Effective plant virus enrichment using carbon nanotubes and microfluidics.

Plant virus detection and identification in crops is a pillar for disease management, import of crop material, production of clean stock plants and basic plant virology studies. In this report, we present a platform for the enrichment and isolation of known or unknown viruses. This platform is based on carbon nanotube arrays inside a microfluidic device that can be a solution for the identification of low titer viruses from plants. Using our microfluidic devices, we achieved enrichment of two economically important viruses, the orthotospovirus, tomato spotted wilt orthotospovirus (TSWV) and the potyvirus, zucchini yellow mosaic virus (ZYMV). The carbon nanotube arrays integrated in these microfluidic devices are capable of trapping viruses discriminated by their size; the virus rich arrays can be then analyzed by common downstream techniques including immunoassays, PCR, HTS and electron microscopy. This procedure offers a simple to operate and portable sample preparation device capable of trapping viruses from raw plant extracts while reducing the host contamination.

Fungicide ingestion reduces net energy gain and microbiome diversity of the solitary mason bee.

Fungicides are frequently used during tree fruit bloom and can threaten insect pollinators. However, little is known about how non-honey bee pollinators such as the solitary bee, Osmia cornifrons, respond to contact and systemic fungicides commonly used in apple production during bloom. This knowledge gap limits regulatory decisions that determine safe concentrations and timing for fungicide spraying. We evaluated the effects of two contact fungicides (captan and mancozeb) and four translaminar/plant systemic fungicides (cyprodinil, myclobutanil, penthiopyrad, and trifloxystrobin) on larval weight gain, survival, sex ratio, and bacterial diversity. This assessment was carried out using chronic oral ingestion bioassays where pollen provisions were treated with three doses based on the currently recommended field use dose (1X), half dose (0.5X), and low dose (0.1X). Mancozeb and penthiopyrad significantly reduced larval weight and survival at all doses. We then sequenced the 16S gene to characterize the larvae bacteriome of mancozeb, the fungicide that caused the highest mortality. We found that larvae fed on mancozeb-treated pollen carried significantly lower bacterial diversity and abundance. Our laboratory results suggest that some of these fungicides can be particularly harmful to the health of O. cornifrons when sprayed during bloom. This information is relevant for future management decisions about the sustainable use of fruit tree crop protection products and informing regulatory processes that aim to protect pollinators.

Systemic pesticides in a solitary bee pollen food store affect larval development and increase pupal mortality.

Solitary bees are often exposed to various pesticides applied for pest control on farmland while providing pollination services to food crops. Increasing evidence suggests that sublethal toxicity of agricultural pesticides affects solitary bees differently than the social bees used to determine regulatory thresholds, such as honey bees and bumblebees. Studies on solitary bees are challenging because of the difficulties in obtaining large numbers of eggs or young larvae for bioassays. Here we show the toxic and sublethal developmental effects of four widely used plant systemic pesticides on the Japanese orchard bee (Osmia cornifrons). Pollen food stores of this solitary bee were treated with different concentrations of three insecticides (acetamiprid, flonicamid, and sulfoxaflor) and a fungicide (dodine). Eggs were transplanted to the treated pollen and larvae were allowed to feed on the pollen stores after egg hatch. The effects of chronic ingestion of contaminated pollen were measured until adult eclosion. This year-long study revealed that chronic exposure to all tested pesticides delayed larval development and lowered larval and adult body weights. Additionally, exposure to the systemic fungicide resulted in abnormal larval defecation and increased mortality at the pupal stage, indicating potential risk to bees from fungicide exposure. These findings demonstrate potential threats to solitary bees from systemic insecticides and fungicides and will help in making policy decisions to mitigate these effects.

Architecture and potential roles of a delta-class glutathione S-transferase in protecting honey bee from agrochemicals.

The European honey bee, Apis mellifera, serves as the principle managed pollinator species globally. In recent decades, honey bee populations have been facing serious health threats from combined biotic and abiotic stressors, including diseases, limited nutrition, and agrochemical exposure. Understanding the molecular mechanisms underlying xenobiotic adaptation of A. mellifera is critical, considering its extensive exposure to phytochemicals and agrochemicals present in the environment. In this study, we conducted a comprehensive structural and functional characterization of AmGSTD1, a delta class glutathione S-transferase (GST), to unravel its roles in agrochemical detoxification and antioxidative stress responses. We determined the 3-dimensional (3D) structure of a honey bee GST using protein crystallography for the first time, providing new insights into its molecular structure. Our investigations revealed that AmGSTD1 metabolizes model substrates, including 1-chloro-2,4-dinitrobenzene (CDNB), p-nitrophenyl acetate (PNA), phenylethyl isothiocyanate (PEITC), propyl isothiocyanate (PITC), and the oxidation byproduct 4-hydroxynonenal (HNE). Moreover, we discovered that AmGSTD1 exhibits binding affinity with the fluorophore 8-Anilinonaphthalene-1-sulfonic acid (ANS), which can be inhibited with various herbicides, fungicides, insecticides, and their metabolites. These findings highlight the potential contribution of AmGSTD1 in safeguarding honey bee health against various agrochemicals, while also mitigating oxidative stress resulting from exposure to these substances.

Ecological Interactions among Thrips, Soybean Plants, and Soybean Vein Necrosis Virus in Pennsylvania, USA.

Analysis of ecological and evolutionary aspects leading to durability of resistance in soybean cultivars against species Soybean vein necrosis orthotospovirus (SVNV) (Bunyavirales: Tospoviridae) is important for the establishment of integrated pest management (IPM) across the United States, which is a leading exporter of soybeans in the world. SVNV is a seed- and thrips- (vector)-borne plant virus known from the USA and Canada to Egypt. We monitored the resistance of soybean cultivars against SVNV, surveyed thrips species on various crops including soybeans in Pennsylvania, and studied thrips overwintering hibernation behavior under field conditions. Field and lab experiments determined disease incidence and vector abundance in soybean genotypes. The impact of the virus, vector, and their combination on soybean physiology was also evaluated. Seed protein, fiber, oil, and carbohydrate content were analyzed using near infra-red spectroscopy. We found that the variety Channel3917R2x had higher numbers of thrips; hence, it was categorized as preferred, while results showed that no variety was immune to SVNV. We found that thrips infestation alone or in combination with SVNV infection negatively impacted soybean growth and physiological processes.

Chemical footprints mediate habitat selection in co-occurring aphids.

Habitat selection is a critical process that shapes the spatial distribution of species at local and regional scales. The mechanisms underlying habitat preference rely on environmental factors, species traits, and ecological interactions with other species. Here, we examined spatial segregation between two co-occurring aphid species (Rhopalosiphum maidis and R. padi) on wheat plants. We hypothesized that spatial segregation between these aphid species was mediated by aphid cuticular compounds left as chemical "footprints" on plant surfaces. Combining field and laboratory experiments, we first examined how plant microsites alter fitness by measuring the fecundity of each species. Next, we tested whether intra- and interspecific pre-inhabitation modified habitat selection in both aphid species. Both aphid species preferred and exhibited higher fecundity on wheat stems versus leaves. Laboratory trials showed that R. maidis pre-inhabitation altered R. padi spatial preference. By gas chromatography-mass spectrometry analysis and bioassays testing the effects of aphid density and footprint extracts, we found a density-dependent response, with R. padi avoiding locations previously inhabited by R. maidis. The chemical analysis of footprint crude extracts revealed a highly abundant compound, 1-hexacosanol, and when presented in the synthetic form, also elicited R. padi displacement. Altogether, it indicated that R. maidis footprints altered R. padi habitat selection with cuticular compounds playing a relevant role in the habitat selection process in co-occurring aphid species.

Tick Control in a Connected World: Challenges, Solutions, and Public Policy from a United States Border Perspective.

Ticks are able to transmit the highest number of pathogen species of any blood-feeding arthropod and represent a growing threat to public health and agricultural systems worldwide. While there are numerous and varied causes and effects of changes to tick-borne disease (re)emergence, three primary challenges to tick control were identified in this review from a U.S. borders perspective. (1) Climate change is implicated in current and future alterations to geographic ranges and population densities of tick species, pathogens they can transmit, and their host and reservoir species, as highlighted by Ixodes scapularis and its expansion across southern Canada. (2) Modern technological advances have created an increasingly interconnected world, contributing to an increase in invasive tick species introductions through the increased speed and frequency of trade and travel. The introduction of the invasive Haemaphysalis longicornis in the eastern U.S. exemplifies the challenges with control in a highly interconnected world. (3) Lastly, while not a new challenge, differences in disease surveillance, control, and management strategies in bordering countries remains a critical challenge in managing ticks and tick-borne diseases. International inter-agency collaborations along the U.S.-Mexico border have been critical in control and mitigation of cattle fever ticks (Rhipicephalus spp.) and highlight the need for continued collaboration and research into integrated tick management strategies. These case studies were used to identify challenges and opportunities for tick control and mitigation efforts through a One Health framework.

Morphology of the Female Reproductive System of the Soybean Thrips, Neohydatothrips variabilis (Beach, 1896) (Thysanoptera: Thripidae).

Soybean thrips (Neohydatothrips variabilis) are an important phytophagous vector of the widely recognized Soybean vein necrosis orthotospovirus (SVNV). Understanding the egg-laying behavior of these thrips could aid in developing strategies for the management of the vector and virus. In this study, we described the egg-laying behavior of N. variabilis and reconstructed the three-dimensional morphology of the female terminalia by using serial block-face scanning electron microscopy (SBFSEM) and confocal laser scanning microscopy (CLSM). The female reproductive system consists of two panoistic ovaries consisting of eight ovarioles. The appendage gland is connected to the ovaries by two muscles, and to the body wall by a single muscle. The spermatheca is connected to the eighth tergum through four branched muscles, to the basivalvulae of the ovipositor by one muscle and to the vagina by a single muscle. The external genitalia are operated by seven muscles. The movement of the eggs inside the ovipositor is achieved by the back and forth "rocking" movement of the first valvulae and valvifer. Eggs are deposited into the parenchymatous tissue alongside leaf veins. To the best of our knowledge, this is the first study describing the internal and external genitalia of N. variabilis.

The Effect of Species Soybean Vein Necrosis Orthotospovirus (SVNV) on Life Table Parameters of Its Vector, Soybean Thrips (Neohydatothrips variabilis Thysanoptera: Thripidae).

Soybean vein necrosis orthotospovirus (SVNV: Tospoviridae: Orthotospovirus), the causal agent of soybean vein necrosis disease, is vectored by soybean thrips Neohydatothrips variabilis (Beach, 1896), and to a lesser extent by five other thrips species. There is increasing incidence of soybean vein necrosis (SVN) disease in all soybean growing states in the United States, Canada, Egypt and Pakistan, necessitating a study of the system's ecology and management. We addressed the effect of SVNV on the life table parameters of the vector. We used an 'age-stage two-sex' life table approach, which provided detailed life stage durations of each larval instar and adults (both sexes). Our results showed that the intrinsic rate of increase (r), finite rate of increase (λ) and mortality index (qx) were higher in the infected population, while the net reproduction rate (Ro), cumulative probability of survival (lx) and gross reproduction rate (GRR) were lower in the uninfected population. Overall, in both infected and uninfected populations, the number of eggs producing haploid males via arrhenotoky ranged from 9-12 per female. Male to female ratio was female biased in the infected population. Overall, our study provided evidence that virus infection, by decreasing the population doubling time, could enhance the virus and vector populations in soybeans.

Potential distribution of fall armyworm in Africa and beyond, considering climate change and irrigation patterns.

The fall armyworm, Spodoptera frugiperda (FAW), first invaded Africa in 2016 and has since become established in many areas across the continent where it poses a serious threat to food and nutrition security. We re-parameterized the existing CLIMEX model to assess the FAW global invasion threat, emphasizing the risk of transient and permanent population establishment in Africa under current and projected future climates, considering irrigation patterns. FAW can establish itself in almost all countries in eastern and central Africa and a large part of western Africa under the current climate. Climatic barriers, such as heat and dry stresses, may limit the spread of FAW to North and South Africa. Future projections suggest that FAW invasive range will retract from both northern and southern regions towards the equator. However, a large area in eastern and central Africa is projected to have an optimal climate for FAW persistence. These areas will serve as FAW 'hotspots' from where it may migrate to the north and south during favorable seasons and then pose an economic threat. Our projections can be used to identify countries at risk for permanent and transient FAW-population establishment and inform timely integrated pest management interventions under present and future climate in Africa.

Anti-Herbivore Resistance Changes in Tomato with Elevation.

Local adaptations of host plants to climatic conditions along an elevation gradient can affect insect-plant interactions. Using local accessions sampled from different elevations within South America, plant defense responses and herbivore growth were evaluated on two host plants: a) cherry tomato, Solanum lycopersicum var. cerasiforme, and b) wild tomato, Solanum pimpinellifolium. The elevational origin of the accessions ranged from 100 to 3000 m above sea level. We hypothesized a higher level of defensive compounds in plants originating from lower elevations and, consequently, stronger resistance to insect herbivory. Interestingly, plant resistance to insect herbivory, as demonstrated by a reduction in Helicoverpa zea growth, was stronger for middle and high-elevation accessions. Total phenolic content increased with elevation in both herbivore-damaged and undamaged leaves, augmenting plant resistance. However, an elevational gradient was not evident for plant defensive proteins (polyphenol oxidase and trypsin protease inhibitors) or the density of leaf trichomes. Tradeoffs between constitutive and induced defenses were evident in both tomato genotypes. Future studies should test the role of plasticity in plant defense systems in restricting or facilitating range expansion of insect herbivores with climate change.

Biology, Genetic Diversity, and Conservation of Wild Bees in Tree Fruit Orchards.

Different species of bees provide essential ecosystem services by pollinating various agricultural crops, including tree fruits. Many fruits and nuts depend on insect pollination, primarily by wild and managed bees. In different geographical regions where orchard crops are grown, fruit growers rely on wild bees in the farmscape and use orchard bees as alternative pollinators. Orchard crops such as apples, pears, plums, apricots, etc., are mass-flowering crops and attract many different bee species during their bloom period. Many bee species found in orchards emerge from overwintering as the fruit trees start flowering in spring, and the active duration of these bees aligns very closely with the blooming time of fruit trees. In addition, most of the bees in orchards are short-range foragers and tend to stay close to the fruit crops. However, the importance of orchard bee communities is not well understood, and many challenges in maintaining their populations remain. This comprehensive review paper summarizes the different types of bees commonly found in tree fruit orchards in the fruit-growing regions of the United States, their bio-ecology, and genetic diversity. Additionally, recommendations for the management of orchard bees, different strategies for protecting them from multiple stressors, and providing suitable on-farm nesting and floral resource habitats for propagation and conservation are discussed.

Insect Herbivore Populations and Plant Damage Increase at Higher Elevations.

Elevation gradients are used as a proxy to simulate climate change effects. A field study was conducted along an elevational gradient in Nepal to understand the effects of abiotic conditions on agriculturally important insect herbivore populations (tobacco caterpillar: Spodoptera litura, tomato fruit worm: Helicoverpa armigera, and South American leaf miner, Tuta absoluta) and herbivory damage on tomatoes. Elevation ranged from 100 m to 1400 m above sea level, representing different climatic zones where tomatoes are grown. Contrary to our hypothesis, natural herbivore populations and herbivory damage significantly increased at higher elevations. Individual insect species responses were variable. Populations of S. litura and T. absoluta increased at higher elevations, whereas the H. armigera population was highest at the mid-elevational range. Temperature variations with elevation also affected insect catch numbers and the level of plant damage from herbivory. In the context of climate warming, our results demonstrate that the interactive effects of elevation and climatic factors (e.g., temperature) will play an important role in determining the changes in insect pest populations and the extent of crop losses.

Fungal infections lead to shifts in thermal tolerance and voluntary exposure to extreme temperatures in both prey and predator insects.

Pathogens can modify many aspects of host behavior or physiology with cascading impacts across trophic levels in terrestrial food webs. These changes include thermal tolerance of hosts, however the effects of fungal infections on thermal tolerances and behavioral responses to extreme temperatures (ET) across trophic levels have rarely been studied. We examined how a fungal pathogen, Beauveria bassiana, affects upper and lower thermal tolerance, and behavior of an herbivorous insect, Acyrthosiphon pisum, and its predator beetle, Hippodamia convergens. We compared changes in thermal tolerance limits (CTMin and CTMax), thermal boldness (voluntary exposure to ET), energetic cost (ATP) posed by each response (thermal tolerance and boldness) between healthy insects and insects infected with two fungal loads. Fungal infection reduced CTMax of both aphids and beetles, as well as CTMin of beetles. Fungal infection modified the tendency, or boldness, of aphids and predator beetles to cross either warm or cold ET zones (ETZ). ATP levels increased with pathogen infection in both insect species, and the highest ATP levels were found in individuals that crossed cold ETZ. Fungal infection narrowed the thermal tolerance range and inhibited thermal boldness behaviors to cross ET. As environmental temperatures rise, response to thermal stress will be asymmetric among members of a food web at different trophic levels, which may have implications for predator-prey interactions, food web structures, and species distributions.

Improving the Phenotypic Properties of the Ceratitis capitata (Diptera: Tephritidae) Temperature-Sensitive Lethal Genetic Sexing Strain in Support of Sterile Insect Technique Applications.

The genetic sexing strain (GSS) of the Mediterranean fruit fly (Ceratitis capitata (Wiedemann)) Vienna 8D53- is based on a male-linked translocation system and uses two selectable markers for male-only production, the white pupae (wp) and the temperature sensitivity lethal (tsl) genes. In this GSS, males emerge from brown pupae and are resistant to high temperatures while females emerge from white pupae, are sensitive to high temperatures. However, double homozygous females (wp tsl/wp tsl) exhibit a slower development rate compared to heterozygous males (wp+tsl+/wp tsl) during the larval stage, which was attributed to the pleiotropic effects of the tsl gene. We present the first evidence that this slower development is due to a different gene, here namely slow development (sd), which is closely linked to the tsl gene. Taking advantage of recombination phenomena between the two loci, we report the isolation of a novel temperature sensitivity lethal strain using the wp mutation as a morphological marker, which showed faster development (wp tsl FD) during the larval stage and increased in its temperature sensitivity compared with the normal tsl strain. Moreover, the introgression of this novel wp tsl FD combined trait into the Vienna 8D53- GSS, resulted in a novel Vienna 8D53- FD GSS, where females showed differences in the thermal sensibility, larval development speed, and productivity profiles. The modification of these traits and their impact on the mass rearing of the GSS for sterile insect technique applications are discussed.

Asymmetric Responses to Climate Change: Temperature Differentially Alters Herbivore Salivary Elicitor and Host Plant Responses to Herbivory.

The effect of temperature on insect-plant interactions in the face of changing climate is complex as the plant, its herbivores and their interactions are usually affected differentially leading to an asymmetry in response. Using experimental warming and a combination of biochemical and herbivory bioassays, the effects of elevated temperatures and herbivore damage (Helicoverpa zea) on resistance and tolerance traits of Solanum lycopersicum var. Better boy (tomato), as well as herbivory performance and salivary defense elicitors were examined. Insects and plants were differentially sensitive towards warming within the experimental temperature range. Herbivore growth rate increased with temperature, whereas plants growth as well as the ability to tolerate stress measured by photosynthesis recovery and regrowth ability were compromised at the highest temperature regime. In particular, temperature influenced the caterpillars' capacity to induce plant defenses due to changes in the amount of a salivary defense elicitor, glucose oxidase (GOX). This was further complexed by the temperature effects on plant inducibility, which was significantly enhanced at an above-optimum temperature; this paralleled with an increased plants resistance to herbivory but significantly varied between previously damaged and undamaged leaves. Elevated temperatures produced asymmetry in species' responses and changes in the relationship among species, indicating a more complicated response under a climate change scenario.

A new ingestion bioassay protocol for assessing pesticide toxicity to the adult Japanese orchard bee (Osmia cornifrons).

Adopting an Integrated Pest and Pollinator Management strategy requires an evaluation of pesticide risk for pollinator species. For non-Apid species, however, the standardized ingestion assays are difficult to implement. This hinders the consideration of non-Apid species in farm management strategies and government regulatory processes. We describe a new method for a mason bee, Osmia cornifrons, which is an important pollinator of apples and other fruit crops. Our approach overcomes high control mortality seen in other bioassay protocols and expands testing to include males as well as females. The new pesticide toxicity assessment protocol showed that (1) a group feeding method is optimum even though there is no trophallaxis, (2) males had better tolerance to pesticides although they are smaller, and (3) pesticides can cause additional mortality after the standard 48 h assessment time specified by European Food Safety Authority and U.S. Environmental Protection Agency.

Pollinator exposure to systemic insecticides and fungicides applied in the previous fall and pre-bloom period in apple orchards.

Pollinators provide a crucial ecosystem service by pollinating commercially cultivated crops, but they are frequently exposed to various agricultural chemicals used for pest management. In this study, we assessed the potential exposure of pollinators to various systemic insecticides and a fungicide used in apple orchards. Residue levels were determined for the whole flower as well as pollen and nectar separately for pre-bloom applications of acetamiprid, imidacloprid, sulfoxaflor, thiacloprid, thiamethoxam, and myclobutanil. Very low pesticide residue levels (2-70 parts per billion, ppb) were found in the whole flower, pollen and nectar samples compared with pesticide concentrations of 60-200 parts per million (ppm) in applied foliarly only 5 days earlier. Insecticide residues from nectar and pollen samples were below the USA EPA classification of No Observable Effect Limit (NOEL) for acute toxicity to honey bees, suggesting that a single foraging visit to flowers may not cause toxicity to bees. However, cumulative acute exposure from multiple flower visits could potentially be harmful to bees, and needs to be studied further. We also examined apple flowers for residues of several systemic insecticides that were applied for brown marmorated stink bug control late in the fall of the previous season. None of the fall sprays that contained premixed insecticide active ingredients (viz., thiamethoxam + lambda-cyhalothrin, and imidacloprid + beta-cyfluthrin), including multiple applications of individual active ingredients (viz., dinotefuran, clothianidin, and sulfoxaflor), persisted until the following spring. Based on these findings, fall applications of insecticides used for controlling invasive pests such as the brown marmorated stink bug (Halyomorpha halys) and the spotted lanternfly (Lycorma delicatula) could be considered safe to pollinator species foraging in apple orchards during the spring bloom the following season.

A Method for a Long-Term Marking of Spotted Lanternfly (Hemiptera: Fulgoridae) Using a Stable Isotope of Nitrogen.

Developing a lifelong marking method for Lycorma delicatula (White, 1845) is crucial to investigate ecological processes. Here we validate a marking method using stable isotope enrichment (15N) of host plants to track spotted lanternfly (SLF), an invasive species causing economic damage on grapes, hardwood forest and landscape tree species. To validate this method, we first determined the isotope dosage to be sprayed on the host plants and subsequently detected in SLF. Second, we examined whether 15N mark remains detectable from the nymphal to adult stage. We demonstrated that two stable isotope dosages applied to the host plants were assimilated by the insect and equally detectable in the exoskeleton, wings, and mature eggs ready to be oviposited. This safe and reliable method can be used to examine fundamental processes of the biology and ecology of SLF that range from dietary resources and resource allocation to food-web structure and dispersal patterns.