Volvox and all volvocine species have a haplontic life cycle Fig. Nitrogen deficiency is the cue for sexual differentiation in most volvocine genera, but in Volvox, a species-specific glycoprotein hormone called sex inducer SI is the trigger for sex. SI production can be induced by heat shock [ 9 ], and when vegetative gonidia are exposed to SI they undergo modified embryogenesis leading to development of sexually dimorphic males or females under the control of a haploid UV sex chromosome system [ 10 , 11 , 12 ].
Fertilization produces diploid zygotes which mature into thick-walled dormant zygospores. Zygospores are environmentally resistant and can remain viable for years in a frozen or desiccated state. Exposure of zygospores to light and nutrients initiates germination and meiosis to produce recombinant haploid progeny that re-enter the vegetative life cycle [ 13 ]. Detailed descriptions of mating and fertilization can be found elsewhere [ 3 , 14 ]. Volvox life cycle. Schematic of the Volvox vegetative and sexual life cycle phases.
Vegetative reproduction grey shaded region occurs in the haploid 1n phase and can be synchronized with a 16 h:8 h light:dark diurnal regime center of diagram , under which one full reproductive cycle is completed every 48 h. After hatching, the parental somatic cells of the previous generation are discarded and undergo senescence and cell death. Sexual development is triggered by exposure to sex inducer and leads to gonidia undergoing modified embryogenesis and development not shown into adult sexual egg-bearing females or adult sexual sperm-packet-bearing males.
Sperm packets are released and swim to females where mating takes place with internal fertilization syngamy , resulting in formation of diploid zygospores. Meiosis occurs upon germination and produces three polar bodies and one haploid progeny that re-enters the vegetative life cycle. This figure was adapted from [ 10 ], and reproduced here with permission from the Annual Review of Microbiology , Vol.
Field samples of Volvox are routinely collected in one of two different ways: Starr and colleagues described a simple means of germinating zygospores from dried soil samples that are usually collected near bodies of water where Volvox has been observed [ 14 ]. Dried soil is advantageous as it can be transported and stored easily, though successful germination is never guaranteed. The alternative is collection of live samples using a phytoplankton sieve as demonstrated in this video from Dr.
A single male—female pair HK9 and HK10 a. Adm and Eve germinated from soil collected near Kobe, Japan, are founders for standard strains still used by most laboratories [ 3 , 14 ]. The HK9 male strain acquired a morphological mutation and was replaced with a normal male progeny b derived from a cross with HK10 [ 16 ]. Additional strains, including newer isolates [ 17 , 18 ], are available from several culture collections see below.
For routine studies female strains e. Because sexual males produce additional SI, a single spontaneous sexual male arising in a culture can sexually induce the entire population of males, leading to its extinction. Cultures are usually maintained in defined liquid media such as Standard Volvox Medium SVM [ 19 ] in a temperature- and light-controlled environment, i.
Maintaining cultures axenically is ideal and requires meticulous sterile technique, but methods exist to remove microbial contaminants. Although there is one report of successful cryopreservation of Volvox [ 20 ], maintenance of Volvox cultures generally requires live passaging every 2 to 4 weeks. Dried or frozen zygospores are an alternative method for long-term storage, but this approach cannot maintain a completely isogenic strain background.
Volvox cultures. Individual spheroids are just barely visible to the naked eye. Culture flasks similar to that in panel b sit in a temperature-controlled acrylic water bath and are lit from below by red and blue LEDs that can be programmed for a diurnal regime. A gas manifold above the flasks provides pressurized air for culture aeration.
Volvox is a rich developmental system where the origins of developmental complexity and innovation can be inferred using comparative methods and direct experimental testing [ 1 , 5 , 21 , 22 ]. The late David Kirk, whose laboratory played a major role in ushering Volvox research into the modern molecular era, summarized 12 key steps or innovations in the progression from a unicellular Chlamydomonas-like ancestor to V.
The earliest innovations include cell adhesion, genetic control of cell number, acquisition of organismal polarity, incomplete cytokinesis with formation of cytoplasmic bridges between embryonic blastomeres, morphogenetic shape changes a precursor to the process of inversion described below , rotation of basal bodies to enable coordinated flagellar motility, and conversion of the cell wall to extracellular matrix ECM and colony boundary.
Deciphering the molecular genetic origins of these steps continues to be an exciting and increasingly approachable goal. A sampling of these innovations and related topics are discussed below in reference to Volvox. Terminally differentiated somatic cells are a hallmark of complex multicellular organisms and are likely to have arisen independently at least three times in volvocine algae [ 5 ].
Germ—soma differentiation in V. Recent cell-type transcriptome studies in Volvox have also shed light on how germ and somatic cells differ, and the potential origins of their specialized differentiation programs [ 29 , 30 , 31 ].
Much remains to be discovered in this system including identifying direct targets of RegA, identification of germ cell-specific differentiation factors, and determining how asymmetric cell divisions governed by the chromatin-associated chaperone proteins glsA, Hsp70A and other factors are specified and executed [ 32 ].
Volvox has evolved some remarkable developmental innovations which have no clear analogs in its unicellular relative, Chlamydomonas. These include asymmetric embryonic cell divisions and a bifurcated embryonic cell division program where both the number of cell divisions and their symmetry differ in a predictable manner among cleaving blastomeres.
The remarkable process of inversion immediately follows embryonic cleavage and involves a sequence of coordinated cell shape changes that enable the embryo to turn itself completely inside-out and assume its adult configuration with outward-oriented somatic precursors and interiorly located gonidial precursors [ 33 ]. Anterior—posterior A-P embryonic polarity and the presence of cytoplasmic bridges connecting post-mitotic blastomeres are key derived features that enable inversion to occur [ 34 , 35 , 36 , 37 ].
Mutants that disrupt inversion and other developmental programming have been isolated and hold great potential for understanding the origin of these innovations reviewed in [ 2 ]. The conserved RWP-RK family transcription factor, Mid minus dominance resides on the male chromosome or minus mating-type locus in isogamous species and controls male or minus sexual differentiation in dioicous species of volvocine algae [ 10 ].
Plus mating type or female are the default differentiation programs when Mid is absent. Monoicy i. In Volvox Mid is inferred to have evolved control of more complex developmental programming than in isogamous volvocine species so that it can promote male sexual differentiation and sperm development [ 38 , 39 ].
Although Mid controls key aspects of Volvox sexual germ cell differentiation, other genes on the Volvox UV sex chromosomes play additional roles in specifying some aspects of male versus female embryonic patterning and in promoting sex-specific reproductive fitness. This finding and others fit predictions of evolutionary theory [ 40 ] where sex chromosomes are postulated to acquire genes required for reproductive fitness of males or females.
Other derived aspects of the Volvox sexual cycle such as SI-triggered signal transduction, internal fertilization, and reduced meiotic products are fascinating and relatively unexplored areas of interest [ 10 , 24 ]. The Chlamydomonas nuclear genome sequence was published in [ 41 ], and the Volvox genome followed in [ 1 , 42 ]. Two other volvocine nuclear genomes for the multicellular genera, Gonium and Tetrabaena , have also been published [ 43 , 44 ], with more likely to follow soon.
Additional genomic resources including some mating loci sequences, unannotated nuclear genome assemblies, and selected genomic regions are also available but have no public browsers or search engines [ 11 , 25 ]. Noncoding genomic regions among the four sequenced species show high divergence, limiting their potential for predicting cis-regulatory elements based on sequence conservation.
Additional regulatory complexity derived from microRNAs or small non-coding RNAs may also exist in Chlamydomonas and Volvox, but little is known about how these pathways are involved in basic cellular processes or development [ 31 , 45 , 46 , 47 ]. Motility in Volvox and other volvocine species poses interesting developmental, evolutionary and biophysical questions [ 48 , 49 , 50 ]. Volvox swims while rotating about its A-P axis which is oriented in the direction of swimming Fig.
Together, the eyespots act as a distributed sensing system allowing the flagella of somatic cells on the side of the spheroid rotating past the path of incoming light to transiently adjust their beat-stroke and effect a turn towards or away from the light [ 53 , 54 , 55 ]. Volvox is amenable to many types of experimental manipulations and methodologies. Because it can be easily synchronized using a diurnal regime, uniform populations from different developmental or life cycle stages can be obtained.
At many but not all stages the germ and somatic cells or cleaving embryos can be physically separated and purified, thus enabling studies of individual cell types [ 29 , 30 ].
Pre-cleavage or cleaving embryos can be released from parental spheroids. They remain viable and can complete development, allowing them to be studied in detail using time lapse imaging, or can be subject to various experimental manipulations [ 56 ].
Volvox is amenable to most standard methods of microscopic imaging including single plane illumination microscopy SPIM, a. Molecular genetics methods for Volvox make it a highly tractable system for developmental genetics. Classical genetics played a critical role in defining different classes of developmental mutants [ 58 , 59 , 60 , 61 , 62 ], but was superseded by the more facile method of transposon tagging which was the method of choice prior to having a genome sequence [ 35 , 63 , 64 , 65 ].
With a sequenced genome, isolates that have abundant sequence polymorphisms [ 66 ], and low-cost resequencing methods, the prospects for using classical developmental genetics are again excellent.
It should be noted that strains tend to lose sexuality during repeated vegetative propagation, but this can be remedied by routine crossing to maintain sexual fitness. Volvox can be transformed using biolistic methods DNA-coated gold particles delivered with a helium gene gun coupled with any of several available selection markers [ 67 , 68 , 69 , 70 , 71 ].
Nuclear transgenes are thought to integrate randomly as they do in Chlamydomonas, and can be subject to silencing and position effects i. During selection the spheroids grow for one or more generations, with non-transformed individuals eventually dying, and stably transformed genetically homogenous transformants emerging.
Regulated promoters have also been described [ 73 , 74 ]. RNAi-mediated knockdowns using antisense [ 75 , 76 ] or hairpin constructs [ 39 ] have been successful for reverse genetics and will likely remain useful for targeting essential genes where a knockout might be lethal, but will likely be replaced by CRISPR—Cas9 or similar genome editing that was successfully adapted for Volvox [ 77 ].
Adapting CRISPR—Cas9-based genome editing to make targeted mutations was a huge breakthrough [ 77 ], and the added ability to do more directed and defined genome editing e.
While Volvox carteri and Chlamydomonas reinhardtii have well-established molecular genetic methods, most other volvocine algae have few or no tools, with successful transformation reported for a handful of other species [ 78 , 79 , 80 , 81 ]. With more interest and improved genomics resources catalyzed by inexpensive genome and transcriptome sequencing, these current limitations can be overcome and will help further elevate the entire clade as a premier model for comparative and mechanistic studies of evolution and development.
Common species are Volvox aureus , Volvox globator , Volvox carteri , and Volvox barberi , etc. Photo source: Microbe wiki. A typical volvox colony consists of a hollow sphere of cells. Each ball, or coenobium , is formed by a single layer of superficial cells joined together.
Each cell is surrounded by a thick mucilaginous wall, forming a gelatinous layer that holds the hollow ball together. In some volvox species, these mucilaginous walls may fill up the internal space of the sphere. These superficial cells are also called vegetative cells or somatic cells.
Each vegetative cell sitting on the surface of the sphere bears two flagella. These flagella face the side of the surrounding water and beat to propel the whole colony through the water. This is why a volvox moves like a rolling ball. The hollow ball consists of a layer of cells. Each cell has a pair of whip-like flagella. The flagella beat in synchrony, allowing the colony of cells to swim.
Image modified from cronodon. Two flagella and one red eyespot are visible. Photo source: microscopy-uk. Other than flagellated somatic cells, a mature Volvox colony also contains reproductive germ cells. The number of germ cells are less than somatic cells and locate in the center of the sphere. Inside the vegetative cell, there is a nucleus, a cup-shaped chloroplast , several contractile vacuoles water-regulating , and other cell organelles.
Each vegetative cell has a red eyespot stigma which can sense light. Volvox, like other green algae, is photosynthetic and it swims toward the light called phototaxis to keep itself illuminated. If the light is too strong, volvoxes also move away from very bright lights that may damage their chloroplasts. The Volvox ball has a preferred front-end and cells in the front of the sphere have larger eyespots than the rest. These eyespots guide the movement of the volvox colony.
The reproductive cells are grouped at the rest side. What new gene functions evolved to permit the evolution of asymmetric division and inversion? How did the other novel developmental traits of Volvox evolve? And are there similarities between the way multicellularity evolved in the volvocine algae and the way it evolved in other kinds of organisms? With the rate of recent progress in this field, answers to these questions, and more, should be on their way soon.
Cheng, Q. The role of GlsA in the evolution of asymmetric cell division in the green alga Volvox carteri. Development Genes and Evolution , — Duncan, L. Journal of Molecular Evolution 65 , 1—11 Herron, M.
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Miller, S. Development , — Nedelcu, A. Environmentally induced responses co-opted for reproductive altruism. Biology Letters 5 , — Nishii, I. A kinesin, invA , plays an essential role in Volvox morphogenesis. Cell , — Peterson, K. Origin of the Eumetazoa: Testing ecological predictions of molecular clocks against the Proterozoic fossil record.
Prochnik, S. Genomic analysis of organismal complexity in the multicellular green alga Volvox carteri. Science , — Sanderson, M. Molecular data from 27 proteins do not support a Precambrian origin of land plants.
American Journal of Botany 90 , — Ueki, N. Controlled enlargement of the glycoprotein vesicle surrounding a Volvox embryo requires the InvB nucleotide-sugar transporter and is required for normal morphogenesis. Plant Cell 21 , — Idaten is a new cold-inducible transposon of Volvox carteri that can be used for tagging developmentally important genes.
Genetics , — What Is a Cell? Eukaryotic Cells. Cell Energy and Cell Functions. Photosynthetic Cells.
Cell Metabolism. The Origin of Mitochondria. Mitochondrial Fusion and Division. The Origin of Plastids. The Origins of Viruses. Discovery of the Giant Mimivirus. Volvox, Chlamydomonas, and the Evolution of Multicellularity. Yeast Fermentation and the Making of Beer and Wine. Dynamic Adaptation of Nutrient Utilization in Humans. Nutrient Utilization in Humans: Metabolism Pathways. An Evolutionary Perspective on Amino Acids. Mitochondria and the Immune Response. Stem Cells in Plants and Animals.
Promising Biofuel Resources: Lignocellulose and Algae. The Discovery of Lysosomes and Autophagy. The Mystery of Vitamin C. Miller, Ph. Citation: Miller, S. Nature Education 3 9 How does multicellularity evolve? Scientists who study a family of green algae that includes unicellular Chlamydomonas and multicellular Volvox are beginning to find answers to this question.
Aa Aa Aa. What Is Multicellularity? Figure 2: Volvox carteri and Chlamydomonas reinhardtii. A Young Volvox adult, with about 2, small somatic cells in a monolayer at the surface, and nineteen large gonidia embedded in the extracellular matrix ECM , just under the somatic cell layer. Multicellularity in the Volvocine Algae.
Strategies for Investigating the Evolution of Multicellularity. From the point of view of geneticists, the somatic cells are seen as mortal and the germ cells as immortal. Although they are composed of individual protists, Volvox colonies can locomote through freshwater habitats, spinning smoothly as all the flagella beat in unison.
Blooms of the chlorophyte occur in "enriched" water bodies or those that are polluted with excess levels of dissolved nitrates and phosphates, and act as an indicator organism.
As primary producers, Volvox colonies produce dissolved oxygen, and as major dietary staples for many aquatic organisms, help support the aquatic food pyramid.
Many types of rotifer thrive by grazing on this green colonial alga, as do other members of the freshwater zooplankton community. Cynthia D.
Kelly , Thomas J. Fellers and Michael W. Visit the Molecular Expressions Website.
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