Number and regulation of protozoan aquaporins reflect environmental complexity.
Protozoa occupy distinct environmental niches, as reflected by their morphology, differentiation during their life cycles, and molecular setup (Ginger, 2006). For instance, protozoan parasites that cause malaria (Plasmodium spp.), toxoplasmosis (Toxoplasma gondii), Chagas' disease (Trypanosoma cruzi), or leishmaniasis (Leishmania spp.) live a sheltered life (Leiriao et al., 2004; Kumar and Valdivia, 2009; Walker et al., 2014). After transmission from the insect vector, they retreat into human cells where they encounter stable conditions, have access to nutrients and metabolic precursors, and are at minimal risk of detection by the immune system (Hviid et al., 2015). Trypanosoma brucei parasites, which cause human African trypanosomiasis, also known as sleeping sickness, swim freely in the bloodstream (Vincent and Barrett, 2015). While there is no shortage of nutrients, mainly glucose from the host, the parasites are exposed to attacks by antibodies and immune cells (Mandal et al., 2014). With respect to the extent and promptness of adaptation, the highest demands are probably imposed on free-living protozoa such as Amoeba proteus (Nishihara et al., 2004) and Dictyostelium discoideum (Plattner, 2013), which encounter severe changes in osmotic conditions, e.g., due to rainfall or drought. AQP water and solute channels are localized at the cellular interface with the surroundings and must be considered players in such adaptation processes (Fadiel et al., 2009).
The tetrameric AQPs share a common protein structure consisting of six transmembrane spans connected by five loops (A-E), with the N-and C-termini located intracellularly. Two half-helices in loops B and E are capped by canonical Asn-Pro-Ala (NPA) signature motifs and form a pseudo seventh transmembrane span (Murata et al., 2000: Fu et al., 2000; Walz et al., 2009). AQP water and solute selectivity, as well as proton exclusion, is based partly on the NPA region plus a second, narrower constriction at the extracellular pore mouth typically holding an arginine in an aromatic environment, the so-called selectivity filter (Murata et al., 2000; de Groot and Grubmuller, 2001; Tajkhorshid et al., 2002; Beitz, 2006; Wu et al., 2009; Yamamoto et al., 2014). The AQP protein family can be functionally divided into water-specific, orthodox AQPs and aquaglyceroporins (Abascal et al., 2014) that are also permeated by small, uncharged solutes such as glycerol, urea, ammonia, and even metalloids such as arsenite. The directionality of water and solute diffusion via AQPs is determined by the prevailing osmotic and chemical gradients, respectively. Based on high-resolution crystal structures, sophisticated molecular dynamic simulations, and functional data obtained from multiple AQP isoforms and point mutants, the basic mechanisms of AQP pore selectivity and cation exclusion are now well understood (Fu et al., 2000; de Groot and Grubmiiller, 2001; Wu et al., 2009).
In mammals, AQPs are involved in the regulation of water homeostasis, in the formation of cerebrospinal fluid, and in epithelial moisturing, for example, of the lung and skin. Several physiological functions and pathophysiological situations in the eye, inner ear, and secretory gland, among others, depend on AQP water permeability. Glycerol permeability is required for maintaining the Cori cycle, i.e., release of glycerol from fatty tissue and uptake in the liver for gluconeogenesis (Agre et al., 2002). Plants express water-specific AQP isoforms in plasma membranes and in organelle membranes of cells in the roots (uptake), stems (transport of solutes such as sugars), and leaves (evaporation). Several plant AQPs possess gating mechanisms that open and close the water channel upon drought, pH shift, or intracellular signaling events.
Regarding protozoa, genome sequencing projects revealed AQPs in almost all species, with the cyst-forming apicomplexan Cryptosporidium spp. representing an exception because they apparently fully lack AQP encoding genes (Beitz, 2005). Can we attribute roles to protozoan AQPs in adaptation to the environment? Is a more complex environment reflected in the functionality or regulation of protozoan AQPs? We summarize the literature on protozoan AQPs with regard to environmental adaptation.
Intracellular Protozoan Parasites
Protozoa of the phylum Apicomplexa, e.g., Plasmodium spp. or Toxoplasma gondii, carry a unique, plastid-like organelle called the apicoplast (Fichera and Roos, 1997). The apicoplast is derived from an endosymbiosis event with a green alga (Huang et al., 2004). It is assumed that about 10% of all genes in the Plasmodium falciparum nucleus seem to be derived from intracellular gene transfer. Plasmodia are pathogenic and cause malaria in mammals, including humans (Beitz, 2007). After a mosquito bite, the parasites undergo one round of development in the liver before the newly formed merozoites retreat into erythrocytes. Within 48 hours, an infected erythrocyte releases up to 32 new merozoites. T. gondii, the causative agent of toxoplasmosis, is capable of invading any nucleated cell (Contreras-Ochoa et al., 2013).
The order Kinetoplastida (Baldauf et al., 2000) includes human-pathogenic parasites such as Leishmania spp. and Trypanosoma cruzi, and is characterized by the presence of a kinetoplast (Shapiro and Englund, 1995). This mitochondrial organelle contains a small genome in a dense granule and is located close to the cell-propelling flagellum. Leishmania are transmitted by the sand fly, retreat into macrophages, and cause leishmaniasis (Pearson and Sousa, 1996). T. cruzi is the causative agent of Chagas' disease in South Africa and in the Latin Americas (Rassi and Marcondes de Rezende, 2012). Triatominae bugs can carry and transfer the parasites to the host, where they can infest any nucleated cell (Couillard et al., 1989; Mandal et al., 2014).
Plasmodium spp. AQPs
The genomes of the malaria parasites encode a single aquaglyceroporin (Beitz, 2005). Having only one AQP is in line with the general reduction in number of proteins at the parasite-host interface of plasmodia (Gardner et al., 2002), probably to reduce antigenic stmctures at the cell surface. AQPs possess antigenic potential, as seen by the human Colton and GIL blood group antigens that are located on erythrocyte AQP1 (Smith et al., 1994) and AQP3 (Roudier et al., 2002). PfAQP from P. falciparum is evenly expressed in asexual blood-stage parasites during development from merozoites via trophozoites to schizonts, as well as in the sexual gametocyte forms. Its localization is restricted to the plasma membrane (Hansen et al., 2002) (Table 1). PfAQP is highly permeable for both water and glycerol. The PfAQP protein sequence is highly similar to that of the Escherichia coli glycerol facilitator GlpF (having 50% similar and 35% identical residues; Hansen et al., 2002), suggesting that in the malaria parasite a bifunctional solute channel of bacterial origin has evolved (Beitz, 2005). Besides glycerol, other solutes pass the PfAQP pore, i.e., ammonia, urea, polyols up to five carbon atoms, arsenite, and the glycolysis-related carbonyl compounds methylglyoxal or dihydroxyacetone (Hansen et al., 2002; Zeuthen et al., 2006; Pavlovic-Djuranovic et al., 2006).
Apicomplexan parasites are strictly intracellular and thus live in a well-defined environment (Beitz et al., 2004) (Fig. 1). Nevertheless, the parasites encounter osmotic stress during kidney passages or during transmission between human and insect hosts (Beitz, 2007). Furthermore, high proliferation rates render Plasmodium parasites dependent on provision of nutrients by the host red blood cells (Beitz, 2007). At the same time, anabolic processes, such as glycerolipid production for the formation of plasma membranes, are increased during cell growth. Reasonably, it is much more efficient for the parasite to use readily available blood glycerol as a precursor to biosynthesis of the glycerolipids than to generate glycerol from glucose, its sole energy source (Beitz, 2007). If high amounts of glycerol were derived from glucose, the NADH pool would suffer due to conversion to [NAD.sup.+]. Replenishing the NADH pool would lead to an accumulation of glutathione in the oxidized form, thus contributing to cell-damaging oxidative stress (Beitz, 2007). Here, high glycerol permeability of PfAQP fits the physiological needs well. The idea that the aquaglyceroporin is a crucial entry route for glycerol into the parasite has been confirmed by a Plasmodium berghei PbAQP deletion strain (Promeneur et al., 2007). PbAQP-null parasites are viable but highly deficient in glycerol transport. Furthermore, they proliferate only about half as fast as wild-type parasites; mice infected with PbAQP-null parasites survived about twice as long. Another function of PfAQP may be in disposal of metabolic waste. As a result of high proliferation rates and massive proteolysis of hemoglobin taken up from infected red blood cells, toxic side products can be expected in large quantities, e.g., methylglyoxal from glycolysis, urea from degradation of arginine, and ammonia from conversion of amino acids to [alpha]-keto acids (Beitz, 2007). PfAQP is permeable for these compounds and may provide an exit pathway to prevent the parasite from self-intoxication.
Toxoplasma gondii AQP
The genome of T. gondii encodes one classical 6-transmembrane domain AQP, TgAQP (Pavlovic-Djuranovic et al., 2003) (Table 1). A second AQP-like sequence is contained in the T. gondii GT1 strain genome annotation (gene ID: TGGT1_267070), which may appear as an unprecedented fusion of two consecutive AQPs. However, functional data on the latter are missing. BLAST analysis suggests a connection between TgAQP and plant tonoplast intrinsic proteins (TIPs) (the BLAST ranking based on the Expect (E) value favors TIP by four and eight orders of magnitude over prototypical AQPs from mammals and bacteria; see Pavlovic-Djuranovic et al., 2003). A phylogenetic relationship to TIPs is further stressed by the presence of a valine instead of the highly conserved arginine in the selectivity filter (Pavlovic-Djuranovic et al., 2003). Furthermore, a novel organelle has been characterized in T. gondii with a composition and function similar to plant vacuoles (for details see Miranda et al., 2010). TgAQP has been localized to the membrane of this organelle, which is suggested to be part of the endocytotic/lysosomal pathway. Expression in Xenopus laevis oocytes showed that TgAQP is an aquaglyceroporin with intermediate water and high glycerol permeability. The single TgAQP may contribute to osmotic and metabolic functions. Hydroxyurea and ammonia also pass the TgAQP pore.
Leishmania spp. AQPs
The L. major genome encodes five AQPs. While LmAQP1 shows strong similarity to bacterial, GlpF-like aquaglyceroporins, the remaining four L. major AQPs (LmAQP[alpha]-[delta]) are closer to plant TIPs (Beitz, 2005) (Table 1). The similarity manifests itself mainly in the amino acid composition of the selectivity filter region; comparable to the Toxoplasma gondii AQP, as mentioned above, LmAQP[alpha]-[delta] contain a lipophilic amino acid residue (valine or leucine) in the otherwise highly conserved arginine position. Only LmAQP1 has been studied in some detail, while the permeability profile of the other LmAQPs has yet to be established.
The initial characterization identified LmAQP1 as a channel conducting trivalent arsenic and antimony metalloids (Gourbal et al., 2004). Expression of LmAQP1 in different Leishmania species produced hypersensitivity to As[(OH).sub.3] and Sb[(OH).sub.3], whereas disruption of one of the two LmAQP1 alleles in L. major conferred a 10-fold increase in resistance to Sb[(OH).sub.3]. This finding relates directly to the first-line treatment of leishmaniasis with pentostam and glucantime, since both drugs release Sb[(OH).sub.3] as the active agent in the macrophage environment (Gourbal et al., 2004). To determine its physiological functions, LmAQP1 was expressed in Xenopus laevis oocytes, rendering them permeable to water, glycerol, methylglyoxal, dihydroxyacetone, and sugar alcohols (Figarella et al., 2007). Urea passed the pore only at a low rate, which may have implications for parasite survival within the host liver. Immunofluorescence microscopy revealed that LmAQP1 is exclusively localized at the flagellum. A physiological function of LmAQP1 was shown to be in volume regulation in response to hypo-osmotic stress. It is not yet clear whether permeability for water or compatible solutes accounts for the osmotic response. Additionally, the parasites migrate faster towards an osmotic gradient. This phenomenon of osmotaxis is thought to drive migration of Leishmania promastigotes during the insect phase, and is proposed to be essential for successful transmission of the parasite to the vertebrate host (Figarella et al., 2007). Phosphorylation of Thr197 by mitogen-activated protein kinase 2 positively regulates the stability of LmAQP1 and alters the parasite's osmoregulatory activity and drug sensitivity (Mandal et al., 2012). Recently, it was also found that the stability of LmAQP1 mRNA in different Leishmania species is regulated by their 3'-untranslated regions, resulting in different sensitivity to antimonite (Mandal et al., 2015). Together, physiological roles in solute transport, volume regulation, and osmotaxis can be attributed to LmAQP1.
The Leishmania donovani genome equally encodes an aquaglyceroporin, LdAQP1, and four TIP-like AQPs (Biyani et al., 2011). LdAQPs with C-terminal GFP-fusions have appeared in the nuclear membrane of L. donovani cells. Indirect phenotypic yeast growth assays seemed to indicate the water and solute permeability of certain LdAQPs. However, these data should probably be viewed as more preliminary and require consolidation.
Trypanosoma cruzi AQPs
Four AQP isoforms, TcAQP and TcAQP[beta]-[delta], can be expected in T. cruzi from the genome annotation (Montalvetti et al., 2004; Song et al., 2014) (Table 1). Thus far, only TcAQP has been functionally characterized. Its protein sequence is closely related to plant TIPs and expression occurs throughout all developmental stages of the parasite (Montalvetti et al., 2004). In Xenopus laevis oocytes, TcAQP yielded poor water permeability; solutes seemed not to pass. TcAQP-GFP fusion proteins were localized in the acidocalcisomes of the parasite and in a contractile vacuole close to the flagellar pocket (Montalvetti et al., 2004). Both acidocalcisomes and the contractile vacuole are parts of the contractile complex that allows various protozoa to cope with hypo-osmotic stress by active expulsion of water (Allen and Naitoh, 2002). In T. cruzi, hypo-osmotic stress leads to an increase in cyclic AMP, swelling of the acidocalcisomes, and displacement of TcAQP from the acidocalcisomes to the contractile vacuole (Rohloff et al., 2004). A contribution of AQPs in the cellular adaptation processes to hypo-osmotic stress in T. cruzi remains to be established (Mandal et al., 2014). AQPs also have been found in the contractile vacuole complexes of Amoeba proteus (Nishihara et al., 2008) and Dictyostelium discoideum (Nolta and Steck, 1994) and are addressed below.
Protozoan Parasites That Live Freely in the Human Blood
Trypanosoma brucei AQPs
Three AQPs have been identified in T. brucei, TbAQP1-3 (Uzcategui et al., 2004) (Table 1). They are of the aquaglyceroporin type and show 40%-45% protein sequence identity to mammalian AQP3 and AQP9 (Uzcategui et al., 2004). For functional analysis, TbAQP1-3 were expressed in yeast and X. laevis oocytes, yielding permeability for water, glycerol, dihydroxyacetone, and trivalent metalloids (Uzcategui et al., 2004). From this finding, one can assume various physiological functions. Glycerol efflux may be important in a unique energy generation mechanism of trypanosomes that involves the glycosome, i.e., a trypanosome-specific organelle (Steinborn et al., 2000). Under anaerobic conditions, glucose degradation within the glycosome results in equimolar amounts of oxidized pyruvate and reduced glycerol to maintain the redox balance (Steinborn et al., 2000). Glycerol is formed from glycerol-3-phosphate by glycerokinase, which transfers the phosphate moiety to ADP, yielding ATP. To drive this energetically highly unfavorable reaction, glycerol must be released swiftly from the chemical equilibrium. Thus, the glycerol efflux capability of the TbAQPs may fulfill a crucial physiological function in anaerobic trypanosome energy metabolism. Yet under aerobic conditions, glycerol influx may equally play a role in providing energy for trypanosomes by serving as an alternative fuel substrate (Eisenthal and Cornish-Bowden, 1998). In fact, glucose uptake is inhibited by 50% at 0.8 mmol [l.sup.1] external glycerol (Bakker et al., 1997). However, in light of 2-5 mmol [l.sup.1] concentrations of blood glucose, it remains to be clarified whether a glycerol concentration in the range of 50-200 [micro]mol [1.sup.-1] is relevant as an energy source (Uzcategui et al., 2004). In a knockdown experiment targeting all three TbAQPs, the cells were able to substitute any missing glycerol uptake; the presence of a glycerol transporter was suggested (Bassarak et al., 2011).
It is generally assumed that Trypanosoma brucei lives under fairly constant osmotic conditions in the bloodstream (Huang et al., 2011) (Fig. 1). However, like other protozoan parasites, trypanosomes may encounter osmotic stress during kidney passages, in the course of transmission, and during progression through different organs of the insect vector. In this context, the intermediate water permeability of TbAQPs may be relevant to osmo-adaptation. Further, passage of As[(OH).sub.3] was found for TbAQP1-3 (Uzcategui et al., 2013), similar to the Leishmania AQPs (Gourbal et al., 2004).
The expression profile of TbAQP transcripts suggests distinct roles of the three TbAQP proteins at the different stages of the life cycle (Uzcategui et al., 2004). TbAQP 1 is the only isoform expressed in procyclic, insect-stage trypanosomes. All three isoforms are present in blood-stage parasites and exhibit distinct subcellular localizations (Bassarak et al., 2011). TbAQP 1 is found in the flagellar membrane. This corresponds to the localization of Leishmania LmAQP1 and may be associated with a function in osmotaxis (Figarella et al., 2007). TbAQP2 is restricted to the flagellar pocket, and TbAQP3 is localized throughout the plasma membrane (Baker et al., 2012). When TbAQP1 or TbAQP3 were knocked down, the cells survived hypo-osmotic stress conditions; AQP2 knockdown parasites did not (Bassarak et al., 2011). TbAQP2 knockout cells also were found to be cross-resistant to melarsoprol and pentamidine (Baker et al., 2012). It was therefore assumed that TbAQP2 is part of the entry mechanism for those drugs (Munday et al., 2014).
Free-Living Amoebae in the Wild
Amoeba proteus lives unsheltered in freshwater and, as such, faces much harsher environmental challenges than protozoa within a human host (Couillard et al., 1989). A. proteus extends tentacles, the so-called pseudopodia, for motility and engulfing prey (Jeon, 1995). Dictyostelium discoideum grows and develops in the soil, where it is subjected to rapidly varying environmental stresses (Flick et al., 1997) (Fig. 1). Under favorable environmental conditions that provide sufficient nutrients or bacterial prey, D. discoideum exists as single-celled amoebae, whereas upon starvation the cells undergo a 24-hour developmental cycle; the amoebae stream together on the order of 50,000 cells to form multicellular aggregates, differentiate into prestalk and prespore cells, generate a fruiting body, and eventually produce spores that persist until the environment is more suitable again (Cotter and Raper, 1968b).
Amoeba proteus AQPs
In the genome of A. proteus, a single AQP gene has been identified (Nishihara et al., 2008) (Table 1). The deduced ApAQP protein appears to be phylogenetically related to plant AQPs of the plasma membrane intrinsic protein type (PIPs) and human water-specific AQPs (Nishihara et al., 2008). Water permeability of ApAQP was relatively low when assayed in Xenopus laevis oocytes; glycerol permeability was not tested for. Immunofluorescence studies localized ApAQP as uniformly distributed on the contractile vacuole and on surrounding vesicles of A. proteus, suggesting a role for ApAQP in osmoregulation, as has been described for TcAQP in Trypanosoma cruzi (Rohloff and Docampo, 2008). Details about the underlying mechanism are not yet known. The plasma membrane of A. proteus seems to be free of AQPs.
Dictyostelium discoideum AQPs
In the D. discoideum genome, four AQP-like genes have been found, AqpA-D, which have the closest phylogenetic relation to plant PIPs and TIPs (Flick et al., 1997; Mitra et al., 2000; Bulow etal., 2012, 2015) (Table 1). Expression of the D. discoideum AQPs is developmentally regulated. AqpB and AqpD are present in amoebae and in all subsequent developmental stages (Bulow et al., 2012, 2015), whereas AqpA (Mitra et al., 2000) and AqpC (Flick et al., 1997) start to appear later in multicellular forms and spores. GFP-fusion proteins of AqpB and AqpD were localized in intracellular structures and in the plasma membrane of D. discoideum (Billow et al., 2012, 2015). AqpB exhibited a punctate pattern throughout the plasma membrane and was found in lamellipodia-like plasma membrane protrusions (Billow et al., 2012 and unpubl. obs.). In situ-hybridization of AqpA (Mitra et al., 2000) and AqpC (Flick et al., 1997) mRNA showed expression in prespore, but not prestalk, cells, matching the localization of the respective proteins in the spores.
Sequence analyses and hydropathy predictions of the four D. discoideum AQPs exposed the typical signature motives and structural features. Hence, it came as quite a surprise that neither of the four AQPs was permeable to water when expressed in Xenopus laevis oocytes (Flick et al., 1997; Mitra et al, 2000; Billow et al., 2012, 2015). The AqpB protein contains an extended intracellular loop D and the N-terminal domain of AqpD is particularly long (Bulow et al., 2012) (Fig. 2). These features are reminiscent of gated AQPs from fungi and plants (Maurel et al., 2002). The water- and glycerol-conducting aquaglyceroporin Fps1 from Saccharomyces cerevisiae closes under hypertonic stress to accumulate intracellular glycerol for osmo-adaptation (Luyten et al., 1995). Elongated N-terminal (Tamas et al., 2003) and C-terminal domains (Hedfalk et al., 2004), as well as two residues in loop B (Karlgren et al., 2004), were required for Fps1 channel gating (Fig. 2). The water-specific Aqyl from Pichia pastoris mediates freeze tolerance and was crystallized in a closed state (Fischer et al., 2009). Here, Tyr31 of the N-terminus appears to be inserted into the pore, occluding it (Fig. 2). Spinach SoPIP2;l closes under conditions of drought stress or anoxia during flooding (Tornroth-Horsefield et al., 2006), and in a pH-dependent manner involving His-193 in loop D (Johansson et al., 1998). Opening is achieved by phosphorylation of Ser-115 in cytoplasmic loop B and Ser-274 in the C-terminal region (Tornroth-Horsefield et al., 2006) (Fig. 2). High resolution x-ray stmctures of SoPIP2;l in closed and open conformations (Tornroth-Horsefield et al., 2006) showed that loop D caps the channel entrance from the cytoplasmic side. Leu-197 of loop D is inserted into a cavity near the entrance, and in combination with other residues it creates a hydrophobic gate (Tornroth-Horsefield et al., 2006).
We generated several truncations and point mutations of AqpB to test for channel gating and found that neither the N-terminal domain nor a putative phosporylation site in loop B is involved in channel opening (Bulow et al., 2012). However, truncation of the extraordinarily long intracellular loop D by 12 amino acids, AqpB[DELTA]208-219, induced water permeability of AqpB (Bulow et al., 2012). Glycerol and urea did not pass the pore. We finally identified a single amino acid of loop D, Tyr216, as a key residue in the gating mechanism, possibly involving phosphorylation (Bulow et al., 2015). Mutation of Tyr216 to aspartate or glutamate initiated water permeability to the same extent as AqpB[DELTA]208-219, whereas neither replacement of Tyr216 by a positive arginine nor introduction of a negative charge in the neighboring position, T217D, opened the channel (Bulow et al., 2015). Expression of the permanently open AqpB[DELTA]208-219 in Dictyostelium amoebae yielded cells with reduced capability to cope with hypotonic stress. Based on the experience with AqpB, re-examination of the other Dictyostelium AQPs with respect to gating may be worthwhile. In the described examples of immobile plants and the single-celled fungi and amoebae, the expression of gated AQPs must be viewed as a common adaptation concept to react to sudden osmotic environmental challenges.
Although there is little information about the permeability profiles of Dictyostelium AQPs, gene knockouts are available for AqpA-C (Flick et al., 1997; Mitra et al., 2000, 2014). The potential physiological roles for AqpA and AqpC in prespore cells expelling water during encapsulation and in spores taking up water during germination (Cotter and Raper, 1968a) were confirmed at least for AqpA. Deletion of the aqpA gene did not affect growth or developmental morphogenesis, but spore dormancy was severely affected (Mitra et al., 2000). However, neither sporulation nor germination suffered when the aqpC gene was deleted (Flick et al., 1997). A potential physiological role for AqpB and AqpD in D. discoideum amoebae is in cell migration (52015). D. discoideum amoebae roam randomly to find food (Wessels et al., 1988). Directed, chemotactic cell migration is observed towards prey bacteria-secreting folate, which is detected by respective D. discoideum receptors (Van Haastert, 1983). Other receptors sense cAMP as a chemotactic signal in the streaming phase of D. discoideum towards cell aggregates (Newell and Mullens, 1978). Similarly, the presence of AQPs of human immune and cancer cells enhances cell motility (Saadoun et al., 2005). D. discoideum is a model organism for cell migration processes (Bozzaro, 2013), and genetic deletion of aqpB yielded a strain with significantly reduced speed of random motility (4.5 [micro]m [min.sup.-1] vs 6.3 [micro]m [min.sup.-1] of wildtype cells) (Billow et al., 2015). Yet chemotaxis towards folate and cAMP was unaffected. In this context, the simultaneously expressed AqpD calls for further studies.
We have discussed protozoan AQPs with regard to three different types of lifestyles: human-pathogenic parasites that live a sheltered life inside other cells (Plasmodia:, Toxoplasma gondii, and Trypanosoma cruzi), parasites that swim freely in the blood of their human host (Trypanosoma brucei), and amoebae that are directly exposed to a changing environment (Amoeba proteus and Dictyostelium discoideum). The number of experimentally characterized protozoan AQPs is still low, and to make general assumptions a larger base of information is needed. It appears, though, that a reduced number of bifunctional AQPs with water and solute permeability seems to be a common theme in the group of intracellular protozoan parasites discussed here. If there is more than one AQP, localization or developmental-stage-dependent expression is highly specialized. Physiologically, water permeability may be important for coping with osmotic stress during transmission between host and vector and during kidney passages in mammals. Further, these AQPs provide access to metabolic precursors and nutrition, e.g., by the uptake of glycerol. Toxic metabolic end products such as methylglyoxal or ammonia are expelled via AQPs. Moreover, and unrelated to metabolism, protozoan parasite AQPs are involved in osmotaxis and shape changes. Contractile vacuoles form a functional link between certain intracellular parasites and free-living amoebae such as A. proteus. Both use this special, AQP-containing organelle complex to deal with hypo-osmotic stress. D. discoideum must adapt to rapidly changing environmental conditions and thus has at least one gated AQP in the amoeboidal stage. From a therapeutic point of view, AQPs may represent interesting new drug targets and drug entry routes (Kun and de Carvalho, 2009; Song et al., 2014). However, as with human AQPs, specific inhibitors with high affinity have yet to be found.
Abascal, F., I. Irisarri, and R. Zardoya. 2014. Diversity and evolution of membrane intrinsic proteins. Biochim. Biophys. Acta 1840: 1468-1481.
Agre, P., L. S. King, M. Yasui, W. B. Guggino, O. P. Ottersen, Y. Fujiyoshi, A. Engel, and S. Nielsen. 2002. Aquaporin water channels--from atomic structure to clinical medicine. J. Physiol. 542: 3-16.
Allen, R. D., and Y. Naitoh. 2002. Osmoregulation and contractile vacuoles of protozoa. Int. Rev. Cytol. 215: 351-394.
Almasalmeh, A., D. Krenc, B. Wu, and E. Beitz. 2014. Structural determinants of the hydrogen peroxide permeability of aquaporins. FEBS J. 281: 647-656.
Baker, N., L. Glover, J. C. Munday, D. Aguinaga Andres, M. P. Barrett, H. P. De Koning, and D. Horn. 2012. Aquaglyceroporin 2 controls susceptibility to melarsoprol and pentamidine in African trypanosomes. Proc. Natl. Acad. Sci. USA 109: 10996-11001.
Bakker, B. M., P. A. Michels, F. R. Opperdoes, and H. V. Westerhoff. 1997. Glycolysis in bloodstream form Trypanosoma brucei can be understood in terms of the kinetics of the glycolytic enzymes. J. Biol. Chem. 272: 3207-3215.
Baldauf, S. L., A. J. Roger, I. Wenk-Siefert, and W. F. Doolittle. 2000. A kingdom-level phylogeny of eukaryotes based on combined protein data. Science 290: 972-977.
Bassarak, B., N. L. Uzcategui, C. Schonfeld, and M. Duszenko. 2011. Functional characterization of three aquaglyceroporins from Trypanosoma brucei in osmoregulation and glycerol transport. Cell. Physiol. Biochem. 27: 411-420.
Beitz, E. 2005. Aquaporins from pathogenic protozoan parasites; structure, function and potential for chemotherapy. Biol. Cell 97: 373-383.
Beitz, E. 2006. Aquaporin water and solute channels from malaria parasites and other pathogenic protozoa. ChemMedChem 1: 587-592.
Beitz, E. 2007. Jammed traffic impedes parasite growth. Proc. Natl. Acad. Sci. USA 104: 13855-13856.
Beitz, E., S. Pavlovic-Djuranovic, M. Yasui, P. Agre, and J. E. Schultz. 2004. Molecular dissection of water and glycerol permeability of the aquaglyceroporin from Plasmodium falciparum by mutational analysis. Proc. Natl. Acad. Sci. USA 101: 1153-1158.
Biyani, N., S. Mandai, C. Seth, M. Saint, K. Natarajan, I. Ghosh, and R. Madhubala. 2011. Characterization of Leishmania donovani aquaporins shows presence of subcellular aquaporins similar to tonoplast intrinsic proteins of plants. PLoS One 6: e24820.
Bozzaro, S. 2013. The model organism Dictyostelium discoideum. Methods Mol. Biol. 983: 17-37.
Bulow, J. von, A. Muller-Lucks, L. Kai, F. Bernhard, and E. Beitz. 2012. Functional characterization of a novel aquaporin from Dictyostelium discoideum amoebae implies a unique gating mechanism. J. Biol. Chem. 287: 7487-7494.
Bulow, J. von, A. Golldack, T. Albers, and E. Beitz. 2015. The amoeboidal Dictyostelium aquaporin AqpB is gated via Tyr216 and aqpB gene deletion affects random cell motility. Biol. Cell 107: 78-88.
Contreras-Ochoa, C. O., A. Lagunas-Martinez, J. Belkind-Gerson, J. Diaz-Chavez, and D. Correa. 2013. Toxoplasma gondii invasion and replication within neonate mouse astrocytes and changes in apoptosis related molecules. Exp. Parasitol. 134: 256-265.
Cotter, D. A., and K. B. Raper. 1968a. Properties of germinating spores of Dictyostelium discoideum. J. Bacteriol. 96: 1680-1689.
Cotter, D. A., and K. B. Raper. 1968b. Spore germination in strains of Dictyostelium discoideum and other members of the Dictyosteliaceae. J. Bacteriol. 96: 1690-1695.
Couillard, P., F. Pothier, and P. Mayers. 1989. The effects of vasopressin and related peptides on osmoregulation in Amoeba proteus. Gen. Comp. Endocrinol. 76: 106-113.
de Groot, B. L., and H. Grubmuller. 2001. Water permeation across biological membranes: mechanism and dynamics of aquaporin-1 and GlpF. Science 294: 2353-2357.
Eisenthal, R., and A. Cornish-Bowden. 1998. Prospects for antiparasitic drugs--the case of Trypanosoma brucei, the causative agent of African sleeping sickness. J. Biol. Chem. 273: 5500-5505.
Fadiel, A., R. D. Isokpehi, N. Stambouli, A. Hamza, A. Benammar-Elgaaied, and T. J. Scalise. 2009. Protozoan parasite aquaporins. Expert Rev. Proteomics 6: 199-211.
Fichera, M. E., and D. S. Roos. 1997. A plastid organelle as a drug target in apicomplexan parasites. Nature 390: 407-409.
Figarella, K., N. L. Uzcategui, Y. Zhou, A. LeFurgey, M. Ouellette, H. Bhattacharjee, and R. Mukhopadhyay. 2007. Biochemical characterization of Leishmania major aquaglyceroporin LmAQP1: possible role in volume regulation and osmotaxis. Mol. Microbiol. 65: 1006-1017.
Fischer, G., U. Kosinska-Eriksson, C. Aponte-Santamaria, M. Palmgren, C. Geijer, K. Hedfalk, S. Hohmann, B. L. de Groot, R. Neutze, and K. Lindkvist-Petersson. 2009. Crystal structure of a yeast aquaporin at 1.15 angstrom reveals a novel gating mechanism. PLoS Biol. 7: el000130.
Flick, K. M., G. Shaulsky, and W. F. Loomis. 1997. The wacA gene of Dictyostelium discoideum is a developmentally regulated member of the MIP family. Gene 195: 127-130.
Fu, D., A. Libson, L. J. Miercke, C. Weitzman, P. Nollert, J. Krucinski, and R. M. Stroud. 2000. Structure of a glycerol-conducting channel and the basis for its selectivity. Science 290: 481-486.
Gardner, M. J., N. Hall, E. Fung, O. White, M. Berriman, R. W. Hyman, J. M. Carlton, A. Pain, K. E. Nelson, S. Bowman et al. 2002. Genome sequence of the human malaria parasite Plasmodium falciparum. Nature 419: 498-511.
Ginger, M. L. 2006. Niche metabolism in parasitic protozoa. Philos. Trans. R. Soc. Lond. B Biol. Sci. 361: 101-118.
Gourbal, B., N. Sonuc, H. Bhattacharjee, D. Legare, S. Sundar, M. Ouellette, B. P. Rosen, and R. Mukhopadhyay. 2004. Drug uptake and modulation of drug resistance in Leishmania by an aquaglyceroporin. J. Biol. Chem. 279: 31010-31017.
Hansen, M., J. F. Kun, J. E. Schultz, and E. Beitz. 2002. A single, bi-functional aquaglyceroporin in blood-stage Plasmodium falciparum malaria parasites. J. Biol. Chem. 277: 4874-4882.
Hedfalk, K., R. M. Bill, J. G. Mullins, S. Karlgren, C. Filipsson, J. Bergstrom, M. J. Tamas, J. Rydstrom, and S. Hohmann. 2004. A regulatory domain in the C-terminal extension of the yeast glycerol channel Fps1p. J. Biol. Chem. 279: 14954-14960.
Huang, G., J. Fang, C. Sant'Anna, Z.-H. Li, D. L. Wellems, P. Rohloff, and R. Docampo. 2011. Adaptor protein-3 (AP-3) complex mediates the biogenesis of acidocalcisomes and is essential for growth and virulence of Trypanosoma brucei. J. Biol. Chem. 286: 36619-36630.
Huang, J., N. Mullapudi, T. Sicheritz-Ponten, and J. C. Kissinger. 2004. A first glimpse into the pattern and scale of gene transfer in the apicomplexa. Int. J. Parasitol. 34: 265-274.
Hviid, L., L. Barfod, and F. J. I. Fowkes. 2015. Trying to remember: immunological B cell memory to malaria. Trends Parasitol. 31: 89-94.
Jeon, K. W. 1995. The large, free-living amoebae: wonderful cells for biological studies. J. Eukaryot. Microbiol. 42: 1-7.
Johansson, I., M. Karlsson, V. K. Shukla, M. J. Chrispeels, C. Larsson, and P. Kjellbom. 1998. Water transport activity of the plasma membrane aquaporin PM28A is regulated by phosphorylation. Plant Cell 10: 451-459.
Karlgren, S., C. Filipsson, J. G. Mullins, R. M. Bill, M. J. Tamas, and S. Hohmann. 2004. Identification of residues controlling transport through the yeast aquaglyceroporin Fps1 using a genetic screen. Eur. J. Biochem. 271: 771-779.
Kumar, Y., and R. H. Valdivia. 2009. Leading a sheltered life: intracellular pathogens and maintenance of vacuolar compartments. Cell Host Microbe 5: 593-601.
Kun, J. F., and E. G. de Carvalho. 2009. Novel therapeutic targets in Plasmodium falciparum: aquaglyceroporins. Expert Opin. Ther. Targets 13: 385-394.
Leiriao, P., C. D. Rodrigues, S. S. Albuquerque, and M. M. Mota. 2004. Survival of protozoan intracellular parasites in host cells. EMBO Rep. 5: 1142-1147.
Luyten, K., J. Albertyn, W. F. Skibbe, B. A. Prior, J. Ramos, J. M. Thevelein, and S. Hohmann. 1995. Fps1, a yeast member of the MIP family of channel proteins, is a facilitator for glycerol uptake and efflux and is inactive under osmotic stress. EMBO J. 14: 1360-1371.
Mandal, G., M. Sharma, M. Kruse, C. Sander-Juelch, L. A. Munro, Y. Wang, J. V. Vilg, M. J. Tamas, H. Bhattacharjee, M. Wiese, and R. Mukhopadhyay. 2012. Modulation of Leishmania major aquaglyceroporin activity by a mitogen-activated protein kinase. Mol. Microbiol. 85: 1204-1218.
Mandal, G., J. Orta, M. Sharma, and R. Mukhopadhyay. 2014. Trypanosomatid aquaporins: roles in physiology and drug response. Diseases 2: 3-23.
Mandal, G., S. Mandal, M. Sharma, K. S. Charrez, B. Papadopoulou, H. Bhattachariee, and R. Mukhopadhyay. 2015. Species-specific antimonial sensitivity in Leishmania is driven by post-transcriptional regulation of AQP1. PLoS Negl. Trop. Dis. 9: e0003500.
Maurel, C., H. Javot, V. Lauvergeat, P. Gerbeau, C. Tournaire, V. Santoni, and J. Heyes. 2002. Molecular physiology of aquaporins in plants. Int. Rev. Cytol. 215: 105-148.
Miranda, K., D. A. Pace, R. Cintron, J. C. Rodrigues, J. Fang, A. Smith, P. Rohloff, E. Coelho, F. de Haas, W. de Souza et al. 2010. Characterization of a novel organelle in Toxoplasma gondii with similar composition and function to the plant vacuole. Mol. Microbiol. 76: 1358-1375.
Mitra, B. N., R. Yoshino, T. Morio, M. Yokoyama, M. Maeda, H. Urushihara, and Y. Tanaka. 2000. Loss of a member of the aquaporin gene family, aqpA affects spore dormancy in Dictyostelium. Gene 251: 131-139.
Montalvetti, A., P. Rohloff, and R. Docampo. 2004. A functional aquaporin co-localizes with the vacuolar proton pyrophosphatase to acidocalcisomes and the contractile vacuole complex of Trypanosoma cruzi. J. Biol. Chem. 279: 38673-38682.
Munday, J. C., A. A. Eze, N. Baker, L. Glover, C. Clucas, D. Aguinaga Andres, M. J. Natto, I. A. Teka, J. McDonald, R. S. Lee et al. 2014. Trypanosoma brucei aquaglyceroporin 2 is a high-affinity transporter for pentamidine and melaminophenyl arsenic drugs and the main genetic determinant of resistance to these drugs. J. Antimicrob. Chemother. 69: 651-663.
Murata, K., K. Mitsuoka, T. Hirai, T. Walz, P. Agre, J. B. Heymann, A. Engel, and Y. Fujiyoshi. 2000. Structural determinants of water permeation through aquaporin-1. Nature 407: 599-605.
Newell, P. C., and I. A. Mullens. 1978. Cell-surface cAMP receptors in Dictyostelium. Symp. Soc. Exp. Biol. 32: 161-171.
Nishihara, E., T. Shimmen, and S. Sonobe. 2004. Functional characterization of contractile vacuole isolated from Amoeba proteus. Cell Struct. Fund. 29: 85-90.
Nishihara, E., E. Yokota, A. Tazaki, H. Orii, M. Katsuhara, K. Kataoka, H. Igarashi, Y. Moriyama, T. Shimmen, and S. Sonobe. 2008. Presence of aquaporin and V-ATPase on the contractile vacuole of Amoeba proteus. Biol. Cell 100: 179-188.
Nolta, K. V., and T. L. Steck. 1994. Isolation and initial characterization of the bipartite contractile vacuole complex from Dictyostelium discoideum. J. Biol. Chem. 269: 2225-2233.
Pavlovic-Djuranovic, S., J. E. Schultz, and E. Beitz. 2003. A single aquaporin gene encodes a water/glycerol/urea facilitator in Toxoplasma gondii with similarity to plant tonoplast intrinsic proteins. FEBS Lett. 555: 500-504.
Pavlovic-Djuranovic, S., J. F. Kun, J. E. Schultz, and E. Beitz. 2006. Dihydroxyacetone and methylglyoxal as permeants of the Plasmodium aquaglyceroporin inhibit parasite proliferation. Biochim. Biophys. Acta 1758: 1012-1017.
Pearson, R. D., and A. Q. Sousa. 1996. Clinical spectrum of leishmaniasis. Clin. Infect. Dis. 22: 1-13.
Plattner, H. 2013. Contractile vacuole complex--its expanding protein inventory. Int. Rev. Cell. Mol. Biol. 306: 371-416.
Promeneur, D., Y. Liu, J. Maciel, P. Agre, L. S. King, and N. Kumar. 2007. Aquaglyceroporin PbAQP during intraerythrocytic development of the malaria parasite Plasmodium berghei. Proc. Natl. Acad. Sci. USA 104: 2211-2216.
Rassi, A., and J. Marcondes de Rezende. 2012. American trypanosomiasis (Chagas disease). Infect. Dis. Clin. North Am. 26: 275-291.
Rohloff, P., and R. Docampo. 2008. A contractile vacuole complex is involved in osmoregulation in Trypanosoma cruzi. Exp. Parasitai. 118: 17-24.
Rohloff, P., A. Montalvetti, and R. Docampo. 2004. Acidocalcisomes and the contractile vacuole complex are involved in osmoregulation in Trypanosoma cruzi. J. Biol. Chem. 279: 52270-52281.
Roudier, N., P. Ripoche, P. Gane, P. Y. Le Pennec, G. Daniels, J. P. Cartron, and P. Bailly. 2002. AQP3 deficiency in humans and the molecular basis of a novel blood group system, GIL. J. Biol. Chem. 277: 45854-45859.
Saadoun, S., M. C. Papadopoulos, M. Hara-Chikuma, and A. S. Verkman. 2005. Impairment of angiogenesis and cell migration by targeted aquaporin-1 gene disruption. Nature 434: 786-792.
Shapiro, T. A., and P. T. Englund. 1995. The structure and replication of kinetoplast DNA. Annu. Rev. Microbiol. 49: 117-143.
Smith, B. L., G. M. Preston, F. A. Spring, D. J. Anstee, and P. Agre. 1994. Human red cell aquaporin CHIP. I. Molecular characterization of ABH and Colton blood group antigens. J. Clin. Invest. 94: 1043-1049.
Song, J., E. Mak, B. Wu, and E. Beitz. 2014. Parasite aquaporins: current developments in drug facilitation and resistance. Biochim. Biophys. Acta 1840: 1566-1573.
Steinborn, K., A. Szallies, D. Mecke, and M. Duszenko. 2000. Cloning, heterologous expression and kinetic analysis of glycerol kinase (TbGLKl) from Trypanosoma brucei. Biol. Chem. 381: 1071-1077.
Tajkhorshid, E., P. Nollert, M. 0. Jensen, L. J. W. Miercke, J. O'Connell, R. M. Stroud, and K. Schulten. 2002. Control of the selectivity of the aquaporin water channel family by global orientational tuning. Science 296: 525-530.
Tamas, M. J., S. Karlgren, R. M. Bill, K. Hedfalk, L. Allegri, M. Ferreira. J. M. Thevelein, J. Rydstrom, J. G. Mullins, and S. Hohmann. 2003. A short regulatory domain restricts glycerol transport through yeast Fps1p. J. Biol. Chem. 278: 6337-6345.
Tornroth-Horsefield, S., Y. Wang, K. Hedfalk, U. Johanson, M. Karlsson, E. Tajkhorshid, R. Neutze, and P. Kjellbom. 2006. Structural mechanism of plant aquaporin gating. Nature 439: 688-694.
Uzcategui, N. L., A. Szallies, S. Pavlovic-Djuranovic, M. Palmada, K. Figarella, C. Boehmer, F. Lang, E. Beitz, and M. Duszenko. 2004. Cloning, heterologous expression, and characterization of three aquaglyceroporins from Trypanosoma brucei. J. Biol. Chem. 279: 42669-42676.
Uzcategui, N. L., K. Figarella, B. Bassarak, N. W. Meza, R. Mukhopadhyay, J. L. Ramirez, and M. Duszenko. 2013. Trypanosoma brucei aquaglyceroporins facilitate the uptake of arsenite and antimonite in a pH dependent way. Cell. Physiol. Biochem. 32: 880-888.
Van Haastert, P. J. 1983. Sensory adaptation of Dictyostelium discoideum cells to chemotactic signals. J. Cell Biol. 96: 1559-1565.
Vincent, I. M., and M. P. Barrett. 2015. Metabolomic-based strategies for anti-parasite drug discovery. J. Biomol. Screen. 20: 44-55.
Walker, D. M., S. Oghumu, G. Gupta, B. S. McGwire, M. E. Drew, and A. R. Satoskar. 2014. Mechanisms of cellular invasion by intracellular parasites. Cell. Mol. Life Sci. 71: 1245-1263.
Walz, T., Y. Fujiyoshi, and A. Engel. 2009. The AQP structure and functional implications. Handb. Exp. Pharmacol. 190: 31-56.
Wessels, D., D. R. Soil, D. Knecht, W. F. Loomis, A. de Lozanne, and J. Spudich. 1988. Cell motility and chemotaxis in Dictyostelium amoebae lacking myosin heavy chain. Dev. Biol. 128: 164-177.
Wu, B., C. Steinbronn, M. Alsterfjord, T. Zeuthen, and E. Beitz. 2009. Concerted action of two cation filters in the aquaporin water channel. EMBO J. 28: 2188-2194.
Wu, B., J. Song, and E. Beitz. 2010. Novel channel enzyme fusion proteins confer arsenate resistance. J. Biol. Chem. 285: 40081-40087.
Yamamoto, E., T. Akimoto, Y. Hirano, M. Yasui, and K. Yasuoka. 2014. 1/f fluctuations of amino acids regulate water transportation in aquaporin 1. Phys. Rev. E Stat. Nonlin. Soft Matter Phys. 89: 022718.
Zeuthen, T., B. Wu, S. Pavlovic-Djuranovic, L. M. Holm, N. L. Uzcategui, M. Duszenko, J. F. Kun, J. E. Schultz, and E. Beitz. 2006. Ammonia permeability of the aquaglyceroporins from Plasmodium falciparum, Toxoplasma gondii and Trypansoma brucei. Mol. Microbiol. 61: 1598-1608.
JULIA VON BULOW AND ERIC BEITZ (*)
Department of Pharmaceutical and Medicinal Chemistry, Christian-Albrechts-University of Kiel, Gutenbergstrasse 76, 24118 Kiel, Germany
Received 28 January 2015; accepted 1 April, 2015.
(*) To whom correspondence should be addressed. E-mail: firstname.lastname@example.org
Table 1 Properties of protozoan AQPs Water Species Isoform permeability Plasmodium falciparum PfAQP High Toxoplasma gondii TgAQP Medium Trypanosoma brucei TbAQPl High TbAQP2 High TbAQP3 High Trypanosoma cruzi TcAQP Very low TcAQP[beta]-[delta] n.d. Leishmania major LmAQP1 High LmAQP[alpha]-[delta] n.d. Amoeba proteus ApAQP Low Dictyostelium discoideum AqpA n.d. AqpB Low AqpC n.d. AqpD n.d. Species Confirmed permeating solutes Plasmodium falciparum Glycerol, short polyols, urea, ammonia, arsenite, methylglyoxal, dihydroxyacetone, hydrogen peroxide Toxoplasma gondii Glycerol, short polyols, urea, ammonia, hydroxyurea Trypanosoma brucei Glycerol, short polyols, urea, ammonia, arsenite, dihydroxyacetone Glycerol, short polyols, urea, ammonia, arsenite, dihydroxyacetone Glycerol, short polyols, urea, ammonia, arsenite, dihydroxyacetone Trypanosoma cruzi - n.d. (not determined) Leishmania major Glycerol, short polyols, urea, arsenite, methylglyoxal, dihydroxyacetone n.d. Amoeba proteus n.d. Dictyostelium discoideum n.d. - n.d. n.d. Localization Ref. Plasmodium falciparum Plasma membrane [a, b, c, d] Toxoplasma gondii Contractile vacuole [b, e] Trypanosoma brucei Flagellar membrane [b, f, g] Flagellar pocket [b, f, g] Plasma membrane [b, f, g] Trypanosoma cruzi Contractile vacuole [h] n.d. [i] Leishmania major Flagellum [j, k] n.d. [i] Amoeba proteus Contractile vacuole [l] Dictyostelium discoideum Pre-spores [m] Plasma membrane, vesicles [n] Multicellular stage [o] n.d. [p] a) Hansen et al., 2002; b) Zeuthen et al., 2006; c) Wu et al., 2010; d) Almasalmeh et al., 2014; e) Pavlovic-Djuranovic et al., 2003; f) Uzcategui et al., 2004; g) Uzcategui et al., 2013; h) Rohloff et al., 2004; i) Beitz 2005; j) Gourbal et al., 2004; k) Figarella et at, 2007; 1) Nishihara et al., 2008; m) Mitra et al., 2000; n) Bulow et al., 2012; o) Flick et al., 1997; p) Bulow et al., 2015.
|Printer friendly Cite/link Email Feedback|
|Author:||Von Bulow, Julia; Beitz, Eric|
|Publication:||The Biological Bulletin|
|Date:||Aug 1, 2015|
|Previous Article:||Involvement of aquaporin channels in water extrusion from biosilica during maturation of sponge siliceous spicules.|
|Next Article:||Aquaporins in the Antarctic midge, an extremophile that relies on dehydration for cold survival.|