CALIFORNIA STATE UNIVERSITY NORTHRIDGE
CHARACTERIZATION OF MOLTING DEFICIENT MUTANTS IN THE
NECROMENIC NEMATODE PRISTIONCHUS PACIFICUS
A thesis submitted in partial fulfillment of the requirements
For the degree of Master of Science in Biology
By
Victor Maxwell Lewis
August 2014
The thesis of Victor Maxwell Lewis is approved:
_______________________________________
_______________
Dr. Chhandak Basu
Date
_______________________________________
_______________
Dr. Cheryl Van Buskirk
Date
_______________________________________
_______________
Dr. Ray Hong, Chair
Date
California State University, Northridge
ii
DEDICATION
I dedicate this first to the man who has probably forgotten more about biology than I will
ever know, my dad. To my brother, thanks for the endless talks and advice, you are my
inspiration. Finally, to my girlfriend who has listened to me blather on about worms and
evolution more than anyone ever should. Thanks for not only putting up with me, but for
supporting me every day.
i
TABLE OF CONTENTS
Signature Page
ii
Dedication
iii
List of Tables
vi
List of Figures
vii
Abstract
viii
Chapter 1: Introduction to the ecdysozoa and molting
1
Chapter 2: The evolution of Hr46/Cel-nhr-23 in ecdysozoans
3
Introduction
3
Methods
8
Results
9
Conclusion
14
Chapter 3: Cholesterol in the nematode molting pathway
16
Introduction
16
Methods
17
Results
19
Conclusion
24
Chapter 4: Pre-hatching development in Pristionchus pacificus
25
Introduction
25
Methods
26
Results
29
Conclusion
46
Chapter 5: Summary
48
ii
References
51
Appendix A: Sequence Data from Chapter 2
55
Appendix B: Distance Matrices from Chapter 2
61
Appendix C: Associated Videos from Chapter 4
63
iii
LIST OF TABLES
Table 3.1. Results of cholesterol deficiency assay
23
Table 4.1. Results of P. pacificus genetic screen
33
Table 4.2. Complementation testing of Ppa-mlt mutant lines
35
Table 4.3. Time to adulthood in Ppa-mlt mutant lines
37
Table 4.4. Frequency of molting deficient phenotype in Ppa-mlt mutants
38
Table 4.5. Pre-hatching molting deficient phenotype in Ppa-mlt mutants
38
iv
LIST OF FIGURES
Figure 2.1. The ecdysozoan superphyla of molting animals
4
Figure 2.2. The ecdysozoan superphyla annotated with molting phenotypes
6
Figure 2.3. Bayesian tree showing gene phylogeny for Cel-nhr-23
13
Figure 3.1. Experimental design for analysis of cholesterol deficiency
20
Figure 3.2. Requirement for dietary and maternally inherited cholesterol
22
Figure 4.1. Pre-hatching juvenile development in P. pacificus
31
Figure 4.2. Molting deficient phenotype of Ppa-mlt mutants
35
Figure 4.3. Decreased cuticle integrity in Ppa-mlt mutants
40
Figure 4.4. Pre-hatching expression of Ppa-pnhr-1
42
Figure 4.5. Unified model of pre-hatching development in P. pacificus
45
v
ABSTRACT
CHARACTERIZATION OF MOLTING IN PRISTIONCHUS PACIFICUS
By
Victor Maxwell Lewis
Master of Science in Biology
In 1997, Aguinaldo et al. proposed that all molting animals be placed in a single superphyla based on a shared attribute of shedding the larval cuticle. Given the recent
integration of all molting animals into the same clade it has become necessary to reevaluate the evolution of this clade-defining characteristic. Two of the most well known
phyla within this group, the arthropoda and the nematoda, have seen extensive studies on
molting in the models Drosophila melanogaster and Caenhorabditis elegans
respectively. In this thesis we show an initial evaluation of the evolution of molting
within ecdysozoans. We begin by evaluating the evolutionary relationships between the
most conserved inaugural member of the ecdysozoan molting pathway, nhr-23/Hr46, in
Chapter 1. From this evaluation we can start to outline the studies necessary to
understand how molting has evolved across the super-phyla. In Chapter 2, we address
the distinct lack of understanding of the hormonal requirements for molting in one of the
two well studied ecdysozoan models, C. elegans. We set in motion the characterization
of endocrine signaling in nematode molting by investigating the source-specific
requirement for cholesterol, an endocrine pre-cursor found within all ecdysozoans.
Finally, in Chapter 3 we initiate an attempt at understanding how evolutionary changes
which affect the process of molting have occurred within the nematoda. We look at
sterotypic molting behaviors, combined with genetic and molecular analysis, to highlight
how heterochronic changes in the relationship between other developmental events can
lead to relative differences in molting between distantly related nematodes.
vi
CHAPTER 1: INTRODUCTION TO THE ECDYSOZOA AND MOLTING
In 1997, Aguinaldo et al. proposed that all molting animals be placed in a single
super-phyla based on a shared attribute of shedding the larval cuticle. Given the recent
integration of all molting animals into the same clade it has become necessary to reevaluate the evolution of this clade-defining characteristic. Two of the most well known
phyla within this group, the arthropoda and the nematoda, have seen extensive studies on
molting in the models Drosophila melanogaster and Caenhorabditis elegans
respectively. Molting has been shown to be a cyclical process where an extra-cellular
matrix -the cuticle- is periodically shed and remade to allow for growth, metamorphosis,
or both. This process always occurs at the termination of each juvenile stage, often into
adulthood, and both the behavioral and physiological processes have been characterized
in D. melanogaster and C. elegans. The hallmark behavior of molting across
ecdysozoans is the cessation of feeding as the previous cuticle is shed and the new cuticle
is synthesized, while physiological changes include increases in the size of feeding
organs relative to body mass, increases in body size relative to body mass, and, in the
case of some hexapods, gross metamorphosis. A greater understanding of the molting
process in members of the ecdysozoa could lead to new targets for drug design against
parasites, as well as further understanding of ECM formation, cell-cell communication.
Although there are known differences at the molecular level between arthropods
and nematodes, such as the co-option of the ancestral molting pathway to include
ecdysone activation in the arthropod D. melanogaster but not the nematode C. elegans,
many areas remain in which little is known. Molting appears to have been adapted in
different lineages to perform different roles. For example, growth is tied to molting in all
ecdysozoan species, however in many hexapods molting is also required for juvenile to
adult metamorphosis, while in tardigrades molting is closely associated with
reproduction. In arthropods the bio-chemical pathway leading to a novel endocrine
signaling pathway has been described, yet nothing is known in nematodes beyond the
requirement for cholesterol, including whether a subsidiary hormone is even present.
Within ecdysozoan phyla little is also known about the evolution of other developmental
traits which interact with the molting pathway. For example, nematodes appear to have a
highly constrained series of 4 juvenile molts, yet nothing is known about how novel
1
developmental events (e.g. the pre-hatching molt in diplogastrid nematodes) occur in
conjunction with this evolutionary constraint. These instances all highlight the need for
the further study of molting in many areas within the ecdysozoan clade.
In this thesis we intend to begin an initial evaluation of the evolution of molting
within ecdysozoans. We begin by evaluating the evolutionary relationships between the
most conserved inaugural member of the ecdysozoan molting pathway, nhr-23/Hr46, in
Chapter 1. From this evaluation we can start to outline the studies necessary to
understand how molting has evolved across the super-phyla. In Chapter 2, we address
the distinct lack of understanding of the hormonal requirements for molting in one of the
two well studied ecdysozoan models, C. elegans. We begin the characterization of
endocrine signaling in nematodes by investigating the source-specific requirement for
cholesterol, the most conserved endocrine pre-cursor found within ecdysozoans. Finally,
in Chapter 3 we initiate an attempt at understanding how evolutionary changes which
affect the process of molting have occurred within the nematoda. We look at sterotypic
molting behaviors, combined with genetic and molecular analysis, to highlight how
heterochronic changes in the relationship between other developmental events can lead to
relative differences in molting between distantly related nematodes.
2
CHAPTER 2: THE EVOLUTION OF HR46/CEL-NHR-23 IN ECDYSOZOANS
Introduction
Prior to 1997, arthropods (e.g. the fruit fly Drosophila melanogaster) and annelids
(e.g. the common earth worm Lumbricus terrestris) were thought to be sister taxa, in part
based upon a common segmented body plan(Aguinaldo et al. 1997). In 1997, Aguinaldo,
et al. proposed the rearrangement of many new groups thought to be only distantly
related, like the nematoda (e.g. the roundworms C. elegans and Pristionchus pacificus)
and the tardigrada (e.g. the waterbear Hypsibius dujardini), into a single clade based on
the shared characteristic of ecdysis, or molting. In the newly proposed ecdysozoan
superphlya the arthropods were assumed to share common ancestry with all other molting
animals: e.g. the nematodes, nematomorphs, kinorhynchs, priapulids, onchyophorans,
and tardigrades (Aguinaldo et al. 1997). A more recent portrayal of the ecdysozoan clade
based on both expressed sequence tag (EST) and microRNA (miRNA) analysis has
shown that within the ecdysozoa there appear to be 2 distinct sub-clades; the
Panarthropoda, comprised of tardigrades, onchyophorans, crustaceans, hexapods,
myriapods, and chelicerates; and the Cycloneuralia, comprised of the nematodes,
nematomorphs, kinorhynchs, and priapulids (Figure 1)(Dunn et al. 2008; Campbell et al.
2011).
3
Figure 2.1 The ecdysozoan superphlya of molting animals. Adapted from (Dunn 2008).
4
While all of the above phyla undergo ecdysis, there appears to be substantial
variation within the clade in regard to the temporal regulation of molting, the number of
molts, and the relationship between molting and growth.
The most parsimonious
analysis indicates that indeterminate molting, or the lack of a set number of molts leading
to maturity or post-maturity, appears to be the ancestral phenotype. While this pattern
appears in most ecdysozoans ((Higgins 1965; Higgins 1989; Neuhaus 1995; Neuhaus and
Higgins 2002; Hutchinson 1969; Newlands and Ruhberg 1978; Wright 2012; Nelson
2002; Suzuki 2003; Hartnoll 2001; Resh and Cardé 2009), determinate molting, or a
constrained series of molts leading to adulthood, has independently evolved both prior to
the nematode/nematomorph split and in chelicerates, while other arthropods show a
mixture of both indeterminate and determinate molting (Figure 2) (Lee 2002; SchmidtRhaesa 1997; Hartnoll 2001; Resh and Cardé 2009). Hexapods are possibly the most
interesting of the arthropod phyla. Although both indeterminate and determinate molting
is represented, within the hexapoda it appears that determinate molting has become
coupled with metamorphosis (e.g. caterpillars and butterflies) (Resh and Cardé 2009).
Fascinatingly, in tardigrades reproduction is tied to molting as eggs are laid within the
space between old and new cuticles (Nelson 2002; Suzuki 2003). As these differences
are almost certainly represented at the molecular level as well, they highlight the
suitability of the ecdysozoa superplylum as a platform for further investigation into the
processes governing evolution at a molecular level.
5
Figure 2.2. The Ecdysozoan superphlya annotated with molting phenotypes. Annotation
estimated based on most parsimonious tree. Adapted from (Dunn 2008).
6
The molecular controls of molting were first investigated in Drosophila in the
1960's and later in C. elegans(Hieb and Rothstein 1968; Gäde, Hoffmann, and Spring
1997; Chitwood 1999). In Drosophila, the ecdysteroid 20-hydroxyecdysone is
synthesized from cholesterol through a biochemical synthesis pathway encoded by the
Halloween family of P450 genes (Gilbert 2004). 20-hydroxyecdysone is responsible for
activating the molting cascade when bound to the protein-dimer EcR/Usp (Hall and
Thummel 1998). This protein-dimer regulates both Hr46 and ftz-f1 expression, which in
turn activates a cascade of early and late elements required for proper molting (Woodard,
Baehrecke, and Thummel 1994; Horner, Chen, and Thummel 1995; Thummel 1995).
Drosophila Hormone receptor-like 46 (Hr46), previously recognized as Drosophila
Hormone Receptor 3 (DHR3), is an orphan hormone receptor (unknown or non-existant
ligand ) which plays an intermediate role in regulating the timing of morphogenesis
(Lam, Jiang, and Thummel 1997; Lam et al. 1999). Hr46 negatively regulates the early
morphogenesis initiation gene EcR through a physical interaction with the protein itself.
Additionally, Hr46 negatively regulates the expression of the EcR activated early
morphogenesis genes BR-C, E74A, E75A and E78B, both through down regulation of
EcR and through inhibition at their promoter regions. While down regulating early
morphogenesis elements, Hr46 simultaneously activates the late morphogenesis gene
cascade through transcriptional activation of ßFTZF1 (Lam, Jiang, and Thummel 1997;
White et al. 1997; Lam et al. 1999; White et al. 1999).
Genes homologous to Hr46 are present in a wide variety of vertebrates and
invertebrates. HR3 proteins have been identified in both closely and distantly related
arthropods, although no formal study of the gene phylogeny has been conducted (Jindra,
Sehnal, and Riddiford 1994; Palli et al. 1996; Lam, Jiang, and Thummel 1997). Outside
arthropods, Hr46 is most closely related to the nematode nuclear hormone receptor 23
(nhr-23) and the vertebrate Retinoic Acid Receptor and Retinoic-Acid-Receptor-related
Orphan Receptor (RAR/ROR) genes (Giguère, McBroom, and Flock 1995; Kostrouchova
et al. 2001). Although nhr-23 function is conserved as a temporal regulator of the
nematode molting pathway qPCR analysis shows that, unlike arthropods in which it is
expressed predominantly during pupation, its expression occurs prior to each molting
event (Kostrouchova et al. 1998; Kostrouchova et al. 2001). This evidence indicates that
7
Hr46/HR3/nhr-23 is a highly conserved gene required for proper temporal regulation of
molting and development in ecdysozoans, although further investigation is required to
validate this in intermediate classes. The mammalian RAR/ROR's are nuclear hormone
receptor/transcription factor class genes which are involved in regulation of CLOCK,
brain and lymphatic development, and embryogenesis. Multiple human, mouse, and
zebrafish studies have shown that disruptions to retinoid genes are implicated in defective
spermatogenesis, embryogenesis, cerebral development, and various carcinomas
(Chambon 1996; Jetten 2009). It appears that the ancestral Hr46/HR3/nhr-23/RAR/ROR
gene was likely involved in regulating proper development, however there has possibly
been a significant divergence of function at the vertebrate/invertebrate split.
Although we have noted the changes to both the trend and the molecular control
of molting in ecdysozoans, there is little information on the evolution of the conserved
genetic backbone in this pathway. DHR3/cel-nhr-23 appears to be the most upstream
conserved element as it is an initial activator of the molting cycle in every ecdysozoan
that has been investigated. Here we intend to evaluate the hypothesis that the expression
of nhr-23 prior to each molt in the ancestral phenotype and that later co-option of this
ancestral pathway into the ecdysone-activated molting pathway seen in arthropods led to
nhr-23/Hr46 expression occuring primarily during pupation. To evaluate this hypothesis
we have created a gene phylogeny from 20 coding sequences representing the nematode
nhr-23 gene family, the arthropod Hr46 gene family, and the vertebrate ROR/RAR gene
family, As our hypothesis suggests, our analysis shows that the nematode nhr-23 gene
family is ancestral to the ecdysone-activated arthropod Hr46 gene family.
Materials and Methods
Sequence Data
Raw sequence data was collected from NCBI, Wormbase, or Flybase databases
(Appendix A). Reference sequence was Cel-nhr-23 as listed in Appendix A. Sequences
were selected from best BLASTn results for NCBI database or from sequences in which
homology was previously annotated in Wormbase or Flybase.
8
Alignment and Model Test
Sequences were aligned using the Bioedit ClustalW Multiple Alignment tool then further
annotated manually to ensure proper reading frame and account for insertions and/or
deletions which might alter the optimal alignment of conserved sequence features.
During the nucleotide alignment process alignments were checked as amino acids for a
correct reading frame, the lack of pre-mature stop codons, and conserved residues. The
modeltest of the aligned sequences was performed using MEGA with the default settings.
Distance Matricies
Distance matrices were generated in MEGA at the amino acid substitution level to
mitigate false indications of phylogenetic distance. Matrices were generated using the
default settings for analyzing amino acid substitutions with the exception of the
substitution model which was varied by matrix. Substitution models used were: pdistance, Poisson, Dayhoff, and Jones-Taylor-Thorton.
Gene Phylogeny
Bayesian trees were generated in MrBayes using 1 million generations, an lset nst of 2, rate
= gamma, and otherwise default settings. Maximum parsimony and maximum likelihood
trees were generated in MEGA using the kimura 2-parameter model for maximum
likelihood and default settings otherwise. Bootstrap analysis was performed for 500
generations and applied to the maximum likelihood and maximum parsimony trees.
Results
Twenty predicted homologs of nhr-23/Hr46, including cel-nhr-23 and Hr46, were
chosen for analysis of evolutionary function and relationship and aligned using the
Bioedit ClustalW Multiple Alignment tool then further annotated manually to ensure
proper reading frame and account for insertions and/or deletions which might alter the
9
optimal alignment of conserved sequence features. The sequences appeared to align in
two general groups, a nematode group and an arthropod/vertebrate group, all of which
had a highly conserved 3' predicted DNA binding domain and a more varied 5' predicted
ligand binding domain. The only noteworthy difference was the D. yakuba sequence
which aligned almost perfectly to the 3' region of the other Drosophila sequences,
however the sequence was terminated at the beginning of the first conserved region.
A modeltest of the aligned sequences was conducted to predict the most likely
substitution model. The most likely model of nucleotide substitution was the K2+G
model, or the Kimura80 ,nst=2, + discrete gamma distribution based on Bayesian
Information Criterion (BIC) scores where lower BIC scores are assumed to produce the
best substitution model. This model presented an Akaike Information Criterion (AICc)
value of 2187.2, Maximum Likelihood value (lnL) of -1053.7, discrete gamma
distribution (+G) of 0.22, a transition/transversion bias (R) of 2.11, and assumed no
evolutionary invariable (+I) sites. The Kimura-2 parameter model was originally
published by Kimura (1980) and assumes an equal distribution of base frequencies and a
transversion and transition rate of one. Our analysis indicates that the Kimura 2parameter model should be used when constructing maximum Likelihood trees and an n st
of 2 for bayesian trees with a gamma distribution rate.
Four pairwise distance matrices were generated in MEGA using the Bioedit
sequence alignment. The sequences show significant divergence between even closely
related species outside the conserved DNA binding domain which raises the concern that
nucleotide-based pairwise values would not accurately indicate the separation between
distantly and closely related species; therefore distance matrices were generated at the
amino acid substitution level to mitigate false indications of phylogenetic distance.
Matrices were generated using the default settings for analyzing amino acid substitutions
with the exception of the substitution model which was varied by matrix. To determine
robust distance relationships between the samples similarities and differences were
evaluated in four different matrices generated by either calculating the number of sites at
which the sequences differ (p-distance), calculating the number of sites at which the
sequences differ while assuming equal substitution rates and correcting for multiple
substitutions at the same loci (Poisson), or by using PAM matrices developed with either
10
small (Dayhoff) or large (Jones-Taylor-Thorton) datasets. While pairwise values differed
depending on the substitution model selected some general similarities can be noted. In
general, distances were lowest when comparisons were within subgroups (i.e. within
nematodes or arthropods) and highest between members of different subgroups, with the
exception of the Caenorhabditis brenneri and Drosophila yakuba sequences. In the case
of C. brenneri the same trend followed, however distances were higher across all
samples. For D. yakuba, distances between other Drosophila species were minimal,
however distances between other species, including other arthropod species, were either
higher than expected or unable to be calculated (indicated by a value of -1.0). These
differences are likely due to the close alignment of the D. yakuba sequence with other
Drosophila species at the 3' end with no alignment in the conserved DNA binding or
ligand binding domains. As is expected, within the subgroups Caenorhabditis species
showed the least divergence between themselves when compared to other nematodes and
the same followed for the Drosophila species and other arthropods.
To further illustrate the results described in the distance matrices, maximum
parsimony, maximum likelihood, and bayesian trees were generated. As both the
maximum parsimony and maximum likelihood trees show a gene phylogeny that appears
to contrast with known phylogenetic relationships (arthropods split two nematode groups)
the bayesian tree was determined to be the most accurate. In the bayesian analysis two
general groups are seen, an ancestral nematode group with a foundation in the
Caenorhabditis species and multiple bifurcations leading to the other nematode groups.
The last bifurcation within the nematode group splits Pristionchus pacificus and the
second general group containing the arthropods and vertebrates. Within this second
group the first bifurcation seperates the vertebrates from the arthropods and the following
bifurcation separates the crustacean (Daphnia magnam) from the insect arthropods.
Within the insect group the most derived cluster contains the Drosophila species. Under
the assumption that probabilities greater than 90% are accurate very few changes are
required to the generated bayesian tree; the only three bifurcations which should be
collapsed are the bifurcation separating C. elegans from the remaining nematode species,
the bifurcation separating P. pacificus and the arthropods from T. spiralis, and the
bifurcation separating P. pacificus from the arthropods. Additionally, the D. sechellia
11
outcropping could be collapsed with the other Drosophila species although this would not
have a large effect on the interpretation.
12
Figure 2.3. Bayesian tree showing gene phylogeny for Cel-nhr-23 and its homologs in related species. After collapsing
unreliable bifurcations arthropods cluster together at the more derived position while the Caenhorabditis species represent the
most basal group. After collapse the separation between the parasitic nematode species and the arthropod species is less
defined.
13
Conclusion
Nhr-23/Hr46 is a well-characterized gene required for proper completion of the
molting cycle in nematodes and arthropods. Twenty predicted homologs of nhr-23/Hr46,
including cel-nhr-23 and Hr46, were chosen for analysis of evolutionary function and
relationship. The sequences appeared to align in two general groups, a nematode group
and an arthropod/vertebrate group, all of which had a highly conserved 3' domain and a
more varied 5' domain. The only noteworthy difference was the D. yakuba sequence that
aligned almost perfectly to the 3' region of the other Drosophila sequences, however the
sequence was terminated at the beginning of the first conserved region. Four different
substitution models were used to generate distance matrices and in all cases distances
were closest when comparisons were within subgroups (i.e. within nematodes or
arthropods) and more distant between members of different subgroups, with the
exception of the Caenorhabditis brenneri and Drosophila yakuba sequences. This
exception in D. yakuba is likely due to the truncated nature of the sequence, while in
C.brenneri there is no clear explanation. To further analyze these results bayesian
phylogenetic tree was produced from the MSA of cel-nhr-23 and its relatives. This tree
validated 1) the predicted phylogeny based on the MSA and distance matrices where the
nematodes grouped together and the arthropods and vertebrates grouped together and 2) a
predicted phylogeny based on known relationships within the species sampled.
The results of my investigation indicate that nhr-23-like genes are closest to their
ancestral form in nematode lineages, and likely diverged in two different directions in
arthropods and vertebrates. This is interesting as it indicates that the ecdysone-activated
molting pathway is the more derived feature; in arthropods the nhr-23 homologs were coopted into the ecdysone activated molting pathway (Lam, Jiang, and Thummel 1997;
Lam et al. 1999) while in vertebrates it appears that they became involved in the retinoic
acid pathway (Giguère, McBroom, and Flock 1995). The function of cel-nhr-23 and its
relatives as a transcriptional activator is relatively assured given the highly conserved
nature of the DNA binding domain within all species and the conserved nature of the
ligand binding domain within sub-clades. Although this is speculation, it seems possible
that co-option of Hr46 into an ecdysone activated pathway in arthropod species was due
to a change in development from the ancestral nematode hemimetabolous mode of
14
development to the metamorphosis-driven holometabolous mode seen in many
arthropods. Future investigations would be required to validate these claims. Further
investigations would also be necessary to determine a possible conserved ligand within
the nematode sub-group and evaluate the extent to which other members of the nematode
molting pathway have been co-opted into the ecdysone-activated arthropod molting
system.
15
CHAPTER 3: CHOLESTEROL IN THE NEMATODE MOLTING PATHWAY
Introduction
Members of the ecdysozoa such as Drosophila melanogaster and Caenorhabditis
elegans share the common developmental trait of periodic molting of the larval
cuticle(Aguinaldo et al. 1997). As cholesterol cannot be synthesized de novo by either
arthropods or nematodes, it is obtained through the diet (Hieb and Rothstein 1968; Gäde,
Hoffmann, and Spring 1997; Chitwood 1999). While in Chapter 1 we evaluated the
evolution of an inaugural member of the ecdysozoan molting pathway, nhr-23/Hr46,
there still remains little investigation into the hormonal control of molting in nonarthropod members of the ecdysozoa (nematodes, etc.). Of particular note, in C. elegans
it has previously been shown that inhibition of the molting cycle through depletion of
cholesterol requires both removal of dietary cholesterol from the media and the reduction
of maternally inherited cholesterol(Meli et al. 2010), however this has never been
quantitatively evaluated.
In Drosophila, the ecdysteroid 20-hydroxyecdysone is synthesized from
cholesterol through a biochemical synthesis pathway encoded by the Halloween family of
P450 genes(Gilbert 2004). 20-hydroxyecdysone is responsible for activating the molting
cascade when bound to the protein-dimer EcR/Usp(Hall and Thummel 1998). This
protein-dimer regulates both DHR3/Hr46 and ftz-f1 expression, which in turn activates a
cascade of early and late elements required for proper molting(Woodard, Baehrecke, and
Thummel 1994; Horner, Chen, and Thummel 1995; Thummel 1995).
Nematodes, unlike arthropods, have a highly conserved series of four juvenile
molts with the last molt occurring at the division between the fourth juvenile stage and
adulthood(Lee 2002). In C. elegans, both NHR-23, homologous to Drosophila DHR3,
and NHR-25, homologous to Drosophila FTZ-F1, are conserved regulators of the
molting cycle between arthropods and nematodes. While NHR-25 shares homology with
FTZ-F1, NHR-25 does not appear to be regulated by NHR-23 in the same manner DHR3
regulates FTZ-F1 function(Kostrouchova et al. 2001). Both NHR-23 and NHR-25 are
likely to be activated by either an unknown cholesterol-synthesized steroid ligand or by
cholesterol directly, although it is unlikely that an ecdysteroid is used for this purpose
16
since no EcR/usp homologs are present in C. elegans(Gissendanner and Sluder 2000;
Kostrouchova et al. 2001; Frand, Russel, and Ruvkun 2005; Parihar et al. 2010).
Moreover, the downstream components of the molting signal pathway in nematodes have
diverged from those in arthropods. NHR-23 and NHR-25 are required for the proper
cyclical expression of mlt-10, which encodes a nematode-specific cholesterol-dependent
structural protein utilized in the removal and rebuilding of the cuticle during
molting(Meli et al. 2010). The transient expression pattern of a mlt-10 transcriptional
reporter in the syncytial hypodermis coincides with the remaking of the nematode
exoskeleton. MLT-10 is the founding member of a large family of proteins characterized
by a DUF644 domain and proline-rich repeats. Because the molting phenotype of mlt-10
mutants is exacerbated by low cholesterol in the media, the transcriptional regulation of
mlt-10 by NHR-23 and NHR-25 may be directly linked to the amount of steroid
hormones biologically available to the two nuclear receptors.
Previous genetic screens for mutants sensitive to low cholesterol required the
replacement of Bacto-agar with agarose in Nematode Growth Media (NGM)(Meli et al.
2010). However, the effects of low-cholesterol NGM have not been quantitatively
evaluated, particularly in wild-type C. elegans. Here we have developed a modified
protocol using phytagar as an alternative to agarose in NGM and quantified the
expression of the cholesterol-dependent molting gene mlt-10p::gfp in the wild-type
background. Prior studies have investigated the effects of MLT-10 disruption under lowcholesterol conditions, but there have been no published studies of mlt-10 expression in
wildtype worms under the same conditions. We hypothesize that mlt-10 expression would
decrease on media without cholesterol supplement. In this study, we sought to deplete
maternally-inherited cholesterol in mlt-10p::gfp worms to determine changes in mlt-10
expression when placed under low-cholesterol conditions over multiple generations.
Materials and Methods
Nematode and Bacterial Strains
C. elegans mlt-10p::gfp-pest(GR1395)(gift from Frand Lab, UCLA) were
cultured at room temperature (~23°C) on E. coli OP50 on Nematode Growth Media as
17
described below (Brenner 1974). OP50 cultures were inoculated in LB at 37°C overnight
and 200 µl of liquid bacteria cultures were seeded onto 6 cm culture plates. For the
cholesterol depletion assay worms were cultured on either culture plates containing
Nematode Growth Media (NGM) or a modified phytagar based NGM (PNGM) without
cholesterol supplement (PNGM-) for two generations before placement of the third
generation on various assay plates, or raised continuously on NGM plates as a control.
Nematode Growth Media and Cholesterol-Deficient Media
Nematode Growth Media was prepared as follows: (1L) 2 g NaCl (Fisher
Scientific #S78449), 4 g Bacto-tryptone (Fisher Scientific #BP1421-500), 3 g KH2PO4
(Acros Organics #205920025), 0.5 g K2HPO4 (Acros Organics #215470010), 20 g Bactoagar (Teknova #A7777), 975 mL distilled H2O. Modified cholesterol-deficient NGM was
prepared as follows: (1L) 2 g NaCl (Fisher Scientific #S78449), 4 g Bacto-tryptone
(Fisher Scientific #BP1421-500), 3g KH2PO4 (Acros Organics #205920025), 0.5 g
K2HPO4 (Acros Organics #215470010), 20 g phytagar (Life Technologies #10695-039),
975 mL distilled H2O. When applicable, cholesterol (Research Organics #1387C) was
added to a 5 µg/mL final concentration. Assay plates consisted of: NGM with the
addition of cholesterol (NGM+), NGM without the addition of cholesterol (NGM-),
PNGM with the addition of cholesterol (PNGM+), and PNGM without the addition of
cholesterol (PNGM-).
Synchronization
C. elegans Mlt-10p::gfp-pest(GR1395) larvae were synchronized at the J2 arrest by
bleaching gravid hermaphrodites and allowing the remaining embryos to hatch on
unseeded plates for 12 hours.
Imaging
A Leica M165 epifluorescence microscope was used for observing mlt-10p::gfppest(GR1395). For image documentation, nematodes were paralyzed with 40 µM sodium
18
azide and mounted on slides with M9 buffer. Images were taken with a Leica DM6000 at
200X or 400X and prepared for publication using Adobe Photoshop.
Statistical Analysis and Graphical Representations
Statistical analysis for maternally inherited cholesterol assay (t test) and
phytagar-based NGM assay (Dunnett’s test) were performed using InStat statistical
software, otherwise analysis was performed using Microsoft Excel. Graphical
representations of collected data were prepared using Microsoft Excel or Powerpoint and
annotated in Adobe Photoshop.
Results
In an effort to enhance the visualization of cholesterol-dependent molting
deficiencies in C. elegans, we have optimized the creation of cholesterol-deficient media
through the replacement of Bacto-agar with Phytagar as a solid substrate (PNGM)
without cholesterol. We found that not adding cholesterol to Bacto-agar NGM did not
result in a significant increase in larvae becoming stuck in cuticle even after two
generations, presumably due to either residual cholesterol or the presence of precursor
sterols convertible to cholesterol in the Bacto-agar (Félix and Braendle 2010). As
cholesterol-deficiency in C. elegans can display a spectrum of associated phenotypes, we
utilized a known cholesterol-dependent molting cycle reporter to quantitatively evaluate
the effects of cholesterol deprivation. We evaluated GFP expression in synchronized
populations of the C. elegans cholesterol-dependent molting reporter line mlt-10p::gfppest(GR1395)(Meli et al. 2010) raised on cholesterol-deficient PNGM (PNGM-) media
for two generations (Fig. 1). Mlt-10p::gfp is expressed in the hypodermis prior to each
larval molt. We observed a significant increase in molting defects associated cholesteroldeficiency, such as unshed larval cuticle and immobility, when worms were raised
continuously without cholesterol supplement compared to the worms rescued with
cholesterol supplement in the third generation on PNGM (Fig.2). The mlt-10(mg364)
loss-of-function mutant line, which has an increased dependency on cholesterol for
19
proper mlt-10 expression, was obtained through a screen for worms that required
exogenous cholesterol to properly complete the molting cycle in the first generation.15
Control Mlt-10p::gfp-pest(GR1395) worms raised continuously on NGM+ showed the
highest 87.8% GFP reporter expression 48 hours after release from L1 arrest. After two
generations without cholesterol supplement, worms transferred to NGM+, NGM-, and
PNGM+ media showed similar GFP reporter expression as each other (53.0%, 49.9%,
and 53.7%, respectively), while worms cultured continuously on PNGM- showed
significantly reduced 32.6% GFP reporter expression (Fig. 2; Table 1).
Figure 3.1. Experimental design for analysis of cholesterol-deficient media and
maternally inherited cholesterol. Mlt-10p::gfp-pest(GR1395) worms were either (A)
raised continuously on NGM with exogenous cholesterol (NGM+) or (B) raised for two
generations on PNGM without the addition of exogenous cholesterol (PNGM-) prior to
transfer to respective assay plate
20
It has previously been shown that the display of of cholesterol-dependent molting
phenotypes requires raising animals on cholesterol-deficient media for two generations,
implying a role for maternally-inherited cholesterol in proper development (Meli et al.
2010). Our assay has allowed us to quantitatively evaluate the extent with which
cholesterol is maternally inherited. Comparison of GFP reporter expression between mlt10p::gfp-pest(GR1395) L4 larvae raised continuously on NGM+ and L4 larvae on
NGM+ media descended from worms raised on PNGM- media for two generations show
a remarkable difference in GFP reporter expression. Specifically, worms raised
continuously on NGM+ showed 34.8% higher GFP reporter expression when compared
to worms initially raised on PNGM- (P<0.01, Fig. 2). This finding also suggests that after
two generations of cholesterol depletion, mlt-10p::gfp expression does not fully recover
even after raising the progeny to the L4 stage on cholesterol-supplemented media made
either with Bacto-agar or phytagar.
21
Figure 3.2. Our analysis shows a requirement for both dietary and maternal cholesterol.
L4 larvae show significantly less GFP expression after 48 hours release from arrest on
PNGM without cholesterol supplement. (A) Representative mlt-10::gfp larva raised on
PNGM- and transferred to PNGM+. Anterior is left. (B) Representative mlt-10p::gfp
larva raised on PNGM- and transferred to PNGM- shows molting deficiency. Black
arrow head shows unshed cuticle on buccal region. (C) Representative GFP expression
from mlt-10p::gfp L4 raised on PNGM- and transferred to PNGM+. (D) Quantitative
analysis of GFP expression in L4 larval mlt-10p::gfp. Error bars show standard error of
the mean (SEM). Multiple comparisons are to worms raised continuously on PNGM(Dunnett’s test). Analysis between NGM+  NGM+ and PNGM-  NGM+ used
unpaired t test (Bracket). *P<0.05; **P<0.01
22
Maternal
Assay
Mean
N(n)
SEM
P
NGM +
NGM +
0.88
10(619)
0.015
<0.01
PNGM NGM +
0.53
10(588)
0.034
<0.01
PNGMNGM 0.50
10(635)
0.024
<0.01
PNGM PNGM +
0.54
10(838)
0.029
<0.01
PNGM PNGM 0.33
10(967)
0.023
NA (Control)
Table 3.1. Results of cholesterol deficiency assay. Data represents 10 technical replicates
from two experiments. Multiple comparisons are to assayed worms raised continuously
on PNGM- (Dunnett’s test). N = Replicates; n = total number of worms sampled; SEM
= standard error of the mean.
23
Conclusion
Cholesterol has been proven to be a necessary requirement for proper
development, including molting, in C. elegans(Chitwood 1999). Phenotypes similar to
those seen from growth of C. elegans on cholesterol-deficient media have been reported
in mutants defective for the LDL receptor protein LRP-1, which has been suggested to be
required for proper endocytosis of cholesterol(Yochem et al. 1999). Furthermore, it has
been shown that this cholesterol deficient phenotype was delayed until the second
generation when N2 wildtype worms were grown on cholesterol deficient media,
implicating a role for maternally inherited cholesterol for proper development(Yochem et
al. 1999). As C. elegans cannot synthesize cholesterol de novo, cholesterol is believed to
be obtained through the worms’ bacterial diet(Hieb and Rothstein 1968; Chitwood 1999).
Dietary restriction results in either L1 diapause or dauer larva formation, the latter of
which has been shown to be repressed through the synthesis of dafachronic acid from
cholesterol(Motola et al. 2006). This implies that there is a requirement for maternallyinherited cholesterol, in addition to the dietary intake of cholesterol, for proper nematode
development.
MLT-10, like other members of the nematode molting pathway, is regulated by
the steroid binding proteins NHR-23 and NHR-25(Meli et al. 2010). Wildtype N2 worms
raised on cholesterol-deficient media appear to be unable to undergo proper ecdysis,
rather than apolysis, as the cuticle appears shed, but not breached. Thus, affected worms
have a visibly detached older cuticle near their head and tail as well as greatly reduced
mobility. This shows that cholesterol deficiency does not appear to cause an immediate
arrest in the activation of the molting cycle. However, a reduction in available cholesterol
may likely decrease activation of the nuclear hormone receptors NHR-23 and NHR-25,
which in turn reduces expression of downstream targets such as mlt-10. The advent of
apolysis, but not ecdysis, in cholesterol-deficient phenotypes therefore also shows that
different levels of expression are required by individual components of the C.
elegans molting pathway for proper development to occur. Our investigation indicates
that robust mlt-10 expression is cholesterol dependent, and that maternally inherited
cholesterol is a necessary requirement in a natural environment, where larval nematodes
may face restrictions on dietary cholesterol intake.
24
CHAPTER 4: PRE-HATCHING DEVELOPMENT IN PRISTIONCHUS
PACIFICUS
Introduction
Although we have previously evaluated the genetic evolution of the molting
pathway in ecdysozoans (see Chapter 1) and the requirement for both maternally
inherited and dietary cholesterol in the model C. elegans (see Chapter 2), there is almost
nothing known about the how evolutionary changes which affect the process of molting
have occurred within the nematoda. In nematodes, molting starts with apolysis or
shedding of the old cuticle, followed by synthesis of the new cuticle, and culminates with
ecdysis, or escape from the old cuticle. Nematodes undergoing molting transition from
active foraging behavior to lethargus, a quiescent period marked by a lack of movement
during which the new cuticle is synthesized. The end of lethargus and the resumption of
active behavior occur just prior to ecdysis, when the worm breaches the old cuticle
(Singh and Sulston 1978). Unlike arthropods, which can vary in molting frequency, larval
development in all nematodes appears to undergo four molts before reaching
reproductive maturity (Lee 2002). This phylum-wide developmental constraint is likely
not limited by genetic restrictions as single gene mutations in the model nematode
Caenorhabditis elegans can cause changes to the molting cycle, such as supernumerary
molts in lin-4, lin-14, and lin-29 mutants (Ambros and Horvitz 1984; Ambros 1989).
Although the four molts after hatching in C. elegans appears to be the ancestral
phenotype, there are several examples that deviate from this pattern in plant and animal
parasites (Felix and Hill 1999). Nematodes that molt prior to hatching include the
pinewood nematode Bursaphelenchus xylophilus and the potato cyst nematode
Globodera pallida (Lee 2002; Oh et al. 2009; Palomares-Rius et al. 2013), as well as the
vertebrate parasites Ascaris lumbricoides and Toxocara cati (Anderson 1992; Lee 2002).
Among non-parasitic nematodes, larvae that undergo the first molt within the eggshell
have been described in the Pristionchus and Oigolaimella species in the family
Diplogastridae (Fürst von Lieven 2005). The strong presence of parasitic lifestyles within
these diverse lineages hints at the possible role a pre-hatching larval stage plays in
driving the evolution of parasitism.
25
Pristionchus pacificus is a nematode with a species-specific association to beetles.
Its unique life style straddles the continuum between free-living and obligate parasitic
nematodes, which invites studies on developmental plasticity, timing, and novelty. In
particular, P. pacificus was once thought to represent a unique group that possess only
three juvenile stages prior to adulthood (Felix and Hill 1999; Hong and Sommer 2006).
Subsequent investigation has since revealed that the first molt actually occurs prior to
hatching in Diplogastrid nematodes, including P. pacificus. Behavioral events
characterizing this as a complete developmental stage have not been shown but is crucial
for the understanding of developmental timing (Fürst von Lieven 2005). In order to
establish behavioral and genetic similarities between the pre-hatching and post-hatching
molts in P. pacificus, we tracked development from late-stage embryos through the J1-J2
transition to hatching. We further corroborated our observations by monitoring
expression changes of a P. pacificus molting marker as well as obtaining general molting
defective mutants that show both pre-hatching and post-hatching molting-defects. Our
findings further supports the hypothesis that the pre-hatching J1 stage in P. pacificus is
homologous to the first post-hatching L1 stage in C. elegans.
Materials and Methods
Nematode and Bacterial Strains
P pacificus wild-type PS312, and Ppa-mlt mutant lines from the PS312
background were cultured at room temperature (~23°C) on E. coli OP50 on Nematode
Growth Media as described below (Brenner 1974). OP50 cultures were inoculated in LB
at 37°C overnight and 200 µl of liquid bacteria cultures were seeded onto 6 cm culture
plates. For the cholesterol depletion assay worms were cultured on either culture plates
containing Nematode Growth Media (NGM). Eggs were cultured at 20°C from
synchronization until observation.
Mutagenesis
26
Mutagenized strains were generated in the PS312 CA background using 50 mM
ENU; 1500 F1 animals were cloned and their F2 progeny inspected for the presence of
molting defects. From plates containing mutants, defective animals were picked again
and screened once more for the mutant phenotype. Mutant lines were backcrossed 3 times
to wild-type PS312 and homozygous molting-defect escapers were established for
detailed analyses
Nematode Growth Media
Nematode Growth Media was prepared as follows: (1L) 2 g NaCl (Fisher
Scientific #S78449), 4 g Bacto-tryptone (Fisher Scientific #BP1421-500), 3 g KH2PO4
(Acros Organics #205920025), 0.5 g K2HPO4 (Acros Organics #215470010), 20 g Bactoagar (Teknova #A7777), 975 mL distilled H2O. Cholesterol (Research Organics
#1387C) was added to a 5 µg/mL final concentration.
Synchronization
C. elegans N2 and P. pacificus PS312 larvae were synchronized at the J2 arrest by
bleaching gravid hermaphrodites and allowing the remaining embryos to hatch on
unseeded plates for 12 hours. Eggs were synchronized by allowing young adult
hermaphrodites to lay eggs for 15 minutes on plates seeded with OP50 prior to removal.
Staining
Hoescht staining was conducted by washing plates with M9 buffer and staining with
1:100 Hoescht 33342 for 12 minutes. Washed worms were then placed on NGM plates
seeded with OP50 for 1 hour to remove excess dye prior to imaging on slides with 2%
Noble Agar pad and 1M sodium azide.
Quantitative real-time PCR (qPCR)
27
J2 larvae were synchronized at the J2 arrest by bleaching gravid hermaphrodites and
allowing the remaining embryos to hatch on unseeded plates for 12 hours. Eggs were
synchronized by allowing young adult hermaphrodites to lay eggs for 15 minutes on
plates seeded with OP50 prior to removal. cDNA was synthesized from RNA in Trizol
with Superscript III First-Strand Synthesis Kit (Invitrogen). qPCR was performed and
analyzed using SYBR chemistry on BioRad CFX96 with primers designed to span exonintron boundaries (forward, reverse):
Ppa-beta-tubulin,
Ppa-pnhr-1,
TCCAAGATCCGTGAGGAGTA, GGAGAGGGTGGCATTGTAG;
CTCTTGAACGGCGTCCCTCTTC, GTGCAGAGTTGCGAAGGCTG.
qPCR data represents 4 technical replicates from two independently isolated RNA
samples at each time-point. Each biological replicate was collected from four pooled
synchronized populations. Relative expression ratio of Ppa-pnhr-1 to the reference gene
Ppa-beta-tubulin was normalized to the no reverse transcriptase negative control.
ANOVA analysis was followed by a Dunn'sTest.
Imaging
Worms were observed using either a Leica S8E stereomicroscope or a Leica DM6000.
For image documentation, nematodes were paralyzed with 40 µM sodium azide and
mounted on slides with M9 buffer. Images were taken with a Leica DM6000 at 200X or
400X and prepared for publication using Adobe Photoshop. Hoescht images were taken
at exposure 350 ms, gain 3, and intensity 3. Eggs were mounted on slides with egg
buffer. Visual tracking of lethargus sequences in P. pacificus were made by observing
synchronized embryos for 10 minutes every hour from 16 hours after egg laying until
hatching. C. elegans embryos were tracked for 10 minutes every 30 minutes from 30
minutes after egg laying until hatching. Tracking of time until adulthood in Ppa-mlt
alleles excludes those individuals that exhibited obvious molting defects. To visualize the
presence of the pre-hatching molt in the mutants, worms from these lines were
synchronized and allowed to lay eggs. When ~80% of larvae had hatched we then
screened the remaining eggs for pre-hatching juveniles which had not properly completed
the molt.
28
Statistical Analysis and Graphical Representations
Statistical analysis cuticle permeability assay (Dunnett's test) was performed
using InStat statistical software, otherwise analysis was performed using Microsoft Excel.
Graphical representations of collected data were prepared using Microsoft Excel or
Powerpoint and annotated in Adobe Photoshop.
Results
To precisely characterize the events leading to the pre-hatching molt in P.
pacificus, we have tracked pre-hatching development from the middle of the first juvenile
stage to hatching in two separate populations (n=15), synchronized to within 15 minutes
at egg-laying. Pre-hatching worms were observed for 10 minutes every hour using light
microscopy. Active worms were defined as individuals to which movement occurred
consistently throughout the 10-minute window, and worms in lethargus were defined as
individuals that exhibited minimal movement during the same time frame. Hatching was
established by visual confirmation of the worm outside the eggshell. We inferred that
ecdysis occurred between the termination of lethargus and the onset of hatching. Active
behavior during the J1 stage of development ended 21.5±0.74 hours post egg-laying,
lethargus 24.1±0.64 hours post egg-laying, and hatching 25.1±0.64 post egg-laying (Fig.
1 A, D). Active behavior consisted of rapid movements, including full body turns,
interspaced by short periods of no movement (Fig. S1). This is in contrast to lethargus
behavior where the worm remained relatively motionless, although there were slight
adjustments made to position during the ~3 hour lethargus period (Fig. S2). Hatching
occurred within one hour of the termination of lethargus.
To determine if the observed pre-hatching behaviors were homologous to the
behaviors associated with molting in post-hatching worms, we tracked an additional
population of worms from the post-hatching J2 arrest through the J2-J3 molt. Active
behavior ended 9.5±0.51 hours after J2 release, lethargus after 11.4±0.81 hours, and
ecdysis after 12.4±0.81 hours (Fig. 1B). Active behavior consisted of typical foraging
29
interspaced by short rests. Lethargus behavior was again characterized by a general lack
of movement although short movements, totaling no more than one body length, were
occasionally observed. Worms undergoing ecdysis were observed to resume activity with
rapid horizontal movement in the anterior region, presumably to further dislodge the
previous cuticle. Exit of the previous cuticle was usually completed around 30 seconds.
To confirm that our observation is indeed a characteristic of Diplogastrid
nematodes, we also tracked a population of C. elegans embryos (n=15) from postelongation embryogenesis through hatching. Active behavior during late-embryogenesis
ended 11.8±0.50 hours post egg laying and hatching occurred 12.3±0.52 hours post egg
laying (Fig. 1C). No lethargus period was observed. In conclusion, we observed the same
stereotyped behavioral events leading to a J1-J2 molt in the late-stage pre-hatching P.
pacificus juveniles, which were observed in newly hatched P. pacificus J2 juveniles
similar to post-hatching molts in C. elegans.
30
Figure 4.1. Pre-hatching juvenile development in P. pacificus. (A) Pre-hatching
development in wild-type P. pacificus PS312 worms. (B) Post-hatching development in
J2 stage wild-type P. pacificus PS312 worms. Note that the sequence of behaviors seen in
(A) is almost identical to those in (B). (C) Late-embryonic development in wild-type N2
C. elegans.
31
To identify molting deficient mutants in P. pacificus, we performed a
nonsaturating genetic screen for juveniles with defective molting. 1500 F1
hermaphrodites were singled to new plates and the resulting F2 and F3 progeny were
screened over one week. We isolated 10 mutant lines in which the mutant phenotype was
a molting deficiency and 1 mutant line (emd-2; csu27) in which the initial observation
showed molting deficiency yet subsequent observation only identified defective pharynx
development and "dumpy" phenotypes (table 4.1). Additional "dumpy" and
"uncoordinated" phenotypes were observed (uncategorized), indicating efficient
mutagenesis.
32
Ppa-mlt
csu26
csu27
csu28
csu29
csu30
csu31
csu32
csu33
csu34
csu36
csu37
emd
emd-1
emd-2
emd-3
emd-4
-
Phenotype
Pre and post-hatching molting defective
"Pug" ; defective pharynx, dumpy
Molting defective, egg-laying defective
Post-hatching molting defective candidate
Post-hatching molt-in-molt phenotype
Post-hatching molting defective candidate
Post-hatching molting defective candidate
Post-hatching molting defective candidate
Post-hatching molting defective candidate
Post-hatching molting defective candidate
Post-hatching molting defective candidate
Outcross
3
3
3
0
1
0
0
0
0
0
0
Table 4.1. Results of P. pacificus genetic screen for molting deficiency.
33
Homozygous
Yes
Yes
Yes
No
No
No
No
No
No
No
No
Two complementing mutant lines (table 4.2), Ppa-mlt(csu26) and Ppa-mlt(csu28),
were selected for further study due to viability for outcrossing and their easily identifiable
phenotype. The molting deficiency is easily distinguished from molting in wild-type
worms through observation of the shed cuticle surrounding both the anterior and posterior
of the worm (Fig. 4.2), whereas the cuticle was only observed to be seen protruding from
the anterior of wild-type worms prior to ecdysis.
34
Replicate
csu26-male x
csu28
0.00
0.00
0.03
csu28-male x
csu26
0.00
0.00
-
csu26 x
csu26
0.23
0.19
0.17
csu28 x
csu28
0.17
0.12
-
1
2
3
Table 4.2. Complementation testing of Ppa-mlt mutant lines showing fraction progeny
exhibiting molting deficiencies. csu26 and csu28 belong to separate complementation
groups. Third replicate tests for csu28-male x csu26 and csu28 x csu28 were not
completed due to a paucity of Ppa-mlt(csu28) males.
Figure 4.2. Molting deficient phenotype of Ppa-mlt mutants. Post-hatching J2-J3 molt
(A) Ppa-mlt(csu26) and (B) Ppa-mlt(csu28). Pre-hatching molt (C) Ppa-mlt(csu26) and
(D) Ppa-mlt(csu28). White arrows indicate unshed cuticle from previous stage. The
35
unshed cuticle in the mutants can be observed in the (B) posterior and (D) mid-body
regions– a phenotype not observed in the wildtype. Scale bars = 20 µm.
Although we were able to observe P. pacificus juveniles in lethargus prior to
hatching, which is indicative of molting, we were unable to directly observe the J1-J2
molt in wild-type worms. To provide additional evidence that molting is a highly
constrained process in which stereotypical gene expression and behavior recur in a
cyclical fashion regardless of its sequence relative to other processes such as hatching, we
surmised that post-hatching molting deficient mutants would also have late-stage eggs
with deficiencies in the pre-hatching molt. The majority of post-hatching molting defects
was observed in the J2-J3 molt, which is expected of mutations in genes responsible for a
general molting process (Table 1).
By subtracting the number of worms that hatched
from those in the J1 stage, we inferred that the frequency of the pre-hatching molting
deficiency is 11.43% and 16.38% for csu26 and csu28, respectively. No wild-type worms
showed pre-hatching molting defects. To assess the possibility of heterochronic changes
in our mutant lines, we tracked Ppa-mlt(csu26) and Ppa-mlt(csu28) from a synchronized
J2 stage to adulthood and saw that although the time required to reach adulthood was
longer in the csu28 line, all worms progressed through every stage and no supernumerary
stages observed.
36
J2
0 hours
Wildtype PS312
Ppa-mlt(csu26)
Ppa-mlt(csu28)
12 hours
Wildtype PS312
Ppa-mlt(csu26)
Ppa-mlt(csu28)
24 hours
Wildtype PS312
Ppa-mlt(csu26)
Ppa-mlt(csu28)
36 hours
Wildtype PS312
Ppa-mlt(csu26)
Ppa-mlt(csu28)
J3
J4
Adul
t
1.00
1.00
1.00
-
-
-
0.05
0.13
0.34
0.95
0.87
0.66
-
-
-
0.86
0.98
1.00
0.14
0.02
-
-
-
0.02
0.09
0.58
0.86
0.87
0.42
0.12
0.04
-
48 hours
Wildtype PS312
Ppa-mlt(csu26)
Ppa-mlt(csu28)
60 hours
Wildtype PS312
Ppa-mlt(csu26)
Ppa-mlt(csu28)
72 hours
Wildtype PS312
Ppa-mlt(csu26)
Ppa-mlt(csu28)
84 hours
Wildtype PS312
Ppa-mlt(csu26)
Ppa-mlt(csu28)
J2
J3
J4
Adul
t
-
0.40
0.72
0.88
0.56
0.28
0.12
0.04
-
0.25
0.64
1.00
1.00
0.11
-
-
0.58
1.00
1.00
0.42
-
-
-
1.00
1.00
1.00
Table 4.3. Time to adulthood in Ppa-mlt mutants. Development from J2 arrest showing
fraction per stage in PS312 (n=55), csu26 (n=53), and csu28 (n=45) after release from J2
arrest at 12-hour intervals.
37
Wild-type PS312
Ppa-mlt(csu26)
Ppa-mlt(csu28)
J2
0.004
0.201
0.092
J3
0.011
0.020
J4
0.006
Total
0.004
0.213
0.118
Table 4.4. Frequency of molting deficient phenotype in Ppa-mlt mutants. Stage specific
frequency of molting deficiency in PS312 (n=1061), csu26 (n=800), and csu28 (n=500).
Wild-type PS312
Ppa-mlt(csu26)
Ppa-mlt(csu28)
Viable J1
0.93
0.71
0.50
Hatched
0.93
0.60
0.33
Inferred Molt Deficient
0.00
0.11
0.17
Table 4.5. Pre-hatching molting deficient phenotype in Ppa-mlt mutants. The prehatching frequency of the molting deficient phenotype was inferred in PS312 (n=235),
csu26 (n=223), and csu28 (n=46).
38
The nematode cuticle functions as both a structural component of the hydrostatic
skeleton and a physical barrier between the worm and its environment. To indicate any
possible deleterious effects the mutations represented in Ppa-mlt(csu26) and Ppamlt(csu28) might have on cuticle function we analyzed cuticle integrity using the
membrane-permeable chromatin stain Hoescht 33342. P. pacificus PS312 wild-type,
Ppa-mlt(csu26), and Ppa-mlt(csu28) adults were stained for 12 minutes and allowed to
remove excess dye by foraging on OP50 seeded NGM plates for 1 hour. Chromatin
staining in the anterior pharyngeal region occurred first in every line and was used as a
positive control for proper staining. Significant differences were seen between P.
pacificus PS312 wild-type, Ppa-mlt(csu26), and Ppa-mlt(csu28) (Figure 3). Staining of
the mid-body region was 15.6% (n = 64; control), 35.4% (n = 65; p = 0.004), and 79.7%
(n=64; p = >0.001) respectively. Unsurprisingly, the Ppa-mlt mutant lines show
decreased cuticle function in our integrity assay when compared to PS312 wild-type.
39
Figure 4.3. Hoescht 33342 staining shows decreased cuticle integrity in Ppa-mlt mutant
lines. (A-C) Staining in the pharyngeal region in PS312 wild-type (A) Ppa-mlt(csu26)
(B) and Ppa-mlt(csu28) (C) was rapid and shows proper staining occurred. (D-F)
Representative images of increased Hoescht staining in Ppa-mlt(csu26) (E) and Ppamlt(csu28) (F) when compared to PS312 wild-type (D). White arrows indicate chromatin
staining in Ppa-mlt lines.
40
Finally, to further substantiate our observations, we looked for gene expression
pattern indicative of molting. Ppa-pnhr-1 is a previously identified homolog of
ultraspiracle (Usp), a well characterized member of the arthropod molting pathway (Yao
et al. 1993; Hannan and Hill 2001; Barchuk, Maleszka, and Simes 2004). Ppa-pnhr-1
has been shown to have cyclical peak expression prior to all three post-hatching molts,
and is the only known marker associated with the onset of molting in P. pacificus
(Parihar et al. 2010). Using quantitative real-time PCR, we measured mRNA expression
and found that Ppa-pnhr-1 level remained low in the early J1 followed by a distinct peak
prior to the J1-J2 molt (figure 3). As a negative control we also assayed the Ppa-pnhr-1
expression profile at the J2 developmental arrest and did not detect a similarly high level
of expression. The timing of the nuclear receptor USP homolog Ppa-pnhr-1 peak
expression coincides with visual marking of lethargus shortly before the first molt inside
the egg. The similarity of lethargus behavior and increased expression of a post-hatching
molting marker strongly suggests that the molt inside the egg is a bonafide molting event.
41
A.
B.
Figure 4.4 Expression of the nuclear receptor USP homolog Ppa-pnhr-1 is expressed
prior to the first molt inside the egg. (A) qPCR of Ppa-pnhr-1. Expression is low in early
J1 (16 hours) and high in late J1 (21 hours), indicative of a pending molt. The J2
developmental arrest serves as the negative control. Data represents eight total qPCR
42
reactions from two independent cDNA samples at each time-point. Each cDNA sample
was collected from RNA from four pooled synchronized populations. The relative
expression ratio of Ppa-pnhr-1 to Ppa-beta-tubulin was normalized to the no reverse
transcriptase negative control. Error bars represent standard error of the mean. **P≤0.01
(Dunn’s Test to J2 arrest). (B) RT-PCR showing no reverse-transcriptase, no template,
and genomic controls for Ppa-pnhr-1 (top) and Ppa-beta-tubulin (bottom).
43
We have taken the data gathered in our characterization of pre-hatching juvenile
development or inferred from our characterization of the Ppa-mlt mutant and combined it
with previously published data to create a unified model of pre-hatching development in
P. pacificus (Figure 4). In our observation eggs were almost exclusively laid at the 2-cell
stage, in contrast to C. elegans where eggs are typically laid sometime during
gastrulation. This difference is likely due to the rapid release of eggs post-fertilization in
P. pacificus whereas substantial stacking occurs within the uterine canal in C. elegans
delaying release. Post-egg laying embryogenesis takes ~13 hours and is terminated at
onset of J1 cuticle synthesis. The first stage juveniles (J1) reside within the egg for ~11
hours during which time all of the typical behaviors leading to molting occur. Second
stage juvenile worms (J2) reside within the egg for ~ 1/2 hour after the completion of the
J1/J2 molt at which time hatching occurs. We observed that, in general, pre-hatching
development in P. pacificus lasts for ~25 hours from egg-laying to hatching.
44
Figure 4.5. Proposed sequence of P. pacificus pre-hatching development unifying
previously published data (Felix and Hill 1999; Fürst von Lieven 2005) and observed
features. Eggs are typically laid at the 2-cell stage (A). Elongation occurs throughout
late-stage embryogenesis including 2-fold (B) and 3-fold (C) embryos.
J1 cuticle
synthesis is completed by the end of elongation and demarks the embryo/J1 division.
Typical morphological features and associated behaviors found in L1 C. elegans occur in
the pre-hatching J1 P. pacificus. Lethargus is defined by a lack of movement and
formation of a buccal cap (D,E; White arrowhead) and is followed by ecdysis (F). Black
arrowhead (F) shows previous cuticle just before ecdysis in Ppa-mlt(csu28). Worms
reach the 2nd juvenile stage just prior to hatching (G,H). White arrow (H) shows remains
of J1 cuticle. Scale bars = 25 µm.
45
Conclusion
We reveal for the first time the behavioral and gene expression events associated
with the first molt inside the eggshell of P. pacificus. Our findings show the highly
stereotypical lethargus behavior occurring in the pre-hatching molt as well as the J2-J3
molt in P. pacificus, and support the hypothesis that the pre-hatching molt is homologous
to the post-hatching molts in P. pacificus and C. elegans. The newly isolated Ppa-mlt
mutants show molting defects both before and after hatching, indicating that the mutation
represented in our screen is required for proper molting in all developmental stages.
Curiously, we were unable to observe a possible molt inside the C. elegans eggshell that
was reported to be limited to the pharyngeal cuticle (Singh and Sulston 1978).
Upregulation of Ppa-pnhr-1 in pre-hatching J1 worms further corroborates our analysis.
Interestingly, no homolog of Ppa-pnhr-1/ultraspiricle has been found in C. elegans and
the functional role of Ppa-pnhr-1 has not been determined. We summarize our results
with previously published data to offer a consensus model of pre-hatching development
in P. pacificus (Felix and Hill 1999; Fürst von Lieven 2005) (Figure 4). Our study
further validates the hypothesis that the pre-hatching molt in P. pacificus is likely due to
the result of a heterochronic shift in juvenile development and the timing of hatching
(Fürst von Lieven 2005).
The evolution of parasitism in nematodes may involve step-wise changes between freeliving, phoretic, necromenic, and parasitic lifestyles (Dillman et al. 2012) and the
occurrence of the first molt prior to hatching in diverse plant and animal parasitic
nematodes suggests multiple independent convergence events. One explanation may be
that prolonging pre-hatching juvenile development before the infective dauer stage would
provide selective advantage to parasitic nematodes. Alternatively, the co-occurrence of
mouth dimorphism and pre-hatching molt in several genera within the Diplogastridae
suggests that replacing the pharyngeal cuticle prior to hatching allows for the elaboration
of the pharynx as a feeding organ during the course of evolution (Fürst von Lieven 2005).
Since J1 larvae are viable without the eggshell, identification of earlier hatching mutants
may be a productive approach toward addressing the consequences of hatching as J1
larvae in P. pacificus.
46
It has also previously been noted that feeding rate in many arthropods is
constrained by the development of structures which grow discontinuously (Dyar 1980).
Similarly, the timing of molts has been directly related to food acquisition during the
following instar (Hutchinson et al. 1997). Nematodes show many characteristics of a
feast-or-famine life-style, e.g. early developmental arrest during famine periods and the
alternative dauer stage when presented with either food shortages or high levels of
competition for available resources. The buccal cavity represents the primary feeding
organ in nematodes and this organ has been shown to increase in size only during molts,
unlike the body which increases linearly within a developmental stage and exponentially
during juvenile development at large. This observation could indicate a selective
advantage in Diplogastrid nematodes where the first molt occurs just prior to hatching,
and therefore the onset of feeding.
Finally, we propose that the term embryonic molt be redefined as the pre-hatching
molt in Diplogastrid nematodes based on our observation that the pre-hatching molt
occurs in the J1-J2 transition just prior to hatching when embryogenesis has already been
completed. Future studies on the genetic mechanisms responsible for the identified
heterochronic changes in the timing of P. pacificus hatching may help to answer how the
presence of a pre-hatching developmental stage might drive the evolution of parasitism
and the Diplogastrid dimorphic mouth formation.
47
CHAPTER 5: SUMMARY
We begin in Chapter 1 by showing that the function of cel-nhr-23 and its relatives
as a transcriptional activator is relatively assured given the highly conserved nature of the
DNA binding domain within all species and the conserved nature of the ligand binding
domain within sub-clades. Furthermore, based on our analysis we hypothesize that the
ecdysone-activated molting pathway is the more derived feature; in arthropods the nhr-23
homologs were likely evolved to facilitate the ecdysone activated molting pathway and
holometabolous development (Lam, Jiang, and Thummel 1997; Lam et al. 1999). One
plausible explanation is that this evolutionary trajectory is responsible for the change in
gene expression of nhr-23from its requirement for all juvenile molts in nematode lineages
to its expression primarily during pupation in holometabolous arthropods.
Further investigations is necessary to evaluate the extent to which other members
of the nematode molting pathway have been co-opted into the ecdysone-iniated arthropod
molting system. Another direction which could lead to promising results would be to
investigate functional changes in the molting pathway between distantly related species.
As we menioned in the introduction, it is interesting that in tardigrades reproduction is
tied to molting as eggs are laid within the space between old and new cuticles (Nelson
2002; Suzuki 2003). Given the recent use of the tardigrade Hypsibius dujardini as a
model for studying evolution and development, including the ability for RNAi, a study
investigating possible co-option of the molting pathway for reproduction could be
feasible.
In Chapter 2 we quantitatively evaluate the role of both environmental and
maternally inherited cholesterol in the C. elegans molting pathway. Based on our
observation of gfp expression in mlt-10p::gfp-pest(GR1395) lines we hypothesize that
maternally inherited cholesterol is a necessary requirement in a natural environment,
where larval nematodes may face restrictions on dietary cholesterol intake. Additionally,
as mlt-10 and other members of the nematode molting pathway are activated by the
steroid binding proteins NHR-23 and NHR-25, we believe that cholesterol is either
modified to become a hormonal signal or used directly in activation of the molting
pathway(Meli et al. 2010).
48
Additional experimentation would support our hypothesis. Gene expression
analysis of other mlt genes known to be activitated NHR-23 and NHR-25 in traditional
and cholesterol depleted media would either support or deny the hypothesis that a
reduction in available cholesterol may likely decrease the activity of the nuclear hormone
receptors NHR-23 and NHR-25. The hypothesis that a cholesterol-derived hormone is
required for activation of the molting pathway is supported by the presence of molting
defects in let-767 mutants, a known steroid dehydrogenase. Ligand binding assays using
NHR-23 or NHR-25 with both cholesterol and previously identified cholesterol
derivatives could determine the endocrine signal necessary for proper activation of the
molting process.
Finally, in Chapter 3 we show the highly stereotypical lethargus behavior
occurring in the pre-hatching molt as well as the J2-J3 molt in P. pacificus. We further
corroborate our observation with both genetic and molecular data. Our observation of
behaviors previously associated with molting shows that the presence of a pre-hatching
developmental stage in P.pacificus is homologous to the first (L1) post-hatching
developmental stage in C. elegans. Our study also further validates the hypothesis that
the pre-hatching molt in P. pacificus is likely due to the result of a heterochronic shift in
juvenile development and the timing of hatching (Fürst von Lieven 2005).
We have raised the possibility that the pre-hatching stage found in the necromenic
nematode P. pacificus is a preadaptation to parasitism. The inherent challenge to an
investigation of this possibility is that study of P. pacificus on its beetle host is difficult as
wild P. pacificus can take up to 14 days to leave the host after its death. An alternative
hypothesis is that the presence of a pre-hatching molt has allowed the development of
dimorphic dentition in diplogastrid nematodes (Fürst von Lieven 2005). The use of
forward genetic screens and chemical mutagenesis is a long-standing tradition in
nematode models. Isolation of a P.pacificus mutant line in which the first developmental
stage occurs post-hatching would both allow the testing of the hypothesis that a prehatching molt is involved in the development of dimorphic dentition as well as shed light
on the molecular mechanisms which are responsible for the observed heterochronic
change. C. elegans hch-1 mutants have been shown to hatch ~24 hours post-fertilization,
a remarkable similarity to the timing of hatching in P. pacificus. One possibility is that a
49
change in the timing of hch-1 expression is responsible for the relative change in the
timing of hatching.
50
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54
APPENDIX A: SEQUENCE DATA FROM CHAPTER 1
Sequence
Accession
Database
Species
Length (bp)
Type
Introns
69-1824
2221-2355
2450-2492
Cel-nhr-23
WBGene00
003622
Wormbase
C. elegans
3991
Genomic
2569-2612
2738-2805
2900-2943
3500-3899
132-483
587-860
933-1251
1348-3397
3493-4180
4297-5912
6119-6475
6607-6712
6771-7123
Ppa-nhr-23
WBGene00
114295
Wormbase
P.
pacificus
16530
Genomic
7225-7434
7503-8107
8254-8509
8632-9128
9245-9956
1005410855
1095211191
1127611583
55
1-2744
2834-3942
4147-4931
Bma-nhr-23
WBGene00
222157
Wormbase
B. malayi
8862
Genomic
5163-5395
5587-8056
8328-8414
8573-8685
59-291
840-1877
Cbn-nhr-23
WBGene00
142815
Wormbase
C.
brenneri
3814
Genomic
2634-2770
2894-2942
3054-3474
1-233
Cbr-nhr-23
WBGene00
040598
Wormbase
C.
briggsae
652-937
2632
Genomic
1499-1534
2347-2632
404-532
625-680
755-3245
Cjp-nhr-23
WBGene00
138394
Wormbase
C.
japonica
6315
Genomic
3367-3638
3690-4675
4937-5476
6159-6224
56
392-1365
1459-2379
2454-4310
Cre-nhr-23
WBGene00
069903
Wormbase
C.
remeneri
8206
Genomic
3532-4253
4346-5182
5837-6705
7000-8115
1-818
157623488
2372723989
2416024330
HR46/DHR3
FBgn00004
48
D.
Flybase
melanogas
2449925800
Genomic
ter
24742
2489424952
2519725255
2540925463
2559225800
Human DNA
AL137018.
NCBI
H. sapiens
57
4980
Genomic
1-452
sequence from
9
543-4799
clone RP11-
4946-4980
133M9 on
chromosome
9q13-21.33,
complete
sequence
143420186755
186906191617
Zebrafish DNA
191767-
sequence from
207380
clone DKEY56E5 in linkage
group 12,
BX890609.
7
NCBI
D. rerio
74958
Genomic
207546211098
complete
211277-
sequence
213327
213534216512
216697218378
Drosophila
sechellia
GM20545
(Dsec\GM20545
XM_00203
3172.1
D.
NCBI
sechellia
2792
mRNA
N/A
2072
mRNA
N/A
), mRNA
Drosophila
XM_00208
NCBI
D.
58
simulans
0840.1
simulans
GD26001
(Dsim\GD26001
), mRNA
Drosophila
yakuba GE21710 XM_00208
(Dyak\GE21710)
9877.1
NCBI
D. yakuba
1524
mRNA
N/A
2009
mRNA
N/A
263
mRNA
N/A
, mRNA
PREDICTED:
Musca domestica
probable nuclear
hormone
XM_00517
receptor HR3-
7878.1
M.
NCBI
domestica
like
(LOC101901236
), mRNA
Culex
quinquefasciatus
retinoic acid
receptor beta,
XM_00186
9307.1
C.
NCBI
quinquefa
sciatus
mRNA
Aedes aegypti
nuclear receptor
AF230281.
3 (HR3) mRNA,
1
NCBI
A. aegypti
1545
mRNA
N/A
NCBI
T. spiralis
1506
mRNA
N/A
complete cds
Trichinella
spiralis nuclear
XM_00337
1268.1
hormone
59
receptor family
member nhr-23
(Tsp_09528)
mRNA,
complete cds
Galleria
mellonella
ecdysteroidinducible
U02621.1
G.
NCBI
mellonella
3413
mRNA
N/A
(GHR3) mRNA,
complete cds
Loa loa steroid
hormone
receptor
XM_00313
(LOAG_02577)
8114.1
NCBI
L. loa
1266
mRNA
N/A
NCBI
D. magna
2055
mRNA
N/A
mRNA,
complete cds
Daphnia magna
HR3 nuclear
receptor mRNA,
FJ755466.1
complete cds
60
APPENDIX B: DISTANCE MATRICIES FROM CHAPTER 1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
1 CelegansCDS
2 CremeneriCDS
0.749
3 CjaponicaCDS
0.709 0.787
4 CBRENNERICDS
22.799 29.286 26.245
5 CbriggsaeCDS
0.906 0.641 0.876 32.071
6 BmalaiCDS
1.668 1.761 1.756 25.011 1.625
7 LLoaMRNA
1.678 1.752 1.810 20.530 1.611 0.060
8 PpacificusCDS
7.812 9.073 8.509 48.042 8.906 5.331 5.183
9 TSpiralisMRNA
3.939 4.432 4.466 31.227 4.305 3.029 2.836 7.450
10 DmelCDS
5.574 6.180 5.933 37.164 5.196 4.055 4.124 9.474 5.219
11 DSECHELLIAMRNA
7.382 8.241 7.667 38.728 6.808 5.575 5.703 15.732 7.147 0.075
12 DYAKUBAMRNA
66.575 -1.000 31.503 -1.000 19.340 40.967 -1.000 -1.000 -1.000 0.555 0.146
13 MDOMESTICAMRNA
4.247 5.243 5.060 22.130 4.000 3.946 3.976 10.860 5.149 0.120 0.124 45.204
14 AAEGYPTIMRNA
4.411 5.115 5.102 32.530 4.277 3.997 4.174 14.373 4.889 0.176 0.188 25.918 0.370
15 DSIMULANSMRNA
2.899 2.641 2.835 21.161 2.752 1.102 1.149 4.766 2.037 0.155 0.023 0.162 0.258 0.187
16 GMELLONELLAMRNA
6.240 7.263 7.292 36.119 6.342 4.393 4.273 13.822 5.926 1.497 1.978 64.774 0.879 1.268 2.954
17 DAPHNIA_MAGNAMRNA
6.102 6.290 6.134 20.684 5.676 4.707 4.621 9.686 5.158 1.519 1.643 -1.000 0.929 0.929 1.966 1.490
18 HumanRARACDS
6.797 7.882 7.367 41.011 6.657 5.252 5.320 8.594 5.764 2.956 3.533 42.840 2.956 3.048 1.480 2.764 2.611
19 DrerioRARACDS
10.633 9.794 10.313 33.241 8.161 6.631 6.397 8.528 10.522 7.488 11.296 -1.000 6.924 6.551 5.768 8.365 7.743 5.631
20 CQUINQUEFASCIATUSMRNA 0.180 0.197 0.197 3.494 0.197 0.236 0.256 0.705 0.147 0.028 0.042 25.439 0.058 0.044 0.044 0.094 0.121 0.405 1.072
Dayoff Model based pairwise distances
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
1 CelegansCDS
2 CremeneriCDS
0.764
3 CjaponicaCDS
0.713 0.791
4 CBRENNERICDS
18.370 25.174 22.562
5 CbriggsaeCDS
0.883 0.626 0.870 27.995
6 BmalaiCDS
1.653 1.685 1.658 18.992 1.583
7 LLoaMRNA
1.658 1.665 1.710 16.298 1.563 0.061
8 PpacificusCDS
7.509 8.706 8.043 40.321 8.526 4.938 4.799
9 TSpiralisMRNA
3.784 4.070 4.255 24.328 4.062 2.894 2.715 6.988
10 DmelCDS
5.603 6.012 6.007 34.785 5.304 3.803 3.859 9.205 5.037
11 DSECHELLIAMRNA
7.440 7.945 7.718 36.500 6.964 5.184 5.287 15.024 6.879 0.078
12 DYAKUBAMRNA
61.426 63.206 32.128 -1.000 20.773 34.254 -1.000 -1.000 -1.000 0.568 0.142
13 MDOMESTICAMRNA
4.264 5.192 5.021 20.169 3.970 3.657 3.676 10.537 4.853 0.122 0.127 24.715
14 AAEGYPTIMRNA
4.303 5.036 4.966 31.521 4.161 3.766 3.938 13.689 4.801 0.180 0.195 21.336 0.377
15 DSIMULANSMRNA
2.907 2.539 2.846 19.977 2.787 1.060 1.140 4.582 1.975 0.162 0.025 0.159 0.263 0.191
16 GMELLONELLAMRNA
5.966 6.922 7.118 34.657 6.245 4.131 4.031 13.314 5.883 1.494 1.962 52.775 0.862 1.213 2.910
17 DAPHNIA_MAGNAMRNA
6.030 6.240 6.250 18.563 5.752 4.438 4.363 9.193 4.921 1.510 1.634 -1.000 0.922 0.893 1.933 1.448
18 HumanRARACDS
6.524 7.441 6.963 35.011 6.456 5.076 5.054 8.067 5.609 2.957 3.567 36.117 2.939 3.038 1.507 2.692 2.545
19 DrerioRARACDS
9.907 9.361 9.656 29.818 7.812 6.432 6.159 8.001 10.000 6.809 10.036 -1.000 6.364 5.990 5.177 7.701 7.344 5.306
20 CQUINQUEFASCIATUSMRNA 0.170 0.186 0.186 2.878 0.186 0.217 0.236 0.704 0.142 0.027 0.041 20.612 0.056 0.043 0.042 0.088 0.109 0.373 1.049
Jones-Taylor-Thorton Model based pairwise distances
61
1
2
3
4
5
6
7
8
9
1 CelegansCDS
2 CremeneriCDS
0.599
3 CjaponicaCDS
0.594 0.632
4 CBRENNERICDS
8.246 9.053 8.690
5 CbriggsaeCDS
0.686 0.483 0.682 9.593
6 BmalaiCDS
1.206 1.181 1.179 7.563 1.153
7 LLoaMRNA
1.172 1.173 1.183 6.491 1.121 0.063
8 PpacificusCDS
3.830 4.270 3.936 11.531 4.193 2.724 2.674
9 TSpiralisMRNA
2.188 2.406 2.443 8.000 2.408 1.911 1.795 3.763
10 DmelCDS
3.214 3.446 3.310 10.034 3.185 2.324 2.394 4.649 3.010
11 DSECHELLIAMRNA
4.043 4.330 3.978 10.778 3.989 3.078 3.189 6.436 3.987
12 DYAKUBAMRNA
12.923 12.083 8.471 14.824 8.250 9.333 17.500 13.111 21.500
13 MDOMESTICAMRNA
2.464 2.942 2.835 8.000 2.495 2.282 2.317 4.946 2.802
14 AAEGYPTIMRNA
2.510 2.940 2.828 9.509 2.510 2.301 2.439 6.143 2.812
15 DSIMULANSMRNA
1.917 1.748 1.809 8.244 1.890 0.809 0.856 2.909 1.433
16 GMELLONELLAMRNA
3.336 3.838 4.082 10.500 3.417 2.416 2.420 6.085 3.077
17 DAPHNIA_MAGNAMRNA
3.273 3.264 3.361 6.947 3.031 2.615 2.683 4.936 2.814
18 HumanRARACDS
3.345 3.563 3.524 11.765 3.306 2.883 2.870 4.157 3.128
19 DrerioRARACDS
4.567 4.639 4.721 9.872 4.194 4.000 4.017 4.677 5.000
20 CQUINQUEFASCIATUSMRNA 0.164 0.182 0.182 1.760 0.182 0.200 0.219 0.560 0.130
10
11
12
13
14
15
0.075
0.487
0.121
0.168
0.151
1.097
1.097
1.915
3.905
0.026
0.143
0.126
0.183
0.026
1.370
1.193
2.269
4.810
0.041
10.500
6.286
0.158
11.276
18.333
9.333
14.667
6.500
0.338
0.246
0.671
0.709
1.953
3.507
0.054
0.164
0.825
0.690
1.977
3.351
0.040
1.839
1.326
1.060
3.047
0.041
16
17
18
19
1.122
1.732 1.752
3.934 3.750 3.176
0.083 0.099 0.345 0.733
Poisson model based pairwise distances
1 CelegansCDS
2 CremeneriCDS
3 CjaponicaCDS
4 CBRENNERICDS
5 CbriggsaeCDS
6 BmalaiCDS
7 LLoaMRNA
8 PpacificusCDS
9 TSpiralisMRNA
10 DmelCDS
11 DSECHELLIAMRNA
12 DYAKUBAMRNA
13 MDOMESTICAMRNA
14 AAEGYPTIMRNA
15 DSIMULANSMRNA
16 GMELLONELLAMRNA
17 DAPHNIA_MAGNAMRNA
18 HumanRARACDS
19 DrerioRARACDS
20 CQUINQUEFASCIATUSMRNA
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
0.375
0.373
0.892
0.407
0.547
0.540
0.793
0.686
0.763
0.802
0.928
0.711
0.715
0.657
0.769
0.766
0.770
0.820
0.141
0.387
0.901
0.326
0.542
0.540
0.810
0.706
0.775
0.812
0.924
0.746
0.746
0.636
0.793
0.765
0.781
0.823
0.154
0.897
0.405
0.541
0.542
0.797
0.710
0.768
0.799
0.894
0.739
0.739
0.644
0.803
0.771
0.779
0.825
0.154
0.906
0.883
0.866
0.920
0.889
0.909
0.915
0.937
0.889
0.905
0.892
0.913
0.874
0.922
0.908
0.638
0.536
0.528
0.807
0.707
0.761
0.800
0.892
0.714
0.715
0.654
0.774
0.752
0.768
0.807
0.154
0.060
0.731
0.656
0.699
0.755
0.903
0.695
0.697
0.447
0.707
0.723
0.742
0.800
0.167
0.728
0.642
0.705
0.761
0.946
0.699
0.709
0.461
0.708
0.728
0.742
0.801
0.179
0.790
0.823
0.866
0.929
0.832
0.860
0.744
0.859
0.832
0.806
0.824
0.359
0.751
0.799
0.956
0.737
0.738
0.589
0.755
0.738
0.758
0.833
0.115
0.070
0.327
0.108
0.143
0.131
0.523
0.523
0.657
0.796
0.026
0.125
0.112
0.155
0.025
0.578
0.544
0.694
0.828
0.039
0.913
0.863
0.136
0.919
0.948
0.903
0.936
0.867
0.253
0.197
0.402
0.415
0.661
0.778
0.051
0.141
0.452
0.408
0.664
0.770
0.038
0.648
0.570
0.514
0.753
0.039
P-value based pairwise distances
62
16
17
18
19
0.529
0.634 0.637
0.797 0.789 0.761
0.077 0.090 0.256 0.423
APPENDIX C: ASSOCIATED VIDEOS FROM CHAPTER 4
Ppa-mlt(csu28) mutant molting phenotype
Z-stack DIC video of the Ppa-mlt(csu28) molting deficiency in the J1 pre-hatching stage.
P. pacificus active behavior
Video of J1 P. pacificus PS312 wild-type during active behavior. Time-lapse video
compiled from images taken over a 1.5 hour timeframe ~6 hours prior to hatching
P. pacificus lethargus behavior
Video of J1 P. pacificus PS312 wild-type during lethargus behavior. Time-lapse video
compiled from images taken over a 1.5 hour timeframe ~3 hours prior to hatching
C. elegans active behavior
Video of C. elegans N2 wild-type embryo during active behavior. Time-lapse video
compiled from images taken over a ~1 hour timeframe
63