The Role of Acidocalcisomes in Parasitic Protists1

ABSTRACT. Acidocalcisomes are acidic organelles with a high concentration of phosphorus present as pyrophosphate (PP_i) and polyphosphate (poly P) complexed with calcium and other cations. The acidocalcisome membrane contains a number of pumps (Ca²⁺‐ATPase, V‐H⁺‐ATPase, H⁺‐PPase), exchangers (Na⁺/H⁺, Ca²⁺/H⁺), and channels (aquaporins), while its matrix contains enzymes related to PP_i and poly P metabolism. Acidocalcisomes have been observed in pathogenic, as well as non‐pathogenic prokaryotes and eukaryotes, e.g. Chlamydomonas reinhardtii, and Dictyostelium discoideum. Some of the potential functions of the acidocalcisome are the storage of cations and phosphorus, the participation of phosphorus in PP_i and poly P metabolism, calcium homeostasis, maintenance of intracellular pH homeostasis, and osmoregulation. In addition, acidocalcisomes resemble lysosome‐related organelles (LRO) from mammalian cells in many of their properties. For example, we found that platelet dense granules, which are LROs, are very similar to acidocalcisomes. They share a similar size, acidic properties, and both contain PP_i, poly P, and calcium. Recent work that indicates that they also share the system for targeting of their membrane proteins through adaptor protein 3 reinforces this concept. The fact that acidocalcisomes interact with other organelles in parasitic protists, e.g. the contractile vacuole in Trypanosoma cruzi, and other vacuoles observed in Toxoplasma gondii, suggests that these cellular compartments may be associated with the endosomal/lysosomal pathway.     

Acidocalcisomes as calcium- and polyphosphate-storage compartments during embryogenesis of the insect Rhodnius prolixus Stahl

Background

The yolk of insect eggs is a cellular domain specialized in the storage of reserve components for embryo development. The reserve macromolecules are stored in different organelles and their interactions with the embryo cells are mostly unknown. Acidocalcisomes are lysosome-related organelles characterized by their acidic nature, high electron density and large content of polyphosphate bound to several cations. In this work, we report the presence of acidocalcisome-like organelles in eggs of the insect vector Rhodnius prolixus.

Methodology/Principal findings

Characterization of the elemental composition of electron-dense vesicles by electron probe X-ray microanalysis revealed a composition similar to that previously described for acidocalcisomes. Following subcellular fractionation experiments, fractions enriched in acidocalcisomes were obtained and characterized. Immunofluorescence showed that polyphosphate polymers and the vacuolar proton translocating pyrophosphatase (V-H⁺-PPase, considered as a marker for acidocalcisomes) are found in the same vesicles and that these organelles are mainly localized in the egg cortex. Polyphosphate quantification showed that acidocalcisomes contain a significant amount of polyphosphate detected at day-0 eggs. Elemental analyses of the egg fractions showed that 24.5±0.65% of the egg calcium are also stored in such organelles. During embryogenesis, incubation of acidocalcisomes with acridine orange showed that these organelles are acidified at day-3 (coinciding with the period of yolk mobilization) and polyphosphate quantification showed that the levels of polyphosphate tend to decrease during early embryogenesis, being approximately 30% lower at day-3 compared to day-0 eggs.

Conclusions

We found that acidocalcisomes are present in the eggs and are the main storage compartments of polyphosphate and calcium in the egg yolk. As such components have been shown to be involved in a series of dynamic events that may control embryo growth, results reveal the potential involvement of a novel organelle in the storage and mobilization of inorganic elements to the embryo cells.     

Proton-pyrophosphatase and polyphosphate in acidocalcisome-like vesicles from oocytes and eggs of Periplaneta americana

Acidocalcisomes are acidic organelles containing large amounts of polyphosphate (poly P), a number of cations, and a variety of cation pumps in their limiting membrane. The vacuolar proton-pyrophosphatase (V-H⁺-PPase), a unique electrogenic proton-pump that couples pyrophosphate (PPi) hydrolysis to the active transport of protons across membranes, is commonly present in membranes of acidocalcisomes. In the course of insect oogenesis, a large amount of yolk protein is incorporated by the oocytes and stored in organelles called yolk granules (YGs). During embryogenesis, the content of these granules is degraded by acid hydrolases. These enzymes are activated by the acidification of the YG by a mechanism that is mediated by proton-pumps present in their membranes. In this work, we describe an H⁺-PPase activity in membrane fractions of oocytes and eggs of the domestic cockroach Periplaneta americana. The enzyme activity was optimum at pH around 7.0, and was dependent on Mg²⁺ and inhibited by NaF, as well as by IDP and Ca²⁺. Immunolocalization of the yolk preparation using antibodies against a conserved sequence of V-H⁺-PPases showed labeling of small vesicles, which also showed the presence of high concentrations of phosphorus, calcium and other elements, as revealed by electron probe X-ray microanalysis. In addition, poly P content was detected in ovaries and eggs and localized inside the yolk granules and the small vesicles. Altogether, our results provide evidence that numerous small vesicles of the eggs of P. americana present acidocalcisome-like characteristics. In addition, the possible role of these organelles during embryogenesis of this insect is discussed.     

Inorganic polyphosphate inhibits an aspartic protease‐like activity in the eggs of Rhodnius prolixus (Stahl) and impairs yolk mobilization in vitro

Inorganic polyphosphate (poly P) is a polymer of phosphate residues that has been shown to act as modulator of some vertebrate cathepsins. In the egg yolk granules of Rhodnius prolixus, a cathepsin D is the main protease involved in yolk mobilization and is dependent on an activation by acid phosphatases. In this study, we showed a possible role of poly P stored inside yolk granules on the inhibition of cathepsin D and arrest of yolk mobilization during early embryogenesis of these insects. Enzymatic assays detected poly P stores inside the eggs of R. prolixus. We observed that micromolar poly P concentrations inhibited cathepsin D proteolytic activity using both synthetic peptides and homogenates of egg yolk as substrates. Poly P was a substrate for Rhodnius acid phosphatase and also a strong competitive inhibitor of a pNPPase activity. Fusion events have been suggested as important steps towards acid phosphatase transport to yolk granules. We observed that poly P levels in those compartments were reduced after in vitro fusion assays and that the remaining poly P did not have the same cathepsin D inhibition activity after fusion. Our results are consistent with the hypothesis that poly P is a cathepsin D inhibitor and a substrate for acid phosphatase inside yolk granules. It is possible that, once activated, acid phosphatase might degrade poly P, allowing cathepsin D to initiate yolk proteolysis. We, therefore, suggest that degradation of poly P might represent a new step toward yolk mobilization during embryogenesis of R. prolixus. J. Cell. Physiol. 222: 606–611, 2010. © 2009 Wiley‐Liss, Inc.     

Dynamics of polymorphism of acidocalcisomes in<Emphasis Type="Italic"> Leishmania</Emphasis> parasites

Growth of Leishmania mexicana amazonensis promastigotes in different culture media resulted in structurally and chemically different acidocalcisomes. When grown in SDM-79 medium, the promastigotes showed large spherical acidocalcisomes of up to 1.2 m diameter distributed throughout the cell. X-ray microanalysis and elemental mapping of the organelles showed large amounts of oxygen, phosphorus, sodium, potassium, magnesium, calcium, and zinc. Immunofluorescence microscopy using antisera raised against a peptide sequence of the vacuolar-type proton pyrophosphatase of Arabidopsis thaliana that is conserved in the Leishmania enzyme, indicated localization in acidocalcisomes. When cells were transferred to Warrens medium, the acidocalcisomes transformed from spherical into branched tubular organelles. The labeling pattern of the vacuolar proton-pyrophosphatase, considered as a marker for the organelle, changed accompanying the structural changes of the acidocalcisomes, and the enzyme showed an apparently lower proton-transporting activity when measured in digitonin-permeabilized promastigotes. X-ray microanalysis and elemental mapping of these structures revealed the additional presence of iron. Together, the results reveal that the morphology and composition of acidocalcisomes are greatly influenced by the culture conditions.Electronic Supplementary Material Supplementary material is available in the online version of this article at     

The Acidocalcisome Vacuolar Transporter Chaperone 4 Catalyzes the Synthesis of Polyphosphate in Insect‐stages of Trypanosoma brucei and T. cruzi

Polyphosphate is a polymer of inorganic phosphate found in both prokaryotes and eukaryotes. Polyphosphate typically accumulates in acidic, calcium‐rich organelles known as acidocalcisomes, and recent research demonstrated that vacuolar transporter chaperone 4 catalyzes its synthesis in yeast. The human pathogens Trypanosoma brucei and T. cruzi possess vacuolar transporter chaperone 4 homologs. We demonstrate that T. cruzi vacuolar transporter chaperone 4 localizes to acidocalcisomes of epimastigotes by immunofluorescence and immuno‐electron microscopy and that the recombinant catalytic region of the T. cruzi enzyme is a polyphosphate kinase. RNA interference of the T. brucei enzyme in procyclic form parasites reduced short chain polyphosphate levels and resulted in accumulation of pyrophosphate. These results suggest that this trypanosome enzyme is an important component of a polyphosphate synthase complex that utilizes ATP to synthesize and translocate polyphosphate to acidocalcisomes in insect stages of these parasites.     

The polyphosphate bodies of Chlamydomonas reinhardtii possess a proton-pumping pyrophosphatase and are similar to acidocalcisomes

Acidocalcisomes are acidic calcium storage compartments described initially in trypanosomatid and apicomplexan parasites. In this work, we describe organelles with properties similar to acidocalcisomes in the green alga Chlamydomonas reinhardtii. Nigericin and NH(4)Cl released (45)Ca(2+) from preloaded permeabilized cells, suggesting the incorporation of a significant amount of this cation into an acidic compartment. X-ray microanalysis of the electron-dense vacuoles or polyphosphate bodies of C. reinhardtii showed large amounts of phosphorus, magnesium, calcium, and zinc. Immunofluorescence microscopy, using antisera raised against a peptide sequence of the vacuolar type proton pyrophosphatase (H(+)-PPase) of Arabidopsis thaliana which is conserved in the C. reinhardtii enzyme, indicated localization in the plasma membrane, in intracellular vacuoles, and the contractile vacuole where it colocalized with the vacuolar proton ATPase (V-H(+)-ATPase). Purification of the electron-dense vacuoles using iodixanol density gradients indicated a preferential localization of the H(+)-PPase and the V-H(+)-ATPase activities in addition to high concentrations of PP(i) and short and long chain polyphosphate, but lack of markers for mitochondria and chloroplasts. In isolated electron-dense vacuoles, PP(i)-driven proton translocation was stimulated by potassium ions and inhibited by the PP(i) analog aminomethylenediphosphonate. Potassium fluoride, imidodiphosphate, N,N'-dicyclohexylcarbodiimide, and N-ethylmaleimide also inhibited PP(i) hydrolysis in the isolated organelles in a dose-dependent manner. These results indicate that the electron-dense vacuoles of C. reinhardtii are very similar to acidocalcisomes with regard to their chemical composition and the presence of proton pumps. Polyphosphate was also localized to the contractile vacuole by 4',6-diamidino-2-phenylindole staining, suggesting, with the immunochemical data, a link between these organelles and the acidocalcisomes.     

Evolution of acidocalcisomes and their role in polyphosphate storage and osmoregulation in eukaryotic microbes

Acidocalcisomes are acidic electron-dense organelles, rich in polyphosphate (poly P) complexed with calcium and other cations. While its matrix contains enzymes related to poly P metabolism, the membrane of the acidocalcisomes has a number of pumps (Ca²⁺-ATPase, V-H⁺-ATPase, H⁺-PPase), exchangers (Na⁺/H⁺, Ca²⁺/H⁺), and at least one channel (aquaporin). Acidocalcisomes are present in both prokaryotes and eukaryotes and are an important storage of cations and phosphorus. They also play an important role in osmoregulation and interact with the contractile vacuole complex in a number of eukaryotic microbes. Acidocalcisomes resemble lysosome-related organelles (LRO) from mammalian cells in many of their properties. They share similar morphological characteristics, acidic properties, phosphorus contents and a system for targeting of their membrane proteins through adaptor complex-3 (AP-3). Storage of phosphate and cations may represent the ancestral physiological function of acidocalcisomes, with cation and pH homeostasis and osmoregulatory functions derived following the divergence of prokaryotes and eukaryotes.     

Acidic vesicles in living hyphae of an arbuscular mycorrhizal fungus, Gigaspora margarita

Localization and movement of organelles in living hyphae of an arbuscular mycorrhizal fungus, Gigaspora margarita, were observed using a combination of fluorescent probes and laser-scanning confocal microscopy. Dense, evenly distributed acidic vesicles were visible in germ tubes and extraradical hyphae using DIC with the fluorescent acidotropic probe LysoTracker. These vesicles were distinct from both tubular vacuoles stained with DFFDA and lipid bodies stained with BODIPY 493/503 or Nile Red. Tubular vacuole bundles appeared to be influenced by the bidirectional cytoplasmic streaming of acidic vesicles and lipid bodies. Movement of the acidic vesicles occurred bidrectionally at different rates. The size and distribution of lipid bodies were variable. Based on our observations, the function of these organelles is discussed in relation to nutrient translocation in arbuscular mycorrhizas. Abbreviations: AM – arbuscular mycorrhiza; DAPI – 4′,6-diamidino-2-phenylindole; DIC – differential interference contrast; BODIPY 493/503 – 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene; DMSO – dimethyl sulfoxide; FITC – fluorescein isothiocynate; caboxy-DFFDA – Oregon Green 488 carboxylic acid diacetate.     

Acidocalcisomes of trypanosomatids have species-specific elemental composition

The elemental composition and stoichiometric profile of elements present in acidocalcisomes of different genera of the Trypanosomatidae family (insect, plant, and mammalian parasites) submitted to parallel cultivation conditions were studied. X-ray microanalysis using transmission electron microscopy in conjunction with a morphometric approach was used to investigate the elemental content, number, distribution, and volumetric density of acidocalcisomes of different species. Microanalytical data showed that the different parasites possess the same elemental composition (oxygen, sodium, magnesium, phosphorus, calcium, iron, and zinc) in their acidocalcisomes. However, the relative concentrations of the elements varied among species, but not within acidocalcisomes of individual species. Iron was detected in acidocalcisomes of all species analyzed, characterizing this element as a constituent of these organelles. Taken together, the results strongly indicate a species-specific composition of acidocalcisomes in trypanosomatid parasites.     

Evidence for the role of vacuolar soluble pyrophosphatase and inorganic polyphosphate in Trypanosoma cruzi persistence

Trypanosoma cruzi infection leads to development of a chronic disease but the mechanisms that the parasite utilizes to establish a persistent infection despite activation of a potent immune response by the host are currently unknown. Unusual characteristics of T. cruzi are that it possesses cellular levels of pyrophosphate (PP_i) at least 10 times higher than those of ATP and molar levels of inorganic polyphosphate (polyP) within acidocalcisomes. We characterized an inorganic soluble EF‐hand containing pyrophosphatase from T. cruzi (TcVSP) that, depending on the pH and cofactors, can hydrolyse either pyrophosphate (PP_i) or polyphosphate (polyP). The enzyme is localized to both acidocalcisomes and cytosol. Overexpression of TcVSP (TcVSP‐OE) resulted in a significant decrease in cytosolic PP_i, and short and long‐chain polyP levels. Additionally, the TcVSP‐OE parasites showed a significant growth defect in fibroblasts, less responsiveness to hyperosmotic stress, and reduced persistence in tissues of mice, suggesting that PP_i and polyP are essential for the parasite to resist the stressful conditions in the host and to maintain a persistent infection.     

A single Na+-Pi cotransporter in Toxoplasma plays key roles in phosphate import and control of parasite osmoregulation

Inorganic ions such as phosphate, are essential nutrients required for a broad spectrum of cellular functions and regulation. During infection, pathogens must obtain inorganic phosphate (P_i) from the host. Despite the essentiality of phosphate for all forms of life, how the intracellular parasite Toxoplasma gondii acquires P_i from the host cell is still unknown. In this study, we demonstrated that Toxoplasma actively internalizes exogenous P_i by exploiting a gradient of Na⁺ ions to drive P_i uptake across the plasma membrane. The Na⁺-dependent phosphate transport mechanism is electrogenic and functionally coupled to a cipargarmin sensitive Na⁺-H⁺-ATPase. Toxoplasma expresses one transmembrane P_i transporter harboring PHO4 binding domains that typify the PiT Family. This transporter named TgPiT, localizes to the plasma membrane, the inward buds of the endosomal organelles termed VAC, and many cytoplasmic vesicles. Upon P_i limitation in the medium, TgPiT is more abundant at the plasma membrane. We genetically ablated the PiT gene, and ΔTgPiT parasites are impaired in importing P_i and synthesizing polyphosphates. Interestingly, ΔTgPiT parasites accumulate 4-times more acidocalcisomes, storage organelles for phosphate molecules, as compared to parental parasites. In addition, these mutants have a reduced cell volume, enlarged VAC organelles, defects in calcium storage and a slightly alkaline pH. Overall, these mutants exhibit severe growth defects and have reduced acute virulence in mice. In survival mode, ΔTgPiT parasites upregulate several genes, including those encoding enzymes that cleave or transfer phosphate groups from phosphometabolites, transporters and ions exchangers localized to VAC or acidocalcisomes. Taken together, these findings point to a critical role of TgPiT for P_i supply for Toxoplasma and also for protection against osmotic stresses.     

Dimensions and composition of eggs from captive bustards (Gruiformes: Otididae): houbara (Chlamydotis undulata), rufous‐crested (Eupodotis ruficrista), and kori (Ardeotis kori)

This study sought to increase understanding of the size and composition of eggs from two subspecies of houbara bustard (Chlamydotis undulata macqueenii and C. u. undulata). Eggs from the rufous‐crested bustard (Eupodotis ruficrista) and kori bustard (Ardeotis kori) were also examined for comparison. Infertile eggs were collected from captive birds; egg mass and linear dimensions were recorded, and egg component masses were determined wet and dry. Significant differences were observed in the composition of the eggs from the two houbara subspecies. Despite being smaller in size, eggs from the macqueenii subspecies had a relatively larger yolk (and relatively less albumen) than eggs from the undulata subspecies. The relative composition of the rufous‐crested and kori bustard eggs showed patterns similar to that of the undulata eggs. For the houbara bustards, changes in initial egg mass (IEM) were associated with changes in egg length more than egg breadth, and changes in egg length and breadth appeared to be due more to increases in albumen mass than to increases in yolk mass. Zoo Biol 21:337–346, 2002. © 2002 Wiley‐Liss, Inc.     

Acidocalcisomes

Acidocalcisomes are acidic organelles containing calcium and a high concentration of phosphorus in the form of pyrophosphate (PP_i) and polyphosphate (poly P). Organelles with these characteristics have been found from bacteria to human cells implying an early appearance and persistence over evolutionary time or their appearance by convergent evolution. Acidification of the organelles is driven by the presence of vacuolar proton pumps, one of which, the vacuolar proton pyrophosphatase, is absent in animals, where it is substituted by a vacuolar proton ATPase. A number of other pumps, antiporters, and channels have been described in acidocalcisomes of different species and are responsible for their internal content. Enzymes involved in the synthesis and degradation of PP_i and poly P are present within the organelle. Acidocalcisomes function as storage sites for cations and phosphorus, and participate in PP_i and poly P metabolism, calcium homeostasis, maintenance of intracellular pH, and osmoregulation. Experiments in which the acidocalcisome Ca²⁺-ATPase of different parasites were downregulated or eliminated, or acidocalcisome Ca²⁺ was depleted revealed the importance of this store in Ca²⁺ signaling needed for host invasion and virulence. Acidocalcisomes interact with other organelles in a number of organisms suggesting their association with the endosomal/lysosomal pathway, and are considered part of the lysosome-related group of organelles.     

Synthesis of 2‐amino‐3‐cyanopyridine derivatives and investigation of their carbonic anhydrase inhibition effects

The conversion of carbon dioxide (CO₂) and bicarbonate (HCO₃⁻) to each other is very important for living metabolism. Carbonic anhydrase (CA, E.C.4.2.1.1), a metalloenzyme familly, catalyzes the interconversion of these ions (CO₂ and HCO₃⁻) and are very common in living organisms. In this study, a series of novel 2‐amino‐3‐cyanopyridines supported with some functional groups was synthesized and tested as potential inhibition effects against both cytosolic human CA I and II isoenzymes (hCA I and II) using by Sepharose‐4B‐l ‐tyrosine‐sulfanilamide affinity chromatography. The structural elucidations of novel 2‐amino‐3‐cyanopyridines were achieved by NMR, IR, and elemental analyses. K _i values of the novel synthesized compounds were found in range of 2.84–112.44 μM against hCA I and 2.56–31.17 μM against hCA II isoenzyme. While compound 7d showed the best inhibition activity against hCA I (K _i: 2.84 μM), the compound 7b demonstrated the best inhibition profile against hCA II isoenzyme (K _i: 2.56 μM).     

Novel N‐propylphthalimide‐ and 4‐vinylbenzyl‐substituted benzimidazole salts: Synthesis,characterization, and determination of their metal chelating effects and inhibition profiles against acetylcholinesterase and carbonic anhydrase enzymes

The novel N‐propylphthalimide‐substituted and 4‐vinylbenzyl‐substituted N‐heterocyclic carbene (NHC) precursors were synthesized by N‐substituted benzimidazolium with aryl halides. The novel N‐propylphthalimide‐substituted and 4‐vinylbenzyl‐substituted NHC precursors have been characterized by using ¹H NMR, ¹³C NMR, FTIR spectroscopy, and elemental analysis techniques. They were tested for the inhibition of AChE and hCA enzymes and demonstrated efficient inhibition profiles with K_i values in the range of 351.0–1269.9 nM against hCA I, 346.6–1193.1 nM against hCA II, and 19.0–76.3 nM against AChE. On the other hand, acetazolamide, a clinically used molecule, utilized as CA inhibitor, obtained a K_i value of 1246.7 nM against hCA I and 1407.6 nM against hCA II. Additionally, tacrine inhibited AChE and obtained a K_i value of 174.6 nM.     

Ultrastructural studies on the formation of yolk granules in the statoblast of a fresh-water bryozoan,Pectinatella gelatinosa

The formation of protein-carbohydrate yolk in the statoblast of a fresh-water bryozoan, Pectinatella gelatinosa, was studied by electron microscopy. Two types (I and II) of yolk cells were distinguished. The type I yolk cells are mononucleate and comprise a large majority of the yolk cells. The type II yolk cells are small in number; they become multinucleate by fusion of cells at an early stage of vitellogenesis. In both types of yolk cells, electron-dense granules (dense bodies) are formed in Golgi or condensing vacuoles, which are then called yolk granules. For the formation of yolk granules, the following processes are considered: 1. Yolk protein is synthesized in the rough-surfaced endoplasmic reticulum (RER) of the yolk cells. 2. The synthesized protein condenses in the cisternal space of the RER and is packaged into small oval swellings, which are then released from the RER as small vesicles (Golgi vesicles, 300-600 A in diameter). 3. The small vesicles fuse with one another to form condensing vacuoles, or with pre-existing growing yolk granules. 4. In the matrix of the condensing vacuoles or growing yolk granules, electron-dense fibers are fabricated and then arranged in a paracrystalline pattern to form the dense body. 5. After the dense body reaches its full size, excess membrane is removed and eventually the yolk granules come to mature. Toward the end of vitellogenesis of the yolk cells, the cytoplasmic organelles are ingested by autophagosomes derived from multivesicular bodies and disappear.     

Size‐dependent reproductive success of wild zebrafish Danio rerio in the laboratory

Size‐dependent reproductive success of wild zebrafish Danio rerio was studied under controlled conditions in the laboratory to further understand the influence of spawner body size on reproductive output and egg and larval traits. Three different spawner size categories attained by size‐selective harvesting of the F₁‐offspring of wild D. rerio were established and their reproductive performance compared during a 5 day period. As to be expected, large females spawned more frequently and had significantly greater clutch sizes than small females. Contrary to expectations, small females produced larger eggs when measured as egg diameter with similar amounts of yolk compared to eggs spawned by large spawners. Eggs from small fish, however, suffered from higher egg mortality than the eggs of large individuals. Embryos from small‐sized spawners also hatched later than offspring from eggs laid by large females. Larval standard length (L_S)‐at‐hatch did not differ between the size categories, but the offspring of the large fish had significantly larger area‐at‐hatch and greater yolk‐sac volume indicating better condition. Offspring growth rates were generally similar between offspring from all size categories, but they were significantly higher for offspring spawned by small females in terms of L_S between days 60 and 90 post‐fertilization. Despite temporarily higher growth rates among the small fish offspring, the smaller energy reserves at hatching translated into lower condition later in ontogeny. It appeared that the influence of spawner body size on egg and larval traits was relatively pronounced early in development and seemed to remain in terms of condition, but not in growth, after the onset of exogenous feeding. Further studies are needed to explore the mechanisms behind the differences in offspring quality between large‐ and small‐sized spawners by disentangling size‐dependent maternal and paternal effects on reproductive variables in D. rerio.     

Volutin granules of Eimeria parasites are acidic compartments and have physiological and structural characteristics similar to acidocalcisomes

The structural organization of parasites has been the subject of investigation by many groups and has lead to the identification of structures and metabolic pathways that may represent targets for anti-parasitic drugs. A specific group of organelles named acidocalcisomes has been identified in a number of organisms, including the apicomplexan parasites such as Toxoplasma and Plasmodium, where they have been shown to be involved in cation homeostasis, polyphosphate metabolism, and osmoregulation. Their structural counterparts in the apicomplexan parasite Eimeria have not been fully characterized. In this work, the ultrastructural and chemical properties of acidocalcisomes in Eimeria were characterized. Electron microscopy analysis of Eimeria parasites showed the dense organelles called volutin granules similar to acidocalcisomes. Immunolocalization of the vacuolar proton pyrophosphatase, considered as a marker for acidocalcisomes, showed labeling in vesicles of size and distribution similar to the dense organelles seen by electron microscopy. Spectrophotometric measurements of the kinetics of proton uptake showed a vacuolar proton pyrophosphatase activity. X-ray mapping revealed significant amounts of Na, Mg, P, K, Ca, and Zn in their matrix. The results suggest that volutin granules of Eimeria parasites are acidic, dense organelles, and possess structural and chemical properties analogous to those of other acidocalcisomes, suggesting a similar functional role in these parasites.