Automated Cell Lineage Reconstruction using Label-Free 4D Microscopy.

Here we describe embGAN, a deep learning pipeline that addresses the challenge of automated cell detection and tracking in label-free 3D time lapse imaging. embGAN requires no manual data annotation for training, learns robust detections that exhibits a high degree of scale invariance and generalizes well to images acquired in multiple labs on multiple instruments.

Influence of environmental temperature on mouth-form plasticity in Pristionchus pacificus acts through daf-11-dependent cGMP signaling.

Mouth-form plasticity in the nematode Pristionchus pacificus has become a powerful system to identify the genetic and molecular mechanisms associated with developmental (phenotypic) plasticity. In particular, the identification of developmental switch genes that can sense environmental stimuli and reprogram developmental processes has confirmed long-standing evolutionary theory. However, how these genes are involved in the direct sensing of the environment, or if the switch genes act downstream of another, primary environmental sensing mechanism, remains currently unknown. Here, we study the influence of environmental temperature on mouth-form plasticity. We find that environmental temperature does influence mouth-form plasticity in most of the 10 wild isolates of P. pacificus tested in this study. We used one of these strains, P. pacificus RSA635, for detailed molecular analysis. Using forward and reverse genetic technology including CRISPR/Cas9, we show that mutations in the guanylyl cyclase Ppa-daf-11, the Ppa-daf-25/AnkMy2, and the cyclic nucleotide-gated channel Ppa-tax-2 eliminate the response to elevated temperatures. Together, our study indicates that DAF-11, DAF-25, and TAX-2 have been co-opted for environmental sensing during mouth-form plasticity regulation in P. pacificus.

Steroid hormone pathways coordinate developmental diapause and olfactory remodeling in Pristionchus pacificus.

Developmental and behavioral plasticity allow animals to prioritize alternative genetic programs during fluctuating environments. Behavioral remodeling may be acute in animals that interact with host organisms, since reproductive adults and the developmentally arrested larvae often have different ethological needs for chemical stimuli. To understand the genes that coordinate the development and host-seeking behavior, we used the entomophilic nematode Pristionchus pacificus to characterize dauer-constitutive mutants (Daf-c) that inappropriately enter developmental diapause to become dauer larvae. We found two Daf-c loci with dauer-constitutive and cuticle exsheathment phenotypes that can be rescued by the feeding of Δ7-dafachronic acid, and that are dependent on the conserved canonical steroid hormone receptor Ppa-DAF-12. Specifically at one locus, deletions in the sole hydroxysteroid dehydrogenase (HSD) in P. pacificus resulted in Daf-c phenotypes. Ppa-hsd-2 is expressed in the canal-associated neurons (CANs) and excretory cells whose homologous cells in Caenorhabditis elegans are not known to be involved in the dauer decision. While in wildtype only dauer larvae are attracted to host odors, hsd-2 mutant adults show enhanced attraction to the host beetle pheromone, along with ectopic activation of a marker for putative olfactory neurons, Ppa-odr-3. Surprisingly, this enhanced odor attraction acts independently of the Δ7-DA/DAF-12 module, suggesting that Ppa-HSD-2 may be responsible for several steroid hormone products involved in coordinating the dauer decision and host-seeking behavior in P. pacificus.

Evolution of neuronal anatomy and circuitry in two highly divergent nematode species.

The nematodes C. elegans and P. pacificus populate diverse habitats and display distinct patterns of behavior. To understand how their nervous systems have diverged, we undertook a detailed examination of the neuroanatomy of the chemosensory system of P. pacificus. Using independent features such as cell body position, axon projections and lipophilic dye uptake, we have assigned homologies between the amphid neurons, their first-layer interneurons, and several internal receptor neurons of P. pacificus and C. elegans. We found that neuronal number and soma position are highly conserved. However, the morphological elaborations of several amphid cilia are different between them, most notably in the absence of 'winged' cilia morphology in P. pacificus. We established a synaptic wiring diagram of amphid sensory neurons and amphid interneurons in P. pacificus and found striking patterns of conservation and divergence in connectivity relative to C. elegans, but very little changes in relative neighborhood of neuronal processes. These findings demonstrate the existence of several constraints in patterning the nervous system and suggest that major substrates for evolutionary novelty lie in the alterations of dendritic structures and synaptic connectivity.

A species-specific nematocide that results in terminal embryogenesis.

Nematode-insect interactions are ubiquitous, complex and constantly changing as the host and nematode coevolve. The entomophilic nematode Pristionchus pacificus is found on a myriad beetle species worldwide, although the molecular dynamics of this relationship are largely unknown. To better understand how host cues affect P. pacificus embryogenesis, we characterized the threshold of sensitivity to the pheromone (Z)-7-tetradecen-2-one (ZTDO) by determining the minimum exposure duration and developmental window that results in P. pacificus embryonic lethality. We found early-stage embryos exposed to volatile ZTDO for as few as 4 h all display terminal embryogenesis, characterized by punctuated development up to 48 h later, with abnormal morphology and limited cavity formation. To determine if the pheromone arrests pre-hatching development by suffocating or permeabilizing the eggshells, we raised embryos under anoxic conditions and also examined eggshell permeability using the lipophilic dye FM4-64. We found that asphyxiating the embryos arrested embryogenesis in a reversible manner but did not phenocopy the effects of ZTDO exposure, whereas the ZTDO-induced disruption of embryogenesis did correlate with increased eggshell permeability. The effects of ZTDO are also highly specific, as other lipid insect compounds do not produce any detectable embryocidal effect. The high specificity and unusual teratogenic effect of ZTDO may be important in mediating the host-nematode relationship by regulating P. pacificus development.

The bacterial community of entomophilic nematodes and host beetles.

Insects form the most species-rich lineage of Eukaryotes and each is a potential host for organisms from multiple phyla, including fungi, protozoa, mites, bacteria and nematodes. In particular, beetles are known to be associated with distinct bacterial communities and entomophilic nematodes. While entomopathogenic nematodes require symbiotic bacteria to kill and reproduce inside their insect hosts, the microbial ecology that facilitates other types of nematode-insect associations is largely unknown. To illuminate detailed patterns of the tritrophic beetle-nematode-bacteria relationship, we surveyed the nematode infestation profiles of scarab beetles in the greater Los Angeles area over a five-year period and found distinct nematode infestation patterns for certain beetle hosts. Over a single season, we characterized the bacterial communities of beetles and their associated nematodes using high-throughput sequencing of the 16S rRNA gene. We found significant differences in bacterial community composition among the five prevalent beetle host species, independent of geographical origin. Anaerobes Synergistaceae and sulphate-reducing Desulfovibrionaceae were most abundant in Amblonoxia beetles, while Enterobacteriaceae and Lachnospiraceae were common in Cyclocephala beetles. Unlike entomopathogenic nematodes that carry bacterial symbionts, insect-associated nematodes do not alter the beetles' native bacterial communities, nor do their microbiomes differ according to nematode or beetle host species. The conservation of Diplogastrid nematodes associations with Melolonthinae beetles and sulphate-reducing bacteria suggests a possible link between beetle-bacterial communities and their associated nematodes. Our results establish a starting point towards understanding the dynamic interactions between soil macroinvertebrates and their microbiota in a highly accessible urban environment.

A host beetle pheromone regulates development and behavior in the nematode Pristionchus pacificus.

Nematodes and insects are the two most speciose animal phyla and nematode-insect associations encompass widespread biological interactions. To dissect the chemical signals and the genes mediating this association, we investigated the effect of an oriental beetle sex pheromone on the development and behavior of the nematode Pristionchus pacificus. We found that while the beetle pheromone is attractive to P. pacificus adults, the pheromone arrests embryo development, paralyzes J2 larva, and inhibits exit of dauer larvae. To uncover the mechanism that regulates insect pheromone sensitivity, a newly identified mutant, Ppa-obi-1, is used to reveal the molecular links between altered attraction towards the beetle pheromone, as well as hypersensitivity to its paralyzing effects. Ppa-obi-1 encodes lipid-binding domains and reaches its highest expression in various cell types, including the amphid neuron sheath and excretory cells. Our data suggest that the beetle host pheromone may be a species-specific volatile synomone that co-evolved with necromeny.

Conserved behavioral and genetic mechanisms in the pre-hatching molt of the nematode Pristionchus pacificus.

BACKGROUND: During development, juvenile nematodes undergo four molts. Although the number of molts appears to be constant within the Nematoda, the timing of the first molt can occur either before or after hatching. A previous study indicates that, as in some parasitic nematode lineages, a pre-hatching juvenile stage also exists in Diplogastrid nematodes. A detailed description of these sequence of events has yet to be shown for any single species. FINDINGS: To delineate the timing of the pre-hatching molt in the beetle-associated Pristionchus pacificus, we tracked individual mid-J1 stage worms inside the eggshell through the J1-J2 transition and hatching. We found that active movement ended 21 hours after egg-laying, followed by lethargus and hatching. We inferred that lethargus behavior represents the onset of the first molt, which precedes each post-hatching molt in C. elegans and P. pacificus. The onset of the J1-J2 molt was also marked by the upregulation of the P. pacificus molting marker Ppa-pnhr-1. We further corroborated the pre-hatching molt with the isolation of two genetic mutants that exhibited aberrant molting both inside the egg and after hatching, as characterized by protracted and often-aborted shedding of the old cuticle. CONCLUSION: Our results describe in detail the pre-hatching juvenile molt in P. pacificus, provide strong visual evidence of a pre-hatching molt, and show support for common genetic mechanisms regulating molting in the pre-hatching and post-hatching developmental stages. Our findings support the hypothesis that the evolution of pre-hatching development in Diplogastrid nematodes is likely due to a heterochronic shift between the timing of the first molt and hatching.

The cGMP signaling pathway affects feeding behavior in the necromenic nematode Pristionchus pacificus.

BACKGROUND: The genetic tractability and the species-specific association with beetles make the nematode Pristionchus pacificus an exciting emerging model organism for comparative studies in development and behavior. P. pacificus differs from Caenorhabditis elegans (a bacterial feeder) by its buccal teeth and the lack of pharyngeal grinders, but almost nothing is known about which genes coordinate P. pacificus feeding behaviors, such as pharyngeal pumping rate, locomotion, and fat storage. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed P. pacificus pharyngeal pumping rate and locomotion behavior on and off food, as well as on different species of bacteria (Escherichia coli, Bacillus subtilis, and Caulobacter crescentus). We found that the cGMP-dependent protein kinase G (PKG) Ppa-EGL-4 in P. pacificus plays an important role in regulating the pumping rate, mouth form dimorphism, the duration of forward locomotion, and the amount of fat stored in intestine. In addition, Ppa-EGL-4 interacts with Ppa-OBI-1, a recently identified protein involved in chemosensation, to influence feeding and locomotion behavior. We also found that C. crescentus NA1000 increased pharyngeal pumping as well as fat storage in P. pacificus. CONCLUSIONS: The PKG EGL-4 has conserved functions in regulating feeding behavior in both C. elegans and P. pacificus nematodes. The Ppa-EGL-4 also has been co-opted during evolution to regulate P. pacificus mouth form dimorphism that indirectly affect pharyngeal pumping rate. Specifically, the lack of Ppa-EGL-4 function increases pharyngeal pumping, time spent in forward locomotion, and fat storage, in part as a result of higher food intake. Ppa-OBI-1 functions upstream or parallel to Ppa-EGL-4. The beetle-associated omnivorous P. pacificus respond differently to changes in food state and food quality compared to the exclusively bacteriovorous C. elegans.

RNAi mediated gene knockdown and transgenesis by microinjection in the necromenic Nematode Pristionchus pacificus.

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression analyses by RNA interference and stable transgenesis remain limited in many species due to the particular anatomy and molecular cellular biology of the organism. For example, outside of the crown group Caenorhabditis that includes Caenorhabditis elegans, stably transmitted transgenic lines in non-Caenorhabditis species have not been reported in this specious phylum (Nematoda), with the exception of Strongyloides stercoralis and Pristionchus pacificus. To facilitate the expanding role of P. pacificus in the study of development, evolution, and behavior, we describe here the current methods to use microinjection for making transgenic animals and gene knock down by RNAi. Like the gonads of C. elegans and most other nematodes, the gonads of P. pacificus is syncitial and capable of incorporating DNA and RNA into the oocytes when delivered by direct microinjection. Unlike C. elegans however, stable transgene inheritance and somatic expression in P. pacificus requires the addition of self genomic DNA digested with endonucleases complementary to the ends of target transgenes and coinjection markers. The addition of carrier genomic DNA is similar to the requirement for transgene expression in Strongyloides stercoralis and in the germ cells of C. elegans. However, it is not clear if the specific requirement for the animals' own genomic DNA is because P. pacificus soma is very efficient at silencing non-complex multi-copy genes or that extrachromosomal arrays in P. pacificus require genomic sequences for proper kinetochore assembly during mitosis. The ventral migration of the two-armed (didelphic) gonads in hermaphrodites further complicates the ability to inject both gonads in individual worms. We also demonstrate the use of microinjection to knockdown a dominant mutant (roller,tu92) by injecting double-stranded RNA (dsRNA) into the gonads to obtain non-rolling F(1) progeny. Unlike C. elegans, but like most other nematodes, P. pacificus PS312 is not receptive to systemic RNAi via feeding and soaking and therefore dsRNA must be administered by microinjection into the syncitial gonads. In this current study, we hope to describe the microinjection process needed to transform a Ppa-egl-4 promoter::GFP fusion reporter and knockdown a dominant roller prl-1 (tu92) mutant in a visually informative protocol.

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).

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.

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.

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.

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.

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.

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.

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.