Prevalence, complete genome, and metabolic potentials of a phylogenetically novel cyanobacterial symbiont in the coral-killing sponge, Terpios hoshinota.

Terpios hoshinota is an aggressive, space-competing sponge that kills various stony corals. Outbreaks of this species have led to intense damage to coral reefs in many locations. Here, the first large-scale 16S rRNA gene survey across three oceans revealed that bacteria related to the taxa Prochloron, Endozoicomonas, SAR116, Ruegeria, and unclassified Proteobacteria were prevalent in T. hoshinota. A Prochloron-related bacterium was the most dominant and prevalent cyanobacterium in T. hoshinota. The complete genome of this uncultivated cyanobacterium and pigment analysis demonstrated that it has phycobiliproteins and lacks chlorophyll b, which is inconsistent with the definition of Prochloron. Furthermore, the cyanobacterium was phylogenetically distinct from Prochloron, strongly suggesting that it should be a sister taxon to Prochloron. Therefore, we proposed this symbiotic cyanobacterium as a novel species under the new genus Candidatus Paraprochloron terpiosi. Comparative genomic analyses revealed that 'Paraprochloron' and Prochloron exhibit distinct genomic features and DNA replication machinery. We also characterized the metabolic potentials of 'Paraprochloron terpiosi' in carbon and nitrogen cycling and propose a model for interactions between it and T. hoshinota. This study builds a foundation for the study of the T. hoshinota microbiome and paves the way for better understanding of ecosystems involving this coral-killing sponge.

Potential of galled leaves of Goji (Lycium chinense) as functional food.

BACKGROUND: Goji (Lycium) is a popular traditional health food, and its fruit and root extracts have been found to possess antioxidant, anti-inflammatory, and hypocholesterolemia-inducing abilities. Goji leaves also contain high amounts of phenolic compounds, similar to its fruit, and their extracts also exhibit several pharmaceutical effects. The induction of galls on Goji leaves reduces their photosynthetic ability and fruit yield, which raise their farming costs, thereby leading to economic loss. However, the defense mechanisms induced by infection may elevate the secondary metabolite content of the leaves, which might provide more nutritive compounds. METHOD: Content of chlorophyll, carotenoids, polyphenols, and flavonoids in the extracts of normal and infected Goji leaves (L. chinense) were analyzed. The relative content of chlorogenic acid and rutin, two major phenolic compounds in Goji leaves, were determined by LC-MS/MS. Antioxidant activity was presented by demonstrating the DPPH scavenging percentage. The extract of Goji fruit (L. barbarum) was also analyzed to show a comparative result. RESULTS: In this study, we found that in infected Goji leaves, the polyphenol content was significantly increased. The level of chlorogenic acid was increased by 36% in galled leaves. The content of rutin in galled leaves was also elevated. Testing the antioxidant activities also showed that the extracts of galled leaves have higher DPPH scavenging abilities. CONCLUSIONS: Our results demonstrated that galled Goji leaves have higher functional value, and may have potential as being consumed as health food.

Transcriptome Profile of the Variegated Ficus microcarpa c.v. Milky Stripe Fig Leaf.

Photosynthetic properties and transcriptomic profiles of green and white sectors of Ficus microcarpa (c.v. milky stripe fig) leaves were examined in naturally variegated plants. An anatomic analysis indicated that chloroplasts of the white sectors contained a higher abundance of starch granules and lacked stacked thylakoids. Moreover, no photosynthetic rate was detected in the white sectors. Transcriptome profile and differential expressed gene (DEG) analysis showed that genes encoding PSII core proteins were down-regulated in the white sectors. In genes related to chlorophyll metabolism, no DEGs were identified in the biosynthesis pathway of chlorophyll. However, genes encoding the first step of chlorophyll breakdown were up-regulated. The repression of genes involved in N-assimilation suggests that the white sectors were deprived of N. The mutation in the transcription factor mitochondrial transcription termination factor (mTERF) suggests that it induces colorlessness in leaves of the milky stripe fig.

Transcriptome profile of cup-shaped galls in Litsea acuminata leaves.

BACKGROUND: Insect galls are atypical plant tissues induced by the invasion of insects. Compared to the host leaf, gall tissues lose photosynthetic ability, but have higher soluble sugar content. Although the physiological and biochemical regulation of gall tissues have been demonstrated, the mechanism of genetic regulation has only been analyzed in few studies. RESULTS: In this study, the transcriptome of cup-shaped galls and its host leaf were de novo assembled. Cellular functional enrichment and differentially expressed gene groups in the gall tissues were analyzed. The genes associated with primary metabolism, including photosynthesis, cell wall turnover, and sugar degradation, were expressed differently in galls and leaves. The examination of gene expression demonstrated that the genes involved in brassinosteroid synthesis and responses exhibited a remarkable modulation in cup-shaped galls, suggesting a potential role of steroid hormones in regulating gall development. CONCLUSIONS: This study revealed the genetic responses, including those involved in source-sink reallocation and phytohormone metabolism, of galls induced by a dipteran insect.

Eliminating interference by anthocyanins when determining the porphyrin ratio of red plant leaves.

Anthocyanins (Ants) are water-soluble secondary metabolites that are responsible for red colour of plant leaves. To determine photosynthetic pigments, 80% acetone was used to extract Ants from Ant-containing leaves of test plants. However, using the 80% acetone extraction method can lead to interference between chlorophylls (Chls) and Ants. Porphyrins, such as protoporphyrin IX (PPIX), Mg-protoporphyrin IX (MgPP), and protochlorophyllide (Pchlide), are Chl biosynthetic intermediates and demonstrate photospectrometric characteristics similar to those of Chl. Although the ether/water extraction method was able to remove Ants interference when detecting porphyrins, the porphyrins extraction efficiency was lower than that of the 80% acetone extraction method. Low Ants levels interfered with individual porphyrin ratios, and the extent of the effect was correlated with Ants concentrations. We developed the three equations could eliminate interference by Ants when determining the porphyrin molecular percentage (%) and were comprehensively applied to all tested species of Ants-containing leaves.

Cortisol Regulates Acid Secretion of H(+)-ATPase-rich Ionocytes in Zebrafish (Danio rerio) Embryos.

Systemic acid-base regulation is vital for physiological processes in vertebrates. Freshwater (FW) fish live in an inconstant environment, and thus frequently face ambient acid stress. FW fish have to efficiently modulate their acid secretion processes for body fluid acid-base homeostasis during ambient acid challenge; hormonal control plays an important role in such physiological regulation. The hormone cortisol was previously proposed to be associated with acid base regulation in FW fish; however, the underlying mechanism has not been fully described. In the present study, mRNA expression of acid-secreting related transporters and cyp11b (encoding an enzyme involved in cortisol synthesis) in zebrafish embryos was stimulated by treatment with acidic FW (AFW, pH 4.0) for 3 d. Exogenous cortisol treatment (20 mg/L, 3 d) resulted in upregulated expression of transporters related to acid secretion and increased acid secretion function at the organism level in zebrafish embryos. Moreover, cortisol treatment also significantly increased the acid secretion capacity of H(+)-ATPase-rich cells (HRCs) at the cellular level. In loss-of-function experiments, microinjection of glucocorticoid receptor (GR) morpholino (MO) suppressed the expression of acid-secreting related transporters, and decreased acid secretion function at both the organism and cellular levels; on the other hand, mineralocorticoid receptor (MR) MO did not induce any effects. Such evidence supports the hypothesized role of cortisol in fish acid-base regulation, and provides new insights into the roles of cortisol; cortisol-GR signaling stimulates zebrafish acid secretion function through transcriptional/translational regulation of the transporters and upregulation of acid secretion capacity in each acid-secreting ionocyte.

Aquaporin 1 Is Involved in Acid Secretion by Ionocytes of Zebrafish Embryos through Facilitating CO2 Transport.

Mammalian aquaporin 1 (AQP1) is well known to function as a membrane channel for H2O and CO2 transport. Zebrafish AQP1a.1 (the homologue of mammalian AQP1) was recently identified in ionocytes of embryos; however its role in ionocytes is still unclear. In this study, we hypothesized that zebrafish AQP1a.1 is involved in the acid secretion by ionocytes through facilitating H2O and CO2 diffusion. A real-time PCR showed that mRNA levels of AQP1a.1 in embryos were induced by exposure to 1% CO2 hypercapnia for 3 days. In situ hybridization and immunohistochemistry showed that the AQP1a.1 transcript was highly expressed by acid-secreting ionocytes, i.e., H+-ATPase-rich (HR) cells. A scanning ion-selective electrode technique (SIET) was applied to analyze CO2-induced H+ secretion by individual ionocytes in embryos. H+ secretion by HR cells remarkably increased after a transient loading of CO2 (1% for 10 min). AQP1a.1 knockdown with morpholino oligonucleotides decreased the H+ secretion of HR cells by about half and limited the CO2 stimulated increase. In addition, exposure to an AQP inhibitor (PCMB) for 10 min also suppressed CO2-induced H+ secretion. Results from this study support our hypothesis and provide in vivo evidence of the physiological role of AQP1 in CO2 transport.

Development in a naturally acidified environment: Na+/H+-exchanger 3-based proton secretion leads to CO2 tolerance in cephalopod embryos.

BACKGROUND: Regulation of pH homeostasis is a central feature of all animals to cope with acid-base disturbances caused by respiratory CO2. Although a large body of knowledge is available for vertebrate and mammalian pH regulatory systems, the mechanisms of pH regulation in marine invertebrates remain largely unexplored. RESULTS: We used squid (Sepioteuthis lessoniana), which are known as powerful acid-base regulators to investigate the pH regulatory machinery with a special focus on proton secretion pathways during environmental hypercapnia. We cloned a Rhesus protein (slRhP), V-type H+-ATPase (slVHA) and the Na+/H+ exchanger 3 (slNHE3) from S. lessoniana, which are hypothesized to represent key players in proton secretion pathways among different animal taxa. Specifically designed antibodies for S. lessoniana demonstrated the sub-cellular localization of NKA, VHA (basolateral) and NHE3 (apical) in epidermal ionocytes of early life stages. Gene expression analyses demonstrated that slNHE3, slVHA and slRhP are up regulated in response to environmental hypercapnia (pH 7.31; 0.46 kPa pCO2) in body and yolk tissues compared to control conditions (pH 8.1; 0.045 kPa pCO2). This observation is supported by H+ selective electrode measurements, which detected increased proton gradients in CO2 treated embryos. This compensatory proton secretion is EIPA sensitive and thus confirms the central role of NHE based proton secretion in cephalopods. CONCLUSION: The present work shows that in convergence to teleosts and mammalian pH regulatory systems, cephalopod early life stages have evolved a unique acid-base regulatory machinery located in epidermal ionocytes. Using cephalopod molluscs as an invertebrate model this work provides important insights regarding the unifying evolutionary principles of pH regulation in different animal taxa that enables them to cope with CO2 induced acid-base disturbances.

Rhcg1 and Rhbg mediate ammonia excretion by ionocytes and keratinocytes in the skin of zebrafish larvae: H+-ATPase-linked active ammonia excretion by ionocytes.

In zebrafish, Rhcg1 was found in apical membranes of skin ionocytes [H⁺-ATPase-rich (HR) cells], which are similar to α-type intercalated cells in mammalian collecting ducts. However, the cellular distribution and role of Rhbg in zebrafish larvae have not been well investigated. In addition, HR cells were hypothesized to excrete ammonia against concentration gradients. In this study, we attempted to compare the roles of Rhbg and Rhcg1 in ammonia excretion by larval skin and compare the capability of skin cells to excrete ammonia against concentration gradients. Using in situ hybridization and immunohistochemistry, Rhbg was localized to both apical and basolateral membranes of skin keratinocytes. A scanning ion-selective electrode technique (SIET) was applied to measure the NH4⁺ flux at the apical surface of keratinocytes and HR cells. Knockdown of Rhbg with morpholino oligonucleotides suppressed ammonia excretion by keratinocytes and induced compensatory ammonia excretion by HR cells. To compare the capability of cells to excrete ammonia against gradients, NH4⁺ flux of cells was determined in larvae exposed to serial concentrations of external NH4⁺. Results showed that HR cells excreted NH4⁺ against higher NH4⁺ concentration than did keratinocytes. Knockdown of the expression of either Rhcg1 or H⁺ -ATPase in HR cells suppressed the capability of HR cells.

Rhcg1 and NHE3b are involved in ammonium-dependent sodium uptake by zebrafish larvae acclimated to low-sodium water.

To investigate whether Na(+) uptake by zebrafish is dependent on NH4(+) excretion, a scanning ion-selective electrode technique was applied to measure Na(+) and NH4(+) gradients at the yolk-sac surface of zebrafish larvae. Low-Na(+) acclimation induced an inward Na(+) gradient (uptake), and a combination of low Na(+) and high NH4(+) induced a larger inward Na(+) gradient. When measuring the ionic gradients, raising the external NH4(+) level (5 mM) blocked NH4(+) excretion and Na(+) uptake; in contrast, raising the external Na(+) level (10 mM) simultaneously enhanced Na(+) uptake and NH4(+) excretion. The addition of MOPS buffer (5 mM), which is known to block NH4(+) excretion, also suppressed Na(+) uptake. These results showed that Na(+) uptake and NH4(+) excretion by larval skin are associated when ambient Na(+) level is low. Knockdown of Rhcg1 translation with morpholino-oligonucleotides decreased both NH4(+) excretion and Na(+) uptake by the skin and Na(+) content of whole larvae. Knockdown of nhe3b translation or inhibitor (5-ethylisopropyl amiloride) treatment also decreased both the NH4(+) excretion and Na(+) uptake. This study provides loss-of-function evidence for the involvement of Rhcg1 and NHE3b in the ammonium-dependent Na(+) uptake mechanism in zebrafish larvae subjected to low-Na(+) water.

Chloride transport in mitochondrion-rich cells of euryhaline tilapia (Oreochromis mossambicus) larvae.

A noninvasive scanning ion-selective electrode technique (SIET) was applied to measure Cl- transport at individual mitochondrion-rich cells (MRCs) in the skin of euryhaline tilapia (Oreochromis mossambicus) larvae. In seawater (SW)-acclimated larvae, outward Cl- gradients (20-80 mM higher than the background) were measured at the surface, indicating a secretion of Cl- from the skin. By serial probing over the surface of MRCs and adjacent keratinocytes (KCs), a significant outward flux of Cl- was detected at the apical opening (membrane) of MRCs. Treatment with 100 microM ouabain or bumetanide inhibited the Cl- secretion by approximately 75%. In freshwater (FW)-acclimated larvae, a lower level of outward Cl- gradients (0.2-1 mM) was measured at the skin surface. Low-Cl- water (<0.005 mM) acclimation increased the apical Na+-Cl- cotransporter (NCC) immunoreactivity of MRCs in the larval skin. An inward flux of Cl- was detected when probing the exterior surface of a group of MRCs (convex-MRCs) that express the NCC. An NCC inhibitor (100 microM metolazone) reduced the flux by approximately 90%. This study provides direct and convincing evidence for Cl- transport by MRCs of SW- and FW-acclimated euryhaline tilapia and the involvement of an apical NCC in Cl- uptake of MRCs of FW-acclimated fish.

Ammonia excretion by the skin of zebrafish (Danio rerio) larvae.

The mechanism of ammonia excretion in freshwater teleosts is not well understood. In this study, scanning ion-selective electrode technique was applied to measure H(+) and NH(4)(+) fluxes in specific cells on the skin of zebrafish larvae. NH(4)(+) extrusion was relatively high in H(+) pump-rich cells, which were identified as the H(+)-secreting ionocyte in zebrafish. Minor NH(4)(+) extrusion was also detected in keratinocytes and other types of ionocytes in larval skin. NH(4)(+) extrusion from the skin was tightly linked to acid secretion. Increases in the external pH and buffer concentration (5 mM MOPS) diminished H(+) and NH(4)(+) gradients at the larval surface. Moreover, coupled decreases in NH(4)(+) and H(+) extrusion were found in larvae treated with an H(+)-pump inhibitor (bafilomycin A1) or H(+)-pump gene (atp6v1a) knockdown. Knockdown of Rhcg1 with morpholino-oligonucleotides also decreased NH(4)(+) excretion. This study demonstrates ammonia excretion in epithelial cells of larval skin through an acid-trapping mechanism, and it provides direct evidence for the involvement of the H(+) pump and an Rh glycoprotein (Rhcg1) in ammonia excretion.