Facultative Vivipary is a Life-History Trait in Caenorhabditis elegans

Organisms partition their resources among growth, maintenance, and reproduction and, when resources become limiting, the allocation to one process necessitates reduced allocation to others. When starved, Caenorhabditis elegans adults retain progeny internally which then consume the parent body contents, and some of those larvae use the resources to reach the resistant, long-lived dauer stage. If starved under similarly extreme conditions, larvae from eggs laid outside of the body are unable to develop into dauers. We interpret this switch from ovipary, or laying eggs, to bearing live young as facultative vivipary. This switch is induced by starvation of late fourth-stage larvae, young adults, or gravid adults. In C. elegans, vivipary is the altruistic allocation of all available parental energy and nutrients to progeny, with the associated costs to adult hermaphrodites of truncated life span and fecundity. As a life-history trait, facultative vivipary is a survival-enhancing response to stress that may provide insights into the evolution of reproduction and longevity.     

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity¹ and vibration². In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus³. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.EIW assays involve counting the number of eggs retained in the uterus of C. elegans⁴. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine^5-9.     

CAENORHABDITIS ELEGANS Fertilization-Defective Mutants with Abnormal Sperm

Seven new fertilization-defective mutants of C. elegans have been isolated and characterized; six are temperature sensitive, one is absolute and all are autosomal recessive. One mutation is in a previously described gene, while the other six define six new fer genes that appear to code for sperm-specific functions necessary for normal fertilization. In all fer mutants, both males and hermaphrodites accumulate sperm in near normal numbers. In hermaphrodites, mutant sperm contact the oocytes, but fail to fertilize them. Instead, the sperm are swept into the uterus by the passing oocytes and are expelled when oocytes are laid. Males of two fer mutants do not transfer sperm during copulation, but the other mutant males transfer sperm that fail to move to the spermatheca. Spermatozoa from fer-1 and fer-4 mutants are motility-defective in vitro as well as in vivo, and their pseudopods have an altered morphology. The period of development during which mutant hermaphrodites are temperature sensitive for fertility overlaps the time of sperm development. Some mutants are temperature sensitive throughout the entire period, and others are temperature sensitive during or just prior to spermiogenesis. In fer-4/+ and fer-7/+ males, the fertility of the mutation-bearing sperm is diminished, reducing the transmission ratio. This implies some post-meiotic expression of these genes.—This set of mutants provides a variety of functional and structural alterations in nematode sperm that should help identify and analyze gene products involved in sperm morphogenesis and motility.     

DNA glycosylase activities in the nematode,Caenorhabditis elegans

DNA glycosylases acting upon uracil- or 3-methyl-adenine-containing DNA have been detected in the sonic extracts of the nematode, Caenorhabditis elegans. 4 types of the asynchronously-growing worms, embryos obtained from gravid hermaphrodites, aseptically-hatched larvae, or dauer larvae. Uracil-DNA glcosylase activity was found in all 4 types of the extracts, and the activity was highest in the embryonic extract. In contrast, 3-methyladenine-DNA glycosylase activity was undetectable in the embryonic extract, while an equal level of activity was found in the other 3 types of the extracts. The results substantiate the ubiquity of base-excision repair in various organisms, and suggest that some of the repair functions may be developmentally regulated in multicellular animals.     

An improved staining procedure for the detection of the peroxidase activity of cytochrome P-450 on sodium dodecyl sulfate polyacrylamide gels

The use of 3,3′,5,5′-tetramethylbenzidine-H₂O₂ as a stain for the peroxidase activity of cytochrome P-450 (or cytochrome P-450 in sodium dodecyl sulfate polyacrylamide gels is described in this report. This reagent can be used to detect very low levels of heme-associated peroxidase activity. The blue-stained bands on polyacrylamide gels are distinet, and the color is stable. The stained gels can be photographed or scanned at 690 nm because the gel background remains clear. The stain is easily removed from the gels to permit subsequent protein staining. Staining first for peroxidase activity has no effect on the subsequent protein staining profile. The peroxidase activity of cytochrome P-450 (or cytochrome P-420) in immunoprecipitates in Ouchterlony double diffusion plates can also be detected using this reagent.     

Mating Damages the Cuticle of C. elegans Hermaphrodites

Lifespan costs to reproduction are common across multiple species, and such costs could potentially arise through a number of mechanisms. In the nematode Caenorhabditis elegans, it has been suggested that part of the lifespan cost to hermaphrodites from mating results from physical damage owing to the act of copulation itself. Here, we examine whether mating damages the surface of the hermaphrodite cuticle via scanning electron microscopy. It is found that mated hermaphrodites suffered delamination of cuticle layers surrounding the vulva, and that the incidence of such damage depends on genetic background. Unmated hermaphrodites demonstrated almost no such damage, even when cultured in soil with potentially abrasive particles. Thus, a consequence of mating for C. elegans hermaphrodites is physical cuticle damage. These experiments did not assess the consequences of cuticle damage for lifespan, and the biological significance of this damage remains unclear. We further discuss our results within the context of recent studies linking the lifespan cost to mating in C. elegans hermaphrodites to male secretions.     

Pharmacological characterization of sodium channels in the primary culture of individual Drosophila embryos: Neurons of a mutant deficient in a putative sodium channel gene

Sodium channels in Drosophila embryonic neurons were characterized pharmacologically in the primary culture of individual gastrulae. In normal cultures, presence of sodium channels was demonstrated by neuronal degeneration in the presence of veratridine and ouabain, which was inhibited by tetrodotoxin. Embryonic neurons of Df(2R)M-c^33a homozygotes that lack a putative sodium channel gene at 60E region showed normal neurotoxin sensitivity. Therefore, sodium channel genes other than 60E must be functional at this developmental stage. We also examined para^ts1 and nap^ts mutants and found that they were also sensitive to the neurotoxins. To determine the genotypes of single embryo cultures by histochemical staining, we utilized a special chromosome bearing a hsp70-lacZ fusion gene. The lacZ expression in the culture was studied in detail.     

Sex Attraction and Mating in Bursaphelenchus okinawaensis and B. xylophilus

The fungal feeding, hermaphroditic Bursaphelenchus okinawaensis is a laboratory model to understand the biology of Bursaphelenchus. The extent to which B. okinawaensis can be used to model Bursaphelenchus xylophilus mating was investigated. A chemotaxis assay was conducted to examine whether B. xylophilus and B. okinawaensis produce and respond to volatile sex attractants. Unmated B. xylophilus females were found to attract B. xylophilus males. Similarly, old (sperm depleted) but not young (sperm repleted) B. okinawaensis hermaphrodites attract B. okinawaensis males. Thus, in both species, sperm status corresponds to its ability to attract males. B. xylophilus males also produce a volatile pheromone that attracts both mated and unmated females. A second assay, in which the behavior of males on petri plates in the presence of different females or hermaphrodites of Bursaphelenchus was observed, revealed that B. xylophilus unmated females attract B. okinawaensis males, and B. okinawaensis old hermaphrodites attract B. xylophilus males. These observations suggested that the pheromones of Bursaphelenchus work to some extent across species. Mating behavior through spicule insertion occurs across species, suggesting that postcopulatory mechanisms prevent production of interspecific progeny. The hermaphroditic B. okinawaensis will be a useful model to conduct genetic studies for the understanding of the molecular mechanisms underlying mating behavior in Bursaphelenchus nematodes.     

Maintenance of androdioecy in the freshwater shrimp, Eulimnadia texana: do hermaphrodites need males for complete fertilization?

Androdioecy (populations comprised of mixtures of males and hermaphrodites) is a rare mating system, found only in a few plants and animals. The rarity of this system stems from the limited benefits to males in an otherwise all-hermaphroditic population. One of the potential benefits to males is typified by the nematode Caenorhabditis elegans, in which hermaphrodites do not produce sufficient sperm to fertilize all of their eggs. Here we explore the possibility that males are needed for complete fertilization of hermaphrodites' eggs in a second androdioecious animal, the clam shrimp Eulimnadia texana. We compare the fertilization rate of outcrossed to selfed eggs to test whether the latter exhibit lower fertilization due to sperm limitation (as in C. elegans). Because this comparison confounds differences in egg fertilization due to sperm limitation with the potential for early inbreeding depression, we also used a third mating treatment, a brother/sister cross, to allow separation of sperm limitation from inbreeding depression. In both populations examined, the proportion of eggs that were fertilized decreased linearly with increasing relatedness: comparing eggs produced by outcrossing, brother/sister, and selfed matings, respectively. This pattern suggests that differences in fertilization among these three treatments were caused solely by inbreeding depression, and therefore that hermaphrodites are not sperm limited. These results are combined with previous data on this species to test whether the maintenance of males can be explained using a population genetics model specifically designed for this species.     

A Novel Gene,fudoh, in the SCCmec Region Suppresses the Colony Spreading Ability and Virulence of Staphylococcus aureus

Staphylococcus aureus colonies can spread on soft agar plates. We compared colony spreading of clinically isolated methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA). All MSSA strains showed colony spreading, but most MRSA strains (73%) carrying SCCmec type-II showed little colony spreading. Deletion of the entire SCCmec type-II region from these MRSA strains restored colony spreading. Introduction of a novel gene, fudoh, carried by SCCmec type-II into Newman strain suppressed colony spreading. MRSA strains with high spreading ability (27%) had no fudoh or a point-mutated fudoh that did not suppress colony spreading. The fudoh-transformed Newman strain had decreased exotoxin production and attenuated virulence in mice. Most community-acquired MRSA strains carried SCCmec type-IV, which does not include fudoh, and showed high colony spreading ability. These findings suggest that fudoh in the SCCmec type-II region suppresses colony spreading and exotoxin production, and is involved in S. aureus pathogenesis.     

Histochemical Detection of Carbohydrates of Blastocystis hominis

The carbohydrates of Blastocystis hominis were detected by histochemical techniques using light and electron microscopy. B. hominis, fixed with various fixatives, followed by treatment with detergents, were stained with periodic acid-Schiff (PAS) or alcian blue (AB). Intense PAS reactions were observed in cells fixed with glutaraldehyde or 1/2 Karnovsky fixative. The cells fixed with other fixatives showed weak or no reactions with PAS staining. Similar results were seen in the case of AB stain. These results indicated that, depending on the fixative used, B. hominis contained PAS- or AB-reactive carbohydrates. At the electron microscopic level, ultrathin sections of B. hominis were stained with periodic acid methenamine silver (PA-MS) or periodic acid thiocarbohydrazide-silver proteinate (PA-TCH-SP) staining techniques. Intense, positive reactions with PA-MS or PA-TCH-SP were observed on the central vacuole, Golgi apparatus, and cytoplasmic vesicles. The filamentous layer showed moderate reactions with PA-MS, whereas in PA-TCH-SP stain, it was stained more densely. The staining intensity of the central vacuole varied from cell to cell. The presence of membrane fusions of the cytoplasmic vesicles with the central vacuole indicated the accumulation of carbohydrates in the central vacuole.     

Localization of Na,K-ATPase in lingual epithelia

Na,K-ATPase was localized in canine fungiform and circumvallfltepapillae by immunocytochemical and histochemical methods. Monoclonalantibodies raised against the -subunit of Na,K-ATPase showedspecific staining in the stratum basale and in the lower layersof the stratum spinosum. Small stained wavy lines, interpretedas trigerrunal fibers, were found in the epithelium near tastebuds. In contrast, conventional histochemical methods showedno staining in the epithelium. In both immunocytochemical and histochemical methods taste budswere densely stained. The histochemical stain in taste budswas essentially eliminated by levamisole and L-cysteine butremained in the presence of 10 mM ouabain or in the absenceof potassium. These data suggest that the majority of the histochemicalstain arises from phosphatases other than Na,K-ATPase.     

Developmental and reproductive consequences of prolonged non-aging dauer in Caenorhabditis elegans

When Caenorhabditis elegans encounters harsh environmental conditions, it enters a non-aging diapause (dauer), an alternative larval stage capable of long-term survival. This replaces the stage of normal development critical for development of the reproductive organs. Here, we report that increased duration of diapause causes a delay in post-dauer development, and also causes severe defects in the reproductive development of males and hermaphrodites. Thus, the dauer state, while allowing for survival under adverse conditions, has important developmental and reproductive consequences. This effect is more pronounced in males, possibly accounting for the increased survival of C. elegans hermaphrodites in challenging environmental conditions.