Air-breathing Walking Catfish Research Articles

Mitochondrial DNA Corroborates the Genetic Variability of Clarias Catfishes (Siluriformes, Clariidae) from Cameroon.

The airbreathing walking catfish (Clariidae: Clarias) comprises 32 species that are endemic to African freshwater systems. The species-level identification of this group is challenging due to their complex taxonomy and polymorphism. Prior to this study, the biological and ecological studies were restricted to a single species, Clarias gariepinus, resulting in a biased view of their genetic diversity in African waters. Here, we generated the 63-mitochondrial Cytochrome c oxidase subunit 1 (COI) gene sequences of Clarias camerunensis and Clarias gariepinus from the Nyong River in Cameroon. Both C. camerunensis and C. gariepinus species maintained adequate intra-species (2.7% and 2.31%) and inter-species (6.9% to 16.8% and 11.4% to 15.1%) genetic distances with other Clarias congeners distributed in African and Asian/Southeast Asian drainages. The mtCOI sequences revealed 13 and 20 unique haplotypes of C. camerunensis and C. gariepinus, respectively. The TCS networks revealed distinct haplotypes of C. camerunensis and shared haplotypes of C. gariepinus in African waters. The multiple species delimitation approaches (ABGD and PTP) revealed a total of 20 and 22 molecular operational taxonomic units (MOTUs), respectively. Among the two Clarias species examined, we found more than one MOTU in C. camerunensis, which is consistent with population structure and tree topology results. The phylogeny generated through Bayesian Inference analysis clearly separated C. camerunensis and C. gariepinus from other Clarias species with high posterior probability supports. The present study elucidates the occurrence of possible cryptic diversity and allopatric speciation of C. camerunensis in African drainages. Further, the present study confirms the reduced genetic diversity of C. gariepinus across its native and introduced range, which might have been induced by unscientific aquaculture practices. The study recommends a similar approach to the same and related species from different river basins to illuminate the true diversity of Clarias species in Africa and other countries.

Genome sequence of walking catfish (Clarias batrachus) provides insights into terrestrial adaptation

BackgroundWalking catfish (Clarias batrachus) is a freshwater fish capable of air-breathing and locomotion on land. It usually inhabits various low-oxygen habitats, burrows inside the mudflat, and sometimes “walks” to search for suitable environments during summer. It has evolved accessory air-breathing organs for respiring air and corresponding mechanisms to survive in such challenging environments. Thereby, it serves as a great model for understanding adaptations to terrestrial life.ResultsComparative genomics with channel catfish (Ictalurus punctatus) revealed specific adaptations of C. batrachus in DNA repair, enzyme activator activity, and small GTPase regulator activity. Comparative analysis with 11 non-air-breathing fish species suggested adaptive evolution in gene expression and nitrogenous waste metabolic processes. Further, myoglobin, olfactory receptor related to class A G protein-coupled receptor 1, and sulfotransferase 6b1 genes were found to be expanded in the air-breathing walking catfish genome, with 15, 15, and 12 copies, respectively, compared to non-air-breathing fishes that possess only 1–2 copies of these genes. Additionally, we sequenced and compared the transcriptomes of the gill and the air-breathing organ to characterize the mechanism of aerial respiration involved in elastic fiber formation, oxygen binding and transport, angiogenesis, ion homeostasis and acid-base balance. The hemoglobin genes were expressed dramatically higher in the air-breathing organ than in the gill of walking catfish.ConclusionsThis study provides an important genomic resource for understanding the adaptive mechanisms of walking catfish to terrestrial environments. It is possible that the coupling of enhanced abilities for oxygen storage and oxygen transport through genomic expansion of myoglobin genes and transcriptomic up-regulation of hemoglobin and angiogenesis-related genes are important components of the molecular basis for adaptation of this aquatic species to terrestrial life.

Unique mitochondrial localization of arginase 1 and 2 in hepatocytes of air-breathing walking catfish, Clarias batrachus and their differential expression patterns under hyper-ammonia stress

Arginase (ARG) catalyzes the final step of ornithine-urea cycle (OUC) leading to a conversion of L-arginine to L-ornithine and urea. Several isoforms of ARG have been reported in vertebrates, out of which the two predominant isoforms are the cytosolic ARG1 and the mitochondrial ARG2. The air-breathing walking catfish (Clarias batrachus) is frequently being challenged by different environmental insults such as hyper-ammonia, dehydration and osmotic stresses in their natural habitats throughout the year. The present study investigated the active presence of ARG1 and ARG2 isoforms in hepatocytes along with unique localization of both the isoforms inside the mitochondria, and also their specific expression patterns under hyper-ammonia stress (5mM NH4Cl) in isolated hepatocytes of walking catfish. Initially, full length sequences of both arg1 and arg2 genes were obtained by RACE-PCR. Studies on molecular characterization demonstrated the presence of all the conserved amino acids required for stability and activity of binuclear metal center in both the isoforms. Phylogenetic analysis of the amino acid sequences of ARG isoforms showed a differentiation of the ARG1 and ARG2 into two distinct clusters with their respective isoforms from other species. Most interestingly, both the isoforms of ARG in hepatocytes were found to be localized inside the mitochondria as evidenced by the presence of mitochondrial target peptide (mTP) in N-terminal of the derived amino acid sequences, and exclusive localization of ARG activity in the mitochondrial fraction. This was additionally confirmed by Western blot analysis of ARGs in mitochondrial and cytosolic fractions, and by immunocytochemical analysis in isolated hepatocytes. Although the possible reasons associated with the presence of both the isoforms of ARGs inside the mitochondria is not clearly understood, perhaps this mitochondrial localization of ARG is functionally advantageous in this catfish for the synthesis of N-acetyl-l-glutamate, the allosteric regulator for the first OUC enzyme, the carbamoyl phosphate synthetase III, and for supplying ornithine required for citrulline synthesis intramitochondrially. Furthermore, the ammonia stress, due to exposure to high external ammonia, led to greater synthesis of urea-N probably as a consequence of induction of ureogenesis, as evidenced by a larger accumulation of urea-N in hepatocytes and higher secretion in culture media parallel to the increased concentration of ammonia-N in hepatocytes. Ammonia stress also led to specific coordinated patterns of induction of both the arg genes in isolated hepatocytes of walking catfish.

Evaluation of extracellular enzyme-producing autochthonous gut bacteria in walking catfish, Clarias batrachus (L.)

The present study was carried out to screen autochthonous gut bacteria in freshwater air breathing walking catfish, Clarias batrachus Linnaeus. Altogether, 100 extracellular enzyme-producing bacteria were isolated from the foregut (FG) and hindgut (HG) regions. Data were presented as log viable counts g-1 gut (LVC). The occurrence of heterotrophic bacterial population was higher in the FG region (LVC = 8.25) than the HG (LVC= 7.3). Similarly, proteolytic, amylolytic and lipolytic bacteria in FG outnumbered (LVC=7.25, 6.77 and 5.23 respectively) the HG (LVC=6.38, 5.58 and 4.04 respectively). However, occurrence of cellulolytic bacteria in both, FG and HG was less (LVC=2.1 and 1.34 respectively) in comparison to the other extracellular enzyme-producing bacteria. Out of the 100 bacterial isolates, 22 isolates were primarily selected through qualitative assay of extracellular enzyme activities. Among them, 3 promising isolates were chosen as potent extracellular enzyme producers on the basis of cumulative scores (≥11) of the qualitative assay and quantitative enzyme assay. Maximum protease activity was revealed by the strain FG10 (201±2.40U), while FG43 exhibited maximum amylase (208.3±10.8U) and lipase (4.73±0.05U) activities. Among the strains isolated from the HG, the highest protease (188.3±1.2U), amylase (97.6±0.46U) and lipase (3.7±0.11U) activities were recorded with the strain HG01. The isolates (FG10, FG43 and HG01) were studied through 16S rRNA partial gene sequence analyses and were identified as Bacillus aryabhattai (KP784311), B. flexus (KR809411), and B. cereus (KR809412), respectively. Further studies are to be conducted to evaluate the efficacy of these strains in vivo to improve the overall health status of the C. batrachus juveniles.

Evaluation of extracellular enzyme-producing autochthonous gut bacteria in walking catfish, Clarias batrachus (L.)

The present study was carried out to screen autochthonous gut bacteria in freshwater air breathing walking catfish, Clarias batrachus Linnaeus. Altogether, 100 extracellular enzyme-producing bacteria were isolated from the foregut (FG) and hindgut (HG) regions. Data were presented as log viable counts g-1 gut (LVC). The occurrence of heterotrophic bacterial population was higher in the FG region (LVC = 8.25) than the HG (LVC= 7.3). Similarly, proteolytic, amylolytic and lipolytic bacteria in FG outnumbered (LVC=7.25, 6.77 and 5.23 respectively) the HG (LVC=6.38, 5.58 and 4.04 respectively). However, occurrence of cellulolytic bacteria in both, FG and HG was less (LVC=2.1 and 1.34 respectively) in comparison to the other extracellular enzyme-producing bacteria. Out of the 100 bacterial isolates, 22 isolates were primarily selected through qualitative assay of extracellular enzyme activities. Among them, 3 promising isolates were chosen as potent extracellular enzyme producers on the basis of cumulative scores (≥11) of the qualitative assay and quantitative enzyme assay. Maximum protease activity was revealed by the strain FG10 (201±2.40U), while FG43 exhibited maximum amylase (208.3±10.8U) and lipase (4.73±0.05U) activities. Among the strains isolated from the HG, the highest protease (188.3±1.2U), amylase (97.6±0.46U) and lipase (3.7±0.11U) activities were recorded with the strain HG01.

Influence of increased environmental water salinity on gluconeogenesis in the air-breathing walking catfish, Clarias batrachus

The present study was aimed at determining the effect of hypertonicity due to increased environmental water salinity on gluconeogenesis in air-breathing walking catfish (Clarias batrachus). In situ exposure to hypertonic saline solution (150 mM NaCl) led to a significant stimulation of glucose efflux due to gluconeogenesis from the liver after 7 days with further elevation after 14 days in the presence of each of the three potential gluconeogenic substrates (lactate, pyruvate, and glutamate). This was accompanied by significant increase of activities of three key gluconeogenic enzymes, namely phosphoenolpyruvate carboxykinase (PEPCK), fructose 1,6-biphosphatase (FBPase), and glucose 6-phosphatase (G6Pase) in liver and kidney by about twofold to threefold. Environmental hypertonicity also led to a significant elevation in the levels of PEPCK, FBPase, and G6Pase enzyme proteins in both the tissues by about 2- to 2.75-fold, accompanied by a significant elevation in the level of PEPCK mRNA by about 2- to 2.5-fold after 7 days, and further enhancement to about 3.5- to 4-fold after 14 days. Thus, the upregulation of PEPCK, FBPase. and G6Pase activities appears to be a result of transcriptional regulation of these genes. The induction of gluconeogenesis under environmental hypertonicity, which this catfish faces regularly in its natural habitat, possibly occurs as a consequence of changes in hydration status/cell volume of different cell types. This would certainly assist in maintaining glucose homeostasis, and also for a proper energy supply to support metabolic demands for ion transport and other altered metabolic processes under various environmental hypertonic stress-related insults.

Influence of cell volume changes on protein synthesis in isolated hepatocytes of air-breathing walking catfish (Clarias batrachus)

The present study aimed at determining the effect of cell volume changes on protein synthesis, measured as the incorporation of [(3)H]leucine into acid-precipitable protein, in isolated hepatocytes of air-breathing walking catfish (Clarias batrachus). The rate of protein synthesis, which was recorded to be 10.02 +/- 0.10 (n = 25) nmoles mg(-1) cell protein h(-1) in isotonic incubation conditions, increased/decreased significantly by 18 and 48%, respectively, following hypo- (-80 mOsmol l(-1))/hypertonic (+80 mOsmol l(-1)) incubation conditions (adjusted with NaCl), with an accompanying increase/decrease of hepatic cell volume by 12 and 20%, respectively. Similar cell volume-sensitive changes of protein synthesis were also observed when the anisotonicity of incubation medium was adjusted with mannitol. Increase of hepatic cell volume by 9%, due to addition of glutamine plus glycine (5 mM each) to the isotonic control incubation medium, led to a significant increase of protein synthesis by 14%. Decrease of hepatic cell volume by 15 and 18%, due to addition of dibutyl-cAMP and adenosine in isotonic control incubation medium, led to a significant decrease of protein synthesis by 30 and 34%, respectively. Thus, it appears that the increase/decrease of hepatic cell volume, caused either by changing the extracellular osmolarity or by the presence of amino acids or certain other metabolites, leads to increase/decrease of protein synthesis, respectively, and shows a direct correction (r = 0.99) between the hepatic cell volume and protein synthesis in walking catfish. These cell volume-sensitive changes of protein synthesis probably help this walking catfish in fine tuning the different metabolic pathways for better adaptation during cell volume changes and also to avoid the adverse affects of osmotic stress. This is the first report of cell volume-sensitive changes of protein synthesis in hepatic cells of any teleosts.

Expression of ornithine–urea cycle enzymes in early life stages of air-breathing walking catfish Clarias batrachus and induction of ureogenesis under hyper-ammonia stress

The air-breathing walking catfish Clarias batrachus is a potential ureogenic teleost with having a full complement of ornithine–urea cycle (OUC) enzymes expressed in various tissues. The present study was aimed at determining the pattern of nitrogenous waste excretion in the form of ammonia-N and urea-N along with the changes of tissue ammonia and urea levels, and the expression of OUC enzymes and glutamine synthetase (GSase) in early life stages of this teleost, and further, to study the possible induction of ureogenesis in 15-day old fry under hyper-ammonia stress. The ammonia and urea excretion was visible within 12 h post-fertilization (hpf), which increased several-fold until the yolk was completely absorbed by the embryo. Although all the early developing stages were primarily ammoniotelic, they also excreted significant amount of nitrogen (N) in the form of urea-N (about 35–40% of total N). Tissue levels of ammonia and urea also increased along with subsequent developmental stages at least until the yolk absorption stage. All the OUC enzymes and GSase were expressed within 4–12 hpf showing an increasing trend of activity for all the enzymes until 350 hpf. There was a significant increase of activity of GSase, carbamyl phosphate synthetase III (CPSase III) and argininosuccinate lyase enzymes (ASL), accompanied with significant increase of enzyme protein concentration of at least two enzymes (GSase and CPSase III) in the 15-day old fry following exposure to 10 mM NH 4Cl as compared to respective controls kept in water over a period of 72 h. Thus, it appears that the OUC enzymes are expressed in early life stages of walking catfish like other teleosts, but at relatively high levels and remain expressed all through the life stages with a potential of stimulation of ureogenesis throughout the life cycle as a sort of physiological adaptation to survive and breed successfully under hyper-ammonia and various other environmental-related stresses.

Role of ureogenesis in tackling problems of ammonia toxicity during exposure to higher ambient ammonia in the air-breathing walking catfish Clarias batrachus.

In the present study, the possible role of ureogenesis to avoid the accumulation of toxic ammonia to a lethal level under hyper-ammonia stress was tested in the air-breathing walking catfish Clarias batrachus by exposing the fish at 25 mM NH4Cl for 7 days. Excretion of ammonia by the NH4Cl-exposed fish was totally suppressed, which was accompanied by significant accumulation of ammonia in different body tissues. The walking catfish, which is otherwise predominantly ammoniotelic, turned totally towards ureotelism from ammoniotelism with a 5- to 6-fold increase of urea-N excretion during exposure to higher ambient ammonia. Stimulation of ureogenesis was accompanied with significant increase of some of the key urea cycle enzymes such as carbamyl phosphate synthetase (urea cycle-related), argininosuccinate synthetase and argininosuccinate lyase both in hepatic and non-hepatic tissues. Due to this unique physiological strategy of turning towards ureotelism from ammoniotelism via the induced urea cycle, this air-breathing catfish is able to survive in very high ambient ammonia, which they face in certain seasons of the year in the natural habitat.

Effect of alkalinity (pH 10) on ureogenesis in the air-breathing walking catfish, Clarias batrachus

Exposure of fish to alkaline conditions inhibits the rate of ammonia excretion, leading to ammonia accumulation and toxicity. The purpose of this study was to determine the role of ureogenesis via the urea cycle, to avoid the accumulation of ammonia to a toxic level during chronic exposure to alkaline conditions, for the air-breathing walking catfish, Clarias batrachus, where a full complement of urea cycle enzyme activity has been documented. The walking catfish can survive in water with a pH up to 10. At a pH of 10 the ammonia excretion rate by the walking catfish decreased by approximately 75% within 6 h. Although there was a gradual improvement of ammonia excretion rate by the alkaline-exposed fish, the rate remained 50% lower, even after 7 days. This decrease of ammonia excretion was accompanied by a significant accumulation of ammonia in plasma and body tissues (except in the brain). Urea-N excretion for alkaline-exposed fish increased 2.5-fold within the first day, which was maintained until day 3 and was then followed by a slight decrease to maintain a 2-fold increase in the urea-N excretion rate, even after 7 days. There was also a higher accumulation of urea in plasma and other body tissues (liver, kidney, muscle and brain). The activity of glutamine synthetase and three enzymes operating in the urea cycle (carbamyl phosphate synthetase, argininosuccinate synthetase, argininosuccinate lyase) increased significantly in hepatic and extra-hepatic tissue, such as the kidney and muscle in C. batrachus, during exposure to alkaline water. A significant increase in plasma lactate concentration noticed during alkaline exposure possibly helped in the maintenance of the acid–base balance. It is apparent that the stimulation of ureogenesis via the induced urea cycle is one of the major physiological strategies adopted by the walking catfish ( C. batrachus) during chronic exposure to alkaline water, to avoid the in vivo accumulation of ammonia to a toxic level in body tissues and for the maintenance of pH homeostasis.

Changes in free amino acid synthesis in the perfused liver of an air-breathing walking catfish, Clarias batrachus infused with ammonium chloride: a strategy to adapt under hyperammonia stress.

The changes in the free amino acid (FAA) levels, the rate of efflux of FAAs from the perfused liver, and the activity of some enzymes related to amino acid metabolism such as glutamate dehydrogenase (GDH, both reductive amination and oxidative deamination), glutamine synthetase (GS), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were studied in the liver of a freshwater air-breathing teleost, the walking catfish, Clarias batrachus, perfused with 5 and 10 mM NH(4)Cl. The level of the various non-essential FAAs increased significantly, with a total increase of about 150%, which was accompanied by a significant increase of both ammonia and urea-N in the perfused liver both with 5 and 10 mM NH(4)Cl. The rate of efflux of these non-essential FAAs from the perfused liver also increased significantly with a total increase of about 115% and 160% at 5 and 10 mM NH(4)Cl, respectively. The activity of the mentioned amino acid metabolism-related enzymes in the perfused liver also got stimulated, except for GDH in the ammonia forming direction and ALT, under a higher ammonia load. The activity (both tissue and specific) of GDH in the glutamate forming direction increased maximally, followed by AST and GS in a decreasing order. Owing to these physiological adaptive strategies related to amino acid metabolism along with the presence of a functional and regulatory urea cycle (reported earlier), it is believed that this catfish is able to survive in very high ambient ammonia or in the air or in the mud during habitat drying.

Types of carbamyl phosphate synthetases and subcellular localization of urea cycle and related enzymes in air-breathing walking catfish,Clarias batrachus

The types of carbamyl phosphate synthetase (CPS), the subcellular localization of urea cycle enzymes and glutamine synthetase (GS) in liver and kidney (potential ureogenic tissues) and also the possible involvement of some extra-hepatic tissues for urea synthesis via the urea cycle were studied in an Indian ureogenic amphibious air-breathing walking catfish, Clarias batrachus. Mitochondrial CPS III [glutamine- and N-acetyl-L-glutamate (NAG)-dependent] and cytosolic CPS II (glutamine-dependent) activities were found to be present in liver, analogous to that described for two other teleosts that have CPS III activity. The same activities and subcellular localization were found in kidney. Unexpectedly, a CPS I-like (ammonia- and NAG-dependent) activity was found to be present at levels higher than the CPS III activity in the mitochondrial fraction of both liver and kidney analogous to another Indian amphibious air-breathing teleost fish, Heteropneustes fossilis. The two other urea cycle enzymes, ornithine transcarbamylase (OTC) and arginase (ARG), and GS were found to be localized primarily in the mitochondria, and argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) in the cytosol analogous to H. Fossilis and elasmobranchs. Significant levels of urea cycle enzymes along with GS activity could also be detected in muscle, intestine and brain tissues, except ASL in muscle and ASS in brain. Our results also indicated that probably all three different types of CPS activities are also expressed in all three extra-hepatic tissues. The urea cycle–related CPS III found in invertebrates and fish is considered to be the evolutionary precursor of the urea cycle–related CPS I in ureotelic mammalian and amphibian species. Whether or not the CPS I-like activity reported here (1) is due to the presence of a separate CPS I gene in addition to a CPS III gene or (2) represents an adapted CPS III activity in C. batrachus, these results suggest that the presence of both CPS I-like and CPS III activities along with the presence of urea cycle enzymes in extra-hepatic tissues may play an important physiological adaptive role for survival of these walking catfish in higher ambient ammonia or living in air or while burrowing inside mud during habitat drying. J. Exp. Zool. 283:121–130, 1999. © 1999 Wiley-Liss, Inc.

Stimulation of ureogenesis in the perfused liver of an Indian air-breathing catfish, Clarias batrachus, infused with different concentrations of ammonium chloride

The pattern of changes of activity of the urea cycle enzymes and the rate of urea-N excretion were studied in the perfused liver of an Indian air-breathing ureogenic walking catfish, Clarias batrachus. The liver was perfused with different concentrations of NH4Cl for a period of 60 min to determine the role of ammonia for stimulation of hepatic ureogenesis and the threshold level of ammonia loading needed to cause such stimulation. Both the urea-N excretion and the ammonia uptake by the perfused liver were found to be a saturable process. Ammonia accumulated significantly in the liver infused with 1.25 μmoles g liver −1 min−1 of NH4Cl, followed by a maximum accumulation of about 28.5 μmoles g wet wt−1 with the infusion of 5.08 μmoles g liver−1 min−1. The Vmax of the urea-N excretion (0.47 μmol g liver−1 min−1) was obtained with the addition of 5.08 μmoles g liver−1 min−1 of NH4Cl. Both the tissue and the specific activity of the urea cycle enzymes, except ornithine transcarbamylase and arginase, were stimulated significantly with the infusion of either 1.25 or 5.08 μmoles g liver−1 min−1 of NH4Cl. Maximum stimulation of tissue activity of carbamoyl phosphate synthetase (about 120%) was seen with the infusion of 5.08 μmol g liver−1 min−1, and for argininosuccinate synthetase (about 135%), and argininosuccinate lyase (about 50%) with the infusion of 10.81 μmol g liver−1 min−1 of NH4Cl. Higher accumulation of ammonia of about 10–15 μmol g wet wt−1 from the physiological level in the perfused liver while infusing with NH4Cl was suggested to be one of the major causes of stimulation of ureogenesis. The presence of such physiological adaptive strategy is probably necessary in this unique group of air-breathing walking catfish to survive under hyper-ammonia stress in their normal habitat or while living outside water or while burrowing inside mud.

Microcirculation of gills and accessory respiratory organs of the walking catfish Clarias batrachus

An ability to extract oxygen directly from the atmosphere enables air-breathing fish to survive otherwise debilitating hypoxic environments. Addition of accessory respiratory organs (ARO) necessitates changes in both the general circulatory system and the microcirculation of the respiratory epithelia. Understanding these modifications provides information on the efficiency of gas exchange organs as well as an indication of the evolutionary processes associated with adaptation to terrestrial habitats. Vascular organization and structure of gills and ARO of the facultative air-breathing walking catfish Clarias batrachus were examined by scanning electron microscopy of vascular replicas and fixed tissue. Well-developed filaments are present on all four pairs of gill arches and they possess three vascular pathways: respiratory (arterioarterial), nutrient (arteriovenous), and interlamellar (arteriovenous), typical of teleosts. ARO, consisting of gill fans, dendritic organs on the second and fourth gill arch, and the suprabranchial epithelium are derived from gill tissue and retain structural features and arterioarterial vessels similar to gill filaments. Gill and ARO vessels are in parallel with each other, and together they are in series with the systemic circulation. Nutrients and interlamellar vessels are reduced in ARO. Other than the presence of multiple ventral aortas, and an additional vessel connecting the suprabranchial epithelium to the dorsal aorta, there are no vascular shunts or anatomical modifications that indicate spatial separation of flow through the heart or between gills and ARO. However, a mechanism is proposed that would prevent unsaturation of dorsal aortic blood by local myogenic vasoconstriction of gill vessels when the fish is in hypoxic water. Despite considerable differences in the gross features of ARO in Clarias and Heteropneustes fossilis (Olson et al. 1990 J. Morphol., 203:165), there are striking similarities in vascular organization and respiratory islet structure that suggest these ARO evolved in a common silurid ancestor and were later modified into an everted arborescent organ or inverted air sac, respectively.