Influence of brood rearing temperature on honey bee development and susceptibility to poisoning by pesticides

Abstract

Adult honey bees (Apis mellifera) usually maintain colony brood rearing temperature between 34-35°C by thermoregulation. The brood may,
however, also be subjected to suboptimal temperature. Here we investigated whether a decrease of brood rearing temperature may have
effects on larval mortality, adult emergence, longevity, morphology and susceptibility to poisoning by pesticides (dimethoate). Using the in
vitro rearing protocol of Aupinel (2005), we were able for the first time to control the brood temperature not only during the pupal stage but
also during the larval stage. Honey bee larvae were reared in vitro at 35°C (optimal) and 33°C (suboptimal) from 12 h after hatching for 15
days. Dimethoate was tested by ingestion either on 4-day old larvae or on 7-day old adults. Our results showed that lower rearing
temperature had no significant effects on larval mortality and adult emergence, but adult bee mortality was strongly affected. Moreover, adult
workers emerging at 33°C were significantly more susceptible to dimethoate. Larval LD50 (48 h) was, however, 28 times higher at 33°C than
at 35°C. The striking differences between larvae and adults may be explained by differential larval metabolism at 33°C and resulting slower
active ingredient absorption. We conclude that adult honey bees reared at even slightly suboptimal brood temperature may be more
susceptible to pesticide poisoning and be characterised by reduced longevity. Thus, low temperature brood rearing could be another stress
factor for colonies.

Full text

ORIGINAL RESEARCH ARTICLE
Influence of brood rearing temperature on honey bee
development and susceptibility to poisoning by pesticides
Piotr Medrzycki1, Fabio Sgolastra2, Laura Bortolotti1, Gherardo Bogo1, Simone Tosi1, Erica Padovani1,
Claudio Porrini2 and Anna Gloria Sabatini1
1Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Unità di Ricerca di Apicoltura e Bachicoltura, Via di Saliceto 80,
40128 Bologna, Italy.
2Dipartimento di Scienze e Tecnologie Agroambientali - Area Entomologia, Università di Bologna, Viale G. Fanin 42,
40127 Bologna, Italy.
Received 30 April 2009, accepted subject to revision 2 December 2009, accepted for publication 13 December 2009.
*Corresponding author: Email: pmedrzycki@inapicoltura.org
Summary
Adult honey bees (
Apis mellifera
) usually maintain colony brood rearing temperature between 34-35°C by thermoregulation. The brood may,
however, also be subjected to suboptimal temperature. Here we investigated whether a decrease of brood rearing temperature may have
effects on larval mortality, adult emergence, longevity, morphology and susceptibility to poisoning by pesticides (dimethoate). Using the
in
vitro
rearing protocol of Aupinel (2005), we were able for the first time to control the brood temperature not only during the pupal stage but
also during the larval stage. Honey bee larvae were reared
in vitro
at 35°C (optimal) and 33°C (suboptimal) from 12 h after hatching for 15
days. Dimethoate was tested by ingestion either on 4-day old larvae or on 7-day old adults. Our results showed that lower rearing
temperature had no significant effects on larval mortality and adult emergence, but adult bee mortality was strongly affected. Moreover, adult
workers emerging at 33°C were significantly more susceptible to dimethoate. Larval LD50 (48 h) was, however, 28 times higher at 33°C than
at 35°C. The striking differences between larvae and adults may be explained by differential larval metabolism at 33°C and resulting slower
active ingredient absorption. We conclude that adult honey bees reared at even slightly suboptimal brood temperature may be more
susceptible to pesticide poisoning and be characterised by reduced longevity. Thus, low temperature brood rearing could be another stress
factor for colonies.
Influencia de la temperatura de la cría en el desarrollo de la
abeja de la miel y susceptibilidad a la intoxicación por los
pesticidas
Resumen
Las abejas adultas (
Apis
mellifera
; Himenoptera: Apidae) mantienen normalmente la temperatura de la cría dentro de un rango estrecho
(34-35°C) mediante la termorregulación. En ciertas situaciones, sin embargo, la cría puede estar sometida a condiciones de temperatura
subóptima. El objetivo de este estudio fue investigar si la disminución de la temperatura de la cría solamente de 2°C puede tener efectos
sobre la mortalidad larvaria y sobre la emergencia de los adultos y otroa parámetros vitales. Por otra parte la susceptibilidad a la intoxicación
por los pesticidas (dimetoato) fue estudiada en larvas y en los adultos emergidos de la cría mantenida en las temperaturas probadas. Para
estos propósitos, las larvas de la abeja de la miel fueron criadas
in vitro
en dos temperaturas distintas: 35°C (óptimo) y 33°C (subóptimo),
desde 12 h después de la incubación hasta los 15 días de edad. En diversos experimentos, el dimetoato fue administrado a las larvas o a los
adultos. La mortalidad larvaria, la emergencia de adultos y la longevidad fueron medidas. Nuestros resultados mostraron que la temperatura
Journal of Apicultural Research
49(1): 52-59 (2010) © IBRA 2010
DOI 10.3896/IBRA.1.49.1.07

Introduction
Since the escalation of honey bee colony loss phenomena and the
definition of “Colony Collapse Disorder” (CCD), many scientific studies
have been carried out each year to determine possible damage
factors and to estimate their relative importance, with the ultimate
aim of establishing preventive and damage limiting measures
(Connor, 2007; Cox-Foster
et
al
., 2007; Henderson
et al
., 2007a; b;
Higes
et al
., 2007; 2009). It is commonly agreed that there is no one
single factor causing bee/colony losses, rather there are many factors
belonging to different categories of stress factors (Oldroyd, 2007; Cox
-Foster and vanEngelsdorp, 2009; Neumann and Carreck, 2010).
Moreover, synergistic effects probably pose obstacles and
complications in experimental procedures, since many factors can
change their importance when administered in combination with
others. Synergism has been studied to test the effects of several
pesticides applied at the same time on honey bees (Colin and
Belzunces, 1992; Pilling and Jepson, 1993; Pilling
et al
., 1995; Meled
et al
., 1998). The sublethal effects of some factors make the problem
complex, because there is often no direct link between the factor and
the damage. The former may cause some behavioural modifications in
workers and, since the honey bee is eusocial, these modifications can
lead to severe damage at the colony level, even if there is no
apparent effect at the individual level (Vandame
et al
., 1995;
Bortolotti
et al
., 2003; Medrzycki
et al
., 2003; Decourtye
et al
., 2004;
2005; Kralj and Fuchs, 2006; Kralj
et al
., 2007; Desneuz
et al
., 2007).
Colony loss is probably often a multifactorial syndrome, and if bees
are stressed by some causes, they may be less able to produce an
effective immune response to pathogens. For example, several studies
(Bowen-Walker and Gunn, 2001; Amdam
et al
., 2004; Yang and Cox-
Foster, 2007) showed the effects of the ectoparasitic mite
Varroa
destructor
(Acari: Varroidae) on honey bee physiology. The altered
bee physiology may increase susceptibility to infective agents or other
stressing factors such as pesticides that may lead the colony to
collapse. The situation is more complicated when the sublethal effect
of one factor acts in synergism with other factors.
Another phenomenon, hypothesised by us and which makes the
problem even more complex, concerns a causal factor which has
invisible direct effects, but may cause delayed indirect damage. In this
case it is much more difficult to demonstrate a link between this
factor and the visible damage.
Brood rearing temperature is one of the most precisely controlled
physiological parameters in a honey bee colony. Adult workers keep
the central brood area at 34-35°C (Himmer, 1927; Seeley and
Heinrich, 1981). In order to maintain the temperature within this
narrow range, the high or low external temperature is contrasted by
thermoregulation behaviours (Kronenberg and Heller, 1982; Jones
et
al
., 2004). Thus, normally only slight deviations from the optimal level
will occur. Nevertheless, in particular situations, the brood may be
subject to conditions of suboptimal temperature. Several studies
(Tautz
et al
., 2003; Groh
et al
., 2004; Jones
et al
., 2005; Becher
et
al
., 2009) report the effects of the temperature of pupal incubation on
adult workers. Negative effects on short-term learning, memory
capacities and orientation were shown when pupae were exposed to
suboptimal temperature instead of the optimal one (34.5°C). In
particular, bees reared at low temperatures showed reduced dance
performances and proboscis extension reflex. McMullan and Brown
(2005) showed that bees newly emerged from brood pupated at lower
temperature (30°C) are more susceptible to tracheal mite infestation
than those emerged from brood pupated at normal temperature. In
all of these studies the brood was incubated at several temperatures
from the moment in which it was capped. We have hypothesised that
incubating the larvae at the suboptimal temperature during the whole
developmental period could have even stronger effects on the
emerging adults. Probably, these weak bees will be more susceptible
to other stressors (even at sublethal level) with slow but important
consequences on the whole colony. Recently, the
in vitro
larvae
rearing method has been established (Aupinel
et al
., 2005), thereby
allowing the exposure of larvae to precisely determined temperatures.
It was hypothesised that a slight poisoning, causing the loss of an
apparently insignificant quantity of adult bees in early spring, i.e. in
conditions of low external temperatures, could potentially reduce the
ability of the colony to maintain brood at constant optimal temperature.
The aim of the present study was therefore to analyse the effects of
suboptimal brood rearing temperature on larvae and emerging adults.
Temperature was studied both as a single factor and in synergism
with poisoning by dimethoate, a commonly used organophosphorous
pesticide and the reference active ingredient for many toxicological
studies (EPPO, 2001).
Effects of brood rearing temperature on honey bees 53
de cría más baja no tiene ningún efecto sobre la mortalidad larvaria, o en la tasa de emergencia de adultos, mientras que la mortalidad de
abeja adulta se vio fuertemente afectada. Por otra parte, las abejas adultas que emergieron de la cría mantenida en la temperatura subóptima
fueron más susceptibles a la intoxicación por el dimetoato. Por el contrario, el LD50 larvario (48 h) fue 28 veces más alto a una temperatura
más baja que la temperatura óptima. Esto se puede explicar por el metabolismo larvario más lento en la temperatura más baja, con la
consecuente absorción más lenta de ingredientes activos. Con este estudio podemos concluir que las abejas adultas que derivaban de la cría
mantenida a temperatura subóptima tienen una aptitud más baja y son más susceptibles a la intoxicación por pesticidas.
Key words: bee losses, brood temperature, CCD, development, mortality, longevity, malformations, pesticide toxicity

Materials and methods
The experiments were carried out by applying the
in vitro
bee brood
rearing protocol of Aupinel
et al
. (2005). The in hive phase consisted
of isolation of the queen on an empty comb for 26 hours. After that,
the larvae (aged about 12 hours) were grafted using a thin paintbrush
(00), into previously prepared 48-well tissue culture plates containing
a plastic queen cell (Nicoplast©) in each well. The appropriate semi-
artificial diet was provided. All the larvae used in one experiment were
taken from the same hive according to the protocol. The plates with
the larvae were then incubated in an airtight Plexiglas desiccator at
95% RH and at the desired temperature, according to the
experimental group. The “warm” larvae were incubated at the optimal
temperature (35°C), while the “cold” larvae were incubated at a
suboptimal temperature (33°C). Some preliminary trials were
54 Medrzycki, Sgolastra, Bortolotti, Bogo, Tosi, Padovani, Porrini, Sabatini
Rearing day "warm" rearing (35°C) "col d" reari ng (33°C)
D1
D3
D4 30µ diet
C
30µ diet
C
D5 40µ diet
C
D6 50µ diet
C
40µ diet
C
D7 50µ diet
C
D8 transfer to 75% R
H
D9 transfer to 75% R
H
D19 emer
g
ence
D20 emergence
graf ting, 20µl diet A, 95% RH
20µl diet B
Table 1.
Brood rearing management according to the incubation
temperature.
%p%p
35 124 43
33 120 48
35 133 50
33 130 65
35 90 79 18
33 183 52 31
35 131 18 77
33 132 23 53
35 40 10 85
33 123 15 26
35 40 20 75
33 81 20 72
35 137 47 50
33 261 28 66
0.000 *
0.876
0.103
-
-
-
-
0.079
0.059
08/08/2008 (2)
05/09/2008 (1)
05/09/2008 (2)
0.824
0.078
0.084
0.678
0.213
0.962
0.092
Dev elopm ent m ortality Em erged adults
G rafting date Brood rearing
te m p .(°C )
n of larvae
on D4
16/05/2008
23/05/2008
25/07/2008
08/08/2008 (1)
Table 2.
Experiment 1. Development mortality (
t
-test) and adult emergence (
c
2
test) related to the brood rearing temperature. Results of 7
separate trials. * significant difference
necessary in order to calibrate Aupinel’s protocol for the conditions of
suboptimal temperature. The “cold” larvae showed slower metabolism
dynamics, thus the diet consumption was slightly slower.
Consequently, one-day delay of the last two feedings was
necessary, while assuring the constant diet availability and the equal
amount of diet consumed for both temperatures (Table 1). Adult
emergence was also delayed by one day, so the longevity
measurements had to take this parameter into account. On the 8th
and 9th day (for “warm” and “cold” larvae respectively), the cells were
moved to another desiccator with the temperature set at 35°C or 33°
C (respectively) and 75% RH. In order to study the influence of brood
temperature on larval development, adult longevity and susceptibility
both of larvae and adults to poisoning by dimethoate, five
experiments were carried out. Experiments 1 and 2 were repeated in
different periods of the year (from spring to late summer).
Experiment 1 Development mortality
During development of both groups of larvae (“warm” and “cold”),
mortality was checked at the end of the larval stage (day 15) and
adult emergence was recorded on day 23, i.e. when no further adult
workers would emerge (Table 2). Since live individuals are in the
pupal stage at day 15 (which corresponds to the 19th day of individual
life), the mortality check was based exclusively on visual assessment.
Pupae appearing normal were considered alive, whilst those which
appeared abnormally developed (shape of larva, lack of appendages,
abnormal colour) were considered dead. Regarding the adult
emergence rate assessment, only workers which had completely left
the cell were considered emerged, and those remaining were
considered non-emerged. The development mortality test was
repeated seven times but only five of the tests were continued until
emergence of adults. The remaining two experiments were
terminated

at the end of the pupal stage, so no emergence data are available
(
Table 2
). The results were processed separately for each experimental
date. The % development mortality (D15) and the % adult
emergence were calculated by referring to the number of larvae alive
at D4. Mortality occurring before D4 was considered to have been
caused
by manipulation during grafting. For the development mortality,
a
t
-test (after arcsine-transformation) was applied, considering single
plates as experimental units. The % adult emergence was compared
by the
c
2
test.
Experiment 2: Adult worker survival
Two groups of grafted larvae were incubated according to the rearing
protocol until D15. After that, the cells containing the pupae were
moved to emergence boxes (16 x 12 x 5cm), equipped with two
feeders: one containing organic
Robinia
honey, and the other one
containing a mix of organic pollen and honey. The pupae and,
subsequently, the emerged adult workers were kept at 35°C and 75%
RH. The experiment was repeated three times in different periods of
the year: in May, July and August, 2008. Survival was assessed
periodically and the results were processed separately for each trial.
The % survival of “cold” and “warm” bees” was compared at different
intervals using the
c
2
test (Table 3).
Effects of brood rearing temperature on honey bees 55
Experiment 3: Larval susceptibility to poisoning
by dimethoate
The larval LD50 (48 h and 72 h) of dimethoate was studied. Both for
the “warm” and the “cold” group, six multiwell plates were prepared,
each containing 48 grafted larvae. On day 4, except for the control
plates, the diet provided was contaminated with progressive amounts
of dimethoate. The quantities of active ingredient provided to the
tested larvae were: 0.83 µg, 1.65 µg, 3.30 µg, 6.60 µg and 13.30 µg;
all 48 larvae on each plate received the same dose (Aupinel
et al
.,
2007). Thus, for each group, one untreated and five treated plates
were obtained. 48 and 72 hours after poisoning, mortality was checked.
The calculation of % mortality was the number of larvae alive at D4.
Subsequently the LD50 value was calculated using probit analysis.
Experiment 4: Adult worker susceptibility to
poisoning by dimethoate
Two groups of adult workers were obtained from the brood reared at
the two temperatures. 144 and 288 larvae were grafted respectively
for the optimal and suboptimal temperature incubation. Before
emergence (day 15), the cells with the pupae were moved to
emergence boxes as described for experiment 2. The adult workers
were then kept at 35°C and 75% RH for seven days and then divided
in groups of 10 individuals which were transferred to Perspex cages
7th day p13th day p20th day p
35 61 93.4 93.4 88.8
33 46 3.2 0.0 0.0
35 16 - 62.5 -
33 56 - 0.0 -
35 101 97.0 97.0 96.0
33 70 0.0 0.0 0.0
0.002 *
-
< 0.001 *
< 0.001 *
< 0.001 *
< 0.001 *
Grafting date
< 0.001 *
-
< 0.001 *
Brood rearing
tem p .(°C ) n
16/05/2008 **
% survival
25/07/2008
08/08/2008
Table 3.
Experiment 2. Adult worker survival (at the 7th, 13th and 20th day of adult life) related to the brood rearing temperature. Results of
three separate trials are shown. *significant difference (
c
2
); ** observations taken on the 8th, 14th, and 20th day.
a b so lu te c o rre c te d
35000
33 0 62 62
35 5 85 84
33 0 100 100
35 5 100 100
33 6 100 100
35 20 20
33 50 50
n of bees
Brood rearing
te m p. (°C )
Tim e (h) p
% control
mortality
% Dim ethoate m ortality
8
24
48
0.001 *
0.673
1.000
Table 4.
Experiment 4. Adult bee mortality 8, 24 and 48 hours after the administration of 0.15µg of dimethoate per bee. Data corrected with
Schneider-Orelli formula. *significant difference (
c
2
).

for the toxicological tests. In order to facilitate handling, the workers
were anesthetised by keeping them at 4°C for 2 hours. The cages
(13 x 6 x 11 cm) had two opposite walls made of transparent Perspex
to allow visual control, and were equipped with a small frame with
bee wax foundation. 200 µl of aqueous sucrose test solution (0.5 g w/w)
was provided to each group of 10 workers, accordingly to EPPO
guidelines. In this way, thanks to trophallaxis, each bee received ~20 µl
of the solution (EPPO, 2001). The test solution was pure for the
control workers, and contaminated with dimethoate for the treated
ones. Every treated worker received in average 0.15 µg of the active
ingredient which is the equivalent of the LD50 (24 h) (Gough
et al
.,
1994). When the test solution was consumed, the cages were
equipped with feeders containing sucrose solution (0.5g w/w)
ad
libitum
. The cages were then kept at 35°C during the test. The
mortality was checked at 8, 24 and 48 hours. The % mortality of
treated workers was corrected with the formula Schneider-Orelli and
analysed with
c
2
- test. The number of treated and control workers
deriving from the “cold” and “warm” rearing is shown in Table 4.
Results
Experiment 1: Development mortality
The decrease of brood temperature by 2°C had no effect either on the
development mortality, or on adult emergence (except for only one of
the five trials; Table 2). Thus from the “cold” brood an apparently
unaffected number of adult workers emerged. The trials conducted in
different periods produced different results in terms of development
mortality. Nevertheless, the difference between the “cold” and “warm”
larvae was never significant.
Experiment 2: Adult worker survival
Workers emerging from brood reared at suboptimal temperature lived
significantly less than the “warm” ones (Table 3, Fig. 1). The group of
“cold” bees reached the 0% survival level very rapidly, while the
“warm” ones were still characterised by low mortality. The differences
were significant for all three trials, even though they were carried out
in different periods.
Experiment 3: Larval susceptibility to poisoning
by dimethoate
The larval LD50 at 48 and 72 hours are shown in Table 5. It is clear
that the “cold” larvae responded more slowly to poisoning by
dimethoate than the “warm” ones. In fact, after 48 hours from
poisoning the mortality was almost stable in the “warm” larvae, while
it was still growing significantly in the “cold” ones.
Medrzycki, Sgolastra, Bortolotti, Bogo, Tosi, Padovani, Porrini, Sabatini
56
A
2 4 6 8 10 12 14 16 18 20
Adult bee age (days)
0
20
40
60
80
100
% survived bees
grafting date: 16/05/08
B
12345678910111213
Adult bee age (days)
0
20
40
60
80
100
% survived bees
grafting date: 25/07/08
C
246810121416182022
Adult bee age (days)
0
20
40
60
80
100
% survived bees
grafting date: 08/08/08
Fig. 1.
Experiment 2. Survival of adult workers emerging from the
brood reared at optimal (35°C; white squares) and suboptimal
temperature (33°C; black stars). Results of three different trials are
shown.

Experiment 4: Adult bee susceptibility to
poisoning by dimethoate
The adult bees originating from the “cold” larvae were significantly
more susceptible to poisoning by dimethoate (Table 4). In dimethoate
treated bees, mortality increased rapidly, reaching 62% after 8 hours.
At that time, bees emerged from the “warm” brood, still had 0%
mortality.
Discussion
Our data suggest that across different experimental time windows
there are significant effects of suboptimal brood rearing temperature
on adult viability (longevity and susceptibility to dimethoate) but not
on bee development. Our results on adult emergence (Experiment 1)
are consistent with earlier findings (Tautz
et al.
, 2003) in that there
were no significant differences in adult emergence between brood
rearing temperatures of 32°C, 34.5°C and 36°C. Due to
in vitro
rearing (Aupinel
et al
., 2005; 2007), we were also able to study the
effects of temperature on larval development, which also yielded no
significant effects. Moreover, larval susceptibility to poisoning by
dimethoate was also not increased by temperature reduction
(Experiment 3). On the contrary, it appeared that lower temperatures
could “protect” the brood against the temporary food contamination,
probably due to reduction of metabolism dynamics with the
consequent slower assimilation of the active ingredient (Petz
et al
.,
2004). Indeed, the LD50 (48h) was 28 times higher when the larvae
were incubated at the suboptimal temperature than at the optimal
temperature. After 72 hours, this difference was much lower. This
result suggests that larval response to dimethoate strongly depends
on the test temperature. In our study, the LD50 (48h) of dimethoate
calculated at 35°C was lower than the value calculated by Aupinel
et
al
. (2007) (0.67
vs.
1.9 µg / larva), although both studies followed the
same protocol. This discrepancy might be explained either by
differences in the quality of royal jelly used for the diets, or by
different honey bee ecotype (French
vs.
Italian) of the
Apis mellifera
ligustica
subspecies used in the tests. In addition, slight deviations
from the fixed temperature (35°C) in both studies may have
significantly influenced the LD50 value.
Although we found no significant effect of the brood rearing
temperature on the worker emergence rate, adult bee viability was
significantly affected. In fact the “cold” adult workers showed a
shorter lifespan (Experiment 2), and were more susceptible to
poisoning by dimethoate (Experiment 4).
These results suggest that the workers emerging from
brood reared at suboptimal temperature are characterised by a lower
viability and might be unable to complete their normal tasks because
of their shortened lifespan. Other authors (Tautz
et al
., 2003; Jones
et
Effects of brood rearing temperature on honey bees 57
al
., 2005) have seen a significant negative effect of lower temperature
during the pupal stage on other aspects of bee life such as waggle
dance performance, learning and memory.
We therefore hypothesise that the studied factor may have severe
negative effects on the entire colony. In the future it will be important
to further study the behavioural effects of the lower brood
temperature in the first hours of larval life. Moreover, it seems that
the effects of the brood temperature on the viability of emerged
adults vary in relationship to the period (Experiment 2). The cold
brood rearing always caused a significant reduction of adult longevity
in comparison to the warm brood rearing. Nevertheless, this reduction
differed according to the season in which the test was performed. In
fact, the “cold” bee longevity seemed to be higher in spring (0%
survival on 14th day) than in summer (0% survival on 7th day). If such
seasonal effect of low brood temperature on adult bees could be
confirmed by further studies, it might well be that brood is able to
better tolerate lower temperatures in colder seasons. Since the
experimental rearing conditions were the same in all seasons it is
feasible that this mechanism should depend on the first 12 h of larval
life (maternal effect, food and temperature conditions in the hive).
Bee mortality and colony losses are complex phenomena and
often it is not easy to find the link between the causes and the
effects. Based on the results of our study, we hypothesize the
following scenario: during early spring, the ratio between adult
workers and reared brood is very delicate due to low external
temperatures at night and the low number of adult workers. In this
period, a moderate bee mortality (e.g. caused by pesticides) could
break this delicate equilibrium and the colony may lose the capacity to
maintain brood temperature at optimal level. The brood would be
reared at suboptimal temperature, producing an apparently normal
quantity of workers, but these new workers will be characterized by
reduced longevity and increased susceptibility to pesticides and,
probably, to other stressors. This will result in the number of new
workers again being insufficient to maintain the brood at the optimal
temperature which, combined with their behavioural dysfunctions
(Tautz
et al
., 2003; Jones
et al
., 2005) will lead to chronic colony
weakening until collapse. This scenario may explain the cases of
mysterious bee losses especially in spring apparently disconnected in
time from the possible cause such as pesticide poisoning.
This is the first investigation of the effects of suboptimal brood
temperature on honey bees, with the stress factor applied since the
first larval instar (12 h age). Future studies are required to analyse
the mechanisms in detail.

Acknowledgements
Research made within the project “APENET: monitoring and research
in apiculture”, funded by the Italian Ministry of Agricultural Food and
Forestry Policies.
We are grateful to Cecilia Costa and Peter Neumann for valuable
comments on the manuscript.
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