MICRURGICAL STUDIES IN CELL PHYSIOLOGY : I. THE ACTION OF THE CHLORIDES OF NA,K, CA,AND MG ON THE PROTOPLASM OF AM?BA PROTEUS.

By means of micro-dissection and injection Amœba proteus was treated with the chlorides of Na, K, Ca, and Mg alone, in combination, and with variations of pH. I. The Plasmalemma. 1. NaCl weakens and disrupts the surface membrane of the ameba. Tearing the membrane accelerates the disruption which spreads rapidly from the site of the tear. KCl has no disruptive effect on the membrane but renders it adhesive. 2. MgCl₂ and CaCl₂ have no appreciable effect on the integrity of the surface membrane of the ameba when applied on the outside. No spread of disruption occurs when the membrane is torn in these salts. When these salts are introduced into the ameba they render the pellicle of the involved region rigid. II. The Internal Protoplasm. 3. Injected water either diffuses through the protoplasm or becomes localized in a hyaline blister. Large amounts when rapidly injected produce a "rushing effect". 4. HCl at pH 1.8 solidifies the internal protoplasm and at pH 2.2 causes solidification only after several successive injections. The effect of the subsequent injections may be due to the neutralization of the cell-buffers by the first injection. 5. NaCl and KCl increase the fluidity of the internal protoplasm and induce quiescence. 6. CaCl₂ and MgCl₂ to a lesser extent solidify the internal protoplasm. With CaCl₂ the solidification tends to be localized. With MgCl₂ it tends to spread. The injection of CaCl₂ accelerates movement in the regions not solidified whereas the injection of MgCl₂ induces quiescence. III. Pinching-Off Reaction. 7. A hyaline blister produced by the injection of water may be pinched off. The pinched-off blister is a liquid sphere surrounded by a pellicle. 8. Pinching off always takes place with injections of HCl when the injected region is solidified. 9. The injection of CaCl₂ usually results in the pinching off of the portion solidified. The rate of pinching off varies with the concentration of the salt. The injection of MgCl₂ does not cause pinching off. IV. Reparability of Torn Surfaces. 10. The repair of a torn surface takes place readily in distilled water. In the different salt solutions, reparability varies specifically with each salt, with the concentration of the salt, and with the extent of the tear. In NaCl and in KCl repair occurs less readily than in water. In MgCl₂ repair takes place with great difficulty. In CaCl₂ a proper estimate of the process of repair is complicated by the pinching-off phenomenon. However, CaCl₂ is the only salt found to increase the mobility of the plasmalemma, and this presumably enhances its reparability. 11. The repair of the surface is probably a function of the internal protoplasm and depends upon an interaction of the protoplasm with the surrounding medium. V. Permeability. 12. NaCl and KCl readily penetrate the ameba from the exterior. CaCl₂ and MgCl₂ do not. 13. All four salts when injected into an ameba readily diffuse through the internal protoplasm. In the case of CaCl₂ the diffusion may be arrested by the pinching-off process. VI. Toxicity. 14. NaCl and KCl are more toxic to the exterior of the cell than to the interior, and the reverse is true for CaCl₂ and MgCl₂. 15. The relative non-toxicity of injected NaCl to the interior of the ameba is not necessarily due to its diffusion outward from the cell. 16. HCl is much more toxic to the exterior of a cell than to the interior; at pH 5.5 it is toxic to the surface whereas at pH 2.5 it is not toxic to the interior. NaOH to pH 9.8 is not toxic either to the surface or to the interior. VII. Antagonism. 17. The toxic effects of NaCl and of KCl on the exterior of the cell can be antagonized by CaCl₂ and this antagonism occurs at the surface. Although the lethal effect of NaCl is thus antagonized, NaCl still penetrates but at a slower rate than if the ameba were immersed in a solution of this salt alone. 18. NaCl and HCl are mutually antagonistic in the interior of the ameba. No antagonism between the salts and HCl was found on the exterior of the ameba. No antagonism between the salts and NaOH was found on the interior or exterior of the ameba. 19. The pinching-off phenomenon can be antagonized by NaCl or by KCl, and the rate of the retardation of the pinching-off process varies with the concentration of the antagonizing salt. 20. The prevention of repair of a torn membrane by toxic solutions of NaCl or KCl can be antagonized by CaCl₂. These experiments show directly the marked difference between the interior and the exterior of the cell in their behavior toward the chlorides of Na, K, Ca, and Mg.     

Balamuthia mandrillaris, Free-Living Ameba and Opportunistic Agent of Encephalitis, Is a Potential Host for Legionella pneumophila Bacteria

Balamuthia mandrillaris is a free-living ameba and an opportunistic agent of granulomatous encephalitis in humans and other mammalian species. Other free-living amebas, such as Acanthamoeba and Hartmannella, can provide a niche for intracellular survival of bacteria, including the causative agent of Legionnaires' disease, Legionella pneumophila. Infection of amebas by L. pneumophila enhances the bacterial infectivity for mammalian cells and lung tissues. Likewise, the pathogenicity of amebas may be enhanced when they host bacteria. So far, the colonization of B. mandrillaris by bacteria has not been convincingly shown. In this study, we investigated whether this ameba could host L. pneumophila bacteria. Our experiments showed that L. pneumophila could initiate uptake by B. mandrillaris and could replicate within the ameba about 4 to 5 log cycles from 24 to 72 h after infection. On the other hand, a dotA mutant, known to be unable to propagate in Acanthamoeba castellanii, also did not replicate within B. mandrillaris. Approaching completion of the intracellular cycle, L. pneumophila wild-type bacteria were able to destroy their ameboid hosts. Observations by light microscopy paralleled our quantitative data and revealed the rounding, collapse, clumping, and complete destruction of the infected amebas. Electron microscopic studies unveiled the replication of the bacteria in a compartment surrounded by a structure resembling rough endoplasmic reticulum. The course of intracellular infection, the degree of bacterial multiplication, and the ultrastructural features of a L. pneumophila-infected B. mandrillaris ameba resembled those described for other amebas hosting Legionella bacteria. We hence speculate that B. mandrillaris might serve as a host for bacteria in its natural environment.     

INTRACELLULAR OXIDATION-REDUCTION STUDIES : I. REDUCTION POTENTIALS OF AM?BA DUBIA BY MICRO INJECTION OF INDICATORS.

Twenty-five oxidation-reduction indicators were injected in oxidized or reduced form into Amoeba dubia and Amœba proteus under controlled conditions of oxygen access. (1) Under anaerobiosis the ameba was able to reduce completely all the reversible oxidation-reduction indicators down to and including indigo disulfonate. (2) Under anaerobiosis the ameba was unable to reoxidize six of the most easily oxidizable indicators. (3) Under aerobiosis the ameba was able to reduce completely all the indicators down to and including 1-naphthol-2-sulfonate indo-2, 6-dichlorophenol. Toluylene blue, methylene blue and indigo tetrasulfonate were sometimes completely and sometimes only partly reduced, depending on the quantity of indicator injected and the duration of observation. (4) The time of reduction varied approximately with the size of the injection. Reduction was more rapid under anaerobiosis than under aerobiosis, more rapid in active than in sluggish cells and was retarded by toxic compounds. (5) Sulfonated compounds were somewhat toxic, as a rule. In interpreting reduction phenomena of micro injection, it is necessary to take into consideration the intensity, capacity and rate factors. It then becomes apparent that the ameba has a high reducing potential lying on the rH scale below the zone of indigo disulfonate. The reducing capacity of the ameba seems to be relatively great in the region of the simple indophenols and of a progressively diminishing magnitude as the zone of the indigos is approached. Material of high reduction potential appears to be generated within the ameba at a measurable rate. These phenomena, observed in the interior of the cell with the aid of indicators, parallel very closely those found in reduction electrode studies on bacterial cultures.     

Mitochondria : II. The Nuclear-Mitochondrial Relationship in Pelomyxa carolinensis Wilson (Chaos chaos L.)

This study has demonstrated that in the ameba, Pelomyxa carolinensis Wilson (Chaos chaos L.) the limiting membranes of mitochondria and postdivision nuclei are often continuous. The morphological relationship may be functional in that it permits an exchange of material resulting directly or indirectly in an increased enzyme content of the mitochondria. It is suggested that through a series of progressive foldings of its envelope, the nucleus may be a site of formation of mitochondria.     

Phosphatase activity in <Emphasis Type="Italic">Amoeba proteus</Emphasis> at low pH values

In free-living Amoeba proteus (strain B), three forms of tartrate-sensitive phosphatase were revealed by using PAGE of supernatant of the ameba homogenate obtained with 1% Triton X-100 or distilled water and subsequent staining of gels with 2-naphthyl phosphate as substrate (pH 4.0). The form with the highest mobility in the gel turned out to be sensitive to all tested phosphatase activity modulators. Two other forms with the lower mobilities were completely or significantly inactivated not only by sodium L-(+)-tartrate, but also by L-(+)-tartaric acid, sodium orthovanadate, ammonium vanadate, ammonium molybdate, EDTA, EGTA, O-phospho-L-tyrosine, DL-dithiothreitol, H₂O₂, 2-mercaptoethanol, and ions of heavy metals—Fe²⁺, Fe³⁺, and Cu²⁺. Based on results of inhibitory analysis, lysosomal location in ameba cells, and wide substrate specificity of these two forms, it was concluded that they belonged to non-specific acid phosphomonoesterases (AcP, EC 3.1.3.2). This AcP is suggested to have both phosphomonoesterase and phosphotyrosylprotein phosphatase activities. Two ecto-phosphatases were revealed in culture medium, in which amebae were cultivated. One of them was inhibited by the same reagent as the ameba tartrate-sensitive AcP and seemed to be the AcP released into the culture medium in the process of exocytosis of the content of food vacuoles. In the culture medium, apart from this AcP, another phosphatase was revealed; it was not affected by any tested inhibitors of AcP and alkaline phosphatase. It cannot be ruled out that this phosphatase belongs to the ecto-ATPases found in many protists; however, so far its ability to hydrolyze ATP has not yet been proven.     

Sonic hedgehog is involved in formation of the ventral optic cup by limiting Bmp4 expression to the dorsal domain

Accumulating evidence suggests that Sonic hedgehog (Shh) signaling plays a crucial role in eye vesicle patterning in vertebrates. Shh promotes expression of Pax2 in the optic stalk and represses expression of Pax6 in the optic cup. Shh signaling contributes to establishment of both proximal–distal and dorsal–ventral axes by activating Vax1, Vax2, and Pax2. In the dorsal part of the developing retina, Bmp4 is expressed and antagonizes the ventralizing effects of Shh signaling through the activation of Tbx5 expression in chick and Xenopus. To examine the roles of Shh signaling in optic cup formation and optic stalk development, we utilized the Smoothened (Smo) conditional knockout (CKO) mouse line. Smo is a membrane protein which mediates Shh signaling into inside of cells. Cre expression was driven by Fgf15 enhancer. The ventral evagination of the optic cup deteriorated from E10 in the Smo-CKO, whereas the dorsal optic cup and optic stalk develop normally until E11. We analyzed expression of various genes such as Pax family (Pax2/Pax6), Vax family (Vax1/Vax2) and Bmp4. Bmp4 expression was greatly upregulated in the optic vesicle by the 21-somite stage. Then Vax1/2 expression was decreased at the 20- to 24-somite stages. Pax2/6 expression was affected at the 27- to 32-somite stages. Our data suggest that the effects of the absence of Shh signaling on Vax1/Vax2 are mediated through increased Bmp4 expression throughout the optic cup. Also unchanged patterns of Raldh2 and Raldh3 suggest that retinoic acid is not the downstream to Shh signaling to control the ventral optic cup morphology.     

A Simplified Overlay Plaque Technic for Evaluating Responses of Small Free-Living Amebae in Grassland Soils*

SYNOPSIS. The responses of amebae and bacteria in a grassland soil were investigated by an overlay plaque technic developed in this laboratory. This procedure, using Aerobacter aerogenes as the food source, allowed convenient assay of significant changes in ameba populations which resulted from additions of nutrient and water. In comparison with controls, when water was added an initial increase occurred in bacterial counts followed by an increase in the numbers of amebae. Upon addition of glucose, ameba populations increased initially and then decreased with time, while populations of bacteria remained constant. The addition of hay resulted in significant increases in populations of bacteria and amebae. Plaque appearance on enumeration plates was most rapid with inocula from nutrient-treated soils. Predominant amebae recovered by this technic were species of Acanthamoeba and Hartmannella. They were estimated to be present in untreated soils at 3.2 × 10³/gram. Ameba feeding experiments were used to evaluate the possible suitability of other bacteria as food. The results indicated that nonpigmented laboratory strains of bacteria were preferred, while pigmented grassland isolates were more rapidly utilized. Small soil amebae appear to be sensitive to minor soil perturbations, and the enumeration procedure developed in this study should aid in following their responses to environmental stresses.     

Orexin受体1对摄食条件反射的调控研究

目的:观察Orexin受体1对摄食条件反射的调控研究。方法:将40只大鼠随机分为四组,分别为NS/NS组,SB/SB组,NS/SB组,SB/NS组,每组10只大鼠,给予大鼠八组"条件刺激-无条件刺激(CS-US)"训练,给予无条件刺激后立即进行条件刺激,每组进行训练前30 min给予SB或生理盐水(NS)。获得性训练后,给予2组反射消失性训练,即给予8次条件刺激。条件刺激是给予大鼠10 s,2kHZ声音刺激,无条件刺激是直接给予大鼠食物。结果:给予条件刺激后,四组大鼠摄食行为均明显增加,摄食间隔均明显缩短,当条件刺激强度增加时,摄食行为也增加,而预先给予SB,与NS/NS组相比,其余组大鼠摄食行为相对减少(P0.05),摄食间隔增加。消失性训练中,与NS/NS,NS/SB组相比,SB/NS组和SB/SB组大鼠摄食行为明显减少(P0.05)。与其他三组大鼠相比,SB/NS组大鼠摄食间隔缩短。预先给予SB使后两次刺激后摄食间隔明显增加(P0.05)。结论:OX1R信号通路调控摄食反射的产生和消失。     

The retinal pigment epithelium of the eye regulates the development of scleral cartilage

The majority of vertebrate species have a layer of hyaline cartilage within the fibrous sclera giving an extra degree of support to the eyeball. In chicks, this is seen as a cuplike structure throughout the scleral layer. However, the mechanisms that control the development of scleral cartilage are largely unknown.Here we have studied the phases of scleral cartilage development and characterised expression profiles of genes activated during the cartilage differentiation programme. CART1 and SOX9, the earliest markers of pre-committed cartilage, are expressed in the mesenchyme surrounding the optic cup. Later AGGRECAN, a matrix protein expressed during chondrocyte differentiation, is also expressed. The expression of these genes is lost following early removal of the optic cup, suggesting a role for this tissue in inducing scleral cartilage. By grafting young retinal pigment epithelium (RPE) and retina into cranial mesenchyme in vivo, it was found that RPE alone has the ability to induce cartilage formation.There are some exceptions within the vertebrates where scleral cartilage is not present; one such example is the placental mammals. However, we found that the cartilage differentiation pathway is initiated in mice as seen by the expression of Cart1 and Sox9, but expression of the later cartilage marker Aggrecan is weak. Furthermore, cartilage forms in mouse peri-ocular mesenchyme micromass culture. This suggests that the process halts in vivo before full differentiation into cartilage, but that murine scleral mesenchyme has retained the potential to make cartilage in vitro.RA, Wnts and Bmps have been linked to the cartilage development process and are expressed within the developing RPE. We find that RA may have a role in early scleral cartilage development but is not likely to be the main factor involved.These data reveal the course of scleral cartilage formation and highlight the key role that the optic cup plays in this process. The driving element within the optic cup is almost certainly the retinal pigmented epithelium.     

Loss and resiliency of social amoeba symbiosis under simulated warming

Anthropogenic global change is increasingly raising concerns about collapses of symbiotic interactions worldwide. Therefore, understanding how climate change affects symbioses remains a challenge and demands more study. Here, we look at how simulated warming affects the social ameba Dictyostelium discoideum and its relationship with its facultative bacterial symbionts, Paraburkholderia hayleyella and Paraburkholderia agricolaris. We cured and cross‐infected ameba hosts with different symbionts. We found that warming significantly decreased D. discoideum''s fitness, and we found no sign of local adaptation in two wild populations. Experimental warming had complex effects on these symbioses with responses determined by both symbiont and host. Neither of these facultative symbionts increases its hosts’ thermal tolerance. The nearly obligate symbiont with a reduced genome, P. hayleyella, actually decreases D. discoideum''s thermal tolerance and even causes symbiosis breakdown. Our study shows how facultative symbioses may have complex responses to global change.     

When maths trumps logic: probabilistic judgements in chimpanzees

When searching for hidden food, do chimpanzees take into account both the number of hidden items and the number of potential hiding locations? We presented chimpanzees with two trays, each of them containing a different food/cup ratio and therefore a different likelihood of finding a baited cup among empty alternatives. Subjects'' performance was directly influenced by the relative difference (probability ratio (PR)) between the two given probabilities. Interestingly, however, they did not appreciate the special value of a truly safe option (with P = 1.0). Instead, they seemed to ‘blindly’ rely on the PR between the two options, systematically preferring the more likely one once a certain threshold had been reached. A control condition ruled out the possibility of low-level learning explanations for the observed performance.     

Prevalence of Entamoeba nuttalli infection in wild rhesus macaques in Nepal and characterization of the parasite isolates

We have recently resurrected the name Entamoeba nuttalli Castellani, 1908 for a potentially virulent ameba isolate, P19-061405, obtained from a rhesus macaque in Kathmandu, Nepal. The ameba was morphologically indistinguishable from Entamoeba histolytica/Entamoeba dispar/Entamoeba moshkovskii, but located phylogenetically between E. histolytica and E. dispar. To evaluate the prevalence of E. nuttalli infection in wild rhesus macaques, 112 fecal samples were collected in four locations of the Kathmandu Valley. PCR analysis of DNA extracted from the feces showed positive rates of E. nuttalli, E. dispar, E. histolytica and E. moshkovskii of 51%, 12%, 0% and 0%, respectively. A total of 14 E. nuttalli isolates were obtained from four locations, of which 6 were established as axenic cultures. The sequences of the serine-rich protein gene of E. nuttalli isolates differed among four locations although no differences were found in the composition of sequence motifs. Isoenzyme pattern was analyzed in 8 isolates obtained from three locations. In hexokinase, the mobility of the slower migrating band was located between E. histolytica and E. dispar regardless of the culture conditions. These results demonstrate that E. nuttalli is highly prevalent in wild rhesus macaques in Nepal. Rhesus macaques appear to be one of the natural hosts and heterogeneity of the serine-rich protein gene might be useful for geographical typing of isolates.     

FURTHER ELECTRON MICROSCOPE STUDIES ON FIBRILLAR ORGANIZATION OF THE GROUND CYTOPLASM OF CHAOS CHAOS

Further evidence for fibrillar organization of the ground cytoplasm of Chaos chaos is presented. Fixations with osmium tetroxide at pH 6 or 8 and with glutaraldehyde at pH 6 or 7 were used on two preparations: (a) single actively streaming cells; (b) prechilled cells treated with 0.05% Alcian blue in the cold and returned to room temperature for 5–10 min. In addition, a 50,000 g pellet of homogenized cells was examined after fixation with glutaraldehyde-formaldehyde alone. In sections from actively streaming cells considerable numbers of filaments were observed in the uroid regions after glutaraldehyde fixation, whereas only traces of filaments were seen after osmium tetroxide fixation at either pH 6 or 8. Microtubules were not seen. In sections from dye-treated cells, filaments (4–6 mµ) and fibrils (12–15 mµ) were found with all three fixatives. The 50,000 g pellet was heterogeneous but contained both clumps of fibrils and single thick fibrils like those seen in the cytoplasm of dye-treated cells. Many fibrils of the same dimensions (12–15 mµ wide, 0.5 µ long) were also seen in the supernatant above the pellet. Negative staining showed that some fibrils separated into at least three strands of 4–6 mµ filaments.     

Scanning Electron Microscope Observations of Amoeba proteus During Phagocytosis*

SYNOPSIS. Phagocytosing Amoeba proteus at different stages of forming foodcups have been observed by scanning electron microscopy. A nonphagocytosing ameba is characterized by dorsal and lateral ridges running longitudinally over the posterior half of the cell and its attachment to the substrate over small areas. When stimulated by prey organisms, the ameba loses polarity and ridges, and adheres to the substrate more firmly over a wider area of contact. Then it forms broad pseudopods to surround its prey and this results in the formation of foodcups. The surface of all amebae is covered with small projections, and membranous blebs are often seen on the surface of phagocytosing organisms.     

THE AMEBA-TO-FLAGELLATE TRANSFORMATION IN TETRAMITUS ROSTRATUS : II. Microtubular Morphogenesis

Tetramitus exhibits independent ameboid and flagellate stages of remarkable morphological dichotomy. Transformation of the ameba involves the formation of four kinetosomes and their flagella. The arrangement of these kinetosomes and associated whorls of microtubules extending under the pellicle establishes the asymmetric flagellate form. While no recognizable kinetosomal precursors have been seen in amebae, and there is no suggestion of self-replication in dividing flagellates, developmental stages of kinetosomes have been identified. These are occasionally seen in association with the nucleus or with dense bodies which lie either inside of or close to the proximal end of the prokinetosome. Outgrowth of flagella involves formation of an axoneme and a membrane. From the distal tip of the kinetosome microtubules grow into a short bud, which soon forms an expanded balloon containing a reticulum of finely beaded filaments. The free ends of the microtubules appear unraveled; they are seen first as single elements, then as doublets, and finally are arranged into a cylinder. Growth in length is accompanied by a reduction in the diameter of the balloon. The concept that the formation of the kinetic apparatus might involve a nuclear contribution, followed by a spontaneous assembly of microtubules, is suggested.     

Control of cell-wall glucan degradation during development inSchizophyllum commune

Cell-free extracts and culture fluids ofSchizophyllum commune were assayed for enzymatic activity effecting the degradation of an alkali-insoluble cell-wall component of this mushroom, a glucan containingβ-(1→3) linkages (R-glucan). The activity of R-glucanase as determinedin vitro with isolated R-glucan as a substrate was found to increase from the onset of pileus formation, a process accompanied by R-glucan degradation in the mycelium. This R-glucanase activity is influenced by the presence of glucose in the culture medium, probably through a mechanism by which glucose represses synthesis of the enzyme. A morphological mutant (cup mutant) producing no pilei and exhibiting a lower degradation of R-glucanin vivo, produced levels of R-glucanase comparable to those of the wild-type stock and gave even higher levels in young cultures. The difference between the wild-type stock and the cup mutant with respect to degradation of R-glucan during development is most probably to be sought in the structure of the cell wall, the R-glucan in isolated cell walls of the cup mutant being less susceptible to enzymatic attack. High resistance to R-glucanase activity was also encountered in certain cell-wall preparations of the wild-type stock e.g. in those prepared from developing pilei. This suggests that cell-wall glucan degradation during pileus formation is controlled by both the level of R-glucanase, as influenced by glucose in the medium, and differences in protection of R-glucan in the cell wall against enzymatic attack.     

Simplified Procedure for the Isolation of Intact Chloroplasts from Chlamydomonas reinhardtii

A simple procedure that yields highly purified intact chloroplasts from Chlamydomonas reinhardtii is described. This procedure involves breakage of cell wall-deficient cells by passing them through a narrow bore syringe needle. The intact chloroplasts are then purified from the crude homogenate by differential centrifugation and Percoll gradient centrifugation. This procedure generates relatively high yields of chloroplasts capable of CO₂ fixation. These chloroplasts were characterized by electron microscopy, marker enzyme analysis, and ferricyanide exclusion. Transmission electron microscopy indicates that these chloroplasts retain their pyrenoids and eyespots. Scanning electron microscopy confirms that the characteristic cup shape of C. reinhardtii chloroplasts persists in vitro. This rapid, inexpensive procedure produces chloroplasts that should be useful for researchers studying the biochemistry and cell biology of C. reinhardtii chloroplasts.     

The effects of a microcystin-producing and lacking strain of Microcystis on the survival of a widespread tropical copepod (Notodiaptomus iheringi)

The tropical copepod Notodiaptomus iheringi (N. iheringi) is an ideal subject for studying zooplankton responses to cyanobacteria because it co-exists with permanent blooms across widespread regions in South America in high abundance. Single and mixed diets containing Cryptomonas and either a microcystin-producing (MC+) or microcystin-lacking (MC?) Microcystis were offered to N. iheringi at different proportions in a 10-day laboratory survival test to distinguish between the effects of toxicity versus nutrition. As expected, the pure MC+ Microcystis diet caused acute toxicity, indicated by high mortality compared to starved copepods. Both Microcystis strains were ingested in a 3-h short-term grazing experiment with pure diets. Despite its toxicity as the sole food source, survival was unaffected by MC+ Microcystis in mixed food diets. Even when MC+ Microcystis was 90% of the total food, survival was similar to the control with 10% Cryptomonas only. Hence, the survival in mixed food diets was controlled by the amount of Cryptomonas, not Microcystis. Previous reports show strong negative effects of Microcystis on copepod survival despite abundant high-quality food. Although this is the first example of copepods avoiding acute Microcystis toxicity in mixed diets, it could be a common trait where permanent blooms dominate the ecosystem.     

Canonical Wnt/β-Catenin Signalling Is Essential for Optic Cup Formation

A multitude of signalling pathways are involved in the process of forming an eye. Here we demonstrate that β-catenin is essential for eye development as inactivation of β-catenin prior to cellular specification in the optic vesicle caused anophthalmia in mice. By achieving this early and tissue-specific β-catenin inactivation we find that retinal pigment epithelium (RPE) commitment was blocked and eye development was arrested prior to optic cup formation due to a loss of canonical Wnt signalling in the dorsal optic vesicle. Thus, these results show that Wnt/β-catenin signalling is required earlier and play a more central role in eye development than previous studies have indicated. In our genetic model system a few RPE cells could escape β-catenin inactivation leading to the formation of a small optic rudiment. The optic rudiment contained several neural retinal cell classes surrounded by an RPE. Unlike the RPE cells, the neural retinal cells could be β-catenin-negative revealing that differentiation of the neural retinal cell classes is β-catenin-independent. Moreover, although dorsoventral patterning is initiated in the mutant optic vesicle, the neural retinal cells in the optic rudiment displayed almost exclusively ventral identity. Thus, β-catenin is required for optic cup formation, commitment to RPE cells and maintenance of dorsal identity of the retina.