Chew, Shit Fun

Suspended animation has long fascinated scientists because of its great application potentials in fields ranging from medicine to space travel. Animals become inactive during suspended animation, with absolutely no intake of food and water, and hence producing minimal or no urine and faecal materials, for an extended period. They enter into a state of torpor, possibly slowing down the biological time in relation to the clock time. If suspended animation can be achieved in humans, surgeons would have more time to operate on patients during critical moments when the blood circulation stops, and the dream of long distance space travel can be realized. In nature, suspended animation is expressed in some adult animals undergoing hibernation or aestivation. ‘Aestivation’ is a loose term that signifies little more than an animal undergoing a state of torpor to survive arid conditions (except for aquatic aestivators like certain sponges and sea cucumbers) at high temperature, in many cases during summer. The term has been used to describe the listless state of endotherms, like ground squirrels and cactus mouse at the height of summer heat, and ectotherms, like amphibians and African lungfishes that make cocoons to encase themselves for weeks to more than a year during the hot dry season.

Giant clams harbor three genera of symbiotic dinoflagellates (Symbiodinium, Cladocopium, Durusdinium) as extracellular symbionts (zooxanthellae). While symbiotic dinoflagellates can synthesize amino acids to benefit the host, they are nitrogen‐deficient. Hence, the host must supply them with nitrogen including urea, which can be degraded to ammonia and carbon dioxide by urease (URE). Here, we report three complete coding cDNA sequences of URE, one for each genus of dinoflagellate, obtained from the colorful outer mantle of the giant clam, Tridacna squamosa. The outer mantle had higher transcript level of Tridacna squamosa zooxanthellae URE (TSZURE) than the whitish inner mantle, foot muscle, hepatopancreas and ctenidium. TSZURE was immunolocalized strongly and atypically in the plastid, moderately in the cytoplasm, and weakly in the cell wall and plasma membrane of symbiotic dinoflagellates. In the outer mantle, illumination upregulated the protein abundance of TSZURE, which could enhance urea degradation in photosynthesizing dinoflagellates. The urea‐nitrogen released could then augment syntheses of amino acids to be shared with the host for its general needs. Illumination also enhanced gene and protein expression levels of TSZURE/TSZURE in the inner mantle and foot muscle, which contain only small quantities of symbiotic dinoflagellate, have no iridocyte, and lack direct exposure to light. With low phototrophic potential, dinoflagellates in the inner mantle and foot muscle might need to absorb carbohydrates in order to assimilate the urea‐nitrogen into amino acids. Amino acids donated by dinoflagellates to the inner mantle and the foot muscle could be used especially for syntheses of organic matrix needed for light‐enhanced shell formation and muscle protein, respectively.

Giant clams are inhabitants of Indo-Pacific coral reefs. They commonly harbor three genera of phototrophic Symbiodiniaceae dinoflagellates (Symbiodinium, Cladocopium, and Durusdinium) as coccoid symbionts (zooxanthellae) mainly in the colorful outer mantle. Photosynthesizing symbionts release photosynthate that may include glycerol to the host. Hence, we cloned and characterized Glycerol Facilitator (GlpF) derived from the coccoid dinoflagellates in the colorful mantle of the giant clam, Tridacna squamosa, and reported herein three major GlpF cDNA sequences, one each for Symbiodinium, Cladocopium, and Durusdinium. The complete cDNA coding sequences of Symbiodinium-GlpF (Symb-GlpF), Cladocopium-GlpF (Clad-GlpF) and Durusdinium-GlpF (Duru-GlpF) comprised 1056, 1047 and 1029 bp, respectively. The respective deduced amino acid sequences consisted of 353 (36.9 kDa), 348 (36.5 kDa) and 342 (36.1 kDa) amino acids. All three encoded proteins had conserved residues associated with low water permeability and high glycerol selectivity, which are characteristics of aquaglyceroporin. Zooxanthellae-GlpF (Zoox-GlpF), which represented GlpFs of Symbiodinium, Cladocopium and Durusdinium phylotypes, was immunolocalized in some vesicles inside the symbionts. Some of these immunolabelled vesicles were located close to the symbiont's plasma membrane, parts of which also displayed immunofluorescence, indicating that these vesicles could probably release their contents through exocytosis. As illumination augmented the gene and protein expression levels of Zoox-GlpF/Zoox-GlpF in the outer mantle of T. squamosa, there could be an increase in the potential of glycerol transport through Zoox-GlpF in the coccoid dinoflagellates during photosynthesis. Future studies should examine whether different phylotypes of Symbiodinium, Cladocopium and Durusdinium would have dissimilar potentials of glycerol transport.

The giant mudskipper, Periophthalmodon schlosseri, is an amphibious fish that builds burrows in the mudflats. It can actively excrete ammonia through its gills, and tolerate high environmental ammonia. This study aimed to examine the effects of seawater (salinity 30; SW) acclimation and/or environmental ammonia exposure on the kinetic properties of Na+/K+-ATPase (Nka) from, and mRNA expression and protein abundance of nka/Nka α–subunit isoforms in, the gills of P. schlosseri pre-acclimated to slightly brackish water (salinity 3; SBW). Our results revealed that the Nka from the gills of P. schlosseri pre-acclimated to SBW for 2 weeks had substantially higher affinity to (or lower Km for) K+ than NH+ 4 , and its affinity to NH+ 4 decreased significantly after 6-days exposure to 75 mmol l−1 NH4Cl in SBW. Hence, Nka transported K+ selectively to maintain intracellular K+ homeostasis, instead of transporting NH+ 4 from the blood into ionocytes during active NH+4 excretion as previously suggested. Two nkaα isoforms, nkaα1 and nkaα3, were cloned and sequenced from the gills of P. schlosseri. Their deduced amino acid sequences had K+ binding sites identical to that of Nkaα1c from Anabas testudineus, indicating that they could effectively differentiate K+ from NH+ 4 . Six days of exposure to 75 mmol l−1 NH4Cl in SBW, or to SW with or without 50 mmol l−1 NH4Cl led to significant increases in Nka activities in the gills of P. schlosseri. However, a significant increase in the comprehensive Nkaα protein abundance was observed only in the gills of fish exposed to 50 mmol l−1 NH4Cl in SW. Hence, post-translational modification could be an important activity modulator of branchial Nka in P. schlosseri. The fast modulation of Nka activity and concurrent expressions of two branchial nkaα isoforms could in part contribute to the ability of P. schlosseri to survive abrupt transfer between SBWand SW or abrupt exposure to ammonia.

The stinging catfish, Heteropneustes fossilis, can tolerate high concentrations of environmental ammonia. Previously, it was regarded as ureogenic, having a functional ornithine-urea cycle (OUC) that could be up-regulated during ammonia-loading. However, contradictory results indicated that increased urea synthesis and switching to ureotelism could not explain its high ammonia tolerance. Hence, we re-examined the effects of exposure to 30 mmol l–1 NH4Cl on its ammonia and urea excretion rates, and its tissue ammonia and urea concentrations. Our results confirmed that H. fossilis did not increase urea excretion or accumulation during 6 days of ammonia exposure, and lacked detectable carbamoyl phosphate synthetase I or III activity in its liver. However, we discovered that it could actively excrete ammonia during exposure to 8 mmol l–1 NH4Cl. As active ammonia excretion is known to involve Na+/K+-ATPase (Nka) indirectly in several ammonia-tolerant fishes, we also cloned various nkaα-subunit isoforms from the gills of H. fossilis, and determined the effects of ammonia exposure on their branchial transcripts levels and protein abundances. Results obtained revealed the presence of five nkaα-subunit isoforms, with nkaα1b having the highest transcript level. Exposure to 30 mmol l–1 NH4Cl led to significant increases in the transcript levels of nkaα1b (on day 6) and nkaα1c1 (on day 1 and 3) as compared with the control. In addition, the protein abundances of Nkaα1c1, Nkaα1c2, and total NKAα increased significantly on day 6. Therefore, the high environmental ammonia tolerance of H. fossilis is attributable partly to its ability to actively excrete ammonia with the aid of Nka.

In light, giant clams can increase rates of shell formation and growth due to their symbiotic relationship with phototrophic zooxanthellae residing extracellularly in a tubular system. Light-enhanced shell formation necessitates increase in the uptake of Ca2+ from the ambient seawater and the supply of Ca2+ through the hemolymph to the extrapallial fluid, where calcification occurs. In this study, the complete coding cDNA sequence of a homolog of voltage-gated calcium channel subunit α1 (CACNA1), which is the pore-forming subunit of L-type voltage-gated calcium channels (VGCCs), was obtained from the ctenidium (gill) of the giant clam, Tridacna squamosa. It consisted of 6081 bp and encoded a 223 kDa polypeptide with 2027 amino acids, which was characterized as the α1D subunit of L-type VGCC. Immunofluorescence microscopy demonstrated that CACNA1 had an apical localization in the epithelial cells of filaments and tertiary water channels in the ctenidium of T. squamosa, indicating that it was well positioned to absorb exogenous Ca2+. Additionally, there was a significant increase in the protein abundance of CACNA1 in the ctenidium of individuals exposed to light for 12 h. With more pore-forming CACNA1, there could be an increase in the permeation of exogenous Ca2+ into the ctenidial epithelial cells through the apical membrane. Taken together, these results denote that VGCC could augment exogenous Ca2+ uptake through the ctenidium to support light-enhanced shell formation in T. squamosa. Furthermore, they support the proposition that light-enhanced phenomena in giant clams are attributable primarily to the direct responses of the host’s transporters/enzymes to light, in alignment with the symbionts’ phototrophic activity.

Giant clams are ecologically and economically relevant reef inhabitants that host photosynthetic dinoflagellates inside tubules located mainly in their colorful outer mantle. This study examined the effects of exposure to darkness for 30 days and the subsequent 11 days of recovery under a normal photoperiod on the outer mantle of the fluted giant clam Tridacna squamosa. Changes in the abundance of iridocytes and symbionts were assessed by fluorescence microscopy. Light microscopy was applied to quantify symbionts isolated from the outer mantle, while chlorophyll was extracted and analyzed by spectroscopy. The transcript levels of the host’s vacuolar-type H+-ATPase subunit A (ATP6V1A) and symbionts’ form II ribulose-1,5-bisphosphate carboxylase/oxygenase (Zoox-rbcII) were determined by quantitative real-time PCR (qPCR) and used as proxies for iridocyte abundance and phototrophic potential, respectively. The protein abundance of ATP6V1A and Zoox-RBCII was quantified by western blotting. After exposure to darkness for 30 days, the outer mantle of T. squamosa individuals lost the distinct multiple color patterns, and the gene and protein expression levels of ATP6V1A/ATP6V1A and Zoox-rbcII/Zoox-RBCII decreased dramatically. Microscopy assessment confirmed the reduction in iridocyte and symbiont populations, and the chlorophyll level also decreased considerably. However, just 11 days after returning to a normal light:dark regimen, the quantity of coccoid dinoflagellates and the expression of Zoox-rbcII/Zoox-RBCII in the outer mantle increased significantly to levels higher than those of the individuals prior to exposure to darkness (control), while the chlorophyll content returned to the control level. Additionally, the outer mantle regained most of its coloration with partial recovery of the iridocyte population. These results are relevant not only for understanding the phenomena of light deprivation and symbiont loss in dinoflagellate-associated reef organisms, but also signify that the giant clam-coccoid dinoflagellate holobiont is phototrophically plastic and particularly tolerant to bleaching.

This study aimed to sequence and characterize two pro-coagulant genes, coagulation factor II (f2) and fibrinogen gamma chain (fgg), from the liver of the African lungfish Protopterus annectens, and to determine their hepatic mRNA expression levels during three phases of aestivation. The protein abundance of F2 and Fgg in the liver and plasma was determined by immunoblotting. The results indicated that F2 and Fgg of P. annectens were phylogenetically closer to those of amphibians than those of teleosts. Three days of aestivation resulted in an up-regulation in the hepatic fgg mRNA expression level, while 6 days of aestivation led to a significant increase (3-fold) in the protein abundance of Fgg in the plasma. Hence, there could be an increase in the blood-clotting ability in P. annectens during the induction phase of aestivation. By contrast, the blood-clotting ability in P. annectens might be reduced in response to decreased blood flow and increased possibility of thrombosis during the maintenance phase of aestivation, as 6 months of aestivation led to significant decreases in mRNA expression levels of f2 and fgg in the liver. There could also be a decrease in the export of F2 and Fgg from the liver to the plasma so as to avert thrombosis. Three to 6 days after arousal from 6 months of aestivation, the protein abundance of F2 and Fgg recovered partially in the plasma of P. annectens; a complete recovery of the transcription and translation of f2/F2 in the liver might occur only after refeeding.

Giant clams harbor dinoflagellates generally of the three genera (Symbiodinium, Cladocopium, and Durusdinium) of phototrophic Symbiodiniaceae. Coccoid dinoflagellates (alias zooxanthellae) are found mainly inside zooxanthellal tubules located in the colorful outer mantle. The symbionts need to obtain carbon, nitrogen and phosphorus from the host for growth and metabolism. The host can absorb exogenous ammonia through the ctenidium and assimilate it into glutamine. Although the host does not normally excrete ammonia, its hemolymph contains only low concentrations of ammonia, indicating that the symbionts can absorb and recycle the ammonia produced metabolically by the host. In this study, we had obtained from the outer mantle of the giant clam, Tridacna squamosa, three major ammonium transporter 2 (AMT2) sequences, one each for Symbiodinium spp. (Symb-AMT2), Cladocopium spp. (Clad-AMT2), and Durusdinium spp. (Duru-AMT2), which comprised 1341 bp, 1308 bp, and 1296 bp, respectively. The respective deduced amino acid sequences contained 447 (~ 46.5 kDa), 436 (~ 45.5 kDa), and 432 (~ 45.0 kDa) residues. Phenogramic and sequence similarity analyses confirmed that these sequences were derived from dinoflagellates. Zooxanthellae-AMT2 (Zoox-AMT2), which represented comprehensively AMT2 of Symbiodinium spp., Cladocopium spp., and Durusdinium spp. was localized at the dinoflagellates’ plasma membranes, indicating that it could partake in the absorption of ammonia from the luminal fluid of the zooxanthellal tubules. Zoox-AMT2 expression was detected in the outer mantle, inner mantle, foot muscle, hepatopancreas and ctenidium of T. squamosa, indicating that the coccoid dinoflagellates residing in all five organs had the potential of ammonia absorption. The outer mantle had the highest transcript level of Zoox-AMT2, and illumination upregulated the protein abundance of Zoox-AMT2 therein. Therefore, it can be deduced that the coccoid dinoflagellates residing in the outer mantle could augment the potential of ammonia absorption in alignment with photosynthesis as the assimilation of ammonia required an increased supply of carbon chains.

This study aimed to obtain the coding cDNA sequences of Na+/K+-ATPase α (nkaα) isoforms from, and to quantify their mRNA expression in, the skeletal muscle (SM), the main electric organ (EO), the Hunter’s EO and the Sach’s EO of the electric eel, Electrophorus electricus. Four nkaα isoforms (nkaα1c1, nkaα1c2, nkaα2 and nkaα3) were obtained from the SM and the EOs of E. electricus. Based on mRNA expression levels, the major nkaα expressed in the SM and the three EOs of juvenile and adult E. electricus were nkaα1c1 and nkaα2, respectively. Molecular characterization of the deduced Nkaα1c1 and Nkaα2 sequences indicates that they probably have different affinities to Na+ and K+. Western blotting demonstrated that the protein abundance of Nkaα was barely detectable in the SM, but strongly detected in the main and Hunter’s EOs and weakly in the Sach’s EO of juvenile and adult E. electricus. These results corroborate the fact that the main EO and Hunter’s EO have high densities of Na+ channels and produce high voltage discharges while the Sach’s EO produces low voltage discharges. More importantly, there were significant differences in kinetic properties of Nka among the three EOs of juvenile E. electricus. The highest and lowest Vmax of Nka were detected in the main EO and the Sach’s EO, respectively, with the Hunter’s EO having a Vmax value intermediate between the two, indicating that the metabolic costs of EO discharge could be the highest in the main EO. Furthermore, the Nka from the main EO had the lowest Km (or highest affinity) for Na+ and K+ among the three EOs, suggesting that the Nka of the main EO was more effective than those of the other two EOs in maintaining intracellular Na+ and K+ homeostasis and in clearing extracellular K+ after EO discharge.