiensis bacteria, a mild response to Helicoverpa armigera nucleopolyhedrovirus, and little-to-no detectable response to either the fungus Beauveria bassiana or Steinernema carpocapsae nematodes.

The global reliance on Bacillus thuringiensis (Bt) proteins for controlling lepidopteran pests in cotton, corn, and soybean crops underscores the critical need to understand resistance mechanisms. Vip3Aa, one of the most widely deployed and currently effective Bt proteins in genetically modified crops, plays a pivotal role in pest management. This study investigates the molecular basis of Vip3Aa resistance in Australian Helicoverpa armigera through genetic crosses, and integrated genomic and transcriptomic analyses. We identified a previously uncharacterized gene, LOC110373801 (designated HaVipR1 ), as potentially important in Vip3Aa resistance in two field-derived resistant lines. Functional validation using CRISPR/Cas9 knockout in susceptible lines confirmed the gene’s role in conferring high-level resistance to Vip3Aa. Despite extensive laboratory selection of Vip3Aa-resistant colonies in Lepidoptera, the biochemical mechanisms underlying resistance have remained elusive. Our research identifies HaVipR1 as a potential contributor to resistance, adding to our understanding of how insects may develop resistance to this important Bt protein. The identification of HaVipR1 contributes to our understanding of potential resistance mechanisms and may inform future resistance management strategies. Future work should explore the biochemical pathways influenced by HaVipR1 and assess its interactions with other resistance mechanisms. The approach utilized here underscores the value of field-derived resistant lines for understanding resistance in agricultural pests and highlights the need for targeted approaches to manage resistance sustainably.

Fall armyworm (FAW) is a global agricultural pest, causing substantial economic losses in corn and many other crops. Complicating efforts to control this pest is its capacity for long distance flights, which has been described in greatest detail for the central and eastern sections of the United States. FAW infestations are also routinely found in agricultural areas in southern Arizona, which lie beyond the western limits of the mapped migratory pathways. Climate suitability analysis found that the affected Arizona locations cannot support permanent FAW populations, indicating that these FAW most likely arise from annual migrations. A better understanding of this migration would provide insights into how large moth populations can move across desert habitats as well as the degree of gene flow occurring between FAW populations across the North American continent. In this study the Arizona populations were genetically characterized and compared to a selection of permanent and migratory FAW from multiple sites in the United States and Mexico. The results are consistent with migratory contributions from permanent populations in the states of Texas (United States) and Sinaloa (Mexico), while also providing evidence of significant barriers to gene flow between populations within Mexico. An unexpected finding was that two genetically distinct FAW subpopulations known as “host strains” have a differential distribution in the southwest that may indicate significant differences in their migration behavior in this region. These findings indicate that the combination of mitochondrial and Z -linked markers have advantages in comparing FAW populations that can complement and extend the findings from other methods.

Background The fall armyworm ( Spodoptera frugiperda (J.E. Smith)) is a highly polyphagous agricultural pest with long-distance migratory behavior threatening food security worldwide. This pest has a host range of > 80 plant species, but two host strains are recognized based on their association with corn (C-strain) or rice and smaller grasses (R-strain). The population genomics of the United States (USA) fall armyworm remains poorly characterized to date despite its agricultural threat. Results In this study, the population structure and genetic diversity in 55  S. frugiperda samples from Argentina, Brazil, Kenya, Puerto Rico and USA were surveyed to further our understanding of whole genome nuclear diversity. Comparisons at the genomic level suggest a panmictic S. frugiperda population, with only a minor reduction in gene flow between the two overwintering populations in the continental USA, also corresponding to distinct host strains at the mitochondrial level. Two maternal lines were detected from analysis of mitochondrial genomes. We found members from the Eastern Hemisphere interspersed within both continental USA overwintering subpopulations, suggesting multiple individuals were likely introduced to Africa. Conclusions Our research is the largest diverse collection of United States S. frugiperda whole genome sequences characterized to date, covering eight continental states and a USA territory (Puerto Rico). The genomic resources presented provide foundational information to understand gene flow at the whole genome level among S. frugiperda populations. Based on the genomic similarities found between host strains and laboratory vs. field samples, our findings validate the experimental use of laboratory strains and the host strain differentiation based on mitochondria and sex-linked genetic markers extends to minor genome wide differences with some exceptions showing mixture between host strains is likely occurring in field populations.

Understanding the genomic effects of prolonged magnetic exposure is essential for ensuring the safety and stability of crop genomes. 3 Chronobiology: timing as a key for pest management Miller et al.observed that allochronic behavior in field populations of the fall armyworm, Spodoptera frugiperda, aligns with previous laboratory findings on mating timing differences between strains. [...]aligning pesticide applications or pheromone trap monitoring times with the active periods of specific pest strains could enhance management efficiency while minimizing chemical use and ecological impact. 4 Driving technological advancements in smart agriculture Gong et al.proposed an advanced version of the YOLOX-Tiny model optimized for maize crop row navigation line recognition. Future research should explore how magnetic and chronobiological factors can be integrated into real-time monitoring and precision intervention systems. d) Ecological and Safety Considerations: The long-term effects of magnetic exposure on ecosystems, soil microbiomes, and non-target organisms remain largely unexplored.

Cultivated cotton, such as Gossypium hirsutum L., produces extrafloral (EF) nectar on leaves (foliar) and reproductive structures (bracteal) as an indirect anti-herbivore defense. In exchange for this carbohydrate-rich substance, predatory insects such as ants protect the plant against herbivorous insects. Some EF nectar-bearing plants respond to herbivory by increasing EF nectar production. For instance, herbivore-free G . hirsutum produces more bracteal than foliar EF nectar, but increases its foliar EF nectar production in response to herbivory. This study is the first to test for systemically induced changes to the carbohydrate composition of bracteal EF nectar in response to foliar herbivory on G . hirsutum . We found that foliar herbivory significantly increased the sucrose content of bracteal EF nectar while glucose and fructose remained unchanged. Sucrose content is known to influence ant foraging behavior and previous studies of an herbivore-induced increase to EF nectar caloric content found that it led to increased ant activity on the plant. As a follow-up to our finding, ant recruitment to mock EF nectar solutions that varied in sucrose content was tested in the field. The ants did not exhibit any preference for either solution, potentially because sucrose is a minor carbohydrate component in G . hirsutum EF nectar: total sugar content was not significantly affected by the increase in sucrose. Nonetheless, our findings raise new questions about cotton’s inducible EF nectar responses to herbivory. Further research is needed to determine whether an herbivore-induced increase in sucrose content is typical of Gossypium spp ., and whether it constitutes a corollary of systemic sucrose induction, or a potentially adaptive mechanism which enhances ant attraction to the plant

Background Molecular genetic approaches have become vital to understanding the evolutionary processes that act on insect pest populations. From mapping the development of resistance to monitoring and predicting pest movement, genomic tools can inform and enhance pest management programs. Here, we used whole genome sequencing population genomics to unravel novel patterns of population structure, linkage, and selection across the genome of a notorious agricultural pest, the fall armyworm. Results Our data strongly support the existence of two genetically distinct strains of fall armyworm in North America, which have previously been referred to as the C-strain and the R-strain. Although these strains have diverged genetically, we find that differentiation is not uniform across the genome. The Z-chromosome appears to drive divergence between strains with high levels of linkage observed across this chromosome. We also show that a region of the Z-chromosome containing a circadian clock gene implicated in allochronic reproductive isolation is under strain-specific selection. Our data indicates that strains differ in their geographic distributions and exhibit distinct patterns of geographic sub-structuring indicative of unique dispersal patterns. We provide the first evidence for nuclear genomic differentiation between the two major overwintering populations of fall armyworm in the US. Finally, our data reveal population-specific patterns of selection on genomic regions containing putative insecticide resistance alleles, which could relate to their biogeography. Conclusions Our results support the existence of the fall armyworm as a pest dyad in the US, with genetically-distinct strains differing in their population structure, dispersal patterns, and genomic signatures of selection on regions likely involved reproductive isolation and insecticide resistance. These differences should be considered when devising and implementing management strategies.

The fall armyworm, Spodoptera frugiperda (J.E. Smith), is comprised of two genetically distinct strains that are morphologically identical yet exhibit differences in their behavior and physiology (C-strain and R-strain). Evidence of ongoing genetic differentiation between strains highlights the importance of considering strain identity in research and management of fall armyworm populations, but the logistical and technical burden of genotyping limits strain-specific applications. Controlled experiments with laboratory colonies have shown that the strains engage in allochronic (“allo” – different, “chronic” – time) mating behavior, with C-strain mating early in the evening (0–5 hours after sunset) and R-strain mating late in the evening (5–10 hours after sunset). Using temporal field collections and genotype data, we show that strain-specific variation in allochronic male mating behavior occurs across Texas and Florida fall armyworm populations, both of which act as primary source populations for annual migrations of this pest into the continental United States. Time of capture in pheromone traps was significantly different between strains in both Texas and Florida, with the R-strain males consistently being collected in the traps late in the night. The C-strain males were generally captured earlier in the night than their R-strain counterparts, though there was notable variation in the timing between nights and across locations. Allochronic behavior in field populations is consistent with previous laboratory studies reporting differences in the timing of mating between the strains, however increased variability in behavior within and across native populations was observed. Although allochronic behavior in local populations may partially contribute to reproductive isolation between the strains, the behavior is not consistent enough to serve as a complete reproductive barrier. Furthermore, the observed variability in behavior both within and between independent sampling events, especially in the C-strain, poses a challenge to the development of models that utilize time of capture as a predictive phenotype for monitoring strain identity in local populations.