Combining environmental DNA and visual surveys can inform conservation planning for coral reefs.

Environmental DNA (eDNA) metabarcoding has the potential to revolutionize conservation planning by providing spatially and taxonomically comprehensive data on biodiversity and ecosystem conditions, but its utility to inform the design of protected areas remains untested. Here, we quantify whether and how identifying conservation priority areas within coral reef ecosystems differs when biodiversity information is collected via eDNA analyses or traditional visual census records. We focus on 147 coral reefs in Indonesia's hyper-diverse Wallacea region and show large discrepancies in the allocation and spatial design of conservation priority areas when coral reef species were surveyed with underwater visual techniques (fishes, corals, and algae) or eDNA metabarcoding (eukaryotes and metazoans). Specifically, incidental protection occurred for 55% of eDNA species when targets were set for species detected by visual surveys and 71% vice versa. This finding is supported by generally low overlap in detection between visual census and eDNA methods at species level, with more overlap at higher taxonomic ranks. Incomplete taxonomic reference databases for the highly diverse Wallacea reefs, and the complementary detection of species by the two methods, underscore the current need to combine different biodiversity data sources to maximize species representation in conservation planning.

Jump dispersal drives the relationship between micro- and macroevolutionary dynamics in the Sicydiinae (Gobiiformes: Oxudercidae) of Sundaland and Wallacea.

Insular biodiversity hotspots of Southeast Asia are remarkable for their biodiverse faunas. With a marine larval phase lasting up to several months, the freshwater fish subfamily Sicydiinae has colonized most islands of these hotspots. However, Sicydiinae diversity is still poorly understood in Southeast Asia. With the objective to estimate intraspecific genetic diversity and infer past demography, we conducted the molecular inventory of Sicydiinae species in Sundaland and Wallacea using 652 bp of the mitochondrial cytochrome oxidase I gene, species delimitation methods and Bayesian Skyline plot reconstructions. In total, 24 Molecular Operational Taxonomic Units are delimited among the 603 sequences belonging to 27 species and five genera. Two cases of discordance between morphology and mitochondrial sequence are observed suggesting ongoing speciation and/or introgression in two genera. Multiple new occurrences are reported, either for a single biodiversity hotspot or both, some of which corresponding to observations of a few individuals far from the range distribution of their conspecifics. Among the ten species or species group whose intraspecific diversity was examined, high levels of genetic diversity and past population expansion are revealed by Tajima's D tests and Bayesian Skyline Plot reconstructions. Together these results indicate that long-distance dispersal is common and suggest that most endemic species originated through founder events followed by population expansion. Patterns of sexual dimorphism and males' coloration among diverging species pair seem to point to sexual selection as an important mechanism contributing to speciation in the Sicydiinae of Sundaland and Wallacea.

Macroalgae exhibit diverse responses to human disturbances on coral reefs.

Scientists and managers rely on indicator taxa such as coral and macroalgal cover to evaluate the effects of human disturbance on coral reefs, often assuming a universally positive relationship between local human disturbance and macroalgae. Despite evidence that macroalgae respond to local stressors in diverse ways, there have been few efforts to evaluate relationships between specific macroalgae taxa and local human-driven disturbance. Using genus-level monitoring data from 1205 sites in the Indian and Pacific Oceans, we assess whether macroalgae percent cover correlates with local human disturbance while accounting for factors that could obscure or confound relationships. Assessing macroalgae at genus level revealed that no genera were positively correlated with all human disturbance metrics. Instead, we found relationships between the division or genera of algae and specific human disturbances that were not detectable when pooling taxa into a single functional category, which is common to many analyses. The convention to use percent cover of macroalgae as an indication of local human disturbance therefore likely obscures signatures of local anthropogenic threats to reefs. Our limited understanding of relationships between human disturbance, macroalgae taxa, and their responses to human disturbances impedes the ability to diagnose and respond appropriately to these threats.

Safeguarding Imperiled Biodiversity and Evolutionary Processes in the Wallacea Center of Endemism.

Wallacea-the meeting point between the Asian and Australian fauna-is one of the world's largest centers of endemism. Twenty-three million years of complex geological history have given rise to a living laboratory for the study of evolution and biodiversity, highly vulnerable to anthropogenic pressures. In the present article, we review the historic and contemporary processes shaping Wallacea's biodiversity and explore ways to conserve its unique ecosystems. Although remoteness has spared many Wallacean islands from the severe overexploitation that characterizes many tropical regions, industrial-scale expansion of agriculture, mining, aquaculture and fisheries is damaging terrestrial and aquatic ecosystems, denuding endemics from communities, and threatening a long-term legacy of impoverished human populations. An impending biodiversity catastrophe demands collaborative actions to improve community-based management, minimize environmental impacts, monitor threatened species, and reduce wildlife trade. Securing a positive future for Wallacea's imperiled ecosystems requires a fundamental shift away from managing marine and terrestrial realms independently.

Social signaling via bioluminescent blinks determines nearest neighbor distance in schools of flashlight fish Anomalops katoptron.

The schooling flashlight fish Anomalops katoptron can be found at dark nights at the water surface in the Indo-Pacific. Schools are characterized by bioluminescent blink patterns of sub-ocular light organs densely-packed with bioluminescent, symbiotic bacteria. Here we analyzed how blink patterns of A. katoptron are used in social interactions. We demonstrate that isolated specimen of A. katoptron showed a high motivation to align with fixed or moving artificial light organs in an experimental tank. This intraspecific recognition of A. katoptron is mediated by blinking light and not the body shape. In addition, A. katoptron adjusts its blinking frequencies according to the light intensities. LED pulse frequencies determine the swimming speed and the blink frequency response of A. katoptron, which is modified by light organ occlusion and not exposure. In the natural environment A. katoptron is changing its blink frequencies and nearest neighbor distance in a context specific manner. Blink frequencies are also modified by changes in the occlusion time and are increased from day to night and during avoidance behavior, while group cohesion is higher with increasing blink frequencies. Our results suggest that specific blink patterns in schooling flashlight fish A. katoptron define nearest neighbor distance and determine intraspecific communication.

Biodiversity inventory of the grey mullets (Actinopterygii: Mugilidae) of the Indo-Australian Archipelago through the iterative use of DNA-based species delimitation and specimen assignment methods.

DNA barcoding opens new perspectives on the way we document biodiversity. Initially proposed to circumvent the limits of morphological characters to assign unknown individuals to known species, DNA barcoding has been used in a wide array of studies where collecting species identity constitutes a crucial step. The assignment of unknowns to knowns assumes that species are already well identified and delineated, making the assignment performed reliable. Here, we used DNA-based species delimitation and specimen assignment methods iteratively to tackle the inventory of the Indo-Australian Archipelago grey mullets, a notorious case of taxonomic complexity that requires DNA-based identification methods considering that traditional morphological identifications are usually not repeatable and sequence mislabeling is common in international sequence repositories. We first revisited a DNA barcode reference library available at the global scale for Mugilidae through different DNA-based species delimitation methods to produce a robust consensus scheme of species delineation. We then used this curated library to assign unknown specimens collected throughout the Indo-Australian Archipelago to known species. A second iteration of OTU delimitation and specimen assignment was then performed. We show the benefits of using species delimitation and specimen assignment methods iteratively to improve the accuracy of specimen identification and propose a workflow to do so.

Assessing species diversity of Coral Triangle artisanal fisheries: A DNA barcode reference library for the shore fishes retailed at Ambon harbor (Indonesia).

The Coral Triangle (CT), a region spanning across Indonesia and Philippines, is home to about 4,350 marine fish species and is among the world's most emblematic regions in terms of conservation. Threatened by overfishing and oceans warming, the CT fisheries have faced drastic declines over the last decades. Usually monitored through a biomass-based approach, fisheries trends have rarely been characterized at the species level due to the high number of taxa involved and the difficulty to accurately and routinely identify individuals to the species level. Biomass, however, is a poor proxy of species richness, and automated methods of species identification are required to move beyond biomass-based approaches. Recent meta-analyses have demonstrated that species richness peaks at intermediary levels of biomass. Consequently, preserving biomass is not equal to preserving biodiversity. We present the results of a survey to estimate the shore fish diversity retailed at the harbor of Ambon Island, an island located at the center of the CT that display exceptionally high biomass despite high levels of threat, while building a DNA barcode reference library of CT shore fishes targeted by artisanal fisheries. We sampled 1,187 specimens and successfully barcoded 696 of the 760 selected specimens that represent 202 species. Our results show that DNA barcodes were effective in capturing species boundaries for 96% of the species examined, which opens new perspectives for the routine monitoring of the CT fisheries.

Aging exacerbates damage and delays repair of alveolar epithelia following influenza viral pneumonia.

BACKGROUND: Influenza virus infection causes significantly higher levels of morbidity and mortality in the elderly. Studies have shown that impaired immunity in the elderly contributes to the increased susceptibility to influenza virus infection, however, how aging affects the lung tissue damage and repair has not been completely elucidated. METHODS: Aged (16-18 months old) and young (2-3 months old) mice were infected with influenza virus intratracheally. Body weight and mortality were monitored. Different days after infection, lung sections were stained to estimate the overall lung tissue damage and for club cells, pro-SPC+ bronchiolar epithelial cells, alveolar type I and II cells to quantify their frequencies using automated image analysis algorithms. RESULTS: Following influenza infection, aged mice lose more weight and die from otherwise sub-lethal influenza infection in young mice. Although there is no difference in damage and regeneration of club cells between the young and the aged mice, damage to alveolar type I and II cells (AT1s and AT2s) is exacerbated, and regeneration of AT2s and their precursors (pro-SPC-positive bronchiolar epithelial cells) is significantly delayed in the aged mice. We further show that oseltamivir treatment reduces virus load and lung damage, and promotes pulmonary recovery from infection in the aged mice. CONCLUSIONS: These findings show that aging increases susceptibility of the distal lung epithelium to influenza infection and delays the emergence of pro-SPC positive progenitor cells during the repair process. Our findings also shed light on possible approaches to enhance the clinical management of severe influenza pneumonia in the elderly.

Molecular analysis of serum and bronchoalveolar lavage in a mouse model of influenza reveals markers of disease severity that can be clinically useful in humans.

BACKGROUND: Management of influenza, a major contributor to the worldwide disease burden, is complicated by lack of reliable methods for early identification of susceptible individuals. Identification of molecular markers that can augment existing diagnostic tools for prediction of severity can be expected to greatly improve disease management capabilities. METHODOLOGY/PRINCIPAL FINDINGS: We have analyzed cytokines, proteome flux and protein adducts in bronchoalveolar lavage (BAL) and sera from mice infected with influenza A virus (PR8 strain) using a previously established non-lethal model of influenza infection. Through detailed cytokine and protein adduct measurements of murine BAL, we first established the temporal profile of innate and adaptive responses as well as macrophage and neutrophil activities in response to influenza infection. A similar analysis was also performed with sera from a longitudinal cohort of influenza patients. We then used an iTRAQ-based, comparative serum proteome analysis to catalog the proteome flux in the murine BAL during the stages correlating with "peak viremia," "inflammatory damage," as well as the "recovery phase." In addition to activation of acute phase responses, a distinct class of lung proteins including surfactant proteins was found to be depleted from the BAL coincident with their "appearance" in the serum, presumably due to leakage of the protein following loss of the integrity of the lung/epithelial barrier. Serum levels of at least two of these proteins were elevated in influenza patients during the febrile phase of infection compared to healthy controls or to the same patients at convalescence. CONCLUSIONS/SIGNIFICANCE: The findings from this study provide a molecular description of disease progression in a mouse model of influenza and demonstrate its potential for translation into a novel class of markers for measurement of acute lung injury and improved case management.

A cellular pathway involved in Clara cell to alveolar type II cell differentiation after severe lung injury.

Regeneration of alveolar epithelia following severe pulmonary damage is critical for lung function. We and others have previously shown that Scgb1a1-expressing cells, most likely Clara cells, can give rise to newly generated alveolar type 2 cells (AT2s) in response to severe lung damage induced by either influenza virus infection or bleomycin treatment. In this study, we have investigated cellular pathway underlying the Clara cell to AT2 differentiation. We show that the initial intermediates are bronchiolar epithelial cells that exhibit Clara cell morphology and express Clara cell marker, Scgb1a1, as well as the AT2 cell marker, pro-surfactant protein C (pro-SPC). These cells, referred to as pro-SPC(+) bronchiolar epithelial cells (or SBECs), gradually lose Scgb1a1 expression and give rise to pro-SPC(+) cells in the ring structures in the damaged parenchyma, which appear to differentiate into AT2s via a process sharing some features with that observed during alveolar epithelial development in the embryonic lung. These findings suggest that SBECs are intermediates of Clara cell to AT2 differentiation during the repair of alveolar epithelia following severe pulmonary injury.

Spatiotemporal quantification of cell dynamics in the lung following influenza virus infection.

Lung injury caused by influenza virus infection is widespread. Understanding lung damage and repair progression post infection requires quantitative spatiotemporal information on various cell types mapping into the tissue structure. Based on high content images acquired from an automatic slide scanner, we have developed algorithms to quantify cell infiltration in the lung, loss and recovery of Clara cells in the damaged bronchioles and alveolar type II cells (AT2s) in the damaged alveolar areas, and induction of pro-surfactant protein C (pro-SPC)-expressing bronchiolar epithelial cells (SBECs). These quantitative analyses reveal: prolonged immune cell infiltration into the lung that persisted long after the influenza virus was cleared and paralleled with Clara cell recovery; more rapid loss and recovery of Clara cells as compared to AT2s; and two stages of SBECs from Scgb1a1⁺ to Scgb1a1⁻. These results provide evidence supporting a new mechanism of alveolar repair where Clara cells give rise to AT2s through the SBEC intermediates and shed light on the understanding of the lung damage and repair process. The approach and algorithms in quantifying cell-level changes in the tissue context (cell-based tissue informatics) to gain mechanistic insights into the damage and repair process can be expanded and adapted in studying other disease models.

Human fetal hepatic progenitor cells are distinct from, but closely related to, hematopoietic stem/progenitor cells.

Much controversy surrounds the identity and origin of human hepatic stem and progenitor cells in part because of a lack of small animal models in which the developmental potential of isolated candidate cell populations can be functionally evaluated. We show here that adoptive transfer of CD34(+) cells from human fetal liver into sublethally irradiated NOD-SCID Il2rg(-/-) (NSG) mice leads to an efficient development of not only human hematopoietic cells but also human hepatocyte-like cells in the liver of the recipient mice. Using this simple in vivo assay in combination with cell fractionation, we show that CD34(+) fetal liver cells can be separated into three distinct subpopulations: CD34(hi) CD133(hi), CD34(lo) CD133(lo), and CD34(hi) CD133(neg). The CD34(hi) CD133(hi) population contains hematopoietic stem/progenitor cells (HSPCs) as they give rise to T cells, B cells, NK cells, dendritic cells, and monocytes/macrophages in NSG mice and colony-forming unit (CFU)-GEMM cells in vitro. The CD34(lo) CD133(lo) population does not give rise to hematopoietic cells, but reproducibly generates hepatocyte-like cells in NSG mice and in vitro. The CD34(hi) CD133(neg) population only gives rise to CFU-GM and burst-forming unit-erythroid in vitro. Furthermore, we show that the CD34(lo) CD133(lo) cells express hematopoietic, hepatic, and mesenchymal markers, including CD34, CD133, CD117, epithelial cell adhesion molecule, CD73, albumin, α-fetal protein, and vimentin and transcriptionally are more closely related to HSPCs than to mature hepatocytes. These results show that CD34(lo) CD133(lo) fetal liver cells possess the hepatic progenitor cell properties and that human hepatic and hematopoietic progenitor cells are distinct, although they may originate from the same precursors in the fetal liver.

Peptide targeting and imaging of damaged lung tissue in influenza-infected mice.

AIM: In this study, we investigate whether pH (low) insertion peptide (pHLIP) can target regions of lung injury associated with influenza infection. MATERIALS & METHODS: Fluorophore-conjugated pHLIP was injected intraperitoneally into mice infected with a sublethal dose of H1N1 influenza and visualized histologically. RESULTS: pHLIP specifically targeted inflamed lung tissues of infected mice in the later stages of disease and at sites where alveolar type I and type II cells were depleted. Regions of pHLIP-targeted lung tissue were devoid of peroxiredoxin 6, the lung-abundant antioxidant enzyme, and were deficient in pneumocytes. Interestingly, a pHLIP variant possessing mutations that render it insensitive to pH changes was also able to target damaged lung tissue. CONCLUSION: pHLIP holds potential for delivering therapeutics for lung injury during influenza infection. Furthermore, there may be more than one mechanism that enables pHLIP variants to target inflamed lung tissue.

Regeneration of alveolar type I and II cells from Scgb1a1-expressing cells following severe pulmonary damage induced by bleomycin and influenza.

The lung comprises an extensive surface of epithelia constantly exposed to environmental insults. Maintaining the integrity of the alveolar epithelia is critical for lung function and gaseous exchange. However, following severe pulmonary damage, what progenitor cells give rise to alveolar type I and II cells during the regeneration of alveolar epithelia has not been fully determined. In this study, we have investigated this issue by using transgenic mice in which Scgb1a1-expressing cells and their progeny can be genetically labeled with EGFP. We show that following severe alveolar damage induced either by bleomycin or by infection with influenza virus, the majority of the newly generated alveolar type II cells in the damaged parenchyma were labeled with EGFP. A large proportion of EGFP-expressing type I cells were also observed among the type II cells. These findings strongly suggest that Scgb1a1-expressing cells, most likely Clara cells, are a major cell type that gives rise to alveolar type I and II cells during the regeneration of alveolar epithelia in response to severe pulmonary damage in mice.

Major shifts in the spatio-temporal distribution of lung antioxidant enzymes during influenza pneumonia.

With the incessant challenge of exposure to the air we breathe, lung tissue suffers the highest levels of oxygen tension and thus requires robust antioxidant defenses. Furthermore, following injury or infection, lung tissue faces the additional challenge of inflammation-induced reactive oxygen and nitrogen species (ROS/RNS). Little is known about the identity or distribution of lung antioxidant enzymes under normal conditions or during infection-induced inflammation. Using a mouse model of influenza (H1N1 influenza virus A/PR/8/34 [PR8]) in combination with bioinformatics, we identified seven lung-abundant antioxidant enzymes: Glutathione peroxidase 3 (Gpx3), Superoxide dismutase 3 (Sod3), Transferrin (Tf), peroxyredoxin6 (Prdx6), glutathione S-transferase kappa 1 (Gstk1), Catalase (Cat), and Glutathione peroxidase 8 (Gpx8). Interestingly, despite the demand for antioxidants during inflammation, influenza caused depletion in two key antioxidants: Cat and Prdx6. As Cat is highly expressed in Clara cells, virus-induced Clara cell loss contributes to the depletion in Cat. Prdx6 is also reduced due to Clara cell loss, however there is a coincident increase in Prdx6 levels in the alveoli, resulting in only a subtle reduction of Prdx6 overall. Analogously, Gpx3 shifts from the basement membranes underlying the bronchioles and blood vessels to the alveoli, thus maintaining balanced expression. Taken together, these studies identify key lung antioxidants and reveal their distribution among specific cell types. Furthermore, results show that influenza depletes key antioxidants, and that in some cases there is coincident increased expression, consistent with compensatory expression. Given that oxidative stress is known to be a key risk factor during influenza infection, knowledge about the antioxidant repertoire of lungs, and the spatio-temporal distribution of antioxidants, contributes to our understanding of the underlying mechanisms of influenza-induced morbidity and mortality.

Double-stranded RNA-activated protein kinase regulates early innate immune responses during respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) is the most common cause of childhood viral bronchiolitis and lung injury. Inflammatory responses significantly contribute to lung pathologies during RSV infections and bronchiolitis but the exact mechanisms have not been completely defined. The double-stranded RNA-activated protein kinase (PKR) functions to inhibit viral replication and participates in several signaling pathways associated with innate inflammatory immune responses. Using a functionally defective PKR (PKR(-/-)) mouse model, we investigated the role of this kinase in early events of RSV-induced inflammation. Our data showed that bronchoalveolar lavage (BAL) fluid from infected PKR(-/-) mice had significantly lower levels of several innate inflammatory cytokines and chemokines. Histological examinations revealed that there was less lung injury in infected PKR(-/-) mice as compared to the wild type. A genome-wide analysis showed that several early antiviral and immune regulatory genes were affected by PKR activation. These data suggest that PKR is a signaling molecule for immune responses during RSV infections.

Induction of lactoferrin gene expression by innate immune stimuli in mouse mammary epithelial HC-11 cells.

Lactoferrin (LF) is a multifunctional protein. While its functions and mechanism of actions are actively being investigated, the cellular signals that regulate LF expression have not been as explored. We have previously demonstrated that LF is upregulated by estrogen in the reproductive system. In this study, we show that the expression of LF was stimulated by bacterial lipopolysaccharide (LPS) and double-stranded RNA (dsRNA) in normal mouse mammalian HC-11 cells. When cells were exposed to either LPS or dsRNA, the mRNA and protein of LF were increased in a dose- and time-dependent manner, yet the kinetics of LF induction by dsRNA or LPS were different. The LPS and dsRNA-induced LF was mainly released into the culture medium where it blocked TNF-alpha production in exposed cells. We explored the mechanisms of LF induction by LPS and dsRNA using specific inhibitors and found that the induction could be attenuated by inhibitors to PKC, NF-kappaB, p38 and JNK, but not by an inhibitor to PKA. Interestingly, ERK inhibitor was effective against dsRNA but not against LPS induction of LF. These data suggest that LF was induced by LPS and dsRNA through PKC, NF-kappaB and MAPK pathways which in turn play an inhibitory role in the continuation of innate inflammation.

Scavenger receptor class-A is a novel cell surface receptor for double-stranded RNA.

Double-stranded RNA (dsRNA) is a potent signal to the host immune system for the presence of an ongoing viral infection. The presence of dsRNA, intracellularly or extracellularly, leads to the induction of innate inflammatory cytokines in many cell types including epithelial cells. However, the cell surface receptor for recognition of extracellular dsRNA is not yet determined. Here, we report that extracellular dsRNA is recognized and internalized by scavenger receptor class-A (SR-A). Treatment of human epithelial cells with specific antagonists of SR-A or with an anti-SR-A antibody significantly inhibited dsRNA induction of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, IL-8, and regulated on activation normal T-cell expressed and secreted (RANTES). Furthermore, intranasal dsRNA treatment of SR-A-deficient (SR-A(-/-)) mice showed a significant decrease in the expression of inflammatory cytokines and a corresponding decrease in the accumulation of polymorphonuclear leukocytes (PMNs) in lungs. These data provide direct evidence that SR-A is a novel cell surface receptor for dsRNA, and therefore, SR-A may play a role in antiviral immune responses.