Respiratory Tree Research Articles

Identification and functional characterization of first alpha‐2‐macroglobulin in sea cucumbers

The broad-spectrum protease inhibitor alpha-2-macroglobulin (α2M) is widely found in vertebrates and invertebrates. However, no research regarding α2M has been described in sea cucumbers to date. In this study, the first sea cucumber α2M named Stmα2M was identified from Stichopus monotuberculatus, a tropical species with high edible and economic values in Southern China. The Stmα2M cDNA is 5276 bp in length, containing a 5′-untranslated region (UTR) of 279 bp, a 3′-UTR of 344 bp and an open reading frame of 4653 bp that encodes a protein of 1550 amino acids. The Stmα2M possesses all the three characteristics of known α2M proteins, including the bait region domain, thioester domain and receptor-binding domain. Higher levels of mRNA expression were noticed in intestine, respiratory tree, coelomocytes and tentacle of S. monotuberculatus. Transcriptional expression of Stmα2M showed the strongest response to lipopolysaccharides (LPS, 8.19-fold up-regulation) after 6 h post-challenge in coelomocytes. In addition, the α2M activity was detected in S. monotuberculatus and protein activity of α2M increased remarkably at 6 h after LPS stimulation. As a whole, this study indicated that Stmα2M is an important immune-related molecule, and the study on functions of Stmα2M may provide some new and valuable ideas for preventing and controlling diseases of cultured sea cucumbers.

A work group report on ultrafine particles (American Academy of Allergy, Asthma & Immunology): Why ambient ultrafine and engineered nanoparticles should receive special attention for possible adverse health outcomes in human subjects

Ultrafine particles (UFPs) are airborne particulates of less than 100nm in aerodynamic diameter. Examples of UFPs are diesel exhaust particles, products of cooking, heating, and wood burning in indoor environments, and, more recently, products generated through the use of nanotechnology. Studies have shown that ambient UFPs have detrimental effects on both the cardiovascular and respiratory systems, including a higher incidence of atherosclerosis and exacerbation rate of asthma. UFPs have been found to alter invitro and invivo responses of the immune system to allergens and can also play a role in allergen sensitization. The inflammatory properties of UFPs can be mediated by a number of different mechanisms, including the ability to produce reactive oxygen species, leading to the generation of proinflammatory cytokines and airway inflammation. In addition, because of their small size, UFPs also have unique distribution characteristics in the respiratory tree and circulation and might be able to alter cellular function in ways that circumvent normal signaling pathways. Additionally, UFPs can penetrate intracellularly and potentially cause DNA damage. The recent advances in nanotechnology, although opening up new opportunities for the advancement of technology and medicine, could also lead to unforeseen adverse health effects in exposed human subjects. Further research is needed to clarify the safety of nanoscale particles, as well as the elucidation of the possible beneficial use of these particulates to treat disease.

Gliotoxin Suppresses Macrophage Immune Function by Subverting Phosphatidylinositol 3,4,5-Trisphosphate Homeostasis.

ABSTRACTAspergillus fumigatus, an opportunistic fungal pathogen, spreads in the environment by releasing numerous conidia that are capable of reaching the small alveolar airways of mammalian hosts. In otherwise healthy individuals, macrophages are responsible for rapidly phagocytosing and eliminating these conidia, effectively curbing their germination and consequent invasion of pulmonary tissue. However, under some circumstances, the fungus evades phagocyte-mediated immunity and persists in the respiratory tree. Here, we report that A. fumigatus escapes macrophage recognition by strategically targeting phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] metabolism through gliotoxin, a potent immunosuppressive mycotoxin. Time-lapse microscopy revealed that, in response to the toxin, macrophages cease to ruffle, undergo abrupt membrane retraction, and fail to phagocytose large targets effectively. Gliotoxin was found to prevent integrin activation and interfere with actin dynamics, both of which are instrumental for phagocytosis; similar effects were noted in immortalized and primary phagocytes. Detailed studies of the underlying molecular mechanisms of toxicity revealed that inhibition of phagocytosis is attributable to impaired accumulation of PtdIns(3,4,5)P3 and the associated dysregulation of downstream effectors, including Rac and/or Cdc42. Strikingly, in response to the diacylglycerol mimetic phorbol 12-myristate 13-acetate, gliotoxin-treated macrophages reactivate beta integrins, reestablish actin dynamics, and regain phagocytic capacity, despite the overt absence of plasmalemmal PtdIns(3,4,5)P3. Together, our findings identify phosphoinositide metabolism as a critical upstream target of gliotoxin and also indicate that increased diacylglycerol levels can bypass the requirement for PtdIns(3,4,5)P3 signaling during membrane ruffling and phagocytosis.

Discovery, structural characterization and functional analysis of alpha-2-macroglobulin, a novel immune-related molecule from Holothuria atra

The non-specific protease inhibitor alpha-2-macroglobulin (A2M) is a key macromolecular glycoprotein that involved in host immune defense against pathogens in vertebrates and invertebrates. However, no research regarding A2M has been developed in echinoderms to date. In this study, the full-length cDNA of A2M was cloned from the sea cucumber (Holothuria atra), which is a tropical species widely distributed along the coasts of the South China Sea and designated HaA2M. HaA2M possesses all three conserved functional domains of known A2M proteins, including the bait region domain, thioester domain and receptor-binding domain. Compared to fish and shrimp A2Ms, the histidine residue from the catalytical regions is well conserved in HaA2M. HaA2M mRNA was predominantly expressed in coelomocytes and, to a lesser extent, in the body wall, intestine and respiratory tree. A2M activity was detected in the coelomic fluids of H. atra. The mRNA expression and activity levels were investigated in the major immune tissues and coelomic fluids of H. atra after challenge with inactivated Vibrio alginolyticus or polyriboinosinic polyribocytidylic acid [Poly (I: C)]. RNA interference (RNAi)-mediated knockdown of HaA2M resulted in a significant reduction of HaA2M gene transcript level (86%). RNAi-mediated silencing of HaA2M gene significantly decreased the A2M activity (38%) and increased the number of viable bacteria (2.8-fold) in the coelomic fluids of H. atra infected by V. alginolyticus. Our study, as a whole, supplied the evidences for HaA2M as an immune-relevant molecule and it might have multiple functions in the innate immune system of H. atra.

Effects of dietary cadmium on growth, antioxidants and bioaccumulation of sea cucumber (Apostichopus japonicus) and influence of dietary vitamin C supplementation

The effects of dietary cadmium (Cd) supplementation on growth, antioxidant capacity and accumulation of Cd in tissues (body wall, digestive tracts, and respiratory tree) of sea cucumber, Apostichopus japonicus, exposed to sub-chronic concentrations (0, 10, 50, 100, and 500mg Cd/kg dry weight) of Cd were investigated. In addition, the potential protective effects of vitamin C (L-ascorbic acid, AsA) against the effects of Cd on sea cucumbers were investigated. Sea cucumbers were exposed to dietary Cd for 30 days, after which another group of healthy sea cucumbers was supplied diet supplemented with mixed Cd and AsA for another 30 days. Cd exposure for 30 days resulted in increased Cd accumulation in tissues of sea cucumbers with exposure time and concentration. The order of Cd accumulation in organs was digestive tracts>respiratory tree>body wall. On day 30, the body weight gain (BWG) and specific growth rate (SGR) decreased significantly (P<0.05) in the 500mg Cd/kg treatment. Superoxide dismutase (SOD) activity, glutathione peroxidase (GSH-Px) activity and catalase (CAT) activity in the coelomic fluid of sea cucumbers decreased with increasing dietary Cd concentration, but malondialdehyde (MDA) content in the coelomic fluid increased. Providing diet supplemented with Cd and AsA indicated that although sea cucumbers exhibited signs of Cd toxicity, no death occurred in response to 50mg Cd/kg for 30 days. Based on these findings, five treatments were provided: 50mg Cd/kg+0mg AsA/kg, 50mg Cd/kg+ 3000mg AsA/kg, 50mg Cd/kg+ 5000mg AsA/kg, 50mg Cd/kg+10,000mg AsA/kg, and 50mg Cd/kg+15,000mg AsA/kg. The BWG and SGR of sea cucumbers fed the AsA supplemented diet mixed with Cd increased. Additionally, MDA levels in coelomic fluid were negatively correlated with dietary AsA levels, while antioxidant capacities (SOD, GSH-Px and CAT) were positively correlated with dietary AsA levels. Moreover, Cd accumulation in tissues decreased in response to dietary AsA supplementation of treatments. Overall, antioxidant capacity and bioaccumulation in sea cucumber was found to decrease and be induced in response to Cd, but vitamin C mitigated these effects, with 5000mg AsA/kg providing the optimum protection against 50mg/kg Cd.

A β-integrin from sea cucumber Apostichopus japonicus exhibits LPS binding activity and negatively regulates coelomocyte apoptosis.

Integrins are a family of membrane glycoproteins, which are the major receptors for extracellular matrix and cell-cell adhesion molecules. In this study, a 1038 bp sequence representing the full-length cDNA of a novel β-integrin subunit (designated as AjITGB) was cloned from Apostichopus japonicas by using combined transcriptome sequencing and RACE approaches. The deduced amino acid sequence of AjITGB shared a conserved tripeptide Arg-Gly-Asp (RGD) binding domain with an S-diglyceridecysteine or N-Palm cysteine residue (C(31)), a transmembrane domain, and a β-integrin cytoplasmic domain. Spatial distribution analysis showed that AjITGB was constitutively expressed in all tested tissues with dominant expression in the muscles and weak expression in the respiratory tree. The pathogen Vibrio splendidus challenge and LPS stimulation could both significantly down-regulate the mRNA expression of AjITGB. Functional investigation revealed that recombinant AjITGB displayed higher LPS binding activity but lower binding activity to PGN and MAN. More importantly, knockdown of AjITGB by specific siRNA resulted in the significant promotion of coelomocyte apoptosis in vitro. Results indicated that AjITGB may serve as an apoptosis inhibitor with LPS binding activity during host-pathogen interaction in sea cucumber.

Thermal stress effects on energy resource allocation and oxygen consumption rate in the juvenile sea cucumber, Holothuria scabra (Jaeger, 1833)

Water temperature is a key factor in aquaculture production of the commercially valuable sea cucumber Holothuria scabra. Knowledge is scarce about actual energetic costs that can be associated with internal acclimatization processes as a response to thermal extremes. In the present study changes in cellular energy allocation, oxygen consumption rate and energy related enzymes' activity (IDH and LDH) were measured in juvenile H. scabra, held at different temperatures: 21, 27 and 33°C. The results showed that the steady temperature change (1°C/day) to both temperature treatments, until reaching the testing temperatures (day 0), clearly affected cellular energy consumption and available energy reserves, measured in the respiratory tree and muscle tissue, respectively. However, 15 and 30days after acclimation, the initial differences in cellular energy allocation between treatments decreased. In contrast to the variations measured in cellular energy allocation, oxygen consumption was highest at 33°C and lowest at 21°C at all three measurement times. Moreover, a significant positive correlation between oxygen consumption rate and temperature was detected at day 15 and day 30. Likewise, a shift from anaerobic to aerobic energy metabolism, indicated by changes in LDH and IDH activities, was observed in the animals from the warm temperature treatment. Results imply that juvenile H. scabra were able to recover from initial disturbances in energy balance, caused by the incremental temperature change of ±6°C. Over the experimental period of 30days, elevated temperature did however, lead to a metabolic shift and more efficient energy turnover, indicated by changes in oxygen consumption rate, LDH and IDH. The synergy of cellular energy allocation and oxygen consumption proved to be a viable indicator to assess the capability of sea cucumbers like H. scabra to cope with extreme temperature conditions. Surprisingly, juvenile H. scabra were able to sustain their energy balance and oxygen consumption rate within the homeostatic range, even at 33°C. Thus, we assume that rearing temperatures of 33°C might be possible, which could improve aquaculture production of H. scabra. However, further research is required to understand the mechanisms and effects of acclimation under aquaculture conditions. Statement of relevanceThe sea cucumber H. scabra is considered a promising aquaculture candidate in the tropics. The commercial interest in H. scabra has led to a great number of attempts to culture this species e.g. in Madagascar, Tanzania, India and Indonesia, where the farming of this species also showed a great potential to increase the livelihood of the local community. Although, considerable research effort has been put into the advancement of the production cycle, fundamental knowledge, e.g. on physiological adjustments due to sub-optimal rearing conditions, remains scarce for H. scabra. Water temperature has been identified as crucial factor, determining production efficiency. In our manuscript we introduce an innovative approach, for the assessment of shifts in the energy budget as well as in metabolic pathways, to detect thermal stress in juvenile H. scabra. Our results show that the combined application of cellular energy allocation (CEA) and measurement of the oxygen consumption rate (OCR) as well as the activity of two metabolic key enzymes: iso-citrate dehydrogenase (IDH) and lactate dehydrogenase (LDH), is a strong analytical tool to evaluate whole animal homeostasis under thermal extremes. To our knowledge this is the first study in which the method of CEA was optimized for the application on isolated muscle and respiratory tree tissue of a sea cucumber, like H. scabra. Hence, our results provide original insights into the thermal acclimation physiology of H. scabra, which provides crucial knowledge for setting up optimal culture conditions for this highly valuable aquaculture candidate.Moreover, our results show that juvenile H. scabra were strongly affected by rapid temperature changes, exhibiting distinct differences in metabolic adjustments as response to short-term cold and warm acclimation, but were able to restore homeostatic conditions at constant cold and warm temperature extremes. Concerning aquaculture practices, these findings imply that CEA might be a good condition indicator to evaluate the overall metabolic status in sea cucumbers. Moreover, rapid temperature fluctuations seem to provoke much higher energetic costs in juvenile H. scabra than constant extreme temperature conditions, which is important to consider in daily management practices.The OCR revealed an enhanced metabolism at warmer temperature conditions, throughout the entire experimental period. Likewise, the warm exposed animals were shifting their energy turnover from a more anaerobic to a more aerobic state, indicated by changes in LDH and IDH activities, and showed increased foraging activity at all times. The combined outcome of CEA, OCR and activities of energy metabolism related enzymes indicates, that the increased metabolic activity in juvenile H. scabra, as response to warmer temperature, was well within the homeostatic range. Thus, contrary to our expectations, a rearing temperature of up to 33°C might presumably be favourable for the aquaculture of H. scabra.

Hemagglutinins on the body surface of sea cucumber, Apostichopus japonicus

The body surface is an indispensable barrier in aquatic animals for protection against diseasecausing microorganisms. However, the defense molecules on the body surface of aquatic invertebrates remain largely unexplored. To examine lectin molecules on the body surface of the Japanese sea cucumber, Apostichopus japonicus, we scraped its integument using a surgical knife. The integument extract showed agglutinating activity against rabbit red blood cells. A 15-kDa protein was successfully isolated by affinity chromatography using a fixed erythrocyte membrane and an acidic buffer as eluant. N-terminal amino acid sequencing, followed by PCR using the degenerated primer based on the N-terminal sequence and RACE PCR, were performed. A fulllength cDNA of the 15-kDa protein contains 1438 bp, including 492 bp of the open reading frame, which encodes 163 amino acid residues. The translated sequence is consistent with that of SJL-1, a C-type lectin, found previously in the whole-body extract of A. japonicus, with the exception of two amino acid residues. Reverse transcription PCR demonstrated that the gene was transcribed in the intestine, respiratory trees, and tentacles, but not in the coelomocytes and body wall, including the body surface. Western blotting, using a specific antibody raised against recombinant SJL-1, showed that the protein exists in the body surface extract and body surface rinsing, but not in the coelomic fluid. These results suggest that SJL-1 was not produced in the integument but transported to the body surface from the synthesis site through an unknown pathway.

P44/42MAPK and p90RSK modulate thermal stressed physiology response in Apostichopus japonicus.

Reversible protein phosphorylation (RPP) plays a key role in signal transduction, enzyme activity and metabolic maintenance in response to extreme changes in the environment. In this study, we identified 55 phosphorylated proteins in Apostichopus japonicus coelomocytes by iTRAQ analysis, and sixteen proteins displayed differently expressed profiles between thermal stressed and control groups. Mitogen-activated protein kinase (MAPK) signaling cascade was further characterized by Western blot. Spatial expression analysis revealed that phospho-p44/42MAPK levels were mainly detected in coelomocytes and muscle, and no signal in intestine and respiratory trees, although the protein were constitutively expressed in all examined tissues. Time-course expression analysis shown that the amount of phospho-p44/42MAPK decreased by 57-60% in coelomocytes and 29-40% in muscle in acute exposure stage of 20°C and 25°C, respectively. In higher temperature remaining stage, phospho-p44/42MAPK was both induced with 1.7-fold increase at 20°C and 5.2-fold at 25°C at the first 12h in coelomocytes, which was consistent with the change of p44/42MAPK. Similarly, 2.5-fold and 2.7-fold increases were detected in muscle at corresponding exposure temperature. In contrast, the p44/42MAPK in muscle showed depressed expression profiles. As time progressed, phospho-p44/42MAPK levels were all decreased significantly both in coelomocytes and in muscles. A reduction in phosphorylation levels was detected at 84h in 20°C exposed coelomocytes with 0.42-fold decrease, and at 36h in 25°C challenged muscle by 0.80-fold decrease. Correlated expression profiles between phospho-p90RSK and phospho-p44/42MAPK suggest that p44/42MAPK may be involved in temperature-induced metabolic suppression through targeting p90RSK in A. japonicus.

Identification and functional characterization of TNF receptor associated factor 3 in the sea cucumber Apostichopus japonicus

TNF receptor associated factors (TRAFs) are a family of proteins primarily involved in both adaptive and innate immunity. In this study, we identified a novel TRAF3 gene in Apostichopus japonicus by transcriptome sequencing and RACE approaches (designated as AjTRAF3). The full-length of AjTRAF3 was of 2796 bp including a 5′ untranslated region (UTR) of 83 bp, a 3′ UTR of 1066 bp and a putative open reading frame of 1647 bp encoding a polypeptide of 548 amino acid residues. The representative domains such as a RING finger domain (residues 54–96), two TRAF domains with zinc finger structure (residues 141–228), a coiled coil and a meprin and TRAF homology (MATH) domain (residues 396–522) were all detected in the deduced amino acids of AjTRAF3. AjTRAF3 was ubiquitously expressed in all examined tissues with predominant expression in the body wall and slightly weaker in intestine, respiratory tree, tube feet, coelomocytes and longitudinal muscle. Time-course expression analysis in coelomocytes revealed that AjTRAF3 was significantly depressed towards Vibrio splendidus infection with a 0.20-fold decrease at 12 h, compared to control levels. AjTRAF3 silencing could elevate intracellular ROS levels by 2.08-fold and 2.09-fold compared to each control group in vitro and in vivo, respectively. Taken together, all these results suggested that AjTRAF3 may play a crucial role in the processes of anti-bacteria response in sea cucumber through regulating ROS production.

Heavy metal accumulation in tissues of two sea cucumbers, Holothuria leucospilota and Holothuria scabra in the northern part of Qeshm Island, Persian Gulf

The concentrations of some heavy metals (Cd, Cu, Zn andPb) were investigated in the sediments and the two species of sea cucumber (Holothuria leucospilota and Holothuria scabra) from northern part of Qeshm Island, Persian Gulf. The levels of Cu, Zn and Pb in sediment varied significantly among sampling sites (p<0.05). The highest levels of Zn and Pb in H. leucospilota were recorded in body wall whereas the highest contents of Zn and Pb in H. scabra were measured in respiratory tree organ, respectively. Cu and Cd were the most abundant elements in gonads of H. leucospilota while highest levels of Cd and Cu in H. scabra were measured in the gonad and derm, respectively. Cu and Zn concentrations were below permissible limits for human consumption while Cd and Pb were above permissible limits for human consumption.

Effects of Acute and Chronic Heavy Metal (Cu, Cd, and Zn) Exposure on Sea Cucumbers (Apostichopus japonicus).

Acute and chronic toxicity tests were conducted with sea cucumber (Apostichopus japonicus) exposed to heavy metals. Acute toxicity values (96 h LC50) were 2.697, 0.133, and 1.574 mg L−1 for Zn, Cu, and Cd, respectively, and were ranked in order of toxicity: Cu > Cd > Zn. Under chronic metal exposure the specific growth rates of sea cucumbers decreased with the increase of metal concentration for all the three metals. After acute metal exposure, the oxygen consumption rate (OCR) decreased. The OCRs in all groups were significantly different than control (P < 0.05) except in the group treated with 1.00 mg L−1 Zn (P < 0.05), where the increase of OCR was observed. The OCRs in groups chronically exposed to metals were significantly lower than that in the control group (P < 0.05). The activity of both pyruvate kinase (PK) and hexokinase (HK) in sea cucumbers followed: respiratory tree > muscle > intestine in natural sea water. After chronic Zn, Cu, and Cd exposure, the change pattern of HK and PK in respiratory tree, muscle, and intestine varied slightly. However, the activity of the enzyme showed a general trend of increase and then decrease and the higher the exposure concentration was, the earlier the highest point of enzyme activity was obtained.

Polycyclic aromatic hydrocarbon pollution affects tissue fatty acid compositions in Holothuria scabra (Jaeger, 1833): A sea cucumber from Qeshm Island, Persian Gulf, Iran

Event Abstract Back to Event Polycyclic aromatic hydrocarbon pollution affects tissue fatty acid compositions in Holothuria scabra (Jaeger, 1833): A sea cucumber from Qeshm Island, Persian Gulf, Iran Elham Kamyab1, 2*, Julian Döring3, Aida Khazaali2 and Andreas Kunzmann2 1 Oldenburg University, Institute for chemistary and marine biology (ICBM), Germany 2 Institute for marine tropical ecology (ZMT), Germany 3 Institute for marine tropical ecology, Germany Tropical sea cucumbers like Holothuria scabra are of increasing commercial importance because of their supposedly beneficial effects on the human health. They are sensitive organisms to any (micro) contaminant accumulated in marine sediments. In spite of their important role in the ecosystem as bioturbators, their physiological performance under anthropogenic environmental pollution is not sufficiently well studied. Polycyclic aromatic hydrocarbons (PAH) in the environment mainly originate from human activities and have negative impacts on the quality of sediments and therefore also on deposit feeders. The aim of the present study was to identify the impact of PAH on the essential fatty acid (EFA) compositions in different tissues (muscle, body wall, and respiratory tree) of H. scabra. EFAs such as Arachidonic acid (20:4(n-3)), Docosahexaenoic acid (22:6(n-3)), and Eicosapentaenoic acid (20:5(n-3)) play important roles in the metabolic pathways of echinoderms and are crucial during the overall physiological performance of a species. Therefore, specimens were sampled at two different sites on Qeshm Island, Iran. At one sampling site, elevated PAH levels could be detected, whereas no PAH pollution was traceable at the other. EFA compositions differed between different tissues within both sampled populations. Also, differences in EFA compositions were observed between tissues of specimens sampled at the polluted and at the not polluted sampling site. This may reveal direct effects of PAH pollution on the benthic primary producer community and/or on the metabolic pathways in H. scabra. Understanding the effects of anthropogenic pollutants on a key species’ EFA composition and thus on its metabolism is crucial to evaluate the impact of man-made pollution on marine ecosystems. Keywords: Fatty Acids, DHA, EPA, PAH, Iran, metabolic pathway, Anthropogenic pollution Conference: IMMR | International Meeting on Marine Research 2016, Peniche, Portugal, 14 Jul - 15 Jul, 2016. Presentation Type: Oral presentation Topic: Biodiversity, Conservation and Coastal Management Citation: Kamyab E, Döring J, Khazaali A and Kunzmann A (2016). Polycyclic aromatic hydrocarbon pollution affects tissue fatty acid compositions in Holothuria scabra (Jaeger, 1833): A sea cucumber from Qeshm Island, Persian Gulf, Iran. Front. Mar. Sci. Conference Abstract: IMMR | International Meeting on Marine Research 2016. doi: 10.3389/conf.FMARS.2016.04.00104 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 19 May 2016; Published Online: 13 Jul 2016. * Correspondence: Ms. Elham Kamyab, Oldenburg University, Institute for chemistary and marine biology (ICBM), Wilhemshaven, Germany, elham.kamyab@zmt-bremen.de Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Elham Kamyab Julian Döring Aida Khazaali Andreas Kunzmann Google Elham Kamyab Julian Döring Aida Khazaali Andreas Kunzmann Google Scholar Elham Kamyab Julian Döring Aida Khazaali Andreas Kunzmann PubMed Elham Kamyab Julian Döring Aida Khazaali Andreas Kunzmann Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Etiology and prevalence of skin disease in Holothuria arguinensis

Event Abstract Back to Event Etiology and prevalence of skin disease in Holothuria arguinensis Fernando G. Canovas1*, Jorge A. Domínguez-Godino1 and Mercedes González-Wangüemert1 1 Centro de Ciências do Mar do Algarve, Fisheries, Biodiversity and Conservation, Portugal Sea cucumbers is seriously over-exploited by fishery pressure, pointing at aquaculture as profitable alternative for a highly demanding market (González-Wangüemert et al. 2015). However, occurrence of diseases could cause serious economic losses and becoming a limiting factor for sustainability of this industry (Yang et al. 2015). Despite that important fact, any study has been performed upon identification of common pathologies and isolation of etiological agents in Atlanto-Mediterranean species, probably because aquaculture of sea cucumber in Europe is still at an early stage. Invertebrates medicine is not easy, but provides an opportunity to contribute to a new and emergent field of animal health (Lewbart 2012). This work addresses the study of etiopathological and epidemiological aspects of diseases, in Holothuria arguinensis. This sea cucumber is becoming target species for aquaculture in Europe (Dominguez-Godino et al., 2015). Sampling was carried out across Ria Formosa Natural Park, where previous information about density, habitat description, human pressures and presence of sick individuals have been reported by our team. This habitat can be considered a semi-closed system, which will allow to infer valuable information about prevalence and morbility of main diseases in wild landscapes with high human influences. Disections were performed and tissue samples were collected from integument, respiratory tree, Polian vesicule and gonads. Microorganisms were cultured in agarose media with sea water and incubated at 18ºC (in dark) until enough growth was noticeable (5-12 days). 16S rRNA gene were amplified using primers 27F/1492R (Muyzer and Marty 2014). Identification was assessed using a metagenomic workflow. Bacterial agents have been identified during outdoor cultivation and wild environment. Etiology of pathological manifestations in integument was associated to bacteria species of the family Bacillaceae, and fungi. In contrast, etiology of internal lesions was associated to species of the families Pseudoalteromonadaceae and Aeromonadaceae. Mortality was described higher in diseases with integumet manifestations (Yang et al. 2015). In contrast, morbility should be higher in diseases with internal pathology, by causing insidious clinical signs (lost of weigth, deformations, atrophia). Acknowledgements This research was supported by CUMARSUR project (ref. PTDC/MAR-BIO/5948/2014) funded by Fundacão para Ciência e Tecnologia (FCT, Portugal). M. González-Wangüemert was funded by FCT Investigator Programme-Career Development (ref. IF/00998/2014).

Expression analysis of microRNAs related to the skin ulceration syndrome of sea cucumber Apostichopus japonicus.

MicroRNAs (miRNAs) are small non-coding RNAs that are involved in many biological processes. To investigate the miRNAs related to skin ulceration syndrome (SUS) of Apostichopus japonicus, small RNA libraries of body wall, intestine, respiratory tree and coelomocytes from healthy and diseased A.japonicus were sequenced on Illumina Hiseq 2000 platform. A total of 247 conserved and 10 novel miRNAs were identified across all libraries. After pair-wise comparisons, 215 miRNAs in body wall, 36 in intestine, 2 in respiratory tree and 38 in coelomocytes showed significant expression differences. Further analyses were conducted on some tissue-specific differentially expressed miRNAs: miR-8 and miR-486-5p in body wall, miR-200-3p, let-7-5p and miR-125 in intestine, miR-278a-3p and bantam in respiratory, miR-10a and miR-184 in coelomocytes. Notably, these miRNAs in some species were reported to function in various physiological or pathological processes associated with immune regulations. Using stem-loop quantitative real time PCR, six representative miRNAs in four tissues were selected to validate the sequencing results. The Pearson's correlation coefficient (R) of the six miRNAs ranged from 0.777 to 0.948, which confirmed the consistency and accuracy between these two approaches. This study provides comprehensive expression and regulation patterns of functional miRNAs in different tissues and gives insights into the tissue-specific immune response mechanisms in SUS-infected A.japonicus.

The Role of the Paratrigeminal Nucleus in Vagal Afferent Evoked Respiratory Reflexes: A Neuroanatomical and Functional Study in Guinea Pigs.

The respiratory tree receives sensory innervation from the jugular and nodose vagal sensory ganglia. Neurons of these ganglia are derived from embryologically distinct origins and as such demonstrate differing molecular, neurochemical and physiological phenotypes. Furthermore, whereas nodose afferent neurons project to the nucleus of the solitary tract (nTS), recent neuroanatomical studies in rats suggest that jugular neurons have their central terminations in the paratrigeminal nucleus (Pa5). In the present study we confirm that guinea pigs demonstrate a comparable distinction between the brainstem terminations of nodose and jugular ganglia afferents. Thus, microinjection of fluorescently conjugated cholera toxin B (CT-B) neural tracers into the caudal nTS and Pa5 resulted in highly specific retrograde labeling of neurons in the nodose and jugular ganglia, respectively. Whereas, nodose neurons more often expressed 160 KD neurofilament proteins and the alpha3 subunit of Na+/K+ ATPase, significantly more jugular neurons expressed the neuropeptides substance P (SP) and, especially, Calcitonin Gene-Related Peptide (CGRP). Indeed, terminal fibers in the Pa5 compared to the nTS were characterized by their significantly greater expression of CGRP, further supporting the notion that jugular afferents project to trigeminal-related brainstem regions. Electrical stimulation of the guinea pig larynx following selective surgical denervation of the nodose afferent innervation to the larynx (leaving intact the jugular innervation) resulted in stimulus dependent respiratory slowing and eventual apnea. This jugular ganglia neuron mediated response was unaffected by bilateral microinjections of the GABAA agonist muscimol into the nTS, but was abolished by muscimol injected into the Pa5. Taken together these data confirm that jugular and nodose vagal ganglia afferent neurons innervate distinct central circuits and support the notion that multiple peripheral and central pathways mediate sensory responses associated with airway irritations.

An adenine nucleotide translocase (ANT) gene from Apostichopus japonicus; molecular cloning and expression analysis in response to lipopolysaccharide (LPS) challenge and thermal stress.

The adenine nucleotide translocases (ANTs) play a vital role in energy metabolism via ADP/ATP exchange in eukaryotic cells. Apostichopus japonicus (Echinodermata: Holothuroidea) is an important economic species in China. Here, a cDNA representing an ANT gene of A. japonicus was isolated and characterized from respiratory tree and named AjANT. The full-length AjANT cDNA is 1924bp, including a 5'-untranslated region (UTR) of 38bp, 3'-UTR of 980bp and an open reading frame (ORF) of 906bp encoding a polypeptide of 301 amino acids. The protein contains three homologous repeat Mito_carr domains (Pfam00153). The deduced AjANT protein sequence has 49-81% in comparison to ANT proteins from other individuals. The predicted tertiary structure of AjANT protein is highly similar to animal ANT proteins. Phylogenetic analysis showed that the AjANT is closely related to Holothuroidea ANT genes. Real-time quantitative reverse transcription-PCR (qPCR) analysis showed that AjANT expression is higher in the respiratory tree than in other examined tissues. After thermal stress or LPS challenge, expression of AjANT was significantly fluctuant compared to the control. These results suggested that changes in the expression of ANT gene might be involved in immune defense and in protecting A.japonicus against thermal stress.

Characterization and expression analysis of a caspase-2 in an invertebrate echinoderm sea cumber Apostichopus japonicus.

Caspase-2 is the most evolutionarily conserved member of the caspase family which mediates the programmed cell death and plays crucial roles in key cellular processes. In this study, a caspase-2 homolog was identified and functionally characterized in sea cucumber Apostichopus japonicus, which we named AjCASP. The full-length cDNA consists of 2100 bp with an ORF encoding a protein of 378 amino acids. The deduced amino acid sequence shows that AjCASP consists of a conserved CARD-CASP2 domain and a CASs domain containing two active residues, two proteolytic cleavage residues, a substrate pocket and a dimer interface as well. In addition, a p20 large subunit with a characteristic five-peptide motif (QACRG) and a p10 small subunit in C-terminal were identified in CASs domain. Above data demonstrated that AjCASP is similar to CED-3 (the caspase-2 homolog of nematode Caenorhabditis elegans), which is further confirmed by phylogenetic tree analysis. AjCASP was ubiquitously expressed in sea cucumber and the obviously higher expression level was observed in coelomocyte, respiratory tree and intestine. Real-time PCR analyses further demonstrated that AjCASP was significantly induced by LPS. Taken together, these results strongly suggest that AjCASP is a caspase-2 homolog and it may be involved in invertebrate immune response, especially in eliminating and degrading invading pathogens.

Protective effects of chicken egg yolk antibody (IgY) against experimental Vibrio splendidus infection in the sea cucumber (Apostichopus japonicus).

Vibrio splendidus is one of the most harmful pathogens associated with skin ulceration syndrome in the sea cucumber (Apostichopus japonicus) due to its high virulence and frequency of appearance. The objective of this study was to determine the effectiveness of chicken egg yolk antibody (IgY) against V.splendidus infection in the sea cucumber. Whole V.splendidus cells were used as an immunogen to immunize 20 White Leghorn hens (25 weeks old). IgY was produced from egg yolks obtained from these immunized hens using water dilution, two-step salt precipitation and ultrafiltration. The purity of the IgY produced was approximately 83%. Enzyme-linked Immunosorbent Assay indicated a high specificity for IgY with a maximum antibody titer of 320,000. The growth of V.splendidus in liquid medium was significantly inhibited by IgY in a dose-dependent manner at concentrations ranging from 1 to 10mg/mL. The protective effects of IgY were evaluated in sea cucumber by intraperitoneally injecting anti-V.splendidus IgY antibodies (10mg/mL) or immersing the sea cucumber in aqueous IgY (1g/L) after an intraperitoneal injection of V.splendidus. Intraperitoneal injection resulted in an 80% survival while immersion resulted in a 75% survival during the 11-day experimental period. The survival rates were significantly higher than the positive control and the non-specific IgY group (P<0.05). As well, the bacterial burden in the respiratory tree, intestine and coelomic liquid was significantly (P<0.05) lower in sea cucumber treated with specific IgY than those treated with non-specific IgY. The phagocytosis of coelomocytes for V.splendidus in the presence of specific IgY was significantly (P<0.05) higher than that obtained with non-specific IgY or without IgY, suggesting that specific IgY enhanced phagocytic activity. The current work suggests that specific IgY has potential for protecting sea cucumbers against V.splendidus infection.

Cleft Palate, Moderate Lung Developmental Retardation and Early Postnatal Lethality in Mice Deficient in the Kir7.1 Inwardly Rectifying K+ Channel.

Kir7.1 is an inwardly rectifying K+ channel of the Kir superfamily encoded by the kcnj13 gene. Kir7.1 is present in epithelial tissues where it colocalizes with the Na+/K+-pump probably serving to recycle K+ taken up by the pump. Human mutations affecting Kir7.1 are associated with retinal degeneration diseases. We generated a mouse lacking Kir7.1 by ablation of the Kcnj13 gene. Homozygous mutant null mice die hours after birth and show cleft palate and moderate retardation in lung development. Kir7.1 is expressed in the epithelium covering the palatal processes at the time at which palate sealing takes place and our results suggest it might play an essential role in late palatogenesis. Our work also reveals a second unexpected role in the development and the physiology of the respiratory system, where Kir7.1 is expressed in epithelial cells all along the respiratory tree.