Analysis of gene expression from the Wolbachia genome of a filarial nematode supports both metabolic and defensive roles within the symbiosis.

The α-proteobacterium Wolbachia is probably the most prevalent, vertically transmitted symbiont on Earth. In contrast with its wide distribution in arthropods, Wolbachia is restricted to one family of animal-parasitic nematodes, the Onchocercidae. This includes filarial pathogens such as Onchocerca volvulus, the cause of human onchocerciasis, or river blindness. The symbiosis between filariae and Wolbachia is obligate, although the basis of this dependency is not fully understood. Previous studies suggested that Wolbachia may provision metabolites (e.g., haem, riboflavin, and nucleotides) and/or contribute to immune defense. Importantly, Wolbachia is restricted to somatic tissues in adult male worms, whereas females also harbor bacteria in the germline. We sought to characterize the nature of the symbiosis between Wolbachia and O. ochengi, a bovine parasite representing the closest relative of O. volvulus. First, we sequenced the complete genome of Wolbachia strain wOo, which revealed an inability to synthesize riboflavin de novo. Using RNA-seq, we also generated endobacterial transcriptomes from male soma and female germline. In the soma, transcripts for membrane transport and respiration were up-regulated, while the gonad exhibited enrichment for DNA replication and translation. The most abundant Wolbachia proteins, as determined by geLC-MS, included ligands for mammalian Toll-like receptors. Enzymes involved in nucleotide synthesis were dominant among metabolism-related proteins, whereas the haem biosynthetic pathway was poorly represented. We conclude that Wolbachia may have a mitochondrion-like function in the soma, generating ATP for its host. Moreover, the abundance of immunogenic proteins in wOo suggests a role in diverting the immune system toward an ineffective antibacterial response.

Pronounced in vivo hemoglobin polymerization in red blood cells of Gulf toadfish: a general role for hemoglobin aggregation in vertebrate hemoparasite defense?

Two human hemoglobin (Hb) variants, Hb C and Hb S, are known to protect against Plasmodium falciparum malaria and have evolved repeatedly in malaria endemic areas. Both aggregate to insoluble crystals (Hb C) or polymers (Hb S) under certain physiological conditions, impair parasite growth, and may facilitate retention of infected red blood cells (RBCs) in the spleen. Given the profound effects of parasites on host evolution in general, and that RBC Hb concentration is often close to its solubility limit throughout vertebrates, similar mechanisms may operate in nonhuman vertebrates. Here we show exercise-induced, profound in vivo Hb polymerization in RBCs of the Gulf toadfish. Hb aggregation was closely associated with the extent of plasma acidosis, fully reversible, and without any signs of hemolysis or anemia. Our literature analysis suggests that aggregation prone Hbs may be relatively old, evolved multiple times in nonhuman vertebrates, show enhanced aggregation during hemoparasite infections, and can be uncovered in vivo by splenectomy. We discuss the working hypothesis that widespread Hb aggregation within several vertebrate groups may be the result of ongoing or past selection pressure against RBC parasites. Further comparative studies of these evolutionary old systems may provide valuable insights into hemoparasite susceptibility and reservoir potential of livestock and companion animals but also into human malaria and sickle cell disease.

Minimal volume regulation after shrinkage of red blood cells from five species of reptiles.

Red blood cells (RBCs) from most vertebrates restore volume upon hypertonic shrinkage and the mechanisms underlying this regulatory volume increase (RVI) have been studied extensively in these cells. Despite the phylogenetically interesting position of reptiles, very little is known about their red cell function. The present study demonstrates that oxygenated RBCs in all major groups of reptiles exhibit no or a very reduced RVI upon approximately 25% calculated hyperosmotic shrinkage. Thus, RBCs from the snakes Crotalus durissus and Python regius, the turtle Trachemys scripta and the alligator Alligator mississippiensis showed no statistically significant RVI within 120 min after shrinkage, while the lizard Tupinambis merianae showed 22% volume recovery after 120 min. Amiloride (10(-4) M) and bumetanide (10(-5) M) had no effect on the RVI in T. merianae, indicating no involvement of the Na(+)/H(+) exchanger (NHE) or the Na(+)/K(+)/2Cl(-) co-transporter (NKCC) or insentive transporters. Deoxygenation of RBCs from A. mississippiensis and T. merianae did not significantly affect RVI upon shrinkage. Deoxygenation per se of red blood cells from T. merianae elicited a slow volume increase, but the mechanism was not characterized. It seems, therefore, that the RVI response based on NHE activation was lost among the early sauropsids that gave rise to modern reptiles and birds, while it was retained in mammals. An RVI response has then reappeared in birds, but based on activation of the NKCC. Alternatively, the absence of the RVI response may represent the most ancient condition, and could have evolved several times within vertebrates.

Genomic diversity of the human intestinal parasite Entamoeba histolytica.

BACKGROUND: Entamoeba histolytica is a significant cause of disease worldwide. However, little is known about the genetic diversity of the parasite. We re-sequenced the genomes of ten laboratory cultured lines of the eukaryotic pathogen Entamoeba histolytica in order to develop a picture of genetic diversity across the genome. RESULTS: The extreme nucleotide composition bias and repetitiveness of the E. histolytica genome provide a challenge for short-read mapping, yet we were able to define putative single nucleotide polymorphisms in a large portion of the genome. The results suggest a rather low level of single nucleotide diversity, although genes and gene families with putative roles in virulence are among the more polymorphic genes. We did observe large differences in coverage depth among genes, indicating differences in gene copy number between genomes. We found evidence indicating that recombination has occurred in the history of the sequenced genomes, suggesting that E. histolytica may reproduce sexually. CONCLUSIONS: E. histolytica displays a relatively low level of nucleotide diversity across its genome. However, large differences in gene family content and gene copy number are seen among the sequenced genomes. The pattern of polymorphism indicates that E. histolytica reproduces sexually, or has done so in the past, which has previously been suggested but not proven.

In vivo red blood cell sickling and mechanism of recovery in whiting, Merlangius merlangus.

Haemoglobin concentrations in vertebrate red blood cells are so high that in human sickle cell disease a single surface amino acid mutation can result in formation of large insoluble haemoglobin aggregates at low oxygen levels, causing peculiar cell deformations or 'sickling'. This may cause vascular occlusion and thereby severe pain, organ failure and death. Here, using light and transmission electron microscopy, we demonstrate extensive in vivo sickling of whiting red blood cells after capture stress without any apparent haemolysis and show its subsequent recovery. We show exceptionally high cooperative proton binding during the sickling process in vitro and identify the reduction of extracellular pH below resting values as the primary cause for in vivo sickling, although the response is modulated to a lesser extent also by oxygen tension. Using isotope tracer fluxes, we further show that beta-adrenergic hormones, which are released under capture stress, activate a powerful endogenous Na/H exchanger in these fish red blood cells, which is known to elevate intracellular pH. beta-adrenergic treatment further leads to a marked reduction of acid-induced in vitro sickling, which is impaired when Na/H exchange is inhibited by amiloride. We propose that this mechanism protects red blood cells of some fishes against the problem of haemoglobin aggregation and red blood cell sickling, except under most severe acidosis. This system offers a unique example of how, over evolutionary time, nature may have overcome what is still a deadly disease in humans.

Oxygen-sensitive regulatory volume increase and Na transport in red blood cells from the cane toad, Bufo marinus.

The red blood cells (RBCs) of cane toad, Bufo marinus, are only partially saturated with oxygen in most of the circulation due to cardiac shunts that cause desaturation of arterial blood. The present study examines the oxygen dependency of RBC ouabain-insensitive unidirectional Na transport, using 22Na, in control cells and in cells exposed to hyperosmotic shrinkage or the beta-adrenergic agonist isoproterenol. Deoxygenation per se induced a slow, but significant Na influx, which was paralleled by a slow increase in RBC volume. Hyperosmotic shrinkage by a calculated 25% activated a robust Na influx that in the first 30 min had a strong PO2 dependency with maximal activation at low PO2 values and a P50 of approximately 5.5 kPa. This activation was completely abolished by the Na/H exchanger (NHE) inhibitor EIPA (10(-4) mol l(-1)). Hyperosmotic shrinkage is particularly interesting in B. marinus as it withstands considerable elevation in extracellular osmolarity following dehydration. Parallel studies showed that deoxygenated B. marinus RBCs had a much faster regulatory volume increase (RVI) response than air-equilibrated RBCs, reflecting the difference in magnitude of Na influxes at the two PO2 values. The extent of RVI ( approximately 60%) after 90 min, however, was similar under the two conditions, reflecting a more prolonged elevation of the shrinkage-induced Na influx in air-equilibrated RBCs. There were no significant differences in the ability to perform RVI between whole blood cells at a PCO2 of 1 and 3 kPa or washed RBCs, and 10(-4) mol l(-1) amiloride reduced the RVI under all conditions, whereas 10(-5) mol l(-1) bumetanide had no effect. Isoproterenol (10(-5) mol l(-1)) induced a significant and prolonged increase in an EIPA-sensitive and bumetanide-insensitive Na influx at low PO2 under iso-osmotic conditions, whilst there was no stimulation by isoproterenol for up to 45 min in air-equilibrated RBCs. The prolonged beta-adrenergic activation of the Na influx at low PO2 is distinctly different from the rapid and transient stimulation in teleost RBCs, suggesting significant differences in the signal transduction pathways leading to transporter activation between vertebrate groups.

Two different oxygen sensors regulate oxygen-sensitive K+ transport in crucian carp red blood cells.

The O2 dependence of ouabain-independent K+ transport mechanisms has been studied by unidirectional Rb+ flux analysis in crucian carp red blood cells (RBCs). The following observations suggest that O2 activates K+-Cl- cotransport (KCC) and deactivates Na+-K+-2Cl- cotransport (NKCC) in these cells via separate O2 sensors that differ in their O2 affinity. When O2 tension (PO2) at physiological pH 7.9 was increased from 0 to 1, 4, 21 or 100 kPa, K+ (Rb+) influx was increasingly inhibited, and at 100 kPa amounted to about 30% of the value at 0 kPa. This influx was almost completely Cl- dependent at high and low PO2, as shown by substituting Cl- with nitrate or methanesulphonate. K+ (Rb+) efflux showed a similar PO2 dependence as K+ (Rb+) influx, but was about 4-5 times higher over the whole PO2 range. The combined net free energy of transmembrane ion gradients favoured net efflux of ions for both KCC and NKCC mechanisms. The KCC inhibitor dihydroindenyloxyalkanoic acid (DIOA, 0.1 mM) abolished Cl- -dependent K+ (Rb+) influx at a PO2 of 100 kPa, but was only partially effective at low PO2 (0-1 kPa). At PO2 values between 0 and 4 kPa, K+ (Rb+) influx was further unaffected by variations in pH between 8.4 and 6.9, whereas the flux at 21 and 100 kPa was strongly reduced by pH values below 8.4. At pH 8.4, where K+ (Rb+) influx was maximal at high and low PO2, titration of K+ (Rb+) influx with the NKCC inhibitor bumetanide (1, 10 and 100 microM) revealed a highly bumetanide-sensitive K+ (Rb+) flux pathway at low PO2, and a relative bumetanide-insensitive pathway at high PO2. The bumetanide-sensitive K+ (Rb+) influx pathway was activated by decreasing PO2, with a PO2 for half-maximal activation (P50) not significantly different from the P50 for haemoglobin O2 binding. The bumetanide-insensitive K+ (Rb+) influx pathway was activated by increasing PO2 with a P50 significantly higher than for haemoglobin O2 binding. These results are relevant for the pathologically altered O2 sensitivity of RBC ion transport in certain human haemoglobinopathies.

Evolution of oxygen secretion in fishes and the emergence of a complex physiological system.

We have reconstructed the events that led to the evolution of a key physiological innovation underpinning the large adaptive radiation of fishes, namely their unique ability to secrete molecular oxygen (O2). We show that O2 secretion into the swimbladder evolved some 100 million years after another O2-secreting system in the eye. We unravel the likely sequence in which the functional components of both systems evolved. These components include ocular and swimbladder countercurrent exchangers, the Bohr and Root effects, the buffering power and surface histidine content of hemoglobins, and red blood cell Na+/H+ exchange activity. Our synthesis reveals the dynamics of gains and losses of these multiple traits over time, accounting for part of the huge diversity of form and function in living fishes.