Non-destructive methods to assess health of wild tropical frogs (túngara frogs: <i>Engystomops pustulosus</i>) in Trinidad reveal negative impacts of agricultural land

Amphibians are threatened globally with at least 43% of species declining and the most important stressor being habitat loss or degradation. Amphibians inhabiting highly biodiverse tropical regions are disproportionately threatened; however, the effects of landscape alterations on amphibian health are virtually unknown. In this study, we utilised non-destructive techniques to compare size (weight, snout-vent length [SVL]), body condition, male secondary sexual features (forelimb width, nuptial pad length) and breeding success (egg number, fertility [percentage fertilised eggs], hatching success) in túngara frogs (Engystomops pustulosus) collected from reference (n = 5), suburban (n = 6) and agricultural (n = 4) sites in Trinidad; characterised by presence/absence of crops/houses. All measured endpoints were negatively impacted in frogs collected from agricultural sites. The largest effect was observed for hatching success (2.77-fold lower) and egg number (2.5-fold lower). Less pronounced effects were observed on male frogs (weight: 1.77-fold lower; SVL: 1.18-fold lower; forelimb width: 1.33-fold lower; nuptial pad length: 1.15-fold lower). Our findings demonstrate negative impacts of agricultural sites on túngara frog health, with the number of viable offspring reduced by almost one third. The methods outlined here are technically simple and low-cost and thereby have potential for application to other species in order to investigate the potential impacts of habitat degradation on amphibian health. Furthermore, as these methods are non-destructive, they could be used to investigate the potential contribution of frog size and/or reproductive capability as a causative factor contributing to population declines in threatened species, which is particularly pressing in tropical regions. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11356-022-20105-4

Local control of intestinal stem cell homeostasis by enteroendocrine cells in the adult <i>Drosophila</i> midgut

Background: Enteroendocrine cells populate gastrointestinal tissues and are known to translate local cues into systemic responses through the release of hormones into the bloodstream.&lt;p&gt;&lt;/p&gt; Results: Here we report a novel function of enteroendocrine cells acting as local regulators of intestinal stem cell (ISC) proliferation through modulation of the mesenchymal stem cell niche in the &lt;i&gt;Drosophila&lt;/i&gt; midgut. This paracrine signaling acts to constrain ISC proliferation within the epithelial compartment. Mechanistically, midgut enteroendocrine cells secrete the neuroendocrine hormone Bursicon, which acts—beyond its known roles in development—as a paracrine factor on the visceral muscle (VM). Bursicon binding to its receptor, DLGR2, the ortholog of mammalian leucine-rich repeat-containing G protein-coupled receptors (LGR4-6), represses the production of the VM-derived EGF-like growth factor Vein through activation of cAMP.&lt;p&gt;&lt;/p&gt; Conclusions: We therefore identify a novel paradigm in the regulation of ISC quiescence involving the conserved ligand/receptor Bursicon/DLGR2 and a previously unrecognized tissue-intrinsic role of enteroendocrine cells.&lt;p&gt;&lt;/p&gt

Pink1 and Parkin regulate Drosophila intestinal stem cell proliferation during stress and aging.

Intestinal stem cells (ISCs) maintain the midgut epithelium in Drosophila melanogaster Proper cellular turnover and tissue function rely on tightly regulated rates of ISC division and appropriate differentiation of daughter cells. However, aging and epithelial injury cause elevated ISC proliferation and decreased capacity for terminal differentiation of daughter enteroblasts (EBs). The mechanisms causing functional decline of stem cells with age remain elusive; however, recent findings suggest that stem cell metabolism plays an important role in the regulation of stem cell activity. Here, we investigate how alterations in mitochondrial homeostasis modulate stem cell behavior in vivo via RNA interference-mediated knockdown of factors involved in mitochondrial dynamics. ISC/EB-specific knockdown of the mitophagy-related genes Pink1 or Parkin suppresses the age-related loss of tissue homeostasis, despite dramatic changes in mitochondrial ultrastructure and mitochondrial damage in ISCs/EBs. Maintenance of tissue homeostasis upon reduction of Pink1 or Parkin appears to result from reduction of age- and stress-induced ISC proliferation, in part, through induction of ISC senescence. Our results indicate an uncoupling of cellular, tissue, and organismal aging through inhibition of ISC proliferation and provide insight into strategies used by stem cells to maintain tissue homeostasis despite severe damage to organelles

Endocrine remodelling of the adult intestine sustains reproduction in Drosophila.

The production of offspring is energetically costly and relies on incompletely understood mechanisms that generate a positive energy balance. In mothers of many species, changes in key energy-associated internal organs are common yet poorly characterised functionally and mechanistically. In this study, we show that, in adult Drosophila females, the midgut is dramatically remodelled to enhance reproductive output. In contrast to extant models, organ remodelling does not occur in response to increased nutrient intake and/or offspring demands, but rather precedes them. With spatially and temporally directed manipulations, we identify juvenile hormone (JH) as an anticipatory endocrine signal released after mating. Acting through intestinal bHLH-PAS domain proteins Methoprene-tolerant (Met) and Germ cell-expressed (Gce), JH signals directly to intestinal progenitors to yield a larger organ, and adjusts gene expression and sterol regulatory element-binding protein (SREBP) activity in enterocytes to support increased lipid metabolism. Our findings identify a metabolically significant paradigm of adult somatic organ remodelling linking hormonal signals, epithelial plasticity, and reproductive output. DOI: http://dx.doi.org/10.7554/eLife.06930.00

Differential compartmentalization of memory B cells versus plasma cells in salmonid fish

The disposition of teleost memory and plasma cells (PCs) has essentially been un-explored. As the organization of the teleost immune system differs significantly from that of mammals (i.e. no bone marrow or lymph nodes, hematopoietic anterior kidney), this disposition could be essential in understanding how comparable functions are achieved. To address this question, the primary and secondary antibody-secreting cell, B memory cell, and antibody responses to T-independent and T-dependent antigens were analyzed in trout. Although the TI and TD antibody responses did not differ substantively from one another, the secondary responses to both were significantly prolonged compared with the primary responses. Logarithmic increases in titer and affinity were achieved for both antigens during the primary, with only modest increases during the secondary response. Antibody-secreting cells, both PCs and plasmablasts, quantitatively paralleled antibody production, with antibody-secreting cells skewing to the hematopoietic anterior kidney for both antigens. However, the enhanced antigen-inducible response of immune fish (indicative of the memory pool) skewed to the peripheral blood and spleen. This pattern of memory versus PC disposition parallels that observed in mammals even though the organization of the respective immune systems differs considerably

Aneuploidy in intestinal stem cells promotes gut dysplasia in Drosophila

Aneuploidy is associated with different human diseases including cancer. However, different cell types appear to respond differently to aneuploidy, either by promoting tumorigenesis or causing cell death. We set out to study the behavior of adult Drosophila melanogaster intestinal stem cells (ISCs) after induction of chromosome missegregation either by abrogation of the spindle assembly checkpoint or through kinetochore disruption or centrosome amplification. These conditions induce moderate levels of aneuploidy in ISCs, and we find no evidence of apoptosis. Instead, we observe a significant accumulation of ISCs associated with increased stem cell proliferation and an excess of enteroendocrine cells. Moreover, aneuploidy causes up-regulation of the JNK pathway throughout the posterior midgut, and specific inhibition of JNK signaling in ISCs is sufficient to prevent dysplasia. Our findings highlight the importance of understanding the behavior of different stem cell populations to aneuploidy and how these can act as reservoirs for genomic alterations that can lead to tissue pathologies.This article is a result of the project Norte Portugal Regional Operational Program (NORTE 2020) Norte-01-0145-FEDER-000029 – Advancing Cancer Research: From basic knowledge to application, under the PORTUGAL 2020 Partnership Agreement through the European Regional Development Fund, and it is also funded by National Funds through Fundação para a Ciência e a Tecnologia under the project PTDC/BEX-BCM/1921/2014

Physiological and stem cell compartmentalization in the adult Drosophila midgut

The precise and systematic digestion and subsequent absorption of nutrients in food is vitally important for the life of all metazoans. The intestine is the organ primarily responsible for absorbing, processing, transporting, and excreting the vast variety of nutrients found in an organism’s diet, and the tissue is maintained throughout its length by multipotent intestinal stem cells. Although previous work identified regions of preferential copper and iron absorption, only recent work is revealing the degree to which regions of the tissue can be specialized. The work presented in this thesis categorizes the extent of physiological, morphological, organizational and molecular heterogeneity along the intestinal anterior-posterior axis. This work is also the first to test stem cells of specific intestinal regions for their tumorigenic capacity and their ability to generate progeny of neighboring regions. I analyzed the properties of intestinal cells along the anterior-posterior axis and found that the tissue contains more specialized regions than the medial zones specialized for iron and copper absorption. By analyzing nutrient staining, cell morphology, and gene expression, I identified at least 10 distinct midgut subregions, and developed a system by which they may be easily identified. Gut regions were precisely isolated using lines with region specific expression that we recovered from the Janelia GAL4 collection, and I performed RNAseq to identify localized candidate digestive pathways. The existence of significant physiological diversity along the length of the midgut raised the question of whether ISCs can support all midgut cells or if they are region specific. Like the differentiated cells of the intestinal epithelium, stem cells also vary regionally in behavior and gene expression, suggesting that they contribute to midgut sub-specialization. Using lineage tracing in regionally restricted GAL4 and protein trap lines I show that stem cells generate progeny located outside their own subregion at only one of six borders tested, suggesting that midgut subregions resemble cellular compartments involved in tissue development. Additionally, tumors generated by disrupting stem cell mediated Notch signaling arose preferentially in three subregions and tumor cells also appeared to respect regional borders. Thus, apparently similar intestinal stem cells differ regionally in cell production, gene expression and in the ability to spawn tumors. This work not only provides new tools for studying the drosophila intestine, but also suggests that heterogeneity and compartmentalization may also exist in mammalian tissues containing many apparently similar stem cells. Such work may enable us to acquire the knowledge needed to better treat digestive diseases, as well as approach similar stem cell potency studies in other stem cell based tissues