Genome-wide data reveal bi-direction and asymmetrical hybridization origin of a fern species Microlepia matthewii.

Introduction: Natural hybridization is common and plays a crucial role in driving biodiversity in nature. Despite its significance, the understanding of hybridization in ferns remains inadequate. Therefore, it is imperative to study fern hybridization to gain a more comprehensive understanding of fern biodiversity. Our study delves into the role of hybridization in shaping fern species, employing Microlepia matthewii as a case study to investigate its origins of hybridization. Methods: We performed double digest Genotyping-by-sequencing (dd-GBS) on M. matthewii and its potential parent species, identifying nuclear and chloroplast SNPs. Initially, nuclear SNPs were employed to construct the three cluster analysis: phylogenetic tree, principal component analysis, and population structure analysis. Subsequently, to confirm whether the observed genetic mixture pattern resulted from hybridization, we utilized two methods: ABBA-BABA statistical values in the D-suite program and gene frequency covariance in the Treemix software to detect gene flow. Finally, we employed chloroplast SNPs to construct a phylogenetic tree, tracing the maternal origin. Results and discussion: The analysis of the nuclear SNP cluster revealed that M. matthewii possesses a genetic composition that is a combination of M. hancei and M. calvescens. Furthermore, the analysis provided strong evidence of significant gene flow signatures from the parental species to the hybrid, as indicated by the two gene flow analyses. The samples of M. matthewii cluster separately with M. hancei or M. calvescens on the chloroplast systematic tree. However, the parentage ratio significantly differs from 1:1, suggesting that M. matthewii is a bidirectional and asymmetrical hybrid offspring of M. hancei and M. calvescens.

Perspectives of pteridophytes microbiome for bioremediation in agricultural applications.

The microbiome is the synchronised congregation of millions of microbial cells in a particular ecosystem. The rhizospheric, phyllospheric, and endospheric microbial diversity of lower groups of plants like pteridophytes, which includes the Ferns and Fern Allies, have also given numerous alternative opportunities to achieve greener and sustainable agriculture. The broad-spectrum bioactivities of these microorganisms, including bioremediation of heavy metals (HMs) in contaminated soil, have been drawing the attention of agricultural researchers for the preparation of bioformulations for applications in climate-resilient and versatile agricultural production systems. Pteridophytes have an enormous capacity to absorb HMs from the soil. However, their direct application in the agricultural field for HM absorption seems infeasible. At the same time, utilisation of Pteridophyte-associated microbes having the capacity for bioremediation have been evaluated and can revolutionise agriculture in mining and mineral-rich areas. In spite of the great potential, this group of microbiomes has been less studied. Under these facts, this prospective review was carried out to summarise the basic and applied research on the potential of Pteridophyte microbiomes for soil bioremediation and other agricultural applications globally. Gaps have also been indicated to present scopes for future research programmes.

Global patterns and climatic determinants of phylogenetic structure of regional fern floras.

Knowledge of relationships between phylogenetic structure of a biological assemblage and ecological factors that drive the variation of phylogenetic structure among regions is crucial for understanding the causes of variation in taxonomic composition and richness among regions, but this knowledge is lacking for the global flora of ferns. Here, we fill this critical knowledge gap. We divided the globe into 392 geographic units on land, collated species lists of ferns for each geographic unit, and used different phylogenetic metrics (tip- vs basal-weighted) reflecting different evolutionary depths to quantify phylogenetic structure. We then related taxonomic and phylogenetic structure metrics to six climatic variables for ferns as a whole and for two groups of ferns (old clades vs polypods) reflecting different evolutionary histories across the globe and within each continental region. We found that when old clades and polypods were considered separately, temperature-related variables explained more variation in these metrics than did precipitation-related variables in both groups. When analyses were conducted for continental regions separately, this pattern holds in most cases. Climate extremes have a stronger relationship with phylogenetic structure of ferns than does climate seasonality. Climatic variables explained more variation in phylogenetic structure at deeper evolutionary depths.

Subgenome evolutionary dynamics in allotetraploid ferns: insights from the gene expression patterns in the allotetraploid species Phegopteris decursivepinnata (Thelypteridacea, Polypodiales).

Allopolyploidization often leads to disruptive conflicts among more than two sets of subgenomes, leading to genomic modifications and changes in gene expression. Although the evolutionary trajectories of subgenomes in allopolyploids have been studied intensely in angiosperms, the dynamics of subgenome evolution remain poorly understood in ferns, despite the prevalence of allopolyploidization. In this study, we have focused on an allotetraploid fern-Phegopteris decursivepinnata-and its diploid parental species, P. koreana (K) and P. taiwaniana (T). Using RNA-seq analyses, we have compared the gene expression profiles for 9,540 genes among parental species, synthetic F1 hybrids, and natural allotetraploids. The changes in gene expression patterns were traced from the F1 hybrids to the natural allopolyploids. This study has revealed that the expression patterns observed in most genes in the F1 hybrids are largely conserved in the allopolyploids; however, there were substantial differences in certain genes between these groups. In the allopolyploids compared with the F1 hybrids, the number of genes showing a transgressive pattern in total expression levels was increased. There was a slight reduction in T-dominance and a slight increase in K-dominance, in terms of expression level dominance. Interestingly, there is no obvious bias toward the T- or K-subgenomes in the number and expression levels overall, showing the absence of subgenome dominance. These findings demonstrated the impacts of the substantial transcriptome change after hybridization and the moderate modification during allopolyploid establishment on gene expression in ferns and provided important insights into subgenome evolution in polyploid ferns.

An open and continuously updated fern tree of life.

Ferns, with about 12,000 species, are the second most diverse lineage of vascular plants after angiosperms. They have been the subject of numerous molecular phylogenetic studies, resulting in the publication of trees for every major clade and DNA sequences from nearly half of all species. Global fern phylogenies have been published periodically, but as molecular systematics research continues at a rapid pace, these become quickly outdated. Here, we develop a mostly automated, reproducible, open pipeline to generate a continuously updated fern tree of life (FTOL) from DNA sequence data available in GenBank. Our tailored sampling strategy combines whole plastomes (few taxa, many loci) with commonly sequenced plastid regions (many taxa, few loci) to obtain a global, species-level fern phylogeny with high resolution along the backbone and maximal sampling across the tips. We use a curated reference taxonomy to resolve synonyms in general compliance with the community-driven Pteridophyte Phylogeny Group I classification. The current FTOL includes 5,582 species, an increase of ca. 40% relative to the most recently published global fern phylogeny. Using an updated and expanded list of 51 fern fossil constraints, we find estimated ages for most families and deeper clades to be considerably older than earlier studies. FTOL and its accompanying datasets, including the fossil list and taxonomic database, will be updated on a regular basis and are available via a web portal (https://fernphy.github.io) and R packages, enabling immediate access to the most up-to-date, comprehensively sampled fern phylogeny. FTOL will be useful for anyone studying this important group of plants over a wide range of taxonomic scales, from smaller clades to the entire tree. We anticipate FTOL will be particularly relevant for macroecological studies at regional to global scales and will inform future taxonomic systems with the most recent hypothesis of fern phylogeny.

Antheridiogen controls spatial dynamics of sex expression in naturally occurring gametophytes of the tree fern Cyathea multiflora.

PREMISE: Antheridiogen systems are a set of pheromonal mechanisms that control sex expression in fern gametophytes. However, antheridiogen has rarely been studied outside of the laboratory, and little is known about its function in natural settings. Combining predictions based on field and laboratory study, we tested whether the sexual structure of gametophytic colonies of a tree fern were attributable to antheridiogen. METHODS: Gametophytic colonies of the antheridiogen-producing tree fern Cyathea multiflora were collected at La Selva Biological Station in Costa Rica in January 2019. The sex of each gametophyte was determined, mapped, and spatial statistic approaches were used to examine the distribution of sex in each colony. RESULTS: In all gametophytic colonies, males were most common, representing 62-68% of individuals. No hermaphroditic gametophytes were identified in any colony. A quadrat-based method showed female gametophytes were not clustered in each colony, while male gametophytes were clustered. In two of the colonies, the K(r) test statistic for males was greater than expected compared to random simulations of sex expression, indicating male sex expression was spatially associated with females. CONCLUSIONS: This study provides the first documentation of spatial sex expression in natural settings of gametophytes of an antheridiogen-producing tree fern species. The profound impact of antheridiogen on gametophytic sex expression in field settings suggests this system is intimately tied to mating system, fitness, and genetic diversity in Cyathea multiflora.

An experimental assessment of competitive interactions between sexual and apomictic fern gametophytes using Easy Leaf Area.

Premise: Few studies have explored competition in fern gametophyte populations. One limiting factor is the tedious measurement of gametophyte size as a proxy for biomass in these small plants. Here, an alternative approach of estimating the number of green pixels from photos was employed to measure the competitive interactions among apomictic and sexual Dryopteris gametophytes. Methods: We cultivated the gametophytes of two apomictic (diploid and triploid) and one sexual (tetraploid) Dryopteris species in monocultures and in two-species mixtures in the ratios 1 : 1 and 1 : 3. The total gametophyte cover of each population originating from 20 spores was assessed using Easy Leaf Area. Assessments were performed weekly between weeks 4 and 10 of cultivation. Additionally, during week 5, the cover of each species in each mixture was estimated separately. Results: We identified a positive correlation between gametophyte size and ploidy level as well as sexual reproduction. The performance of the tested species in mixtures was dependent on the competitor species identity, indicating the importance of competition between gametophytes. Discussion: The methods outlined can be used for a rapid assessment of fern gametophyte cover in large gametophyte populations. Ploidy level and reproduction type seem to play a major role in the competitive abilities of fern gametophytes, but more research is needed on this topic.

Antibacterial and antitumor activities of a lectin-rich preparation from Microgramma vacciniifolia rhizome.

The rhizome of Microgramma vacciniifolia contains a lectin (carbohydrate-binding protein) called MvRL. Studies demonstrated that a MvRL-rich fraction did not show in vivo genotoxicity and acute toxicity in mice. This study aimed to evaluate the MvRL-rich fraction from M. vacciniifolia rhizome for antibacterial activity in vitro and in vivo as well as antitumor effect in vivo using the Ehrlich carcinoma model in mice. The fraction showed antibacterial activity against Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus with minimal inhibitory concentrations ranging from 31.2 to 125.0 â€‹µg/mL and minimal bactericidal concentrations from 62.5 to 200 â€‹µg/mL. The fraction was also effective in vivo against infection caused by these bacteria on Tenebrio molitor larvae considering the parameters evaluated. In regard to the antitumor activity, the treatments of Ehrlich carcinoma-bearing mice with the fraction at 100 and 200 â€‹mg/kg per os resulted in 62.58% and 75.43% of tumor inhibition, respectively. In conclusion, the MvRL-rich fraction showed in vivo antibacterial and antitumor activities and thus can be considered as an alternative of natural origin for the development of candidates for therapy.

Antinociceptive and Anti-Inflammatory Effects of Saline Extract and Lectin-Rich Fraction from Microgramma vacciniifolia Rhizome in Mice.

Previous studies have characterized a saline extract from Microgramma vacciniifolia rhizome and its lectin (MvRL)-rich fraction with low acute toxicity. In the present study, we evaluated these preparations for acute toxicity (1,000 mg/kg) and antinociceptive and anti-inflammatory activities (100-400 mg/kg for the extract and 25-50 mg/kg for the fraction). No signs of toxicity were observed. Both the extract and fraction increased the latency period for nociception in the hot plate assay, decreased writhing induced by acetic acid, and promoted analgesic effects in phases 1 and 2 of the formalin test. The antinociceptive mechanism was attributed to interactions with opioid receptors and K+ ATPase channels. The extract and fraction decreased carrageenan-induced paw edema in 46.15 % and 77.22 %, respectively, at the highest doses evaluated. Furthermore, the fraction was shown to act on the bradykinin pathway. The ability to decrease leukocyte migration after treatment was also verified in the peritonitis and air pouch models. In exudates collected from air pouches, decreased tumor necrosis factor (TNF)-α and increased interleukin (IL)-10 levels were noted. Both the extract and fraction also effectively inhibited the development of granulomatous tissue. In conclusion, the substances investigated in this study can be used for the development of novel therapeutic options for pain and inflammatory processes.

Light- and hormone-mediated development in non-flowering plants: An overview.

MAIN CONCLUSION: Light, hormones and their interaction regulate different aspects of development in non-flowering plants. They might have played a role in the evolution of different plant groups by conferring specific adaptive evolutionary changes. Plants are sessile organisms. Unlike animals, they lack the opportunity to abandon their habitat in unfavorable conditions. They respond to different environmental cues and adapt accordingly to control their growth and developmental pattern. While phytohormones are known to be internal regulators of plant development, light is a major environmental signal that shapes plant processes. It is plausible that light-hormone crosstalk might have played an important role in plant evolution. But how the crosstalk between light and phytohormone signaling pathways might have shaped the plant evolution is unclear. One of the possible reasons is that flowering plants have been studied extensively in context of plant development, which cannot serve the purpose of evolutionary comparisons. In order to elucidate the role of light, hormone and their crosstalk in the evolutionary adaptation in plant kingdom, one needs to understand various light- and hormone-mediated processes in diverse non-flowering plants. This review is an attempt to outline major light- and phytohormone-mediated responses in non-flowering plant groups such as algae, bryophytes, pteridophytes and gymnosperms.

Toxicity assessment of saline extract and lectin-rich fraction from Microgramma vacciniifolia rhizome.

Microgramma vacciniifolia is broadly used in folk medicine but safety information is unavailable. Therefore, we evaluated the toxicity of a saline extract and a lectin-rich fraction of M. vacciniifolia rhizome. The extract showed hemolytic activity on mice erythrocytes at 1000 µg/mL, whereas the fraction promoted hemolysis (8.57-26.15%) at all tested concentrations (10-1000 µg/mL). Acute toxicity test in mice indicated an LD50 of >5000 mg/kg. Hematological alterations and increased serum alkaline phosphatase level were observed in the treated animals. Transaminases and urea levels increased in the groups treated with the extract or fraction at 5000 mg/kg. Leukocyte infiltration was observed in the liver of extract-treated animals and in the liver and lungs of mice treated with the fraction. The kidneys of animals treated with the fraction at 5000 mg/kg presented hydropic degeneration. The extract and fraction did not induce oxidative stress in the liver and did not show genotoxicity, as examined by micronucleus and comet assays. In conclusion, the preparations were not lethal to mice but caused some signs of toxicity, mainly the fraction. The results indicated the need to evaluate the toxicity of M. vacciniifolia rhizome in other models and in chronic assays.

Fern gametophytes of Angiopteris lygodiifolia and Osmunda japonica harbor diverse Mucoromycotina fungi.

Mycorrhizal symbiosis between plants and fungi is ubiquitous, and has been played key roles in plant terrestrialization and diversification. Although arbuscular mycorrhizal (AM) symbioses with Glomeromycotina fungi have long been recognized as both ancient and widespread symbionts, recent studies showed that Mucoromycotina fungi were also ancestral symbionts and would thus be expected to co-exist with many land plants. To explore whether Mucoromycotina colonize fern gametophytes, we subjected fungal associations with gametophytes of two distantly related ferns, Angiopteris lygodiifolia (Marattiales) and Osmunda japonica (Osmundales), to molecular analysis. Direct PCR amplification from intracellular hyphal coils was also performed. We detected Mucoromycotina sequences in the gametophytes of A. lygodiifolia and O. japonica at rates of 41% (7/17) and 50% (49/98) of gametophytes, respectively, and assigned them to 10 operational taxonomic units of Endogonales lineages. In addition, we used AM fungal-specific primers and detected Glomeromycotina sequences in all individuals examined. The results suggest that Glomeromycotina and Mucoromycotina colonized fern gametophytes simultaneously. We found that Mucoromycotina were present in fern gametophytes of Marratiales and Osmundales, which implies that a variety of fern taxa have Mucoromycotina associations.

Taxonomic studies of pteridophytes of Ambon and Seram (Moluccas) collected on Indonesian-Japanese botanical expeditions 1983-1986. XIII. Hymenophyllaceae.

Identifications are given for 713 specimens of Hymenophyllaceae collected on Ambon and Seram islands, the Moluccas, Indonesia, during 1983-86. The collection is composed of forty-seven species and one variety belonging to seven genera. The dataset is deposited in GBIF and available at https://www.gbif.jp/ipt/resource?r=seram_hymen.

Effects of graywater on the growth and survival of ornamental plants in nature-based systems.

The current paper investigates the development of two ornamental plants, canna lily (Canna x generalis) and giant horsetail (Equisetum giganteum), at both bench and pilot scale. Combinations of gravel-filled mesocosm, planted and unplanted (control), irrigated with light greywater (GWL) or tap water (WT), were used. Both species were able to grow under the tested conditions with no indication of toxicity that could affect the development. Irrigation with GWL, resulted in higher evapotranspiration (2.2 mm-2.8 mm) in canna lily than giant horsetail (1.7 mm-2.3 mm) in mesocosm system. When the plants were mature and the season was more humid and warmer, canna lily and giant horsetail irrigated with GWL evapotranspirated 69.23% and 30.77%, respectively as compared to the unplanted GWL-irrigated-mesocosm. Principal components and cluster analysis identified similarity between evapotranspiration (ET) and the characteristics of the plants. Both species can thus be used in constructed wetlands taking into consideration elements such as the space available, level of water and solar incidence so as to allow the full development of the plants. The roots of giant horsetail require high water availability. Low solar incidence is indicated for giant horsetail, and the opposite for canna lily, if flowering is desired.

Unraveling metabolic mechanisms behind chloroplast desiccation tolerance: Chlorophyllous fern spore as a new promising unicellular model.

Fern spores are unicellular structures produced by the sporophyte generation that give rise to the haploid gametophyte. When released from the sporangium, spores are desiccation tolerant (DT) in the royal fern (Osmunda regalis) and contain fully developed chloroplasts. As a consequence, this type of spores is called chlorophyllous spores (CS). Upon transfer to germination conditions, CS initiate a process of imbibition that suppresses DT in 72 h, before the germination starts. In parallel to such change in DT, thylakoids undergo a profound remodelling in composition and function. Firstly, sustained quenching of chlorophyll fluorescence is relaxed, giving rise to photochemically active CS, while lipid composition shifts from that of a resting structure to a metabolically active cell. Basically trigalactolipids decreased in favour of monogalactolipids, with a parallel desaturation of fatty acids. Storage lipids such as triacylglycerol were quickly depleted. These results highlight the importance of the structure of thylakoids lipid as a key to protect membrane integrity during desiccation, together with the saturation of fatty acids and the constitutive chlorophyll quenching to prevent oxidative damage. The CS used here, in which the same cell shifts from DT to sensitive strategy in 72 h, reveal their potential as unicellular models for future studies on DT.

Surface treated Pteris vittata L. pinnae powder used as an efficient biosorbent of Pb(II), Cd(II), and Cr(VI) from aqueous solution.

Biosorption is a surface-dependent phenomenon. Surface modifications by chemical treatment methods could either improve or reduce the biosorption capacity of potential biosorbents. In the present work, pristine Pteris vittata L. pinnae (PPV) powder was treated separately with sodium hydroxide (NaOH), calcium chloride (CaCl2), and nitric acid (HNO3). The pristine and treated biosorbents were used to assess the biosorption of Pb(II), Cd(II), and Cr(VI) as a function of pH. Kinetics and adsorption isotherms were studied. Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy and scanning electron microscope combined with energy dispersive x-ray (SEM-EDX) spectroscopic techniques were used to characterize the biosorbents before and after chemical treatments. The possible functional groups contributing to the metal sorption were identified. Results revealed favorable biosorption of Pb(II), Cd(II), and Cr(VI) described by pseudo-second order kinetics. NaOH-treated P. vittata (NPV) showed higher biosorption capacity for Pb(II) and Cd(II) compared to that of PPV. ATR-FTIR studies indicated that -OH, -COOH, and -NH2 groups were mainly involved in Cr(VI) and -OH in Pb(II) and Cd(II) biosorption. The enhanced efficiency of NPV and CaCl2 treated P. vittata (CPV) in the uptake of Pb(II) and Cd(II) compared to PPV can be associated with their altered physicochemical characters.

A review of the use of pteridophytes for treating human ailments.

The aim of this review was to explore the pharmacological activity of early tracheophytes (pteridophytes) as an alternative medicine for treating human ailments. As the first vascular plants, pteridophytes (aka, ferns and fern allies) are an ancient lineage, and human beings have been exploring and using taxa from this lineage for over 2000 years because of their beneficial properties. We have documented the medicinal uses of pteridophytes belonging to thirty different families. The lycophyte Selaginella sp. was shown in earlier studies to have multiple pharmacological activity, such as antioxidant, anti-inflammatory, anti-cancer, antidiabetic, antiviral, antimicrobial, and anti-Alzheimer properties. Among all the pteridophytes examined, taxa from the Pteridaceae, Polypodiaceae, and Adiantaceae exhibited significant medicinal activity. Based on our review, many pteridophytes have properties that could be used in alternative medicine for treatment of various human illnesses. Biotechnological tools can be used to preserve and even improve their bioactive molecules for the preparation of medicines against illness. Even though several studies have reported medicinal uses of ferns, the possible bioactive compounds of several pteridophytes have not been identified. Furthermore, their optimal dosage level and treatment strategies still need to be determined. Finally, the future direction of pteridophyte research is discussed.

A review of the use of pteridophytes for treating human ailments / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

The aim of this review was to explore the pharmacological activity of early tracheophytes (pteridophytes) as an alternative medicine for treating human ailments. As the first vascular plants, pteridophytes (aka, ferns and fern allies) are an ancient lineage, and human beings have been exploring and using taxa from this lineage for over 2000 years because of their beneficial properties. We have documented the medicinal uses of pteridophytes belonging to thirty different families. The lycophyte Selaginella sp. was shown in earlier studies to have multiple pharmacological activity, such as antioxidant, anti-inflammatory, anti-cancer, antidiabetic, antiviral, antimicrobial, and anti-Alzheimer properties. Among all the pteridophytes examined, taxa from the Pteridaceae, Polypodiaceae, and Adiantaceae exhibited significant medicinal activity. Based on our review, many pteridophytes have properties that could be used in alternative medicine for treatment of various human illnesses. Biotechnological tools can be used to preserve and even improve their bioactive molecules for the preparation of medicines against illness. Even though several studies have reported medicinal uses of ferns, the possible bioactive compounds of several pteridophytes have not been identified. Furthermore, their optimal dosage level and treatment strategies still need to be determined. Finally, the future direction of pteridophyte research is discussed.

A free-access online key to identify Amazonian ferns.

There is urgent need for more data on species distributions in order to improve conservation planning. A crucial but challenging aspect of producing high-quality data is the correct identification of organisms. Traditional printed floras and dichotomous keys are difficult to use for someone not familiar with the technical jargon. In poorly known areas, such as Amazonia, they also become quickly outdated as new species are described or ranges extended. Recently, online tools have allowed developing dynamic, interactive, and accessible keys that make species identification possible for a broader public. In order to facilitate identifying plants collected in field inventories, we developed an internet-based free-access tool to identify Amazonian fern species. We focused on ferns, because they are easy to collect and their edaphic affinities are relatively well known, so they can be used as an indicator group for habitat mapping. Our key includes 302 terrestrial and aquatic entities mainly from lowland Amazonian forests. It is a free-access key, so the user can freely choose which morphological features to use and in which order to assess them. All taxa are richly illustrated, so specimens can be identified by a combination of character choices, visual comparison, and written descriptions. The identification tool was developed in Lucid 3.5 software and it is available at http://keyserver.lucidcentral.org:8080/sandbox/keys.jsp.

Microgramma vacciniifolia (Polypodiaceae) fronds contain a multifunctional lectin with immunomodulatory properties on human cells.

In this study, we report the purification and characterization of a multifunctional lectin (MvFL) from Microgramma vacciniifolia fronds as well as its immunomodulatory properties on human peripheral blood mononuclear cells (PBMCs). MvFL (pI 4.51; 54kDa) is a glycoprotein able to inhibit trypsin activity and that has sequence similarities (32% coverage) with a plant RNA-binding protein. Hemagglutinating activity of MvFL was not altered by heating at 100°C for 30min, but was reduced in alkaline pH (8.0 and 9.0). Fluorimetric analyses showed that this lectin did not undergo marked conformational changes when heated. However, the MvFL conformation changed depending on the pH. MvFL at 6.25-25µg/mL was not cytotoxic to lymphocytes present among PBMCs. The PBMCs incubated for 24h with the lectin (12.5µg/mL) showed increased TNF-α, IFN-γ, IL-6, IL-10, and nitric oxide production. MvFL also stimulated T lymphocytes from PBMCs to differentiate into CD8+ cells. The activation (indicated by CD28 expression) of these cells was also stimulated. In conclusion, MvFL is a heat-stable and multifunctional protein, with both lectin and trypsin inhibitor activities, and capable of inducing predominantly a Th1 response in human PBMCs as well as activation and differentiation of T lymphocytes.