Higher Vertebrates Research Articles

Regulatory roles of cytokines in T and B lymphocytes-mediated immunity in teleost fish

T and B lymphocytes (T and B cells) are immune effector cells that play critical roles in adaptive immunity and defend against external pathogens in most vertebrates, including teleost fish. In mammals, the development and immune response of T and B cells is associated with cytokines including chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors during pathogenic invasion or immunization. Given that teleost fish have evolved a similar adaptive immune system to mammals with T and B cells bearing unique receptors (B-cell receptors (BCRs) and T-cell receptors (TCRs)) and that cytokines in general have been identified, whether the regulatory roles of cytokines in T and B cell-mediated immunity are evolutionarily conserved between mammalians and teleost fish is a fascinating question. Thus, the purpose of this review is to summarize the current knowledge of teleost cytokines and T and B cells as well as the regulatory roles of cytokines on these two types of lymphocytes. This may provide important information on the parallelisms and dissimilarities of the functions of cytokines in bony fish versus higher vertebrates, which may aid in the evaluation and development of adaptive immunity-based vaccines or immunostimulants.

Isolation of Antimicrobial Peptides (AMPs) From Different Sources: A Review

Antimicrobial peptides (AMPs) are small peptide molecules produced by a wide range of species, from bacteria to higher vertebrates, as a part of the innate immune system. These naturally produced peptides fight against intrusive microbes like viruses, bacteria, and fungi. AMPs are produced in various body parts by different organisms, such as skin secretion of amphibians; leaf, stem, fruits, or whole parts of plants; glandular secretion of mammals; the venom of reptiles and arthropods, etc. Due to this source variation, there is no universal method for the isolation, i.e., extraction and purification of the AMPs. Therefore, several techniques have been established to extract and purify these natural peptides from various sources. Generally, solid-phase extraction and chemical extraction techniques are the most common methods used for the extraction of AMPs. For purification and identification purposes, chromatography, gel electrophoresis, filtration, Edman degradation, circular dichroism, mass spectrometry, etc. are mainly adopted. In this review, we provide a brief discussion on the isolation process of AMPs based on their sources. This study would be helpful in isolating newer AMPs that could be used as potential antimicrobial agents. Bangladesh Pharmaceutical Journal 26(1): 99-111, 2023 (January)

Immunohistochemistry of the Gut-Associated Lymphoid Tissue (GALT) in African Bonytongue (Heterotis niloticus, Cuvier 1829).

Heterotis niloticus is a basal teleost, belonging to the Osteoglossidae family, which is widespread in many parts of Africa. The digestive tract of H. niloticus presents similar characteristics to those of higher vertebrates, exhibiting a gizzard-like stomach and lymphoid aggregates in the intestinal lamina propria. The adaptive immune system of teleost fish is linked with each of their mucosal body surfaces. In fish, the gut-associated lymphoid tissue (GALT) is generally a diffuse immune system that represents an important line of defense against those pathogens inhabiting the external environment that can enter through food. The GALT comprises intraepithelial lymphocytes, which reside in the epithelial layer, and lamina propria leukocytes, which consist of lymphocytes, macrophages, granulocytes, and dendritic-like cells. This study aims to characterize, for the first time, the leukocytes present in the GALT of H. niloticus, by confocal immuno- fluorescence techniques, using specific antibodies: toll-like receptor 2, major histocompatibility complex class II, S100 protein, serotonin, CD4, langerin, and inducible nitric oxide synthetase. Our results show massive aggregates of immune cells in the thickness of the submucosa, arranged in circumscribed oval-shaped structures that are morphologically similar to the isolated lymphoid follicles present in birds and mammals, thus expanding our knowledge about the intestinal immunity shown by this fish.

Molecular characterization and expression analysis of a novel cold-inducible RNA-binding protein (CIRBP) gene in lamprey (Lethenteron reissneri).

Cold-inducible RNA-binding protein (CIRBP) responds to a wide array of cellular stresses such as cold shock, hypoxia, and inflammatory responses. However, functional studies of CIRBP in jawless vertebrates are limited. In this study, a CIRBP homolog from the jawless vertebrate lamprey (Lethenteron reissneri) was cloned and characterized (named Lr-CIRBP). The cDNA fragment of Lr-CIRBP has a 516bp open reading frame (ORF) that encodes 171 amino acids, comprising a glycine-rich region at the C-terminal, similar to higher vertebrates but slightly shorter, and an RNA recognition motif (RRM) domain at the N-terminus. The predicted Lr-CIRBP sequence had 51.4 ~ 70.6% similarity with CIRBPs from other vertebrates. Further phylogenetic analysis revealed that Lr-CIRBP is located in the outgroup of vertebrates and is the ancestor of vertebrates. Based on real-time quantitative PCR experimental analysis, Lr-CIRBP expression was highest in leukocytes and increased significantly after multi-stimulation, peaking at 12h. RNA interference showed that Lr-CIRBP knockdown can down-regulate the expression of inflammatory factors in Lethenteron reissneri. In conclusion, our study successfully clarifies the ancestral features and functions of CIRBP, while revealing valuable insight into how the protein is involved in the immune responses of a jawless vertebrate.

Eccentric contraction-induced strength loss in dystrophin-deficient muscle: Preparations, protocols, and mechanisms.

The absence of dystrophin hypersensitizes skeletal muscle of lower and higher vertebrates to eccentric contraction (ECC)-induced strength loss. Loss of strength can be accompanied by transient and reversible alterations to sarcolemmal excitability and disruption, triad dysfunction, and aberrations in calcium kinetics and reactive oxygen species production. The degree of ECC-induced strength loss, however, appears dependent on several extrinsic and intrinsic factors such as vertebrate model, skeletal muscle preparation (in vivo, in situ, or ex vivo), skeletal muscle hierarchy (single fiber versus whole muscle and permeabilized versus intact), strength production, fiber branching, age, and genetic background, among others. Consistent findings across research groups show that dystrophin-deficient fast(er)-twitch muscle is hypersensitive to ECCs relative to wildtype muscle, but because preparations are highly variable and sensitivity to ECCs are used repeatedly to determine efficacy of many preclinical treatments, it is critical to evaluate the impact of skeletal muscle preparations on sensitivity to ECC-induced strength loss in dystrophin-deficient skeletal muscle. Here, we review and discuss variations in skeletal muscle preparations to evaluate the factors responsible for variations and discrepancies between research groups. We further highlight that dystrophin-deficiency, or loss of the dystrophin-glycoprotein complex in skeletal muscle, is not a prerequisite for accelerated strength loss-induced by ECCs.

Advances in Vertebrate (Cyto)Genomics Shed New Light on Fish Compositional Genome Evolution.

Cytogenetic and compositional studies considered fish genomes rather poor in guanine-cytosine content (GC%) because of a putative "sharp increase in genic GC% during the evolution of higher vertebrates". However, the available genomic data have not been exploited to confirm this viewpoint. In contrast, further misunderstandings in GC%, mostly of fish genomes, originated from a misapprehension of the current flood of data. Utilizing public databases, we calculated the GC% in animal genomes of three different, technically well-established fractions: DNA (entire genome), cDNA (complementary DNA), and cds (exons). Our results across chordates help set borders of GC% values that are still incorrect in literature and show: (i) fish in their immense diversity possess comparably GC-rich (or even GC-richer) genomes as higher vertebrates, and fish exons are GC-enriched among vertebrates; (ii) animal genomes generally show a GC-enrichment from the DNA, over cDNA, to the cds level (i.e., not only the higher vertebrates); (iii) fish and invertebrates show a broad(er) inter-quartile range in GC%, while avian and mammalian genomes are more constrained in their GC%. These results indicate no sharp increase in the GC% of genes during the transition to higher vertebrates, as stated and numerously repeated before. We present our results in 2D and 3D space to explore the compositional genome landscape and prepared an online platform to explore the AT/GC compositional genome evolution.

Short- and long-term polystyrene nano- and microplastic exposure promotes oxidative stress and divergently affects skin cell architecture and Wnt/beta-catenin signaling

Nano- and microplastic particles (NMP) are strong environmental contaminants affecting marine ecosystems and human health. The negligible use of biodegradable plastics and the lack of knowledge about plastic uptake, accumulation, and functional consequences led us to investigate the short- and long-term effects in freshly isolated skin cells from mice. Using fluorescent NMP of several sizes (200 nm to 6 µm), efficient cellular uptake was observed, causing, however, only minor acute toxicity as metabolic activity and apoptosis data suggested, albeit changes in intracellular reactive species and thiol levels were observed. The internalized NMP induced an altered expression of various targets of the nuclear factor-2-related transcription factor 2 pathway and were accompanied by changed antioxidant and oxidative stress signaling responses, as suggested by altered heme oxygenase 1 and glutathione peroxide 2 levels. A highly increased beta-catenin expression under acute but not chronic NMP exposure was concomitant with a strong translocation from membrane to the nucleus and subsequent transcription activation of Wnt signaling target genes after both single-dose and chronic long-term NMP exposure. Moreover, fibroblast-to-myofibroblast transdifferentiation accompanied by an increase of α smooth muscle actin and collagen expression was observed. Together with several NMP-induced changes in junctional and adherence protein expression, our study for the first time elucidates the acute and chronic effects of NMP of different sizes in primary skin cells' signaling and functional biology, contributing to a better understanding of nano- and microplastic to health risks in higher vertebrates.

A soluble TLR5 is involved in PBLs activation and antibacterial immunity via TLR5M-MyD88-signaling pathway in tongue sole Cynoglossus semilaevis

In higher vertebrates, there is only a membranal TLR5 (TLR5M), which is crucial for host defense against microbes via MyD88 signaling pathway. In teleost, both TLR5M and soluble TLR5 (TLR5S) are identified, whereas the antibacterial mechanism of TLR5S is largely unknown. In this study, we studied the immune antibacterial mechanism of Cynoglossus semilaevis TLR5S homologue (named CsTLR5S). CsTLR5S, a 71.1 kDa protein, consists of 649 amino acid residues and shares 41.7 %–57.8 % overall sequence identities with teleost TLR5S homologues. CsTLR5S contains a single extracellular domain (ECD) composed of 12 leucine-rich repeats. CsTLR5S expression was constitutively identified and upregulated by bacterial infection in tissues. In vitro recombinant CsTLR5S (rCsTLR5S) could interact with bacteria and tongue sole rTLR5M (rCsTLR5M). Furthermore, rCsTLR5S could bind to the membranal CsTLR5M of peripheral blood leukocytes (PBLs), which led to enhancing the activity and the antibacterial role of PBLs via Myd88-NF-κB pathway. In vivo rCsTLR5S could activate the Myd88-NF-κB pathway, facilitate the release of proinflammatory cytokines, and enhance the host antibacterial response against Vibrio harveyi. Moreover, the knockdown of CsTLR5M or the Myd88 inhibitor could significantly suppress the antibacterial effect of rCsTLR5S. Collectively, our findings added important insights into the TLR5S immune antibacterial property in a TLR5M-MyD88-dependent manner.

Localized Infections with P. aeruginosa Strains Defective in Zinc Uptake Reveal That Zebrafish Embryos Recapitulate Nutritional Immunity Responses of Higher Eukaryotes.

The innate immune responses of mammals to microbial infections include strategies based on manipulating the local concentration of metals such as iron (Fe) and zinc (Zn), commonly described as nutritional immunity. To evaluate whether these strategies are also present in zebrafish embryos, we have conducted a series of heart cavity-localized infection experiments with Pseudomonas aeruginosa strains characterized by a different ability to acquire Zn. We have found that, 48 h after infection, the bacterial strains lacking critical components of the Zn importers ZnuABC and ZrmABCD have a reduced colonization capacity compared to the wild-type strain. This observation, together with the finding of a high level of expression of Zur-regulated genes, suggests the existence of antimicrobial mechanisms based on Zn sequestration. However, we have observed that strains lacking such Zn importers have a selective advantage over the wild-type strain in the early stages of infection. Analysis of the expression of the gene that encodes for a Zn efflux pump has revealed that at short times after infection, P. aeruginosa is exposed to high concentrations of Zn. At the same time, zebrafish respond to the infection by activating the expression of the Zn transporters Slc30a1 and Slc30a4, whose mammalian homologs mediate a redistribution of Zn in phagocytes aimed at intoxicating bacteria with a metal excess. These observations indicate that teleosts share similar nutritional immunity mechanisms with higher vertebrates, and confirm the usefulness of the zebrafish model for studying host-pathogen interactions.

Single-cell transcriptomic analysis reveals diversity within mammalian spinal motor neurons

Spinal motor neurons (MNs) integrate sensory stimuli and brain commands to generate movements. In vertebrates, the molecular identities of the cardinal MN types such as those innervating limb versus trunk muscles are well elucidated. Yet the identities of finer subtypes within these cell populations that innervate individual muscle groups remain enigmatic. Here we investigate heterogeneity in mouse MNs using single-cell transcriptomics. Among limb-innervating MNs, we reveal a diverse neuropeptide code for delineating putative motor pool identities. Additionally, we uncover that axial MNs are subdivided into three molecularly distinct subtypes, defined by mediolaterally-biased Satb2, Nr2f2 or Bcl11b expression patterns with different axon guidance signatures. These three subtypes are present in chicken and human embryos, suggesting a conserved axial MN expression pattern across higher vertebrates. Overall, our study provides a molecular resource of spinal MN types and paves the way towards deciphering how neuronal subtypes evolved to accommodate vertebrate motor behaviors.

Confocal and Electron Microscopic Structure of the Cornea from Three Species of Penguin

Corneal confocal microscopy has not previously been performed in penguins, despite recognition of its unusually flat shape. To identify features that the penguin shares with other birds and or mammals and those specific to penguins, we undertook confocal microscopic examination of two little (Eudyptula minor), four gentoo (Pygoscelis papua) and five king (Aptenodytes patagonicus) penguin corneas. Transmission electron microscopy was performed on one gentoo and one king penguin, for finer details. Features shared with other higher vertebrates included a five-layered cornea and a similar limbus. Typically avian were a lower density of stromal cells, a more regular arrangement of collagen bands and an absent basal nerve plexus. Features unique to penguins included a flattened superficial epithelium (king penguin), stromal myofibroblasts (all) and an irregular endothelium (little penguin). Other features uniquely identified by confocal microscopy in birds include epithelial and stromal nerves, guttata and stromal imprints on Descemet's membrane. Transmission electron microscopy identified a lack of wing cells (king penguin), greater posterior collagen lamellae thickness (gentoo penguin) and significantly less interlacing of collagen lamellae in the central cornea (king and gentoo). Most of these unique features are yet to be explained, but some could be adaptations to diving.

Genome-wide identification of olive flounder (Paralichthys olivaceus) SOCS genes: Involvement in immune response regulation to temperature stress and Edwardsiella tarda infection

The suppressors of cytokine signaling (SOCS) gene family participates in development and immunity through negative regulation of cytokine signaling pathways. Although the immune response of SOCS gene family members has been extensively characterized in teleost, no similar study has been reported in olive flounder yet. In our present study, a total of 13 SOCSs in olive flounder were identified and characterized systematically. By querying the SOCS sequences of ten teleost fish species, we found there were exactly more members of SOCSs in fish than mammals, which indicated that there were more duplication events occurred in fish than in higher vertebrates. Phylogenetic analysis clearly illuminated that SOCS genes were highly conserved. The analysis of gene structure and motif showed SOCS proteins of olive flounder shared a high level of sequence similarity strikingly. The expression profiles of tissues and developmental stages indicated that SOCS members had a kind of specificity in temporality and spatiality. RNA–Seq analysis of temperature stress and E. Tarda infection demonstrated SOCS members were involved in inflammatory response. In a word, our results would provide a further reference for understanding the mechanism of SOCS genes in olive flounder.

Developmental stage-specific exposure and neurotoxicity evaluation of low-dose clothianidin during neuronal circuit formation.

Neonicotinoid pesticides (NN) were recently reported to exhibit adverse effects in higher vertebrates. Moreover, NNs are routinely transferred from mother to offspring, raising concerns about their effects on future generations. The fetal and neonatal periods are the most critical to the formation of neural circuits in the brain through neurogenesis and differentiation, neuronal migration, axon guidance, and synaptogenesis. NN exposure throughout the fetal and neonatal periods was found to affect the neurobehavior of the offspring, but the stage-specific neurobehavioral effects are unclear. We exposed fetal and neonatal mice to a no-observed-adverse-effect level (NOAEL) of clothianidin (CLO) for 4 days during each of four developmental stages: neurite proliferation and differentiation (fetal days 9-12, CLO-1), neurite outgrowth (fetal days 15-18, CLO-2), synapse formation and astrocyte differentiation (days 1-4 after birth, CLO-3), and synapse remodeling (days 11-14 after birth, CLO-4). CLO's neurobehavioral effects were evaluated in juveniles and adults, revealing that CLO-1 and CLO-2 caused behavioral abnormalities in adult mice. CLO-3 significantly increased locomotor activity and decreased juvenile neurons in the hippocampal dentate gyrus in adulthood. Comprehensive gene analysis of CLO-3 revealed high expression of genes related to neurite outgrowth and axonal branching in the hippocampus in juveniles and adults. These results revealed developmental stage-specific effects of a NOAEL of CLO in the fetal and neonatal periods, suggesting that the susceptibility of the fetus and neonate to CLO varies by developmental stage.

Molecular Characterization of High Mobility Group Box 1a (HMGB1a) Gene in Red-Bellied Pacu, Piaractus brachypomus.

High mobility group box 1 (HMGB1) is a chromosomal protein in the nucleus and a potent extracellular proinflammatory cytokine, widely described in mammals, nevertheless, with scarce reports in fish. In this study, full open reading frame of HMGB1a gene from Piaractus brachypomus is reported as well as its molecular characterization, including tissue gene expression. At predicted protein level, HMGB1a showed similarities with its orthologs in teleosts and higher vertebrates. The relative gene expression of HMGB1a mRNA was measured in several tissues including the brain, where a differential expression appeared in brain regions, i.e., higher expression in the cerebellum and telencephalon. In addition, in an assay of sublethal exposure to chlorpyrifos, upregulation of HMGB1a was detected in optic chiasm. Furthermore, in a traumatic brain injury model, upregulation of HMGB1a expression was evident 24 hours after lesion and remained higher up to 14 days. These findings suggest a role for HMGB1a in brain damage and its candidature as biomarker of brain injury; however, more studies are required to elucidate the functions of HMGB1a and its regulation in P. brachypomus.

Biology of amphibian granulocytes - From evolutionary pressures to functional consequences

Granulocyte-lineage cells are important innate immune effectors across all vertebrates. Named for conspicuous secretory granules, granulocytes have historically been studied for their antimicrobial roles. Although versions of these cells are found in all vertebrate species examined to date, disparate environmental and physiological pressures acting on distinct vertebrate classes have shaped many of the facets dictating granulocyte biology. Immune pressures further determine granulopoietic constraints, ultimately governing granulocyte functions. For amphibians that inhabit pathogen-rich aquatic environments for some or all their lives, their unique granulocyte biologies satisfy many of their antimicrobial needs. Amphibians also occupy an intermediate position in the evolution of vertebrate immune systems, using combinations of primitive (e.g., subcapsular liver) and more recently evolved (e.g., bone marrow) tissue sites for hematopoiesis and specifically, granulopoiesis. The last decade of research has revealed vertebrate granulocytes in general, and amphibian granulocytes in particular, are more complex than originally assumed. With dynamic leukocyte phenotypes, granulocyte-lineage cells are being acknowledged for their multifaceted roles beyond immunity in other physiological processes. Here we provide an overview of granulopoiesis in amphibians, highlight key differences in these processes compared to higher vertebrates, and identify open questions.

Research Note: Development of a chicken experimental model platform for induced pluripotent stem cells by using CRISPR/Cas9-mediated NANOG knock-in reporter DF1 cells

NANOG, as a transcription factor, plays a key role in maintaining pluripotency in higher vertebrates. Thus, NANOG gene expression is a critical index for the transition from somatic cells to the pluripotent stage. Here, we established chicken knock-in DF1 cells in which the red fluorescent protein (RFP) gene was specifically inserted into the transcriptional start site of the NANOG gene through the CRISPR‒Cas9 (clustered regularly interspaced short palindromic repeat-CRISPR associated protein 9) technical platform. Subsequently, 4 transcription factors (Pou5f3, Sox2, Nanog, and Lin28A) were introduced into the NANOG-RFP DF1 cells, and finally, the induced pluripotent cells were established and examined by endogenous NANOG promoter-controlled RFP gene expression. The development of induced pluripotent stem cells (iPSCs) in avians would be useful for practical applications in the field of avian biotechnology, including biobanking genetic materials and restoring endangered species. In this study, a reporter cell line system was established to efficiently identify the induced pluripotent stage, and it will facilitate potential use for various purposes in the field of avian experimental models.

Genome-wide characterization of the Triplophysa dalaica slc4 gene family and expression profiles in response to salinity changes

BackgroundThe solute carrier 4 (SLC4) gene family is involved in a variety of physiological processes in organisms and is essential for maintaining acid-base balance in the body. The slc4 genes have been extensively studied in mammals, and they play important roles in intracellular and extracellular pH regulation and in the secretion and uptake of HCO3− and other ions (Na+ and Cl−) between transepithelial cells in different tissues. This study identified and characterized the entire slc4 gene family of Triplophysa dalaica.ResultsFifteen slc4 genes were identified in the whole genome of Triplophysa dalaica in this study, including five copies of Na+-independent Cl−/HCO3− transporters, eight members of Na+-dependent HCO3− transporters, and two genes coding Na+-coupled borate transporters. The chromosomal location information, isoelectric points, and molecular weights of the 15 slc4 genes were analyzed. The results for gene structure, domain analysis, and phylogenetic relationships of this gene family showed that the slc4 genes (except for slc4a9, which is missing in teleosts) are significantly expanded in teleosts compared to higher vertebrates. This phenomenon suggests that the slc4 gene family played an important role in the transition from aquatic to terrestrial animals. RT-PCR results showed that different slc4 genes showed diversified expression patterns in the tissues of T. dalaica. For osmotic pressure regulating organs, slc4a1b, slc4a4b, slc4a7, and slc4a11a were highly expressed in gills. In the kidney, slc4a1a, slc4a3, and slc4a10b were highly expressed, suggesting that the slc4 genes play a specific role in the salinity adaptation of T. dalaica. Our study has deciphered the biological roles of the slc4 genes in maintaining ionic and acid-base homeostasis in teleost fishes and provides a foundation for future exploration of the highly differentiated gene family in Triplophysa.ConclusionsThe results are relevant for the breeding of alkali-tolerant varieties in saline-alkali areas for aquaculture. Our findings have important implications for the adaptation process of freshwater species to saline-alkali water.

Characterization and role of connective tissue growth factor gene in collagen synthesis in swim bladder of chu's croaker (Nibea coibor)

Connective tissue growth factor (Ctgf) is a matricellular protein with diverse biological function. It is regarded as a central regulator of tissue fibrosis and collagen synthesis in mammals. However, its roles in fish remain elusive. Here, a ctgf gene was cloned (NcCtgf), characterized and functionally studied in the chu's croaker (Nibea coibor). NcCtgf encoded a protein containing 346 amino acids, 38 conserved cysteine residues, 4 functional domains and a signal peptide. NcCtgf shared highest identity (99.4 %) to the Larimichthys crocea Ctgf protein. Phylogenetic tree revealed that NcCtgf clustered with the teleost Ctgfa and Ctgf of higher vertebrates, instead of teleost Ctgfb. NcCtgf was expressed with higher level in gonad, spleen, gill and swimming bladder than other tissues, and was up-regulated in swim bladder synchronously with collagen I genes by hydroxyproline and TGF-β1 treatment. NcCtgf knockdown/overexpression inhibited/promoted collagen synthesis in swim bladder cell, respectively. Notably, NcCtgf protein could be secreted to cell culture medium and up-regulated collagen I expression in swim bladder cell. These findings indicate NcCtgf plays vital roles in collagen synthesis in swim bladder of Nibea coibor, and provide basis for further understanding of ctgf evolution and exploring new approach for enhancing collagen deposition in fish products during aquaculture.

Preservation of Membrana Testacea in titanosaurid dinosaur eggshells from the Upper Cretaceous Deccan volcano-sedimentary strata of Central India

AbstractThe calcitic eggshell units of amniote eggs are underlain by a soft organic layer, the Membrana Testacea (MT), which has a mesh-like texture consisting of fibrils of organic material. Because of its soft anatomy, the MT is rarely preserved in fossils and only a few fossil reports of MT are known so far. Here we report the preservation of a mineralized MT layer in titanosaur eggshells recovered from a marlstone facies interbedded with the Deccan lava flows exposed near Piplanarayanwar village of Chhindwara District, Madhya Pradesh state in Central India. The MT layer is mesh-like, resembling protein membranes of extant reptiles and the MT reported in titanosaurid eggshells of the Upper Cretaceous Anacleto Formation at Auca Mahuevo, Argentina. The presence of tendrils and fibres of calcite in the MT layer testifies to the fact that the calcium layer represents the original fibrous MT. It also supports the view that fossilization of soft tissues like MT is possible because of the inferred anaerobic conditions that prevailed during the deposition of Piplanarayanwar intertrappean sediments in lacustrine or paludal bodies in a coastal-plain setting.

Implications of zero-deforestation palm oil for tropical grassy and dry forest biodiversity.

Many companies have made zero-deforestation commitments (ZDCs) to reduce carbon emissions and biodiversity losses linked to tropical commodities. However, ZDCs conserve areas primarily based on tree cover and aboveground carbon, potentially leading to the unintended consequence that agricultural expansion could be encouraged in biomes outside tropical rainforest, which also support important biodiversity. We examine locations suitable for zero-deforestation expansion of commercial oil palm, which is increasingly expanding outside the tropical rainforest biome, by generating empirical models of global suitability for rainfed and irrigated oil palm. We find that tropical grassy and dry forest biomes contain >50% of the total area of land climatically suitable for rainfed oil palm expansion in compliance with ZDCs (following the High Carbon Stock Approach; in locations outside urban areas and cropland), and that irrigation could double the area suitable for expansion in these biomes. Within these biomes, ZDCs fail to protect areas of high vertebrate richness from oil palm expansion. To prevent unintended consequences of ZDCs and minimize the environmental impacts of oil palm expansion, policies and governance for sustainable development and conservation must expand focus from rainforests to all tropical biomes.