Applying dimorphic yeasts as model organisms to study mycelial growth: Part 1. Experimental investigation of the spatio-temporal development of filamentous yeast colonies

Colony development of the dimorphic yeasts Yarrowia lipolytica and Candida boidinii on solid agar substrates under glucose limitation served as a model system for mycelial development of higher filamentous fungi. Strong differences were observed in the behaviour of both yeasts: C. boidinii colonies reached a final colony extension which was small compared to the size of the growth field. They formed cell-density profiles which steeply declined along the colony radius and no biomass decay processes could be detected. The stop of colony extension coincided with the depletion of glucose from the growth substrate. These findings supported the hypothesis that glucose-limited C. boidinii colonies can be regarded as populations of single cells which grow according to a diffusion-limited growth mechanism. Y. lipolytica colonies continued to extend after the depletion of the primary nutrient resource, glucose, until the populations covered the entire growth field which was accomplished by utilization of mycelial biomass.     

Forest fragmentation and colony performance of forest tent caterpillar

We monitored survival and egg production in colonies of Malacosoma disstria in the understorey to examine relationships between colony performance and forest structure at several spatial scales. As forest cover increased, fewer moths survived. Increased mortality from a virus and a parasitoid assemblage were the main causes of this result. Forest cover measured at the smallest spatial scale (53 × 53 m) was the best predictor of colony performance. We attribute these effects of fragmentation to fine-scale differences in microclimate betweeen edge and interior habitats (edge effects).     

Persisting Hyper-abundance of Leaf-cutting Ants (Atta spp.) at the Edge of an Old Atlantic Forest Fragment

Leaf-cutting ants (LCAs) profoundly benefit from edge creation in Neotropical forests, where they act as a keystone species and disturbance agent. In view of their poorly explored population dynamics, the question arises whether high densities of LCAs are a transitional or a persisting phenomenon. We studied the temporal variation of LCA colony densities at the edge of the Brazilian Atlantic forest. At physically stable edges of an old forest fragment, densities of Atta cephalotes and Atta sexdens (11 and five times higher in a 50 m edge zone in comparison with the forest interior) persisted over a 4-yr interval (2001–2005) with no significant difference in densities between years. Species-specific per colony growth rates ranged from 12 to −5 percent/yr, suggesting that populations were approximately at equilibrium. High rates of colony turnover (little less than 50% in 4 yr) indicated an average colony life span of about 7 yr—a life expectancy considerably lower than previous estimates for Atta colonies. Stable, hyper-abundant populations of LCAs accord with the constantly high availability of palatable pioneer vegetation (the preferred food source of LCAs) at forest edges and are expected to persist in time as long as forests are characterized by high edge to interior ratios, with potentially long-lasting consequences for the ecosystem.     

Cutting More from Cut Forests: Edge Effects on Foraging and Herbivory of Leaf-Cutting Ants in Brazil

Edge-mediated changes in species composition are known to result in modified species interactions. Because of the crucial trophic position of herbivores and their far-reaching impact on plant communities, it is important to understand how edge influences herbivory. In the present paper, we investigated whether and how leaf-cutting ant foraging is altered in the forest edge, as this habitat is characterized by an increased proportion of pioneer species. We assessed basic foraging data as well as the herbivory rate ( i.e. , the proportion of the leaf material harvested by a colony in relation to the available leaf area in the foraging area) of Atta cephalotes colonies at the edge versus interior sites of a large remnant of the Atlantic forest in Northeast Brazil. Our results indicated clear edge effects on leaf-cutting ants: equally sized A. cephalotes colonies located at the forest edge removed about twice as much leaf area from their foraging grounds than interior colonies (14.3 vs. 7.8%/col/yr). This greater colony-level impact within the forest edge zone was a consequence of markedly reduced foraging areas (0.9 vs. 2.3 ha/col/yr) and moderately lower leaf area index in this habitat, whereas harvest rates were the same. Our results suggest that forest edges induce increased leaf-cutting ant herbivory, probably via the release of resource limitation. Together with the increase of leaf-cutting ant populations along forest edges, this may amplify environmental changes induced by habitat fragmentation.     

Differential growth patterns between successive litters of the eusocial Damaraland mole-rat, Cryptomys damarensis, from Namibia

Differential mean rates of growth were calculated for successive litters of mole-rats born to genetically unrelated pairs of C. damarensis. The Logistic equation provided the closest fit to the growth data.
Both intra- and inter-colonial variation in mean maximum growth rate between successive litters incorporated into the natal colonies were determined.
The mean asymptote (A), growth rate constant (K) and inflection time (I) were modelled for the first five litters of pups born to pairs of mole-rats. Inter-colonial analysis of recruitment to colonies revealed litters 1, 2, and 5 to grow faster than litters 3 and 4. Litters 1 and 2 had significantly higher asymptotes and inflection times than litters 3, 4, and 5 ( P < 0.001). All litters were assimilated into their natal colonies. There were no significant differences between males and females for the asymptote, growth rate constant, or inflection time ( P < 0.05).
Intra-colonial variation of litters 1 to 4 born to a single colony revealed comparable results to that obtained for inter-colonial comparisons. Multiple comparisons showed litters 1 and 2 to have a significantly higher absolute growth rate than litters 3 and 4. The lowest rate of growth being for litter 4.
The patterns of mean differential growth are discussed in the light of the social organization of the colony. It is postulated that growth rates in litters 1, 2, and 3 are more rigid since they constitute the main functional unit of colony organization. Litters 4 and 5 show a greater plasticity in growth; it is speculated that these colony members become incorporated into the various worker and defender groupings which are characteristic of C. damarensis colonies.     

pH gradients through colonies of Bacillus cereus and the surrounding agar.

pH-sensitive microelectrodes, constructed with a tip diameter of about 4 microns, were deployed through 24 h and 48 h colonies of Bacillus cereus incubated on CYS medium (Casamino acids, yeast extract, salts), with and without glucose. Measurements of pH were used to construct pH profiles through the colony and the surrounding agar. pH gradients could be detected for at least 800 microns into the agar beneath a 24 h colony, and to approximately 10 mm horizontally away from the edge of the colony. In older colonies, the lateral gradient extended for over 20 mm. The pH of the underlying agar was increased by up to 1.45 pH units after 48 h growth without glucose. When colonies were grown with glucose, a significant area of acidification was observed within the colony in addition to a zone of alkalinization present at its periphery. Acidification was thought to be due to the anaerobic fermentation of glucose producing organic acids whilst alkalinization was due to the aerobic oxidation of amino acids releasing ammonia.     

Determinate growth and modularity in a gorgonian octocoral

Growth rates of branches of colonies of the gorgonian Pseudopterogorgia elisabethae were monitored for 2 years on a reef at San Salvador Island, Bahamas. Images of 261 colonies were made at 6-month intervals and colony and branch growth analyzed. Branch growth rates differed between colonies and between the time intervals in which the measurements were made. Colonies developed a plumelike morphology through a pattern of branch origination and determinate growth in which branch growth rates were greatest at the time the branch originated and branches seldom grew beyond a length of 8 cm. A small number of branches had greater growth rates, did not stop growing, and were sites for the origination of subsequent "generations" of branches. The rate of branch origination decreased with each generation of branching, and branch growth rates were lower on larger colonies, leading to determinate colony growth. Although colonial invertebrates like P. elisabethae grow through the addition of polyps, branches behave as modules with determinate growth. Colony form and size is generated by the iterative addition of branches.     

Delayed maturation in the colonial coralGoniastrea aspera (Scleractinia): whole-colony mortality, colony growth and polyp egg production

The present study examines (1) the cost of reproduction on colony growth, and (2) relationships among sexual maturity, whole-colony mortality rate and colony growth rate inGoniastrea aspera free from external influences by macrobenthos. Survival of colonies in permanent plots was followed for two years. Egg production by polyps in colonies collected just before the first spawning of a year was estimated by dissecting the polyps. Growth of the colonies (increase in number of polyps) was followed over one annual reproductive cycle. The cost of egg production on colony growth was apparent through colony ontogeny: (1) immature colonies had a greater annual growth rate than mature colonies, but produced almost no eggs; (2) in mature colonies, growth rate was negatively correlated with NE/PV (number of eggs per polyp volume mm^-3). Annual whole-colony mortality was high in colonies with fewer than11 polyps in initial colony size, while mortality was extremely low once a colony grew beyond this size. This critical size for low whole-colony mortality was much smaller than the colony size (40 polyps) which would attain maturity one year later. Age at maturity was estimated as six years. While survival to maturity may be a selective force for the evolution of delayed maturation, the present data suggest that high colony fecundity, achieved after a long growth period as an immature colony, and an abrupt decrease of colony growth rate after maturation are the crucial forces.     

Functional differentiation in bryozoan colonies: a proteomic analysis

Bryozoans are typical modular organisms. They consist of repetitive structural units, the zooids. Bryozoan colonies grow by zooidal budding, with the distribution pattern of the budding loci underlying the diversity of colony forms. Budding is usually restricted to the colony periphery, where a “growing edge” or local terminal growth zones are formed. Non-budding parts of the colony can be functionally subdivided, too. In many species colonies consist of regular, often repetitive zones of feeding and non-feeding modules, associated with a periodical degeneration and regeneration of the polypide retractile tentacle crown with a gut and the accompanying musculature. The mechanisms of functional differentiation in bryozoan colonies are unknown. Presumably, budding and/or polypide recycling are induced or inhibited by certain determinants of functional specialization in different colony parts. An effective tool of their identification is the comparison of proteomes in functionally different zones. Here we report the results of proteomic analysis of three bryozoan species from the White Sea with a different colony form: Flustrellidra hispida, Terminoflustra membranaceotruncata and Securiflustra securifrons. Using differential two-dimensional electrophoresis (2D-DIGE), we compared proteomes of the growing edge, the zone with polypides and the zone without polypides. We assessed the general level of differences between the zones and revealed proteins whose relative abundance changed gradually along the proximal-distal colony axis. These proteins might be involved in the determination of the functional differentiation of the colony.     

Growing Yeast into Cylindrical Colonies

Microorganisms often form complex multicellular assemblies such as biofilms and colonies. Understanding the interplay between assembly expansion, metabolic yield, and nutrient diffusion within a freely growing colony remains a challenge. Most available data on microorganisms are from planktonic cultures, due to the lack of experimental tools to control the growth of multicellular assemblies. Here, we propose a method to constrain the growth of yeast colonies into simple geometric shapes such as cylinders. To this end, we designed a simple, versatile culture system to control the location of nutrient delivery below a growing colony. Under such culture conditions, yeast colonies grow vertically and only at the locations where nutrients are delivered. Colonies increase in height at a steady growth rate that is inversely proportional to the cylinder radius. We show that the vertical growth rate of cylindrical colonies is not defined by the single-cell division rate, but rather by the colony metabolic yield. This contrasts with cells in liquid culture, in which the single-cell division rate is the only parameter that defines the population growth rate. This method also provides a direct, simple method to estimate the metabolic yield of a colony. Our study further demonstrates the importance of the shape of colonies on setting their expansion. We anticipate that our approach will be a starting point for elaborate studies of the population dynamics, evolution, and ecology of microbial colonies in complex landscapes.     

Growing Yeast into Cylindrical Colonies

Microorganisms often form complex multicellular assemblies such as biofilms and colonies. Understanding the interplay between assembly expansion, metabolic yield, and nutrient diffusion within a freely growing colony remains a challenge. Most available data on microorganisms are from planktonic cultures, due to the lack of experimental tools to control the growth of multicellular assemblies. Here, we propose a method to constrain the growth of yeast colonies into simple geometric shapes such as cylinders. To this end, we designed a simple, versatile culture system to control the location of nutrient delivery below a growing colony. Under such culture conditions, yeast colonies grow vertically and only at the locations where nutrients are delivered. Colonies increase in height at a steady growth rate that is inversely proportional to the cylinder radius. We show that the vertical growth rate of cylindrical colonies is not defined by the single-cell division rate, but rather by the colony metabolic yield. This contrasts with cells in liquid culture, in which the single-cell division rate is the only parameter that defines the population growth rate. This method also provides a direct, simple method to estimate the metabolic yield of a colony. Our study further demonstrates the importance of the shape of colonies on setting their expansion. We anticipate that our approach will be a starting point for elaborate studies of the population dynamics, evolution, and ecology of microbial colonies in complex landscapes.     

Spatiotemporal dynamics of growth and death within spherical bacterial colonies

Bacterial growth within colonies and biofilms is heterogeneous. Local reduction of growth rates has been associated with tolerance against various antibiotics. However, spatial gradients of growth rates are poorly characterized in three-dimensional bacterial colonies. Here, we report two spatially resolved methods for measuring growth rates in bacterial colonies. As bacteria grow and divide, they generate a velocity field that is directly related to the growth rates. We derive profiles of growth rates from the velocity field and show that they are consistent with the profiles obtained by single-cell-counting. Using these methods, we reveal that even small colonies initiated with a few thousand cells of the human pathogen Neisseria gonorrhoeae develop a steep gradient of growth rates within two generations. Furthermore, we show that stringent response decelerates growth inhibition at the colony center. Based on our results, we suggest that aggregation-related growth inhibition can protect gonococci from external stresses even at early biofilm stages.     

Smaller colonies and more solitary living mark higher elevation populations of a social spider

1. There appears to be a pattern of decreasing sociality with increasing elevation across social spider species in the genus Anelosimus at tropical latitudes. Our data suggest that this pattern holds within a single species, Anelosimus eximius, on a smaller altitudinal gradient. 2. In comparing colony size at six different altitudes in north-eastern Ecuador, we find that the lowland A. eximius populations tend to have larger colonies and few solitary females. At higher elevations, many of the colonies are small and the proportion of solitary females is greater. 3. Contrary to expectation, we also found no difference in spider density between the upper elevation and lowland populations. This result may be partly due to the fact that upper elevation populations occur only at the forest edge (as opposed to both edge and interior) where populations at all elevations appear more robust.     

Uptake of Glucose and Phosphorus by Growing Colonies of Fusarium oxysporum as Quantified by Image Analysis

Olsson, S. 1994. Uptake of glucose and phosphorus by growing colonies of Fusarium oxysporum as quantified by image analysis. Experimental Mycology 18, 33-47. The simplest of all heterogeneous environments for fungal colony growth is the petri dish with an agar medium. As the colony grows there will be a depression of nutrient concentrations under the colony caused by the uptake of nutrients by the growing colony. Image analysis methods have been developed for measuring medium concentrations of glucose and phosphorus with simultaneous biomass density determinations in agar systems. Maps of the concentrations in the agar medium under the colony and of colony biomass density were produced. A new method for weighing fungal colonies grown on agar is also presented. For Fusarium oxysporum phosphorus and glucose uptake from the medium was the same irrespective of the C/mineral ratios in the medium within the measured range of ratios. Even the concentration profiles of the nutrients under the colony were the same irrespective of nutrient ratios. Distribution of biomass density was affected by differences in glucose concentrations, being highest at the colony margin at the lower concentrations. The results indicate that the fungal colony is able to take up nutrients at the margin in excess of the local needs.     

Effect of cloning rate on fitness-related traits in two marine hydroids

Hydractinia symbiolongicarpus and Podocoryna carnea are colonial marine hydroids capable of reproducing both sexually and asexually. Asexual reproduction, by colony fragmentation, produces a genetic clone of the parent colony. This study examines the effect of very different cloning rates on colony growth rate, oxygen uptake rate, and colony morphology. Colonies of one clone of each species were maintained for an extended time in two treatments: in a state of constant vegetative growth by repeated cloning, and in a state restricted from vegetative growth (no cloning). For both species, tissue explants taken from the growing colonies grew more slowly than similar explants taken from the restricted colonies. For one species, tissue explants from the growing colonies used oxygen at a higher rate than similar explants from restricted colonies; for the other species, no difference was detected, although the sample size was small. For both species, tissue explants from restricted colonies formed more circular, "sheet-like" shapes, whereas those from their growing counterparts formed more irregular, "runner-like" shapes. After these experiments, in the third winter of treatment, all colonies experienced a severe tissue regression. Within 6 months after this event, the colonies had regrown to their former sizes. A growth assay at this point revealed no difference in growth rate, possibly suggesting an epigenetic basis for these results. Changes in clonal growth rates and morphology correlated with variation in fragmentation rate might affect the ecology of these and other clonal organisms.     

Cell proliferation and growth inZoothamnium niveum (Oligohymenophora, Peritrichida) — Thiotrophic bacteria symbiosis

Chemolithoautotrophic, sulphide-oxidizing (thiotrophic) symbioses represent spectacular adaptations to fluctuating environmental gradients and survival is often accomplished when growth is fuelled by sufficient nourishment through the symbionts leading to fast cell proliferation. Here we show 5′-bromo-2′ deoxyuridine (BrdU) pulse labelling of vegetative growingZoothamnium niveum, a colonial ciliate obligately associated with thiotrophic ectosymbionts, and demonstrate age related growth profiles in three heteromorphic host cell types. At the colony’s apex, a large top terminal zooid performed high proliferation activity, which decreased significantly with increasing colony age but was still present in old colonies indicating that this cell possesses lifelong cell division potential. In contrast, terminal branch zooids proliferated independent of colony age but appeared to be limited by their cell division capacity predetermined by branch size, thus leading to the strict, feather-shaped colony form. Appearance of labelled terminal branch zooids allowed us to distinguish a highly proliferating apical colony region from an almost inactive, senescent basal region. In macrozooids attached to the colony, extensive BrdU labelling suggests that DNA synthesis occurs in preparation for a new generation. As motile swarmers, the macrozooids seem to be arrested in the cell cycle and mitosis and cell division occur when the swarmer settles and transforms into a top terminal zooid buildingup a new colony.     

ARRESTED GROWTH OF CHINESE HAMSTER OVARY (CHO) CELL COLONIES ON AGAR

Chinese hamster ovary (CHO) cells plated on agar form two classes of colonies; those which increase continuously in diameter and those which become arrested in outward growth. All colonies continue to increase in mass and thickness as demonstrated by computer-assisted analysis of time sequence photographs of several thousand colonies and by examination of histological sections. Colonies which shift from a monolayer to a mounded morphology at fairly large colony diameter (?1 mm) continue to increase in diameter. Colonies in which mounding occurs at smaller colony diameter (?1 mm) cease to increase in diameter but continue to increase in thickness, as demonstrated by histological examination and by computer-assisted analysis. Rapid cell division occurs at the edge of all colony classes, as shown by the distribution of mitotic figures. In arrested colonies these dividing cells must move toward the colony core to compensate for dying cells. Necrotic cells are found as a discrete zone at the air-colony interface in all cases for CHO cell colonies growing on agar. As such, necrosis is probably due to limitations in nutrient diffusion upward from the agar rather than oxygen diffusion downward.     

A comparison of craniofacial growth between two colonies of marmosets (Callithrix jacchus)

General and craniofacial measurements from young marmosets in a recently established Australian colony are presented and compared with similar data published for a long-established colony in the United Kingdom. Statistically significant differences between the colonies were those associated with large error variances, suggesting difficulties in locating certain landmarks rather than real differences in growth. Thus, direct comparison can be made between craniofacial growth studies conducted at these two different marmoset colonies.     

Edge‐mediated reduction of phorid parasitism on leaf‐cutting ants in a Brazilian Atlantic forest

Previous studies have shown that leaf‐cutting ant populations benefit greatly from living in or near the edges of the Brazilian Atlantic forest. One of the mechanisms responsible for this rise in population density is an edge‐mediated increase of pioneer plants, resulting in increased food availability for the ants (i.e., less bottom‐up control). Here, we hypothesized that the release from natural enemies (i.e., less top‐down control) may also contribute to the phenomenon. We investigated whether parasitism of phorid flies on leaf‐cutting ants decreases in colonies located along the forest edge vs. the interior of a large tract of Atlantic forest in northeastern Brazil. For this, we assessed abundance and rates of oviposition attack by phorids in bimonthly intervals over a period of 1 year in 10 adult colonies of Atta cephalotes (L.) (Hymenoptera: Formicidae: Myrmicinae), five at the forest edge and five in the forest interior. The number of phorids attracted by ants at edge colonies was 40% lower than that at interior colonies. The temporal variation in phorid attraction was also significant, with approximately 35% fewer flies in the dry months as compared to the rainy months. As a result of lower phorid abundance, ant workers of edge colonies suffered three times fewer oviposition attacks than those of interior colonies. There was a tendency for fewer attacks during dry months, but the difference in the temporal variation was not significant. Our findings suggest that edge creation contributes to increased leaf‐cutting ant abundance, not only via the attenuation of bottom‐up forces, but also through an environmentally triggered depression of parasitoid abundance/efficiency, possibly because of adverse environmental conditions in edge habitats.     

Effects of colony size on larval performance in a processionary moth

1. Some lepidopteran species have larvae that live gregariously, especially in early instars. Colony‐living species may benefit from improved protection from predators, thermoregulation, and feeding facilitation, for example. 2. While many studies have compared solitary and gregarious life styles, few data exist as to the relationship between size of the larval colony and larval performance in gregarious species. The present study was aimed at understanding the importance of colony size for growth and survival of the northern pine processionary moth (Thaumetopoea pinivora) larvae. 3. Field studies, comparing three different sizes of colonies of T. pinivora larvae, showed that individuals in larger colonies had a higher survival rate compared with those living in smaller colonies and also a faster growth rate. 4. The higher survival rate of large colonies was attributed to improved protection from predacious arthropods. 5. In early spring, the young larvae bask in the sun to increase their body temperature. In field experiments the thermal gain was higher in large colonies, and individuals in such colonies also grew faster. As growth rate was not affected by colony size when the ability to bask was experimentally removed in a laboratory experiment, the higher growth rate of the larger colonies was probably due to improved thermoregulation rather than feeding facilitation. 6. The size of larval colonies of gregarious insects depends on natural mortality events as well as on female oviposition strategy. Our results show that decreasing colony size can lead to a reduction in growth rate and survival. It is therefore important to understand whether or not small colonies will benefit equally from the gregarious behaviour.