Are hydrothermal vents oases for parasitic protists?

Two recent 18S ribosomal RNA-based surveys of protist diversity near hydrothermal vents in the Pacific and Atlantic oceans allowed the identification of a variety of sequences related to several parasitic protist lineages. These include the Apicomplexa, Perkinsozoa, Syndiniales and Kinetoplastida. This diversity of parasitic protists could be hosted by the dense animal populations that thrive around these hydrothermal vents which contrasts with the scarcely populated cold deep-sea waters. These protist parasites might explain some of the mysterious sudden mortality episodes affecting the hydrothermal vent fauna.     

Mitochondrial translation in absence of local tRNA aminoacylation and methionyl tRNAMet formylation in Apicomplexa

Apicomplexans possess three translationally active compartments: the cytosol, a single tubular mitochondrion, and a vestigial plastid organelle called apicoplast. Mitochondrion and apicoplast are of bacterial evolutionary origin and therefore depend on a bacterial‐like translation machinery. The minimal mitochondrial genome contains only three ORFs, and in Toxoplasma gondii the absence of mitochondrial tRNA genes is compensated for by the import of cytosolic eukaryotic tRNAs. Although all compartments require a complete set of charged tRNAs, the apicomplexan nuclear genomes do not hold sufficient aminoacyl‐tRNA synthetase (aaRSs) genes to be targeted individually to each compartment. This study reveals that aaRSs are either cytosolic, apicoplastic or shared between the two compartments by dual targeting but are absent from the mitochondrion. Consequently, tRNAs are very likely imported in their aminoacylated form. Furthermore, the unexpected absence of tRNA^Met formyltransferase and peptide deformylase implies that the requirement for a specialized formylmethionyl‐tRNA^Met for translation initiation is bypassed in the mitochondrion of Apicomplexa.     

Hydrogenosomes and Mitosomes: Conservation and Evolution of Functions1

ABSTRACT. The field studying unusual mitochondria in microbial eukaryotes has come full circle. Some 10–15 years ago it had the evangelical task of informing the wider scientific community that not all eukaryotes had mitochondria. Advances in the field indicated that although some protists might not have mitochondria, the presence of genes of mitochondrial ancestry suggested their lineage once had. The subsequent discovery of mitochondrial compartments in all supposedly amitochondriate protists studied so far indicates that all eukaryotes do have mitochondria indeed. This assertion has fuelled novel eukaryotic origin theories and weakened others. But what do we know about these unusual mitochondria from anaerobic protists? Have they all converged onto similar roles? Iron–sulphur cluster assembly is often hailed as the unifying feature of these organelles. However, the iron–sulphur protein that is so important that a complete organelle is being maintained has not been identified. Is it to be expected that all unusual mitochondria perform the same physiological role? These organelles have been found in numerous protists occupying different ecological niches. Different selection pressures operate on different organisms so there is no reason to suspect that their mitochondria should all be the same.     

Phosphatidylglycerophosphate synthase associates with a mitochondrial inner membrane complex and is essential for growth of Trypanosoma brucei

Maintenance of the lipid composition is important for proper function and homeostasis of the mitochondrion. In Trypanosoma brucei, the enzymes involved in the biosynthesis of the mitochondrial phospholipid, phosphatidylglycerol (PG), have not been studied experimentally. We now report the characterization of T. brucei phosphatidylglycerophosphate synthase (TbPgps), the rate‐limiting enzyme in PG formation, which was identified based on its homology to other eukaryotic Pgps. Lipid quantification and metabolic labelling experiments show that TbPgps gene knock‐down results in loss of PG and a reduction of another mitochondria‐specific phospholipid, cardiolipin. Using immunohistochemistry and immunoblotting of digitonin‐isolated mitochondria, we show that TbPgps localizes to the mitochondrion. Moreover, reduced TbPgps expression in T. brucei procyclic forms leads to alterations in mitochondrial morphology, reduction in the amounts of respiratory complexes III and IV and, ultimately, parasite death. Using native polyacrylamide gel electrophoresis we demonstrate for the first time in a eukaryotic organism that TbPgps is a component of a 720 kDa protein complex, co‐migrating with T. brucei cardiolipin synthase and cytochrome c1, a protein of respiratory complex III.     

Toxoplasma gondii Toc75 Functions in Import of Stromal but not Peripheral Apicoplast Proteins

Apicomplexa are unicellular parasites causing important human and animal diseases, including malaria and toxoplasmosis. Most of these pathogens possess a relict but essential plastid, the apicoplast. The apicoplast was acquired by secondary endosymbiosis between a red alga and a flagellated eukaryotic protist. As a result the apicoplast is surrounded by four membranes. This complex structure necessitates a system of transport signals and translocons allowing nuclear encoded proteins to find their way to specific apicoplast sub‐compartments. Previous studies identified translocons traversing two of the four apicoplast membranes. Here we provide functional support for the role of an apicomplexan Toc75 homolog in apicoplast protein transport. We identify two apicomplexan genes encoding Toc75 and Sam50, both members of the Omp85 protein family. We localize the respective proteins to the apicoplast and the mitochondrion of Toxoplasma and Plasmodium. We show that the Toxoplasma Toc75 is essential for parasite growth and that its depletion results in a rapid defect in the import of apicoplast stromal proteins while the import of proteins of the outer compartments is affected only as the secondary consequence of organelle loss. These observations along with the homology to Toc75 suggest a potential role in transport through the second innermost membrane.     

A mitochondrial Hsp70 orthologue in Vairimorpha necatrix: molecular evidence that microsporidia once contained mitochondria

Microsporidia are small (1–20 μm) obligate intracellular parasites of a variety of eukaryotes, and they are serious opportunistic pathogens of immunocompromised patients [1]. Microsporidia are often assigned to the first branch in gene trees of eukaryotes [2] and [3], and are reported to lack mitochondria [2] and [4]. Like diplomonads and trichomonads, microsporidia are hypothesised to have diverged from the main eukaryotic stock prior to the event that led to the mitochondrion endosymbiosis [2] and [4]. They have thus assumed importance as putative relics of premitochondrion eukaryote evolution. Recent data have now revealed that diplomonads and trichomonads contain genes that probably originated from the mitochondrion endosymbiont [5], [6], [7], [8] and [9], leaving microsporidia as chief candidates for an extant primitively amitochondriate eukaryote group. We have now identified a gene in the microsporidium Vairimorpha necatrix that appears to be orthologous to the eukaryotic (symbiont-derived) Hsp70 gene, the protein product of which normally functions in mitochondria. The simplest interpretation of our data is that microporidia have lost mitochondria while retaining genetic evidence of their past presence. This strongly suggests that microsporidia are not primitively amitochondriate and makes feasible an evolutionary scenario whereby all extant eukaryotes share a common ancestor which contained mitochondria.     

Role of ATG3 in the parasite Toxoplasma gondii

Toxoplasma gondii belongs to the phylum Apicomplexa, a diverse group of early branching unicellular eukaryotes related to dinoflagellates and ciliates. Like several other Apicomplexa such as Plasmodium (the causative agent of malaria), T. gondii is a human pathogen responsible for a potentially lethal disease called toxoplasmosis. Most Apicomplexa have complex life cycles, involving intermediate hosts and vectors, which include obligatory intracellular developmental stages. In the case of malaria and toxoplasmosis, it is that replicative process, leading to the ultimate lysis of the host cell, which is causing the symptoms of the disease. For Toxoplasma, the invasive and fast-replicating form of the parasite is called the tachyzoite. While autophagy has been a fast-growing field of research in recent years, not much was known about the relevance of this catabolic process in medically important apicomplexan parasites. Vesicles resembling autophagosomes had been described in drug-treated Plasmodium parasites in the early 1970s and a potential role for autophagy in organelle recycling during differentiation between Plasmodium life stages has also been recently described. Interestingly, recent database searches have identified putative orthologs of the core machinery responsible for the formation of autophagosomes in several protists, including Toxoplasma. In spite of an apparently reduced machinery (only about one-third of the yeast ATG genes appear to be conserved), T. gondii seemed thus able to perform macroautophagy, but the cellular functions of the pathway for this parasite remained to be demonstrated.     

Leishmania mitochondrial tRNA importers

The RNA Import Complex (RIC) is a multi-subunit protein complex from the mitochondria of the kinetoplastid protozoon Leishmania tropica that induces transport of tRNA across natural and artificial membranes. Leishmania, Trypanosoma and related genera of the order Kinetoplastidae are early diverging, atypical eukaryotes with unique RNA metabolic pathways, including the import of nucleus-encoded tRNAs into the mitochondrion to complement the deletion of all organelle-encoded tRNA genes. Biochemical and genetic studies of RIC are contributing to greater understanding of the mechanism of import. Additionally, RIC was shown to act as an efficient delivery vehicle for tRNA and other small RNAs into mitochondria within intact mammalian cells, indicating its applicability to the management of diseases caused by mitochondrial mutations.     

How do endosymbionts become organelles? Understanding early events in plastid evolution

What factors drove the transformation of the cyanobacterial progenitor of plastids (e.g. chloroplasts) from endosymbiont to bona fide organelle? This question lies at the heart of organelle genesis because, whereas intracellular endosymbionts are widespread in both unicellular and multicellular eukaryotes (e.g. rhizobial bacteria, Chlorella cells in ciliates, Buchnera in aphids), only two canonical eukaryotic organelles of endosymbiotic origin are recognized, the plastids of algae and plants and the mitochondrion. Emerging data on (1) the discovery of non‐canonical plastid protein targeting, (2) the recent origin of a cyanobacterial‐derived organelle in the filose amoeba Paulinella chromatophora, and (3) the extraordinarily reduced genomes of psyllid bacterial endosymbionts begin to blur the distinction between endosymbiont and organelle. Here we discuss the use of these terms in light of new data in order to highlight the unique aspects of plastids and mitochondria and underscore their central role in eukaryotic evolution. BioEssays 29:1239–1246, 2007. © 2007 Wiley Periodicals, Inc.     

Mock communities highlight the diversity of host‐associated eukaryotes

Host‐associated microbes are ubiquitous. Every multicellular eukaryote, and even many unicellular eukaryotes (protists), hosts a diverse community of microbes. High‐throughput sequencing (HTS) tools have illuminated the vast diversity of host‐associated microbes and shown that they have widespread influence on host biology, ecology and evolution (McFall‐Ngai et al. 2013 ). Bacteria receive most of the attention, but protists are also important components of microbial communities associated with humans (Parfrey et al. 2011 ) and other hosts. As HTS tools are increasingly used to study eukaryotes, the presence of numerous and diverse host‐associated eukaryotes is emerging as a common theme across ecosystems. Indeed, HTS studies demonstrate that host‐associated lineages account for between 2 and 12% of overall eukaryotic sequences detected in soil, marine and freshwater data sets, with much higher relative abundances observed in some samples (Ramirez et al. 2014 ; Simon et al. 2015 ; de Vargas et al. 2015 ). Previous studies in soil detected large numbers of predominantly parasitic lineages such as Apicomplexa, but did not delve into their origin [e.g. (Ramirez et al. 2014 )]. In this issue of Molecular Ecology, Geisen et al. ( 2015 ) use mock communities to show that many of the eukaryotic organisms detected by environmental sequencing in soils are potentially associated with animal hosts rather than free‐living. By isolating the host‐associated fraction of soil microbial communities, Geisen and colleagues help explain the surprisingly high diversity of parasitic eukaryotic lineages often detected in soil/terrestrial studies using high‐throughput sequencing (HTS) and reinforce the ubiquity of these host‐associated microbes. It is clear that we can no longer assume that organisms detected in bulk environmental sequencing are free‐living, but instead need to design studies that specifically enumerate the diversity and function of host‐associated eukaryotes. Doing so will allow the field to determine the role host‐associated eukaryotes play in soils and other environments and to evaluate hypotheses on assembly of host‐associated communities, disease ecology and more.     

Diversity and reductive evolution of mitochondria among microbial eukaryotes

All extant eukaryotes are now considered to possess mitochondria in one form or another. Many parasites or anaerobic protists have highly reduced versions of mitochondria, which have generally lost their genome and the capacity to generate ATP through oxidative phosphorylation. These organelles have been called hydrogenosomes, when they make hydrogen, or remnant mitochondria or mitosomes when their functions were cryptic. More recently, organelles with features blurring the distinction between mitochondria, hydrogenosomes and mitosomes have been identified. These organelles have retained a mitochondrial genome and include the mitochondrial-like organelle of Blastocystis and the hydrogenosome of the anaerobic ciliate Nyctotherus. Studying eukaryotic diversity from the perspective of their mitochondrial variants has yielded important insights into eukaryote molecular cell biology and evolution. These investigations are contributing to understanding the essential functions of mitochondria, defined in the broadest sense, and the limits to which reductive evolution can proceed while maintaining a viable organelle.     

The Golgi apparatus in parasitic protists

This review summarizes the current reports on the Golgi apparatus of parasitic protists. Numerous recent publications have demonstrated that studies on intracellular traffic in parasites essentially advanced our knowledge on the Golgi structure and function, which has been traditionally based on research on yeast and mammalian cultured cells. It has been reported that the parasitic lifestyle determines the functional and structural peculiarities of the secretory systems in unrelated groups of unicellular parasites that make them different from those in mammalian and yeast cells. This review covers the best-studied protists, predominantly those of high medical importance, belonging to the following taxa: Parabasalia (Trichomonas), Diplomonada (Giardia), Entamoebidae (Entamoeba), parasitic Alveolata of the phyllum Apicomplexa (Toxoplasma, Plasmodium), and Kinetoplastida (Trypanosoma, Leishmania). The morphology of the Golgi organelle in eukaryotes from various taxonomic groups has been compared. Within three of the six highest taxa of Eukaryota (Adl et al., 2005) a minimum of eight groups are represented by species lacking Golgi dictiosomes. However, biochemical and/or molecular (genomic) evidence indicate that an organelle with the functions of the Golgi was present in every lineage of eukaryotes studied thus far. Loss of the Golgi organelle is a secondary event as proven by identification of Golgi genes in the genomes of Golgi-lacking lineages. The loss might have occurred independently several times in evolution. Neither the number of stacks, nor the size of the organelle correlates with the intensity of secretion or the position of the species on the evolutionary tree (in terms of presumably early/lately diverged lineages).     

Killing the Dead: Chemotherapeutic Strategies Against Free‐Living Cyst‐Forming Protists (Acanthamoeba sp. and Balamuthia mandrillaris)

The opportunist free‐living protists such as Acanthamoeba spp. and Balamuthia mandrillaris have become a serious threat to human life. As most available drugs target functional aspects of pathogens, the ability of free‐living protists to transform into metabolically inactive cyst forms presents a challenge in treatment. It is hoped, that the development of broad spectrum antiprotist agents acting against multiple cyst‐forming protists to provide target‐directed inhibition will offer a viable drug strategy in the treatment of these rare infections. Here, we present a comprehensive report on upcoming drug targets, with emphasis on cyst wall biosynthesis along with the related biochemistry of encystment pathways, as we strive to bring ourselves a step closer to being able to combat these deadly diseases.     

An ADP/ATP-specific mitochondrial carrier protein in the microsporidian Antonospora locustae

The mitochondrion is one of the defining characteristics of eukaryotic cells, and to date, no eukaryotic lineage has been shown to have lost mitochondria entirely. In certain anaerobic or microaerophilic lineages, however, the mitochondrion has become severely reduced that it lacks a genome and no longer synthesizes ATP. One example of such a reduced organelle, called the mitosome, is found in microsporidian parasites. Only a handful of potential mitosomal proteins were found to be encoded in the complete genome of the microsporidian Encephalitozoon cuniculi, and significantly no proteins of the mitochondrial carrier family were identified. These carriers facilitate the transport of solutes across the inner mitochondrial membrane, are a means of communication between the mitochondrion and cytosol, and are abundant in organisms with aerobic mitochondria. Here, we report the characterization of a mitochondrial carrier protein in the microsporidian Antonospora locustae and demonstrate that the protein is heterologously targeted to mitochondria in Saccharomyces cerevisiae. The protein is phylogenetically allied to the NAD⁺ transporter of S. cerevisiae, but we show that it has high specificity for ATP and ADP when expressed in Escherichia coli. An ADP/ATP carrier may provide ATP for essential ATP-dependent mitosomal processes such as Hsp70-dependent protein import and export of iron-sulfur clusters to the cytosol.     

Evolution of energy metabolism and its compartmentation in Kinetoplastida

Kinetoplastida are protozoan organisms that probably diverged early in evolution from other eukaryotes. They are characterized by a number of unique features with respect to their energy and carbohydrate metabolism. These organisms possess peculiar peroxisomes, called glycosomes, which play a central role in this metabolism; the organelles harbour enzymes of several catabolic and anabolic routes, including major parts of the glycolytic and pentosephosphate pathways. The kinetoplastid mitochondrion is also unusual with regard to both its structural and functional properties.     

The incredible shrinking organelle

The mitochondrion is probably the evolutionary remnant of a bacterial symbiont, yet contemporary mitochondria are nothing like contemporary bacteria. Evolutionary shrinkage of the mitochondrial genome is well documented, but what about wholesale shrinkage of the organelle itself?Considering its central role in energy metabolism in almost all eukaryotes, the mitochondrion is an amazingly plastic organelle, both evolutionarily and functionally. The few genes that the mitochondrial genome (mitochondrial DNA; mtDNA) encodes are clearly bacterial in origin—emanating from the α-proteobacterial lineage—supporting the widely held view that the mitochondrion is the evolutionary remnant of a bacterial symbiont (Gray et al, 2001). However, contemporary mitochondria are nothing like contemporary bacteria. For one thing, even the most gene-rich mtDNA encodes far less genetic information than the most gene-poor bacterial genome, and mitochondrial genomes are different from bacterial genomes in form, organization and mode of expression; these features vary tremendously among diverse eukaryotes. Mitochondrial genomes might be circular, linear or even highly fragmented, and they might contain highly fragmented and rearranged genes. Only within a poorly studied group of eukaryotic microbes—protists—known as jakobid flagellates does the mtDNA resemble a typical, albeit highly reduced, bacterial genome.In addition, the mitochondrial proteome is not only overwhelmingly (>90%) encoded in the nucleus, but only a small proportion (10–15%) is demonstrably α-proteobacterial in evolutionary affiliation. Thus, in the evolutionary transition from bacterial symbiont to integrated organelle, the mitochondrion has undergone an impressive degree of re-tailoring, shedding the bulk of its genetic information and taking on proteins of diverse evolutionary origins. Moreover, this re-tailoring is highly variable within different eukaryotic lineages, with an intriguing chunk of the mitochondrial proteome seeming to be organism-specific—lacking demonstrable sequence homologues other than in very close evolutionary relatives.Although the evolutionary shrinkage of the mitochondrial genome is well-documented, what is less widely appreciated is the wholesale shrinkage of the organelle itself in certain anaerobic eukaryotes. Taken to its extreme, such shrinkage involves complete loss of the mitochondrial genome, with a consequent reduction in the structural complexity and biochemical versatility of the organelle. This simplification might include elimination of the electron-transport chain (ETC) and thus lead to inability of the resulting mitochondrion-related organelle (MRO) to carry out a key function of aerobic mitochondria: ATP synthesis through coupled oxidative phosphorylation (for a full account, see Hjort et al, 2010).One such MRO, the hydrogenosome, is a hydrogen-producing organelle that was originally characterized in an anaerobic protist, Trichomonas vaginalis. The T. vaginalis hydrogenosome lacks mtDNA as well as components of the classic mitochondrial ETC, relying instead on substrate-level phosphorylation to generate ATP. Initially, the resemblance between the anaerobic biochemistry of the T. vaginalis MRO and that of anaerobic bacteria such as Clostridia raised the possibility that the hydrogenosome might have a different evolutionary origin than the classic aerobic mitochondrion. However, studies of hydrogenosomal proteins have demonstrated that the hydrogenosome is an evolutionarily derived (remnant) mitochondrion. Hydrogenosomes have been found in eukaryotes that are widely separated in phylogenetic trees, and in such trees, anaerobic, hydrogenosome-containing eukaryotes are often interspersed with close relatives that grow aerobically and contain conventional mitochondria. This punctate phylogenetic distribution suggests that the transition from mitochondrion to hydrogenosome has happened repeatedly and independently throughout eukaryotic evolution.The mitosome, an even more shrunken MRO that has not only dispensed entirely with a genome, but also has no ATP-generating capacity. This MRO was discovered in anaerobic eukaryotes that were initially thought to lack mitochondria entirely, the postulate being that they diverged away from the main line of eukaryotic evolution prior to the symbiosis that led to the mitochondrion. However, in all supposedly amitochondriate protists that have been examined, a candidate mitosome has been identified. As with hydrogenosomes, a punctate phylogenetic distribution of mitosomes is emerging.Recently, intermediate forms of ''shrinking organelle'' have been identified in the anaerobic protists Nyctotherus ovalis, Blastocystis sp. and Proteromonas lacertae (Hjort et al, 2010; Pérez-Brocal et al, 2010; de Graaf et al, 2011), relatives of brown algae and diatoms. In these cases, regions of the mtDNA that code for terminal portions of the ETC and for the mitochondrial ATP synthase have been discarded. The remaining DNA specifies genes for components of a mitochondrial translation system, as well as subunits of a proton-pumping complex I (NADH:ubiquinone oxidoreductase); a remarkable example—comparing the ciliate Nyctotherus with the stramenopiles Blastocystsis or Proteromonas—of convergent mtDNA evolution. These observations suggest that the transitional MROs of Nyctotherus, Blastocystis and Proteromonas retain a partial ETC, as well as the ability to synthesize protein, whereas other data (EST surveys) indicate that they are metabolically more complex than either hydrogenosomes or mitosomes. The discovery of these particular MROs is important because their existence argues that the transition from fully fledged aerobic mitochondrion to fully fledged anaerobic mitosome proceeds through, and might stop at, several intermediate stages: a realization that not only dramatically emphasizes the evolutionary and functional versatility of the mitochondrion, but also opens the possibility that we might yet uncover still other variations of this incredible shrinking organelle.     

Morphological changes of giant mitochondria in the unicellular to multicellular phase during parthenogenesis of Ulva partita (Ulvophyceae) revealed by expression of mitochondrial targeting GFP and PEG transformation

A giant mitochondrion that branches and connects as a single mitochondrion in a cell has been observed during specific phases of the cell cycle of unicellular green algae, but has not been observed in multicellular algae. The genus Ulva is a green macroalga in which the haploid and diploid phases are isomorphic and its gametes develop parthenogenetically. The existence or absence of the giant mitochondrion, and its behavior in Ulva partita, were investigated using a parthenogenesis system. To observe the parthenogenesis of gametes and the dynamics of mitochondria by fluorescence microscopy, we developed an experimental system for culturing and observing U. partita on cover slips: gametes were suspended in 6‐well plates filled with artificial seawater, and cover slips were placed on the well bottoms. The gametes settled on the cover slips as spherical cells (1‐cell S phase). These cells grew into larger cells, losing their eyespot (1‐cell L phase), and developed into multicellular thalli. Gene introduction using the polyethylene glycol (PEG) method is available with transformation efficiencies of 9.0–15.1%. Transformation was performed using a plasmid encoding green fluorescent protein (GFP) fused to the mitochondrial targeting sequence, and mitochondria were labeled by GFP fluorescence. This revealed a string‐shaped giant mitochondrion in a cell of the 1‐cell S phase. In the 1‐cell L phase, a reticular mitochondrion was observed. After the initiation of cell division, the reticular mitochondrion was fragmented, and small oval mitochondria were observed in the 5‐cell phase.     

History assignment: when was the mitochondrion founded?

The near simultaneous radiation of the major eukaryotic evolutionary assemblages — plants, animals, fungi, and at least three other complex protist assemblages worthy of ‘kingdom level’ status — was preceded by the divergence of many independent protist lineages. The earliest branches are represented by organisms that do not contain mitochondria or plastids, suggesting that the primitive eukaryotic state did not include these organelles. New information about nuclear-coded proteins that localize in the mitochondrion, however, suggests that the ancestral symbionts for mitochondria were present in the first eukaryotes. Phylogenetic support for this hypothesis is persuasive but it is not possible to account for the relative times of divergence for mitochondria and their ancestral symbionts relative to eukaryotic branching patterns inferred from nuclear genes.     

Mitochondrial deformation and apocrine secretory mechanism in the rabbit submandibular organ as revealed by electron microscopy

Summary The submandibular organ (a sort of apocrine sweat glands) of the rabbit was observed with the electron microscope. The cell structure of glandular tubules varies depending upon the secretory activity; there are three functional stages. The secretory cells at the resting stage are characterized by low height, absence of secretory substance, and presence of small and slender mitochondria.In the synthesizing stage, enlargement and peculiar deformation of mitochondria are observed. Secretory substance always occurs near the deformed mitochondria. The part of a mitochondrion closely abutting on the secretion mass is extremely thin, and contains longitudinally oriented cristae. Sometimes a direct continuity is observed between the thinned portion of the deformed mitochondria and the mass of secretory substance. It is presumed that the secretion is initially produced in the mitochondria and then discharged from them. The Golgi apparatus and the rough surfaced endoplasmic reticulum may be involved indirectly. Smooth surfaced vesicles, probably related to the transport of raw material, are extremely abundant in the cells of this stage.The development of a generally homogeneous projection into the gland lumen is characteristic of the stage of secretion discharge. The mitochondria are again small and slender, and the secretion is liquefied. At the base of the full-grown projection, cytoplasm is condensed to form a demarcation zone from which the projection may become detached. This mechanism of release of secretory product is quite the same as the so-called apocrine secretory process long postulated by light microscopists.