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Investigating the mechanisms of threshold weight in the tobacco
hornworm, Manduca sexta
A Thesis by Zhou Wang
Faculty Advisor: Yuichiro Suzuki
Submitted in Partial Fulfillment
of the
Prerequisite for Honors
in Biological Sciences, Wellesley College
May 2016
© 2016 Zhou Wang and Yuichiro Suzuki
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Acknowledgements
This thesis is dedicated to my grandmother and my parents, who have given me everything.
I want to thank the many people who made this thesis possible and encouraged me in this
incredible process. My thesis advisor, Yui Suzuki, not only provided his brilliant mind as a
scientist and professor, but also provided me with the inspiration and courage to be the best
scientist I can be. Thank you for everything you have done for me and all my words in this
short section cannot begin to describe how grateful I am to have you as a mentor. Thank you for
believing in me when I didn’t believe in myself.
I want to thank my thesis committee: Kimberly O’Donnell, Melissa Beers, and Sarah Wall-
Randell. I am grateful for your insightful feedback and comments on this project. Thank you for
keeping me on track and your wise words!
I would also like to thank the entire Suzuki lab for supporting my efforts in this thesis, whether
they provided words of encouragement or helped me with my lab experiments. This was such a
wonderful and supportive research community to have. In particular, I want to thank Victoria
Wang for showing me the YAS cat, Prioty Sarwar for providing me with her realness, and Mia
Accomando who was always willing to get coffee with me.
To my family at Wellesley: Beba Cibralic, Eloisa Cleveland, Houda Khaled, Anat Mano, Natalie
Oppenheimer, Shweta Patwardhan, Jordan Sessa, Rose Trilesskaya, and Helen Walsh. Thank
you for hearing me out and selflessly giving me your friendship when I needed it. Particularly, I
want to thank Rose and Houda for supporting me during some last minute technology troubles!
To my family back home, for their constant source of love and support in more ways than I can
imagine. I wouldn’t be at Wellesley without you guys, and I am so grateful for your belief in my
success.
Last but not least, I would like to thank the Department of Biological Sciences at Wellesley
College for its support in funding this project. Thanks for making this possible!
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Table of Contents
Abstract ....................................................................................................................................................... 5
Introduction ............................................................................................................................................... 6
Insects and their development cycles ...........................................................................................6
Holometabolous insects and their growth ....................................................................................7
Endocrine processes that regulate holometabolous insect development ......................................7
Prothoracicotropic hormone (PTTH) .............................................................................................. 8
Juvenile hormone (JH) ....................................................................................................................... 8
Ecdysone ............................................................................................................................................. 9
Mechanisms of body size regulation ..........................................................................................10
Mechanisms regulating the decision to stop growth (critical weight)........................................11
Potential physical cues for sensing size .....................................................................................12
Mechanisms regulating growth rate ...........................................................................................12
Terminal growth period ..............................................................................................................12
Threshold weight ........................................................................................................................13
Genes known to influence instar number ...................................................................................15
Krüppel homolog 1 (kr-h1) ............................................................................................................. 15
Methoprene tolerant (Met) ............................................................................................................. 15
Ventral veins lacking (vvl) .............................................................................................................. 16
Investigating the characteristics of threshold weight ................................................................17
Materials and Methods .......................................................................................................................... 19
Animal rearing ............................................................................................................................19
Hypoxia treatment ......................................................................................................................19
RNA isolation .............................................................................................................................20
c-DNA synthesis ........................................................................................................................21
Quantitative RT-PCR .................................................................................................................21
Results ....................................................................................................................................................... 23
Hypoxia generates supernumerary larvae ..................................................................................23
Threshold weight and timing of decision to undergo a supernumerary molt .............................25
Threshold weight determination in JH-deficient black mutants ................................................28
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Duration of instar correlates with the decision to enter the supernumerary stage .....................30
Manduca deficient in JH do not enter the supernumerary stage as often ...................................32
qPCR analysis of candidate genes correlated with threshold weight .........................................34
Discussion ................................................................................................................................................ 40
Hypoxia on threshold weight .....................................................................................................40
Timing of threshold weight decision ..........................................................................................41
The role of JH on threshold weight ............................................................................................42
Molecular correlates of the threshold weight .............................................................................43
Potential model for the specification of molt identity ................................................................44
References................................................................................................................................................. 47
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ABSTRACT
How organisms grow to the correct adult size is one of the most puzzling and
interesting questions in developmental biology, yet one that is poorly understood. Two
main parameters determine the final adult size: the growth rate and the duration of
growth. In holometabolous insects, such as Manduca sexta, an organism widely used to
study insect physiology, the size attained at the final instar is the main determinant of
adult size after metamorphosis. A size threshold called the “threshold size” was first
identified when starvation during the penultimate instar led to a “supernumerary”
instar stage before entering the final instar. Despite its importance in lengthening the
duration of growth and hence the final adult size in Manduca, the mechanisms that
govern the developmental fate at the threshold size is not well understood. Here, I
show that hypoxia during the third instar can successfully generate supernumerary and
final instar larvae. In Manduca deficient in JH, however, few larvae entered the
supernumerary stage and had a prolonged fourth instar duration. This finding suggests
a potential role for JH titers in stimulating PTTH/ecdysteroid production in wildtype
Manduca to facilitate entry into the supernumerary stage. The expression levels of the
ventral veins lacking (vvl), krüppel homolog 1 (kr-h1) and Methoprene tolerant (Met) in the
brain were compared between animals under and above the threshold size. Met
expression did not differ, although kr-h1 expression was higher in supernumerary
animals. Higher kr-h1 expression may suggest more active JH signaling, which might be
associated with the development of a supernumerary instar. Vvl expression was also
elevated in larvae undergoing a supernumerary molt. A model is proposed to explain
how a supernumerary larva might arise.
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INTRODUCTION
One of the fundamental questions of developmental biology is how organisms control
their growth to the right size. Body size is one of the key distinguishing features of a species, yet
very little is known about the mechanisms that regulate body size (Nijhout 2014). While size
variation is most obvious between species, variation in body size between individuals has
implications on life history traits; body size has been correlated with fecundity, survival, and
mate selection. For example, a strong positive correlation exists between adult female mass and
number of eggs or larvae reproduced (Honek 1993, Berger et al. 2008). In addition to increasing
reproductive capacity, the relationship between larger insects and longevity has also been shown
across several taxa (Calabi and Porter, 1989; Taylor et al. 1998).
Insects and their developmental cycles
Insects can be categorized into three groups based on how their morphology and
behaviors change during development. A small number of insect orders belong to the Ametabola,
which do not undergo metamorphosis. In this group, insects undergo three life history stages, the
egg, nymphs, and adult. The juvenile morphology does not differ from the adult body structure
other than size and sexual maturation of genitalia. Insects that undergo ametabolous development
are found in primitive insect orders, such as Archeognatha and Collembola, and are wingless.
Hemimetabola refers to insects that go through “incomplete metamorphosis” that differs from
ametabolous insects because hemimetabolous insects usually develop wings postembryonically,
and the nymph is the “juvenile” stage before the final adult. A range of insects are categorized in
this type of development, and include orders, such as Odonata (damselflies), Orthoptera
(grasshoppers), Hemiptera (stink bugs), Homoptera (aphids), and Mantodea (praying mantis).
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The adult form typically resembles that of the nymphal stages with the exception of wing
structures. Holometabolous insects differ greatly from the other types of insects because they
undergo complete metamorphosis and have four stages of development: egg, larva, pupa, and
adult. Holometabolous insects are distinct from the other groups because the adult form differs
completely morphologically, behaviorally, and physiologically from the previous developmental
stage. The larval stages exhibit tremendous amount of growth for the taxing pupal stage later in
development. Holometabolous insects include orders Lepidoptera (butterflies and moths),
Diptera (flies), Hymenoptera (bees and ants), and Coleoptera (beetles).
Holometabolous insects and their growth
Many studies on regulation of body size have been conducted on holometabolous insects.
Larval growth stages determine the final adult size because the pupa does not feed and adult
insects are constrained by a hard exoskeleton after metamorphosis (Nijhout 2003); therefore,
growth occurs exclusively during the larval stages. After reaching the end of feeding, growth is
stopped. Much of the physiological aspects of body size variation have been studied in Manduca
sexta, which typically has five larval instars in standard laboratory conditions (Kingsolver 2007).
Larval growth in M. sexta is exponential; up to 90% of body mass is gained during the final
instar (D’Amico et al., 2001).
Endocrine processes that regulate holometabolous insect development
In holometabolous insects, ecdysteroids and juvenile hormone regulate numerous aspects of
physiology, such as the transition of life history stages, reproduction, and development
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(Riddiford 1996, Nijhout 1994, Wyatt and Davey, 1996). These two classes of hormones can
interact to alter developmental timing and ultimately final body size.
PTTH
Molting is a crucial process that arthropods regularly go through in order to
accommodate body growth. The production of ecdysteroids initiates the molting process, which
is in turn regulated by prothoracicotropic hormone, or PTTH. PTTH was first found in the brains
of the silk moth Bombyx mori; its presence induced ecdysone biosynthesis in the prothoracic
gland (Kataoka et. al, 1991). PTTH is secreted from the brain and is received by the prothoracic
gland, which in turn initiates the production of ecdysone; therefore, the timing of PTTH secretion
affects when ecdysone is produced. During the larval stages of Manduca and Drosophila, pulses
of PTTH are produced at the end of each instar which in turn stimulates the production of
ecdysone, allowing the larvae to molt to the next instar (Henrich et al., 1999). In Drosophila,
PTTH is not necessary for molting or metamorphosis; however, the ablation of PTTH producing
cells results in delayed metamorphosis and larger body size through lengthening the feeding
period (McBrayer et al., 2007).
Juvenile hormone
Juvenile hormones are a class of acyclic sesquiterpenoids that is important in the
developmental regulation of insects. They are often nicknamed the “status quo hormone”,
because ectopic application of JH mimics suppresses metamorphosis in the final instar and
causes the larva to recapitulate the current life history stage when it molts again. The discovery
of JH as the status quo hormone came from parabiosis experiments of Rhodnius prolixus , or the
kissing bug (Wigglesworth 1964). The role of JH and metamorphic timing is interesting because
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of the change in the interaction between JH and PTTH at the end of the penultimate instar stage.
In earlier instars, the production of PTTH is not inhibited by JH, which allows larvae to molt in
the presence of high JH titers. In the final instar, a switch in JH sensitivity occurs such that the
brain can no longer release PTTH in the presence of JH. Consequently, the production of
ecdysone and the onset of metamorphosis is delayed when ectopic JH is applied (figure 1).
Ecdysone
Production of ecdysteroids is needed to transition an organism from one molt to the next,
as the removal of ecdysteroids results in disrupted molting and metamorphosis (figure 1).
However, levels of JH determine the nature of the molt and whether the organism progresses
from its previous life history stage. At high levels of JH, production of ecdysteroids allows the
organism to molt into another larval instar; however, ecdysteroids in the absence of JH allow the
organism to transition from the larval instar to a pupa.
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Figure 1. Hormone interactions and metamorphosis. Juvenile hormone (JH, purple) is
maintained at a high level throughout the larval stages. Prothoracicotropic hormone (PTTH,
orange) is produced to stimulate ecdysone production, which then allows the instar to molt into
the next instar. After the final instar has reached the critical weight, JH levels begin to decline.
This decline in JH allows PTTH to be produced and the final peak of ecdysone (blue) to initiate
pupation. Figure adapted from Truman and Riddiford (1999).
Mechanism of body size regulation
Mechanisms controlling body size must somehow assess size upstream of growth
mechanisms (Nijhout 2014), and the rate of growth and cessation of growth are the two key
aspects that determine size. Environmental and genetic conditions can influence molecular
mechanisms that regulate growth; many studies have shown how temperature, nutrition, and
competition affects body size (Conlon and Raff, 1999; Atkinson 1994; Chapman 1998). Genetic
variation can also result in intraspecific size variation (Davidowitz et al. 2003). Insects provide a
tractable model for studying the regulation of size as the majority of size physiology studies have
been conducted in Drosophila melanogaster and Manduca sexta (Chown and Gaston, 2010;
Nijhout 2014).
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Although the mechanism underlying life history transitions in each taxa may be different,
the parameters that regulate these changes in developmental stages are similar. Various life
history parameters regulate body size and growth, which include a) the decision to stop growth,
b) the period between the decision to stop growth and when the body actually stops growing,
otherwise known as the terminal growth period (TCG), c) the time point when growth stops, and
d) the growth rate (Davidowitz and Helm, 2015). The first three deal with the duration of growth,
which requires knowledge of how organisms regulate the termination of growth. The growth rate
affects, but cannot alone explain for the attainment of final body size; slow growth rates can
achieve a bigger body size by elongating the growth period while fast growing animals can reach
a smaller size by shortening this period.
Mechanisms regulating the decision to stop growth (Critical weight)
The decision point to stop growth occurs first, which can be triggered endogenously or by
environmental factors. For many insects, the decision point is called the critical weight, the
weight at which starvation no longer delays metamorphosis (Nijhout and Williams, 1974a). The
attainment of this size threshold leads to a decline in juvenile hormone titers, which allows for
PTTH secretion and subsequent ecdysone synthesis needed to metamorphose. Although the
mechanism that regulates critical weight has not been elucidated, factors, such as nutrition and
genetic variation, have been found to shift the critical weight (Davidowitz et al., 2003).
The critical weight of Drosophila melanogaster is regulated differently. In this species,
JH plays minimal roles and the attainment of the critical weight is marked by a pulse of
ecdysteroid production (Koyama et al., 2014). This pulse of ecdysteroid is driven by nutrient-
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based insulin/TOR signaling, which influences prothoracic gland size production (Mirth et al.
2005).
Potential physical cues for sensing size
Previous research has also suggested that a physical constraint can assist in sensing size;
for example, stretch receptors in Oncopeltus fasciatus sense size and can be artificially activated
using saline or air injections, resulting in precocious molting (Nijhout 1979). Additionally,
previous research has shown that hypoxic conditions cause smaller molt sizes (Callier and
Nijhout, 2011) and suggested that oxygen levels might act as a size sensing mechanism. They
proposed that because the tracheal system cannot grow during a larval instar, as the larva grows,
the concentration of oxygen delivered would decrease.
Mechanisms regulating growth rate
Food limitation and temperature, along with oxygen deprivation, can suppress the growth
rate (Blanckenthorn 1999, Atkinson 1994, Davidowitz 2004, Callier and Nijhout 2011) and
affect the final body size. However, by lengthening the duration of growth, organisms can reach
a bigger final body size despite being reared in lower temperatures (Atkinson 1994).
The Terminal Growth Period
Once the decision to stop somatic growth has been reached, the actual timing of when
somatic growth stops does not occur immediately. The delay period between the decision to stop
growing and the point of cessation of growth is called the terminal growth period or the interval
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of cessation of growth (ICG), and lengthening or shortening this period can alter final body size.
The critical weight marks the onset of the clearing of juvenile hormone in M. sexta, and complete
clearance of JH in the final instar allows for the production of PTTH, and subsequent release of
ecdysteroids, which marks the end of the ICG. Factors, such as the rate of JH clearance, PTTH
photogate period, and temperature, affect the duration of the ICG. The rate of JH clearance is
dependent on levels of JH degradation enzymes, such as Juvenile hormone esterase (JHE). Once
JH is cleared, PTTH secretion is dependent on an eight hour photoperiod (Truman 1972, Truman
and Riddiford 1974); if JH clearance has not occurred during this 8 hour period, then PTTH is
not released until the next time window, lengthening the ICG and therefore increasing body size.
Increases in temperature increase the growth rate, but also increase the rate of the breakdown of
JH, resulting in a shorter ICG and smaller overall body size (Davidowitz et al., 2004; Davidowitz
et al., 2005). Once ecdysteroids are produced in Manduca, larvae stop feeding, gut purge, and
enter the wandering stage, characterized by a search for a pupation site (Chown and Gaston,
2010). During this period, the final size of the larvae is reached, and is a reliable predictor for
final body size as the pupal stage does not feed.
Threshold Weight
All of the above are events that occur in the final instar. However, there is in fact another
size assessment point, the threshold weight, which takes place during the earlier instar. The
threshold weight must be attained in order for a larva to enter the final larval instar; when a
penultimate instar larva fails to attain the threshold weight, it undergoes a supernumerary molt
and can subsequently molt into a very large final instar.
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By starving larva at various sizes of the penultimate instar, a range of fifth instar sizes
can be generated (Nijhout 1975) based on head capsule size, which serves as a proxy for body
size. At the final instar, larvae with a head capsule size below 5.1mm molts to a supernumerary
instar, while those above that size proceed to the pupal stage. This experiment set the concept of
a “threshold size”: in order to progress from one developmental stage to the other, a decision
must be made to enter the final instar, regulated by a size limit (figure 2). Body weight can also
serve as a proxy for determining the threshold weight (Grunert et al., 2015). However, how does
the organism actually assess its size to make the switch from one developmental pathway to the
other? Because of the increased time to feed and grow, the threshold weight has profound effects
on the final body size, yet little is known about how it is sensed.
Figure 2. Threshold weight is an absolute weight minimum size needed to progress into the
final instar. Nijhout (1975) first identified the threshold weight by starving penultimate instar
larvae and found larvae under a weight molted into an additional “supernumerary” instar. Those
above the weight minimum progressed to the final instar and pupated. However, genes involved
in the nature of the subsequent molt are unknown.
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Genes known to influence instar number
To date, only a handful of genes have been shown to alter the instar when the larva
initiates metamorphosis: Here, I focus on JH signaling and ventral veins lacking (vvl).
krüppel homolog 1 (kr-h1)
The transcription factor Kr-h1 has been shown to mediate JH signaling in Drosophila and
Tribolium castaneum (Minakuchi et al. 2007, Minakuchi et al. 2009). The expression of kr-h1 is
maintained during embryonic and larval stages, but begins to decline during the pre-pupal stage
and disappears in the adult stage, appearing briefly during reproduction (Minakuchi et al, 2007).
Knockdown of JH acid O-methyltransferase 3 (JHAMT3) in Tribolium results in decreased
expression of kr-h1, suggesting that the kr-h1 transcript level is dependent on JH biosynthesis
levels since TcJHAMT is the rate limiting enzyme for JH production (Minakuchi et al., 2007).
An important piece of evidence for Kr-h1 as a transcription factor of JH production came from
knockdown experiments of kr-h1, which resulted in precocious metamorphosis; furthermore,
addition of exogenous JH in combination with jhamt3 knockdown restored kr-h1expression and
prevented precocious metamorphosis (Minakuchi et al., 2009). This result suggested the role of
kr-h1 as a mediator of JH signaling.
Methoprene-tolerant (Met)
The gene Met encodes a transcription factor that has been thought to act as the receptor
for JH; a single ortholog of Met knocked down in Tribolium results in precocious metamorphosis
(Konopova and Jindra, 2007), which suggests that Met is involved in sensing JH levels to
prevent early metamorphosis until the organism has reached the correct size. In addition to the
results of Met knockdown leading to the expected phenotype of early metamorphosis, Met has
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been found to have high affinity binding to JH (Shemshedini and Wilson 1990, Miura et al.,
2005).
Ventral Veins Lacking (vvl)
Vvl is a POU transcription factor that has been known to regulate timing of
metamorphosis in beetles. Vvl is expressed in several tissues, including the CNS in Tribolium.
Knockdown experiments of vvl in the penultimate instar of Tribolium resulted in precocious
metamorphosis without molting and failure to complete molt. Precocious metamorphosis as a
result of vvl knockdown suggests that JH signaling no longer maintains the juvenile stage when
vvl is absent. Comparing knockdowns of Met and vvl, Cheng et al. (2014) found no significant
difference between the two experimental treatments in rates of precocious metamorphosis, but in
both cases, larvae metamorphosed earlier than the controls. A difference between the Met and vvl
knockdown animals was that Met knockdown animals were able to complete a molt before
metamorphosis, while vvl knockdowns entered precocious metamorphosis without molting.
Furthermore, vvl knockdown of fifth instar larvae resulted in reduced JHAMT3 expression,
suggesting that vvl plays a role in regulating JH biosynthesis instead of JH sensitivity. POU
factors have also been shown to impact the timing of puberty in mammals. Thus, POU factors
may play critical roles in regulating the onset of metamorphic transitions in a variety of
organisms.
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Figure 3. Genes involved in regulating the timing of metamorphosis. Vvl knockdown has
been shown to induce precocious metamorphosis, mediated via reduction of production of JH
and JH response gene kr-h1. Vvl knockdown also led to reduced ecdysteroid titers, suggesting its
role in ecdysone biosynthesis. Figure adapted from Cheng et al. (2014).
Investigating characteristics of the threshold weight
The objective of this study was to investigate characteristics of the threshold weight and
how it is regulated. Although previous experiments have shown starvation to be a successful
method in determining threshold weight, could nutrient independent methods generate larvae
below and above the threshold weight? Since a sensitivity switch in JH levels during the final
instar determines the nature of its molt, how does JH impact threshold weight? How would the
threshold weight differ in a black mutant strain deficient in JH levels? Finally, how are known
genes involved in metamorphic timing such as Met, kr-h1, and vvl expressed below and above
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threshold weight? Investigating changes in expression levels of these candidate genes could
provide clues into the regulatory mechanism of threshold weight and body size.
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MATERIALS AND METHODS
Animal rearing
Wildtype Manduca sexta were obtained from Carolina Biological Supply Company
(www.carolina.com). black mutant Manduca were obtained from Dr. H. Frederik Nijhout (
Department of Biological Sciences, Duke University, Durham, North Carolina). All larvae were
raised on an artificial diet (Kemirembe et al., 2012) unless noted otherwise. Each organism was
raised at 26.5ºCin a 1 oz. soufflé cup until the end of the 4th instar and a 5 oz. soufflé cup at the
beginning of the fifth instar.
Hypoxia treatment
On day 0 of the third instar, larvae entering head capsule slippage (HCS) - a sign of
initiation of molting - were placed in a sealed cell culture chamber in cups with tops that had
multiple holes. A 5% oxygen/carbon dioxide mixture was delivered into the oxygen chamber.
The oxygen concentration of the chamber was kept around 4+/-1% throughout the experiment.
Larvae were kept in hypoxic conditions until the end of the third instar when the head capsule
began to slip. Once taken out of the chamber, organisms were weighed, and new artificial diet
was given to each organism. The tops of the cups were replaced with ones that had just one or
two holes. Weight was recorded every day, and the duration of the instar was also determined.
The fate of the larvae, whether they entered the final instar or the supernumerary instar, was
determined by tracking the fate of the larvae that emerged after the molt at the end of the fourth
instar: those that gut purged were determined to have molted into a fifth (and final) instar; those
that initiated head capsule slippage again were determined to have molted into a supernumerary
fourth instar.
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RNA isolation
The brain and the central nervous system (CNS) were dissected from wild type hypoxia-
treated larvae at the end of the fourth instar and on day zero after molting into either
supernumerary or final instar larvae. Two classes of larvae were identified: those that were
entering the supernumerary instar and those entering the final (fifth) instar. Tissues were
homogenized using 500 µL trizol reagent to isolate RNA. One hundred µl of chloroform was
added to the tissues and centrifuged at 11,500 rpm at 4 ºC for 15 min to separate RNA from the
rest of the organic matter. The top aqueous layer was then transferred in a separate RNAse-free
tube and suspended in 250 µL isopropanol to precipitate RNA. Centrifugation at 11,500 rpm at 4
ºC for 10 min allowed for RNA to form a pellet, and the supernatant was discarded. The pellet
was washed with 500 µL 80% ethanol/DEPC water with another round of centrifugation at 7400
rpm for 10 min at 4ºC. The supernatant was removed, and the pellet was allowed to fully dry
before resuspending in 13 µL DEPC water. The resulting solution was incubated at 60 ºC for 5
min to dissolve the pellet. Promega RQ1 RNAse-free DNAse kit was used to remove any DNA
from the RNA solution. Twenty µL of isopropanol and 2 µL of 3M sodium acetate (pH: 5.2) was
added to precipitate the remaining RNA, and the mixture was kept at -20ºC overnight.
Centrifugation at 14,000 rpm at 4ºC for 10 min allowed the RNA pellet to form. The supernatant
was discarded and the pellet was washed with 75% ethanol and centrifuged using the same
parameters as the previous step. Pellets were allowed to completely dry before resuspending in
10 µL DEPC water. Concentrations were recorded using a Nanodrop 2000 spectrophotometer.
Final RNA was stored in -80ºC until cDNA synthesis.
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cDNA synthesis
cDNA was synthesized using 1 µg of RNA via reverse transcription using Thermo Fisher
cDNA synthesis kit. One µL of oligo dT primer was added to a volume totaling 1 µg RNA for
each sample, and total volume was brought up to 12 µL. The solution was heated at 65ºC for 5
min. Four µL of 5X reaction buffer, 2 µL of 10 mM dNTP mix, 1 µL of nuclease inhibitor, and 1
µL of reverse transcriptase enzyme was added, and the mix was incubated at 42ºC for 1 hour for
cDNA synthesis. All samples were then incubated at 70 ºC for 5 min to inactivate enzyme. Final
cDNA product was then stored at -20 ºC and used for quantitative real-time polymerase chain
reaction (qRT-PCR).
Quantitative RT-PCR
Hypoxia-treated larvae were classified as supernumerary-destined and final instar-
destined Manduca larvae based on the weight at time of fourth instar head capsule slippage
(HCS), the onset of a molt. RNA was isolated from these larvae at the fourth instar HCS and day
0 supernumerary or final instar. For the HCS larvae, the RNA were collected while the larval
head capsule was liquid filled, within the first half of the molt period. Ten brain samples were
collected for expression measurements, while three samples were collected for the central
nervous system. SsoAdvanced SYBR green supermix (Bio-rad) was used to qRT-PCR analyses.
To look at mechanisms of how threshold weight might be regulated, expressions of JH receptor
Met, JH response gene kr-h1, and vvl were measured. rpL17A was used as an internal control
gene, and a 1:5 fold dilution of cDNA was used to generate a standard curve. Primers used for
the qPCR are shown in Table 1.
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Gene Forward (FW)/reverse (RV) Sequence (5’-3’)
Msvvl FW GCTCTACGGCAATGTGTTCT
RV CATGCGCTTCTCCTTCTGT
Mskrh-1 FW GCATCGTTCACAACCTACACC
RV TCCGAGTGGAAAGCGTCAA
Met FW ATAAGGAGGCAGAGGGTCAG
RV TCAAATGGCGAGTCCAATAC
MsrpL17A* FW TCCGCATCTCACTGGGTCT
RV CACGGCAATCACATACAGGTT
Table 1: Primer sequences used for qPCR. *Primer sequences obtained from Ono et al (2006).
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RESULTS
Hypoxia generates supernumerary larvae
Starvation or providing diminished nutrition during the fourth instar has been shown to
generate supernumerary fourth instar Manduca larvae (Nijhout 1975). To determine whether
hypoxia could also generate supernumerary larvae, day 0 third instar larvae were subjected to
hypoxic conditions until the onset of the molt to the fourth instar when the larvae began to
undergo head capsule slippage. Weights were recorded every day until end of final instar or until
time of gut purge, an indication of the onset of metamorphosis. Larvae reared in hypoxic
conditions had two distinct fates at the end of the fourth instar: 43.2% of the larvae (n=16)
molted into a supernumerary fourth instar and the remaining larvae entered the final instar (n=21)
(Fig. 4 and 5).
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Figure 4. Hypoxia generates two developmental trajectories for Manduca sexta. A) Average
growth trajectory of final instar destined larvae (blue) and supernumerary destined larvae (red)
until the end of the fourth instar. Larvae were subjected to hypoxic conditions from the
beginning to the end of the third instar. Weights were recorded at 3rd
HCS and every day after. B)
Individual growth trajectories averaged in A.
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Threshold weight and timing of decision to undergo a supernumerary molt
To determine when the decision is made to enter the supernumerary stage, the fate of the
larva (final instar or supernumerary instar) was plotted against the fourth instar day 1 larval
weights. The fourth instar day 1 larval weights were also plotted against the third instar HCS
weights. Larvae that enter the supernumerary stage have weights that overlap with larvae that
enter the final instar stage (Fig. 5A), suggesting that the fourth day 1 weight does not explain the
decision to enter the supernumerary stage. Only the extremely small or extremely large fourth
instar day 1 sizes are predictive of the nature of the molt. This finding suggests that the decision
to enter the supernumerary stage is not made at day 1 of the fourth instar.
Instead, the differences in masses between supernumerary destined larvae and final instar
larvae treated with hypoxia at the fourth instar head capsule slip (HCS) suggested that the
decision to enter the supernumerary stage is made towards the end of the 4th instar. Weights at 4
HCS could clearly predict whether hypoxia treated larvae would enter the supernumerary or final
instar (Fig. 6A). Those that weighed below approximately 0.75 g at the fourth head capsule slip
underwent a supernumerary molt, while those that weighed above approximately 0.75 g at
molted into a final instar larva.
26
Figure 5. Weight at first day of the fourth instar is a poor predictor of nature of subsequent
molt. Larvae were subjected to hypoxic conditions during the third instar and were weighed at
day 1 of the 4th instar and then tracked for supernumerary molt/gut purge. Hypoxia-treated
supernumerary animals (green triangle) underwent two larval molts after entering the fourth
instar. Hypoxia-treated normal (red square) animals molted into the final instar at the end of the
fourth instar. Normoxia animals (blue diamond) were kept at normal oxygen levels throughout
the larval stage.
27
Figure 6. The decision to enter
supernumerary stage is made at the
end of the fourth instar. A) Manduca
were weighed at fourth instar HCS and
then tracked for supernumerary molt/gut
purge. Hypoxia-treated supernumerary
animals (green triangle) underwent
two larval molts after entering the
fourth instar. Hypoxia-treated normal
(red square) animals molted into the
final instar at the end of the fourth
instar. Normoxia animals (blue
diamond) were kept at normal oxygen
levels throughout the larval stage. B)
Fourth HCS weights plotted against
weight at third HCS.
28
Threshold weight determination in JH-deficient black mutants
Because juvenile hormone (JH) is ubiquitously present during the larval stages of
Manduca sexta until the fourth instar, we wondered whether a reduction in JH titers would affect
the threshold weight. To determine the role of JH and its effect on threshold weight, black
mutant larvae deficient in JH production were subjected to hypoxia during the entire third instar,
and then switched to normoxic conditions at the end of the third instar. Supernumerary instar-
destined larvae had a fourth HCS weight below around 0.5 g, ranging from 0.3 g to 0.51 g; final
instar-destined larvae had a fourth HCS weight above 0.8 g, with a range between 0.8 g and 1.23
g (Fig. 7A). Given that the threshold weight is 0.75 g in wild type animals, and given the broad
gap in fourth instar HCS weights between 0.51 g and 0.8 g, it is unclear whether JH plays a role
in shifting the threshold weight.
29
Figure 7. Threshold weight
determination in black mutant
Manduca. A) Graph of larvae
weights at 4th
HCS and nature of
molt. Black mutant larvae were
subjected to hypoxic conditions
during the third instar and tracked
until the second molt or gut purge.
Hypoxia-treated supernumerary
animals (green triangle) underwent
two larval molts after entering the
fourth instar. Hypoxia-treated
normal (red circle) animals molted
into the final instar at the end of
the fourth instar. Normoxia animals
(blue diamond) were kept at
normal oxygen levels throughout
the larval stage. A few of these
molted into supernumerary instars
(blue square). B) Plot of fourth
HCS weights against third HCS
weights.
30
Duration of instar correlates with the decision to enter the supernumerary stage
The growth trajectories of the supernumerary destined larvae and the final instar-destined
larvae show that one possible contributor to the differences in the final weight is the duration of
the feeding period. Thus, I determined whether supernumerary larvae have a shorter fourth instar
duration (Fig. 8). Larvae exposed to hypoxic condition during the third instar and entered the
final instar after the fourth molt had a feeding period of 2.91 days, similar to the average time of
normoxic control larvae, which took 2.90 days. Supernumerary-destined larvae had a
significantly reduced feeding period of 2.2 days before entering head capsule slippage (ANOVA,
p<0.0001). This finding suggests that a shorter feeding period prevents supernumerary larvae
from attaining the threshold weight. The extended duration of the fourth instar can compensate
for any size deficiencies at the onset of the fourth instar, hence explaining the lack of correlation
between the weight at the onset of the fourth instar and the nature of the fourth instar molt.
31
Figure 8. Fourth instar duration (mean± SE) in wildtype and black mutant Manduca.
Fourth instar larvae either treated with hypoxia or normoxia, and the developmental fate was
determined. Days were counted from day 0 of the fourth instar. Green=wildtype, gray=black
mutant. ANOVA, p<0.0001. Means not sharing the same letter are statistically significant
(Tukey HSD, P<0.05).
32
Manduca deficient in JH do not enter the supernumerary stage as often
The majority of black mutant larvae did not enter the supernumerary stage (Fig. 9). In the
wildtype larvae, 42.9% of the third instar HCS larvae weighing 0.12 - 0.17 g underwent a
supernumerary molt. In contrast, almost none (2.5%) of the black mutant larvae in this weight
range underwent a supernumerary molt. The duration of the fourth instar was 3.9 days in black
mutant larvae in normoxia conditions, longer than the wildtype larvae in normoxia conditions.
This finding suggests that JH deficiency somehow allows for a longer fourth instar duration,
during which smaller black mutant larvae may have extended feeding time to obtain the
unknown threshold weight needed to enter the final instar.
33
Figure 9. Wildtype larvae weighing 0.12-0.17 g at the third instar HCS enter the
supernumerary stage more frequently than black mutant larvae in the same weight
category. Shown are the percentages of wildtype and black mutant larvae that undergo a
supernumerary molt.
34
qPCR analysis of candidate genes correlated with threshold weight
Previous research has observed that when vvl is knocked down, Tribolium larvae undergo
precocious metamorphosis (Cheng et al., 2014). One explanation for this observation is that the
absence of vvl is associated with the onset of metamorphosis. In addition to vvl, I also looked at
the JH response gene kr-h1 and the JH receptor Met to determine whether JH signaling might be
involved in determining the threshold size. Since the size at HCS is correlated with the nature of
the molt, I investigated the expression of vvl, kr-h1, and Met in the brain and the CNS at HCS.
Larvae weighing less than 0.7 g, were identified as those undergoing a supernumerary molts, and
those weighing over 0.8 g were identified as undergoing a final molt. For each of these two
groups of larvae, the brains and the CNS were dissected and pooled.
The following were the general trends observed for qPCR based on one single replicate.
Met expression was found to be very slightly upregulated in the brain and CNS of supernumerary
destined animals at the end of the fourth instar although this within the margin of error seen in
qPCR data (Fig. 10). Therefore, we do not think that Met expression changes significantly
between the two samples.
35
Figure 10. Expression of Met in
final instar larvae and
supernumerary destined larvae. Ten Manduca were dissected for
brains and three for central nervous
systems (CNS). Larvae were weighed
prior to dissection to determine the
nature of the molt. Met expression
was quantified using qPCR using
rpL17A as an internal control. Each
bar represents the mean of triplicates
for one biological sample.
36
Kr-h1 was upregulated in the brain and the CNS at the end of the fourth instar larvae
undergoing a supernumerary molt, relative to those undergoing a final molt (Fig. 11). In addition,
the brain and CNS of the day 0 supernumerary fourth instar larvae also had upregulated kr-h1
expression. Upregulation of kr-h1 at the end of the fourth instar suggests that high JH signaling
at the end of the fourth instar may be correlated with a supernumerary molt.
37
Figure 11. Expression of kr-h1
in final instar larvae and
supernumerary destined larvae. Ten Manduca were dissected for
brains and three for central
nervous systems (CNS). Larvae
were weighed prior to dissection
to determine the nature of the
molt. kr-h1 expression was
quantified using qPCR using
rpL17A as an internal control.
Each bar represents the mean of
triplicates for one biological
sample.
38
Vvl was upregulated in the brain and the CNS in larvae undergoing a supernumerary
molt. Its increase in expression during the end of the fourth instar coincides with the decision to
enter the supernumerary stage (Fig. 12). Taken together, these results show that vvl expression is
increased during the decision to enter the supernumerary stage, and its expression may prevent
the larvae from entering the final instar when they are below the threshold weight. Although
these trends are interesting and suggestive, these qPCR results are based on one single biological
replicate; thus, these data should be considered preliminary.
39
Figure 12. Expression of vvl in
final instar larvae and
supernumerary destined larvae. Ten Manduca were dissected for
brains and three for central nervous
systems (CNS). Larvae were
weighed prior to dissection to
determine the nature of the molt. vvl
expression was quantified using
qPCR using rpL17A as an internal
control. Each bar represents the
mean of triplicates for one biological
sample.
40
DISCUSSION
In this study, we investigated several aspects of the threshold weight: whether alternative
methods could generate the supernumerary stage previously observed, when the decision to enter
the supernumerary stage is made, the role of JH in threshold weight determination, and candidate
genes that can possibly regulate threshold weight. By exposing larvae to hypoxic conditions, we
were able to generate supernumerary destined and final instar destined Manduca and determined
that the threshold weight is 0.75 g in wildtype Manduca, which corroborates previous findings
(Grunert et al., 2015; Nijhout 1975). In contrast, when JH titers were reduced, very few molted
into the supernumerary stage. The size at the end of the fourth instar determines the nature of the
molt. In addition, vvl and kr-h1 expression in supernumerary-destined larvae were found to be
elevated relative to the final instar-destined larvae. Although these expression changes in vvl and
kr-h1 were determined from one biological replicate and further research is needed to confirm
this expression trend, these expression differences between the two different types of larvae
could be a possible proximate mechanism of how threshold weight is regulated.
Hypoxia on threshold weight
Previous work has shown that oxygen can modify final adult size in Drosophila and
Manduca (Frazier et al., 2004; Klok and Harrison, 2009; Callier and Nijhout, 2011). Callier and
Nijhout (2011) found that the tracheal system only increased in area between each larval instar,
suggesting that as the body cavity grows within an instar, oxygen tissue delivery decreases as the
tracheal system fails to keep up with the growing volume of the larva. The authors suggested that
this reduction in oxygen delivery to surrounding tissues serves as a cue that the animal is big
enough to molt or initiate metamorphosis. Thus, Manduca subjected to hypoxic conditions on
average had reduced fourth and fifth instar body sizes (Callier and Nijhout, 2011). Similarly, the
41
final body size of Drosophila reared in hypoxic environmental conditions was significantly
reduced compared to those reared under normoxic conditions (Callier et al., 2013).
We reasoned that hypoxia would trigger a fourth instar molt at a smaller weight if third
instar larvae were reared under hypoxic conditions. These larvae would then molt at a smaller
fourth instar body size, generating larvae that molt into supernumerary instars. Hypoxia as a
treatment during the third instar successfully generated both supernumerary and final instar
animals (Fig. 4). The weight at the end of the third instar can only predict the nature of the fourth
instar molt (supernumerary destined and final instar destined larvae) if they fall outside the
normal size range of the third HCS instars. If the weights of larvae between 0.13-0.17g, the
nature of the fourth instar molt is not predictable. The fact that we can obtain both
supernumerary and final instar destined Manduca using the hypoxia treatment shows that this is
a useful way to generate supernumerary larvae that avoids having to use starvation and other
strategies that might add additional confounding variables.
Timing of threshold weight decision
Nijhout (1975) first discovered that threshold weight is an absolute size minimum
obtained in order to enter the final the instar. This finding suggested that rather than counting the
number of instars until hatching to enter metamorphosis, the size of the larvae is sufficient to
make this decision.
Under laboratory conditions, we observed that hypoxic conditions could generate
supernumerary fourth instar larvae when third instar larvae were exposed to hypoxic conditions.
On day 1 of the fourth instar, the weight serves as a poor predictor of the nature of the molt.
However, the weight at the fourth HCS does, supporting the notion that an absolute weight
governs the decision to enter the supernumerary stage. Additionally, we found that
42
supernumerary-destined larvae have a shorter instar duration relative to the final instar-destined
larvae (Fig. 8). Interestingly, black mutants have longer instar duration relative to the wildtype,
and this led to most of the larvae molting into final instar larvae.
These observations suggest that JH may play a critical role in the timing of molting, and
this in turn may underlie the differences between supernumerary and final instars and their
growth trajectories. Based on the black mutant data, we speculate that JH might promote the
earlier release of ecdysteroids, causing larvae to molt sooner.
The role of JH in threshold weight
In wild type Manduca, levels of JH are relatively high until the end of the fourth instar,
when JH levels decrease and rise again after ecdysis into the fifth instar (Fain and Riddiford,
1975). To understand how JH could affect the threshold weight, black mutant Manduca,
deficient in levels of JH, were subjected to the same hypoxic method shown to generate
supernumerary instars in the wildtype larvae. Surprisingly, fewer black mutant larvae entered the
supernumerary stage, suggesting that the reduced levels of JH prevent larvae from entering the
supernumerary stage (Fig.9). We propose that supernumerary-destined animals have higher
levels of JH compared to final instar-destined larvae, shortening the fourth instar duration by
releasing ecdysteroids relatively early. The most well-studied interaction between JH and
ecdysteroids occurs in the final instar when the presence of JH delays the production and
response to PTTH in Bombyx mori and in Manduca (Sakuri et al., 1989; Rountree and
Bollenbacher 1986). Although these findings highlight the role of JH suppressing PTTH
response and production in the feeding final instar larvae, it is possible that JH might have a
different role in earlier instars to stimulate PTTH production and enter the supernumerary stage.
43
In fact, JH appears to have stimulatory role on ecdysteroidogenesis during the prepupal period
(Gruetzmacher et al., 1984). Furthermore, previous experiments suggest that higher JH titers may
be correlated with reduced duration of earlier instars (Suzuki and Nijhout, 2008). However,
additional experiments are needed to test whether differences in JH levels during the penultimate
instar makes a difference in the nature of the molt.
Molecular correlates of the threshold weight
To begin to understand the molecular mechanisms that sense threshold weight and
mediate the decision to enter the threshold weight, we decided to look at differences in
expression of candidate genes, vvl, Met, and kr-h1, during the HCS and day 0 after ecdysis in
supernumerary and final instar destined wildtype Manduca. As expected, Met gene expression
did not dramatically differ between supernumerary and final instars during HCS and day 0
stages. Given that JH binding leads to changes in phosphorylation states (Cai et al., 2014,
Konopova and Jindra, 2008), it is not surprising that gene expression of Met did not change; it is
still possible that differences in Met reception during the two developmentally different larvae
can mediate downstream molecular mechanisms of threshold weight. Recent studies have also
suggested that JH may bind to a cell surface receptor instead of Met. Therefore, we cannot rule
out a difference in JH sensitivity based on our expression analysis.
qPCR results suggested that kr-h1 expression is elevated during the supernumerary
fourth stage, suggesting that higher levels of JH signaling may lead to a supernumerary molt
(Fig. 11). As we previously mentioned, the decision to undergo the supernumerary stage is made
at the end of the fourth HCS stage which correlates with increased kr-h1 expression in the brain
44
and the CNS. These observations suggest that elevated JH at the end of the fourth instar may be
associated with a supernumerary molt.
Our results also suggest that vvl expression is elevated in the CNS and the brain of the
fourth HCS of supernumerary-destined larvae. Since this is based on one biological replicate;
further replicates are needed to confirm this increase in expression. This increase in expression is
correlated with the timing of the decision to enter the supernumerary instar, along with elevated
JH signaling seen based on kr-h1 expression during the supernumerary stage.
Potential model for the specification of molt identity
Based on the findings, a potential model is proposed to explain how the distinct molt
fates are specified (Fig. 13). The sensitive period for specifying the nature of the molt occurs at
the end of the fourth instar. One possible difference between supernumerary and final instar
larvae is the relative levels of JH and ecdysteroids present at the time of ecdysis. In normoxia
larvae, JH levels are high at a constant level during the earlier larval stages, with the exception of
a dip in JH levels prior to the final instar (Fain and Riddiford, 1975). Given that black mutants
delay molting, JH likely acts to stimulate ecdysteroid production. Because supernumerary
destined Manduca molt at a reduced weight, it is possible that it has higher JH levels that
stimulate ecdysteroid production and initiate molting before JH levels fall during the fourth
instar. This early ecdysteroid production may lead to higher JH and ecdysteroid titers during the
sensitive period, leading to the specification of a supernumerary molt. This would be correlated
with a higher expression of JH response gene.
45
Figure 13. A proposed model for the decision to enter the supernumerary stage in Manduca. At the end of the fourth instar, a decrease in JH levels coincides with a peak in ecdysteroid
production during the sensitive period (shown in gray), allowing entry into the final instar.
Supernumerary destined larvae have high JH levels coinciding with an ecdysone peak from a
truncated fourth instar duration, during which kr-h1 and vvl expression is increased. At a low JH
level, the decrease in JH levels at the end of the fourth instar is much lower than the JH threshold
sensed to enter the supernumerary instar; therefore, the subsequent molt is final.
46
In contrast, larvae destined to undergo a final molt would have lower JH titers, delaying
the peak of ecdysteroid production until the next day. During the sensitive period at the end of
the instar, JH levels dip and along with the ecdysteroid peak, they may then downregulate gene
expression, such as kr-h1 and vvl .
This model suggests that JH may be a stimulator of PTTH/ecdysteroid production. The
findings of this study suggests that JH level differences during the sensitive period, along with
timing of the ecdysteroid peak, may be coordinated by genes such as kr-h1 and vvl which
ultimately regulate the mechanism that determines the nature of a molt. The threshold weight
then may be set by the relative amounts of JH and ecdysteroids circulating at the end of the
instar.
47
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