Orchids reduce attachment of herbivorous snails with leaf trichomes.

Protective structures in the epidermis are essential for land plants to defend themselves against herbivores. In this study, we investigated the effect of different types of trichomes of three orchids, Calanthe triplicata, Dendrochilum pallidiflavens and Trichotosia ferox, on attachment of herbivorous land snails, using histochemistry and centrifuge experiments. Size, ornamentation and histochemistry of epicuticular trichomes on the orchid leaves were assessed with light microscopy, scanning electron microscopy and transmission electron microscopy. Total forces needed to detach two differently shaped snail species, Subulina octona and Pleurodonte isabella, were measured using a turntable equipped with a synchronized strobe. Snails were placed in two positions, either perpendicular or parallel to the main veins on the orchid leaves, both on the adaxial (= upper) or abaxial (= lower) side. The results obtained provided three new insights. First, a perpendicular or parallel position of the snails to the main veins did not significantly affect the attachment performance of either species tested. Secondly, snails detached significantly easier on leaf sides covered with a high density of lignin filled epicuticular trichomes. Thirdly, the removal of glandular trichomes did not affect the attachment forces; however, the absence of lignified trichomes increased the attachment of the snails. Our study highlights the importance of studying micro-ornamentation in combination with performance for obtaining a better understanding of the defense mechanisms employed by different species of orchids to deter herbivorous snails.

Translational characterization of the temporal dynamics of metabolic dysfunctions in liver, adipose tissue and the gut during diet-induced NASH development in Ldlr-/-.Leiden mice.

Background: NAFLD progression, from steatosis to inflammation and fibrosis, results from an interplay of intra- and extrahepatic mechanisms. Disease drivers likely include signals from white adipose tissue (WAT) and gut. However, the temporal dynamics of disease development remain poorly understood. Methods: High-fat-diet (HFD)-fed Ldlr-/-.Leiden mice were compared to chow-fed controls. At t = 0, 8, 16, 28 and 38w mice were euthanized, and liver, WAT depots and gut were analyzed biochemically, histologically and by lipidomics and transcriptomics together with circulating factors to investigate the sequence of pathogenic events and organ cross-talk during NAFLD development. Results: HFD-induced obesity was associated with an increase in visceral fat, plasma lipids and hyperinsulinemia at t = 8w, along with increased liver steatosis and circulating liver damage biomarkers. In parallel, upstream regulator analysis predicted that lipid catabolism regulators were deactivated and lipid synthesis regulators were activated. Subsequently, hepatocyte hypertrophy, oxidative stress and hepatic inflammation developed. Hepatic collagen accumulated from t = 16 w and became pronounced at t = 28-38 w. Epididymal WAT was maximally hypertrophic from t = 8 w, which coincided with inflammation development. Mesenteric and subcutaneous WAT hypertrophy developed slower and did not appear to reach a maximum, with minimal inflammation. In gut, HFD significantly increased permeability, induced a shift in microbiota composition from t = 8 w and changed circulating gut-derived metabolites. Conclusion: HFD-fed Ldlr-/-.Leiden mice develop obesity, dyslipidemia and insulin resistance, essentially as observed in obese NAFLD patients, underlining their translational value. We demonstrate that marked epididymal-WAT inflammation, and gut permeability and dysbiosis precede the development of NAFLD stressing the importance of a multiple-organ approach in the prevention and treatment of NAFLD.

Sepal Identity of the Pappus and Floral Organ Development in the Common Dandelion (Taraxacum officinale; Asteraceae).

The dry one-seeded fruits (cypselae) of the Asteraceae are often crowned with a pappus, an appendage of hairs or scales that assists in dispersal. It is generally assumed, but little investigated, that the pappus represents the outer floral whorl where the sepals are usually located. We analysed pappus-sepal homology in dandelions using micromorphological and floral gene expression analyses. We show that the pappus initiates from a ring primordium at the base of the corolla, heterochronic to the petals. Pappus parts form from this ring, with those in the alternipetalaous position usually being ahead in growth, referring to sepal identity. Tof-APETALLA1 expression increased during floret development and was highest in mature pappus. Tof-PISTILLATA expression was high and confined to the floral tissues containing the petals and stamens, consistent with expectations for sepals. Apart from the pappus, the dispersal structure of dandelion consists of the upper part of the fruit, the beak, which originates from the inner floral whorl. Thus, our results support the homology of the pappus with the sepals, but show that it is highly derived. Together with our floral stage definitions and verified qPCR reference genes, they provide a basis for evolution and development studies in dandelions and possibly other Asteraceae.

Pitfall Flower Development and Organ Identity of Ceropegia sandersonii (Apocynaceae-Asclepiadoideae).

Deceptive Ceropegia pitfall flowers are an outstanding example of synorganized morphological complexity. Floral organs functionally synergise to trap fly-pollinators inside the fused corolla. Successful pollination requires precise positioning of flies headfirst into cavities at the gynostegium. These cavities are formed by the corona, a specialized organ of corolline and/or staminal origin. The interplay of floral organs to achieve pollination is well studied but their evolutionary origin is still unclear. We aimed to obtain more insight in the homology of the corona and therefore investigated floral anatomy, ontogeny, vascularization, and differential MADS-box gene expression in Ceropegia sandersonii using X-ray microtomography, Light and Scanning Electronic Microscopy, and RT-PCR. During 10 defined developmental phases, the corona appears in phase 7 at the base of the stamens and was not found to be vascularized. A floral reference transcriptome was generated and 14 MADS-box gene homologs, representing all major MADS-box gene classes, were identified. B- and C-class gene expression was found in mature coronas. Our results indicate staminal origin of the corona, and we propose a first ABCDE-model for floral organ identity in Ceropegia to lay the foundation for a better understanding of the molecular background of pitfall flower evolution in Apocynaceae.

Ontogeny and Anatomy of the Dimorphic Pitchers of Nepenthes rafflesiana Jack.

An enigmatic feature of tropical pitcher plants belonging to the genus Nepenthes is their dimorphic prey-capturing pitfall traps. In many species, the conspicuously shaped upper and lower pitchers grow from a swollen leaf tendril tip until finally opening as insect-alluring devices. Few have studied the ontogeny of these traps from an anatomical and quantitative morphological perspective. We investigated whether the anatomy and development of lower and upper type pitchers of N. rafflesiana differ or overlap in terms of 3D geometric morphology and microstructure progression and presence. We hypothesized that there is an overlap in the initial, but not all, developmental stages of the two pitcher types and that one pitcher type is suspended in development. We identified four important morphological changes of pitcher ontogeny and defined these as curvation, elongation, inflation and maturation phases. Pitcher length indicated progress through developmental phases, and we propose to use it as a tool for indication of developmental stage. Microstructure development coincided with the developmental phases defined. Additionally, we discovered a new anatomical feature of extrafloral nectariferous peristomal glands between the inner peristome ridges of upper and lower pitchers being hollow and analyze the chemistry of the sugars on the outside of these glands. Ontogenetic shape analysis indicated that upper and lower pitcher types develop with similar phase progression but have no directly overlapping morphology. This means that upper pitchers are not a derived state from lower pitchers. Independent developmental programs evolved to produce distinctly shaped upper and lower pitchers in Nepenthes, likely to exploit different food sources.

Morphological and Molecular Characterization of Orchid Fruit Development.

Efficient seed dispersal in flowering plants is enabled by the development of fruits, which can be either dehiscent or indehiscent. Dehiscent fruits open at maturity to shatter the seeds, while indehiscent fruits do not open and the seeds are dispersed in various ways. The diversity in fruit morphology and seed shattering mechanisms is enormous within the flowering plants. How these different fruit types develop and which molecular networks are driving fruit diversification is still largely unknown, despite progress in eudicot model species. The orchid family, known for its astonishing floral diversity, displays a huge variation in fruit dehiscence types, which have been poorly investigated. We undertook a combined approach to understand fruit morphology and dehiscence in different orchid species to get more insight into the molecular network that underlies orchid fruit development. We describe fruit development in detail for the epiphytic orchid species Erycina pusilla and compare it to two terrestrial orchid species: Cynorkis fastigiata and Epipactis helleborine. Our anatomical analysis provides further evidence for the split carpel model, which explains the presence of three fertile and three sterile valves in most orchid species. Interesting differences were observed in the lignification patterns of the dehiscence zones. While C. fastigiata and E. helleborine develop a lignified layer at the valve boundaries, E. pusilla fruits did not lignify at these boundaries, but formed a cuticle-like layer instead. We characterized orthologs of fruit-associated MADS-domain transcription factors and of the Arabidopsis dehiscence-related genes INDEHISCENT (IND)/HECATE 3 (HEC3), REPLUMLESS (RPL) and SPATULA (SPT)/ALCATRAZ (ALC) in E. pusilla, and found that the key players of the eudicot fruit regulatory network appear well-conserved in monocots. Protein-protein interaction studies revealed that MADS-domain complexes comprised of FRUITFULL (FUL), SEPALLATA (SEP) and AGAMOUS (AG) /SHATTERPROOF (SHP) orthologs can also be formed in E. pusilla, and that the expression of HEC3, RPL, and SPT can be associated with dehiscence zone development similar to Arabidopsis. Our expression analysis also indicates differences, however, which may underlie fruit divergence.

Evolution of wood anatomical characters in Nepenthes and close relatives of Caryophyllales.

Background and Aims: Nepenthes attracts wide attention with its spectacularly shaped carnivorous pitchers, cultural value and horticultural curiosity. Despite the plant's iconic fascination, surprisingly little anatomical detail is known about the genus beyond its modified leaf tip traps. Here, the wood anatomical diversity of Nepenthes is explored. This diversity is further assessed with a phylogenetic framework to investigate whether the wood characters within the genus are relevant from an evolutionary or ecological perspective, or rather depend on differences in developmental stages, growth habits, substrates or precipitation. Methods: Observations were performed using light microscopy and scanning electron microscopy. Ancestral states of selected wood and pith characters were reconstructed using an existing molecular phylogeny for Nepenthes and a broader Caryophyllales framework. Pairwise comparisons were assessed for possible relationships between wood anatomy and developmental stages, growth habits, substrates and ecology. Key Results: Wood anatomy of Nepenthes is diffuse porous, with mainly solitary vessels showing simple, bordered perforation plates and alternate intervessel pits, fibres with distinctly bordered pits (occasionally septate), apotracheal axial parenchyma and co-occurring uni- and multiseriate rays often including silica bodies. Precipitation and growth habit (stem length) are linked with vessel density and multiseriate ray height, while soil type correlates with vessel diameter, vessel element length and maximum ray width. For Caryophyllales as a whole, silica grains, successive cambia and bordered perforation plates are the result of convergent evolution. Peculiar helical sculpturing patterns within various cell types occur uniquely within the insectivorous clade of non-core Caryophyllales. Conclusions: The wood anatomical variation in Nepenthes displays variation for some characters dependent on soil type, precipitation and stem length, but is largely conservative. The helical-banded fibre-sclereids that mainly occur idioblastically in pith and cortex are synapomorphic for Nepenthes , while other typical Nepenthes characters evolved convergently in different Caryophyllales lineages.

Dextral and sinistral Amphidromus inversus (Gastropoda: Pulmonata: Camaenidae) produce dextral sperm.

Coiling direction in pulmonate gastropods is determined by a single gene via a maternal effect, which causes cytoskeletal dynamics in the early embryo of dextral gastropods to be the mirror image of the same in sinistral ones. We note that pulmonate gastropod spermatids also go through a helical twisting during their maturation. Moreover, we suspect that the coiling direction of the helical elements of the spermatozoa may affect their behaviour in the female reproductive tract, giving rise to the possibility that sperm chirality plays a role in the maintenance of whole-body chiral dimorphism in the tropical arboreal gastropod Amphidromus inversus (Müller, 1774). For these reasons, we investigated whether there is a relationship between a gastropod's body chirality and the chirality of the spermatozoa it produces. We found that spermatozoa in A. inversus are always dextrally coiled, regardless of the coiling direction of the animal itself. However, a partial review of the literature on sperm morphology in the Pulmonata revealed that chiral dimorphism does exist in certain species, apparently without any relationship with the coiling direction of the body. Though our study shows that body and sperm chirality follows independent developmental pathways, it gives rise to several questions that may be relevant to the understanding of the chirality of spermatid ultrastructure and spermatozoan motility and sexual selection.