PHYSIOLOGICAL ECOLOGY

Effect of Temperature on Post-Wintering Development and Total

Lipid Content of Alfalfa Leafcutting Bees
KEVIN M. O’NEILL,1,2 RUTH P. O’NEILL,3 WILLIAM P. KEMP,4 AND CASEY M. DELPHIA1

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Environ. Entomol. 40(4): 917Ð930 (2011); DOI: 10.1603/EN10320
ABSTRACT Temperature plays an important role in effective management of the alfalfa leafcutting
bee [Megachile rotundata (F.); Megachilidae], the major commercial pollinator of seed alfalfa [Medi-
cago sativa (L.); Fabaceae] in North America. To improve our understanding of threshold and
optimum rearing temperatures of M. rotundata, we examined the effect of temperature on postwin-
tering development by using a greater number of temperature treatments than applied in previous
studies (19 versus eight or fewer) and analytical tools formulated to model nonlinear relationships
between temperature and insect development rates. We also tested the hypothesis that rearing
temperature inßuences adult body lipid content at emergence, which could affect adult survival,
establishment and performance as a pollinator, and reproductive success. We found that the Lactin-2
and Briere-2 models provided the best Þts to data and gave reasonable estimates of lower (16 Ð18⬚C)
and upper (36 Ð39⬚C) developmental thresholds and optimum (33Ð34⬚C) rearing temperatures for
maximizing development rate. Bees successfully emerged over a broad range of temperatures (22Ð
35⬚C), but variation in development rate among individuals reared at the same temperature was lowest
at 31Ð33⬚C. The optimum rearing temperature to maximize the proportion of body lipids in adults was
27Ð29⬚C. Our results are discussed in relation to previous Þndings and speak to the difÞculties in
designing practical rearing guidelines that simultaneously maximize development rate, survival, and
adult condition, while synchronizing adult emergence with alfalfa bloom.

KEY WORDS pollinators, alfalfa leafcutting bee, Megachile rotundata, thermal biology, lipids

The alfalfa leafcutting bee (Megachile rotundata F.;
Megachilidae), a solitary cavity-nesting species, is the
major commercial pollinator of alfalfa (Medicago sa-
tiva L.) grown for seed production in western North
America (Pitts-Singer 2008). Within nests, females
construct a linear series of cells, each lined and capped
with leaf pieces cut from nearby vegetation and pro-
visioned with a mass of pollen and nectar. In managed
populations, females are provided with large, high-
density arrays of nest tunnels within “bee shelters”.
Producers that maintain large populations of bees
across growing seasons can forego the need to pur-
chase bees from commercial suppliers each year, and
may even be able to sell bees to other growers.
In western North America, the life cycle of M. ro-
tundata starts in late June to early July when females
begin laying eggs on pollen-nectar masses within leaf
cells. After completing Þve larval stages, the bees ei-
ther emerge several weeks later as second generation
adults (nondiapausing bees) or enter diapause in the
prepupal stage within their leaf cells sometime in the
1 Department of Land Resources and Environmental Sciences,
Montana State University, Bozeman, MT 59717.
2 Corresponding author, e-mail: koneill@montana.edu.
3 Department of Plant Sciences and Plant Pathology, Montana State
University, Bozeman, MT 59717.
4 USDA, Agricultural Research Service, Red River Valley Agricultural
Research Center, 1605 Albrecht Blvd. North, Fargo, ND 58102-2765.
fall. Cells of diapausing bees that provide pollinators
for the next growing season, may be left by producers
within the artiÞcial nesting materials or removed and
stored as “loose cells”. After overwintering, bees are
then reared to the adult stage and released back into
Þelds (Delaplane and Mayer 2000, Pitts-Singer 2008,
Pitts-Singer and Cane 2011).
Effective management of M. rotundata includes
rearing procedures that attempt to maximize survival
of the immature stages while timing development so
that adults emerge in synchrony with crop bloom.
Thus, because of its potential importance to both the
survival and development rate of bees, researchers
have studied effects of temperature on all stages of M.
rotundataÕs life cycle, looking at both short- and long-
term exposure to alternative temperature regimes.
Different studies have addressed the relationship of
long-term rearing temperatures to development and
survival before (WhitÞeld and Richards 1985, Te-
pedino and Parker 1986, (Richards et al. 1987), Kemp
and Bosch 2001, Pitts-Singer and James 2009); during;
and after (Stephen and Osgood 1965, Rank and Go-
erzen 1982, Richards and WhitÞeld 1988, Kemp and
Bosch 2000) overwintering. Others have examined the
effects of short-term exposure to high (Undurraga and
Stephen 1980a, Barthell et al. 2002) and low temper-
atures (Undurraga and Stephen 1980b); or to condi-
tions that affect survival of immature stages during the

0046-225X/11/0917Ð0930$04.00/0 䉷 2011 Entomological Society of America

918 ENVIRONMENTAL ENTOMOLOGY
summer within Þelds (Peterson et al. 1994, Richards
1996, Pitts-Singer and James 2008). The role of tem-
perature in mediating speciÞc factors that affect bee
mortality and production have been studied for chalk-
brood fungus (Ascosphaera aggregata Skou) (Vanden-
berg and Goettel 1995, James 2005); parasitoid wasps
(Pteromalus venustus Walker) (WhitÞeld and Rich-
ards 1985); and “pollen ball” (Pitts-Singer and James
2008). Because researchers can work with large pop-
ulations, M. rotundata also provides a convenient
model organism for studies of the thermal biology of
solitary bees.
The goals of research on the effects of temperature
on bees being reared from the overwintering prepupal
phase to the adult stage in late spring for release into
Þelds have included determining: 1) the lower and
upper threshold temperatures between which devel-
opment occurs, 2) the range of rearing temperatures
in which survival of immature and postemergent
adults is maximized, 3) the temperatures at which the
rate of development is maximized and the emergence
of the sexes is most synchronized, and 4) the effect of
rearing temperature on subsequent success of adults
(Stephen and Osgood 1965, Rank and Goerzen 1982,
Richards and WhitÞeld 1988, Kemp and Bosch 2000).
Despite progress already made, however, there is po-
tential for reÞning our knowledge of threshold and
optimum rearing temperatures, and the effects of tem-
perature on adult condition. One important question
is whether one can simultaneously maximize devel-
opment rate, survival, and adult condition, without
being forced to choose which outcomes are most im-
portant and which have to be compromised.
Our overall goal was to further explore the effect of
temperature on postwintering development under ex-
perimental conditions in which we could impose a
greater number of temperature treatments than in
previous studies. By rearing bees on a laboratory ther-
mal gradient, we were able to use 19 different tem-
peratures, which exceeds the maximum number of
treatments (eight; Richards and WhitÞeld 1988) in
previous studies of postwintering of M. rotundata. The
greater number of treatments allowed us to more
carefully deÞne nonlinear relationships between rear-
ing temperature and the biological parameters of in-
terest. We also used development-rate models (Lactin
et al. 1995, Briere et al. 1999) that have not been
applied previously to this species, but which have
been effective when used for other insects (Kontodi-
mas et al. 2004). The nonlinear relationship between
temperature and the developmental rate of M. rotun-
data was recognized previously (Stephen and Osgood
1965, Richards and WhitÞeld 1988), but not modeled
quantitatively for postwintering development. A sec-
ond novel aspect of our study is that we used the
method described by Strohm (2000) to test the hy-
pothesis that rearing temperature inßuences the total
amount of body lipids carried by adults upon emer-
gence; we assume that lipids (which are primarily
from fat bodies) affect posteclosion survival and re-
productive success. Our speciÞc objectives were,
therefore, to estimate for each sex: 1) the lower and
Vol. 40, no. 4
upper threshold temperatures for development, 2) the
rate of development at different rearing temperatures,
3) the range of temperatures associated with the high-
est proportion of successfully emerging adults, and 4)
the temperature that produces adult bees with the
highest proportion of body lipids at emergence.
Materials and Methods
Sources of Bees. Bees used in the experiments were
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obtained from three sources. In 2007, we used bees
reared in polystyrene laminate bee boards (Beaver
Plastics, Ltd., Acheson, Alberta, Canada) within bee
shelters we had placed on an alfalfa (Medicago sativa
L.) seed farm 3 km south of Rockvale, MT in summer
2006. The next 2 yr, we obtained bee cells after they
had been removed from boards by cooperating pro-
ducers, in 2008 from a seed alfalfa farm near Laurel,
MT and in 2009 from JWM Leafcutters, Nampa, ID.
Each year, bee cells were placed in winter storage in
an incubator set at 8⬚C, 80% RH at the end of October.
Rearing Bees on Thermal Gradient. In June each
year (2007Ð2009), we placed prepupae (still in cells
made of their cocoons and the leaf pieces that sur-
rounded them) into gelatin capsules for rearing on a
laboratory thermal gradient consisting of four parallel
15- by 100- by 0.8-cm-thick aluminum bars, the ends of
which rested upon two 2.5-cm-wide, hollow aluminum
rods (square cross-section) oriented perpendicular to
the long axis of the bars. Water from a hot water bath
was pumped in a loop through the two rods beneath
one end of the bars, while another pumped a water and
propylene glycol mixture from a cold bath through the
rods at the other end. Temperature (Tbar) was mea-
sured every 10 cm along each bar with thermocouples
imbedded from below into the center of the bar. To
help stabilize temperature, each bar was enclosed
within a 2.5-cm high clear Plexiglas cover (Altuglas
International, Philadelphia, PA), except when
emerged bees were being removed; the covers Þt
loosely over the bar to allow air exchange. Before the
Þrst emergence of adult bees and during nights there-
after, the entire apparatus sat within an opaque en-
closure. Tbar ranged from 14 to 41⬚C, though there was
some variation between years. In linear regressions of
the Tbar on the linear position along the bar, r2 values
ranged from 0.96 to 0.99.
At the beginning of each experiment in mid-June of
each year, bee cells were removed from winter stor-
age. Within 2 h, we placed 760 cells within no. 00
gelatin capsules that had been punctured twice on
each end with a pin to provide ventilation. Before
being placed into the capsules, cells were examined to
help assure that they had been capped by the provi-
sioning females and were intact; cells in which a bee
does not develop to the overwintering stage tend to be
fragile because of the lack of cocoon silk that strength-
ens the cell. At each 5-cm interval along the length of
a bar (from the 5-cm to the 95-cm positions), 10 cells
were placed in a row across the bar; each of the four
bars thus had 19 rows of cells experiencing 19 tem-
perature treatments. Gelatin capsules were anchored
August 2011 OÕNEILL ET AL: TEMPERATURE AND M. rotundata DEVELOPMENT
to the bars with double-sided cellophane tape, so that
the center of each cell sat exactly on a line marking a
5-cm interval. Bees were present on the gradient from
14 June to 5 October 2007 (113 d), 5 June to 11
September 2008 (101 d), and 12 June to 21 September
in 2009 (98 d). The gradient was monitored multiple
times each day until such time as 1 wk passed without
adult emergence; when adult bees emerged, they
were placed in a freezer (⬇⫺22⬚C) within their gel-
atin capsules.
To determine actual rearing temperature (Trt) ex-
perienced by developing bees, we conducted mea-
surements on a set of capsules, each containing a dried
bee cell (including its leaf-capsule) into which a 0.3-
mm-diameter hypodermic thermocouple probe
(Omega Engineering, Inc., Stamford, CT) had been
inserted with its tip within the center of the bee cell;
dried bee cells were used to prevent evaporative water
loss from affecting temperature measurements when
the cell was punctured by the thermocouple probe.
The bee cells were moved to different positions on the
gradient (with the Plexiglas lid in place) where tem-
perature (Tcell) was allowed to stabilize before re-
cording. Two measurements (using different leaf cap-
sules) were undertaken at each 10-cm interval on all
four bars. By regressing capsule temperature Tcell on
Tbar, we created an equation for converting bar tem-
perature to the temperature experienced by develop-
ing bees (r2 ⫽ 0.995, N ⫽ 72):
T rt ⫽ 2.7045 ⫹ 0.8818 ⫻ T bar
Development Rates and Developmental Threshold
Temperature. The relationship between days to emer-
gence (D) and Trt for each sex and each of the 3 yr was
characterized with a quadratic regression by using
SigmaStat (San Jose, CA). To examine whether vari-
ation in the rate of development (1/D) changed with
Trt, we calculated the coefÞcient of variation (⫽[stan-
dard deviation/mean] ⫻ 100; Zar 1999) of 1/D after
combining data into 3⬚C intervals (over the Trt range
from 22 to 36⬚C).
By examining the relationship of 1/D to Trt, one can
determine the lower developmental threshold (Tmin),
the temperature above which development proceeds.
In linear models, Tmin is estimated as the x-intercept
value when 1/D ⫽ 0. In some nonlinear models, Tmin
is the lower intercept value when the regression line
intercepts the x-axis twice. Nonlinear models may also
estimate Tmax, the temperature (i.e., the upper x-in-
tercept value) above which the “rate of development
is zero or life cannot be maintained for a prolonged
period” (Kontodimas et al. 2004). Based on visual
examination of our data, previous studies of develop-
ment rate in M. rotundata (Stephen and Osgood 1965,
Richards and WhitÞeld 1988), and studies comparing
the value of alternative temperature-development
models (Lactin et al. 1995, Briere et al. 1999, Kontodi-
mas et al. 2004), we examined the nonlinear relation-
ship of 1/D to Trt by using three models: quadratic
model, the Lactin-2 model (Lactin et al. 1995), and the
Briere-2 (Briere et al. 1999), all of which estimate Tmin,
Tmax, and Topt (i.e., the temperature optimum ⫽ Trt at
919
which developmental rate is the highest). The models
were assessed using TableCurve 2D, version 4.06
(SPSS Inc. 1996) and six different data sets, one from
each year for the two sexes.
The quadratic model is of the form 1/D ⫽ a ⫹ bTrt ⫹
cTrt2, where a, b, and c are Þtted coefÞcients and the
roots of the quadratic equation provide estimates of
Tmin and Tmax. The Lactin-2 model takes the form:
1/D ⫽ e␳Trt Ð e关␳Tm Ð (␳Tm Ð Trt)/⌬] ⫹ ␭
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where ␳, Tm, ⌬, and ␭ are Þtted coefÞcients; Tmin and
Tmax are determined from the two x-intercepts. The
Briere-2 model, which provides direct estimates of
Tmin and Tmax as well as standard errors for both
variables, is of the form:
1/D ⫽ aTrt(Trt - Tmin)(Tmax - Trt)1/m
where a, Tmin, Tmax, and m are Þtted coefÞcients. Both
the Lactin-2 and Briere-2 models are appropriate for
data sets in which 1/D declines relatively sharply after
Trt exceeds Topt, as was the case for some of our data
sets. A model was considered superior to the other
nonlinear models if it produced a higher r2 and lower
residual sum of squares (RSS) than alternative models
(Kontodimas et al. 2004).
Several earlier studies of postwintering develop-
ment in M. rotundata either provide linear models of
the relationship of 1/D to Trt (Richards and WhitÞeld
1988) or present data over ranges of Trt in which a
linear relationship can be examined (Rank and Go-
erzen 1982, Kemp and Bosch 2000). Therefore, we also
constructed linear models (for each year ⫻ sex com-
bination) across the range of rearing temperatures
(Trt ⱕ 32⬚C) when the relationship of 1/D to Trt was
clearly linear. This approach follows that of Richards
and WhitÞeld (1988) who examined developmental
rate across a Trt range of 20 Ð32⬚C. For comparison to
the results of Rank and Goerzen (1982) and Kemp and
Bosch (2000), we conducted our own analyses of their
published mean development times at different tem-
peratures. The linear regression model was of the form
1/D ⫽ aTrt ⫹ b, where a ⫽ the slope and b ⫽ the
y-intercept. Linear models cannot determine Tmax and
Topt, but can estimate degree-days (dd) for develop-
ment from the period of removal of the bees from
winter storage to adult emergence (i.e., as the recip-
rocal of the slope of the regression line; Richards and
WhitÞeld 1988).
Adult Emergence Success and Sex Ratio. Each year,
we immediately froze all intact cells (from which no
adults emerged) after 1 wk had passed without any
adults emerging; the latter occurred from mid-Sep-
tember to early October. We later dissected each cell
to determine the stage to which the bee had devel-
oped at the time at which it was removed from the
gradient; stage was recorded as prepupa, pupa, or
unemerged adult male or female. We also recorded
whether the cell contained evidence of chalkbrood
disease or parasitoids, or whether it could be catego-
rized as having a condition referred to as “pollen ball”.
Pollen ball occurs when a cell contains a mass of pollen
920 ENVIRONMENTAL ENTOMOLOGY
and nectar, but either no egg or an egg that never
hatched (Pitts-Singer 2008).
During the 3 yr, adult bees emerged from 10 to 12
of the 19 bands of cells (temperature treatments) on
the gradient bars. For each temperature band (N ⫽ 4),
we determined: 1) the mean Tbar while cells were
present on the gradient, based on weekly measure-
ments taken each year; 2) the mean proportion of cells
that contained prepupae, pupae, and unemerged
adults at the end of observations; 3) the mean pro-
portion of cells that produced adult bees that emerged
from their leaf cells; and 4) the proportion of females
among emerged adults. All nonviable cells were ex-
cluded from analyses, so that the total number of cells
associated with a temperature band ranged from 29 to
40, though only three of 57 sets had fewer than 35
viable cells; all means are presented with associated
standard errors. To test the hypothesis that the de-
velopmental stage reached by bees was temperature-
dependent, we compared the proportion of bees in a
given stage across temperatures by using KruskalÐ
Wallis tests. To test the hypothesis that mortality was
sex-biased, we regressed the arcsine-square root trans-
formed proportion of females (combined across bars
for each band of cells on the gradient) on the mean
temperature for that band across the four bars.
One potential limitation of our study is that we did
not know the cell contents when they were placed on
the gradient. All cells were stored at room tempera-
ture (⬇25Ð26⬚C) between removal from the Þeld and
winter storage several months later, so any healthy
nondiapausing bees should have completed develop-
ment and emerged before wintering took place. Some
nondiapausing bees might have died during develop-
ment in the fall, and have been misdiagnosed as dying
during postwintering development because cells were
not opened for inspection until after the latter period.
However, because cells were randomly assigned to
positions on the bars, this would be unrelated to the
temperature treatments received. The development
patterns we observed across temperatures suggest that
few nondiapausing bees remained in the cells we in-
cubated. Each year, of the 19 sets of cells on each bar,
5Ð 6 sets produced no adults (eclosed or uneclosed).
Temperature, Adult Body Size, and Lipid Content.
For each adult bee that emerged (2007 and 2008 stud-
ies only), we determined its head width to the nearest
0.04 mm with a microscope equipped with an ocular
micrometer. Each bee was placed in a numbered glass
vial that was then put into a 55⬚C drying oven for 5 d,
at which time their dry mass had stabilized. The dry
mass of each bee was then determined to the nearest
0.01 mg on a Sartorius TE64 balance. Following the
method given by Strohm (2000) to extract body fat
from dry insects, we immediately added 5 ml (2007)
or 10 ml (2008) of petroleum ether (EMD Chemicals
Inc, Gibbstown, NJ) to each vial and capped the vial.
After six days, the petroleum ether extract was de-
canted and the vial was air dried for 1 h under a
laboratory hood. The vials were then placed back in
the oven for 24 h before a postextraction body mass
was obtained. The difference between the two masses
Vol. 40, no. 4
for each bee was considered to be the amount of fat
extracted; we then calculated the proportion of dry
mass comprised of body fat (Pf). Each year, all bees of
both sexes were subjected to the drying, weighing, and
fat extraction protocol at the same time.
Emergence Success and Lipid Content in Response
to Short-term Exposure to 38ⴗC. Because no adults
emerged from cells in which Trt exceeded 36⬚C, we
conducted another experiment in which sets of de-
veloping bees were subjected to a temperature of 38⬚C
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for durations of 0.5Ð 48 h. The experiment was con-
ducted at four different times after removal of cells
from overwintering storage in June 2009. The four
times (age treatments) were designed to correspond
to different stages in the postwintering development
of bees: 2 d and 7 d (prepupae); 13 d (early pupae,
when most have white body color, but dark eyes); and
17 d (late pupae, when most have dark body and eye
color) (International Pollination Systems 2011). At
the beginning of the experiment, we prepared 32 sets
of 50 bee cells each within ventilated gelatin capsules
(as described above); each set was placed in a clear
plastic 500-ml cup. These sets were randomly divided
into four sets of eight cups to be used at the four
designated ages. To start incubation, all 32 sets were
placed in an incubator set at a constant 29⬚C. At 0800
hours, 2 d after incubation began, seven sets were
moved to an incubator set at 38⬚C (range: 37Ð 40⬚C).
An open pan of water in each incubator maintained
relative humidity at ⬇25%. After being placed at the
higher temperature, one set of cells was removed and
returned to 29⬚C after each of the following times:
0.5 h, 1 h, 2 h, 4 h, 8 h, 24 h, and 48 h (exposure-duration
treatments); the one set that remained in the 29⬚C
incubator during the entire experiment acted as the
control for each age treatment. The cells were then
monitored for adult emergence and the total number
of emerged adults was recorded daily; all bees emerg-
ing were immediately frozen for later analysis of body
fat mass (of females only) by using the same methods
described above. The same protocol was followed for
each of the four age treatments.
To test the hypothesis that mortality varied with
exposure-duration treatment in each age treatment,
the frequency distributions of the total number of bees
emerged across the eight exposure times were ana-
lyzed with ␹2 goodness-of-Þt tests, with the null hy-
pothesis being equal emergence across exposure-du-
rations. To test the hypothesis that mortality varied
with age treatment within each exposure-duration
treatment, the frequency distributions of the total
number of bees emerged across the four ages were
analyzed with ␹2 goodness-of-Þt tests, with the null
hypothesis being equal emergence across ages. Within
each age treatment group, we tested the hypothesis
that Pf of females after emergence varied among ex-
posure-duration treatments by using linear regression,
regressing the arcsine square-root transformed Pf on
duration of exposure to 38⬚C. We also used a Kruskal-
Wallis test (with DunnÕs test for multiple compari-
sons) to determine whether the mean proportion fat
varied among age treatments.
August 2011 OÕNEILL ET AL: TEMPERATURE AND M. rotundata DEVELOPMENT 921

Table 1. Quadratic regressions of time to emergence (D) on rearing temp (Trt) for M. rotundata; all regressions significant at P < 0.001

Study Sex N Regression of D on Trt F d.f. r2

2007 Female 138 D ⫽ 508.54 ⫺ 31.324Trt ⫹ 0.502Trt2 640.2 2, 135 0.91
Male 288 D ⫽ 376.04 ⫺ 22.393Trt ⫹ 0.349Trt2 3022.9 2, 285 0.96
2008 Female 177 D ⫽ 561.46 ⫺ 33.580Trt ⫹ 0.522Trt2 1364.8 2, 174 0.94
Male 163 D ⫽ 495.68 ⫺ 29.243Trt ⫹ 0.448Trt2 1422.8 2, 160 0.95
2009 Female 136 D ⫽ 581.88 ⫺ 35.463Trt ⫹ 0.562Trt2 1191.5 2, 133 0.95
Male 314 D ⫽ 462.92 ⫺ 27.943Trt ⫹ 0.439Trt2 1774.2 2, 311 0.92

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Results
Development Rates and Developmental Threshold
Temperature. Temperatures within gel caps (Trt)
ranged from 15.1 to 38.4⬚C in 2007, 16.8 Ð39.1⬚C in 2008,
and 18.1Ð39.3⬚C in 2009. Trt differences between ad-
jacent positions on bars averaged 1.2 ⫾ 0.01⬚C in 2007,
1.2 ⫾ 0.0⬚C in 2008, and 1.2 ⫾ 0.0⬚C in 2009. The
relationship of days to adult emergence (D) to Trt
followed a quadratic relationship each year in both
sexes (Table 1; Fig. 1). In four of the 6 yr ⫻ sex
combinations, a small number of bees reared at Trt ⱖ
34⬚C emerged later, as much as 8 wk later, than other
bees reared at the same or slightly lower temperatures.
As a result, the coefÞcient of variation of the rate of
development (1/D) in those four data sets was higher
for bees reared at Trt ⱖ 34⬚C than at any other tem-
perature (Table 2); variation was lowest in the 31Ð
33⬚C range, but became increasingly higher at lower
temperatures.
All four types of regression models of development
rate provide highly signiÞcant Þts to the data for all
year ⫻ sex combinations (Table 3). The linear models,
which were restricted to Trt ⱕ 32⬚C (to mirror similar
analyses in earlier studies), provide estimates of Tmin
from 16.6 to 18.1⬚C for females and 16.3Ð19.3⬚C for
males. The linear model estimates for degree-day ac-
cumulation required to complete postwintering de-
velopment ranged from 309 to 326 dd for females and
264 Ð282 for males.
Most of the six data sets were clearly nonlinear upon
visual inspection (Fig. 2). The quadratic models cap-
tured some essence of the fact that some bees reared
at Trt ⱖ 34⬚C had unusually long developmental pe-
riods. The quadratic models predicted Tmin values of
18.5Ð20.0⬚C, Topt values of 33.5Ð36.3⬚C, and Tmax val-
ues 46.9 Ð 66.2⬚C. The Lactin-2 and Briere-2 models
provided superior results compared with the qua-
dratic models, always having higher r2 and lower RSS
values. They matched each other most closely in pre-
dictions of Topt (mean absolute difference ⫽ 0.2 ⫾
0.1⬚C, N ⫽ 6), but the Briere-2 models gave consis-
tently lower values for both Tmin (1.0 ⫾ 0.2⬚C) and
Tmax (1.5 ⫾ 0.2⬚C). The Briere-2 models had higher r2
and lower RSS values than the Lactin-2 models in four
of six comparisons. Comparing the unweighted mean
values of the nonlinear models, the Lactin-2 and Bri-
ere-2 models gave Tmin estimates lower than the qua-
dratic models (Table 4). Mean Topt estimates were all Fig. 1. Relationship of days to adult emergence to rearing
within 1.5⬚C of one another, but the quadratic models temperature for M. rotundata after removal from winter stor-
gave estimates of Tmax 13Ð23⬚C higher than Lactin-2 age. Females: closed circles, upper regression line; males:
and Briere-2 models. open squares.
922 ENVIRONMENTAL ENTOMOLOGY Vol. 40, no. 4

Table 2. Relationship of rearing temp to the coefficient of the gradient (Fig. 3). Adults emerged from cells over
variation in development rate for M. rotundata Trt ranges of 20 Ð35⬚C in 2007, 21Ð36⬚C in 2008, and
CoefÞcient of variation of
20 Ð36⬚C in 2009. In 2007, the proportion of cells from
Rearing development rate (1/D) which adults emerged peaked at ⬎90% across a broad
temp (⬚C) Females Males range of Trt, from 23 to 34⬚C. A similar broad plateau
2007 2008 2009 2007 2008 2009 of high emergence success was observed in 2008
(Trt ⫽ 22Ð35⬚C) and 2009 (22Ð32⬚C), although overall
22Ð24 16.8 17.3 19.1 14.7 21.5 18.8
25Ð27 15.2 11.1 14.5 10.3 15.6 13.6 emergence success was lower in 2008. After all cells
28Ð30 10.7 9.7 11.7 8.1 12.8 9.7 were removed from the gradient and dissected, most
31Ð33 6.8 8.2 7.9 5.5 8.3 7.6 that had been at Trt ⱖ 36 Ð37⬚C contained dead, brown,

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34Ð35 25.9 20.5 21.1 4.9
Protandry occurred at all temperatures, though it
seems to be greatest at extreme temperatures (Fig. 1).
Based on the values of 1/D at Topt in the Briere-2
models, the number of days until emergence at Topt for
females was 20 in 2007 and 22 in both 2008 and 2009.
For males, the values were 17 in 2007, 19 in 2008, and
18 in 2009.
Adult Emergence Success and Sex Ratio. Because of
its effect on development rate, rearing temperature
affected the stage to which bees developed while on
7.1 21.6
desiccated prepupae. The majority of those cells at
Trt ⱕ 18⬚C also contained prepupae, but most of those
were white, turgid, and apparently still alive. At Trt of
18 Ð19⬚C each year, ⬇40 Ð90% of the cells still con-
tained pupae by the end of rearing, apparently alive,
but stalled in that stage. In 2009, a small number of cells
containing pupae also was observed at Trt ⫽ 37⬚C.
Finally, a substantial number of intact leaf cells con-
tained apparently fully-developed, but unemerged,
adults that may have died in the cells before being
frozen. The frequency of these cells peaked at Trt ⫽
19 Ð21⬚C, with smaller peaks at Trt ⫽ 36 Ð38⬚C in 2007

Table 3. Coefficients and parameters for four models of developmental rate (1/D) as a function of rearing temp (Trt) for M. rotundata;
all regressions significant at P < 0.001. Top row represents linear regression analysis by using data for Trt < 33°C to match similar analysis
by Richards and Whitfield (1988). Sample sizes for quadratic, Lactin-2, and Briere-2 analyses same as in Table 1

Females Males
Model
2007 2008 2009 2007 2008 2009
Linear (Trt ⱕ 32⬚C)
N 127 145 114 254 144 274
a 0.00322 ⫾ 0.0001 0.00324 ⫾ 0.0001 0.00307 ⫾ 0.0001 0.00357 ⫾ 0.0000 0.00379 ⫾ 0.0001 0.00355 ⫾ 0.0000
b ⫺0.0533 ⫾ 0.0020 ⫺0.0588 ⫾ 0.0016 -0.0532 ⫾ 0.0020 ⫺0.0583 ⫾ 0.0012 ⫺0.0703 ⫾ 0.0021 ⫺0.0613 ⫾ 0.0013
Tmin 16.6 18.1 17.3 16.3 19.3 17.3
deg-days 311 309 326 280 264 282
F 2,018.3 3,278.3 1,747.5 7,025.3 2,477.0 6,628.8
r2 0.942 0.958 0.940 0.965 0.946 0.958
RSS 0.00110 0.000840 0.000942 0.00168 0.00136 0.00197
Quadratic
a ⫺0.1669 ⫾ 0.0183 ⫺0.1907 ⫾ 0.0178 ⫺0.2080 ⫾ 0.0182 ⫺0.1572 ⫾ 0.0073 ⫺0.1486 ⫾ 0.0150 ⫺0.1761 ⫾ 0.0122
b 0.0119 ⫾ 0.0013 0.0133 ⫾ 0.0013 0.0149 ⫾ 0.0013 0.0111 ⫾ 0.0005 0.0098 ⫾ 0.0011 0.0125 ⫾ 0.0009
c ⫺0.0002 ⫾ 0.0000 ⫺0.0002 ⫾ 0.0000 ⫺0.0002 ⫾ 0.0000 ⫺0.0001 ⫾ 0.0000 ⫺0.0001 ⫾ 0.0000 ⫺0.0002 ⫾ 0.0000
Tmin 18.9 20.0 19.9 18.5 19.7 19.0
Tmax 53.8 50.7 46.9 59.6 66.2 54.6
Topt 35.6 35.3 33.5 34.1 36.3 36.2
F 419.0 493.0 342.2 3,728.3 1,058.1 1,034.1
r2 0.861 0.850 0.837 0.963 0.930 0.870
RSS 0.002993 0.003960 0.002994 0.002150 0.002161 0.007971
Lactin-2
␳ 0.0032 ⫾ 0.0001 0.0032 ⫾ 0.0002 0.0031 ⫾ 0.0001 0.0037 ⫾ 0.0001 0.0036 ⫾ 0.0001 0.0030 ⫾ 0.0001
Tm 40.9702 ⫾ 0.9525 43.0583 ⫾ 1.1222 41.9131 ⫾ 0.6307 45.5023 ⫾ 0.9990 42.6699 ⫾ 1.2615 41.0933 ⫾ 1.3423
⌬ 1.4411 ⫾ 0.2551 1.8580 ⫾ 0.3085 1.6857 ⫾ 0.1844 2.4805 ⫾ 0.2682 1.6628 ⫾ 0.3106 1.1657 ⫾ 0.3036
␭ ⫺1.0567 ⫾ 0.0037 ⫺1.0614 ⫾ 0.0041 ⫺1.0580 ⫾ 0.0032 ⫺1.0658 ⫾ 0.0022 ⫺1.0693 ⫾ 0.0031 ⫺1.0523 ⫾ 0.0024
Tmin 17.1 18.5 18.0 17.1 18.7 18.0
Tmax 37.0 39.1 37.1 39.1 38.2 37.8
Topt 33.2 33.5 33.0 33.8 34.1 34.5
F 416.3 467.0 542.5 3751.1 997.7 786.9
r2 0.904 0.890 0.925 0.975 0.949 0.884
RSS 0.002091 0.002902 0.001380 0.001440 0.001581 0.007179
Briere-2
a 0.0001 ⫾ 0.0000 0.0001 ⫾ 0.0000 0.0001 ⫾ 0.0000 0.0001 ⫾ 0.0000 0.0001 ⫾ 0.0000 0.0001 ⫾ 0.0000
m 5.0192 ⫾ 0.7429 3.4385 ⫾ 0.4558 3.1710 ⫾ 0.2686 3.1827 ⫾ 0.3415 4.3334 ⫾ 0.6673 5.4612 ⫾ 0.3098
Tmin 15.8 ⫾ 0.7 18.0 ⫾ 0.5 17.7 ⫾ 0.4 16.2 ⫾ 0.3 17.9 ⫾ 0.3 16.0 ⫾ 0.3
Tmax 35.7 ⫾ 0.1 36.7 ⫾ 0.2 36.6 ⫾ 0.1 37.5 ⫾ 0.4 36.8 ⫾ 0.5 36.2 ⫾ 0.01
Topt 33.4 33.6 33.0 33.8 34.3 34.0
F 429.4 464.9 524.4 3823.7 993.1 1308.0
r2 0.906 0.890 0.923 0.976 0.949 0.927
RSS 0.002033 0.002913 0.001424 0.001411 0.001557 0.004465
August 2011 OÕNEILL ET AL: TEMPERATURE AND M. rotundata DEVELOPMENT 923

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Fig. 2. Relationship of the rate of postwintering development to rearing temperature for M. rotundata. Dashed line:
Lactin-2 model; solid line: Briere-2 model.

and 2008. The proportion of females among emerged
adults was unrelated to Trt in 2007 (linear regression,
F1, 11 ⫽ 0.006, r2 ⫽ 0.02, P ⫽ 0.94); 2008 (F1, 10 ⫽ 0.051,
r2 ⫽ 0.07, P ⫽ 0.83); and 2009 (F1, 11 ⫽ 3.17, r2 ⫽ 0.21,
P ⫽ 0.10).
Temperature, Adult Body Size, and Lipid Content.
Females were signiÞcantly larger than males in both
head width and dry mass (Table 5), but head width did
not vary with Trt by using either linear or quadratic
regression models (Table 6). In linear models, total
dry mass (before fat extraction) varied inversely with
Trt in three of the four data sets, but variation in Trt
explained ⱕ5% of the variation in body mass. Qua-
dratic models fared slightly better, but with r2 values
of just 0.05Ð 0.08.
Females emerged with signiÞcantly more body fat
than males in 2007, but not in 2008. However, the mean
proportion of body fat in males exceeded that in females
in both years (Table 5). Variation in Trt explained 13Ð
27% of variation in the proportion body fat in newly-
emerged adults (Table 7; Fig. 4). Based on these models,
the optimum Trt with regards to maximization of pro-
portion body fat, ranges from ⬇27Ð29⬚C.
Emergence Success and Lipid Content in Response
to Short-term Exposure to 38ⴗC. When developing
bees were moved from the 29⬚C incubator and ex-
posed to 38⬚C in another incubator for durations of
time from 0 to 48 h, we observed no signiÞcant dif-
ferences in the number of bees emerging either: 1)
across exposure-duration treatments within age treat-
ments or 2) across age treatments within exposure-
duration treatments (Table 8). The 342 females that
emerged as adults in this experiment (all treatments
combined) had a mean dry mass of 139.1 ⫾ 0.1 mg, a

Table 4. Unweighted mean (ⴞ SE) values for Tmin, Topt, and Tmax for each of the four model types (from Table 3). Top row represents
linear regression analysis using data for Trt < 32°C to match similar analysis by Richards and Whitfield (1988)

Females Males
Tmin Topt Tmax Tmin Topt Tmax
Linear 17.3 ⫾ 0.4 Ð Ð 17.6 ⫾ 0.9 Ð Ð
Quadratic 19.9 ⫾ 0.4 34.8 ⫾ 0.7 50.5 ⫾ 2.0 19.1 ⫾ 0.3 35.5 ⫾ 0.7 60.1 ⫾ 3.4
Lactin-2 17.9 ⫾ 0.4 33.2 ⫾ 0.1 37.7 ⫾ 0.7 17.9 ⫾ 0.5 34.1 ⫾ 0.2 38.4 ⫾ 0.4
Briere-2 17.2 ⫾ 0.7 33.3 ⫾ 0.2 36.3 ⫾ 0.3 16.7 ⫾ 0.6 34.0 ⫾ 0.1 36.8 ⫾ 0.4
924 ENVIRONMENTAL ENTOMOLOGY Vol. 40, no. 4

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Fig. 3. Relationship of rearing temperature to the stage reached by developing M. rotundata at time when all adult bees
had either emerged or when leaf cells were dissected at end of experiment and their contents designated either as prepupae,
pupae, or uneclosed adults. Number of days that cells were on gradient is indicated in parentheses after year designation.
The proportion of bees reaching a stage varied signiÞcantly with temperature for each stage each year (KruskalÐWallis tests,
P ⬍ 0.001 for all tests). Sample sizes for each one-degree increment range from 8 to 38 in 2007, 4Ð51 in 2008, and 3Ð38 in 2009.

mean body fat mass of 20.4 ⫾ 0.05 mg, and a mean
proportion body fat of 0.143 ⫾ 0.003 (range: 0.030 Ð
0.257). Pf varied across time treatments only in the
Þnal age treatment (Table 9); in that group, Pf de-
clined with increasing duration of exposure, but ex-
posure time explained just 11% of the variation in Pf.
Discussion
Research on the effects of temperature on devel-
opment of postwintering M. rotundata has been un-
dertaken with the goal of designing practical rearing
guidelines that maximize development rate, survival,
and adult condition, while synchronizing adult female
activity with alfalfa bloom (Stephen and Osgood 1965,
Richards and WhitÞeld 1988, Kemp and Bosch 2000,
Yocum et al. 2010). The research also points to the
value of using M. rotundata as an easily-manipulated
subject for Þeld and lab studies of the developmental
thermal biology of solitary aculeate Hymenoptera.
Several previous studies of the effect of constant
rearing temperature on the development of postwin-
tering M. rotundata reported that bees reared at 35 and
37⬚C sometimes emerged much later than those bees
at lower temperatures (Stephen and Osgood 1965,
Richards and WhitÞeld 1988); however, those higher
temperatures were not included in their calculations
of Tmin. In addition, the restricted ranges of temper-
ature treatments in some studies and the use of linear
regression models precluded quantitative estimates of
Topt or Tmax (Kontodimas et al. 2004). Therefore, we
decided to further explore the effect of temperature

Table 5. Mean head width, dry body mass, body fat mass, and proportion fat mass for female and male M. rotundata at the time of
emergence on thermal gradient; sample sizes same as those in Table 1

Head Body dry mass at Fat Proportion Range in

Year Sex
width (mm) emergence (␮g) mass (␮g) body fat proportion body fat
2007 Female 3.24 ⫾ 0.01a 142.4 ⫾ 2.1a 26.3 ⫾ 0.7b 0.182 ⫾ 0.0003 0.051Ð0.283
male 3.09 ⫾ 0.01 107.0 ⫾ 1.1 22.5 ⫾ 0.4 0.207 ⫾ 0.0003d 0.086Ð0.307
2008 Female 3.10 ⫾ 0.01a 132.2 ⫾ 1.6a 19.6 ⫾ 0.6c 0.145 ⫾ 0.0003 0.017Ð0.233
Male 3.03 ⫾ 0.01 108.8 ⫾ 1.5 19.2 ⫾ 0.6 0.172 ⫾ 0.0004d 0.031Ð0.277

a
MannÐWhitney test; female value signiÞcantly greater than male value (P ⬍ 0.001).
b
MannÐWhitney test; female value signiÞcantly greater than male value (P ⬍ 0.05).
c
MannÐWhitney test; female value not signiÞcantly different from male value (P ⫽ 0.65) .
d
MannÐWhitney test; male value signiÞcantly greater than female value (P ⬍ 0.001).
August 2011 OÕNEILL ET AL: TEMPERATURE AND M. rotundata DEVELOPMENT 925

Table 6. Regressions of head width and dry body mass of emerged Richards and WhitÞeld (1988) reported that 0.5%
adults on rearing temp; sample sizes same as those in Table 1 prepupae develop to the adult stage at 15⬚C; and the
Size Model
bees were apparently unharmed, because when
Year Sex F d.f. r2 P moved to a 30⬚C incubator after 120 d, the remaining
variable type
prepupae successfully completed development. Kemp
Head width Linear 2007 Female 2.63 1, 136 0.019 0.11
Male 2.91 1, 286 0.010 0.09 and Bosch (2000) observed that ⬇4% of postwintering
2008 Female 1.35 1, 175 0.008 0.25 bees incubated at 18⬚C for 4 mo reached the adult
Male 0.15 1, 161 0.001 0.70 stage. We also saw some development at ⬍19⬚C. In
Quadratic 2007 Female 1.49 2, 135 0.022 0.22
Male 1.71 2, 285 0.012 0.19
2007, for example, no bees reared at 15Ð17⬚C range
2008 Female 1.40 2, 174 0.016 0.24 pupated, but at 18⬚C, 40% pupated and one bee

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Male 1.21 2, 160 0.015
Dry mass Linear 2007 Female 7.58 1, 136 0.053a
Male 5.21 1, 286 0.018a
2008 Female 10.02 1, 175 0.054a
Male 0.96 1, 161 0.006a
Quadratic 2007 Female 5.79 2, 135 0.079b
Male 10.28 2, 285 0.067d
2008 Female 6.63 2, 174 0.071
Male 4.31 2, 160 0.051c
a
Sign of correlation coefÞcient is negative.
b
Quadratic model provides a signiÞcantly better Þt to the data than
linear model) at P ⬍ 0.05.
c
Quadratic model provides a signiÞcantly better Þt to the data than
linear model) at P ⬍ 0.001.
d
Quadratic model provides a signiÞcantly better Þt to the data than
linear model) at P ⬍ 0.01.
on developmental rate in M. rotundata by using a
greater number of temperature treatments and ana-
lytical tools formulated to model nonlinear relation-
ships of Trt to development rate in other insects (Lac-
tin et al. 1995, Briere et al. 1999). Previous studies
either provide estimates of Tmin for postwintering M.
rotundata, or data that we can use to calculate Tmin
(and sometimes Topt and Tmax) (Table 10).
The earliest attempt to estimate Tmin for M. rotun-
data was that of Stephen and Osgood (1965) who
stated that “below 19⬚C development does not occur”.
They were not explicit in how they arrived at this
value, but did show that they were able to rear prepu-
pae to adults at 22⬚C, but not at 17⬚C, the next lowest
treatment in their study. Our quadratic models give
Tmin values in a similar range (18.5Ð20.0⬚C). Other
estimates of Tmin range from 15.7 to 18.1⬚C for linear
models and from 16.5 to 17.9⬚C for nonlinear models
(Table 10). The latter includes our reanalysis of data
in Richards and WhitÞeld (1988) by using the Briere-2
model, which resulted in Tmin estimates nearly iden-
tical to those researchers using our data and the same
model.
The 19⬚C Tmin estimate of Stephen and Osgood
(1965) is higher than that in other studies (Table 10).
0.27 reached the adult stage, without emerging. In 2008,
0.01 one bee reached the pupal stage and one the adult
0.02
0.002
stage (without emerging) at 17⬚C. Thus, though there
0.33 is no doubt that development is either very slow or
0.02 stalls below 19⬚C, it is not completely halted in all bees
0.001 at Trt from 15 to 18⬚C.
0.01
0.04 Our linear models provided estimates of the degree-
days required for completion of postwintering devel-
opment that were similar to those estimates of Rich-
ards and WhitÞeld (1988) and to those estimates
calculated from data in Rank and Goerzen (1982), but
much lower than those estimates based on data in
Kemp and Bosch (2000) (Table 10). The midpoint
value (dd ⫽ 295) between the unweighted means for
females (dd ⫽ 315) and males (dd ⫽ 275) is the same
as the combined-sexes value given by Richards and
WhitÞeld (1988).
Various sources give guidelines for prerelease op-
timal (rate-maximizing) rearing temperatures for M.
rotundata at 29 Ð32⬚C (Stephen and Osgood 1965,
Richards 1984, Baird et al. 1991, Delaplane and Mayer
2000, Pitts-Singer 2008) often with the stated goal of
maximizing the rate of development. Stephen and
Osgood (1965) recommended 32⬚C as the best tem-
perature for rearing, relative to the next closest rearing
temperatures in their experiments (29 and 35⬚C). A
similar conclusion could be made from Richards and
WhitÞeldÕs (1988) study, relative to their 30 and 35⬚C
treatments. We came to similar conclusions, though
our estimates for Topt were slightly higher (33Ð34⬚C).
Reassessment of the data of Richards and WhitÞeld
(1988) by using a Briere-2 model gives Topt values of
32Ð34⬚C. Studies of other megachilids, however, indi-
cate that Topt varies within the family. Kapil and Sihag
(1985) reared Megachile flavipes Spinola, a subtropical
species also used for commercial alfalfa pollination, at
six temperatures from 24.0 to 31.5⬚C. Those insects
reared at 28.5⬚C, the fourth highest temperature
tested, developed most rapidly and had the lowest
overall mortality.

Table 7. Regressions of arcsine square-root proportion body fat on rearing temp for M. rotundata adults; sample sizes same as those
in Table 1

Optimum
Year Sex Regression of proportion fat (Pf) on Trt F r2 P
rearing temp.a
2007 Female Pf ⫽ ⫺0.364 ⫹ 0.0609Trt ⫺ 0.00113Trt2 11.98a 0.15 ⬍0.001 26.9
Male Pf ⫽ ⫺0.777 ⫹ 0.0929Trt ⫺ 0.00169Trt2 52.34a 0.27 ⬍0.001 27.5
2008 Female Pf ⫽ ⫺0.919 ⫹ 0.0941Trt ⫺ 0.00166Trt2 16.43a 0.16 ⬍0.001 28.4
Male Pf ⫽ ⫺1.005 ⫹ 0.0102Trt ⫺ 0.00179Trt2 12.36a 0.13 ⬍0.001 28.5

a
Value of T for max Pf value in quadratic and refers to optimum rearing temp if maximizing proportion fat is the only issue of concern.
926 ENVIRONMENTAL ENTOMOLOGY Vol. 40, no. 4

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Fig. 4. Relationship of proportion body fat of newly emerged M. rotundata to rearing temperature.

At 38⬚C, which is 4 Ð5⬚C above Topt, we found no
effects on developmental mortality of M. rotundata
even with exposures as long as 48 h, and detected very
little effect on adult fat stores. Other studies also in-
dicate that immature M. rotundata can be fairly robust
in the face of short-term exposure to temperatures
⬎Topt. In experiments exposing prepupae and pupae
to high temperatures for up to 3 h, Undurraga and
Stephen (1980a) observed 100% mortality at 50⬚C, but
none at 45⬚C. The 45⬚C treatment, however, did result
in accelerated development of prepupae and retarded
development of pupae. Similarly, 3-h exposure of
prepupae to 35Ð 48⬚C caused no appreciable prepupal
mortality, but mortality was 100% at 53Ð55⬚C (Barthell
et al. 2002). Over 90% of prepupae survived the 49⬚C
treatment, but only 4% later completed development
and emerged at 30⬚C.
Longer-term exposure to temperatures just several
degrees above Topt is, however, clearly problematic
for postwintering bees. Stephen and Osgood (1965)
stated that “above 38⬚C mortality is complete” (over 19
wk of rearing), and Richards and WhitÞeld (1988)
observed only 14% emergence among bees reared at
37⬚C, their hottest treatment (over 17 wk). Our study
gave similar results (over 14 Ð16 wk). Of the cells kept
at 35⬚C, 48% produced adults in 2007, and 64% did so

Table 8. Number of adult bees emerging (out of 50) within each exposure-duration treatment/age treatment group

Age treatment (no. of days following wintering

Exposure-duration treatment when high temp treatment began) ␹2
P
(no. of h at 38⬚C) 2 7 13 17 value
(Prepupae) (Prepupae) (Early pupae) (Late pupae)
0 (control) 43 44 47 46 0.222 0.974
0.5 44 41 43 46 0.299 0.960
1 45 42 44 45 0.136 0.987
2 46 44 46 45 0.061 0.996
4 42 44 42 43 0.064 0.996
8 48 41 46 50 0.968 0.809
24 43 44 42 47 0.318 0.957
48 43 41 43 50 1.056 0.788
␹2 value 0.621 0.372 0.609 0.903
P 0.999 0.999 0.999 0.996
August 2011 OÕNEILL ET AL: TEMPERATURE AND M. rotundata DEVELOPMENT 927

Table 9. Proportion body fat among adult M. rotundata fe- is probable that many females would emerge too late
males in each age treatment and results of linear regressions of in the season to reproduce successfully; they would
arcsine square root-transformed Pf on duration of exposure to
38°C treatment within each age treatment group not be synchronized with many of the ßoral resources
they evolved to exploit and would run into declining
Age treatment (day Pf temperatures later in the summer. Such lower tem-
following wintering peratures could reduce development rates for brood
N r2 F P
when high temp
treatment began) (mean ⫾ SE)a and shorten the daily period over which adults could
forage. In managed populations of M. rotundata, cells
2 0.134 ⫾ 0.004 89 0.01 0.94 0.336
7 0.134 ⫾ 0.006 76 0.03 2.16 0.146
are removed from winter storage, and rearing is com-
13 0.157 ⫾ 0.005 90 0.01 1.30 0.257 menced, with the goal of synchronizing bee emer-

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17 0.144 ⫾ 0.005 87 0.11
a
Comparison of Pf among age treatments, Kruskal-Wallis Test, P ⬍
0.001 (in multiple comparisons using DunnÕs Test (␣ ⫽ 0.05) Pf for day
13 ⬎ days 2 and 7, but not ⬎ day 17.
in 2008; in 2009, development was successful in 55% of
cells reared at 36⬚C. However, at 37Ð38⬚C no adults
emerged from leaf cells in any year, although 16 Ð25%
of bees reached the pupal or unemerged adult stage.
Given these observations, the Lactin-2 (Tmax ⫽ 37Ð
39⬚C) and Briere-2 model (Tmax ⫽ 36 Ð38⬚C) gave
reasonable Tmax values, but the quadratic models
clearly overestimated Tmax (at 47Ð 66⬚C). The Tmax
values that we generated using Briere-2 models for the
data of Richards and WhitÞeld (1988) also seem too
high (Table 10).
The proportion of cells that produce emerging
adults can be high over a relatively wide temperature
range, and sex ratio is apparently unaffected by Trt.
Emergence success was ⱖ88% in Þve temperature
treatments from 20 to 32⬚C in the study of Richards
and WhitÞeld (1988), and high in both the 26 and 29⬚C
treatments of Kemp and Bosch (2000). In our study,
a high proportion of cells successfully produced adults
over rearing temperatures ranging from ⬇22Ð35⬚C,
depending on the year and source of bees. However,
if such temperature variation occurred during
postwintering development in natural populations, it
10.91 0.001 gence with alfalfa bloom or that of other crops (De-
laplane and Mayer 2000). An associated goal is to have
bees develop quickly, so as to shorten the period
during which immature bees are exposed to parasi-
toids or uncontrollable temperature extremes before
emergence. Thus, data on emergence success at dif-
ferent Trt is only relevant when considered in com-
bination with information on the rate of development.
The Þnal point to consider is whether Trt affects the
condition and performance of adult bees. On this
matter, the published evidence is scant for M. rotun-
data, though there is relevant comparative evidence
from other megachilids. Kemp and Bosch (2000) de-
termined that adult bees that had been reared at 26
and 29⬚C survived longer than for those bees reared at
22⬚C. Because adults in that study remained unfed
after emergence, these results are an indication of
condition upon emergence, though not necessarily an
indicator of how well bees would do in the Þeld where
they have access to nectar and pollen that could help
dampen temperature effects experienced earlier in
life. In another study of M. rotundata, Tepedino and
Parker (1986) reared bees (starting with prepupae) at
different temperatures during late summer and fall,
and then reared bees from all treatments at identical
temperatures postwintering. Females reared at 29⬚C
during the prewintering period weighed 13% more
than those females reared at 24⬚C and 44% more than

Table 10. Comparison of parameter estimates for developmental rate models of different studies of post-wintering development in
M. rotundata

Range (⬚C) (no.) of temp Model Source of

Reference Sex Tmin Topt Tmax dd
treatments in analysis type parameter values
Linear models
Rank and Goerzen (1982) 20Ð30 (3) Both Lineara Our calculation 18.1 Ð Ð 290
Richards and WhitÞeld (1988) 20Ð32 (5) Both Linearb Original paper 15.7 Ð Ð 295
Kemp and Bosch (2000) 22Ð29 (3) Female Linearc Our calculation 16.3 Ð Ð 441
Male Linearc Our calculation 15.8 Ð Ð 427
OÕNeill et al. (present study) 20Ð32 (12Ð13) Female Lineard Present study 17.3 Ð Ð 315
Male Lineard Present study 17.6 Ð Ð 275
Nonlinear models
Richards and WhitÞeld (1988) 20Ð37 (7) Female Briere-2e Our calculation 17.1 32.3 40.1 Ð
Male Briere-2e Our calculation 16.5 34.2 45.0 Ð
OÕNeill et al. (present study) 20Ð38 (17Ð19) Female Lactin-2f Present study 17.9 33.2 37.7 Ð
Male Lactin-2f Present study 17.9 34.1 38.4 Ð
Female Briere-2f Present study 17.2 33.3 36.3 Ð
Male Briere-2f Present study 16.7 34.0 36.8 Ð

a
Using mean no. of days to emergence at each temp (r2 ⫽ 0.99, P ⬍ 0.05).
b
Using day on which 50% of adults emerged at each temp.
c
Using mean no. of days to emergence at each temp (r2 for females ⫽ 0.98, P ⫽ 0.10; for males ⫽ 0.99, P ⫽ 0.06).
d
See Table 4; degree-day (dd) values are unweighted means of values in Table 3.
R for females ⫽ 0.98, P ⫽ 0.006, for males ⫽ 0.99; P ⫽ 0.001.
e 2
f
See Table 4.
928 ENVIRONMENTAL ENTOMOLOGY
those females kept at 16⬚C; equivalent values for males
were 2% and 37%. In Osmia bicornis (L.) after being
reared as larvae at 20, 25, and 30⬚C, adults were smaller
from higher temperature treatments partially because,
as larvae, they consumed a smaller proportion of the
provisions provided by their mothers (Radmacher and
Strohm 2010).
In addressing the hypothesis that postwintering Trt
affects body size and body fat stores of M. rotundata
adults, we found that temperature was unrelated to
the head width of emerging adults and had a small, but
signiÞcant effect on body mass. Apparently, linear
dimensions of adults are determined either by factors
other than temperature, or are affected by tempera-
ture only before the end of the overwintering period.
The effect of temperature on body mass may be ex-
plained by its effect on fat stores. The proportion of
petroleum ether-extracted lipids in dry body mass of
newly emerged M. rotundata adults (most of which
should come from fat bodies; Buckner et al. 2004) was
as high as 0.31. In 2007, 30% of females and 62% of
males had body fat proportions ⱖ0.20. In 2008, the
values were 10% of females and 33% of males. Buckner
et al. (2004) reported that internal lipids, primarily
triacylglycerols, comprised 21% of wet weight and 34%
of dry weight of diapausing M. rotundata prepupae
that had been wintering at 4⬚C for 7 mo. The fact that
this value is higher than that observed in most other
insects, including honey bees (Apis mellifera L.)
(Buckner et al. 2004), suggests that lipids are partic-
ularly important to the survival and reproductive suc-
cess of M. rotundata. Buckner et al. (2004) proposed
a dual function for stored lipids in M. rotundata, as
cryoprotectants critical to winter survival and as high
density energy stores used during postwintering de-
velopment to the adult stage. During prerelease rear-
ing, as bees complete development, their rate of me-
tabolism increases dramatically and they lose about a
quarter of their body mass (Kemp et al. 2004).
Lipids remaining at the time of adult emergence
likely are essential to adult reproductive success, es-
pecially that of females. After emerging as adults, M.
rotundata can fuel some of their activities (e.g., ßight)
by feeding on nectar. For female insects, however, fat
bodies are an important source of materials for egg
development (Pan et al. 1969, Keely 1985). In M.
rotundata, the major increase in the volume of devel-
oping oocytes does not occur until the second week
after adult emergence (Richards 1994), though in
Megachile flavipes Spinola, vitellogenins that will even-
tually provide proteins for eggs appear in hemolymph
on the second day after emergence (Sihag 1985). In
honey bee workers (Human et al. 2007) and M. ro-
tundata (Richards 1994), pollen consumed after emer-
gence also seems to be important for oocyte devel-
opment (Richards 1994). In solitary nest-provisioning
Hymenoptera, eggs are larger relative to body size
than they are in related parasitoid and social species,
and each female lays few eggs in her lifetime (Iwata
1955, 1960; Iwata and Sakagami 1966; Alexander and
Rozen 1987; OÕNeill 2001), so each egg requires sub-
stantial investment from females. For M. rotundata
Vol. 40, no. 4
females, substantial parental investment in individual
offspring (including large eggs) results in their having
relatively low fecundity; when nesting in alfalfa Þelds,
they typically provision 12Ð16 cells during their lives
(Gerber and Klostermeyer 1972). Thus, assuming the
importance of stored lipids to adult reproductive suc-
cess and the fact that the rate at which body fats are
metabolized during postwintering development is
temperature-dependent, our results suggest that rear-
ing temperature may have an indirect effect on adult
Downloaded from https://academic.oup.com/ee/article/40/4/917/383408 by University of Torino user on 20 November 2020
Þtness, perhaps limiting the size and number of eggs
laid by females. The low r2 values in the regressions of
Pf on Trt indicate that the amount of fat remaining at
emergence also is inßuenced by other factors, includ-
ing provision quality and quantity (Klostermeyer et al.
1973, Horne 1995, Kim 1999, Peterson and Roitberg
2006); maternal effects (OÕNeill et al. 2010); and tem-
perature experienced at other times in the beeÕs life
cycle (e.g., because of variation in temperature within
nesting materials; Peterson et al. 1994).
In conclusion, our models suggest that Topt (for
rapid rearing) is 33Ð34⬚C for M. rotundata. However,
it may not be possible to maximize the rate of devel-
opment, while also maximizing: 1) emergence success,
which declined above 32⬚C in the study by Richards
and WhitÞeld (1988) and in our 2009 experiment and
2) adult fat content, which may affect establishment
and thus reproductive performance. The signiÞcance
of the latter depends upon whether the observed dif-
ferences in body fat content across rearing tempera-
tures have a signiÞcant effect on the survival, polli-
nation efÞciency, and reproductive success of adult
bees. The rapid decline of both mean development
rate and percent survival above 33Ð34⬚C (reßected in
the shapes of Lactin-2 and Briere-2 models) also sug-
gests that it might be risky to try to Þne-tune rearing
temperatures to the lipid-optimum, especially if bees
are maintained in facilities where temperature control
is not accurate to a sufÞcient level. Finally, the tem-
peratures at which each variable is maximized may
vary with the source and condition of the prepupae
being overwintered, and levels of extrinsic mortality
factors, especially parasitoids (Vandenberg and Goet-
tel 1995) that emerge Þrst and then attack developing
bees during incubation.
Acknowledgments
We thank James Buckner and Theresa Pitts-Singer for
reviewing the manuscript, Samuel OÕNeill and Richard S.
Miller for assisting with the research, and David Weaver
for providing technical advice; Charles and Robert Wil-
liams, and John and Shelley Wold kindly provided bees for
use in the experiments. Funding for the research was
provided by the Montana Agricultural Experiment Station,
the Montana Alfalfa Seed Growers Association, the Mon-
tana Department of Agriculture, the Western Alfalfa Seed
Growers Association, and the USDAÐARS Bee Biology and
Systematics Laboratory.
August 2011 OÕNEILL ET AL: TEMPERATURE AND M. rotundata DEVELOPMENT
References Cited
Alexander, B., and J. G. Rozen. 1987. Ovaries, ovarioles, and
oocytes in parasitic bees (Hymenoptera, Apoidea). Pan-
Pac. Entomol. 63: 155Ð164.
Baird, C. R., D. F. Mayer, and R. M. Bitner. 1991. Pollina-
tors, pp. 1Ð8. In Alfalfa Seed Production and Pest Man-
agement. Western Regional Extension Publication 12.
Barthell, J. F., J. M. Hranitz, R. W. Thorp, and M. K. Shue.
2002. High temperature responses in two exotic leafcut-
ting bee species: Megachile apicalis and M. rotundata (Hy-
menoptera: Megachilidae). Pan-Pac. Entomol. 78: 235Ð
246.
Briere, J.-F., P. Pracros, A.-Y. Le Roux, and J.-S. Pierre. 1999.
A novel rate model of temperature-dependent develop-
ment for arthropods. Environ. Entomol. 28: 22Ð29.
Buckner, J. S., W. P. Kemp, and J. Bosch. 2004. Character-
ization of triacylglycerols from overwintering prepupae
of the alfalfa pollinator Megachile rotundata (Hymenop-
tera: Megachilidae). Arch. Insect Biochem. Physiol. 57:
1Ð14.
Delaplane, K. S., and D. F. Mayer. 2000. Crop pollination by
bees. CABI, Wallingford, United Kingdom.
Gerber, H. S., and E. C. Klostermeyer. 1972. Factors affect-
ing the sex ratio and nesting behavior of the alfalfa leaf-
cutter bee. Washington Experiment Station Technical
Bulletin 73, Washington State University, Pullman, Wash-
ington.
Horne, M. 1995. Pollen preference and its relationship to
nesting success of Megachile rotundata (Hymenoptera:
Megachilidae). Ann. Entomol. Soc. Am. 88: 862Ð867.
Human, H., S. W. Nicolson, K. Strauss, C.W.W. Pirk, and V.
Dietemann. 2007. Inßuence of pollen quality on ovarian
development in honeybee workers (Apis mellifera scute-
llata). J. Insect Physiol. 53: 649Ð655.
International Pollination Systems. 2011. A calendar of
incubation for alfalfa leafcutting bees. (http://www.
pollination.com/managed/albincubationcalendar.cfm).
Iwata, K. 1955. The comparative anatomy of the ovary in
Hymenoptera. Part I. Aculeata. Mushi 29: 17Ð34.
Iwata, K. 1960. The comparative anatomy of the ovary in
Hymenoptera. Supplement on Aculeata with descriptions
of ovarian eggs of certain species. Acta Hymenopterol. 1:
91Ð97.
Iwata, K., and S. F. Sakagami. 1966. Gigantism and dwarÞsm
in bee eggs in relation to modes of life, with notes on the
number of ovarioles. Jpn. J. Ecol. 16: 4 Ð16.
James, R. R. 2005. Temperature and chalkbrood develop-
ment in the alfalfa leafcutting bee, Megachile rotundata.
Apidologie 36: 15Ð23.
Kapil, R. P., and R. C. Sihag. 1985. Storage and incubation
temperatures for artiÞcial rearing of the subtropical leaf-
cutting bee Megachile flavipes. J. Agric. Res. 24: 199 Ð202.
Keely, L. L. 1985. Physiology and biochemistry of the fat
body, pp. 211Ð248. In G. A. Kerkut and L. I. Gilbert (eds.),
Comprehensive Insect Physiology, Biochemistry and
Pharmacology, vol. 3. Pergamon, Oxford, United King-
dom.
Kemp, W. P., and J. Bosch. 2000. Development and emer-
gence of the alfalfa pollinator Megachile rotundata (Hy-
menoptera: Megachilidae). Ann. Entomol. Soc. Am. 93:
904 Ð911.
Kemp, W. P., and J. Bosch. 2001. Postcocooning tempera-
tures and diapause in the alfalfa pollinator Megachile ro-
tundata (Hymenoptera: Megachilidae). Ann. Entomol.
Soc. Am. 94: 244 Ð250.
Kemp, W. P., J. Bosch, and B. Dennis. 2004. Oxygen con-
sumption during the life cycles of the prepupa-wintering
bee Megachile rotundata and the adult-overwintering bee
929
Osmia lignaria (Hymenoptera: Megachilidae). Ann. En-
tomol. Soc. Am. 97: 161Ð170.
Kim, J. Y. 1999. Inßuence of resource level on maternal
investment in a leaf-cutter bee (Hymenoptera: Megachi-
lidae). Behav. Ecol. 10: 552Ð556.
Klostermeyer, E. C., S. J. Mech, Jr., and W. B. Rasmussen.
1973. Sex and weight of Megachile rotundata (Hymenop-
tera: Megachilidae) progeny associated with provision
weights. J. Kans. Entomol. Soc. 46: 536 Ð548.
Kontodimas, D. C., P. A. Eliopoulos, G. J. Stathas, and L. P.
Economou. 2004. Comparative temperature-dependent
Downloaded from https://academic.oup.com/ee/article/40/4/917/383408 by University of Torino user on 20 November 2020
development of Nephus includens (Kirsch) and Nephus
bisignatus (Boheman) (Coleoptera: Coccinellidae) prey-
ing on Planococcus citri (Risso) (Homoptera: Pseudococ-
cidae): evaluation of linear and various non-linear models
using speciÞc criteria. Environ. Entomol. 33: 1Ð11.
Lactin, D. J., N. J. Holliday, D. L. Johnson, and R. Craigen.
1995. Improved rate model of temperature-dependent
development by arthropods. Environ. Entomol. 24: 68 Ð75.
O’Neill, K. M. 2001. Solitary Wasps: Natural History and
Behavior. Cornell University Press, Ithaca, New York.
O’Neill, K. M., A. M. Pearce, R. P. O’Neill, and R. S. Miller.
2010. Offspring size and sex ratio variation in a feral
population of alfalfa leafcutting bees (Hymenoptera:
Megachilidae; Megachile rotundata F.). Ann. Entomol.
Soc. Am. 103: 775Ð784.
Pan, M. L., W. J. Bell, and W. H. Telfer. 1969. Vitellogenic
blood protein synthesis by insect fat body. Science 165:
393Ð394.
Peterson, J. H., and B. D. Roitberg. 2006. Impact of resource
levels on sex ratio and resource allocation in the solitary
bee, Megachile rotundata. Environ. Entomol. 35: 1404 Ð
1410.
Peterson, S. S., C. R. Baird, and R. M. Bitner. 1994. Heat
retention during incubation in nests of the alfalfa leaf-
cutting bee (Hymenoptera: Megachilidae). J. Econ. En-
tomol. 87: 345Ð349.
Pitts-Singer, T. L. 2008. Past and present management of
alfalfa bees, pp. 105Ð123. In R. James and T. L. Pitts-Singer
(eds.), Bee Pollination in Agricultural Ecosystems. Ox-
ford University Press, New York.
Pitts-Singer, T. L., and R. R. James. 2008. Do weather con-
ditions correlate with Þndings in failed, provision-Þlled
nest cells of Megachile rotundata (Hymenoptera: Mega-
chilidae) in western North America? J. Econ. Entomol.
101: 674 Ð 685.
Pitts-Singer, T. L., and R. R. James. 2009. Prewinter man-
agement affects Megachile rotundata (Hymenoptera:
Megachilidae) prepupal physiology and adult emergence
and survival. J. Econ. Entomol. 102: 1407Ð1416.
Pitts-Singer, T. L., and J. H. Cane. 2011. The alfalfa leafcut-
ting bee, Megachile rotundata: the worldÕs most inten-
sively managed solitary bee. Annu. Rev. Entomol. 56:
221Ð237.
Radmacher, S., and E. Strohm. 2010. Factors affecting off-
spring body size in the solitary bee Osmia bicornis (Hy-
menoptera, Megachilidae). Apidologie 41: 169 Ð177.
Rank, G. H., and D. W. Goerzen. 1982. Effect of incubation
temperatures on emergence of Megachile rotundata (Hy-
menoptera: Megachilidae). J. Econ. Entomol. 75: 467Ð
471.
Richards, K. W. 1984. Alfalfa leafcutter bee management in
western Canada. Agriculture Canada Publication 1495E,
Ottawa, ON.
Richards, K. W. 1994. Ovarian development in the alfalfa
leafcutter bee, Megachile rotundata. J. Apic. Res. 33: 199 Ð
203.
930 ENVIRONMENTAL ENTOMOLOGY
Richards, K. W. 1996. Effect of environment and equipment
on production of alfalfa leafcutter bees (Hymenoptera:
Megachilidae) in southern Alberta, Canada. Can. Ento-
mol. 128: 47Ð56.
Richards, K. W., and G. H. Whitfield. 1988. Emergence and
survival of leafcutter bees, Megachile rotundata, held at
constant incubation temperatures (Hymenoptera: Mega-
chilidae). J. Apic. Res. 27:. 197: 204.
Richards, K. W., G. H. Whitfield, and G. B. Schaalje. 1987.
Effects of temperature and duration of winter storage on
survival and period of emergence for the alfalfa leafcut-
ting bee (Hymenoptera: Megachilidae). J. Kans. Ento-
mol. Soc. 60: 70 Ð76.
Sihag, R. C. 1985. Vitellogenin of alfalfa pollinating bee
Megachile flavipes. Current Sci. 54: 92Ð94.
SPSS Inc. 1996. TableCurve 2D, version 4.06. AISN Soft-
ware, Inc.
Stephen, W. P., and C. E. Osgood. 1965. The induction of
emergence in the leaf-cutter bee, Megachile rotundata, an
important pollinator of alfalfa. J. Econ. Entomol. 58: 284 Ð
286.
Strohm, E. 2000. Factors affecting the body size and fat
content in a digger wasp. Oecologia 123: 184 Ð191.
Tepedino, V. J., and F. D. Parker. 1986. Effect of rearing
temperature on mortality, second-generation emer-
gence, and size of adult is Megachile rotundata (Hyme-
noptera: Megachilidae). J. Econ. Entomol. 79: 974 Ð977.
Vol. 40, no. 4
Undurraga, J. M., and W. P. Stephen. 1980a. Effect of tem-
perature on development and survival in post-diapausing
alfalfa leafcutting bee prepupae and pupae (Megachile
rotundata (F.): Hymenoptera: Megachilidae). I. High
temperatures. J. Kans. Entomol. Soc. 53: 669 Ð 676.
Undurraga, J. M., and W. P. Stephen. 1980b. Effect of tem-
perature on development and survival in post-diapausing
leafcutting bee pupae (Megachile rotundata (F)). II. Low
temperatures. J. Kans. Entomol. Soc. 53: 669 Ð 676.
Vandenberg, J. D., and M. S. Goettel. 1995. Chalkbrood sus-
ceptibility among alfalfa leafcutting bee (Hymenoptera:
Downloaded from https://academic.oup.com/ee/article/40/4/917/383408 by University of Torino user on 20 November 2020
Megachilidae) larvae reared at different temperatures. J.
Econ. Entomol. 88: 810 Ð 814.
Whitfield, G. H., and K. W. Richards. 1985. Inßuence of
temperature on survival and rate of development of Pte-
romalus venustus (Hymenoptera: Pteromalidae), a para-
site of the alfalfa leafcutter bee (Hymenoptera: Mega-
chilidae). Can. Entomol. 117: 811Ð 818.
Yocum, G. D., J. P. Rinehart, M. West, and W. P. Kemp. 2010.
Interrupted incubation and short-term storage of the al-
falfa pollinator Megachile rotundata (Hymenoptera:
Megachilidae): a potential tool for synchronizing bees
with bloom. J. Econ. Entomol. 103: 234 Ð241.
Zar, J. H. 1999. Biostatistical Analysis (4th ed.). Prentice
Hall: Englewood Cliffs, New Jersey.
Received 29 December 2010; accepted 23 April 2011.