Intracellular symbiotes of the pea aphid,Acyrthosiphon pisum

Histological and ultrastructural studies on the mycetome of the pea aphid, Acyrthosiphon pisum, disclosed two types of symbiotes. The more common primary symbiotes were oval in shape and were found in large mycetocytes making up the bulk of the mycetome. The secondary symbiotes were smaller, rod-shaped, and were restricted to an apparently syncytial sheath partially enclosing the primary mycetocytes. Extensive rough endoplasmic reticulum and Golgi occurred in the sheath but not in the primary mycetocytes. Lysosomal breakdown occurred in both primary and secondary symbiotes but the two processes differed markedly. In the primary mycetocytes, a small number of symbiotes were broken down individually to form small, compact residual bodies. In the sheath, breakdown of secondary symbiotes was more extensive: large numbers were broken down within cytolysomes.     

Ultrastructure of Symbiotic Bacteria in Normal and Antibiotic-Treated Blastocrithidia culicis and Crithidia oncopelti*

SYNOPSIS. An electron microscope study of diplosomes in Blastocrithidia culicis and bipolar bodies in Crithidia oncopelti has shown that both entities appear to be intracellular symbiotes and have a similar fine structure. They are enclosed by 2 unit membranes which are separated by a large space of very low density. The outer membrane is derived probably from the host cell. The matrix of the symbiotes is composed of dense ribosome-like particles and of areas of low density containing fine fibrillae. The particles are of the same size as ribosomes in bacteria and the fibrils have the characteristics of bacterial DNA. Thus, the lucid areas with fibrillae correspond to the nucleoids in bacteria. These observations suggest that the symbiotes are bacteria. The effect of chloramphenicol (CAP) and penicillin G (PCL) on these symbiotic bacteria was studied by culturing the host flagellates in media containing the antibiotics. The effect was analyzed at different intervals after the treatment by electron microscopy. After single treatment in the blood broth containing 0.08% (w/v) CAP, symbiotes appeared to have enlarged nucleoids, became deformed and eventually degenerated. In Grace's medium (supplemented with 10% fetal bovine serum) containing 0.6 or 2.4% (w/v) PCL, symbiotes of C. oncopelti remained unaltered, whereas some symbiotes of B. culicis became pleomorphic. Symbiotes of both species persisted after repeated transfers in PCL media and reverted to normal forms when transferred to PCL-free media. Sensitivity of symbiotes to CAP provides further evidence of their bacterial nature. The effect of PCL on the symbiotes of B. culicis suggests the presence in their cell envelopes of mucopeptide, which probably provides rigidity for maintaining the bacterial shape of the symbiotes.     

Isolation and characterization of symbiotes from the Rocky Mountain wood tick, Dermacentor andersoni

Wolbachia-like symbiotes in the Rocky Mountain wood tick, Dermacentor andersoni, were isolated repeatedly by injection of ovarial tissues into 5-day-old chick embryos. In Giemsa-stained smears of infected embryo tissues, the organisms appeared as blueish or pinkstained coccal bodies indistinguishable from those seen in the ovaries of ticks, where they are located in the luminal epithelium and funicle cells, as well as in oocytes.Electron microscopy revealed that these symbiotes are highly pleomorphic and vary in size from 0.6 to 3.4 μm in diameter. Their fine structure in tissue cells is differentiated into a granular, cortical region, which contains densely stained ribosomes, and a medullary region consisting of a diffuse reticulum partially or completely devoid of granular material or ribosomes. Multiplication is by binary fission. Each organism is delimited by a distinct plasmalemma; a cell wall as in bacterial and rickettsial agents was not observed in organisms from ovarial tissues.Symbiotes cultivated in chick embryos and then injected intracoelomically into adult D. andersoni, developed rapidly and produced massive infestations in hemocytes, hypodermal tissues, salivary glands, and in connective tissues surrounding midgut, Malpighian tubules, and ovary. In hypodermal tissue, organisms with a distinct bilayered cell envelope were occasionally detected. The massive invasion of tissues by injected symbiotes invariably proved fatal for ticks.Results of complement-fixation tests and of fluorescent antibody staining indicated that symbiotes in D. andersoni are closely related to Wolbachia persica, previously isolated from Argas arboreus.     

Vitamin requirements of larvae of Sitophilus oryzae

Improvements were made in the meridic larval diet for Sitophilus oryzae by replacing the minerals and vitamins supplied by dietary brewer's yeast and wheat germ with mineral and vitamin mixtures. The effects of different concentrations of individual vitamins were studied with the improved diet containing 20% casein. In later tests the dietary casein was replaced with a mixture of 15 amino acids. The results indicated that these larvae, which contain an associated bacteria-like micro-organism, required thiamine, nicotinic acid, pyridoxine, folic acid, and biotin in the diet. No requirement for riboflavin or pantothenic acid could be demonstrated on either the 20% casein diet or the amino acid diet possibly due to contamination of the cornstarch with these two vitamins. In addition, the larvae did not require choline or inositol for the growth of one larval generation.The asymbiotic larvae of S. granarius failed to develop on the improved casein diet indirectly implicating the symbiotes in a nutritional rôle; however, the symbiotes present in S. oryzae apparently do not provide B vitamins. Larvae of S. oryzae failed to develop when the concentration of casein was reduced to 10% while growth was maintained with a 10% concentration of the amino acid mixture. Casein is not an optimal source of amino acids for this species.     

First cytochemical study of haemocytes from the crab Carcinus aestuarii (Crustacea,Decapoda)

For the first time, a morphological study of haemocytes from the crab Carcinus aestuarii was carried out by means of light microscopy and differing cytochemical assays. Analysis of haemocyte size frequency distribution (performed by means of a Coulter Counter) revealed the presence of two distinct haemocyte fractions in C. aestuarii haemolymph, depending on cell size. The first fraction was of about 3–5 µm in diameter and 30–50 fL in volume, the second was of about 6–12 µm in diameter and over 200 fL in volume. Mean cell diameter and volume were 8.20±1.7 µm and 272.30±143.5 fL, respectively. Haemocytes observed under light microscope were distinguished in three cell types: granulocytes (28%; 11.94±1.43 µm in diameter) with evident cytoplasmic granules, semigranulocytes (27%; 12.38±1.76 µm in diameter) with less granules than granulocytes, and hyalinocytes (44%; 7.88±1.6 µm in diameter) without granules. In addition, a peculiar cell type was occasionally found (about 1%): it was 25–30 µm in diameter and had a great vacuole and a peripheral cytoplasm with granules. Granulocyte and semigranulocyte granules stained in vivo with Neutral Red, indicating that they were lysosomes. Giemsa’s dye confirmed that granulocytes and semigranulocytes were larger than hyalinocytes. Pappenheim’s panoptical staining and Ehrlich’s triacid mixture allowed to distinguish granule-containing cells (including semigranulocytes) in acidophils (64%), basophils (35%) and neutrophils (1%). Hyalinocytes showed always a basophilic cytoplasm. Haemocytes were positive to the PAS reaction for carbohydrates, even if cytoplasm carbohydrate distribution varied among cell types. Lastly, lipids were found on cell membrane and in cytoplasm of all haemocyte types in the form of black spots produced after Sudan Black B staining. The morphological characterisation of C. aestuarii haemocytes by light microscopy was necessary before performing both ultrastructural and functional studies of circulating cells.Key words: Carcinus aestuarii, crab, haemocytes, light microscopy, cytochemical assays, morphological characterisation.     

Resuscitation of amebae deprived of essential symbiotes: Micrurgical studies*

SYNOPSIS. The effect of depletion and restoration of obligatory bacterial endosymbiotes on Amoeba proteus strain xD was studied. Removal of the symbiotes by culturing the amebae at 26.5 C resulted in loss of viability of the host cells, indicating that this strain is dependent on its endosymbiotes for survival. Amebae depleted of bacteria could initially be resuscitated by injection of isolated symbiotes, but prolonged deprivation led to irreversible changes. Nuclei of aposymbiotic amebae were viable when transplanted into the cytoplasm of normal cells, but the symbiote-depleted cytoplasm of heat-treated amebae could not be resuscitated by renucleation. No immediate ultrastructural changes were detected in aposymbiotic amebae except for clumping of nucleoli. Thus it appears that the symbiote performs an essential function as a cytoplasmic constituent.     

Hydroxycinnamoyl acid amides and aromatic amines in the inflorescences of some araceae species

Hydroxycinnamoyl acid amides (HCA's) were found to be important components in the inflorescences of different Araceae species. HCA's occurred in large amount in spathes and in the male and female flowers, and were totally absent from the sterile flowers, commonly found on Araceae spadices. Differences in the distribution of HCA's were noted between male and female flowers. Thus the amount of neutral HCA's was always greater in the male than in the female flowers and the female flowers generally contained more basic HCA'S. In the inflorescences of some Araceae species in the Monsteroideae and Philodendroideae (genera Monstera, Raphidophora and Philodendron), the aromatic amines tyramine and dopamine were very abundant, with concentrations ranging from 1 to 4 mg of each amine per g fr. wt.     

In vivo sterol biosynthesis by pea aphid symbiotes as determined by digitonin and electron microscopic autoradiography

Summary Pea aphid primary symbiotes have previously been shown to synthesize cholesterol in vitro. Two electron microscopic techniques were used here to determine whether the symbiotes also synthesize cholesterol in vivo and whether this cholesterol is made available to the aphid. We also inquired into a possible role of secondary symbiotes in cholesterol biosynthesis. Treatment of aphids with digitonin resulted in significant alteration of ultrastructural sites in primary and secondary symbiote membranes. We concluded that these sites are areas of high cholesterol concentration in the symbiotes.Electron microscopic autoradiography with ³H-mevalonate precursor indicated that both primary and secondary symbiotes synthesize cholesterol; in both cases, the majority of grains were associated with the symbiote membranes. While the frequency of grains on the symbiotes remained constant, irrespective of incubation time in labelled media, the frequency of grains over surrounding tissues increased exponentially as the time of incubation was increased from 30 min to 8 h, indicating that symbiote cholesterol is transported to other tissues. High voltage electron microscopic autoradiography permitted thick section autoradiography, reducing the time of emulsion exposure from 54 days (thin section) to 12 days (0.5 m sections).Research supported by the College of Agricultural and Life Sciences, University of Wisconsin, and by a research grant (PCM 74-2401 A01) from The National Science FoundationThe authors wish to thank Dr. G.A. DeZoeten for his invaluable advice and assistance with the autoradiographic techniques, Mr. Gary Gaard for his help with electron microscopy, and Dr. Dale Johnston and Dr. Damien Neuberger for their generous help in the use of the high voltage EM     

Omikron,ein essentieller Endosymbiont von Euplotes aediculatus*†

ZUSAMMENFASSUNG. Der Süßwasserciliat Euplotes aediculatus beherbergt in seinem Cytoplasma 900–1000 stäbchenförmige Symbionten, die für seine Vermehrung essentiell zu sein scheinen. Durch Wachstum in Gegenwart von Penicillin kann Euplotes von seinen Symbionten befreit werden, es führt dies jedoch stets auch zu einem Verlust der Teilungsfähigkeit. Eine Reinfektion ist bei etwa 4% der Zellen möglich, die dann nach 4–5 Tagen sich wieder zu vermehren beginnen. Die Endosymbionten wurden sowohl lichtmikroskopisch als auch elektronenmikroskopisch untersucht. In ihrer Feinstruktur und Färbbarkeit gleichen sie, wie kappa und andere von Paramecium her bekannte “killer”-Partikel, gramnegativen Bakterien. Sie unterscheiden sich von diesen Symbionten jedoch dadurch, daß ihre DNA im Cytoplasma nicht homogen verteilt ist, sondern sich in 3–9 schon lichtmikroskopisch erkennbaren Zentren konzentriert. Die in Euplotes vorkommenden Symbionten lassen keine “killer”-Aktivität erkennen. Sie werden omikron-Partikel genannt. SYNOPSIS. The fresh water ciliate Euplotes aediculatus contains in its cytoplasm 900–1000 rod-shaped symbiotes which appear to be essential for division. Growth of Euplotes in the presence of penicillin results in loss of these symbiotes and simultaneously in a loss of the ciliate's ability to divide. Reinfection with the symbiotes can be achieved in 4% of the cells which then resume growth after a lag period of 4–5 days. The endosymbiotes have been studied by light and electron microscopy. In their fine structure and staining reaction they resemble gram-negative bacteria as do kappa and other killer particles of Paramecium. The symbiotes of Euplotes, however, are unusual in that their DNA is not distributed throughout the cytoplasm but is localized in 3–9 areas (nucleoids), which are visible even in the light microscope. No killing activity seems to be associated with the symbiotes. Following the practice of referring to those endosymbiotes by Greek letters they are here designated omikron particles.     

Histochemical Detection of Acetogenins and Storage Molecules in the Endosperm of Annona Macroprophyllata Donn Sm. Seeds

Acetogenins (ACGs) are bioactive compounds with cytotoxic properties in different cell lines. They are antitumoural, antiparasitic, antimalarial, insecticidal, antimicrobial, anti-fungal and antibacterial. These secondary metabolites function in plant defence and are found in specific organelles and specific cells, thereby preventing toxicity to the plant itself and permitting site-specific defence. The aim of this work was to histochemically determine the in situ localisation of ACGs in the endosperm of Annona macroprophyllata seeds using Kedde’s reagent. Additionally, the co-localisation of ACGs with other storage molecules was analysed. The seeds were analysed after 6 and 10 days of imbibition, when 1 or 2 cm of the radicle had emerged and metabolism was fully established. The seeds were then transversally cut in half at the midline and processed using different histological and histochemical techniques. Positive reactions with Kedde’s reagent were only observed in fresh, unfixed sections that were preserved in water, and staining was found only in the large cells (the idioblasts) at the periphery of the endosperm. The ACGs’ positive reaction with Sudan III corroborated their lipid nature. Paraffin sections stained with Naphthol Blue Black showed reactions in the endosperm parenchyma cells and stained the proteoplasts blue, indicating that they might correspond to storage sites for albumin-like proteins. Lugol’s iodine, which is similar in chemical composition to Wagner’s reagent, caused a golden brown reaction product in the cytoplasm of the idioblasts, which may indicate the presence of alkaloids.Based on these results, we propose that Kedde’s reagent is an appropriate histochemical stain for detecting ACGs in situ in idioblasts and that idioblasts store ACGs and probably alkaloids. ACGs that are located in idioblasts found in restricted, peripheral areas of the endosperm could serve as a barrier that protects the seeds against insects and pathogen attack.Key words: Annona, Kedde’s reagent, acetogenin histochemistry, storage reserves histochemistry, acetogenin idioblast     

Flask cells and flask-shaped glandular cells of amphibian skin specifically produce fucose-rich glycoproteins

A battery of horseradish peroxidase-conjugated lectins has been employed as a cytochemical tool for the labelling of specific cell types in amphibian epidermis. Among the lectins used, onlyUlex europaeus I (UEA I) showed specific reaction with the cytoplasm of flask cells. In addition, UEA I stained flask-shaped secretory cells in dermal glands and a reaction on glandular ductal cells was also observed. At the electron microscopic level, lect-in binding was found in granules distributed among mitochondria in the cytoplasm of flask cells and in larger mucous granules of flask-shaped glandular cells, which were released into the lumen. UEA I also stained the extracellular space above flask cells. The labelling was due mainlty to a glycoprotein of mol. wt. approx. 27 kDa. Structural and cytochemical similarities between flask cells and flask-shaped cells of dermal glands could be a consequence of a common secretory role of both cell types.     

Symbiosis in the Larger Foraminiferan Sorites marginalis (with Notes on Archaias spp.)

SYNOPSIS. It was demonstrated with the aid of light and electron microscope studies that Sorites marginalis (Lamarck) harbors zooxanthellae. The hosts were scraped from Thalassia testudinum Konig growing in Key Largo Sound (Florida, U.S.A.) and immediately preserved in appropriate fixatives. Zooxanthellae were distributed unevenly throughout all the chamberlets; only a few symbiotes were found in the embryonic chambers and the inner or outer chambers, but the intermediate chambers were packed with symbiotes. The outer chambers contained many food vacuoles in addition to symbiotes. Some zooxanthellae might have been in the process of degeneration or digestion. The symbiotes were found to have a typical dinoflagellate nucleus, a single large lobate cortical chloroplast with one stalked pyrenoid, an accumulation body, and many starch granules. The nonmotile stage of the zooxanthella was similar, but perhaps not identical, to Symbiodinium microadriaticum Freudenthal from various hosts.
The foraminiferan host is heterokaryotic with hundreds of generative (small) nuclei and scores of vegetative (large) nuclei. Most of the generative nuclei were found in the embryonic apparatus and the inner chambers. Most of the vegetative nuclei were found in the inner and outer chambers.     

中华绒螯蟹血细胞的显微、亚显微形态结构及其分类

通过相差显微镜和电镜观察,根据中华绒螯蟹血细胞胞质内有无颗粒以及颗粒大小、染色反应、折光性和形成方式的特点,血细胞分为胞质内无颗粒的无颗粒细胞、胞质内只有具折光性和呈淡红色反应大颗粒的大颗粒细胞、胞质内只有无折光性和呈淡蓝色染色反应小颗粒的小颗粒细胞以及胞质内同时具有大颗粒和小颗粒二种颗粒特性的大小颗粒中间型细胞.小颗粒的形成方式是高尔基体成熟面小泡脱离后直接成为小颗粒,而大颗粒的形成方式是高尔基体成熟面小泡脱离后,数个小泡逐渐聚集成蜂窝状大颗粒,进一步发育成熟为均质大颗粒.实验结果表明:三种有颗粒的细胞是互相独立的,可能分别由无颗粒细胞分化而成.       

Isolation of intracellular symbiotes by immune lysis of flagellate protozoa and characterization of their DNA

A new method dependent on immune lysis is described for the isolation of intracellular symbiotes from two species of flagellate protozoa Blastocrithidia culicis and Crithidia oncopelti. The symbiote- containing flagellates are exposed to complement and antisera prepared in rabbits against symbiote-free organisms. The immune lysis seems to weaken the plasma membranes of the flagellates so that subsequent application of gentle shearing force liberates the intracellular entities in an undamaged condition. The symbiotes are then separated from other cellular components by DNAse digestion and differential centrifugation. The average recovery of symbiotes isolated by this method is 20%. Light and electron microscopy establishes the structural integrity and numerical abundance of isolated symbiotes in the final fractions. Integrity of symbiotes is further indicated by the high activity of a marker enzyme, uroporphyrinogen I synthetase. The DNA's of symbiote-containing and symbiote-free flagellates, and of isolated symbiotes were purified and compared after isopycnic centrifugation. The comparison establishes the presence of DNA's in symbiotes of both species. The guanine-cytosine (G-C) content of symbiote DNA differs from that of host DNA's in C. oncopelti, but resembles that of kinetoplast DNA in B. culicis. The latter observation was further shown by heat denaturation study. Renaturation kinetics indicate that the genome complexity of symbiote DNA in B. culicis is similar to that of bacteria.     

The mechanics of budding and copulation in saccharomyces

The yeast cell contains a nucleus whose rigid centrosome carries a band of Feulgen-positive chromatin (centrochromatin) on its surface. The first step in budding is the formation of the bud by an extension of the centrosome over which the cell wall persists. Next the nuclear vacuole extends a process into the bud which contains the chromosomes. Finally the centrochromatin divides directly and the cells separate; a plug either of centrosome or cytoplasm sealing the bud pore. The cytoplasm, the centrosome, the centrochromatin and the nuclear wall are autonomous non genic organelles which never originate de novo.Copulation is the reverse of budding. The centrosomes fuse first; the cytoplasms mix; the nuclear vacuoles fuse by processes which travel along the fused centrosomes; and finally the centrochromatins fuse to form a single band.Figures 1–12. Drawings of budding yeast cells fixed in Schaudinn's fluid and stained with iron alum hemotoxylin, mounted in balsam. The cell wall is not visible due to the clearing action of the balsam. Except for Figure 5, the chromosomes and the nucleolus in the nuclear vacuole have been completely destained. The bud scar described by Barton is shown clearly at the end of the cell distal from the centrosome. The nuclear vacuole is usually forced into the extrusion formed by the bud scar. Since the cell wall is not visible, the plug of material connecting bud and mother cell as shown in Figure 12, fits into the cell wall and probably corresponds to the plug in the bud scar described by Barton. The details of the budding process are described in the text.Figures 13–18. Copulating yeast cells stained with Barrett's hemotoxylin and aceto-orcein and mounted in the stain. Chromosomes are visible in the nuclear vacuoles. The centrosome is usually visible and often appears to have a core which stains differentially. Except in Figure 16, the centrochromatin is visible as darkly stained material; in some cases surrounded by a clear zone. The “thick waisted” form of the cells identifies them as derived from recent copulations and distinguishes them from budding cells. The process of copulation is discussed in the text.     

In Situ Fluorescence Imaging of Myelination

We describe a novel fluorescent dye, 3-(4-aminophenyl)-2H-chromen-2-one (termed case myelin compound or CMC), that can be used for in situ fluorescent imaging of myelin in the vertebrate nervous system. When administered via intravenous injection into the tail vein, CMC selectively stained large bundles of myelinated fibers in both the central nervous system (CNS) and the peripheral nervous system (PNS). In the CNS, CMC readily entered the brain and selectively localized in myelinated regions such as the corpus callosum and cerebellum. CMC also selectively stained myelinated nerves in the PNS. The staining patterns of CMC in a hypermyelinated mouse model were consistent with immunohistochemical staining. Similar to immunohistochemical staining, CMC selectively bound to myelin sheaths present in the white matter tracts. Unlike CMC, conventional antibody staining for myelin basic protein also stained oligodendrocyte cytoplasm in the striatum as well as granule layers in the cerebellum. In vivo application of CMC was also demonstrated by fluorescence imaging of myelinated nerves in the PNS. (J Histochem Cytochem 58:611–621, 2010)     

Cytochemical analysis of the haplosporosomes and vesicle-like droplets of Haplosporidium lusitanicum (Haplosporida,Haplosporidiidae), parasite of Helcion pellucidus (Prosobranchia)

A cytochemical study of the spore of Haplosporidium lusitanicum, a haplosporidian parasite recently found in Helcion pellucidus, is described. Cytochemical analysis with Sudan Black B at the light microscope level revealed that the vesicle-like droplets (VLD) situated in the apical and basal zones of the endosporoplasm in close contact with the external membrane is strongly stained dark blue. These structures are partially digested with lipase. Both reactions suggest the presence of lipoid components. The dense bodies of the exosporoplasm seem to be analogous in chemical composition. On the other hand, these two distinct structures, when subjected to Thiéry's test for glycoproteins, gave positive results. We think that these materials are complex structures simultaneously containing lipids and glycoproteins. They may be involved in the formation of the complex membranous system (“spherule”) that develops during spore maturation in this species. The matrix of haplosporosomes submitted to Thiéry's test for glycoproteins was also positive. A comparative cytochemical analysis has revealed that the external membrane of the haplosporosomes is more glycoproteinaceous than the internal one, which is more lipoidal.     

Localization and Solubilization of the Iron(III) Reductase of Geobacter sulfurreducens

The iron(III) reductase activity of Geobacter sulfurreducens was determined with the electron donor NADH and the artificial electron donor horse heart cytochrome c. The highest reduction rates were obtained with Fe(III) complexed by nitrilotriacetic acid as an electron acceptor. Fractionation experiments indicated that no iron(III) reductase activity was present in the cytoplasm, that approximately one-third was found in the periplasmic fraction, and that two-thirds were associated with the membrane fraction. Sucrose gradient separation of the outer and cytoplasmic membranes showed that about 80% of the iron(III) reductase was present in the outer membrane. The iron(III) reductase could be solubilized from the membrane fraction with 0.5 M KCl showing that the iron(III) reductase was weakly bound to the membranes. In addition, solubilization of the iron(III) reductase from whole cells with 0.5 M KCl, without disruption of cells, indicated that the iron(III) reductase is a peripheral protein on the outside of the outer membrane. Redox difference spectra of KCl extracts showed the presence of c-type cytochromes which could be oxidized by ferrihydrite. Only one activity band was observed in native polyacrylamide gels stained for the iron(III) reductase activity. Excision of the active band from a preparative gel followed by extraction of the proteins and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of high-molecular-mass, cytochrome-containing proteins in this iron(III) reductase activity band. From these experimental data it can be hypothesized that the iron(III) reductase of G. sulfurreducens is a peripheral outer membrane protein that might contain a c-type cytochrome.     

Pathological changes in the nephrocytes of the shore crab, Carcinus maenas, following injection of bacteria

The gills of Carcinus maenas were examined by light and electron microscopy following injection of either sterile saline or the bacteria Bacillus cereus and Moraxella sp., to determine any role(s) for the nephrocytes in the host defense reactions. The results showed that although intact bacteria were not sequestered to the nephrocytes, these cells were active in the removal of large quantities of cell debris from the hemolymph. Much of this material was derived from the breakdown of the hemocytes in response to the presence of bacteria and it's accumulation in the central vacuoles of the nephrocytes resulted in the degradation of these cells. It is proposed that while nephrocytes do not phagocytose intact bacteria, they augment the host defenses by clearing much of the hemocyte and associated bacterial debris from the gills, thus preventing blockage of the lamellar sinuses and subsequent impairment of respiration.