Modulating responses of indicator genes in cellular homeostasis, immune defense and apoptotic process in the Macrophthalmus japonicus exposed to di(2-ethylhexyl) phthalate as a plastic additive.

Di(2-ethylhexyl) phthalate (DEHP), have been increasingly used as plasticizers to manufacture soft and flexible materials and ubiquitously found in water and sediments in the aquatic ecosystem. The aim of the present study was to evaluate the effect of DEHP exposure on cellular homeostasis (HSF1 and seven HSPs), immune responses (ILF), and apoptotic responses (p53, BAX, Bcl-2). DEHP exposure upregulated the expression of HSF1 and ILF. Moreover, it altered the expression levels of HSPs (upregulation of HSP70, HSP90, HSP40, HSP83, and HSP67B2 and downregulation of HSP60 and HSP21) in conjunction with HSF1 and ILF in the gills and hepatopancreas of M. japonicus exposed to DEHP. At the protein level, DEHP exposure changed apoptotic signals in both tissues of M. japonicus. These findings indicate that chronic exposures to several DEHP concentrations could disturb cellular balance, damage the inflammatory and immune systems, and induce apoptotic cell death, thereby affecting the survival of M. japonicus.

Morphological and genetic analysis for the diversity conservation of rare species, Thamnaconus multilineatus (Tetraodontiformes: Monacanthidae).

Climate changes have altered biodiversity and ultimately induced community changes that have threatened the survival of certain aquatic organisms such as fish species. Obtaining biological and genetic information on endangered fish species is critical for ecological population management. Thamnaconus multilineatus, registered as an endangered species by the IUCN in 2019, is a Data Deficient (DD) species with a remarkably small number of habitats worldwide and no known information other than its habitat and external form. In this study, we characterized the external and osteological morphology of a T. multilineatus specimen collected from eastern Jeju Island, South Korea, in 2020. We also investigated the phylogenetic relationships among related fish species through complete mitochondrial DNA (mtDNA) analysis of the T. multilineatus specimen. The external and skeletal characteristics of T. multilineatus were similar to those of previous reports describing other fish of the genus Thamnaconus, making it difficult to classify T. multilineatus as a similar species based only on morphological characteristics. As a result of analyzing the complete mtDNA of T. multilineatus, the length of the mtDNA was determined to be 16,435 bp, and the mitochondrial genome was found to have 37 CDCs, including 13 PCGs, 22 tRNAs, and 2 rRNAs. In the phylogenetic analysis within the suborder Balistoidei, T. multilineatus mtDNA formed a cluster with fish of the genus Thamnaconus. This study is the first to report on the skeletal structure and complete mtDNA of T. multilineatus. Since the current research on T. multilineatus has only been reported on morphology, the results of this study will be utilized as important information for the management and restoration of T. multilineatus as an endangered species and significant fishery resource.

Responses of multifunctional immune complement component 1q (C1q) and apoptosis-related genes in Macrophthalmus japonicus tissues and human cells following exposure to environmental pollutants.

Apoptosis is a key defense process for multiple immune system functions, playing a central role in maintaining homeostasis and cell development. The purpose of this study was to evaluate the effects of environmental pollutant exposure on immune-related apoptotic pathways in crab tissues and human cells. To do this, we characterized the multifunctional immune complement component 1q (C1q) gene and analyzed C1q expression in Macrophthalmus japonicus crabs after exposure to di(2-ethylhexyl) phthalate (DEHP) or hexabromocyclododecanes (HBCDs). Moreover, the responses of apoptotic signal-related genes were observed in M. japonicus tissues and human cell lines (HEK293T and HCT116). C1q gene expression was downregulated in the gills and hepatopancreas of M. japonicus after exposure to DEHP or HBCD. Pollutant exposure also increased antioxidant enzyme activities and altered transcription of 15 apoptotic signaling genes in M. japonicus. However, patterns in apoptotic signaling in response to these pollutants differed in human cells. HBCD exposure generated an apoptotic signal (cleaved caspase-3) and inhibited cell growth in both cell lines, whereas DEHP exposure did not produce such a response. These results suggest that exposure to environmental pollutants induced different levels of immune-related apoptosis depending on the cell or tissue type and that this induction of apoptotic signaling may trigger an initiation of carcinogenesis in M. japonicus and in humans as consumers.

Growth retardation and suppression of ubiquitin-dependent catabolic processes in the brackish water clam Corbicula japonica in response to salinity changes and bioaccumulation of toxic heavy metals.

The brackish water clam (Corbicula japonica) is constantly exposed to stressful salinity gradients and high levels of heavy metals in the freshwater-saltwater interface of estuary environments, which are introduced from upstream regions and land. To identify the key molecular pathways involved in the response to salinity changes and heavy metal bioaccumulation, we obtained the transcriptomes of C. japonica inhabiting different salinities and heavy metal distributions in Gwangyang Bay (Korea) using RNA sequencing. Among a total of 404,486 assembled unigenes, 5534 differentially expressed genes were identified in C. japonica inhabiting different conditions, 1549 of which were significantly upregulated and 1355 were significantly downregulated. Correlation analyses revealed distinct gene expression patterns between the low and high conditions of salinity and heavy metal bioaccumulation. Functional annotation revealed significant downregulation of genes involved in "ubiquitin-dependent protein catabolic process," "tricarboxylic acid cycle," and "intracellular protein transport" in C. japonica from the high condition compared to the low condition. Transcription and translation pathways were significantly enriched in the high condition. Additionally, upon comparison of the low and high conditions by qRT-PCR and proteasome enzyme activity analyses, our findings demonstrated that environmental stress could suppress the ubiquitin-proteasome complex (UPC). Additionally, transcriptomic changes under high salinity stress conditions may be related to an increase in cellular protection by defense enzymes, which leads to more energy being required and a disruption of energy homeostasis. Ultimately, this could cause growth retardation in the clam C. japonica. In summary, this study provides the first evidence of UPC suppression induced by a combination of high salinity and heavy metal bioaccumulation stress in C. japonica, which could compromise the survival and growth of estuarine bivalves.

Surveillance spilled Chironomidae (Diptera) larvae from drinking water treatment plants in South Korea using morphogenetic species analysis and eDNA metabarcoding.

Chironomid larvae (Diptera: Chironomidae) are tremendous indicator species that can tolerate a broad range of environmental conditions, from polluted to unimpaired water ecosystems. These species are ubiquitously observed in all bioregions and can even be found in drinking water treatment plants (DWTPs). Detection of chironomid larvae in DWTPs is a critical issue because their presence may be indicative of the water quality in the supply of tap water for human consumption. Therefore, the aim of the present study was to identify the chironomid communities that reflect the water quality of DWTPs and develop a biomonitoring tool to detect biological contamination of the chironomids in DWTPs. To do so, we investigated the identity and distribution of chironomid larvae in seven DWTP areas using morphological identification, DNA barcoding, and sediment environmental DNA (eDNA) analysis. A total of 7924 chironomid individuals encompassing three subfamilies and 25 species of 19 genera were identified in 33 sites within the DWTPs. The Gongchon and Bupyeong DWTPs were dominated by Chironomus spp. larvae, which were correlated with low levels of dissolved oxygen in the water. In the Samgye DWTP and Hwajeong DWTP, Chironomus spp. were almost absent, and instead, Tanytarsus spp. were abundant. Additionally, the Gangjeong DWTP was dominated by a Microtendipes sp., and two species of Orthocladiinae (a Parametriocnemus sp. and a Paratrichocladius sp.) were found only in the Jeju DWTP. We also identified the eight most abundant Chironomidae larvae found in the DWTPs. Furthermore, eDNA metabarcoding of DWTP sediment indicated the presence of different eukaryotic fauna and confirmed the presence of chironomids in DWTPs. These data provide useful morphological and genetic information regarding chironomid larvae that can be used for the water quality biomonitoring of DWTPs to support the supply of clean drinking water.

A Review on the Involvement of Heat Shock Proteins (Extrinsic Chaperones) in Response to Stress Conditions in Aquatic Organisms.

Heat shock proteins (HSPs) encompass both extrinsic chaperones and stress proteins. These proteins, with molecular weights ranging from 14 to 120 kDa, are conserved across all living organisms and are expressed in response to stress. The upregulation of specific genes triggers the synthesis of HSPs, facilitated by the interaction between heat shock factors and gene promoter regions. Notably, HSPs function as chaperones or helper molecules in various cellular processes involving lipids and proteins, and their upregulation is not limited to heat-induced stress but also occurs in response to anoxia, acidosis, hypoxia, toxins, ischemia, protein breakdown, and microbial infection. HSPs play a vital role in regulating protein synthesis in cells. They assist in the folding and assembly of other cellular proteins, primarily through HSP families such as HSP70 and HSP90. Additionally, the process of the folding, translocation, and aggregation of proteins is governed by the dynamic partitioning facilitated by HSPs throughout the cell. Beyond their involvement in protein metabolism, HSPs also exert a significant influence on apoptosis, the immune system, and various characteristics of inflammation. The immunity of aquatic organisms, including shrimp, fish, and shellfish, relies heavily on the development of inflammation, as well as non-specific and specific immune responses to viral and bacterial infections. Recent advancements in aquatic research have demonstrated that the HSP levels in populations of fish, shrimp, and shellfish can be increased through non-traumatic means such as water or oral administration of HSP stimulants, exogenous HSPs, and heat induction. These methods have proven useful in reducing physical stress and trauma, while also facilitating sustainable husbandry practices such as vaccination and transportation, thereby offering health benefits. Hence, the present review discusses the importance of HSPs in different tissues in aquatic organisms (fish, shrimp), and their expression levels during pathogen invasion; this gives new insights into the significance of HSPs in invertebrates.

Exploring the Antimicrobial Potential and Biofilm Inhibitory Properties of Hemocyanin from Hemifusus pugilinus (Born, 1778).

The seafood industry plays a huge role in the blue economy, exploiting the advantage of the enriched protein content of marine organisms such as shrimps and molluscs, which are cultured in aquafarms. Diseases greatly affect these aquatic organisms in culture and, hence, there is need to study, in detail, their innate immune mechanisms. Hemocyanin is a non-specific innate defense molecule present in the blood cells of several invertebrates, especially molluscs, arthropods, and annelids. It is concerned with oxygen transport, blood clotting, and immune enhancement. In the present study, this macromolecular metalloprotein was isolated from the hemolymph of the marine snail Hemifusus pugilinus (Born, 1778) using Sephadex G-100 gel filtration column chromatography. It occurred as a single band (MW 80 kDa) on SDS-PAGE. High-performance liquid chromatography (HPLC) of the purified hemocyanin showed a single peak with a retention time of 4.3 min. The secondary structure and stability of the protein were detected using circular dichroism (CD), and the spectra demonstrated negative ellipticity bands close to 208 nm and 225 nm, indicating ß-sheets. Further exploration of the purified hemocyanin revealed remarkable antimicrobial and antibiofilm activities against Gram-positive (Enterococcus faecalis and Staphylococcus aureus) and Gram-negative bacteria (Pseudomonas aeruginosa and Proteus vulgaris) at a concentration of 1-5 µg/mL. Spectrophotometric and in situ microscopic analyses (CLSM) unveiled the potential of the purified hemocyanin to inhibit biofilm formation in these bacteria with a minimal inhibitory concentration of 40 µg/mL. Furthermore, H. pugilinus hemocyanin (10 µg/mL concentration) displayed antifungal activity against Aspergillus niger. The purified hemocyanin was also assessed for cytotoxicity against human cancer cells using cell viability assays. Altogether, the present study shows that molluscan hemocyanin is a potential antimicrobial, antibiofilm, antifungal, anticancer, and immunomodulatory agent, with great scope for application in the enhancement of the immune system of molluscs, thereby facilitating their aquaculture.

Transcriptional Responses of Stress-Related Genes in Pale Chub (Zacco platypus) Inhabiting Different Aquatic Environments: Application for Biomonitoring Aquatic Ecosystems.

Pale chub (Zacco platypus) is a dominant species in urban rivers and reservoirs, and it is used as an indicator to monitor the effects of environmental contaminants. Gene responses at the molecular level can reflect the health of fish challenged with environmental stressors. The objective of this study was to identify correlations between water quality factors and the expression of stress-related genes in Z. platypus from different lake environments (Singal and Juam Lakes). To do so, transcriptional responses of genes involving cellular homeostasis (heat-shock protein 70, HSP70; heat-shock protein 90, HSP90), metal detoxification (metallothionein, MT), and antioxidation (superoxide dismutase, SOD; catalase, CAT) were analyzed in the gill and liver tissues of Z. platypus. HSP70, HSP90, and MT genes were overall upregulated in Z. platypus from Singal Lake, which suffered from poorer water quality than Juam Lake. In addition, gene responses were significantly higher in Singal Lake outflow. Upregulation of HSP70, HSP90, and MT was significantly higher in Z. platypus gills than in the liver tissue. In addition, integrated biomarker response and heatmap analysis determined correlations between expression of biomarker genes or water quality factors and sampling sites of both lakes. These results suggest that stress-related genes used as multiple biomarkers may reflect spatial characteristics and water quality of different lake environments, and they can be used for biomonitoring and ecological risk assessment.

Complete mitochondrial genome sequence of a brackish water clam Corbicula japonica (Cyrenidae: Bivalvia) collected from an estuary of Gwangyang Bay in Korea.

The complete mitochondrial genome of the clam Corbicula japonica is 17,432 bp in length. The sequence consists of 13 protein-coding, 2 ribosomal RNAs, and 22 transfer RNA genes (GenBank accession no. MZ895053). The proportion of base-pairs in C. japonica are A + T (70.5%) and G + C (29.5%). Phylogenetic analysis reveal C. japonica to be sister species to C. fluminea within the monophyletic genus Corbicula, with high support. This study is helpful to the classification of the brackish water clam C. japonica, which is difficult to identify during early development owing to variation of shell morphology.

Environmental co-exposure of high temperature and Cu induce hormonal disturbance of cortisol signaling and altered responses of cellular defense genes in zebrafish.

Global warming is causing a continuous increase in environmental temperatures, which simultaneously activates toxic environmental stresses, such as heavy metal exposure, in aquatic ecosystems. The present study aimed at evaluating the effects of Cu toxicity along with increased temperature during zebrafish embryogenesis. Decreased survival rates were observed following combined exposure to high temperature and Cu. Heart rates of zebrafish embryos were significantly increased only during heat stress. An abnormal morphology with curved body shape was induced by exposure to a combination of Cu and heat stress. Furthermore, heat stress also triggered Cu-induced intracellular reactive oxygen species (ROS) production, with upregulation of superoxide dismutase (SOD) and glutathione s-transferase (GST) expression, and cell death with modified expression of p53 and B-cell lymphoma-2 (Bcl-2) in zebrafish embryos. Finally, increased cortisol levels and altered expression of cortisol-signaling genes were observed following exposure to Cu and high temperatures. These results highlight that realistic exposure to combined stressors induces developmental disturbances via stress-induced responses involving oxidative stress and cell death as well as transcriptional alterations leading to cortisol signaling in fish.

Apoptotic p53 Gene Expression in the Regulation of Persistent Organic Pollutant (POP)-Induced Oxidative Stress in the Intertidal Crab Macrophthalmusjaponicus.

Persistent organic pollutants (POPs), some of the most dangerous chemicals released into the aquatic environment, are distributed worldwide due to their environmental persistence and bioaccumulation. In the study, we investigated p53-related apoptotic responses to POPs such as hexabromocyclododecanes (HBCDs) or 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) in the mud crab Macrophthalmus japonicus. To do so, we characterized M. japonicus p53 and evaluated basal levels of p53 expression in different tissues. M. japonicus p53 has conserved amino acid residues involving sites for protein dimerization and DNA and zinc binding. In phylogenetic analysis, the homology of the deduced p53 amino acid sequence was not high (67−70%) among crabs, although M. japonicus p53 formed a cluster with one clade with p53 homologs from other crabs. Tissue distribution patterns revealed that the highest expression of p53 mRNA transcripts was in the hepatopancreas of M. japonicus crabs. Exposure to POPs induced antioxidant defenses to modulate oxidative stress through the upregulation of catalase expression. Furthermore, p53 expression was generally upregulated in the hepatopancreas and gills of M. japonicus after exposure to most concentrations of HBCD or BDE-47 for all exposure periods. In hepatopancreas tissue, significant increases in p53 transcript levels were observed as long-lasting apoptotic responses involving cellular defenses until day 7 of relative long-term exposure. The findings in this study suggest that exposure to POPs such as HBCD or BDE-47 may trigger the induction of cellular defense processes against oxidative stress, including DNA repair, cell cycle arrest, and apoptosis through the transcriptional upregulation of p53 expression in M. japonicus.

Integrated analysis of exoskeletal surface profile and chitin-related gene expression on Macrophthalmus japonicus mud crabs exposed to hexabromocyclododecane.

Hexabromocyclododecanes (HBCDs), widely used brominated flame retardants, easily accumulate in aquatic organisms such as Macrophthalmus japonicus crabs, which inhabit tidal flat sediments. To analyze the effects of HBCD exposure in chitin-formed exoskeleton, we investigated molecular responses of chitin-related genes as well as physical changes of the exoskeletal surface form as a new biological end-point on M. japonicus. The expression patterns of chitin biosynthesis-, modification-, and degradation-related genes in the gills and hepatopancreases of M. japonicus were also analyzed. Additionally, the survivability and exoskeleton surface profiles of M. japonicus crabs were evaluated. M. japonicus chitin synthase expression was significantly downregulated, whereas that of the chitinase transcript was significantly upregulated upon exposure to all HBCD concentrations on day 7. Contrastingly, the gene expression of chitin deacetylase 1 significantly increased upon exposure to all HBCD concentrations on day 1, and this increase was significantly elevated on day 4. The expression of chitin deacetylase 1 was dose-dependent. Additionally, decreased survival and exoskeleton surface profile changes were observed in M. japonicus crabs exposed to all HBCD concentrations. These results suggest that exposure to HBCD induces changes in the synthesis, modification, and degradation of chitin, a pivotal component of the cuticular exoskeleton, and may disrupt the exoskeletal surface structure in M. japonicus crabs.

Multi-Level Gene Expression in Response to Environmental Stress in Aquatic Invertebrate Chironomids: Potential Applications in Water Quality Monitoring.

In freshwater ecosystems, aquatic invertebrates are influenced continuously by both physical stress and xenobiotics. Chironomids (Diptera; Chironomidae), or non-biting midges, are the most diverse and abundant invertebrates in freshwater habitats. They are a fundamental link in food chains of aquatic ecosystems. Chironomid larvae tolerate stress factors in their environments via various physiological processes. At the molecular level, environmental pollutants induce multi-level gene responses in Chironomus that regulate cellular protection through the activation of defense processes. This paper reviews literature on the transcriptional responses of biomarker genes to environmental stress in chironomids at the molecular level, in studies conducted from 1991 to 2020 (120 selected literatures of 374 results with the keywords "Chironomus and gene expression" by PubMed search tool). According to these studies, transcriptional responses in chironomids vary depending on the type of stress factor and defensive responses associated with antioxidant activity, the endocrine system, detoxification, homeostasis and stress response, energy metabolism, ribosomal machinery, apoptosis, DNA repair, and epigenetics. These data could provide a comprehensive overview of how Chironomus species respond to pollutants in aquatic environments. Furthermore, the transcriptomic data could facilitate the development of genetic tools for water quality and environmental monitoring based on resident chironomid species.

Differential Expression of the Apolipoprotein AI Gene in Spotnape Ponyfish (Nuchequula nuchalis) Inhabiting Different Salinity Ranges at the Top of the Estuary and in the Deep-Bay Area of Gwangyang Bay, South Korea.

Spotnape ponyfish (Nuchequula nuchalis) is a dominant species that is broadly distributed from estuarine to deep-bay areas, reflecting a euryhaline habitat. Apolipoprotein AI (ApoAI) is a main component of plasma lipoproteins and has crucial roles in lipid metabolism and the defense immune system. In this study, we characterized the N. nuchalis ApoAI gene and analyzed the expression of the ApoAI transcript in N. nuchalis collected at various sites in the estuary and the deep-bay area which have different salinities. Owing to the fish's mobility, we conducted stable isotope analyses to confirm the habitat characteristics of N. nuchalis. Carbon and nitrogen isotope ratios (δ13C and δ15N) from N. nuchalis indicated different feeding sources and trophic levels in the estuarine and deep-bay habitats. The characterized N. nuchalis ApoAI displayed residual repeats that formed a pair of alpha helices, indicating that the protein belongs to the apolipoprotein family. In the phylogenetic analysis, there was no sister group of N. nuchalis ApoAI among the large clades of fish species. The transcriptional expression level of ApoAI was higher in N. nuchalis inhabiting the deep-bay area with a high salinity (over 31 psu) than in N. nuchalis inhabiting the top of the estuary with a low salinity (6~15 psu). In addition, the expression patterns of N. nuchalis ApoAI were positively correlated with environmental factors (transparency, pH, TC, and TIC) in the high salinity area. These results suggest that ApoAI gene expression can reflect habitat characteristics of N. nuchalis which traverse broad salinity ranges and is associated with functional roles of osmoregulation and lipid metabolism for fish growth and development.

Complete mitochondrial genome of Chironomus flaviplumus (Diptera: Chironomidae) collected in Korea.

The complete mitochondrial genome of Chironomus flaviplumus was sequenced. The circular mitochondrial genome is 15,739 bp and consists of 13 protein-coding, two ribosomal RNAs, and 22 transfer RNA genes (GenBank accession no. MW770891). Results of phylogenetic analysis indicate that the species clustered with other species of the family Chironomidae. This study is helpful to the identification of C. flaviplumus larvae, which is difficult to be identified by morphology.

Purification and partial characterization of carbohydrate-recognition protein C-type lectin from Hemifusus pugilinus.

A mannose binding lectin (C-type lectin) was detected in a molluscan snail Hemifusus pugilinus, this lectin molecule was isolated and purified from the plasma using mannose-fixed sepharose CL-4B column affinity chromatography. The purified protein corresponds to the molecular weight of 118 kDa on an SDS-PAGE gel. The divalent cation-dependent nature of the H. pugilinus lectin (Hp-Lec) evidenced through pH and thermal stability analysis using Circular Dichroism (CD) and Surface Plasmon Resonance (SPR) respectively. Functional investigations of the Hp-Lec reveal a broad spectrum of bacterial agglutination activity against wide range of Gram-positive and Gram-negative bacterial strains. Furthermore, Hp-Lec displayed the haemo agglutination activity against vertebrate red blood cells (RBCs) and its titers were recorded. Excitingly, microbial virulent pathogens such as fungal strains tested against the purified Hp-Lec (25 and 50 µg/ml), which exhibits the effective antifungal activity against tested fungal pathogens such as Aspergillus niger and A. flavus.

Expression Levels of the Immune-Related p38 Mitogen-Activated Protein Kinase Transcript in Response to Environmental Pollutants on Macrophthalmus japonicus Crab.

Environmental pollution in the aquatic environment poses a threat to the immune system of benthic organisms. The Macrophthalmus japonicus crab, which inhabits tidal flat sediments, is a marine invertebrate that provides nutrient and organic matter cycling as a means of purification. Here, we characterized the M. japonicus p38 mitogen-activated protein kinase (MAPK) gene, which plays key roles in the regulation of cellular immune and apoptosis responses. M. japonicusp38 MAPK displayed the characteristics of the conserved MAPK family with Thr-Gly-Tyr (TGY) motif and substrate-binding site Ala-Thr-Arg-Trp (ATRW). The amino acid sequence of the M. japonicus p38 MAPK showed a close phylogenetic relationship to Eriocheir sinensis MAPK14 and Scylla paramamosainp38 MAPK. The phylogenetic tree displayed two origins of p38 MAPK: crustacean and insect. The tissue distribution patterns showed the highest expression in the gills and hepatopancreas of M. japonicus crab. In addition, p38 MAPK expression in M. japonicus gills and hepatopancreas was evaluated after exposure to environmental pollutants such as perfluorooctane sulfonate (PFOS), irgarol, di(2-ethylhexyl) phthalate (DEHP), and bisphenol A (BPA). In the gills, p38 MAPK expression significantly increased after exposure to all concentrations of the chemicals on day 7. However, on day 1, there were increased p38 MAPK responses observed after PFOS and irgarol exposure, whereas decreased p38 MAPK responses were observed after DEHP and BPA exposure. The upregulation of p38 MAPK gene also significantly led to M. japonicus hepatopancreas being undertested in all environmental pollutants. The findings in this study supported that anti-stress responses against exposure to environmental pollutants were reflected in changes in expression levels in M. japonicusp38 MAPK signaling regulation as a cellular defense mechanism.

Effects of thermal stress-induced lead (Pb) toxicity on apoptotic cell death, inflammatory response, oxidative defense, and DNA methylation in zebrafish (Danio rerio) embryos.

Lead (Pb) is a toxic environmental pollutant that is frequently present in effluents from urban, mining, and industrial sources. The combinatorial effects of heavy metal exposure and temperature in aquatic organisms have received considerable attention as heat stress occurs simultaneously in conjunction with several contaminants in a natural environment. In this study, we examined the potential effects of Pb exposure in conditions of thermal stress (34 °C) in zebrafish (Danio rerio) embryos. Thermal stress at 34 °C induced a dramatic decrease in the survival rate, although exposure to Pb at 26 °C decreased the survival rate of the embryos. Malformations, such as the curved body shape, were increased in response to exposure to a combination of Pb and heat stress. The combination of Pb and heat stress also caused a decrease in the heart rate. Moreover, Pb and high-temperature exposure induced the upregulation of SOD, CAT, TNF-α, IL-1ß, p53, and BAX transcripts, and downregulation of Dnmt1 and Dnmt3b transcripts. Thermal stress enhanced transcriptional responses of eight indicator genes following Pb toxicity. The induction of cell death in response to combined exposures was also confirmed in the body of zebrafish by fluorescence intensity image analysis. These data indicated that thermal stress enhanced the poisonous effects of Pb exposure on antioxidant defense, inflammation, and apoptotic mechanisms. Transcriptional inhibition of DNA methylation-related genes might serve as a crucial factor contributing to the possibility of epigenetic adaptation by altering combined stress. We suggest that a careful evaluation of the potential effects of climate change (especially temperature) should be considered when investigating the toxic levels of metal pollution, such as Pb, in an aquatic environment.

First Gut Content Analysis of 4th Instar Midge Larvae (Diptera: Chronomidae) In Large-Scale Weirs Using a DNA Meta-Barcoding Approach.

Chironomidae larvae play an important role in the food chain of river ecosystems in Korea, where it is dominant. However, detailed information on the diet of Chironomidae larvae are still lacking. The purpose of this study was to identify the gut contents of 4th instar larvae of a Chironomidae inhabiting four large-scale weirs (Sejong Weir, Juksan Weir, Gangjeong-Goryeong Weir, and Dalseong Weir) using a DNA meta-barcoding approach. We found that dominant Operational Taxonomic Unit (OUT) was assigned to Paractinolaimus sp. (Nematoda), and the sub-dominant OTU was assigned to Dicrotendipes fumidus (Chironomidae). The most common OTUs among the individuals included phytoplankton, such as Tetrahymena sp., D. armatus, Pseudopediastrum sp., Tetradesmus dimorphus, Biddulphia tridens, and Desmodesmus spp. We calculated the selectivity index (E') and provided scientific evidence that Chironomidae larvae have a significant preference (E' > 0.5) for Desmodesmus armatus, E. minima, and T. dimorphus, while it does not show preference for other species found in its gut. Differences in physico-chemical factors, such as water quality, nutrients, Chl-a, and carbon concentrations, resulting from anthropogenic impacts (i.e., construction of large-scale weirs) as well as the particle size of prey organisms (small-sized single cell) and effects of chemicals (chemokinesis) could affect the feeding behavior of Chironomidae larvae.

Morphological and functional characterization of circulating hemocytes using microscopy techniques.

In the present study, Microscopy studies were performed to characterize the blood cells of the mangrove crab Episesarma tetragonum. Three types of hemocytes were observed: granulocytes, semi-granulocytes, and hyalinocytes or agranulocytes. Hyalinocytes have a distinguished nucleus surrounded by the cytoplasm, and a peculiar cell type was present throughout the cytosol, lysosomes with hemocyte types (granules) stained red (pink). Giemsa staining was used to differentiate between the large and small hemocytes. Ehrlich's staining was used to differentiate granule-containing cells in acidophils (55%), basophils (44%), and neutrophils (<1%). Periodic acid-Schiff staining was used to identify the sugar molecules in the cytoplasm. Cell-mediated immune reactions including phagocytosis, encapsulation, agglutination, and peroxidase-mediated cell adhesion are the functions of hemocytes. Agglutination reaction involves both kind of cells involved in yeast and heme-agglutination responses in invertebrates. The beta glucan outer layer of yeast cells was recognized by hemocyte receptors. Human RBC cells were agglutinated via granulocytes. E. tetragonum hemocytes are an important animal model for studying both ultrastructural and functional activity of circulating cells. In addition, E. tetragonum hemocytes exhibited excellent antibacterial and antibiofilm activities were studied through plating and microplate assays. Biofilm inhibition was also visualized through changes in biochemical assays and morphological variations were visualized through levels in in situ microscopy analysis.