Macro-Rheology Characterization of Gill Raker Mucus in the Silver Carp, Hypophthalmichthys molitrix.

The silver carp, Hypophthalmichthys molitrix, is an invasive planktivorous filter feeder fish that infested the natural waterways of the upper Mississippi River basin due to its highly efficient filter feeding mechanism. The characteristic organs called gill rakers (GRs), found in many such filter feeders, facilitate the efficient filtration of food particles such as phytoplankton that are of a few microns in size. The motivation to investigate the rheology of the GR mucus stems from our desire to understand its role in aiding the filter feeding process in the silver carp. The mucus-rich fluid, in a 'thick and sticky' state may facilitate the adhesion of food particulates. The permeation and transport through the GR membrane are facilitated by the action of external shear forces that induce varying shear strain rates. Therefore, mucus rheology can provide a vital clue to the tremendous outcompeting nature of the silver carp within the pool of filter feeding fish. Based on this it was posited that GR mucus may provide an adhesive function to food particles and act as a transport vehicle to assist in the filter feeding process. The main objective of the protocol is to determine the yield stress of the mucus, attributed to the minimum shear stress required to initiate flow at which irreversible plastic deformation is first observed across a structured viscoelastic material. Accordingly, rheological properties of the GR mucus, i.e., viscosity, storage, and loss moduli, were investigated for its non-Newtonian, shear-thinning nature using a rotational rheometer.   A protocol presented here is employed to analyze the rheological properties of mucus extracted from the gill rakers of a silver carp, fished at Hart Creek location of the Missouri River. The protocol aims to develop an effective strategy for rheological testing and material characterization of mucus assumed to be a structured viscoelastic material.

Regeneration of the gill filaments and replacement of serotonergic neuroepithelial cells in adult zebrafish (Danio rerio).

Respiratory epithelia and chemoreceptors of the gills and mammalian lung derive from the same embryonic structures. While the lung is limited to facultative regeneration, the regenerative capacity of the gill has not been adequately explored. We report regeneration of gill filaments and respiratory lamellae in adult zebrafish (Danio rerio). Gill filaments retained a constitutive population of mitotic cells identified by the proliferating cell nuclear antigen (PCNA). Within 24 h of resection, a new mass of PCNA-positive cells appeared at the filament tip. At 40 days post-resection, approximately half of resected tissue was replaced; and at 160 days post-resection, regeneration was nearly complete. Chemoreceptive neuroepithelial cells, identified by serotonin immunohistochemistry, were present in regenerates and established innervation by nerve fibres. Use of the transgenic zebrafish line Tg(fli1a:EGFP), in which the gill vasculature was labelled with enhanced green fluorescent protein, indicated that angiogenesis occurred during the regenerative process. Thus, the zebrafish is capable of substantive gill regeneration and replacement of respiratory chemoreceptors.

Corrosion casting of the cardiovascular structure in adult zebrafish for analysis by scanning electron microscopy and X-ray microtomography.

Zebrafish have come to the forefront as a flexible, relevant animal model to study human disease, including cardiovascular disorders. Zebrafish are optically transparent during early developmental stages, enabling unparalleled imaging modalities to examine cardiovascular structure and function in vivo and ex vivo. At later stages, however, the options for systematic cardiovascular phenotyping are more limited. To visualise the complete vascular tree of adult zebrafish, we have optimised a vascular corrosion casting method. We present several improvements to the technique leading to increased reproducibility and accuracy. We designed a customised support system and used a combination of the commercially available Mercox II methyl methacrylate with the Batson's catalyst for optimal vascular corrosion casting of zebrafish. We also highlight different imaging approaches, with a focus on scanning electron microscopy (SEM) and X-ray microtomography (micro-CT) to obtain highly detailed, faithful three-dimensional reconstructed images of the zebrafish cardiovascular structure. This procedure can be of great value to a wide range of research lines related to cardiovascular biology in small specimens.

Ecotoxicological Analyses of Springs of a Brazilian Northeast Conservation Unit.

Ecotoxicological assessment is an useful tool to evaluate toxicity of pollutants on aquatic organisms. In the present study, we evaluated the lethal effect, micronucleus frequency and histological changes in gills of fish species Danio rerio exposed to water from two sources of a Conservation Unit. We performed acute tests, Micronucleus Test in blood cells and histological analysis of gills. As results, the springs had no lethal toxic effects on the species. Nuclear alterations were present in erythrocytes in organisms exposed to water from the two sampled sites. As well, we found moderate to severe gill abnormalities generating the impairment of respiratory vital functions of these organisms. This study indicates that the water of the springs does not present lethal toxic effect but damage in gills for species from short exposure time.

Lysosomal Membrane Stability in Hemocytes and Micronuclei in Gills of Perumytilus purpuratus Lamarck 1819 (Bivalvia: Mytilidae) Exposed to Copper.

The aim of this study was to determine the cytotoxic and genotoxic effects of copper on the bivalve Perumytilus purpuratus. The individuals were exposed to three copper concentrations: 1, 30 and 45 µg L-1 for 24, 48 and 96 h. Lysosomal membrane stability in hemocytes was determined through the neutral red retention time (NRRT) and micronucleus (MN) frequency tests in hemocytes and gills. The results show that the NRRT decreased significantly at 30 µg L-1 after 48 h of exposure. The frequency of MN was significantly greater in gills after 24 h in all concentrations tested. Copper is cytotoxic from 30 µg L-1 and genotoxic from 1 µg L-1. The use of these biomarkers of effects in P. purpuratus is proposed as an early warning tool for monitoring in environmental assessment of coastal ecosystems impacted by mining activities.

Histopathological effects of Pedunsaponin A on Pomacea canaliculata.

Pedunsaponin A, a novel molluscicidal compound isolated from Pueraria peduncularis, exhibits strong toxicity against Pomacea canaliculata. To determine the mechanisms of Pedunsaponin A toxicity, its effects on the organs and hemocytes of P. canaliculata were examined in this study. The results showed that Pedunsaponin A had significant toxic effects on different organs of the snail, including the lungs, gills, mantle, siphon tube, ventricle, pericardial cavity, hepatopancreas, kidneys, and the major symptom of this toxicity was the loss of cilia in the lungs and gills. Additionally, in further studies on the effects of Pedunsaponin A treatment, we found that the hemocyte count was changed and hemocyte morphology was damaged, which was primarily reflected by cytoplasm leakage, nuclei deformation, and significant reductions in the number of ribosomes and granulocyte mitochondria. Based on these results and considering that blood vessels are distributed in the lungs and gills, we hypothesized that Pedunsaponin A would first destroy the cilia, which disrupt physiological activities such as respiration, excretion and feeding, and then enter the hemolymph through blood vessels, disrupt the normal function of the hemocytes and destroy the snail immune system, eventually resulting in the death of the snail.

Study on DNA damaging effects of 4-nonylphenol using erythrocytes from peripheral circulation, gill and kidney of fish Channapunctatus.

The present study aimed to evaluate the genotoxic effect of 4-nonylphenol (NP) on blood cells of fish Channapunctatus. Fish were exposed to three sublethal concentrations (0.15 mg l⁻¹; 0.31 mg l⁻¹ and 0.63 mg l⁻¹) of 4-NP for 24, 48, 72 and 96 hrs. Blood cells from kidney, gills and peripheral circulation were analyzed for the presence of micronuclei and other changes in the erythrocytes. Significant changes were observed in all the experimental groups tested when compared with control. Highest genotoxicity was observed in blood cells obtained from gills (MN-2.92%, aberrant cell- 70.64%), followed by kidney (MN-1.34%, aberrant cells-64.94%), were least effect was observed in blood cells obtained from peripheral circulation (MN-0.88%, aberrant cells-46.27%).Therefore, micronucleus test performed on blood cells obtained from different sources showed that gills were more sensitive as compared to peripheral blood and kidney revealing genotoxic effect of 4-NP on fish C. punctatus.

Gill reaction to pollutants from the Tamis River in three freshwater fish species, Esox lucius L. 1758, Sander lucioperca (L. 1758) and Silurus glanis L. 1758: a comparative study.

The study evaluated the effects of waterborne pollutants from the Tamis River on gill histology and possible differences in gill reaction patterns between three freshwater fish species, pike Esox lucius L. 1758, pike-perch Sander lucioperca (L. 1758) and wels catfish Silurus glanis L. 1758 from the Tamis River. Gills from analysed fish species showed moderate to intense histopathological alterations. The most frequent progressive alteration was hyperplasia of epithelium, whereas the most frequent regressive alteration was epithelial lifting. Circulatory disturbances were most often manifested in the form of hyperaemia. During comparative analysis, differences in gill indices, reaction and alteration indices, as well as in gill and filament prevalence between analysed species, were observed. Although all analysed fish species did show both progressive and regressive alterations, there was a significant difference in the level of expression of these reaction patterns. Gill index obtained for pike clearly stands out as the lowest. Wels catfish showed the highest progressive reaction index, significantly higher in comparison with the other two species (P < 0.05), while pike-perch showed the highest regressive reaction index, also significantly higher in comparison with the other species (P < 0.001). These results may implicate species-specific gill reactions and thus present a useful tool for better understanding toxic mechanisms of various pollutants.

The peptide hormone cholecystokinin modulates the tonus and compliance of the bulbus arteriosus and pre-branchial vessels of the rainbow trout (Oncorhynchus mykiss).

The bulbus arteriosus is a compliant structure between the ventricle and ventral aorta of teleost fish. It serves as a "wind-kessel" that dampens pressure variations during the cardiac cycle allowing a continuous flow of blood into the gills. The bulbus arteriosus receives sympathetic innervation and is affected by several circulating substances, indicating neurohumoral control. We have previously shown that the peptide hormone, cholecystokinin (CCK), affects the hemodynamics of the cardiovascular system in rainbow trout (Oncorhynchus mykiss) by increasing flow pulse amplitude without affecting cardiac output. We hypothesized that this could be explained by an altered tonus or compliance/distensibility of the bulbus arteriosus. Our results show that there is a substantial effect of CCK on the bulbus arteriosus. Concentrations of CCK that altered the cardiac function of in situ perfused hearts also contracted the bulbus arteriosus in vitro. Pressure-volume curves revealed a change in both the tonus and the compliance/distensibility of this structure. Furthermore, the stimulatory (constricting) effect of CCK was also evident in the ventricle and vasculature leading to the gills, but absent in the atrium, efferent branchial arteries and dorsal aorta. In conclusion, CCK alters the mechanical properties of the ventricle, bulbus arteriosus, ventral aorta and afferent gill vasculature, thus maintaining adequate branchial and systemic blood flow and pressure when cardiorespiratory demands change, such as after feeding.

Optimal lamellar arrangement in fish gills.

Fish respire through gills, which have evolved to extract aqueous oxygen. Fish gills consist of filaments with well-ordered lamellar structures, which play a role in maximizing oxygen diffusion. It is interesting that when we anatomically observe the gills of various fish species, gill interlamellar distances (d) vary little among them, despite large variations in body mass (Mb). Noting that the small channels formed by densely packed lamellae cause significant viscous resistance to water flow, we construct and test a model of oxygen transfer rate as a function of the lamellar dimensions and pumping pressure, which allows us to predict the optimal interlamellar distance that maximizes the oxygen transfer rate in the gill. Comparing our theory with biological data supports the hypothesis that fish gills have evolved to form the optimal interlamellar distances for maximizing oxygen transfer. This explains the weak scaling dependence of d on Mb: d ∼ Mb(1/6).

The paracrine role of 5-HT in the control of gill blood flow.

Storage of serotonin in teleost gill cells has been detected in neurons, polymorphous granular cells and in neuroepithelial cells. Innervation from the glossopharyngeal nerve (first gill arch) and the vagus nerve (all gill arches) carries afferent as well as efferent fibers. This innervation extends to the efferent filament artery, including the sphincter muscle associated with the efferent filament artery, but except for the Antarctic fish does not reach the afferent filament artery. Serotonergic nerves as well as neuroepithelial cells have been shown to release serotonin, while very little is known about the polymorphous granular cells. The paracrine action of the released serotonin may affect vascular smooth muscle cells and pillar cells, which also contain contractile filaments. Already the earliest functional studies revealed a severe increase in branchial resistance as a result of serotonin application, combined with an increase in the perfusion of the arterio-venous path and the central sinus spaces of the gills. Pharmacological analysis demonstrated that this is a serotonin specific effect, which in Antarctic fish is due to activation of the 5-HT(2) receptor, while inhibition of the 5-HT(1) receptor does not reduce the serotonin induced vasoconstriction of gill blood vessels. Hypoxic degranulation of serotonergic cells evoked the hypothesis that serotonergic vasoconstriction might result in more even and overall better perfusion of gill lamellae. Microscopic analysis indicated, however, that perfusion of distal lamellae was reduced after serotonin application. Furthermore, a serotonergic increase in branchial resistance caused a decrease in dorsal arterial oxygen saturation, not an increase as would be expected as a result of a better perfusion of gill lamellae. A detailed analysis of hypoxic effects on gill perfusion revealed that hypoxia induced changes in gill blood flow are due to cholinergic effects, but serotonergic influences could not be detected. These observations contradict the hypothesis that serotonergic vasoconstriction might support hypoxic gas exchange. The functional significance of the serotonergic control of gill blood flow therefore is not yet totally clear. Recent observations indicate that specific inhibitors of serotonin re-uptake accumulate in freshwater and in estuaries. Considering the negative effect of serotonin on arterial blood oxygenation this may become a threat to teleost species.

Development of embryonic gill vasculature in the yellow stingray, Urobatis jamaicensis.

Corrosion casting was utilized to examine the development of gill vasculature in embryonic yellow stingrays, Urobatis jamaicensis (formerly Urolophus jamaicensis). The most marked changes in vascular configuration of the gills occur in the earliest castable stages of gestation. These changes included development of afferent external gill filament vessels and progression from paired dorsal aortae to a single fused dorsal aorta. Internal gill vasculature was found to nearly match that of an adult by the time the external gill filaments had fully regressed and yolk sac had been exhausted (>47 mm disc width). Examination of embryo casts also revealed characteristics of the branchial vasculature not previously reported in adult specimens. These include the presence of pre-lamellar sphincters, intertrematic branches, afferent distributing arteries, which supply blood to many afferent filament arteries resulting in greater interconnection of the filaments, and observation that the afferent branchial artery in the first hemibranch supplies blood directly to afferent filament arteries on the dorsal half of this arch.

Effects of hypoxia and petroleum on the genotoxic and morphological parameters of Hippocampus reidi.

Hypoxia events are common in many aquatic systems, which may be a natural event or provoked by anthropogenic actions, as well as accidents involving oil occurring throughout the world are frequent. Thus, through the possibility of occurrence of these two situations in same place the purpose of this study was to evaluate if damage caused by crude oil on genotoxic and morphological parameters in the marine fish species Hippocampus reidi will be aggravated by events of severe hypoxia. Sea horses were exposed during 8h to the following conditions: crude oil (OIL), severe hypoxia (HYP), association of severe hypoxia and crude oil (HYP+OIL) and normoxia without contaminant (CONT). An increase in micronuclei observed in OIL and HYP+OIL groups indicates that the crude oil exposure was a determining factor in the micronuclei induction and hypoxia did not intensify this result. In comet assays, both petroleum and hypoxia provoke DNA damage. The most frequent histopathology in the control groups and in those exposed to OIL and HYP+OIL groups were: hypertrophy and capillary dilation; hypertrophy and hyperplasia; hypertrophy, epithelial "lifting" and epithelial hyperplasia. An elongation of the lamellae was observed in fish from the two groups exposed to hypoxia, probably due to the fact that these groups required a greater flow of blood in the gills to increase the efficiency of gas exchange, since they were in a hypoxic environment. In summary, the micronuclei test and comet assay can be used as a good biomarker of contamination by petroleum. The association of hypoxia with crude oil in some aspects may exacerbate the responses of fish, in the light of the increase in DNA damage and the alterations in thickness of the gill epithelium.

Gross and fine anatomy of the respiratory vasculature of the mudskipper, Periophthalmodon schlosseri (Gobiidae: Oxudercinae).

To illustrate vascular modification accompanying transition from aquatic to amphibious life in gobies, we investigated the respiratory vasculatures of the gills and the bucco-opercular cavities in one of the most terrestrially-adapted mudskippers, Periophthalmodon schlosseri, using the corrosion casting technique. The vascular system of Pn. schlosseri retains the typical fish configuration with a serial connection of the gills and the systemic circuits, suggesting a lack of separation of O(2)-poor systemic venous blood and O(2)-rich effluent blood from the air-breathing surfaces. The gills appear to play a limited role in gas exchange, as evidenced from the sparsely-spaced short filaments and the modification of secondary lamellar vasculature into five to eight parallel channels that are larger than red blood cell size, unlike the extensive sinusoidal system seen in purely water-breathing fishes. In contrast, the epithelia of the bucco-opercular chamber, branchial arches, and leading edge of the filaments are extensively laden with capillaries having a short (<10 µm) diffusion distance, which strongly demonstrate the principal respiratory function of these surfaces. These capillaries form spiral coils of three to five turns as they approach the epithelial surface. The respiratory capillaries of the bucco-opercular chamber are supplied by efferent blood from the gills and drained by the systemic venous pathway. We also compared the degree of capillarization in the bucco-opercular epithelia of Pn. schlosseri with that of the three related intertidal-burrowing gobies (aquatic, non-air-breathing Acanthogobius hasta; aquatic, facultative air-breathing Odontamblyopus lacepedii; amphibious air-breathing Periophthalmus modestus) through histological analysis. The comparison revealed a clear trend of wider distribution of denser capillary networks in these epithelia with increasing reliance on air breathing, consistent with the highest aerial respiratory capacity of Pn. schlosseri among the four species.

Comparative physiology of the pulmonary circulation.

Two selective pressures have shaped the evolution of the pulmonary circulation. First, as animals evolved from heterothermic ectotherms to homeothermic endoderms with their corresponding increase in the ability to sustain high oxygen consumptions, the blood-gas barrier had to become successively thinner, and also provide an increasingly large area for diffusive gas exchange. Second, the barrier had to find a way to maintain its mechanical integrity in the face of extreme thinness, and this was assisted by the increasing separation of the pulmonary from the systemic circulation. A remarkable feature throughout the evolution of air-breathing vertebrates has been the tight conservation of the tripartite structure of the blood-gas barrier with its three layers: capillary endothelium, extracellular matrix, and alveolar epithelium. The strength of the barrier can be ascribed to the very thin layer of type IV collagen in the extracellular matrix. In the phylogenic progression from amphibia and reptiles to mammals and birds, the blood-gas barrier became successively thinner. Also, the area increased greatly reflecting the greater oxygen demands of the organism. The gradual separation of the pulmonary from the systemic circulation continued from amphibia through reptiles to mammals and birds. Only in the last two classes are the circulations completely separate with the result that the pulmonary capillary pressures can be maintained low enough to avoid stress failure of the blood-gas barrier. Remarkably, the barrier is generally much thinner in birds than mammals, and it is also much more uniform in thickness. These advantages for gas exchange can be explained by the support of avian pulmonary capillaries by the surrounding air capillaries. This arrangement was made possible by the adoption of the flow-through system of ventilation in birds as opposed to the reciprocating pattern in mammals.

Functional morphology of the gills of the shortfin mako, Isurus oxyrinchus, a lamnid shark.

This study examines the functional gill morphology of the shortfin mako, Isurus oxyrinchus, to determine the extent to which its gill structure is convergent with that of tunas for specializations required to increase gas exchange and withstand the forceful branchial flow induced by ram ventilation. Mako gill structure is also compared to that of the blue shark, Prionace glauca, an epipelagic species with lower metabolic requirements and a reduced dependence on fast, continuous swimming to ventilate the gills. The gill surface area of the mako is about one-half that of a comparably sized tuna, but more than twice that of the blue shark and other nonlamnid shark species. Mako gills are also distinguished from those of other sharks by shorter diffusion distances and a more fully developed diagonal blood-flow pattern through the gill lamellae, which is similar to that found in tunas. Although the mako lacks the filament and lamellar fusions of tunas and other ram-ventilating teleosts, its gill filaments are stiffened by the elasmobranch interbranchial septum, and the lamellae appear to be stabilized by one to two vascular sacs that protrude from the lamellar surface and abut sacs of adjacent lamellae. Vasoactive agents and changes in vascular pressure potentially influence sac size, consequently effecting lamellar rigidity and both the volume and speed of water through the interlamellar channels. However, vascular sacs also occur in the blue shark, and no other structural elements of the mako gill appear specialized for ram ventilation. Rather, the basic elasmobranch gill design and pattern of branchial circulation are both conserved. Despite specializations that increase mako gill area and efficacy relative to other sharks, the basic features of the elasmobranch gill design appear to have limited selection for a larger gill surface area, and this may ultimately constrain mako aerobic performance in comparison to tunas.

Cardiovascular system of the Majidae (Crustacea: Decapoda).

The cardiovascular system of majid crabs was mapped using corrosion casting techniques. The general form of the circulatory system was comparable to that of other malacostracan crustaceans, but with distinct differences between several arterial systems. The anterior aorta exited from the anterior surface of the heart supplying hemolymph to the antennae, eyestalks, gastric muscles and brain. This artery was more complex compared with other decapods. The anterolateral arteries exited from the anterior dorsal surface of the heart and supplied hemolymph to the hypodermis, stomach, antennal gland and mandibular muscles. The hepatic arteries were larger and more complex compared with other decapod families, branching profusely within the hepatopancreas and gonads. The small posterior aorta exited from the posterior-ventral surface of the heart. Standard sex-specific differences in this artery were observed. Exiting from the ventral surface of the heart, the sternal artery supplied each pereiopod in a segmental arrangement. The sternal artery arrangement was different to other brachyuran crabs, possibly a symplesiomorphy with segmented ancestors. In accordance with anatomical descriptions of blue crabs and Cancer crabs it would also seem appropriate to classify the circulatory system of the Majidae as one that is "incompletely closed".

A new species of Cardicola Short, 1953 (Digenea: Aporocotylidae) from the heart and branchial vessels of two surfperches (Perciformes: Embiotocidae) in the eastern Pacific Ocean off California.

Cardicola nonamo n. sp. (Digenea: Aporocotylidae) infects the heart of white seaperch, Phanerodon furcatus Girard, 1854 (Perciformes: Embiotocidae) (type host), in Monterey Bay, California, and the branchial vessels of rubberlip seaperch, Rhacochilus toxotes Agassiz, 1854 (Embiotocidae), from Naples Reef, Santa Barbara Channel, off Santa Barbara, California. It is most easily distinguished from other species of Cardicola Short, 1953 by the combination of having (1) rows of minute tegumental spines distributing along the entire ventrolateral body margin; (2) an aspinous anterior sucker comprising a nearly indistinct spheroid structure centering on the mouth; (3) an esophagus 17-21% of the total body length; (4) convoluted posterior ceca 51-65% of the total body length and 9-18x length of the anterior ceca; (5) a rectangular, intercecal testis not extending posteriad beyond the posterior ceca; (6) a post-testicular and post-cecal ovary; (7) an oviduct emanating from the posterodextral margin of the ovary; (8) a post-ovarian uterus coiling twice just posterior to the ovary; and (9) male and female genital pores opening dorsomedially and posterior to the uterus and ootype. The new species most closely resembles Cardicola ambrosioi Braicovich, Etchegoin, Timi, and Sardella, 2006 , which infects the blood vessels of the gill and liver of Brazilian flathead, Percophis brasiliensis Quoy and Gaimard, 1825 (Perciformes: Percophidae), in the southwestern Atlantic Ocean off Argentina; however, C. ambrosioi differs morphologically from the new species at least by lacking post-ovarian spine rows and by having posterior ceca that are 4x length of the anterior ceca. This is the first published record of an aporocotylid from a surfperch (Embiotocidae) as well as that of a species of Cardicola from the Pacific Ocean east of the Hawaiian Islands.

The relationship between O2 chemoreceptors, cardio-respiratory reflex and hypoxia tolerance in the neotropical fish Hoplias lacerdae.

The localization, distribution and orientation of O(2) chemoreceptors associated with the control of cardio-respiratory responses were investigated in the neotropical, Hoplias lacerdae. Selective denervation of the cranial nerves (IX and X) was combined with chemical stimulation (NaCN) to characterize the gill O(2) chemoreceptors, and the fish were then exposed to gradual hypoxia to examine the extent of each cardio-respiratory response. Changes in heart rate (f(H)) and ventilation amplitude (V(amp)) were allied with chemoreceptors distributed on both internal and external surfaces of all gill arches, while ventilation rate (f) was allied to the O(2) chemoreceptors located only in the internal surface of the first gill arch. H. lacerdae exposed to gradual hypoxia produced a marked bradycardia (45%) and 50% increase in V(amp), but only a relatively small change in f (32%). Thus, the low f(R) response yet high V(amp) were in accord with the characterization of the O(2) chemoreceptors. Comparing these results from H. lacerdae with hypoxia-tolerant species revealed a relationship existent between general oxygenation of the individual species environment, its cardio-respiratory response to hypoxia and the characterization of O(2) chemoreceptors.