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Effect of temperature regime on diapause intensity in an adult-wintering
Hymenopteran with obligate diapause
F. Sgolastra
a,
*, J. Bosch
b,c
, R. Molowny-Horas
b
, S. Maini
a
, W.P. Kemp
d
a
Dipartimento di Scienze e Tecnologie Agroambientali, Area Entomologia, Universita
`di Bologna, viale G. Fanin 42, 40127 Bologna, Italy
b
CREAF, Universitat Auto
`noma de Barcelona, Bellaterra, Spain
c
Biology Department, Utah State University, Logan, UT, USA
d
USDA-ARS Red River Valley Agricultural Research Center, Fargo, ND, USA
1. Introduction
Diapause is the primary mechanism whereby insects of
temperate zones synchronize their life cycle with seasonal
changes. Diapause may be succinctly defined as a genetically
programmed, neurohormonally mediated, dynamic state of low
metabolic activity during which morphogenesis ceases or sig-
nificantly slows down (Tauber et al., 1986; Danks, 1987). To
emphasize diapause as a process rather than as a status,
Andrewartha (1952) coined the term diapause development.
However the use of the word ‘‘development’’ to describe a process
of ‘‘arrest of development’’ has caused some confusion (Hodek,
2002), and we thus use the more intuitive terms diapause initiation,
maintenance and termination (Kostal, 2006). In principle, the
physiological differences between these phases are clear, but
researchers do not always agree on how to characterize their
transitions, especially as it regards to diapause termination (Kostal,
2006). This is so for several reasons. First, diapause does not entail a
complete cessation of development (Hahn and Denlinger, 2007).
Response to environmental factors such as temperature, photo-
period and humidity, are far from constant during the course of
diapause (Gray et al., 1995; Kostal, 2006) and even at the lowest
metabolic rates some degree of biochemical activity occurs (Danks,
1987). Although morphogenesis is arrested, growth, mobility and
feeding may sometimes take place during diapause (Hodek, 2002;
Kostal et al., 2008). Second, the physiological mechanisms under-
lying diapause are highly variable. This is not surprising, given that
diapause has appeared many times independently during the
course of arthropod evolution (Tauber et al., 1986), and may occur
at different developmental stages, even among closely related
species (Tauber et al., 1986; Danks, 1987). This variability makes it
difficult to establish general principles, and to apply results from
one taxon to another. For example, respiration rates during
diapause vary widely between species, both in absolute terms and
in relation to respiration rates during non-diapause (Table 5 in
Danks, 1987). Similarly, chilling (exposure to ‘‘cold’’ temperatures)
Journal of Insect Physiology 56 (2010) 185–194
ARTICLE INFO
Article history:
Received 8 July 2009
Received in revised form 8 October 2009
Accepted 8 October 2009
Keywords:
Respiration rate
Metabolic rate
Over-wintering
Diapause development
Weight loss
Osmia lignaria
Hymenoptera: Megachilidae
Respiratory quotient
ABSTRACT
Osmia lignaria is a solitary bee that over-winters as a fully eclosed, cocooned, unfed adult. Our objective is
to understand the effect of wintering temperature on diapause maintenance and termination in this
species. We measure respiration rates and weight loss in individuals exposed to various wintering
temperatures (0, 4, 7, 22 8C, outdoors) and durations (28, 84, 140, 196, 252 days). We use time to emerge
and respiration response (respiration rate measured at 22 8C) as indicators of diapause intensity. Adults
spontaneously lower their respiration rates to 0.1 ml/g h within 1 month after adult eclosion,
indicating obligatory diapause. Non-wintered individuals main tain low respiration rates, but lose weight
rapidly and die by mid-winter. In wintered adults, two phases can be distinguished. First, respiration
response undergoes a rapid increase and then reaches a plateau. This phase is similar in bees wintered at
0, 4 and 7 8C. In the second phase, respiration response undergoe s an exponential increase, which is more
pronounced at the warmer temperatures. Composite exponential functions provide a good fit to the
observed respiration patterns. Adults whose respiration response has reached 0.45 ml/g h emerge
promptly when exposed to 20 8C, indicating diapaus e completion. Individuals wintered for short periods
do not reach such respiration levels. When exposed to 20 8C these individuals lower their metabolic rate,
and their emergence time is extended. The relationship between respiration rates and emergence time
follows a negative exponential function. We propose two alternative models of diapause termination to
interpret these results.
ß2009 Elsevier Ltd. All rights reserved.
* Corresponding author. Fax: +39 0512096281.
E-mail address: fabio.sgolastra2@unibo.it (F. Sgolastra).
Contents lists available at ScienceDirect
Journal of Insect Physiology
journal homepage: www.elsevier.com/locate/jinsphys
0022-1910/$ – see front matter ß2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jinsphys.2009.10.001
is a prerequisite for diapause development in some species but not
in others (Hodek and Hodkova
´, 1988; Tables 2 and 3 in Hodek,
2002). Third, it is difficult to extrapolate results obtained in the
laboratory (with a clear demarcation of environmental factors), to
field conditions, with environmental factors changing and
fluctuating in complex ways (Hodek, 2002; Kostal, 2006). It is
not always clear whether observed physiological responses are
attributable to diapause termination or to the experimental
manipulations themselves (Ragland et al., 2009). Many tempe-
rate-zone insects are known to terminate diapause by mid-winter,
and then remain in a state of post-diapause quiescence during
which the insect has the potential to resume morphogenesis,
which at that point is inhibited by low temperature (Tauber et al.,
1986; Table 1 in Hodek, 2002). The insect is then able to respond
immediately to development- or activation-promoting conditions.
For this reason, it is usually assumed that diapause under field
conditions is completed 4–5 months after its initiation (Hodek,
2002). However, there is some debate as to whether the transition
between diapause and post-diapause quiescence is gradual or
abrupt (Sawyer et al., 1993; Gray et al., 1995).
In this study we investigate the effect of temperature on
diapause initiation, maintenance and termination in the solitary
bee Osmia lignaria (Hymenoptera, Megachilidae). This species
shows several biological traits that make it an interesting model
for a study of this sort. First, in contrast to most other species in
which diapause has been studied, O. lignaria is an obligate
diapauser. Second, development from egg to adult in this species
takes place inside a sealed nest in complete darkness. Therefore, as
opposed to other diapause models (Tables 21 and 22 in Danks,
1987; Kostal et al., 2000, 2008), photoperiod does not play a key
role in diapause maintenance or termination in O. lignaria.Third,
O. lignaria over-winter as fully eclosed adults within their natal
cocoon and therefore have no access to food during pre-wintering.
This is in contrast to most other adult-wintering insects whose
diapause has been studied (including a bumblebee, Bombus
terrestris), in which pre-wintering adults ingest food and build up
their metabolic reserves in preparation for winter (Tauber et al.,
1986; Hodkova
´and Hodek, 1989; Hodek and Honek, 1996;
Beekman et al., 1998).
Most studies use the time required for overt resumption of
development or reproduction when exposed to diapause-termi-
nating conditions as a measure of diapause intensity and
termination (Kostal, 2006). Fewer studies (e.g., Gray et al., 1995;
Yaginuma and Yamashita, 1999; Singtripop et al., 2007; Ragland
et al., 2009) use respiration rates. In this study we measure
respiration rates and time required to emerge in O. lignaria females
exposed to various wintering temperatures, including field
temperatures, and various wintering durations. Our objective is
to understand the effect of temperature regime on winter diapause
initiation, maintenance and termination in this species. O. lignaria
reaches the adult stage towards the end of summer. Then, until the
onset of winter, fully formed adults within their cocoons undergo a
period during which temperatures are still appropriate for
development (pre-wintering period). Previous studies (Kemp
et al., 2004; Bosch et al., 2008) have shown that during this
period adults lower their respiration rates from 0.20 to 0.25 ml/g h
in the newly eclosed adult to 0.1 ml/g h. We address the following
questions: (1) given that pre-wintering adults have already
lowered their respiration rates, what is their respiratory response
to chilling temperatures? (2) Can diapause be completed without
chilling? (3) How does diapause maintenance proceed at different
wintering temperatures? How do adults wintered for short periods
respond to warm (incubation) temperatures? (4) Is it possible to
establish a clear transition between diapause and post-diapause?
(5) Can we use respiration rates during winter as a predictor of
emergence time in the spring?
The answers to these questions are interpreted in the light of
results on survival, vigour and emergence time obtained in a
previous study in which similar temperature regimes were used
(Bosch and Kemp, 2003).
2. Materials and methods
2.1. Life cycle of O. lignaria
O. lignaria is a spring-flying, solitary bee native to North
America. Adults fly for about a month in early spring (March–
April), during which time females build nests in pre-established
cavities (typically, abandoned beetle burrows in dead wood). These
nests are provisioned with pollen mixed with nectar as food for the
progeny. Eggs hatch within 1–2 weeks, and larval development
takes place through five instars. By early summer, the last instar
finishes up the pollen-nectar provision, spins a thick cocoon and
enters a short summer (prepupal) diapause (Kemp et al., 2004;
Kemp and Bosch, 2005; Bosch et al., 2008). Pupation takes place 1–
2 months after cocoon spinning, and adult eclosion occurs by late
summer or early autumn (Bosch and Kemp, 2000). Fully eclosed
adults harden their cuticle for about a day, but do not emerge out of
the cocoon. They enter diapause in autumn (Kemp et al., 2004;
Bosch et al., 2008), only to emerge in the following spring. Adult-
wintering appears to be a derived state within the Megachilidae,
most of which winter in the prepupal stage (Bosch et al., 2001).
Wintering in the prepupal stage is also the prevalent state in most
bees (Apiformes) and other Hymenoptera (Stephen et al., 1969;
Gauld and Bolton, 1988). O. lignaria is an excellent pollinator of
fruit tree flowers. For this reason, management methods have been
developed to use populations of this species for commercial
orchard pollination (Torchio, 1985; Bosch and Kemp, 2002).
2.2. Experiment 1: effect of wintering temperatures
2.2.1. Populations and rearing methods
We used the progeny of an O. lignaria population released in
early May 2002 in an apple orchard in North Logan, Utah, USA.
Drilled wood blocks with inserted paper straws (length: 15 cm;
diameter: 7.5 mm) were used as nesting materials. By the end of
the nesting period (20 June), some straws in which nests had been
built were pulled out of the wood blocks and brought to the
laboratory, where they were kept in a 22 8C cabinet. Other nests
were reinserted in wood blocks and stored in a North-facing open
barn in the same apple orchard. Temperature in the barn was
recorded hourly with a temperature logger. Beginning on 5 August,
when bees started to reach adulthood, we X-rayed all nests
(Stephen and Undurraga, 1976) every 3 days. We used X-ray plates
to sex individuals (females are usually larger than males and are
located in the innermost cells within a nest; Torchio, 1989; Bosch
and Kemp, 2001), and to establish adult eclosion dates.
From the group of nests kept at 22 8C, we selected 28 females (7
per treatment)that reached adulthood in midAugust. These females
(within their cocoons) were extracted from paper straws and placed
individually in clear gel capsules and pre-wintered at 22 8C for 30
days, then acclimatized for 7 days at 14 8C, and finally transferred to
wintering cabinets at 0, 4, and 7 8C, respectively. These bees were
held at their respective winter temperatures until May or until
individuals started emerging. In a fourth treatment, intended to test
the effect of absence of wintering, bees were left at 22 8C throughout
pre-wintering and wintering. An additional group of 7 females were
selected from the nests kept in the orchard. These females were also
placed individually in clear gel capsules and then kept within a
ventilated plastic food container within the barn in the apple
orchard (outdoors treatment). Temperature was measured with a
data logger adjacent to the wintered cocoons.
F. Sgolastra et al. / Journal of Insect Physiology 56 (2010) 185–194
186
2.2.2. Respiration rates and weight loss
The first respiration measurements were taken in mid Septem-
ber, 1 month after adult eclosion,when respiration rates had reached
their minimum values (Sgolastra, 2007; Bosch et al., 2008). From
then on, measurements were taken once a week until1 October, and
then once every 2 weeks until emergence or until 12 May.
Oxygen consumption and CO
2
production were measured
using cons tant volume respirom etry. We used a Sable Sys tems FC-
1O
2
Analyzer
1
and a Li-Cor CO
2
Analyzer
1
operating in
differential mode with a 100 ml/min flow rate (http://www.sa-
blesys.com/index.php). This allowed accuracy of measurement
that exceeded 0.001% in detecting departures from an undepleted
air stream that had been scrubbed of CO
2
and water vapour with a
Drierite
1
- Ascarite
1
column. At each sample date, we measured
the O
2
consumed and CO
2
produced by each of 7 individual bees
for 2 h at 22 8C in a Peltier cabinet. Data were collected via the
Sable Systems data acquisition program DATACAN
1
following
manufacturer’s protocols. Upon completion of a respirometry
session, individual bees were weighed. For comparison purposes,
O
2
and CO
2
levels were adjusted for the weight of each individual
and expressed as ml/g h. The ratio between CO
2
production and O
2
consumption (respiratory quotient, RQ) was calculated for each
respiration measurement. Weight loss was calculated as the
percentage of fresh body weight after each respiration measure-
ment in reference to the initial body weight measured at the
beginning of wintering.
It is important to note that respiration measurements were
conducted at 22 8C for all treatments. Thus, we did not measure
actual respiration rates during diapause, but the metabolic
response of diapausing bees (wintered at different temperatures)
when exposed to 22 8C (the temperature at which respiration
measurements were conducted). We therefore use the term
‘‘respiration response’’ throughout our study, and use the
magnitude of this response as a measure of diapause intensity.
This approach was chosen because we knew from previous studies
that even in populations wintered for long periods, incubation at
20 8C was required for female emergence (Bosch and Kemp,
2001; unpublished data), and we were interested in exploring the
relationship between a measurable metabolic response and time to
emerge following incubation (objective 5). We believe the short
(2 h) exposure of bees to 22 8C during respiration measurements
did not significantly affect the general course of diapause because
our results are consistent with those obtained in a previous study
in which new individuals were used in each respiration measure
(Kemp et al., 2004).
2.2.3. Statistical analysis
We used repeated-measures ANOVA to analyze differences in
weight loss (arcsine-transformed) and RQ among treatments
(excluding the 22 8C and outdoors treatments) throughout the
wintering period.
Visual inspection of the respiration data of treatments 0, 4 and
78C, showed an initial increase followed by a plateau, and then a
second increase. We fitted a five-parameter composite function to
describe the observed pattern:
R¼ða
1
a
2
e
a
3
t
Þþa
4
e
a
5
t
where Ris the respiration rate, tis the number of wintering days
and a
1
to a
5
are fitted parameters. The first term of the equation
describes the first phase as an inverted exponential rapidly
reaching a plateau, and the second term describes the second
exponential increase. The variance of O
2
and CO
2
data increased
with time, especially at t>100 days. For this reason, we log-
transformed the data, which not only increased homoscedasticity
but also markedly improved the convergence of the regression
procedure.
2.3. Experiment 2: effect of wintering duration
2.3.1. Population and rearing methods
We used the progeny of an O. lignaria population released in a
cherry orchard in North Ogden, Utah, USA, in April 2004. Nesting
materials were similar to those used in experiment 1. Nests
obtained were brought to the laboratory in early May, and kept in a
temperature cabinet simulating Logan (50 Km from North Ogden)
daily temperatures with a 12:12 h thermoperiod (May = 18:11 8C;
June = 21:15 8C; July = 24:18 8C; August = 22:15 8C). The high
temperature of each monthly thermoperiod was obtained as the
average between the maximum and the mean monthly tempera-
tures of the month. Similarly, the low temperature of each monthly
thermoperiod was obtained as the average between the minimum
and the mean monthly temperatures of each month.
As in experiment 1, nests were X-rayed every 3 days to monitor
individual adult eclosion. On 23 August, approximately in the
middle of the adult eclosion period, 50 newly eclosed females
(within their cocoons) were selected. These females were placed in
clear gel capsules and distributed among 5 wintering duration
treatments: 28, 84, 140, 196 and 252 days. Bees of all treatments
were pre-wintered at 22:15 8C (12:12 h) for 2 weeks, then
acclimatized to 10 8C for 1 week, and finally wintered at 4 8C.
Upon completion of each wintering treatment, adults within their
cocoons were placed individually in glass vials and incubated at
20 8C. Cocoons were monitored daily for emergence. For each
treatment, we calculated percent survival (individuals emerging
completely out of the cocoon) and emergence time (interval
between incubation date and emergence date).
2.3.2. Respiration rates and weight loss
Respiration rates and weight were measured on 7 females per
treatment, following the same protocol as in experiment 1.
Respiration measurements were taken at least at five selected
intervals: the day after adult eclosion, at the end of the pre-
wintering, 7 days after wintering (4 8C) initiation; and the day
before and the day after beginning of incubation (20 8C). From then
on, respiration rates were measured every 2 weeks until
emergence. As in experiment 1, RQs were calculated for each
respiration measurement. Weight loss during wintering was
calculated as the difference between the weight at adult eclosion
and at the end of wintering. Weight loss during incubation was
calculated as the difference between weight at the end of wintering
and at the last respiration measurement before emergence (or
death in females not emerging).
2.3.3. Statistical analysis
Percent weight loss (arcsine-transformed) across wintering
duration treatments was analyzed using one way-ANOVA. One
way-ANOVA was also used to analyze the effect of wintering
duration on emergence time (time between incubation and
emergence) and RQ. One of our objectives was to predict
emergence time as a function of respiration rates during wintering.
Thus, we fit negative exponential models to describe the relation-
ship between respiration rates at the end of wintering and
incubation time required for emergence.
3. Results
3.1. Experiment 1: wintering temperature
3.1.1. Respiration rates
Respirometry measurements started 1 month after adult
eclosion, when respiration rates were at minimum values (about
0.10 ml/g h). Following transfer from 14 8C to wintering tempera-
tures (0, 4 and 7 8C, respectively), bees responded with a rapid
F. Sgolastra et al. / Journal of Insect Physiology 56 (2010) 185–194
187
increase of their respiration response (Fig. 1). Within 2 weeks,
respiration response reached a plateau during which CO
2
production remained approximately stable and O
2
consumption
increased slowly. During this period, diapause intensity was
similar in bees wintered at 0, 4 and 7 8C(Fig. 1). Then, in mid
December (100 days of wintering) respiration responses started
to diverge among treatments, showing an exponential increase
that was more pronounced (greater a
5
parameter in our non-linear
model) at the warmer temperatures (Fig. 1). By mid April, bees of
the 7 8C treatment started to emerge during the respiration
measurement at 22 8C. Our model provided a good fit to
the described relationship between respiration response and
wintering days (Fig. 2,Table 1). RQ values first decreased, reaching
minimum values towards mid-winter, and then increased in late-
winter (Fig. 1;F
(16,288)
= 13.68; P<0.0001). RQ values differed
among temperature treatments (F
(2,18)
= 13.52; P<0.001),
with a significant wintering duration–temperature interaction
(F
(32,288)
= 5.23; P<0.0001).
Fig. 1. Mean
SE O
2
consumption (A), CO
2
production (B) and respiratory quotient (C) in O. lignaria females wintered at 0, 4, 7, and 22 8C. The first measurements were taken 1
month after adult eclosion, when respiration rates are at their lowest.
Fig. 2. Fitted non-linear regression of O
2
consumption (A) and CO
2
production (B) in O. lignaria females wintered at 0 (circles), 4 (triangles), and 7 8C (squares). Goodness-of-fit
parameters are shown in Table 1.
F. Sgolastra et al. / Journal of Insect Physiology 56 (2010) 185–194
188
The respiration response of bees wintered outdoors is shown in
Fig. 3. As for bees wintered at 0, 4 and 7 8C, outdoors bees also
increased their respiration response coinciding with an important
temperature drop in early September, and then reached a plateau
with a very slow increase. This plateau was longer than in the 0, 4
and 7 8C treatments, and then in early April, as soon as ambient
temperatures reached 20 8C, respiration response skyrocketed and
bees started to emerge. RQ levels in bees wintered outdoors
followed a pattern similar to bees reared under artificial conditions
reaching minimum values (0.7) from December to February.
Bees of the 22 8C treatment were never exposed to cold
temperatures. These bees never expressed the initial increase in
respiration rates observed in the other treatments. Thus, respira-
tion rates of bees kept at 22 8C remained low throughout the
winter (Fig. 1).
3.1.2. Weight loss
The low respiration rates expressed by bees of the 22 8C
treatment did not prevent them from loosing weight dramatically
(Fig. 4). Within 3 months these bees had lost over 50% of their
weight, and eventually all of them died within their cocoons. Bees
of the other treatments also lost weight throughout the winter
(F
(15,270)
= 863.64; P<0.0001), but at a much lower rate. Bees
wintered at 7 8C lost significantly more weight (0.18 mg/day) than
those wintered at 0 and 4 8C (0.06–0.07 mg/day; F
(2,18)
= 31.59;
P<0.0001), and there was a significant interaction between
temperature and wintering days (F
(30,270)
= 16.24; P<0.0001).
3.2. Experiment 2: wintering duration
3.2.1. Respiration rates
O
2
consumption and CO
2
production of newly eclosed adults
were close to 0.25 ml/g h (Fig. 5). During pre-wintering (14 days at
Table 1
Goodness-of-fit parameters of the non-linear regression models of Fig. 2.
Temperature R
2
FP
O
2
consumption
08C 80.1 1964.4 <0.0001
48C 74.6 781.1 <0.0001
78C 78.8 332.5 <0.0001
CO
2
production
08C 68.3 2623.2 <0.0001
48C 76.1 1067.1 <0.0001
78C 80.8 444.2 <0.0001
Fig. 4. Mean percent of initial body weight in O. lignaria females wintered at 0, 4, 7,
22 8C and outdoors. Error bars not shown for clarity.
Fig. 3. Mean
SE O
2
consumption (A), CO
2
production (B) and respiratory quotient (C) of O. lignaria females wintered outdoors. The first measurements were taken 1 month after
adult eclosion, when respiration rates are at their lowest. Dotted line indicates mean daily ambient temperature.
F. Sgolastra et al. / Journal of Insect Physiology 56 (2010) 185–194
189
22–15 8C, 12:12 h), respiration response declined to 0.15 ml/g h.
As in experiment 1, as soon as bees were transferred to 10 8C,
respiration response initiated a logistic climb and then continued
to increase throughout wintering at a much slower rate. In
treatments with short wintering durations (28, 84 days), bees were
incubated at 20 8C while respiration response was still low. Upon
incubation, females of these two treatments showed a tendency to
increase their metabolic rates, but then rapidly lowered their O
2
consumption and CO
2
production to levels similar to those
expressed during wintering (Fig. 5). Instead, bees wintered for
longer periods (approaching natural wintering duration in north-
ern Utah) did not lower their respiration rates during incubation
(Fig. 5). RQ values followed a similar pattern to experiment 1,
reaching minimum values towards mid-winter (84–140 days)
(Fig. 6;F
(6,40)
= 6.52; P<0.001).
3.2.2. Weight loss
Bees lost weight much more rapidly during incubation than
during wintering (Fig. 6). Because bees wintered for shorter
periods required longer incubation periods to emerge, total weight
loss diminished with wintering duration (Fig. 6;F
(4,21)
= 36.29;
P<0.0001). Total weight loss was 10% in bees wintered for the
longest periods (196 and 252 days) compared to 29% in females
wintered for 28 days.
3.2.3. Survival and emergence time
Nine of the 10 females wintered for 28 days failed to emerge
from the cocoon, and the one that emerged died the day after. In
the other treatments, survival was high: 9 of 10 females in the 196-
day treatment and 7 of 10 females in the remaining treatments.
Emergence time declined with wintering duration (Fig. 6;
Fig. 5. Mean
SE O
2
consumption, CO
2
production and weight during pre-wintering (PW), wintering (W) and incubation (I) in O. lignaria females wintered for 28 (A), 84 (B), 140 (C),
196 (D) and 252 (E) days at 4 8C. The first measurements were taken on the day of adult eclosion.
F. Sgolastra et al. / Journal of Insect Physiology 56 (2010) 185–194
190
F
(3,26)
= 74.153; P<0.001; 28-day-treatment excluded). Bees
wintered for 84 days required 48.00
3.57 days of incubation to
emerge versus 2.86 0.83 days in bees wintered for 252 days. The
relationship between respiration levels at the end of wintering and
emergence time followed a negative exponential curve (Fig. 7).
4. Discussion
4.1. Diapause initiation and maintenance
Respiration rates in newly eclosed O. lignaria were 0.25 ml/
g h, and then dropped to 0.15 ml/g h within 2 weeks, and to
0.10 ml/g h within 4 weeks (Fig. 5). These results are in
agreement with previous studies, which showed a similar pattern
in individuals reared either at 22 8C or under natural temperature
regimes (Kemp et al., 2004; Sgolastra, 2007). Diapause initiation
occurs in individuals reared at constant temperatures in complete
darkness, indicating that winter diapause in O. lignaria is a fixed
component of the ontogenic program, requiring no external cue
(obligatory diapause; Tauber et al., 1986; Kostal, 2006).
If not chilled, diapausing O. lignaria adults maintain minimum
respiration levels (0.10 ml/g h) until they die. Similar results have
been obtained in insects diapausing in different developmental
stages, such as larvae of the drosophilid fly Chymomyza costata
(Kostal et al., 2000), or embryos of the katydid Eobiana engelhardti
(Higaki and Ando, 2005), showing that a period of cold
temperatures is necessary to complete the diapause process. In
nature, O. lignaria populations reach the adult stage in late
summer, shortly before the onset of winter temperatures. Given
that diapause initiation (time during which respiration rates drop
and reach minimum values) lasts 2–4 weeks, the effective period of
diapause maintenance is rather short in this species (Fig. 8).
4.2. Response to chilling and diapause termination
As soon as diapausing adults are chilled, even for periods as
short as 7 days, they respond by quickly increasing their
respiration rates (measured at 22 8C), showing that the ‘‘metabolic
brake’’ (low respiration rates) operating while temperatures were
high (pre-winter) has been eased off. Following Kostal (2006), and
given that diapause cannot be terminated without chilling, we
interpret this response as the beginning of the process of diapause
termination (Fig. 8). Interestingly, individuals chilled 5 days after
adult eclosion (before they have had the time to reach minimum
[0.10 ml/g h] respiration levels) also respond to cold temperature
by raising their respiration rate (Sgolastra, 2007). Thus, chilling
acts as a synchronizing stimulus among individuals initiating
diapause on different dates (Kostal, 2006). The period over which
individuals within a population reach adulthood may span for as
long as a month (Bosch et al., 2001; Sgolastra, 2007), but the period
of emergence lasts only 1–2 weeks (Bosch et al., 2001; Sgolastra,
2007). These results are different from results obtained on another
Fig. 6. Mean
SE respiratory quotient (A) and percentage of weight loss, and emergence time (B) in O. lignaria females wintered for 28, 84, 140, 196 and 252 days at 4 8C and
incubated at 20 8C.
Fig. 7. Relationship of O
2
consumption (A) and CO
2
production (B) measured at the end of wintering with emergence time following incubation at 20 8C (time to
emerge = 623.745*exp(9.902*O
2
); R
2
= 0.86; P<0.001; time to emerge = 1709.638*exp(18.977*CO
2
); R
2
= 0.89; P<0.001).
F. Sgolastra et al. / Journal of Insect Physiology 56 (2010) 185–194
191
Megachilid, Megachile rotundata. In this species, which enters
diapause in summer in the prepupal stage, respiration response
remains at 0.1–0.2 ml/g h throughout the autumn and winter, and
does not increase until prepupae are exposed to incubation
temperatures (Kemp et al., 2004).
Following the initial response to cold temperatures, our
respirometry results show two distinct phases (Fig. 8). In the first
phase, diapause intensity follows a rapid decrease and then
reaches a plateau, during which diapause intensity remains stable
or decreases slowly. This first phase lasts 100 days, and appears
to be independent of wintering temperature (within a certain
range) (Fig. 2). RQ values, which were as high as 0.9 during
diapause initiation reach minimum levels (0.7) towards the end
of this phase (Figs. 1 and 6), indicating a gradual change in
metabolic energy substrates from carbohydrates early on, to lipids
(and possibly proteins) in mid-winter (Adedokun and Denlinger,
1985; Hahn and Denlinger, 2007). Also during this first phase,
individuals moved from wintering to incubation temperatures
(20 8C) respond by lowering their respiration rates (Fig. 5). Instead,
in the second phase, diapause intensity decreases exponentially,
and this decrease diverges among temperature treatments, being
faster at warmer temperatures (Fig. 2). RQ levels increase again,
and reach values of 0.9 when bees approach their emergence
time (150 days in the 7 8C treatment, 200 days in the 4 8C
treatment, April in the outdoors treatment) (Figs. 1, 3 and 6).
During this second phase, O. lignaria females no longer lower their
respiration rates when exposed to 20 8C(Fig. 5).
Following the general diapause model proposed by Kostal
(2006), the first phase would correspond to the period of diapause
termination, and the second phase to the period of post-diapause
quiescence, during which low metabolic rates are maintained
exogenously while temperatures are still too cold for morphogen-
esis (or emergence in O. lignaria). According to this interpretation,
diapause in O. lignaria would be characterized by an early (mid-
winter) diapause termination, and a long post-diapause quies-
cence, in agreement with findings in many other temperate-zone
insects (Tauber et al., 1986; Hodek, 2002). This view is supported
by results obtained in a previous study (Bosch and Kemp, 2003; see
also Bosch and Kemp, 2004) showing reduced winter survival in
bees wintered for <90 days, and thus indicating that diapause is
clearly not completed at these wintering durations, together with
lack of a consistent effect of wintering temperature on emergence
time in bees wintered for <90 days. The same study shows that
males start emerging without incubation at wintering >150 days
at 7 8C, an unequivocal sign that diapause has been completed at
this temperature (although males wintered at 0 or 4 8C require
incubation temperatures to emerge, even after 270 days of
chilling).
The above interpretation, however, does not provide a
satisfactory explanation for the relatively long period of incubation
required for emergence in bees wintered for 100–150 days. If O.
lignaria were in post-diapause at such wintering durations, we
would expect individuals to respond immediately to development-
or activation-promoting conditions (Tauber et al., 1986; Kostal,
2006). Yet, O. lignaria individuals wintered at 4 8C for 100–150 days
and then incubated at 20–22 8C take 7–15 days to emerge,
compared to 2–7 days in individuals wintered for 190–240 days
(Bosch and Kemp, 2000, 2003; and unpublished data; Bosch et al.,
2000; this study). Studies on other Osmia species show similar
patterns (Tase
´i, 1973; van der Steen and de Ruijter, 1991; Bosch
and Blas, 1994; Bosch and Kemp, 2004; Maeta et al., 2006). Based
on these results, 2 days appears to be the shortest possible
emergence time for O. lignaria females incubated at 20 8C.
Emergence times of this sort are achieved in individuals whose
respiration response have reached levels of 0.45 ml/g h of CO
2
production and 0.55 ml/g h of O
2
consumption (Fig. 7). These
respiration levels could thus be considered indicators of diapause
completion in O. lignaria. If incubated when respiration rates have
not reached levels close to 0.4 ml/g h (84-day and 140-day
treatments), emergence periods are significantly extended, indi-
cating that diapause is still not completed. Unlike males, females
do not emerge until exposed to temperatures of 20 8C, even in
fully wintered populations (Bosch and Kemp, 2001; and unpub-
lished data). Females having reached levels of CO
2
production of
0.45 ml/g h would be in post-diapause quiescence until exposed to
temperatures eliciting emergence. According to this second
interpretation, after 100 days of wintering, O. lignaria would
have attained the potential to terminate diapause, but diapause
completion would not be reached until early spring, and the
duration of the termination period would be dependent on
temperature (shorter at warmer temperatures). In the field, the
respiration response followed a pattern similar to that of treatment
08C, and never reached levels close to 0.3 ml/g h until late-March,
when temperatures started increasing (Fig. 3). Then, as soon as
mean ambient temperatures reached values close to 20 8C in April,
the respiration response skyrocketed. The low respiration response
recorded until March, and the long emergence periods (15 days)
expressed by populations wintered outdoors for insufficiently long
periods (117 days) (Bosch et al., 2000), are in agreement with this
second interpretation of the second phase.
Fig. 8. Schematic depiction (following Kostal, 2006) of diapause intensity during pre-winter (20 8C), winter (0, 4 or 7 8C), and incubation (20 8C) in O. lignaria females. Arrow
tips indicate emergence out of the cocoon. See Section 4for interpretation of 1st and 2nd phases.
F. Sgolastra et al. / Journal of Insect Physiology 56 (2010) 185–194
192
Future studies should attempt to elucidate which of the two
proposed diapause models (early diapause termination followed
by a long post-diapause quiescence or late-diapause termination
followed by a much abbreviated post-diapause quiescence) is valid
for O. lignaria. Because physiological processes are usually gradual,
our two hypotheses may in fact be two extremes of a continuum.
As noted in the introduction, it is often difficult to characterize the
transition between the various phases of diapause (Kostal, 2006).
This is especially so in O. lignaria for two reasons. First, being an
obligate diapauser, we cannot compare the response of diapausing
and non-diapausing individuals. Second, because diapause occurs
in complete darkness, we cannot measure the response to
photoperiod throughout wintering. External and internal mor-
phological changes have often been used as indicators of diapause
completion (e.g. Kostal et al., 2000; Ragland et al., 2009). Because O.
lignaria over-winters as a fully formed adult, external morpholo-
gical changes are not readily apparent, but ovary maturation is far
for complete in wintered females (Sgolastra, 2007). Thus, oocyte
size could provide a good indicator of morphogenesis resumption
in this species. Future studies should also analyze molecular
markers (Denlinger, 2002). Patterns of expression during diapause
and post-diapause of genes enconding various families of heat-
shock proteins have shown promising results in two flies and
another Megachilid, the alfalfa leafcutting bee M. rotundata
(Hayward et al., 2005; Tachibana et al., 2005; Yocum et al.,
2005, 2006). In the Heteropteran Pyrrhocoris apterus, which
winters as an emerged, active adult, levels of transcripts of genes
coding for enzymes involved in polyol biosynthesis, were found to
closely match diapause intensity and time to oviposition (Kostal
et al., 2008).
4.3. Survival and weight loss
Irrespective of the precise timing of diapause termination,
exposure to cold temperature is necessary to complete diapause in
O. lignaria. Non-chilled bees, as well as bees chilled for very short
periods, respond to warm temperatures by lowering their
respiration, and thus never reach levels eliciting emergence
(Figs. 1 and 5). However, even while maintaining low respiration
rates, diapausing individuals kept at high temperatures incur in
high metabolic cost, resulting in extensive fat body depletion, loss
of vigour and increased mortality (Bosch and Kemp, 2004;
Sgolastra, 2007; Bosch et al., 2008). Of course, these losses would
be much greater did bees not keep their metabolic rates low under
these conditions. At any rate, none of the bees exposed to the no-
wintering treatment survived. In insects diapausing as feeding
stages, increased metabolic activity during periods of warm
weather in the initiation phase is compensated by feeding and
building up of energy reserves (Tauber et al., 1986; Kostal, 2006;
Hahn and Denlinger, 2007), a possibility not available to pre-
wintering Osmia adults. Both in O. lignaria and in O. cornuta, weight
loss rates are much higher during pre-wintering (0.2–0.4 mg/day)
than during wintering (0.05–0.09 mg/day) (Bosch and Kemp, 2003,
2004; Kemp et al., 2004).
Only one of the females exposed to a very short winter (28 days)
survived to emergence. This result is consistent with other Osmia
studies in which survival of bees wintered for 30 days, ranged
between 0 and 40% (Bosch and Kemp, 2003, 2004; Maeta et al.,
2006). In addition, those individuals that manage to emerge
following short wintering periods are weak and have short post-
emergence longevity (Bosch and Kemp, 2003, 2004). Conversely, in
the bumblebee B. terrestris survival was not negatively affected by
short wintering periods (1 month; Beekman et al., 1998). O. lignaria
incubated following a short wintering period respond by rapidly
lowering their metabolism, but nonetheless loose weight rapidly at
a rate similar to that expressed during pre-wintering (Fig. 6).
Because these bees require long incubation periods to emerge
(Fig. 6), the end result is a very high weight loss. Metabolic
depression is a common response to environmental stress, but is
usually accompanied by a decrease in fitness (Parsons, 1996;
Chown and Gaston, 1999).
In addition to wintering duration, winter temperature also
affects O. lignaria fitness. Bees exposed to milder winter
temperatures emerge sooner, but express increased weight loss
and fat body depletion, resulting in decreased survival and poor
vigour at emergence (Bosch and Kemp, 2003). Shorter diapause
duration at warmer temperatures has been attributed to the
greater metabolic rate and increased catabolism of nutrient
resources (Hahn and Denlinger, 2007). Studies on the ladybird
beetle Coleomegilla maculata and the gall fly Eurosta solidaginis
have also shown reduced survival and vigour in individuals
wintered at warm temperatures (Jean et al., 1990; Irwin and Lee,
2000). Conversely, wintering temperature did not affect winter
survival in B. terrestris (Beekman et al., 1998). In nature, earlier
emergence in years with milder winters could serve as a
mechanism to synchronize O. lignaria emergence with advanced
blooming time of its vernal host plants. However, increased body
weight loss and fat body depletion during mild winters may
compromise post-diapause performance. A trade-off between
diapause and reproductive success has been reported in several
insects (Bradshaw et al., 1998; Irwin and Lee, 2000; Ellers and van
Alphen, 2002; Musolin and Numata, 2003), but losses incurred
during suboptimal winters may be off-set in species whose
individuals feed before reproducing, as is the case in O. lignaria
(Peferoen et al., 1981; Jansson et al., 1989; Ishihara and Shimada,
1995).
Management methods have been developed to use O. lignaria
and other Osmia species for orchard pollination (Bosch and Kemp,
2002). By improving our understanding of the ecophysiological
processes underlying winter diapause, our results should help to
improve temperature regimes currently used to rear Osmia
populations. In addition, the use of respiration rates as an indicator
of emergence time may help to improve synchronization between
bee emergence and blooming of the target crop.
Acknowledgements
We are grateful to G. Trostle and S. Kalaskar (USDA-ARS, Logan)
for their invaluable help in all phases of the study, and to J. Rinehart
and K. Anderson (USDA-ARS, Fargo) for reviewing the manuscript.
We very much appreciate the constructive and helpful comments
of V. Kostal and an anonymous reviewer. This study was partially
supported by a Ph.D. Scholarship from the University of Bologna to
F.S. and the Spanish program Consolider-Ingenio MONTES to J. B.
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