Endophytic ancestors of modern leaf miners may have evolved in the Late Carboniferous.

Endophytic feeding behaviors, including stem borings and galling, have been observed in the fossil record from as early as the Devonian and involve the consumption of a variety of plant (and fungal) tissues. Historically, the exploitation of internal stem tissues through galling has been well documented as emerging during the Pennsylvanian (c. 323-299 million years ago (Ma)), replaced during the Permian by galling of foliar tissues. However, leaf mining, a foliar endophytic behavior that today is exhibited exclusively by members of the four hyperdiverse holometabolous insect orders, has been more sparsely documented, with confirmed examples dating back only to the Early Triassic (c. 252-250 Ma). Here, we describe a trace fossil on seed-fern foliage from the Rhode Island Formation of Massachusetts, USA, representing the earliest indication of a general, endophytic type of feeding damage and dating from the Middle Pennsylvanian (c. 312 Ma). Although lacking the full features of Mesozoic leaf mines, this specimen provides evidence of how endophytic mining behavior may have originated. It sheds light on the evolutionary transition to true foliar endophagy, contributes to our understanding of the behaviors of early holometabolous insects, and enhances our knowledge of macroevolutionary patterns of plant-insect interactions.

A new fossil Acmopyle with accessory transfusion tissue and potential reproductive buds: Direct evidence for ever-wet rainforests in Eocene Patagonia.

PREMISE: Acmopyle (Podocarpaceae) comprises two extant species from Oceania that are physiologically restricted to ever-wet rainforests, a confirmed fossil record based on leaf adpressions and cuticles in Australia since the Paleocene, and a few uncertain reports from New Zealand, Antarctica, and South America. We investigated fossil specimens with Acmopyle affinities from the early Eocene Laguna del Hunco site in Patagonia, Argentina. METHODS: We studied 42 adpression leafy-shoot fossils and included them in a total evidence phylogenetic analysis. RESULTS: Acmopyle grayae sp. nov. is based on heterophyllous leafy shoots with three distinct leaf types. Among these, bilaterally flattened leaves uniquely preserve subparallel, linear features that we interpret as accessory transfusion tissue (ATT, an extra-venous water-conducting tissue). Some apical morphologies of A. grayae shoots are compatible with the early stages of ovuliferous cone development. Our phylogenetic analysis recovers the new species in a polytomy with the two extant Acmopyle species. We report several types of insect-herbivory damage. We also transfer Acmopyle engelhardti from the middle Eocene Río Pichileufú flora to Dacrycarpus engelhardti comb. nov. CONCLUSIONS: We confirm the biogeographically significant presence of the endangered West Pacific genus Acmopyle in Eocene Patagonia. Acmopyle is one of the most drought-intolerant genera in Podocarpaceae, possibly due to the high collapse risk of the ATT, and thus the new fossil species provides physiological evidence for the presence of an ever-wet rainforest environment at Laguna del Hunco during the Early Eocene Climatic Optimum.

Spatiotemporal remote sensing of ecosystem change and causation across Alaska.

Contemporary climate change in Alaska has resulted in amplified rates of press and pulse disturbances that drive ecosystem change with significant consequences for socio-environmental systems. Despite the vulnerability of Arctic and boreal landscapes to change, little has been done to characterize landscape change and associated drivers across northern high-latitude ecosystems. Here we characterize the historical sensitivity of Alaska's ecosystems to environmental change and anthropogenic disturbances using expert knowledge, remote sensing data, and spatiotemporal analyses and modeling. Time-series analysis of moderate-and high-resolution imagery was used to characterize land- and water-surface dynamics across Alaska. Some 430,000 interpretations of ecological and geomorphological change were made using historical air photos and satellite imagery, and corroborate land-surface greening, browning, and wetness/moisture trend parameters derived from peak-growing season Landsat imagery acquired from 1984 to 2015. The time series of change metrics, together with climatic data and maps of landscape characteristics, were incorporated into a modeling framework for mapping and understanding of drivers of change throughout Alaska. According to our analysis, approximately 13% (~174,000 ± 8700 km2 ) of Alaska has experienced directional change in the last 32 years (±95% confidence intervals). At the ecoregions level, substantial increases in remotely sensed vegetation productivity were most pronounced in western and northern foothills of Alaska, which is explained by vegetation growth associated with increasing air temperatures. Significant browning trends were largely the result of recent wildfires in interior Alaska, but browning trends are also driven by increases in evaporative demand and surface-water gains that have predominately occurred over warming permafrost landscapes. Increased rates of photosynthetic activity are associated with stabilization and recovery processes following wildfire, timber harvesting, insect damage, thermokarst, glacial retreat, and lake infilling and drainage events. Our results fill a critical gap in the understanding of historical and potential future trajectories of change in northern high-latitude regions.

Real-time, in vivo intracellular recordings of caterpillar-induced depolarization waves in sieve elements using aphid electrodes.

Plants propagate electrical signals in response to artificial wounding. However, little is known about the electrophysiological responses of the phloem to wounding, and whether natural damaging stimuli induce propagating electrical signals in this tissue. Here, we used living aphids and the direct current (DC) version of the electrical penetration graph (EPG) to detect changes in the membrane potential of Arabidopsis sieve elements (SEs) during caterpillar wounding. Feeding wounds in the lamina induced fast depolarization waves in the affected leaf, rising to maximum amplitude (c. 60 mV) within 2 s. Major damage to the midvein induced fast and slow depolarization waves in unwounded neighbor leaves, but only slow depolarization waves in non-neighbor leaves. The slow depolarization waves rose to maximum amplitude (c. 30 mV) within 14 s. Expression of a jasmonate-responsive gene was detected in leaves in which SEs displayed fast depolarization waves. No electrical signals were detected in SEs of unwounded neighbor leaves of plants with suppressed expression of GLR3.3 and GLR3.6. EPG applied as a novel approach to plant electrophysiology allows cell-specific, robust, real-time monitoring of early electrophysiological responses in plant cells to damage, and is potentially applicable to a broad range of plant-herbivore interactions.

Evaluation of Lipid Quality in Fruit: Utilizing Lipidomic Approaches for Assessing the Impact of Biotic Stress on Pecans (Carya illinoinensis).

There is a scarcity of data on how the lipid composition of oily seeds changes in response to biotic stress. Yellow peach moth (Conogethes punctiferalis) has caused massive economic losses on the pecan (Carya illinoinensis) industry. Lipidomics is used in this study to determine the lipid composition of pecan and how it changes in response to insect attack. Pecan had 167 lipids, including 34 glycerolipids (GL), 62 glycerophospholipids (GP), 17 fatty acyls (FA), 41 sphingolipids (SP), and 13 saccharolipids (SL). The effects of biotic stress on lipids, particularly GL and GP, were significant. Biotic stress significantly reduced the lipid content of chains longer than 48. Forty-four significantly different lipids were discovered as potential biomarkers for distinguishing non-infected pecans from infested pecans. In addition, we used bioinformatics to identify the five most important metabolic pathways in order to investigate the processes underlying the changes. Our discoveries may offer valuable insights for enhancing pecan production in the future and contribute novel perspectives towards enhancing the nutritional value of pecans.

A new North American species of Etainia (Lepidoptera, Nepticulidae), feeding on Arbutus and Arctostaphylos species (Ericaceae).

Etainiathoraceleuca van Nieukerken, Epstein & Davis, sp. nov. is the second native American species of Etainia Beirne, 1945, and the second known Etainia species feeding on Ericaceae. The species is known from light-collected adults in the USA (California, Arizona) and Canada (Ontario). These were linked via DNA barcodes to larvae that make short leafmines on Arbutus and Arctostaphylos species, then continue feeding in stems and branches, causing damage in nurseries and planted trees in Sonoma and Marin Counties, California. The holotype was accidentally reared from Arbutusarizonica, without observing the damage. Life history and damage are described in detail. Damage in Arctostaphylosuva-ursi found in Washington State probably belongs to E.thoraceleuca, which is a sister species to the European E.albibimaculella (Larsen, 1927).

Rapid character scoring and tabulation of large leaf-image libraries using Adobe Bridge.

Premise: Digital image libraries are an integral part of specimen-based research. However, coding and extracting metadata for hundreds of specimens on a personal computer can be complex. In addition, most existing workflows require downsampling or platform switching and do not link character data directly to the images. Methods and Results: We demonstrate a method to code and embed into images the standard leaf architecture and insect-damage characters that are widely used in paleobotany. Using the visual file browser Adobe Bridge, customizable and searchable keywords can be applied directly and reversibly to individual full-resolution images, and the data can be extracted and formatted into a matrix using scripts. Conclusions: Our approach is intuitive and acts as a digital mimic and complement to the experience of sorting and analyzing specimens in-person. Keywords can be easily customized for other data types that require visual sorting using image libraries.

Modulation of Aleurone Peroxidases in Kernels of Insect-Resistant Maize (Zea mays L.; Pob84-C3R) After Mechanical and Insect Damage.

Peroxidases (PODs) have many biological functions during the plant life cycle. In maize kernels, endosperm PODs have been identified as biochemical contributors to resistance against Sitophilus zeamais, but their identities have not been determined. In this study, we identified these PODs and determined whether their contributions are basal or inducible. Semi-purification and LC-MS/MS analyses showed that the protein ZmPrx35 is the predominant soluble endosperm POD from kernels of Pob84-C3R. Subsequent time-course analyses after mechanical damage showed that POD activity was regulated in a fluctuating kinetics pattern and that zmprx35 mRNA expression levels reflected this pattern. After 48 h of infestation with S. zeamais or Prostephanus truncatus, soluble endosperm POD activities were 1.38- or 0.85-fold, respectively. Under the same conditions, zmprx35 expression was induced 1.61-fold (S. zeamais infestation) and 1.17-fold (P. truncatus infestation). These findings suggest that ZmPrx35 contributes to the protective responses of aleurone cells against wounding and pest attacks, which could be enhanced/repressed by insect factors. Our data also provide evidence that the mechanisms of resistance of maize Pob84-C3R kernels toward the insect pests S. zeamais and P. truncatus are independent.

Plant and insect herbivore community variation across the Paleocene-Eocene boundary in the Hanna Basin, southeastern Wyoming.

Ecosystem function and stability are highly affected by internal and external stressors. Utilizing paleobotanical data gives insight into the evolutionary processes an ecosystem undergoes across long periods of time, allowing for a more complete understanding of how plant and insect herbivore communities are affected by ecosystem imbalance. To study how plant and insect herbivore communities change during times of disturbance, we quantified community turnover across the Paleocene--Eocene boundary in the Hanna Basin, southeastern Wyoming. This particular location is unlike other nearby Laramide basins because it has an abundance of late Paleocene and Eocene coal and carbonaceous shales and paucity of well-developed paleosols, suggesting perpetually high water availability. We sampled approximately 800 semi-intact dicot leaves from five stratigraphic levels, one of which occurs late in the Paleocene-Eocene thermal maximum (PETM). Field collections were supplemented with specimens at the Denver Museum of Nature & Science. Fossil leaves were classified into morphospecies and herbivore damage was documented for each leaf. We tested for changes in plant and insect herbivore damage diversity using rarefaction and community composition using non-metric multidimensional scaling ordinations. We also documented changes in depositional environment at each stratigraphic level to better contextualize the environment of the basin. Plant diversity was highest during the mid-late Paleocene and decreased into the Eocene, whereas damage diversity was highest at the sites with low plant diversity. Plant communities significantly changed during the late PETM and do not return to pre-PETM composition. Insect herbivore communities also changed during the PETM, but, unlike plant communities, rebound to their pre-PETM structure. These results suggest that insect herbivore communities responded more strongly to plant community composition than to the diversity of species present.

Quantification of Thrips Damage Using Ilastik and ImageJ Fiji.

Quantification of insect damage is an essential measurement for identifying resistance in plants. In screening for host plant resistance against thrips, the total damaged leaf area is used as a criterion to determine resistance levels. Here we present an objective novel method for analyzing thrips damage on leaf disc using the freely available software programs Ilastik and ImageJ. The protocol was developed in order to screen over 40 Capsicum lines for resistance against Frankliniella occidentalis (Western Flower Thrips) and Thrips tabaci (Onion thrips).

Effect of micro-encapsulated antioxidant formulations on mycobiota, residual levels, sensory analyses and insect pest attack in stored peanuts.

The in situ effect of microencapsulated 2(3)-tert-butyl-4 hydroxyanisole (BHA) on stored peanuts (Arachis hipogaea) intended for human consumption was evaluated. Peanut were stored unshelled in flexible containers called "big bags" that were made of polypropylene raffia. 100 kg of peanuts were used in each big bag and stored in refrigerated cells (<18 °C) for about 5 months in two different peanut processing companies during 2015/2016 period. Fungal populations, aflatoxin accumulation, BHA residues, acidity and fatty acid profile, sensory analyses, insect damage and environmental factors variation, were evaluated. At the end of the storage period, significant (p < 0.05) fungitoxic effects of the BHA formulation were observed in the order of 30 and 15% for the first and second company, respectively. Cladosporium, yeasts, Penicillium, Fusarium, Alternaria and Aspergillus were the main fungal isolates. No aflatoxins were found for both companies and years evaluated. In addition, taste of the peanuts was not significantly affected (p < 0.05) by formulation used and insect damage was always lower than 3%. However, different levels of BHA were detected throughout the experiment in the two companies, with final levels of 2.5 for the C1 and 275 ng BHA/g peanuts in C2. Formulation did not affect acidity and organoleptic properties of peanuts. These results show that BHA formulation could be used as part of alternative strategy for control of fungal contamination storage period.

Vegetation recovery in an oil-impacted and burned Phragmites australis tidal freshwater marsh.

In-situ burning of oiled marshes is a cleanup method that can be more effective and less damaging than intrusive manual and mechanical methods. In-situ burning of oil spills has been examined for several coastal marsh types; however, few published data are available for Phragmites australis marshes. Following an estimated 4200gallon crude oil spill and in-situ burn in a Phragmites tidal freshwater marsh at Delta National Wildlife Refuge (Mississippi River Delta, Louisiana), we examined vegetation impacts and recovery across 3years. Oil concentrations in marsh soils were initially elevated in the oiled-and-burned sites, but were below background levels within three months. Oiling and burning drastically affected the marsh vegetation; the formerly dominant Phragmites, a non-native variety in our study sites, had not fully recovered by the end of our study. However, overall vegetation recovery was rapid and local habitat quality in terms of native plants, particularly Sagittaria species, and wildlife value was enhanced by burning. In-situ burning appears to be a viable response option to consider for future spills in marshes with similar plant species composition, hydrogeomorphic settings, and oiling conditions. In addition, likely Phragmites stress from high water levels and/or non-native scale insect damage was also observed during our study and has recently been reported as causing widespread declines or loss of Phragmites stands in the Delta region. It remains an open question if these stressors could lead to a shift to more native vegetation, similar to what we observed following the oil spill and burn. Increased dominance by native plants may be desirable as local patches, but widespread loss of Phragmites, even if replaced by native species, could further acerbate coastal erosion and wetland loss, a major concern in the region.

Aflatoxins, discolouration and insect damage in dried cowpea and pigeon pea in Malawi and the effectiveness of flotation/washing operation in eliminating the aflatoxins.

Aflatoxin contamination and biodeterioration were examined in 302 samples of dry cowpeas and pigeon peas that were randomly purchased from 9 districts of the Southern Region of Malawi during July and November 2015. Further, the impact of flotation/washing on aflatoxin levels on the pulses was elucidated. Aflatoxin analyses involved immunoaffinity column (IAC) clean-up and HPLC quantification with fluorescence detection (FLD) while legume biodeterioration assessments were done by visual inspection. Aflatoxins were frequently detected in cowpea (24%, max., 66 µg/kg) and pigeon pea (22%, max., 80 µg/kg) samples that were collected in the month of July. Lower aflatoxin incidence of 15% in cowpeas (max., 470 µg/kg) and 14% in pigeon peas (max., 377 µg/kg) was recorded in the November collection. Overall, aflatoxin levels were significantly higher in the pulses that were collected in November. However, there were no significant differences in the total aflatoxin (aflatoxin B1 (AFB1) + AFB2 + AFG1 + AFG2) levels between the two types of pulses. Remarkably, in 76.2% of the aflatoxin positive cowpea and in 41.7% of the aflatoxin positive pigeon pea samples, aflatoxin G1 concentration exceeded aflatoxin B1. Insect damage percentage averaged at 18.1 ± 18.2% (mean ± SD) in the cowpeas and 16.1 ± 19.4% in pigeon peas. Mean discolouration percentage (number of pulses) of the cowpeas and pigeon peas was found to be at 6.7 ± 4.9 and 8.7 ± 6.2%, respectively. Washing and discarding the buoyant fraction was highly efficient in reducing aflatoxin levels; only 5.2 ± 11.1% of the initial aflatoxin level was found in the cleaned samples. In conclusion, cowpeas and pigeon peas sold on the local market in Malawi may constitute a hazard especially if floatation/washing step is skipped.

Spatial patterns of aflatoxin levels in relation to ear-feeding insect damage in pre-harvest corn.

Key impediments to increased corn yield and quality in the southeastern US coastal plain region are damage by ear-feeding insects and aflatoxin contamination caused by infection of Aspergillus flavus. Key ear-feeding insects are corn earworm, Helicoverpa zea, fall armyworm, Spodoptera frugiperda, maize weevil, Sitophilus zeamais, and brown stink bug, Euschistus servus. In 2006 and 2007, aflatoxin contamination and insect damage were sampled before harvest in three 0.4-hectare corn fields using a grid sampling method. The feeding damage by each of ear/kernel-feeding insects (i.e., corn earworm/fall armyworm damage on the silk/cob, and discoloration of corn kernels by stink bugs), and maize weevil population were assessed at each grid point with five ears. The spatial distribution pattern of aflatoxin contamination was also assessed using the corn samples collected at each sampling point. Aflatoxin level was correlated to the number of maize weevils and stink bug-discolored kernels, but not closely correlated to either husk coverage or corn earworm damage. Contour maps of the maize weevil populations, stink bug-damaged kernels, and aflatoxin levels exhibited an aggregated distribution pattern with a strong edge effect on all three parameters. The separation of silk- and cob-feeding insects from kernel-feeding insects, as well as chewing (i.e., the corn earworm and maize weevil) and piercing-sucking insects (i.e., the stink bugs) and their damage in relation to aflatoxin accumulation is economically important. Both theoretic and applied ramifications of this study were discussed by proposing a hypothesis on the underlying mechanisms of the aggregated distribution patterns and strong edge effect of insect damage and aflatoxin contamination, and by discussing possible management tactics for aflatoxin reduction by proper management of kernel-feeding insects. Future directions on basic and applied research related to aflatoxin contamination are also discussed.