Morphological Characterization and Functional Assessment of Trichomes in Solanaceae

Chapter 1: This chapter provides an overview of various trichome types in plants, and also empirically examines their variation on abaxial and adaxial leaf surface of 14 Solanum species. Detailed nomenclature, density, and dimension measurements of each trichome type has been provided using scanning electron-microscopy. Chapter 2: Scanning electron microscopy was major component of methodology used in my thesis for scanning leaf samples to estimate trichome traits. In this chapter, a more efficient and cost-effective methodology for scanning electron microscopy has been explored. Chapter 3: This chapter examines the relationship of trichome density and herbivore feeding behavior using abaxial and adaxial leaf surfaces of 11 species in Solanaceae. The study concludes that trichome density is higher on abaxial leaf surface of Solanaceae species, and despite the negative consequences of higher trichome density, caterpillars tend to stay and feed more on abaxial leaf surface. Chapter 4: This chapters provides the findings of two foliar surface defenses present in two Solanaceae species with significant variation in trichomes (epidermal hairs) and waxes (hydrophobic layer) and their interactions with a Solanaceae specialist herbivore. Overall, the study concludes that waxes alone can act as a great surface barrier for herbivores. Chapter 5: This chapter provides an overview of my major finding and also suggests future directions for research on plant surface defenses and their functional roles in deterring insect herbivory. To summarize, our results indicate that there is enormous diversity for trichomes in Solanaceae. The trichomes vary in their shape, density, and dimensions within and among the species. The higher trichome density has negative consequences for caterpillar feeding, but caterpillars still tend to feed on abaxial leaf surface with higher trichome density. Additionally, no-prep desktop scanning electron microscopy is as much effective in capturing details of samples as regular scanning electron microscopy is both tedious and expensive. And finally, we also show that epicuticular waxes are also an important component of plant defenses, and should be studied in tandem with trichomes to understand the multi-layered surface defense barrier that restricts herbivory in plants

Are epicuticular waxes a surface defense comparable to trichomes? A test using two Solanum species and a specialist herbivore.

Although plants possess a suite of structural defenses, most studies have focused on trichomes. Trichomes can have both pre- and post-ingestive effects and have been consistently found to reduce herbivory. Along with trichomes, a few studies have focused on epicuticular waxes as an important defense; however, manipulated comparisons examining herbivore growth and development is limited. In this study, using two Solanum species (Solanum glaucescens and Solanum macrocarpon) that vary in both defenses, we tested the hypothesis that variation in defenses will affect herbivore feeding, primarily by restricting feeding commencement. We used electron microscopy together with a series of plant- and diet-based manipulative experiments, using tobacco hornworm (Manduca sexta; Lepidoptera: Sphingidae) as the herbivore. We found that S. glaucescens leaves had significantly fewer trichomes and significantly higher wax content when compared to S. macrocarpon. We also found that S. glaucescens waxes acted as a strong physical barrier resulting in lower mass gain and higher mortality of caterpillars compared to S. macrocarpon. Artificial diet manipulation experiments also suggested the possible toxicity of waxes. Collectively, we show that epicuticular waxes can play a significant role as a strong surface barrier and should be examined further

Morphological characterization of trichomes shows enormous variation in shape, density and dimensions across the leaves of 14 Solanum species

Trichomes are the epidermal appendages commonly observed on plant surfaces including leaves, stem and fruits. Plant trichomes have been well studied as a structural plant defence designed to protect plants against abiotic and biotic stressors such as UV rays, temperature extremities and herbivores. Trichomes are primarily classified into glandular and non-glandular trichomes, based on the presence or absence of a glandular head. The plant genus Solanum is the largest genus of family Solanaceae that houses ~3500 species of ecological and economic importance have a diverse set of trichomes that vary in density and morphology. However, due to the incomplete and contradictory classification system, trichomes have subjective names and have been largely limited to be grouped into glandular or non-glandular types. Through this study, we did a complete workup to classify and characterize trichomes on both adaxial and abaxial leaf surface of 14 wild and domesticated species of the genus Solanum. Using electron microscopy, statistical analyses and artistic rendition, we examined finer details of trichomes and measured their density and dimensions to compile a detailed data set which can be of use for estimating the variation in trichome types, and their density, with consequences for understanding their functional roles. Our study is the first of its kind that provides us with a better and well-defined classification, density and dimension analysis to complete the morphological classification of trichomes on both leaf surfaces of a diverse range of members in Solanum genus

Revisiting plant defense-fitness trade-off hypotheses using Solanum as a model genus

Plants possess physical and chemical defenses which have been found to deter herbivores that feed and oviposit on them. Despite having wide variety of defenses which can be constitutive or induced, plants are attacked and damaged by insects associated with different mouthparts and feeding habits. Since these defenses are costly, trade-offs for growth and defense traits play an important role in warding off the herbivores, with consequences for plant and herbivore growth, development and fitness. Solanum is a diverse and rich genus comprising of over 1,500 species with economic and ecological importance. Although a large number of studies on Solanum species with different herbivores have been carried out to understand plant defenses and herbivore counter defenses, they have primarily focused on pairwise interactions, and a few species of economic and ecological importance. Therefore, a detailed and updated understanding of the integrated defense system (sum of total defenses and trade-offs) is still lacking. Through this review, we take a closer look at the most common plant defense hypotheses, their assumptions and trade-offs and also a comprehensive evaluation of studies that use the genus Solanum as their host plant, and their generalist and specialist herbivores from different feeding guilds. Overall, review emphasizes on using ubiquitous Solanum genus and working toward building an integrated model which can predict defense-fitness-trade-offs in various systems with maximum accuracy and minimum deviations from realistic results

Desktop scanning electron microscopy in plant-insect interactions research: A fast and effective way to capture electron micrographs with minimal sample preparation

The ability to visualize cell and tissue morphology at a high magnification using scanning electron microscopy (SEM) has revolutionized plant sciences research. In plant-insect interactions studies, SEM based imaging has been of immense assistance to understand plant surface morphology including trichomes (plant hairs; physical defense structures against herbivores (Kaur and Kariyat, 2020a, 2020b; Watts and Kariyat, 2021), spines, waxes, and insect morphological characteristics such as mouth parts, antennae, and legs, that they interact with. While SEM provides finer details of samples, and the imaging process is simpler now with advanced image acquisition and processing, sample preparation methodology has lagged. The need to undergo elaborate sample preparation with cryogenic freezing, multiple alcohol washes and sputter coating makes SEM imaging expensive, time consuming, and warrants skilled professionals, making it inaccessible to majority of scientists. Here, using a desktop version of Scanning Electron Microscope (SNE- 4500 Plus Tabletop), we show that the “plug and play” method can efficiently produce SEM images with sufficient details for most morphological studies in plant-insect interactions. We used leaf trichomes of Solanum genus as our primary model, and oviposition by tobacco hornworm (Manduca sexta; Lepidoptera: Sphingidae) and fall armyworm (Spodoptera frugiperda; Lepidoptera: Noctuidae), and leaf surface wax imaging as additional examples to show the effectiveness of this instrument and present a detailed methodology to produce the best results with this instrument. While traditional sample preparation can still produce better resolved images with less distortion, we show that even at a higher magnification, the desktop SEM can deliver quality images. Overall, this study provides detailed methodology with a simpler “no sample preparation” technique for scanning fresh biological samples without the use of any additional chemicals and machinery

Sex and stress modulate pupal defense response in tobacco hornworm

In insects, larval and adult defenses against predators have been well studied. However, pupal (also known as resting stage) defenses have been overlooked and not examined thoroughly. Although some pupa possess antipredator strategies such as hairs, spines, cryptic coloration, and exudation of chemicals, few studies have tested these responses and the factors affecting them. Here, we investigated the behavioral responses in tobacco hornworm Manduca sexta that pupates in soil by introducing an external stimulus using vibrations from an electric toothbrush to mimic predation. We observed that M. sexta made violent wriggling (twitching), followed by pulsating movements in response to the vibrational stimulus. Detailed examination showed that these twitches and pulsating events occurred more frequently and for longer periods of time in male pupa and were dependent on the magnitude of the stress (high and low frequency). However, when we estimated the angular force exerted by pupa using radian and angular momentum of twitches, it was found to be independent of pupal sex. A follow-up experiment on possible cascading effects of stress exposure on eclosion success revealed that low- and high-frequency stress exposure didn’t cause any of the common defects in eclosed adults. Our study clearly demonstrates that the so-called defenseless pupal stage uses a wide range of measurable defense behaviors that can actively defend against predators and should be examined further-linking observed behavior with underlying mechanisms

Phylogenetic ctDNA analysis depicts early-stage lung cancer evolution.

The early detection of relapse following primary surgery for non-small-cell lung cancer and the characterization of emerging subclones, which seed metastatic sites, might offer new therapeutic approaches for limiting tumour recurrence. The ability to track the evolutionary dynamics of early-stage lung cancer non-invasively in circulating tumour DNA (ctDNA) has not yet been demonstrated. Here we use a tumour-specific phylogenetic approach to profile the ctDNA of the first 100 TRACERx (Tracking Non-Small-Cell Lung Cancer Evolution Through Therapy (Rx)) study participants, including one patient who was also recruited to the PEACE (Posthumous Evaluation of Advanced Cancer Environment) post-mortem study. We identify independent predictors of ctDNA release and analyse the tumour-volume detection limit. Through blinded profiling of postoperative plasma, we observe evidence of adjuvant chemotherapy resistance and identify patients who are very likely to experience recurrence of their lung cancer. Finally, we show that phylogenetic ctDNA profiling tracks the subclonal nature of lung cancer relapse and metastasis, providing a new approach for ctDNA-driven therapeutic studies

Picking sides: feeding on the abaxial leaf surface is costly for caterpillars

Main conclusion The study provides us with the evidence that caterpillars tend to feed on the abaxial leaf surface despite the damage caused to them because of higher trichome density. Abstract To defend against herbivory, plants have evolved physical and chemical defense mechanisms, including trichomes (hair like appendages on leaves and stem) being one of them. Caterpillars, a major group of insect herbivores are generally found to occupy the abaxial (underside) leaf surface, considered as an avoidance mechanism from biotic and abiotic stresses. Since trichomes are a first line of defense, we examined the correlation between abaxial vs adaxial (above side) trichomes and caterpillar feeding, behavior, and growth. A combination of field, lab and microscopy experiments were performed using tobacco hornworm, Manduca sexta (Lepidoptera: Sphingidae), a Solanaceae specialist caterpillar, and multiple host species. We found that M. sexta caterpillars overwhelmingly preferred to stay and feed on the abaxial leaf surface, but the abaxial leaf surface also had significantly more trichomes, and consequently, caterpillars took significantly longer to commence feeding. In addition, lab-based diet experiment containing shaved trichomes showed that feeding on the abaxial leaf surface with more trichomes also affected caterpillar growth. Taken together, our study shows that although caterpillars prefer to feed on the abaxial leaf surface, they accrue feeding delays and developmental constraints, indicating tradeoffs affecting performance, and exposure to predation and abiotic stressors

Revisiting plant defense-fitness trade-off hypotheses using Solanum as a model genus

Plants possess physical and chemical defenses which have been found to deter herbivores that feed and oviposit on them. Despite having wide variety of defenses which can be constitutive or induced, plants are attacked and damaged by insects associated with different mouthparts and feeding habits. Since these defenses are costly, trade-offs for growth and defense traits play an important role in warding off the herbivores, with consequences for plant and herbivore growth, development and fitness. Solanum is a diverse and rich genus comprising of over 1,500 species with economic and ecological importance. Although a large number of studies on Solanum species with different herbivores have been carried out to understand plant defenses and herbivore counter defenses, they have primarily focused on pairwise interactions, and a few species of economic and ecological importance. Therefore, a detailed and updated understanding of the integrated defense system (sum of total defenses and trade-offs) is still lacking. Through this review, we take a closer look at the most common plant defense hypotheses, their assumptions and trade-offs and also a comprehensive evaluation of studies that use the genus Solanum as their host plant, and their generalist and specialist herbivores from different feeding guilds. Overall, review emphasizes on using ubiquitous Solanum genus and working toward building an integrated model which can predict defense-fitness-trade-offs in various systems with maximum accuracy and minimum deviations from realistic results