Using Caenorhabditis elegans To Study Bioactivities of Natural

Chapter 14

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Using Caenorhabditis elegans To Study Bioactivities of Natural Products from Small Fruits Linking Bioactivity and Mechanism in Vivo Mark A. Wilson, Piper R. Hunt, and Catherine A. Wolkow* Laboratory of Neurosciences, National Institute on Aging, Intramural Research Program, Biomedical Research Center, 251 Bayview Blvd., Baltimore, MD 21224, USA *[email protected]

Natural products from small fruits elicit a variety of bioactivities studied primarily in vitro. Although rigorous, these approaches fail to replicate the inherent complexity of the whole organism, where uptake and detoxification processes limit interactions with the target of interest. Currently, most in vivo studies use rodent models, such as mice and rats. Invertebrates, such as Caenorhabditis elegans nematodes, offer an efficient alternative to rodents. C. elegans nematodes are cheap to maintain, easily cultivated in large numbers and amenable to genetic and cellular studies. This chapter provides a brief introduction to the use of C. elegans for in vivo studies of natural products. In addition, we describe our own use of C. elegans to elucidate bioactivities of blueberry proanthocyanins and a collection of resveratrol analogs, with the goal of encouraging new investigations of natural products using C. elegans.

There is great interest in potential health benefits of natural products. Small fruits are particularly rich sources of promising compounds. Realizing this promise will require efficient innovative approaches for identifying and characterizing natural product bioactivities. The free-living nematode, © 2010 American Chemical Society In Flavor and Health Benefits of Small Fruits; Qian, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

Caenorhabditis elegans, provides a powerful, but underutilized, resource for such investigations. The power of the C. elegans model is illustrated by the fact that major cellular processes, such as apoptosis and microRNA-based gene regulation, were originally described in this organism.

Introduction to Caenorhabditis elegans


Laboratory Equipment and Supplies C. elegans is within the Rhabditidae family of free-living nematodes that feed on bacteria and slime molds. In the laboratory, C. elegans populations can be easily grown on solid agar medium supplemented with a food source of E. coli bacteria. In fact, growth and maintenance of C. elegans in the laboratory requires minimal investment in specialized materials and equipment, other than for items common to a standardly-equipped biological laboratory (Table 1). The animals grow well within the range of temperatures between 15°C to 25°C. Many studies can be conveniently conducted at room temperatures of 20-23°C, if controlled within this range. Otherwise, a refrigerated incubator set at the desired temperature provides a standardized cultivation environment. Nematodes are usually cultivated on solid agar medium in plastic petri dishes with a lawn of E. coli bacteria as a food source (1). Methodologies for growing large C. elegans populations in solid and liquid media have been developed (2, 3). These conditions may be suitable for studies of natural products. In addition, several Internet resources are now available for obtaining information on a variety of aspects of C. elegans biology and methodology (Table 2).

Anatomy The C. elegans anatomy contains most major tissues and cell types found in mammals. The cylindrical body is radially symmetric. A collagenous cuticle surrounding the body protects against abrasion and dehydration. The cuticle is open to the environment at the head for food ingestion and sensation, at the vulva for egg laying and at the anus for defecation and sensation. The cuticle is synthesized by epidermal cells that lie just beneath it. Interspersed within the epidermal layer are four longitudinal rows of body wall muscles that promote locomotion and movement. In the head, the major medial structures are the pharynx, the head nerve ganglia and nerve ring. The posterior end of the pharynx joins the intestine, which proceeds for the length of the body to the anus. Food is drawn into the body by the pumping action of the pharynx muscles (4). Pharynx muscles also concentrate the food particles, expeling excess liquid, and crush food particles for passage to the intestine. Within the intestine, food is further broken down and the components are absorbed.

228 In Flavor and Health Benefits of Small Fruits; Qian, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

Table 1. Equipment and Supplies Needed for C. elegans Research


Item

*

Approximate Cost

Purpose

Refrigerated incubator

Growth and maintenance of worm strains within appropriate temperature range (15°-25°C)

$4,000

Stereodissecting microscope

Visualizing worms for manipulation and phenotypic scoring

$3,000

Glass pasteur pipets, platinum wire, alcohol lamps, wooden toothpicks

Supplies for manipulating worms during strain maintenance and growth

Growth medium and petri dishes

Worm growth

Autoclave, clinical centrifuge, refrigerator

Standard laboratory equipment for media preparation, worm collection

*

Ultra low temperature freezer

Long-term storage of worm strains

*

$200

≤ $1.00/ plate

Costs not included for equipment considered to be standard for biological research.

Table 2. Caenorhabditis elegans Internet Resources Title Caenorhabditis Genetics Center

URL www.cbs.umn.edu/CGC/

Resources Caenorhabditis strain collection

WormBase

www.wormbase.org

Curated C. elegans Genome resource

Caenorhabditis elegans WWW Server

elegans.swmed.edu

Links to many C. elegans-related websites

Worm Book

www.wormbook.org

Worm biology and methodology articles

Worm Atlas

www.wormatlas.org

Detailed C. elegans anatomical descriptions

Worm Classroom

www.wormclassroom.org

Basic C. elegans methodology information

The hermaphrodite nervous system consists of 302 neurons, which utilize most of the major neurotransmitters, including acetylcholine, GABA, serotonin and dopamine (5). The nervous system allows the animal to perform several well-characterized quantifiable behaviors, such as locomotion, chemotaxis, mechanosensation and foraging. The male nervous system contains additional neurons which regulate male mating behavior (6).



The gonad consists of somatic and germline structures. C. elegans are hermaphroditic, with the adult hermaphrodite producing both sperm and oocytes for self-fertilization. In addition, X-chromosome non-disjunction results in XO zygotes which develop into males that produce sperm for cross-fertilization. The hermaphrodite gonad consists of two symmetric U-shaped arms which begin at the vulva, extend towards the head and tail and then reflex back toward the midbody. The distal end of each arm contains the germline stem cells which divide and differentiate progressively along the length of each gonad arm. Spermatogenesis occurs during late larval development and the gonad switches to oogenesis during adulthood. Fertilization occurs in the spermatheca, where the sperm are collected, when a mature oocyte is extruded from the proximal gonad. The fertilized egg passes from the spermatheca into the uterus. Within a few hours of fertilization, a protective eggshell matures around each embryo and the eggs are passed out of the vulva and laid.

The C. elegans Life Cycle C. elegans development proceeds through four distinct larval stages (Figure 1). Between each larval stage, the prior-stage cuticle is shed. Cuticle shedding occurs during the inter-molt lethargus when feeding and movement cease. Developmental age can be determined by the presence of stage-specific anatomical features. Such developmentally informative features include the presence or absence of cuticular alae, which are longitunial ridges in the cuticle that are present in first-stage L1 larvae, dauer larvae and adults. Gonadal development can also be a useful marker of developmental age, particularly during the early larval stages. Late-stage larvae can be identified by the presence of a developing vulva. Following the 4th larval molt, mature adults emerge with a fully-differentiated vulva at the midbody. Additionally, the adult gonad is mature and reproduction may commence. In the presence of signals reflecting environmental conditions unsuitable for continued reproduction, larvae enter an alternative developmental pathway to form dauer larvae, which arrest development as an alternative third larval stage (7). The environmental triggers for dauer diapause include limited food availability, high population density signaled by high levels of a constitutively-secreted pheromone, and high temperature. These cues may only trigger dauer arrest if detected in the first larval stage (L1). If appropriately detected, then L2 larvae develop into a special “predauer” form, called the L2d, which molts into the dauer larvae. Dauer larvae possess specialized features that promote survival and dispersal, such as plugged cuticle openings to prevent dehydration, adaptive behaviors to enhance dispersal, and upregulation of antioxidant and heat shock proteins. In addition to increased stress resistance, metabolism is shifted in dauer larvae to utiize fat preferentially. Dauer larvae may resume normal development, molting into the fourth larval stage, upon transfer to a more hospitable environment.



Figure 1. The C. elegans life cycle. Embryonic development occurs in the egg until a mature first stage larva (L1) hatches. Larval development proceeds through four larval stages (L1-L4) before the final molt into the adult hermaphrodite. Adults reproduce and lay eggs for 3-5 days, then enter a postreproductive phase for approximately 2 weeks before death. Inhospitable environments trigger developmental arrest at the dauer larval stage. Dauer larvae are long-lived and stress resistant. Dauers resume development as L4s after return to favorable conditions.

Genetic Analysis in C. elegans The C. elegans genome was the first metazoan genome to be fully sequenced (8). The genome annotations have been compiled into Wormbase (www.wormbase.org). Wormbase provides a wealth of information about gene conservation, structure and function in this organism. In addition, information obtained from standard forward genetic analysis is also included. Wormbase provides a comprehensive resource on C. elegans biology for current knowledge about a protein or pathway of interest. Several of the signaling pathways that are important for human development or disease are conserved in C. elegans. For example, Notch signaling regulates several cell fate decisions in the embryo and developing larvae (9, 10). A C. elegans homolog of mammalian insulin/IGF-I receptors, together with a p110 PI3K, regulate dauer arrest and adult longevity (11). The Ras GTPase homolog, LET-60, couples signaling by FGF- and EGF-related receptors to intracellular MAPK pathways to regulate a variety of developmental events (12). The Nrf-family transcription factor, SKN-1, carries out dual functions in embryonic mesodermal specification and oxidative stress resistance in larvae and adults (13). Natural products interacting with any of these, or other, conserved signaling 231 In Flavor and Health Benefits of Small Fruits; Qian, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

pathways should produce visible phenotypes that may constitute the basis for further mechanistic study.


Considerations for Studying Natural Products in C. elegans A variety of approaches are available for in vivo studies of natural products in C. elegans. Usually, the first goal is to identify quantifiable phenotypes that result from treatment with the compounds of interest. Vehicle controls are essential. Some examples of easily-scored C. elegans phenotypes include embryonic lethality, constitutive dauer arrest, sterility, egg-laying defects, locomotory defects and changes in adult survival. Many investigators begin by screening for phenotypes affecting biological processes within their areas of expertise. For example, this laboratory’s specialization in the biology of C. elegans adults informs our studies of adult longevity and stress resistance after treatment with small fruit botanicals. However, any strong, scorable phentoype is useful for follow-up studies. The ease of working with C. elegans makes it feasible to screen compounds for a variety of phenotypes, without particular bias to specific processes. The thick cuticle presents a major challenge for chemical interventions in C. elegans. This permeability barrier can make the effective dose in C. elegans many times higher than that needed for cultured cells. Our investigations usually involve chronic treatment with compounds supplemented in the growth medium. We prefer to use solid growth medium whenever possible, due to the ease of worm manipulation and visualization. On solid medium, compounds may enter the body through an oral route during food uptake. Exposure can also occur through contact with the sensory cilia, which are nerve terminals open to the environment through pores in the cuticle. At least one group of compounds likely acts by altering the function of these neurons (14). Exposure efficiency might be increased by the use of genetic or environmental conditions which weaken the cuticle, although this area has not been heavily explored. One problem with delivering treatments on solid medium is inefficient delivery due to uncertain availability in the agar surface. Some of these concerns can be alleviated by using liquid growth conditions, rather than solid agar. One approach is to use a defined worm culture medium, with necessary nutrients added from purified sources (3, 15). Alternatively, a standard buffer (S buffer) can be used as the medium and heat-killed bacteria, at a standardized concentration, can be provided as a food source (2). Natural products in the medium may be more efficiently ingested under liquid conditions due to the increased exposure of the worms. However, phenotypic characterization can be more difficult than on solid medium. Fungal and bacterial contamination can also be more serious problems when growing worms under liquid conditions. An additional concern for both solid and liquid medium is the possibility that natural products will be subject to metabolic degradation by the bacteria provided as a food source. To alleviate this problem, the bacteria can be neutralized, either by UV irradiation or antibiotic treatment. Secondary effects on bacterial metabolism have been determined to affect some worm phenotypes (16). 232 In Flavor and Health Benefits of Small Fruits; Qian, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


Figure 2. The prolongevity activity of blueberry polyphenols cofractionated with proanthocyanidins. Total blueberry polyphenols, collected in the C18 eluate of blueberry juice, extended C. elegans adult lifespan (0 µg/ml, mean lifespan 12.0 days, n=97; 200 µg/ml, mean lifespan 14.8 days, n=88, p vs 0 µg/ml

Using Caenorhabditis elegans To Study Bioactivities of Natural

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