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In JoVE (1)
Other Publications (8)
- The Plant Cell
- Journal of Experimental Zoology. Part A, Comparative Experimental Biology
- BioEssays : News and Reviews in Molecular, Cellular and Developmental Biology
- Current Biology : CB
- Zoological Science
- Evolution & Development
- Proceedings of the National Academy of Sciences of the United States of America
- Development (Cambridge, England)
Articles by Ray L. Hong in JoVE
JoVE General
RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus
Biology Department, California State University
In model organisms, transgenesis can manipulate gene functions while RNAi can knockdown specific mRNA transcripts 1-2. This protocol aims to illustrate the techniques needed to introduce stably transmitted DNA and transient double stranded RNA into the necromenic nematode Pristionchus pacificus for studies in evolutionary, developmental, and behavioral biology.
Other articles by Ray L. Hong on PubMed
Regulatory Elements of the Floral Homeotic Gene AGAMOUS Identified by Phylogenetic Footprinting and Shadowing
In Arabidopsis thaliana, cis-regulatory sequences of the floral homeotic gene AGAMOUS (AG) are located in the second intron. This 3-kb intron contains binding sites for two direct activators of AG, LEAFY (LFY) and WUSCHEL (WUS), along with other putative regulatory elements. We have used phylogenetic footprinting and the related technique of phylogenetic shadowing to identify putative cis-regulatory elements in this intron. Among 29 Brassicaceae species, several other motifs, but not the LFY and WUS binding sites identified previously, are largely invariant. Using reporter gene analyses, we tested six of these motifs and found that they are all functionally important for the activity of AG regulatory sequences in A. thaliana. Although there is little obvious sequence similarity outside the Brassicaceae, the intron from cucumber AG has at least partial activity in A. thaliana. Our studies underscore the value of the comparative approach as a tool that complements gene-by-gene promoter dissection but also demonstrate that sequence-based studies alone are insufficient for a complete identification of cis-regulatory sites.
Cultivation of the Rhabditid Poikilolaimus Oxycercus As a Laboratory Nematode for Genetic Analyses
Vulva formation is a paradigm for evolutionary developmental biology in nematodes. Not only do the number of vulval precursor cells (VPCs) differ between members in the Rhabditidae and Diplogastridae, they are also sculpted via different developmental mechanisms, either by cell fusion in most Rhabditidae or by programmed cell death in the Diplogastridae. In this context, the species Poikilolaimus oxycercus is the only known species in the family Rhabditidae to have a subset of the Pn.p cells commit programmed cell death during the patterning of the VPCs. Our current study introduces P. oxycercus as a new laboratory organism. There are discrete laboratory strains that are genetically polymorphic from each other as well as heterogeneous within each strain. In order to cultivate this gonochoristic nematode into an experimental model with a tractable genetic system, we produced two inbreeding tolerant, near-isogenic strains capable of producing viable progeny with each other. We also described P. oxycera's morphology by scanning electron microscopy (SEM), basic life history traits, hybrid viability, and mating behavior. P. oxycercus females have no preference for inter- or intra-strain matings, and can mate with multiple males in a relatively short time period, suggesting a propensity for maintaining heterozygosity through promiscuity. Interestingly, all sexes from three species in the genus Poikilolaimus show five 4',6-diamidino-2-phenylindole (DAPI) staining bodies in their germ line cells. This could indicate that Poikilolaimus species possess five bivalent chromosomes in their germ lines, in contrast to the hermaphroditic Caenorhabditis elegans or Pristionchus pacificus, which have six chromosomes.
Pristionchus Pacificus: a Well-rounded Nematode
Nematodes pervade Earth's biosphere and occupy innumerable ecological niches. The role of Caenorhabditis elegans as a model for developmental processes has encouraged us to cultivate a second nematode, Pristionchus pacificus, as a comparative counterpoint to address questions in development, behavior and ecology in nematode evolution. We hope that this endeavor, now more than a decade underway, will allow us to project findings onto other comparative models for biological processes. To this end, our laboratory has made an extensive genetic map and mutant screens to understand changes in developmental programs. Recently, we have been capitalizing on the whole genome sequence of P. pacificus to describe more thoroughly the molecular basis for these changes, as well as to better integrate our molecular knowledge with the biodiversity of Pristionchus species.
Chemoattraction in Pristionchus Nematodes and Implications for Insect Recognition
Nematodes and insects are the two dominant animal taxa in species numbers, and nematode-insect interactions constitute a significant portion of interspecies associations in a diversity of ecosystems. It has been speculated that most insects represent mobile microhabitats in which nematodes can obtain food, mobility, and shelter. Nematode-insect associations can be classified as phoretic (insects used for transportation, not as food), necromenic (insect used for transportation, then carcass as food), and entomopathogenic (insect is killed and used as food). To determine how nematodes target their hosts, we analyzed the chemosensory response and behavioral parameters of closely related Pristionchus nematodes that form species-specific necromenic associations with scarab beetles and the Colorado potato beetle. We found that all four studied Pristionchus species displayed unique chemoattractive profiles toward insect pheromones and plant volatiles with links to Pristionchus habitats. Moreover, chemoattraction in P. pacificus differs from that of C. elegans not only in the types of attractants, but also in its tempo, mode, and concentration response range. We conclude that Pristionchus olfaction is highly diverse among closely related species and is likely to be involved in shaping nematode-host interactions.
The Nematode Pristionchus Pacificus (Nematoda: Diplogastridae) is Associated with the Oriental Beetle Exomala Orientalis (Coleoptera: Scarabaeidae) in Japan
Pristionchus pacificus has been developed as a nematode satellite organism in evolutionary developmental biology. Detailed studies of vulva development revealed multiple differences in genetic and molecular control in P. pacificus compared to the model organism Caenorhabditis elegans. To place evolutionary developmental biology in a comprehensive evolutionary context, such studies have to be complemented with ecology. In recent field studies in western Europe and eastern North America we found 11 Pristionchus species that are closely associated with scarab beetles and the Colorado potato beetle. However, P. pacificus was not commonly found in association with scarab beetles in these studies. Here, we describe the results of a similar survey of scarab beetles in Japan. Pristionchus pacificus was the most common Pristionchus species on scarab beetles in Japan, with 40 out of 43 (93%) isolates. The other Pristionchus isolates represent three novel species, which we refer to as Pristionchus sp. 11, Pristionchus sp. 14, and Pristionchus sp. 15. Thirty-seven of the established P. pacificus strains were found on the oriental beetle Exomala orientalis. Laboratory studies with the sex pheromone (Z)-7-tetradecen-2-one of the oriental beetle revealed that P. pacificus shows strong olfactory attraction to the beetle's sex pheromone, which provides a potential mechanism for the recognition and interaction of P. pacificus and E. orientalis. Together, this study identifies P. pacificus as the most common Pristionchus nematode in field studies in Japan, identifies E. orientalis as an important host species, and provides the basis for the ecology of P. pacificus.
Species-specific Recognition of Beetle Cues by the Nematode Pristionchus Maupasi
The environment has a strong effect on development as is best seen in the various examples of phenotypic plasticity. Besides abiotic factors, the interactions between organisms are part of the adaptive forces shaping the evolution of species. To study how ecology influences development, model organisms have to be investigated in their environmental context. We have recently shown that the nematode Pristionchus pacificus and its relatives are closely associated with scarab beetles with a high degree of species specificity. For example, P. pacificus is associated with the oriental beetle Exomala orientalis in Japan and the northeastern United States, whereas Pristionchus maupasi is primarily isolated from cockchafers of the genus Melolontha in Europe. Here, we investigate how Pristionchus nematodes identify their specific insect hosts by using chemotaxis studies originally established in Caenorhabditis elegans. We observed that P. maupasi is exclusively attracted to phenol, one of the sex attractants of Melolontha beetles, and that attraction was also observed when washes of adult beetles were used instead of pure compounds. Furthermore, P. maupasi chemoattraction to phenol synergizes with plant volatiles such as the green leaf alcohol and linalool, demonstrating that nematodes can integrate distinct chemical senses from multiple trophic levels. In contrast, another cockchafer-associated nematode, Diplogasteriodes magnus, was not strongly attracted to phenol. We conclude that interception of the insect communication system might be a recurring strategy of Pristionchus nematodes but that different nematodes use distinct chemical cues for finding their beetle hosts.
Natural Variation in Pristionchus Pacificus Insect Pheromone Attraction Involves the Protein Kinase EGL-4
The geographical mosaic theory of coevolution predicts that different local species interactions will shape population traits, but little is known about the molecular factors involved in mediating the specificity of these interactions. Pristionchus nematodes associate with different scarab beetles around the world, with Pristionchus pacificus isolated primarily from the oriental beetle in Japan. In particular, the constituent populations of P. pacificus represent a rare opportunity to study multiple specialized interactions and the mechanisms that influence population traits at the genetic level. We identified a component of the cGMP signaling pathway to be involved in the natural variation for sensing the insect pheromone ETDA, using targeted introgression lines, exogenous cGMP treatment, and a null egl-4 allele. Our data strongly implicate egl-4 as one of several loci involved in behavioral variation in P. pacificus populations. That EGL-4 homologs have been independently implicated for behavioral variations in other invertebrate models suggests that EGL-4 may act as a modulator for interspecies behavioral repertoires across large phylogenetic distances.
Dual Roles of the BZIP Transcription Factor PERIANTHIA in the Control of Floral Architecture and Homeotic Gene Expression
Flowers develop from floral meristems, which harbor stem cells that support the growth of floral organs. The MADS domain transcription factor AGAMOUS (AG) plays a central role in floral patterning and is required not only for the specification of the two reproductive organ types, but also for termination of stem cell fate. Using a highly conserved cis-regulatory motif as bait, we identified the bZIP transcription factor PERIANTHIA (PAN) as a direct regulator of AG in Arabidopsis. PAN and AG expression domains overlap, and mutations in either the PAN-binding site or PAN itself abolish the activity of a reporter devoid of redundant elements. Whereas under long-day conditions pan mutants have merely altered floral organ number, they display in addition typical AG loss-of-function phenotypes when grown under short days. Consistently, we found reduced AG RNA levels in these flowers. Finally, we show that PAN expression persists in ag mutant flowers, suggesting that PAN and AG are engaged in a negative-feedback loop, which might be mediated by the stem-cell-inducing transcription factor WUSCHEL (WUS).
