Drosophila embryogenesis scales uniformly across temperature in
developmentally diverse species

Steven G. Kuntz1,2∗, Michael B. Eisen1,2,3,4

1 QB3 Institute for Quantitative Biosciences, University of California,
Berkeley, California, United States of America
2 Department of Molecular and Cell Biology, University of California,
Berkeley, California, United States of America
3 Howard Hughes Medical Institute, University of California, Berkeley,
California, United States of America
4 Department of Integrative Biology, University of California, Berkeley,
California, United States of America
$\ast$ E-mail<EMAIL_ADDRESS>
## Abstract

Temperature affects both the timing and outcome of animal development, but the
detailed effects of temperature on the progress of early development have been
poorly characterized. To determine the impact of temperature on the order and
timing of events during Drosophila melanogaster embryogenesis, we used time-
lapse imaging to track the progress of embryos from shortly after egg laying
through hatching at seven precisely maintained temperatures between 17.5∘C and
32.5∘C. We employed a combination of automated and manual annotation to
determine when 36 milestones occurred in each embryo. D. melanogaster
embryogenesis takes $\sim$33 hours at 17.5∘C, and accelerates with increasing
temperature to a low of 16 hours at 27.5∘C, above which embryogenesis slows
slightly. Remarkably, while the total time of embryogenesis varies over two
fold, the relative timing of events from cellularization through hatching is
constant across temperatures. To further explore the relationship between
temperature and embryogenesis, we expanded our analysis to cover ten
additional Drosophila species of varying climatic origins. Six of these
species, like D. melanogaster, are of tropical origin, and embryogenesis time
at different temperatures was similar for them all. D. mojavensis, a sub-
tropical fly, develops slower than the tropical species at lower temperatures,
while D. virilis, a temperate fly, exhibits slower development at all
temperatures. The alpine sister species D. persimilis and D. pseudoobscura
develop as rapidly as tropical flies at cooler temperatures, but exhibit
diminished acceleration above 22.5∘C and have drastically slowed development
by 30∘C. Despite ranging from 13 hours for D. erecta at 30∘C to 46 hours for
D. virilis at 17.5∘C, the relative timing of events from cellularization
through hatching is constant across all species and temperatures examined
here, suggesting the existence of a previously unrecognized timer controlling
the progress of embryogenesis that has been tuned by natural selection as each
species diverges.

## Author Summary

Temperature profoundly impacts the rate of development of “cold-blooded”
animals, which proceeds far faster when it is warm. There is, however, no
universal relationship. Closely related species can develop at markedly
different speeds at the same temperature. This creates a major challenge when
comparing development among species, as it is unclear whether they should be
compared at the same temperature or under different conditions to maintain the
same developmental rate. Facing this challenge while working with flies
(Drosophila species), we found there was little data to inform this decision.
So, using time-lapse imaging, precise temperature-control, and computational
and manual video-analysis, we tracked the complex process of embryogenesis in
11 species at seven different temperatures. There was over a three-fold
difference in developmental rate between the fastest species at its fastest
temperature and the slowest species at its slowest temperature. However, our
finding that the timing of events within development all scaled uniformly
across species and temperatures astonished us. This is good news for
developmental biologists, since we can induce species to develop nearly
identically by growing them at different temperatures. But it also means flies
must possess some unknown clock-like molecular mechanism driving embryogenesis
forward.

## Introduction

It has long been known that Drosophila, like most poikilotherms, develops
faster at higher temperatures, with embryonic [1], larval [1, 2], and pupal
stages [3, 4], as well as total lifespan [5, 6] showing similar logarithmic
trends. While genetics, ecology, and evolution of this trait have been
investigated for over a century [2, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17],
the effects of temperature on the order and relative timing of developmental
events, especially within embryogenesis, are poorly understood.

We became interested in the relationship between species, temperature, and the
cadence of embryogenesis for practical reasons. Several years ago, we
initiated experiments looking at the genome-wide binding of transcription
factors in the embryos of divergent Drosophila species: D. melanogaster, D.
pseudoobscura, and D. virilis. With transcription factor binding a highly
dynamic process, we tried to match both the conditions (especially
temperature, which we believed would affect transcription factor binding) in
which embryos were collected and the developmental stages we analyzed.
However, our initial attempts to collect D. pseudoobscura embryos at 25∘C —the
temperature at which we collect D. melanogaster —were unsuccessful, with large
numbers of embryos failing to develop, likely a consequence of D.
pseudoobscura’s alpine origin. While D. virilis lays readily at 25∘C, we found
that their embryos develop more slowly than D. melanogaster, complicating the
collection of developmental stage-matched samples.

Having encountered such challenges with just three species, and planning to
expand to many more, we were faced with several important questions. Given
that embryogenesis occurs at different rates in different species [8, 18], how
should we time collections to get the same mix of stages we get from our
standard 2.5 –3.5 hour collections in D. melanogaster, or any other stage we
study in the future? Is it better to compare embryos collected at the same
temperature even if it is not optimal for, or even excludes, some species; or,
should we collect embryos from each species at their optimal temperature, if
such a thing exists? Should we select a temperature for each species so that
they all develop with a similar velocity? Or should we find a set of species
that develop at the same speed at a common temperature? And even if we could
match the overall rate of development, would heterochronic effects mean that
we could not get an identical mix of stages?

We found a woeful lack in the kind of data needed to answer these questions.
Powsner precisely measured the effect of temperature on the total duration of
embryogenesis in D. melanogaster [1], and Markow made similar measurements for
other Drosophila species at a fixed temperature (24∘C) [18], but the precise
timing of events within embryogenesis had been described only for D.
melanogaster at 25∘C [19, 20].

The work described here was born to address this deficiency. We used a
combination of precise temperature control, time-lapse imaging, and careful
annotation to catalog the effects of a wide range of temperatures on embryonic
development in 11 Drosophila species from diverse climates. We focused on
species with published genome sequences [21] (Table 1), as these are now
preferentially used for comparative and evolutionary studies. Of the species
we studied D. melanogaster, D. ananassae, D. erecta, D. sechellia, D.
simulans, D. willistoni, and D. yakuba are all native to the tropics, though
D. melanogaster, D. ananassae, and D. simulans have spread recently to become
increasingly cosmopolitan [17]. D. mojavensis is a sub-tropical species, while
D. virilis is a temperate species that has become holarctic and D. persimilis
and D. pseudoobscura are alpine species (Figure 1A).

## Results

### Time-lapse imaging tracks major morphological events

We used automated, time-lapse imaging to track the development of embryos held
at a constant and precise temperature from early embryogenesis (pre-
cellularization) to hatching. We maintained the temperature at $\pm$0.1∘C
using thermoelectric Peltier heat pumps. Different sets of embryos were
analyzed at temperatures ranging from 17.5∘C to 32.5∘C, in 2.5∘C increments.
Images were taken every one to five minutes, depending on the total time of
development. A minimum of four embryos from each species were imaged for each
temperature, for a total of 77 conditions. In total, time-lapse image series
were collected and analyzed from over 1000 individual embryos.

We encountered, and solved, several challenges in designing the experimental
setup, including providing the embryos with sufficient oxygen [22, 23] and
humidity. We found that glass slides were problematic due to a lack of
oxygenation and led to a $\sim$28% increase in developmental time, so we
instead employed an oxygen-permeable tissue culture membrane, mounted on a
copper plate to maintain thermal conduction. At higher temperatures, we found
that the embryos dehydrated, so humidifiers were used to increase ambient
humidity. Detailed photos of the apparatus and descriptions can be found in
Figure S1.

We used a series of simple computational transformations (implemented in
Matlab) to orient each embryo, correct for shifting focus, and adjust the
brightness and contrast of the images, creating a time-lapse movie for each
embryo. We manually examined images from 60 time-lapse series in D.
melanogaster and identified 36 distinct developmental stages [19, 20] that
could be recognized in our movies (Table 2,
http://www.youtube.com/watch?v=dYSrXK3o86I and
http://www.youtube.com/watch?v=QKVmRy3dDR0 or “D. melanogaster with labelled
stages” and “D. melanogaster with labelled stages at reduced framerate” in
DOI:10.5061/dryad.s0p50”). Due to the volume of images collected, we
implemented a semi-automated system to annotate our entire movie collection.
Briefly, images from matching stages in manually annotated D. melanogaster
movies were averaged to generate composite reference images for each stage
(Figure 2). We then used a Matlab script to find the image-matrix correlation
between each of these composite reference images to the images in each time-
lapse to estimate the timing of each morphological stage via the local
correlation maximum (Figure S2A).

Of the 36 events, the eight most unambiguous events (Figure S3), identifiable
regardless of embryo orientation, were selected for refinement and further
analysis (pole bud appears, membrane reaches yolk, pole cell invagination,
amnioproctodeal invagination, amnioserosa exposed, clypeolabrum retracts,
heart-shaped midgut, and trachea fill) (Figure S2B,C). Using a Python-scripted
graphical user interface, each of the eight events in every movie was manually
examined and the algorithm prediction adjusted when necessary. Timing of
hatching was excluded from these nine primary events because it was highly
variable, likely due to the assay conditions following dechorionation, and
suitable only as an indication of successful development, not as a reliable
and reproducible time point. The “membrane reaches yolk stage” was used
throughout as a zero point due to the precision with which the stage could be
identified in all species and from all orientations.

Links to representative time-lapse videos are provided in Table 3.

###  D. melanogaster embryogenesis scales uniformly with temperature

As expected, the total time of embryogenesis of D. melanogaster had a very
strong dependence on temperature (Figure 3,
http://www.youtube.com/watch?v=-yrs4DcFFF0 or “D. melanogaster at 7
temperatures” in DOI:10.5061/dryad.s0p50). From 17.5∘C to 27.5∘C, there was a
two-fold acceleration in developmental rate, matching the previously observed
doubling of total lifespan with a 10∘C change in temperature [6]. The velocity
of embryogenesis at 30∘C is roughly the same as at 27.5∘C, and is appreciably
slower at 32.5∘C, likely due to heat stress. At 35∘C, successful development
becomes extremely rare.

To examine how these temperature-induced shifts in the total time of
embryogenesis were reflected in the relative timing of individual events, we
rescaled the time series data for each embryo so that the time from our most
reliable early landmark (the end of cellularization) to our most reliable late
landmark (trachea filling) was identical, and examined where each of the
remaining landmarks fell (Figure 3C). We were surprised to find that D.
melanogaster exhibited no major changes in its proportional developmental time
under any of the non-stressful temperature conditions tested. Therefore, at
least as far as most visually evident morphological features go, embryogenesis
scales uniformly across a two-fold range of total time.

When the embryos were under heat stress ($>$30∘C), we observed a very slight
contraction in the proportion of time between early development (pole bud
appears) to the end of cellularization (membrane reaches yolk), and a slight
contraction between the end of cellularization and mid-germ band retraction
(amnioserosa exposure).

### Embryogenesis scales uniformly across species despite significant
differences in temperature dependent developmental rate

In each of the ten additional Drosophila species we examined we observed all
of the 36 developmental landmarks we identified in D. melanogaster in the same
temporal order (Figure 4A). However, there was marked interspecies variation
in both the total time of embryogenesis at a given temperature (Figure 4B-E,
Table 3) and the way embryogenesis time varied with temperature (Figure 5).

When we examined the 10 remaining species, we found not only that the relative
timing of events was constant across temperature within a species, as observed
in D. melanogaster, but that landmarks occurred at the same relative time
between species at all non-stressful temperatures (Figures 6, Table 4).

### Developmental time is exponentially related to $\alpha/T$

Between 17.5∘C and 27.5∘C the total developmental time for all species can be
approximated relatively accurately by an exponential regression ($R^{2}>0.9$).
For all species we find that temperature T can be related to developmental
time $t_{dev}$, agreeing with a long history of temperature-dependent rate
modeling [24]:

$t_{dev}\approx e^{\frac{\alpha}{T}}$

and developmental rate v:

$ln(v)\approx-\frac{\alpha}{T}$

The parameters of these relations for each species, which includes two
independent coefficients, are included in Table 5. Also included in Table 5 is
the $Q_{10}$, an empirical description of biological rate change from a 10∘C
temperature change, for the 17.5∘C to 27.5∘C interval. At higher temperatures,
heat stress appears to counter the logarithmic trend and lengthens
developmental time. Since the temperature responses are highly reproducible,
the developmental time for each species can be modeled and predictions made
for future experiments (Figure S4).

### Effect of temperature on developmental rate is coupled to climatic origin

Seven of the eleven species we examined were of tropical origin, with only two
alpine, one subtropical and one temperature species. At mid-range temperatures
(22.5∘C - 27.5∘C), the tropical species developed the fastest, followed by the
subtropical D. mojavensis, the alpine D. pseudoobscura and D. persimilis, and
the temperate D. virilis (Figure 5), in accord with [18].

Some tropical species have expanded into temperature zones and a variety of
wild strains have been collected from a variety of climates. We examined nine
additional strains of D. melanogaster collected along the eastern United
States [25, 26]. Though collected along a tropical to temperate cline and
there was some variation between strains, no trends were seen (Figure S5A,B).

The tropical species all showed highly similar responses to temperature, even
though they originate from different continents (Africa, Asia and South
America) and are not closely related (five of the species are in the
melanogaster subgroup, but D. ananassae and D. willistoni are highly diverged
from both D. melanogaster and each other). Though they possess similar
temperature-responses, these species possess significantly different and
independent temperature response curves ($p<0.05$) and the differences are
large enough to be relevant for precise developmental experiments. These
cross-species differences tend to be, but are not necessarily, larger than
those seen between D. melanogaster strains (Figure S5C). The embryogenesis
rate for these species increases rapidly with temperature ($Q_{10}\sim 2.2$)
before slowing down at and above 30∘C (Figure S6A-F,
http://www.youtube.com/watch?v=vy6L4fmWkso or “D. ananassae at 7 temperatures”
in DOI:10.5061/dryad.s0p50). The two closely related alpine species (D.
pseudoobscura and D. persimilis) match the embryogenesis rate of the tropical
species at 17.5∘C, but accelerate far less rapidly with increasing temperature
($Q_{10}\sim 1.6$), especially at 25∘C and above (Figure S6I,J,
http://www.youtube.com/watch?v=sYi-FUXpv4Q or “D. pseudoobscura at 6
temperatures” in DOI:10.5061/dryad.s0p50). These species also show a sharp
increase in embryogenesis rate and low viability above 27.5∘C, consistent with
their cooler habitat. The subtropical D. mojavensis (Figure S6H,
http://www.youtube.com/watch?v=XWMs4oUx_mU or “D. mojavensis at 6
temperatures” in DOI:10.5061/dryad.s0p50) and temperate D. virilis (Figure
S6G, http://www.youtube.com/watch?v=eyr4ckDb0kM or “D. virilis at 6
temperatures” in DOI:10.5061/dryad.s0p50) both develop very slowly at low
temperature, but accelerate rapidly as temperature increases ($Q_{10}$ of
$\sim 2.5$ and $\sim 2.2$ respectively). D. virilis remains the slowest
species up to 30∘C, while D. mojavensis is as fast as the tropical species at
high temperatures. These species are both members of the virilis-repleta
radiation and it remains to be seen if this growth response is characteristic
of the group as a whole, independent of climate.

### Effects of heat stress

Under heat-stress, the proportionality of development is disrupted in some
embryos (Figure S7A). The effect is not uniform, as some embryos developed
proportionally under heat-stress and others exhibited significant aberrations,
largely focused in post-germband shortening stages. This can be most clearly
seen in individuals of D. ananassae, D. mojavensis, D. persimilis, and D.
pseudoobscura. We did not identify any particular stage as causing this delay,
but rather it appears to reflect a uniform slowing of development.

Early heat shock significantly disrupts development enough to noticeably
affect morphology in yolk contraction, cellularization, and gastrulation
(Figure S7B). Syncytial animals are the most sensitive to heat-shock (Figure
S7C). In D. melanogaster and several other species we observed a slight
contraction of proportional developmental time between early development (pole
bud appears) and the end of cellularization (membrane reaches yolk) under
heat-stress ($>$ 30∘C, Figure S7D). While all later stages following
cellularization maintain their proportionality even at very high temperatures,
the pre-cellularization stages take proportionally less and less time. This
indicates that at higher temperatures, some pre-cellularization kinetics scale
independently of later stages, possibly leading to mortality as the
temperature becomes more extreme.

## Discussion

We have addressed the lack of good data on the progress of embryogenesis in
different species and at different temperatures with a carefully collected and
annoted series of time-lapse movies in 11 species at seven temperatures that
span most of the viable range for Drosophila species. From a practical
standpoint, the predictable response of each species to temperature, and the
uniform scaling of events between species and temperature, provides a
relatively simple answer to the question that motivated this study - to
determine how to obtain matched samples for genomic studies: simply choose the
range of stages to collect in one strain or species, and scale the collection
and aging times appropriately. The fact that development scales uniformly over
non-extreme temperatures would seem to give some leeway in the choice of
temperature, so long as heat-stress is avoided, though it remains unclear how
molecular processes are affected by temperature.

### Uniform scaling and the timing of embryogenesis

In carrying out this survey, we were surprised to find that the relative
timing of landmark events in Drosophila embryogenesis is constant across
greater than three-fold changes in total time, spanning 15∘C and over 100
million years of independent evolution. And the fact that the same holds true
for 34 developmental landmarks at two temperatures in the zebrafish Danio
rerio [27], (the only other species for which we were able to locate similar
data), suggests that this phenomenon may have some generality. But why is this
so?

Drosophila development involves a diverse set of cellular processes including
proliferation, growth, apoptosis, migration, polarization, differentiation,
and tissue formation. One might expect (we certainly did) these different
processes to scale independently with temperature, much as different chemical
reactions do, and as a result, different stages of embryogenesis or parts of
the developing embryo would scale differentially with temperature. But this is
not the case. The simplest explanation for this observation is that a single
shared mechanism controls timing across embryogenesis throughout the genus
Drosophila. But what could such a mechanism be? One possibility is that there
is an actual clock —some molecule or set of molecules whose abundance or
activity progresses in a clocklike manner across embryogenesis and is read out
to trigger the myriad different processes that occur in the transition from a
fertilized egg to a larvae. However there is no direct evidence that such a
clock exists (although we note that there is a pulse of ecdysone during
embryogenesis with possible morphological functions [28, 29]). A more likely
explanation is that there is a common rate limiting process throughout
embryogenesis. Our data are largely silent on what this could be, but we know
from other experiments that it is cell, or at least locally, autonomous [30,
31, 32] and would have to limit processes like migration that do not require
cell division (we also note that cell division has been excluded as a
possibility in zebrafish [32]). However, energy production, yolk utilization,
transcription or protein synthesis are reasonable possibilities.

Although there are very few comparisons of the relative timing of events
during development, it has long been noted that various measurements of
developmental timing scale exponentially with $\alpha/T$ [5, 24, 6, 1, 33],
but no good explanation for this phenomenon has been uncovered. Perhaps
development is more generally limited by something that scales exponentially
with $\alpha/T$, like metabolic rate, which, we note, has been implicated
numerous times in lifespan, which is, in some ways, a measure of developmental
timing.

Gillooly and co-workers, noting the there was a relationship between metabolic
rate, temperature and animal size, have proposed a model that incorporates
mass into the Arrhenius equation to explain the relationship between these
factors in species from across the tree of life [34, 35]. We, however, do not
find that mass can explain the differences in temperature-dependence between
species. Even closely-related species, with nearly 2-fold differences in their
mass (e.g. D. melanogaster, D. simulans, D. sechellia, D. yakuba, and D.
erecta), have significant divergence in their proportionality coefficients
that do not converge at all when correcting for differences in mass through
the one quarter power scaling proposed by Gillooly, et al. This suggests that
some other factor is responsible for the differences, as has been argued by
other groups [36, 37, 18]. The relationship between climate and temperature
response raises the possibility that whatever this factor is has been subject
to selection to tune the temperature response to each species’ climate.
However, without additional data this is purely a hypothesis.

Although a common rate-limiting step is simplest explanation for uniform
scaling, it is certainly not the only one. It is possible that different rate
limiting steps or other processes control developmental velocity at different
times or in different parts of the embryo, and that they scale identically
with temperature either coincidentally, or as the result of selection (it is
important to remember that, as per Arrhenius, one does not expect different
reactions to scale identically with temperature). If this is the result of
selection, what is the selection pressure? Evolutionary developmental
biologists, perhaps most notably Stephen J. Gould, have long written about how
changes in either the absolute or relative timing of different events during
development have had significant effects on morphology throughout animal
evolution [38, 39, 40, 41]. Perhaps this is also true for fly embryogenesis,
but that any such changes in morphology are selectively disadvantageous and
have been strongly selected against. It is also likely that many developing
fly embryos experience significant changes in temperature while developing, so
there may be strong selection to maintain uniform development across
temperature to ensure normal progression while the temperature is changing.

Finally, we note that there are limits to this uniformity. At extreme
temperatures, especially high ones, things no longer scale uniformly, likely
reflecting the differential negative effects of high temperature at different
stages of embryogenesis as well as the differential ability of the embryo to
compensate for them. There are also clearly checkpoints in place that, while
not triggered during normal embryogenesis, are important in extreme or unusual
circumstances. Most strikingly, when Lucchetta et al. and Niemuth et al.
examined embryos developing in chambers that allowed for independent
temperature control of the anterior and posterior portions of the embryo, the
two parts of the embryo developed at different velocities for much of
embryogenesis [30, 31]. They found that embryos are robust to asynchrony in
timing across the embryo, though there are critical periods that, once passed,
do not permit re-synchronization of development [30], hinting at some specific
checkpoints or feedback.

### Climate and the rate of embryogenesis

The clustering of developmental timing and its temperature response with
climate —especially amongst tropical species from different continents and
parts of the Drosophila tree —suggests that this is an adaptive, or in some
cases permissive, phenotype, although with only 11 species and poor coverage
of non-tropical species this has to remain highly speculative. There are
necessarily additional components to the temperature response, as significant
variation exists within the tropical species and between D. melanogaster
strains. The virilis-repleta radiation, which includes both D. virilis and D.
mojavensis may have a climate-independent adaptation that leads to slowed
development at cooler temperatures, a feature that is hard to rationalize. The
poor response of the alpine D. pseudoobscura and D. persimilis to high
temperature is consistent with their cool climate. Nevertheless, little is
known about when and where most of these species lay their eggs and their
natural microclimates.

The clustering of developmental responses in species by their native climates
rather than their climates of collection suggests that if climate adaptation
is a contributing factor, the response arises slowly or rarely. The tested
strains of D. melanogaster were collected in temperate, subtropical, and
tropical climates and the D. simulans strain was collected in a sub-tropical
climate. Nevertheless, both species performed qualitatively like other
tropical species and unlike native species collected nearby. This suggests
that temperature responses are neither rapidly evolving (with D. melanogaster
being present in the temperate United States for over 130 years [42]) nor
primed for change in tropical species.

## Materials and Methods

### Rearing of Drosophila

Drosophila strains were reared and maintained on standard fly media at 25∘C,
except for D. persimilis and D. pseudoobscura which were reared and maintained
at 22∘C. D. melanogaster lines were raised at 18∘C and 22∘C for several years
and their temperature response profiles were observed, verifying that
transferring embryos from the ambient growth temperature for a line to the
experimental temperature did not lead to heat-shock responses and had
relatively little impact on the temperature response (Figure S8A,B). Egg-lays
were performed in medium cages on 10 cm molasses plates for 1 hour at 25∘C
after pre-clearing for all species except D. persimilis, which layed at 22∘C.
Comparisons to D. melanogaster raised and laying at 22∘C confirmed that growth
at lower temperatures does not account for all of the differences between the
tropical and alpine species (Figure S8C).To encourage egg-lay, cornmeal food
media was added to plates for D. sechellia and pickled cactus was added to
plates for D. mojavensis. Embryos were collected and dechorionated with fresh
50% bleach solution (3% hypochlorite final) for 45 to 90 seconds (based on the
species) in preparation for imaging. Dechorionation timing was selected as the
time it took for 90% of the eggs to be successfully dechorionated. This
prevented excess bleaching, as many species, such as D. mojavensis, are more
sensitive than D. melanogaster. Strains used were D. melanogaster, OreR, DGRP
R303, DGRP R324, DGRP R379, DGRP R380, DGRP R437, DGRP R705, Schmidt Ln6-3,
Schmidt 12BME10-24, and Schmidt 13FSP11-5; D. pseudoobscura, 14011-0121.94,
MV2-25; D. virilis, 15010-1051.87, McAllister V46; D. yakuba, 14021-0261.01,
Begun Tai18E2; D. persimilis, 14011-0111.49,(Machado) MSH3; D. simulans,
14021-0251.195, (Begun) simw501; D. erecta, 14021-0224.01, (TSC); D.
mojavensis wrigleyi, 15081-1352.22, (Reed) CI 12 IB-4 g8; D. sechellia,
14021-0248.25, (Jones) Robertson 3C; D. willistoni, 14030-0811.24, Powell
Gd-H4-1; D. ananassae, 14024-0371.13, Matsuda (AABBg1).

### Time-lapse Imaging

Embryos were placed on oxygen-permeable film (lumox, Greiner Bio-one), affixed
with dried heptane glue and then covered with Halocarbon 700 oil (Sigma) [43].
The lumox film was suspended on a copper plate that was temperature-regulated
with two peltier plates controlled by an H-bridge temperature controller
(McShane Inc., 5R7-570) with a thermistor feedback, accurate to $\pm$0.1∘C.
Time-lapse imaging with bright field transmitted light was performed on a
Leica M205 FA dissecting microscope with a Leica DFC310 FX camera using the
Leica Advanced Imaging Software (LAS AF) platform. Greyscale images were saved
from pre-cellularization to hatch. Images were saved every one to five
minutes, depending on the temperature. A humidifier was used to mitigate
fluctuations in ambient humidity, though fluctuations did not affect
developmental rate. Due to fluctuations in ambient temperature and humidity,
the focal plane through the halocarbon oil varied significantly. Therefore,
z-stacks were generated for each time-lapse and the most in-focus plane at
each time was computationally determined for each image using an algorithm
(implemented in Matlab) through image autocorrelation [44, 45]. Time-lapse
videos available from Dryad Digital Repository: doi:10.5061/dryad.s0p50

### Event estimation

A subset of time-lapses in D. melanogaster were analyzed to obtain a series of
representative images for each of the 36 morphological events, selected as all
events defined by [19, 46] that were reproducibly identifiable under our
conditions, described. These images were sorted based on embryo orientation
and superimposed to generate composite reference images. Images from each
time-lapse to be analyzed were manually screened to determine the time when
the membrane reaches the yolk, the time of trachea filling, and the
orientation of the embryo (Figure S3. This information was fed into a Matlab
script, along with the time-lapse images and the set of 34 composite reference
images, to estimate the time of 34 morphological events during embryogenesis
via image correlation. The same D. melanogaster reference images were used for
all species for consistency. A correlation score was generated for each frame
of the time-lapse. The running score was then smoothed (Savitzky-Golay
smoothing filter) and the expected time window was analyzed for local maxima.
The error in event calling for the computer is very large (greater than what
we see for the overall spread across individuals of a single species at a
given temperature), necessitating manual verification or correction of events.
Many of these errors are due to aberrations in the image that confuse the
computer but would not confuse a person. This results in a few bad images
having a very negative effect of the overall accuracy of the computer
analysis, but permits a significant improvement with just a little user input.
The error in manual calls is very small compared to the variation between
individuals. Computer-aided estimates were individually verified or corrected
using a python GUI for all included data.

### Statistical analysis

Statistical significance of event timing was determined by t-test with
Bonferonni multiple testing corrections. Median correction to remove outliers
was used in determining the mean and standard deviation of each developmental
event. Least-squares fitting was used to determine the linear approximation of
log-corrected developmental time for each species. Python and Matlab scripts
used in the data analysis are available at
github.com/sgkuntz/TimeLapseCode.git.

## Acknowledgments

We obtained flies from the Bloomington and UCSD stock centers. We thank Paul
Schmidt for the clinal fly lines. We would also like to thank T. Kaplan and P.
Combs for their advice and assistance on data analysis and programming, C.
Bergman for comments, Ng Wei Tian for his work on event verification, and BCK
and EDKK for their support.

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## Figures

Figure 1: Geographic and climatic origin and phylogeny of analyzed Drosophila
species (A) Ancestral ranges are shown for each species [47, 17, 48]. While D.
melanogaster and D. simulans are now cosmopolitan and D. ananassae is
expanding in the tropics (green), their presumed ancestral ranges are shown.
D. virilis is holarctic (gray) and restricted from the tropics, with a poor
understanding of its ancestral range. Other species are more or less found in
their native ranges, covering a variety of climates. Sites of collection are
noted by arrows. (B) The phylogeny of the sequenced Drosophila species. Many
of the tropical species are closely related, though D. willistoni serves as a
tropical out-group compared to the melanogaster and obscura groups. Branch
lengths are based on evolutionary divergence times [49]. (C) Range sizes vary
considerably between the species. Figure 2: Developmental landmarks used in
study Many images of each stage (examples on the left) were averaged to
generate composite images (lateral view on the right) for each of the
developmental stages, of which 29 are shown. Figure 3: Developmental time of
D. melanogaster varies with temperature (A) Images of developing D.
melanogaster embryos at each temperature are shown for a selection of stages
to highlight the overall similarity of development. (B) The time individual
animals reached the various time-points are shown, with each event being a
different color. Time 0 is defined as the end of cellularization, when the
membrane invagination reaches the yolk. Between 17.5∘C and 27.5∘C the total
time of embryogenesis, $t_{dev}$ measured as the mean time between
cellularization and trachea fill, has a logarithmic relationship to
temperature described by $t_{dev}=4.02e^{37.31/T}$ where T is temperature in
∘C ($R^{2}=0.963$). (C) The developmental rate in D. melanogaster changes
uniformly with temperature, not preferentially affecting any stage. Timing
here is normalized between the end of cellularization and the filling of the
trachea.  Figure 4: Drosophila species develop at different rates and respond
to temperature in distinct ways (A) Images of developing embryos of each
species are shown to scale. All species go through the same stages in the same
order at all viable temperatures. (B) At 17.5∘C all species show uniformly
long developmental times, with D. virilis and D. mojavensis being
significantly longer than other species. (C) At 22.5∘C and (D) 27.5∘C there is
considerably more variation between species. While developmental times
decrease with increasing temperature across all species, the effect is muted
in the alpine species. (E) At 30∘C, developmental rate has stopped
accelerating and the alpine species are seeing considerable slow-down in
development time.  Figure 5: Temperature dependent developmental rates are
climate specific The time between the end of cellularization and trachea fill
are shown for all species at a range of temperatures. The climatic groups –
tropical (warm colors), alpine (blues), temperate (purple), and sub-tropical
(green) – clearly stand out from one another to form four general trends.
Figure 6: Proportionality of developmental stages is not affected by non-heat-
stress temperatures (A) Across species, development maintains the same
proportionality. D. pseudoobscura stands out as not being co-linear at higher
temperatures. Instead, the later part of its development is slowed and takes
up a disproportionally long time. (B) Plotting proportionality across all
species and all temperatures reveals the approximately normally distributed
proportionality of all morphological stages.

## Tables

Table 1: Drosophila species and strains Species | Stock number | Strain | Collection site
---|---|---|---
D. melanogaster |  | OreR | Oregon, USA
D. pseudoobscura | 14011-0121.94 | MV2-25 | Mesa Verde, Colorado, USA
D. virilis | 15010-1051.87 | McAllister V46 | unknown, possibly Asia
D. yakuba | 14021-0261.01 | Begun Tai18E2 | Liberia
D. persimilis | 14011-0111.49 | Machado MSH3 | Mt. St. Helena, California, USA
D. simulans | 14021-0251.195 | Begun simw501 | Mexico City, Mexico
D. erecta | 14021-0224.01 | (TSC) | unknown, probably Africa
D. mojavensis wrigleyi | 15081-1352.22 | Reed CI 12 IB-4 g8 | Catalina Island, California, USA
D. sechellia | 14021-0248.25 | (Jones) Robertson 3C | Cousin Island, Seychelles
D. willistoni | 14030-0811.24 | Powell Gd-H4-1 | Guadeloupe Island, France
D. ananassae | 14024-0371.13 | Matsuda (AABBg1) | Hawaii, USA
Table 2: Major morphological events in Drosophila development Event | Stage [19, 46] |  Notes
---|---|---
Posterior gap appears | 2 |  Gap between yolk and vitelline membrane
Pole bud appears | 3 |  Cells migrate into the posterior gap
Nuclei at periphery | 4 |  Cells migrate to edges
Pole cells form | 4 |  Replication of the pole cells
Yolk contraction | 4 |  Light edge of embryo expands
Cellularization begins | 5 |  Cell cycle 14
Membrane reaches the yolk | 5 |  This is regarded as the zero time-point
Pole cells migrate | 6 |  Pole cells begin anterior movement
Cephalic furrow forms | 6 |  Dorsal and ventral furrows form
Pole cells invaginate | 7 |  Pole cells enter dorsal furrow
Transversal fold formation | 7 |  Dorsal furrows between amnioproctodeum and cephalic furrow
Cephalic furrow reclines | 8 |  Dorsal furrow moves posteriorly
Amnioproctodeal invagination | 8 |  Invagination approaches cephalic fold
Anterior midgut primordial | 8 |  Tissue thickens at anterior ventral edge
Stomodeal plate forms | 9 |  Ventral gap anterior to cephalic fold
Stomodeum invagination | 10 |  Ventral furrow anterior to cephalic fold
Clypeolabral lobe forms | 10 |  Dorsal, ventral furrows both present
Germ band maxima | 11 |  Maximum extension of germband
Clypeolabrum rotates | 11 |  Clypeolabrum shifts dorsally
Posterior gap | 11 |  Gap forms before germband shortening
Gnathal bud appears | 12 |  Ventral tissue between the clypeolabrum and cephalic folds moves anteriorly
Germband retraction begins | 12 |  Movement begins mid-germband
Amnioserosa exposed | 12 |  Germband retracted to the posterior 30% of the embryo
Germband retracted | 13 |  Germband fully retracted
Dorsal divot | 14 |  Dorsal gap between head and amnioserosa
Clypeolabrum retracts | 14 |  Clypeolabrum pulls away from anterior vitelline membrane
Anal plate forms | 14 |  Posterior depression forms
Midgut unified | 14 |  Dark circle forms at embryo’s center
Heart-shaped midgut | 15 |  Triangular midgut
Clypeolabrum even with ventral lobes | 16 |  Ventral lobes move anteriorly to be even with clypeolabrum
Gnathal lobes pinch | 16 |  Gnathal lobes meet
Convoluted gut | 16 |  Separation between sections of the midgut
Head involution done | 17 |  Head lobes complete anterior migration
Muscle contractions | 17 |  Head begins twitching
Trachea fills | 17 |  Developmental end point
Hatch | 17 |  Highly variable
Table 3: Drosophila development videos

Subject | Link
---|---
D. melanogaster with labelled stages | http://www.youtube.com/watch?v=dYSrXK3o86I
D. melanogaster with labelled stages at reduced framerate | http://www.youtube.com/watch?v=QKVmRy3dDR0
D. melanogaster at 7 temperatures | http://www.youtube.com/watch?v=-yrs4DcFFF0
11 species at 17.5∘C | http://www.youtube.com/watch?v=HId_Idz-GhQ
11 species at 22.5∘C | http://www.youtube.com/watch?v=jO6JfgwMaH4
11 species at 27.5∘C | http://www.youtube.com/watch?v=vlYeuFqKQhI
D. ananassae at 7 temperatures | http://www.youtube.com/watch?v=vy6L4fmWkso
D. mojavensis at 6 temperatures | http://www.youtube.com/watch?v=XWMs4oUx_mU
D. virilis at 6 temperatures | http://www.youtube.com/watch?v=eyr4ckDb0kM
D. pseudoobscura at 6 temperatures | http://www.youtube.com/watch?v=sYi-FUXpv4Q

* All videos available at DOI:10.5061/dryad.s0p50

Table 4: The timing of specific developmental events can be predicted as a function of total developmental time Stage |  Event Timing (hours post cellularization) | Percent Error
---|---|---
Pole bud appears |  $t_{pba}\approx-0.093t_{dev}$ | 8%
Pole cells invaginate |  $t_{pci}\approx 0.018t_{dev}$ | 40%
Amnioproctodeal invagination |  $t_{api}\approx 0.035t_{dev}$ | 18%
Amnioserosa exposed |  $t_{ase}\approx 0.35t_{dev}$ | 6%
Clypeolabrum retracts |  $t_{clr}\approx 0.49t_{dev}$ | 4%
Heart-shaped midgut |  $t_{hsm}\approx 0.57t_{dev}$ | 12%
Table 5: The developmental time of embryos between 17.5∘C and 27.5∘C is a
species-specific function of temperature

Species | Developmental Time* | $R^{2\dagger}$ |  95% Confidence Prediction Interval for Future Observations |  $Q_{10}^{\ddagger}$ (27.5:17.5)
---|---|---|---|---
D. virilis# | $t_{Dvir}=5.64e^{37.08/T}$ | 0.989 |  $t_{Dvir}\pm 31.937\sqrt{1.00+(\frac{1}{T}-0.04)^{2}}$ |  2.2
D. mojavensis# | $t_{Dmoj}=3.67e^{43.81/T}$ | 0.983 |  $t_{Dmoj}\pm 54.263\sqrt{1.00+(\frac{1}{T}-0.05)^{2}}$ |  2.5
D. willistoni | $t_{Dwil}=3.63e^{40.50/T}$ | 0.944 |  $t_{Dwil}\pm 3.122\sqrt{1.00+(\frac{1}{T}-0.04)^{2}}$ |  2.3
D. pseudoobscura | $t_{Dpse}=7.61e^{25.95/T}$ | 0.903 |  $t_{Dpse}\pm 39.257\sqrt{1.00+(\frac{1}{T}-0.05)^{2}}$ |  1.7
D. persimilis | $t_{Dper}=9.31e^{21.20/T}$ | 0.961 |  $t_{Dper}\pm 22.598\sqrt{1.00+(\frac{1}{T}-0.05)^{2}}$ |  1.6
D. ananassae | $t_{Dana}=2.94e^{42.68/T}$ | 0.979 |  $t_{Dana}\pm 1.440\sqrt{1.00+(\frac{1}{T}-0.05)^{2}}$ |  2.4
D. yakuba | $t_{Dyak}=4.67e^{33.08/T}$ | 0.943 |  $t_{Dyak}\pm 2.203\sqrt{1.00+(\frac{1}{T}-0.05)^{2}}$ |  2.0
D. erecta | $t_{Dere}=5.21e^{32.97/T}$ | 0.937 |  $t_{Dere}\pm 2.689\sqrt{1.00+(\frac{1}{T}-0.04)^{2}}$ |  2.0
D. melanogaster | $t_{Dmel}=4.02e^{37.31/T}$ | 0.963 |  $t_{Dmel}\pm 1.281\sqrt{1.00+(\frac{1}{T}-0.05)^{2}}$ |  2.2
D. sechellia | $t_{Dsec}=4.47e^{34.67/T}$ | 0.957 |  $t_{Dsec}\pm 2.386\sqrt{1.00+(\frac{1}{T}-0.04)^{2}}$ |  2.1
D. simulans | $t_{Dsim}=3.50e^{39.14/T}$ | 0.960 |  $t_{Dsim}\pm 1.883\sqrt{1.00+(\frac{1}{T}-0.05)^{2}}$ |  2.3

* End of cellularization to trachea fill in hours, where T is in ∘C

$\dagger R^{2}$, the Pearson Product-Moment’s Correlation Coefficient of
determination, is calculated following a least-squares regression across all
data points to a curve of the form ln(developmental time) = b($1\over{T}$)+a.

$\ddagger$ $Q_{10}$ is the ratio between developmental times across a 10
degree interval, in this case between 27.5∘C and 17.5∘C. A value of 2.2 would
indicate that development takes 2.2 times as long at 17.5∘C than at 27.5∘C.

$\\#$ Curve fit through 30∘C

## Supplementary Figures

Figure S1: Microscopy imaging setup (A) The imaging setup, showing the
dissecting microscope with temperature control apparatus on the automated
stage. (B) A close-up view of the temperature controlled platform flanked by
heat-sinks (blue) that sit atop the Peltier thermoelectric controllers. In the
center is a copper plate, with a thermister at the bottom to monitor plate
temperature. The holes in the green masking tape line up with holes drilled
through the copper plate and lined with a gas-permeable membrane. The masking
tape helps retain the halocarbon oil. (C) A closer view of the setup. (D) A
schematic of the setup demonstrates the temperature control and imaging
apparatus in cross-section. Figure S2: Events were predicted by computational
analysis before manual verification (A) For every time-lapse, each frame was
correlated to each of the 34 composite images. (B) The running scores for 6
different events, with their maxima (black arrows) highlighted to reflect the
estimated event time. (C) The time of amnioserosa exposure is estimated by the
strong correlation at about 450 frames into the time-lapse. Figure S3:
Identifying morphological stages (A) ‘Pole bud appears’ stage is identified by
the first appearance of cells migrating into the posterior gap of the embryo
(black arrow). (B) ‘Membrane reaches yolk’ stage is identified by the
converging of the leading edge of the invaginating cytoplasmic membrane on the
dark yolk. (C) ‘Pole cell invagination’ is identified by the completion of the
fold (black arrow) that encapsulates the pole cells (yellow arow). (D)
‘Amnioproctodeal invagination’ is identified by the point when the leading
edge of the posterior invagination (black arrow) has covered $\sim$80% of the
distance to the leading edge of the cephalic furrow (vertical yellow line) and
the pole cells have reached the interior of the embryo. (E) ‘Amnioserosa
exposed’ is identified by the point when the trailing edge of the germ band
has retracted to the posterior 30% of the embryo. (F) ‘Clypeolabrum retracts’
is identified by the withdrawal of the ventral edge of the clypeolabrum (black
arrow) from the gnathal buds and vitelline membrane to create a gap (black
arrowhead). (G) ‘Heart-shaped midgut’ is identified by the posterior
elongation of the formerly spherical developing midgut and residual yolk (dark
mass in the center of the embryo) to form a contiguous dark teardrop or heart-
shaped mass (delimited with yellow lines). (H) ‘Trachea fill’ is identified by
the rapid darkening of the trachea as they fill. The primary branches of the
trachea run along the both the left and right dorsal sides, originating at the
posterior of the embryo. Figure S4: Prediction of future observations of
development at different temperatures The behavior of developing embryos can
be predicted. The mean line (green) generated from least-squares curve-fitting
(Table 5) and the 95% confidence prediction interval for future observations
(dashed orange line) are shown for each species. Figure S5: Different D.
melanogaster wild isolate strains exhibit a limited range of temperature
responses (A) Lines (R303, R324, R379, R380, R437, and R705) collected near
Raleigh, North Carolina [25] exhibit a range of temperature responses. (B)
Clinal lines from Florida (DmelFL), Pennsylvania (DmelPA), and Maine (DmelME)
[26] exhibit a range of responses similar to those of the Raleigh lines.
Despite their clinal distribution, no trends are seen, with flies from Florida
and Maine being virtually indistinguishable. This is possibly due to their
relatively recent introduction across the cline. (C) Despite the differences
between the D. melanogaster lines above, they all (seen here grouped together
as light blue points) lie within the response range seen for the melanogaster
species subgroup, mainly falling between the responses of D. melanogaster
Ore-R and D. erecta. Like Ore-R, their growth is significantly slower than D.
yakuba, D. ananassae, D. simulans, and D. sechellia, but obeys the same
general trend. Figure S6: Ten species of Drosophila exhibit dynamic response
to temperature changes (A-F) There is some variation species to species, but
all tropical Drosophila exhibit a similar temperature response-curve to D.
ananassae. (G) Temperate D. virilis also has a steep response, though
intermediate to the previous two groups. (H) Sub-tropical D. mojavensis has a
steeper temperature response, though a similar high temperature developmental
time. (I,J) Alpine D. pseudoobscura and D. persimilis have a cold response
like the tropical species, but longer developmental times at warmer
temperatures. Figure S7: Heat-stress affects syncytial developmental
proportionality and morphology (A) At heat-stress temperatures, the
proportionality of developmental stages is affected in some, but not all,
embryos. (B) Heat stress in D. melanogaster at 32.5∘C affects morphology
during yolk contraction and gastrulation. Embryos may exhibit asynchronous
yolk-contraction (first image), uneven nuclear distribution during
cellularization (second image), or disrupted morphology during gastrulation
(third image). (C) Heat shock at 37.5∘C for 30 minutes reveals embryos
sensitivity prior to the completion of cellularization. Most animals that had
completed cellularization survived heat-shock and continued to develop
properly (blue diamonds), while no animals that had not completed
cellularization prior to heat-shock survived. All embryos that died (orange
stars) exhibited severe morphological disruptions. (D) Linear regression of
stages across different temperatures reveals that, despite significant
variance in later stages (shown in colored bars), only the pre-cellularization
time point is affected by heat-stress enough to exhibit a significantly
different slope between higher temperatures (27.5∘C and above, yellow bar) and
lower temperatures (25∘C and below, red bar).  Figure S8: Temperature
conditioning of adult flies leads to some heat tolerance (A) D. melanogaster
raised for many generations at 25 ∘C, 22 ∘C, and 18 ∘C produce embryos that
show similar temperature responses, though there is some accelerated growth
when acclimatized to higher temperatures. There is no indication of severe
heat shock as embryos are moved from the acclimatized temperature to the
experimental temperature. (B) D. mojavensis and D. virilis exhibit a similar
trend of only minor differences between strains acclimatized at 25 ∘C and 22
∘C. (C) The difference between D. melanogaster raised at 22 ∘C and D.
persimilis also raised at 22 ∘C remains significant, indicating that the heat-
stress response of D. persimilis is not due simply to its being raised at 22
∘C.