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.

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], 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 SEM (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.

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.

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. 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.