Phenotypic plasticity and the possible role of genetic assimilation: Hypoxia-induced trade-offs in the morphological traits of an African cichlid

In this study we investigate the possible role of phenotypic plasticity and genetic assimilation in the process of adaptation and evolutionary change in the cichlid Pseudocrenilabrus multicolor victoriae . In the field we compared a population of a stable hypoxic habitat with one of a stable well-oxygenated habitat. In the laboratory, we compared individuals from the same mother raised under hypoxic or well-oxygenated conditions to examine phenotypic plasticity. Morphological parameters of three categories were measured: (a) the gill apparatus, (b) the surrounding structural elements, and (c) the outer shape of the fish. Swamp-dwelling fish had a 29% greater total gill surface area than fish from the well-oxygenated habitat due to their larger gill filament length and greater lamellar area. In the plasticity experiment, total gill surface area was 18% greater in the hypoxia group due to a larger number of longer filaments. Surrounding elements and outer shape also differed between the field populations and between fish grown under hypoxic and well-oxygenated conditions, but there was disparity between the field results and the plasticity experiment. The disparity between field and experimental fish may be due to: (a) differences in selection pressures between populations, (b) different constraints for genetic and plasticity changes, or (c) selection against plastic responses to hypoxia. Our results suggest that both (a) and (c) are involved.     

Brood protection at a cost: mouth brooding under hypoxia in an African cichlid

This study quantifies the behavioral response of the widespread mouth brooding African cichlid Pseudocrenilabrus multicolor victoriae to progressive hypoxia. We exposed four gender/stage classes of P. multicolor (males, brooding females, females that had just released young, and non-brooding females) to progressive hypoxia and recorded the percent time spent using aquatic surface respiration (surface skimming, ASR) and gill ventilation rates. This was done for fish collected from three sites in Uganda (lake, swamp, and river) after long-term acclimation to normoxia. There was no effect of site of origin on response to hypoxia, but ASR thresholds did differ between gender/stage classes. The oxygen level (threshold) at which spent 10, 50, and 90% of their time at the surface using ASR was much higher for brooding females than for males, whereas ASR thresholds did not differ between non-brooding females and males. Similarly, the level at which ASR was initiated was much higher in brooding females than males, but did not differ between males and non-brooders, or between males and females than had just released young. The rate of gill ventilation dropped significantly in males and all stages of females after initiation of ASR, suggesting that surface skimming increases efficiency of oxygen acquisition. These results suggest that mouth brooding in female P. multicolor ASR improves oxygen uptake but imposes a cost in terms of time spent at the water surface, and this may affect maternal predation risk in low-oxygen habitats.     

Hypoxia and male behaviour in an African cichlid Pseudocrenilabrus multicolor victoriae

This study tested the prediction that hypoxia may reduce the frequency of energetically expensive behaviours by quantifying male mating and aggressive displays in the cichlid Pseudocrenilabrus multicolor victoriae after long-term acclimation (5 months) to either high dissolved oxygen (DO) or low DO. Regardless of DO treatment, males engaged in more aggressive displays than mating displays; however, males acclimated to low DO reduced their total number of displays compared to high DO-acclimated males.     

Respiratory oxygen consumption among groups of Pseudocrenilabrus multicolor victoriae subjected to different oxygen concentrations during development

The routine metabolic rate of Pseudocrenilabrus multicolor victoriae , laboratory-reared, F1 offspring of parents collected from a hypoxic swamp, did not differ between sibs raised under normoxia and hypoxia. In the offspring of parents originating from a river where the oxygen levels were generally high but fluctuating, the metabolic rate of fish raised under normoxia was higher than for sibs raised under extreme hypoxia. Although further experiments with a larger random sample of the parental stock will be necessary to validate the patterns, the difference between the experiments may indicate the selective environment of the parents.     

The effect of hypoxia on sex hormones in an African cichlid Pseudocrenilabrus multicolor victoriae

Human activities increase the occurrence of aquatic hypoxia (low dissolved oxygen) globally. In fishes, short term hypoxia impairs multiple stages of reproduction (e.g., behavior, hormones, development), but no studies have investigated a species that lives and reproduces under hypoxia. This study examines the effects of hypoxia on sex hormones in the mouth brooding African cichlid Pseudocrenilabrus multicolor victoriae. Non-invasive measures of testosterone and estradiol levels in females were collected across the reproductive cycle in the laboratory, and at the time of capture in the field. In the laboratory, hormone levels were higher during pre-brooding (T=1.06, E2=1.62pg/mL/h) than brooding (T=0.61, E2=0.34pg/mL/h) or post-brooding (T=0.53, E2=0.51pg/mL/h) phases, but did not differ between hypoxic (1.2±0.0mg/L) and normoxic (7.3±0.1mg/L) populations. In the field, females were sampled from one low and one high oxygen population in two regions in Uganda (Mpanga River, Nabugabo Region). In both regions, hypoxic populations exhibited higher levels of testosterone than well-oxygenated populations, although there was no population level difference in estradiol levels. Hypoxic sites were also characterized by a higher testosterone/estradiol ratio and a lower proportion of brooding females. These results provide field evidence of hypoxia-mediated endocrine disruption in a fish species that experiences lifelong hypoxia.     

Environmental determination of sex in Apistogrammai (Cichlidae) and two other freshwater fishes (Teleostei)

Environmental sex determination by temperature could be revealed significantly in 33 Apistogramma-species and in Poecilia melanogaster . In some, but not all, Apistogramma-species pH also influences the sex ratio, whereas neither temperature nor pH affect the sex ratio of Pseudocrenilabrus multicolor victoriae . The sex in offspring of A. trifasciata is determined within a sensitive period of about 30 to at least 40 days after spawning.     

Hypoxia tolerance of two centrarchid sunfishes and an introduced cichlid from karstic Everglades wetlands of southern Florida, U.S.A.

In this study, the hypoxia tolerance of three Everglades fishes, two native centrarchids ( Lepomis gulosus and Lepomis marginatus ) and a recently introduced cichlid ( Hemichromis letourneuxi ), were documented. Aquatic surface respiration (ASR) thresholds were lowest for H. letourneuxi , followed by L. gulosus , then L. marginatus . The ASR thresholds for L. marginatus were within ranges reported for small, freshwater tropical fishes, while those for L. gulosus were similar to swamp-adapted fishes. For H. letourneuxi , ASR thresholds were some of the lowest reported. All three species showed excellent tolerance of low dissolved oxygen levels when allowed access to the surface. When denied surface access, L. marginatus lost equilibrium at a higher oxygen tension than the other species. Overall, although all species easily tolerated hypoxia, H. letourneuxi appeared to be best equipped to deal with hypoxia, followed by L. gulosus , then L. marginatus . Hemichromis letourneuxi also exhibited more aggressive behaviours than the centrarchids. These results suggest that hypoxia is not likely to prevent H. letourneuxi from exploiting the seasonally inundated wetlands of south Florida while expanding its range there.     

The influence of aquatic surface respiration (ASR) on cardio-respiratory function of the serrasalmid fish Piaractus mesopotamicus

When exposed to severely hypoxic water, many teleosts skim the better oxygenated surface layer (aquatic surface respiration, ASR). Information is scarce concerning the thresholds triggering ASR and its cardio-respiratory consequences. To assess the ambient conditions leading to ASR and to evaluate its effects on cardio-respiratory function, we exposed specimens of Piaractus mesopotamicus to gradual hypoxia (water oxygen tension ranging from 120 to 10 torr) with or, alternatively, without access to the surface. Concurrently, ASR, cardiac and respiratory frequencies, O₂ uptake and gill ventilation were monitored. With surface access, ASR developed below the critical tension for O₂ uptake (34 torr) by normal gill ventilation. Moreover, the time spent in ASR increased with prolonged hypoxic exposure to a maximum of 95% of total time. Without surface access, the species exhibited hypoxic bradycardia, that had not occurred in the group with fully developed ASR. Even without ASR, P. mesopotamicus recovered readily from hypoxic exposure, showing that this species possesses a number of mechanisms to cope with environmental hypoxia.     

Evolution of the ribosomal RNA internal transcribed spacer one (ITS-1) in cichlid fishes of the Lake Victoria region

The nucleotide sequences of the first internal transcribed spacer (ITS-1) of the ribosomal RNA gene cluster have been determined for 11 species of closely related endemic cichlid fishes of the Lake Victoria region (LVR) and 6 related East African cichlids. The ITS-1 sequences confirmed independently derived basal phylogenies, but provide limited insight within this species flock. The line leading to Pseudocrenilabrus multicolor arose early, close to the divergence event that separated the tilapiine and haplochromine tribes of the "African Group" of the family Cichlidae. In this phylogeny, Astatoreochromis alluaudi and the riverine Astatotilapia burtoni are sister taxa, which together are a sister group to a monophyletic assemblage including both Lake Victoria and Lake Edward taxa. The ITS-1 data support the monophyly of haplochromine genera across lakes. Since Lake Victoria is believed to have been dry between 14, 500 and 12,400 BPE, the modern assemblage must have been derived from reinvasion by the products of earlier cladogenesis events. Thus, although the regional superflock is monophyletic, the haplochromines of Lake Victoria itself did not evolve in situ from a single ancestor.     

Geographic variation in phenotypic plasticity in response to dissolved oxygen in an African cichlid fish

Genetic adaptation and phenotypic plasticity are two ways in which organisms can adapt to local environmental conditions. We examined genetic and plastic variation in gill and brain size among swamp (low oxygen; hypoxic) and river (normal oxygen; normoxic) populations of an African cichlid fish, Pseudocrenilabrus multicolor victoriae. Larger gills and smaller brains should be advantageous when oxygen is low, and we hypothesized that the relative contribution of local genetic adaptation vs. phenotypic plasticity should be related to potential for dispersal between environments (because of gene flow’s constraint on local genetic adaptation). We conducted a laboratory‐rearing experiment, with broods from multiple populations raised under high‐oxygen and low‐oxygen conditions. We found that most of the variation in gill size was because of plasticity. However, both plastic and genetic effects on brain mass were detected, as were genetic effects on brain mass plasticity. F₁ offspring from populations with the highest potential for dispersal between environments had characteristically smaller and more plastic brains. This phenotypic pattern might be adaptive in the face of gene flow, if smaller brains and increased plasticity confer higher average fitness across environment types.     

Population genetic structure across dissolved oxygen regimes in an African cichlid fish

Ecological isolation is a process whereby gene flow between selective environments is reduced due to selection against maladapted dispersers, migrant alleles, or hybrids. Although ecological isolation has been documented in several systems, gene flow can often be high among selective regimes, and more studies are thus needed to better understand the conditions under which ecological gradients or divergent selective regimes should influence population structure. We test for ecological isolation in a system in which high plasticity occurs with respect to traits that are adaptive in alternate forms under different environmental conditions. Pseudocrenilabrus multicolor victoriae is a widespread haplochromine cichlid fish in East Africa that exploits both normoxic (normal oxygen) rivers/lakes and hypoxic (low oxygen) swamps. Here, we examine population structure, using mitochondrial DNA and microsatellites, to determine if genetic divergence is significantly increased between dissolved oxygen regimes relative to within them, while controlling for geographical structure. Our results indicate that geographical separation influences population structure, while no effects of divergent selection with respect to oxygen regimes were detected. Specifically, we document (i) genetic clustering according to geographical region, but no clustering according to oxygen regime; (ii) higher genetic variation among than within regions, but no effect of oxygen regime on genetic variation; (iii) isolation by distance within one region; and (iv) decreasing genetic variability with increasing geographical distance from Lake Victoria. We speculate that plasticity may be facilitating gene flow between oxygen regimes in this system.     

Metabolic and cardiorespiratory responses of summer flounder Paralichthys dentatus to hypoxia at two temperatures

To quantify the tolerance of summer flounder Paralichthys dentatus to episodic hypoxia, resting metabolic rate, oxygen extraction, gill ventilation and heart rate were measured during acute progressive hypoxia at the fish's acclimation temperature (22° C) and after an acute temperature increase (to 30° C). Mean ±s.e. critical oxygen levels (i.e. the oxygen levels below which fish could not maintain aerobic metabolism) increased significantly from 27 ± 2% saturation (2·0 ± 0·1 mg O(2) l(-1) ) at 22° C to 39 ± 2% saturation (2·4 ± 0·1 mg O(2) l(-1) ) at 30° C. Gill ventilation and oxygen extraction changed immediately with the onset of hypoxia at both temperatures. The fractional increase in gill ventilation (from normoxia to the lowest oxygen level tested) was much larger at 22° C (6·4-fold) than at 30° C (2·7-fold). In contrast, the fractional decrease in oxygen extraction (from normoxia to the lowest oxygen levels tested) was similar at 22° C (1·7-fold) and 30° C (1·5-fold), and clearly smaller than the fractional changes in gill ventilation. In contrast to the almost immediate effects of hypoxia on respiration, bradycardia was not observed until 20 and 30% oxygen saturation at 22 and 30° C, respectively. Bradycardia was, therefore, not observed until below critical oxygen levels. The critical oxygen levels at both temperatures were near or immediately below the accepted 2·3 mg O(2) l(-1) hypoxia threshold for survival, but the increase in the critical oxygen level at 30° C suggests a lower tolerance to hypoxia after an acute increase in temperature.     

Systemic Respiratory Adaptations to Air Exposure in Intertidal Decapod Crustaceans

SYNOPSIS: The responses of intertidal decapods to emersion areclosely related to the particular conditions of emersion, yetall members of this group of animals face the problems of watershortage and internal hypoxia during air exposure. Several speciesexhibit modification of normal ventilatory activity and thisresponse seems to enable these crabs to take up seawater fromthe substrate. Other crabs have specific morphological adaptationspermitting recirculation of water from the exhalent aperturesback into the gill chamber. The hemocyanin of some species hasa higher affinity for oxygen, and this difference may be moreprevalent in tropical animals. The higher oxygen affinity undoubtedlycompensates in part for the lower internal oxygen tensions duringair exposure. Structural specialization of the branchial apparatusmay prevent the gill lamellae from adhering together, a processwhich reduces the surface available for gas exchange. Thereis a wide range of responses to emersion and yet relativelyfew specific adaptations. Some species are able to merely tolerateair exposure, while others are able to more fully exploit thehabitat.     

Hypoxia tolerance of three native and three alien species of bitterling inhabiting Lake Kasumigaura,Japan

Differences in hypoxia tolerance among three native and three alien bitterling species were examined by means of field surveys and aquarium experiments. I caught fish in minnow traps and measured environmental factors such as dissolved oxygen and current velocity once a month between June and November 2006 at 30 points around Lake Kasumigaura, Japan. In addition, aquarium experiments were carried out to compare aquatic surface respiration thresholds among the bitterling species. Much more individuals of an alien species, Rhodeus ocellatus ocellatus, was caught at the least oxygen range than expected in the field, and showed the highest hypoxia tolerance in the laboratory experiments. Another alien bitterling, Acheilognathus rhombeus, also showed higher hypoxia tolerance than the three native bitterling species. Lake Kasumigaura is well known for eutrophication and water masses with low dissolved oxygen have often been observed there. Differences in hypoxia tolerance among bitterling species might have affected bitterling assemblage structure in Lake Kasumigaura, which is currently characterized by the dominance of R. o. ocellatus.     

Physiological refugia: swamps,hypoxia tolerance and maintenance of fish diversity in the Lake Victoria region

In Lake Nabugabo, Uganda, a satellite of Lake Victoria, approximately 50% of the indigenous fishes disappeared from the open waters subsequent to the establishment of the introduced predatory Nile perch, Lates niloticus. This pattern is similar to the faunal loss experienced in the much larger Lake Victoria. Several of these species persisted in wetland refugia (e.g. ecotonal wetlands, swamp lagoons); however, deep swamp refugia (habitats lying well within the dense interior of fringing wetlands), are available only to a subset of the basin fauna with extreme tolerance to hypoxia. Although air-breathers are common in deep swamp refugia; we also documented a surprisingly high richness and abundance of non-air-breathing fishes. We describe several mechanisms that may facilitate survival in deep swamp refugia including high hemoglobin concentration, high hematocrit, large gill surface area and a low critical oxygen tension (P(c)). In addition, swamp-dwelling fishes showed lower PO(2) thresholds for onset of aquatic surface respiration than the lake-dwelling fishes. This suggests higher tolerance to hypoxia in the swamp fishes because they are able to withstand a lower oxygen tension before approaching the surface. We suggest that physiological refugia may be important in modulating the impact of Nile perch and indigenous fishes in the Lake Nabugabo region; this highlights the need to evaluate relative tolerance of introduced predators and indigenous prey to environmental stressors.     

Behavioural responses of a south-east Australian floodplain fish community to gradual hypoxia

1. Hypoxic conditions occur frequently during hot, dry summers in the small lentic waterbodies (billabongs) that occur on the floodplains of the Murray‐Darling River system of Australia. Behavioural responses to progressive hypoxia were examined for the native and introduced floodplain fish of the Ovens River, an unregulated tributary of the Murray River in south‐east Australia. 2. Given the high frequency of hypoxic episodes in billabongs on the Ovens River floodplain, it was hypothesised that all species would exhibit behaviours that would confer a degree of hypoxia‐tolerance. Specifically, it was hypothesised that as hypoxia progressed, gill ventilation rates (GVRs) would increase and aquatic surface respiration (ASR) would become increasingly frequent. Fish were subjected to rapid, progressive hypoxia from normoxia to anoxia in open tanks. 3. All tested species exhibited behaviours consistent with their use of potentially hypoxic habitats. As hypoxia progressed, GVRs increased and all species, with the exception of oriental weatherloach, began to switch increasingly to ASR with 90% of individuals using ASR at various oxygen concentrations below 1.0 mg O₂ L⁻¹. Australian smelt, redfin perch and flat‐headed galaxias were the first three species to rise to ASR, with 10% of individuals using ASR by 2.55, 2.29 and 2.21 mg O₂ L⁻¹ respectively. Goldfish and common carp were the last two species to rise to ASR, with 10% of individuals using ASR by 0.84 and 0.75 mg O₂ L⁻¹ respectively. In contrast to other species, oriental weatherloach largely ceased gill ventilation and used air‐gulping as their primary means of respiration during severe hypoxia and anoxia. 4. Australian smelt, redfin perch and flat‐headed galaxias were unable to maintain ASR under severe hypoxia, and began exhibiting erratic movements, termed terminal avoidance behaviour, and loss of equilibrium. All other species continued to use ASR through severe hypoxia and into anoxia. Following a rise to ASR, GVRs either remained steady or decreased slightly indicating partial or significant relief from hypoxic stress for these hypoxia‐tolerant species. 5. Behavioural responses to progressive hypoxia amongst the fish species of the Ovens River floodplain indicate a generally high level of tolerance to periodic hypoxia. However, species‐specific variation in hypoxia‐tolerance may have implications for community structure of billabong fish communities following hypoxic events.     

Hypoxia and interdemic variation in Poecilia latipinna

Variation in respiratory traits was quantified between two populations of the sailfin molly Poecilia latipinna (one from a periodically hypoxic salt marsh, Cedar Key, and one from a chronically normoxic river site, Santa Fe River). Two suites of characters were selected: traits that may show both short‐term acclimation response and interdemic variation in acclimation response (metabolic rate, critical oxygen tension and respiratory behaviour), and those that are not likely to respond to short‐term acclimation but may vary among populations (gill morphometric characters). Sailfin mollies from the salt marsh, acclimated to hypoxia (1 mg l⁻¹, c . 20 mmHg) for 6 weeks, spent less time conducting aquatic surface respiration and had lower gill ventilation rates than hypoxia‐acclimated conspecifics from the well‐oxygenated river site. Poecilia latipinna acclimated to hypoxia exhibited a lower critical oxygen tension ( P _c) than fish acclimated to normoxia; however, there was also a significant population effect. Poecilia latipinna from Cedar Key exhibited a lower P _c than fish from the Santa Fe River, regardless of acclimation. Cedar Key fish had a 14% higher mean gill surface area relative to fish from the Santa Fe River, a character that could account, at least in part, for their greater tolerance to hypoxia.     

Salinity effects on behavioural response to hypoxia in the non-native Mayan cichlid Cichlasoma urophthalmus from Florida Everglades wetlands

This study quantified the hypoxia tolerance of the Mayan cichlid Cichlasoma urophthalmus over a range of salinities. The species was very tolerant of hypoxia, using aquatic surface respiration (ASR) and buccal bubble holding when oxygen tensions dropped to <20 mmHg ( c. 1·0 mg l⁻¹) and 6 mmHg, respectively. Salinity had little effect on the hypoxia tolerance of C. urophthalmus , except that bubble holding was more frequent at the higher salinities tested. Levels of aggression were greatest at the highest salinity. The ASR thresholds of C. urophthalmus were similar to native centrarchid sunfishes from the Everglades, however, aggression levels for C. uropthalmus were markedly higher.     

Gill surface area provides a clue for the respiratory basis of brain size in the blacktip shark (Carcharhinus limbatus)

Brain size varies dramatically, both within and across species, and this variation is often believed to be the result of trade-offs between the cognitive benefits of having a large brain for a given body size and the energetic cost of sustaining neural tissue. One potential consequence of having a large brain is that organisms must also meet the associated high energetic demands. Thus, a key question is whether metabolic rate correlates with brain size. However, using metabolic rate to measure energetic demand yields a relatively instantaneous and dynamic measure of energy turnover, which is incompatible with the longer evolutionary timescale of changes in brain size within and across species. Morphological traits associated with oxygen consumption, specifically gill surface area, have been shown to be correlates of oxygen demand and energy use, and thus may serve as integrated correlates of these processes, allowing us to assess whether evolutionary changes in brain size correlate with changes in longer-term oxygen demand and energy use. We tested how brain size relates to gill surface area in the blacktip shark Carcharhinus limbatus. First, we examined whether the allometric slope of brain mass (i.e., the rate that brain mass changes with body mass) is lower than the allometric slope of gill surface area across ontogeny. Second, we tested whether gill surface area explains variation in brain mass, after accounting for the effects of body mass on brain mass. We found that brain mass and gill surface area both had positive allometric slopes, with larger individuals having both larger brains and larger gill surface areas compared to smaller individuals. However, the allometric slope of brain mass was lower than the allometric slope of gill surface area, consistent with our prediction that the allometric slope of gill surface area could pose an upper limit to the allometric slope of brain mass. Finally, after accounting for body mass, individuals with larger brains tended to have larger gill surface areas. Together, our results provide clues as to how fishes may evolve and maintain large brains despite their high energetic cost, suggesting that C. limbatus individuals with a large gill surface area for their body mass may be able to support a higher energetic turnover, and, in turn, a larger brain for their body mass.     

Isolation and characterization of tetranucleotide microsatellite markers in a mouth‐brooding haplochromine cichlid fish (Pseudocrenilabrus multicolor victoriae) from Uganda

Eight tetranucleotide microsatellite loci were isolated from the haplochromine cichlid fish, Pseudocrenilabrus multicolor victoriae, an important model species for studies in respiratory ecology, conservation, and evolution. We surveyed variation at these loci in 23 individuals from western Uganda, finding four to 19 alleles per locus and an average expected heterozygosity of 0.8575. These microsatellite loci will be used to examine gene flow and population structure in Ugandan P. m. victoriae.