Toxicity evaluation and microbiota response of the lined sole Achirus lineatus (Chordata: Achiridae) exposed to the light petroleum water-accommodated fraction (WAF).

Exposure to contaminants might directly affect organisms and alter their associated microbiota. The objective of the present study was to determine the impact of the petroleum-water-accommodated fraction (WAF) from a light crude oil (API gravity 35) on a benthic fish species native from the Gulf of Mexico (GoM). Ten adults of Achirus lineatus (Linnaeus, 1758) were exposed to a sublethal WAF/water solution of 50% v/v for 48 hr. Multiple endpoints were measured including tissue damage, presence of polycyclic aromatic hydrocarbons (PAHs) metabolites in bile and gut microbiota analyses. Atrophy and fatty degeneration were observed in livers. Nodules and inflammation were detected in spleen, and structural disintegration and atrophy in the kidney. In gills hyperplasia, aneurysm, and gills lamellar fusion were observed. PAHs metabolites concentrations in bile were significantly higher in exposed organisms. Gut microbiome taxonomic analysis showed significant shifts in bacterial structure and composition following WAF exposure. Data indicate that exposure to WAF produced toxic effects in adults of A. lineatus, as evidenced by histological alterations and dysbiosis, which might represent an impairment to long-term subsistence of exposed aquatic organisms.

Biological responses of shoal flounder (Syacium gunteri) to toxic environmental pollutants from the southern Gulf of Mexico.

The Gulf of Mexico (GoM) is exposed to a diversity of contaminants, such as hydrocarbons and heavy metal(oid)s, either from natural sources or as a result of uncontrolled coastal urbanisation and industrialisation. To determine the effect of these contaminants on the marine biota along the Mexican GoM, the biological responses of the shoal flounder Syacium gunteri, naturally exposed, were studied. The study area included all the Mexican GoM, which was divided into three areas: West-southwest (WSW), South-southwest (SSW) and South-southeast (SSE). The biological responses included the global DNA methylation levels, the expression of biomarker genes related to contaminants (cytochrome P450 1A, glutathione S-transferase, glutathione reductase, glutathione peroxidase, catalase, and vitellogenin), histopathological lesions and PAH metabolites in bile (hydroxynaphthalene, hydroxyphenanthrene, hydroxypyrene and Benzo[a]pyrene). The correlation between the biological responses and the concentration of contaminants (hydrocarbons and metal(oid)s), present in both sediments and organisms, were studied. The shoal flounders in WSW and SSW areas presented higher DNA hypomethylation, less antioxidative response and biotransformation gene expression and a higher concentration of PAH metabolites in bile than SSE area; those responses were associated with total hydrocarbons and metals such as chromium (Cr). SSE biological responses were mainly associated with the presence of metals, such as cadmium (Cd) and copper (Cu), in the tissue of shoal flounders. The results obtained on the physiological response of the shoal flounder can be used as part of a permanent active environmental surveillance program to watch the ecosystem health of the Mexican GoM.

Evaluation of two independent protocols for the extraction of DNA and RNA from different tissues of sea cucumber Isostichopus badionotus.

Isostichopus badionotus is a sea cucumber species of great ecological and economic relevance for Mexico and Central American and Caribbean countries; however, the protocols for the extraction of the nucleic acids have not yet been published. In this study, we describe the first protocols to obtain DNA and RNA from different tissues of I. badionotus, which include the respiratory tree, gonad, longitudinal muscle bands, anterior intestine and cloaca. The extraction of high-quality DNA was performed using the DNeasy Blood & Tissue kit (Qiagen, Valencia, CA, USA) with minor modifications in different points of the protocol. Concerning the RNA, the method of TRIzol was used. This method is particularly advantageous in situations where cells or tissues are enriched for endogenous RNases or when the separation of cytoplasmic RNA from nuclear RNA is impractical. The methodologies used in this study allowed us to obtain DNA and RNA of high quality and integrity in the different tissues of I. badionotus, which will be the basis for future genomic and transcriptomic studies. •The successful extraction of DNA and RNA was achieved in the different tissues of I. badionotus.•The concentrations of DNA and RNA obtained were adequate for a diversity of analyses at a molecular level.

Metazoan parasite infracommunities of the dusky flounder (Syacium papillosum) as bioindicators of environmental conditions in the continental shelf of the Yucatan Peninsula, Mexico.

BACKGROUND: We assessed metrics of the metazoan parasite infracommunities of the dusky flounder (Syacium papillosum) as indicators of aquatic environmental health of the Yucatan Shelf (YS) prior to oil extraction. We sampled the dusky flounder and its parasites along the YS, mostly during the 2015 north wind season (November-April). Our aims were: (i) to determine whether the parasite infracommunity metrics of S. papillosum exhibit significant differences among YS subregions; (ii) to determine whether the probability of the occurrence of its parasite species and individuals were affected by environmental variables, nutrients, heavy metals and hydrocarbons at the seascape level; and (iii) to determine whether there were statistical differences between the parasite infracommunity metrics of S. papillosum from YS and those of Syacium gunteri from the Campeche Sound. Multivariate statistical analyses and generalised additive models (GAMs) were used to examine the potential statistical associations between the contaminants, environmental variables and parasite community metrics, and the maximum entropy algorithm (MaxEnt) was used to characterise the habitat's suitability for the parasite's probability of occurrence. RESULTS: We recovered 48 metazoan parasite species from 127 S. papillosum, with larval cestodes and digeneans being the most numerically-dominant. Multivariate analyses showed significant differences in parasite infracommunity metrics among Western YS, Mid YS and Caribbean subregions, with the latter being the richest in species but not in individuals. The GAM and MaxEnt results indicated a negative effect of top predators (e.g. sharks and rays) removal on parasite metrics. The parasite infracommunities of S. papillosum were twice as rich in the number of species and individuals as those reported for S. gunteri from the Campeche Sound. CONCLUSIONS: The significant differences among subregions in parasite metrics were apparently due to the interruption of the Yucatan current during the north wind season. The fishing of top predators in combination with an influx of nutrients and hydrocarbons in low concentrations coincides with an increase in larval cestodes and digeneans in S. papillosum. The dusky flounder inhabits a region (YS) with a larger number of metazoan parasite species compared with those available for S. gunteri in the Campeche Sound, suggesting better environmental conditions for transmission in the YS.

Gene expression profile and molecular pathway datasets resulting from benzo(a)pyrene exposure in the liver and testis of adult tilapia.

Benzo(a)pyrene (BaP), the prototype of polycyclic aromatic hydrocarbons, is known to exhibits genotoxic and carcinogenic effects promoting molecular impacts. The dataset presented here is associated with the research article paper entitled "Transcriptome Analysis Reveals Novel Insights Into the Response of Low-dose Benzo(a)pyrene Exposure in Male Tilapia". In this article, we presented a transcriptomic characterization of male tilapia exposure to BaP in the short term. This data provides an extended analysis of changes in the gene expression and identification of pathways in the liver and testis of male tilapia exposure to BaP. We used gene set enrichment analysis (GSEA) and sub-network enrichment analysis (SNEA) to identify gene networks and pathways associated with molecular adverse effects of BaP exposure. The data indicates that target pathways related to promoting carcinogenesis such as DNA repair and DNA replication were affected as well as other crucial biological processes. Moreover, to determine whether some of the key reported genes of DNA damage are affected by BaP exposure, Quantitative PCR (qPCR) was performed. Gene set categories and sub-networks are provided and the corresponding signature differences from BaP exposure are listed. The information in these datasets may contribute to understanding the potential carcinogenesis mechanism of action from low BaP exposure.

Genes coding for transporters showed a rapid and sharp increase in their expression in response to lead, in the aquatic fern (Salvinia minima Baker).

Salvinia minima was assessed for its ability to accumulate lead (Pb) by exposing it to concentrations of 40µM Pb(NO3)2 during 24h. At the same time, the expression levels were quantified, of four genes coding for transporters: SmABCC (ABCC-MRP), SmATPase (ATPase-P3A), SmNhaD (Type-Na+/H+) and SmABCG (ABCG-WBC). In the absence of lead, S. minima had very low expression of those genes, when plants were exposed to the metal however, those genes showed a rapid (in just three hours or less) and sharp increase (up to 60 times) in their expression, particularly the SmNhaD (Type-Na+/H+) gene. This sharp increase in expression levels of the genes studied, occurred at the same time that the plant accumulated the highest content of lead in its tissues. The first two genes, are apparently implicated in detoxification and lead accumulation mechanisms, while the other two genes are apparently involved in maintaining cell balance (homeostatic control) and membrane integrity. Our results confirmed that S. minima is efficient for phytoremediation of water bodies contaminated by lead, as it is efficient in accumulating this metal in its tissues (bioconcentration factor; BCF) values greater than 1000, in short times of exposure. More importantly, our data on the expression profiles of four genes coding for transporters, represent a first sight scenario of the molecular basis for understanding the different mechanism of detoxification, apparently present in this aquatic fern.

Transcriptome analysis reveals novel insights into the response of low-dose benzo(a)pyrene exposure in male tilapia.

Despite a wide number of toxicological studies that describe benzo[a]pyrene (BaP) effects, the metabolic mechanisms that underlie these effects in fish are largely unknown. Of great concern is the presence of BaP in aquatic systems, especially those in close proximity to human activity leading to consumption of potentially contaminated foods. BaP is a known carcinogen and it has been reported to have adverse effects on the survival, development and reproduction of fish. The purpose of this study was to investigate if a low dose of BaP can alter genes and key metabolic pathways in the liver and testis in male adult tilapia, and whether these could be associated with biological endpoints disruption. We used both high-throughput RNA-Sequencing to assess whole genome gene expression following repeated intraperitoneal injections of 3 mg/kg of BaP (every 6 days for 26 days) and morphometric endpoints as indicators of general health. Condition factor (K) along with hepatosomatic and gonadosomatic indices (morphometric parameters) were significantly lower in BaP-treated fish than in controls. BaP exposure induced important changes in the gene expression pattern in liver and testis as revealed by both Pathway and Gene Ontology (GO) analyses. Alterations that were shared by both tissues included arachidonic acid metabolism, androgen receptor to prostate-specific antigen signaling, and insulin-associated effects on lipogenesis. The most salient liver-specific effects included: biological processes involved in detoxification, IL6-associated insulin resistance, mTOR hyperactivation, mitotic cytokinesis, spindle pole and microtubule binding. BaP effects that were confined to the testis included: immune system functions, inflammatory response, estrogen and androgen metabolic pathways. Taken together, gene expression and morphometric end point data indicate that the reproductive success of adult male tilapia could be compromised as a result of BaP exposure. These results constitute new insights on the mechanism of action of low dose BaP in a non-model organism (tilapia).

Partial mRNA Sequences of the Biomarkers CYP1A, GST, CAT, GR, SOD, GPx, VTG and p53 in Flatfish Syacium gunteri from Gulf of Mexico.

The present study reports partial sequences of a group of genes used as exposure and effect biomarkers of organic contaminants and/or heavy metals in Syacium gunteri. In order to isolate these sequences, cDNA was used to amplify fragments between 200 and 800 bp, which were then cloned and sequenced. The sequences presented high percentages of identity with genes involved in the metabolism of xenobiotic biotransformation (cytochrome P4501A and glutathione S-transferase), oxidative stress (catalase, glutathione reductase, glutathione peroxidase and superoxide dismutase), reproductive system (vitellogenin) and with the tumor suppressor protein p53 reported in the GenBank database. Subsequently, from the deduced sequence of amino acids of each fragment, their tridimensional structure was predicted, using homologous proteins from the Protein Data Base. This study generates an important base of molecular biomarkers for the monitoring of environmental health in the Gulf of Mexico.

Phenotypic plasticity in gene expression and physiological response in red drum Sciaenops ocellatus exposed to a long-term freshwater environment.

Red drum (Sciaenops ocellatus) is a euryhaline fish commonly found in the Gulf of Mexico and along the Atlantic coast of North America. Because of high commercial demand and its euryhaline characteristics, aquaculture of this species has diversified from marine to low-salinity aquaculture systems. In recent years, interest in the feasibility of producing red drum in inland freshwater systems has grown and this prompted us to investigate its osmoregulatory capacity after rearing for 8 months in a freshwater aquaculture system. We compared the activities of several genes and enzymes involved in the osmoregulatory process in freshwater-acclimatized (FW) and seawater (SW) red drum. The gene expression profiles were variable: the expression of genes encoding Na+/K+-ATPase (NKA) and the cystic fibrosis transmembrane regulator (CFTR) was slightly higher in SW than FW fish, while phosphoenolpyruvate carboxykinase (PEPCK) and the glucocorticoid receptor messenger RNA (mRNA) levels were higher in FW red drum. The total plasma K concentration was 60.3% lower, and gill NKA activity was 63.5% lower in FW than in SW fish. PEPCK activity was twofold higher in FW than in SW red drum. Similarly, liver glycogen was 60% higher in FW fish. In summary, both gene expression and the enzyme activity data support the phenotypic plasticity of red drum and suggest that the limited capacity for ion homeostasis observed, in particular the low plasma K concentration, was due to the composition of freshwater and does not necessarily reflect a physiological inability to osmoregulate.

Genetic variation, multiple paternity, and measures of reproductive success in the critically endangered hawksbill turtle (Eretmochelys imbricata).

The Yucatán Peninsula in Mexico contains some of the largest breeding groups of the globally distributed and critically endangered hawksbill turtle (Eretmochelys imbricata). An improved understanding of the breeding system of this species and how its genetic variation is structured among nesting areas is required before the threats to its survival can be properly evaluated. Here, we genotype 1195 hatchlings and 41 nesting females at 12 microsatellite loci to assess levels of multiple paternity, genetic variation and whether individual levels of homozygosity are associated with reproductive success. Of the 50 clutches analyzed, only 6% have multiple paternity. The distribution of pairwise relatedness among nesting localities (rookeries) was not random with elevated within-rookery relatedness, and declining relatedness with geographic distance indicating some natal philopatry. Although there was no strong evidence that particular rookeries had lost allelic variation via drift, younger turtles had significantly lower levels of genetic variation than older turtles, suggesting some loss of genetic variation. At present there is no indication that levels of genetic variation are associated with measures of reproductive success such as clutch size, hatching success, and frequency of infertile eggs.

Copper stress on photosynthesis of black mangle (Avicennia germinans).

The effects of copper toxicity on the photosynthetic activities of Avicennia germinans was investigated using two CuSO4 concentrations (0.062 and 0.33 M) added in Hoagland's solution in an aerated hydroponic system. Photosynthesis and chlorophyll fluorescence were measured after 30 h of copper stress. Results obtained in this study show that increasing levels of Cu(+2) of 0.062 and 0.33 M Cu(+2) resulted in a general reduction of the stomatal conductance (28 and 18%, respectively) and 100% of inhibition of net photosynthesis. Additionally, at these concentrations of Cu(+2), reductions of chlorophyll fluorescence parameters were also observed. These changes suggested that the photosynthetic apparatus of Avicennia germinans was the primary target of the Cu(+2) action. It is concluded that Cu(+2) ions causes a drastic decline in photosynthetic gas exchange and Chlorophyll fluorescence parameters in A. germinans leaves.

Bile benzo[a]pyrene concentration and hepatic CYP1A induction in hypoxic adult tilapia (Oreochromis niloticus).

Aquatic hypoxia is a seasonal condition in some coastal and continental wetlands where co-exposure with polycyclic aromatic hydrocarbons (PAHs) pollution may be detrimental to the biota. In the present study, adult tilapia, an euryoxic fish of high economic importance, were intraperitoneally injected with benzo[a]pyrene (BaP) (20 mg kg(-1)) and then exposed to graded hypoxia to assess combined effects on some detoxification and fitness parameters. Seventy-two hours after a stepped decrease in dissolved oxygen (<2 mg L(-1)), BaP treatment resulted in a significant diminution on the biliary BaP concentration (70% of normoxic group) and an increase in blood glucose levels (2.17-fold compared with normoxic group). These effects returned to control values in the following 48 h of hypoxia exposure. BaP-induced CYP1A mRNA levels were unaffected by hypoxia, suggesting that reduced bile BaP concentration may be related with effects on protein amount or enzyme activities. LDH mRNA levels, blood lactate and hematocrit remained without change, suggesting no extreme detrimental effects for tilapia in the short-term of the BaP-hypoxia challenge. Our results indicate that BaP treatment and hypoxia targeted glucose metabolism and biliary BaP elimination, probably by favoring the storage of BaP in tilapia tissues.

Benzo(a)pyrene induces hepatic AKR1A1 mRNA expression in tilapia fish (Oreochromis niloticus).

AKR1A1 or aldehyde reductase is a member of the aldo-keto reductases superfamily that is evolutionarily conserved among species. AKR1A1 is one of the five AKRs (AKR1A1 and 1C1-1C4) implicated in the metabolic benzo(a)pyrene (BaP) activation to reactive BaP 7,8-dione. BaP is a polycyclic aromatic hydrocarbon (PAH) widely distributed in aquatic ecosystems and its metabolic activation is necessary to produce its toxic effects. Although the presence of AKR1A1 in fish has been reported, its tissue distribution in tilapia (Oreochromis niloticus) and AKR1A1 inducibility by BaP are not known yet. Moreover, cytochrome P4501A (CYP1A) mRNA expression in fish has been used as a PAH biomarker of effect. Therefore, BaP effects on AKR1A1 and CYP1A gene expressions in tilapia, a species of commercial interest, were investigated by real-time RT-PCR. A partial AKR1A1 cDNA was identified, sequenced and compared with AKR1A1 reported sequences in the GenBank DNA database. Constitutive AKR1A1 mRNA expression was detected mainly in liver, similarly to that of CYP1A. BaP exposure resulted in statistically significant AKR1A1 and CYP1A mRNA induction in liver (20- and 120-fold, respectively) at 24 h. On the other hand, ethoxyquin (EQ) was used as control inducer for AKR1A1 mRNA. Interestingly, EQ also induced CYP1A mRNA levels in tilapia liver. Our results suggest that teleost AKR1A1, in addition to CYP1A, are inducible by BaP. The mechanism of AKR1A1 induction by BaP and its role in fish susceptibility to BaP toxic effects remains to be elucidated.

Response of CYP1A Gene expression in fish liver of catfish (Ariopsis felis) from Gulf of Mexico and their relationship with the genetic variability.

We determined the hepatic Cytochrome P4501A (CYP1A) mRNA and Ethoxyresorufin-O-deethylase (EROD) activities in the fish, Ariopsis felis, from highly polluted to relatively pristine regions in the southwest Gulf of Mexico and their relationship with the genetic polymorphisms of the same fish. We hypothesized that a high genetic variation reflects interindividual variability in levels of CYP1A mRNA underlying the pathway culminating in EROD induction caused by the environmental contaminants. Catfish from Laguna de Mecoacan exhibited marked induction of CYP1A mRNA and high levels of hepatic EROD activities, whereas fish from Laguna de Celestun showed no induction of CYP1A mRNA and moderately low levels of EROD activities. In contrast, the similarity index considering all samples varied from 0.4 to 0.87, showing a wide range of variation. A dendrogram showed a clear grouping of fish collected from the Laguna de Terminos, Rio Coatzacoalcos and Laguna de Celestun, with discrete subgroups according to region. In contrast, fish from Laguna de Mecoacan were grouped together completely separate from the rest of the fish. Despite the low number of fish from Mecoacan (a high bootstrap support was observed in this group), the results indicated a significant genetic variability in comparison with the other ecosystems included. The differential level of expression of CYP1A and the EROD activity observed among the ecosystems analyzed could be due to the high range of genetic variation, with special emphasis on fish collected in Mecoacan where it is possible to find a subspecies of Ariopsis felis.

Cell viability and leakage of electrolytes in Avicennia germinans exposed to heavy metals.

The effect of heavy metal stress on the cell viability and leakage of electrolytes of Avicennia germinans leaf discs was investigated by the tissue tolerance test. Foliar discs were incubated with different Cd2+ or CU2+ concentrations for 24 h; thereafter, the cell membrane stability of the tissue was assayed by the cell viability Evans blue and leakage electrolytes methods. The results indicated that electrolyte leakage of the leaf discs increased 24 h after exposure to heavy metal stress, as shown by a reduction of the cell viability by 30% in discs exposed to higher doses of Cd2+ (0.546 M) and Cu2+ (0.7 M), respectively. Additionally, the histological analysis of the leaf discs exposed to heavy metal stress revealed that at higher Cd2+ and/or Cu2+ concentrations an increase in the intercellular spaces and destruction of mesophyll cells was observed 24 h after exposure. In summary, the biochemical and structural changes observed in foliar tissues of A. germinans suggest that higher cadmium and copper concentrations may result in structural changes and altered physiological characters in leaves.

Identification of genes expressed as a result of lindane exposure in Oreochromis niloticus using differential display.

In order to assess the effect of lindane exposure on gene expression in tilapia (Oreochromis niloticus), twenty male fish were individually weighted and injected intraperitoneally with a single dose of lindane (19.09 mg/kg bw) using corn oil as a carrier vehicle, while a second group of twenty male fish (controls) was only injected with the carrier vehicle. Groups of four fish each were then sacrificed at 3, 6, 12, 18 and 24h after treatment application and total RNA was extracted from liver tissue. The differential display (DD) technique was then used to identify differentially expressed cDNA fragments between treatment and control fish. A total of fifty cDNA fragments were isolated and sequenced, from which only four showed homology with genes previously described in other fish species, namely the immunoglobulin heavy chain (IgH), coagulation factor V (FV), casein kinase 2 alpha (CK2a), and the receptor protein-tyrosine-like phosphatase (RPT-LP). The expression of such genes was confirmed using quantitative real time-polymerase chain reaction (QRT-PCR). Results showed that lindane exposure triggered the differential expression of these genes during the first 6, 18 and 24h subsequent to treatment application, suggesting that lindane exposure can trigger a rapid immune system response in tilapias.

The checkered puffer (Spheroides testudineus) and its helminths as bioindicators of chemical pollution in Yucatan coastal lagoons.

The suitability of using helminth communities as bioindicators of environmental quality of the Yucatan coastal lagoons status was tested on the checkered puffer (Spheroides testudineus) in four coastal lagoons along the Yucatan coast. The concentration of chemical pollutants in sediments, water quality parameters, helminth infracommunity characteristics, as well as fish physiological biomarkers, including EROD (7-ethoxyresorufin-O-deethylase) and catalase activities, were measured. Results from sediment analyses demonstrated the presence of hydrocarbons, organochlorine pesticides and polychlorinated biphenyls at varying concentrations, some of which exceeded the Probability Effect Level (PEL). Significant negative associations among organochlorine pesticides, infracommunity characteristics and fish physiological responses were observed in most of the lagoons. Results suggest that EROD activity and parasite infracommunity characteristics could be useful tools to evaluate the effects of chemical pollutants on the fish host and in the environment. Importantly, certain parasites appear to influence biomarker measurements, indicating that parasites should be considered in ecotoxicological studies.

Changes in gene expression induced by polycyclic aromatic hydrocarbons in the human cell lines HepG2 and A549.

Polycyclic aromatic hydrocarbons (PAH) are the main components of emissions generated by coke oven factories and many of these chemicals are carcinogenic. The goal of this study was to examine changes in gene expression in two human cell lines, HepG2 and A549, induced by exposure to a soil extract containing PAH using microarry technology. Soil samples were obtained from the vicinity of a coke oven factory in northeastern Mexico. For comparison, the gene expression pattern induced by Benz[a]pyrene (BaP) was also analyzed. The number of altered genes by both treatments was 2-fold higher in hepatic than in pulmonary cells. Differentially-modulated genes in the two cell lines were identified and grouped by biological function using genomic databases. A group of nine genes up- and down-regulated by either the PAH extract or BaP were selected for validation by real-time PCR. The cellular functions of these PAH-responsive genes included: xenobiotic metabolism (CYP1A1 and CYP1B1), DNA repair (ERCC5), oxidative stress response and cell proliferation (FTH1 and PRDX1), protein degradation (PSMD7), ion transport (FXYD3), steroid biosynthesis (FDFT1), and signaling pathways (PTGER3). The real-time PCR analysis confirmed most of the microarray data with significant correlation. Additional studies are required to determine the mechanisms involved in the PAH-mediated modulation of these genes and to associate these changes with human health.

An improved method for the isolation of total RNA from Avicennia germinans leaves.

Isolation of high-quality RNA of Avicennia germinans L. tissue is difficult due to high levels of phenols and other substances that interfere when using conventional procedures for the isolation. These substances not only decrease the yield but also the quality of RNA is almost poor. We present here a simple RNA protocol and fast methodology that effectively removes these contaminating substances without affecting the yield. The protocol developed is based on the SDS/phenol method with modifications including beta-mercaptoethanol to prevent oxidation of phenolic complexes, and phenol/chloroform extraction is introduced to remove proteins, genomic DNA, and secondary metabolites, and co-precipitated polysaccharides. Both A260/A230 and A260/A280 absorbance ratios of isolated RNA were around 2 and the yield was about 0.3 mg g(-1) fresh weight. Good-quality total RNA from leaves of Avicennia germinans could be easily isolated within 2 h by this protocol which avoided the limitation of plant materials and could provide total RNA for all kinds of further molecular studies.