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Abstract

Hygienic behavior in honey bees can be tested by piercing the brood with a pin; however, there is concern that variability in the quantity of fluids that leaks from the pupae could influence test results. Colonies of Apis mellifera carnica were tested to evaluate this possibility. We made four repetitions of four treatments and one control in each of three colonies. The order of degree of hygienic behavior was: pin-killed capped worker brood with a drop of body fluid injected underneath the cell capping > pin-killed capped worker brood > undamaged capped brood with a drop of body fluid injected underneath the cell capping > control or a drop of pupal body fluid placed on the cell cappings. All of the differences were significant (Tukey test, P < 0.05) except the body fluid on the cell cap, which gave the same results as the control. The addition, inside worker brood cells, of pupal body fluid had a significant effect on honey bee hygienic behavior, both in normal brood and in pin-killed brood. (C) Inra/DIB/AGIB/Elsevier, Paris.
Original
article
Influence
of
body
fluid
from
pin-killed
honey
bee
pupae
on
hygienic
behavior
Kátia
Peres
Gramacho
Lionel
Segui
Gonçalves
a
Peter
Rosenkranz
David
De
Jong’
a
Biology
Departament,
Faculty
of
Philosophy,
Science
and
Letters
of
Ribeirao
Preto,
University
of
Sao
Paulo,
14040-901
Ribeirao
Preto,
SP,
Brazil
b
Universität
Hohenheim,
Landesanstalt
für
Bienenkunde,
August-von-Hartmann
Stra&szlig;e
13,
70593
Stuttgart,
Germany
c
Genetics
Department,
Faculty
of
Medicine,
University
of
São
Paulo,
14049-900
Ribeirão
Preto,
SP,
Brazil
(Received
24
October
1998;
revised
5
March
1999;
accepted
18
May
1999)
Abstract -
Hygienic
behavior in
honey
bees
can
be
tested
by
piercing
the
brood
with
a
pin;
however,
there
is
concern
that
variability
in
the
quantity
of fluids
that leaks
from
the
pupae
could
influence
test
results.
Colonies
of Apis
mellifera
carnica
were
tested
to
evaluate
this
possibility.
We
made
four
repetitions
of
four
treatments
and
one
control
in
each
of
three
colonies.
The
order
of
degree
of
hygienic
behavior
was:
pin-killed
capped
worker
brood
with
a
drop
of body
fluid
injected
underneath
the
cell
capping
> pin-killed
capped
worker
brood
> undamaged
capped
brood
with
a
drop
of
body
fluid
injected
underneath
the
cell
capping
> control
or a
drop
of
pupal
body
fluid
placed
on
the
cell
cappings.
All
of the
differences
were
significant
(Tukey
test,
P
<
0.05)
except the
body
fluid
on
the
cell
cap,
which
gave
the
same
results
as
the
control.
The
addition,
inside
worker
brood
cells,
of
pupal
body
fluid
had
a
significant
effect
on
honey
bee
hygienic
behavior,
both
in
normal
brood
and
in
pin-killed
brood.
&copy;
Inra/DIB/AGIB/Elsevier,
Paris
Apis
mellifera
carnica
/
hygienic
behavior
/
body
fluid
/
pin-killing
method
*
Correspondence
and
reprints
E-mail:
gramacho@usp.br
1.
INTRODUCTION
Honey
bee
workers
(Apis
mellifera
L.)
are
able
to
recognize
diseased,
damaged
or
dead
brood
inside
capped
cells
and
remove
them
through
hygienic
or
cleaning
behav-
ior
[4,
5].
Gramacho
and
Gonçalves
[2],
working
with
Africanized
honey
bees
Apis
mellifera
scutellata,
made
a
comparative
study
of
the
freeze-killed
and
pin-killed
brood
assays
to
test
hygienic
behavior
of
workers.
They
found
no
significant
differ-
ences
between
the
two
methods.
They
con-
sidered
the
pin-killing
method
developed
by
Newton
and
Ostasiewski
[3]
more
useful
than
the
freeze-killing
method
because
it
is
easier
to
use
in
the
field
and
in
the
laboratory
and
less
expensive
to
implement
than
the
freezing
method.
Newton
and
Ostasiewski
[3]
had
suggested
the
pin-killing
assay
as
a
means
to
test
for
honey
bee
colony
resis-
tance
to
American
foulbrood
(Paenibacil-
lus
larvae).
However,
Spivak
and
Downey
[6]
prefer
the
freeze-killing
method,
because
they
found
that
the
pin-killed
brood
was
quickly
removed
by
virtually
all
colonies,
and
was
therefore
not
a
good
discrimina-
tory
test.
Taber
and
Gilliam
[11],
Spivak
and
Gilliam
[7]
and
Spivak
and
Downey
[6]
made
important
contributions
and
improve-
ments
to
the
methodology
and
field
assays
of
hygienic
behavior
of
honey
bees.
Spivak
and
Reuter
[8]
compared,
through
the
freeze-
killing
method,
colonies
with
naturally
mated
queens
from
a
hygienic
line
of
Italian
honey
bees
to
colonies
from
a
commercial
line
of
Italian
bees
not
selected
for
hygienic
behavior.
They
showed
that
the
hygienic
colonies
removed
significantly
more
freeze-
killed
brood
and
had
less
chalkbrood
(a
brood
disease
caused
by
the
fungus
Ascos-
phaera
apis)
than
the
commercial
colonies.
This
was
the
first
study
carried
out
in
the
USA
to
evaluate
hygienic
stock
in
large
field
colonies.
Also
Spivak
and
Reuter
[9,
10]
recently
described
a
method
of
freezing
brood
with
liquid
nitrogen
and
suggested
that
this
is
the
best
screening
procedure
for
hygienic
behavior
studies.
However,
we
did
not
find
this
method
convenient
or
simple
for
routine
use.
It
is
necessary
to
carry
a
liq-
uid
nitrogen
tank
and
to
take
several
safety
precautions
(protective
clothing,
heavy
gloves,
boots,
glasses,
etc.)
to
avoid
acci-
dents
with
this
extremely
cold
liquid.
The
main
advantage
is
that
it
does
not
require
the
cutting
out
and
removal
of
pieces
of
brood
comb,
to
be
frozen
overnight
and
later
returned
to
the
colony.
The
pin-killing
method
kills
or
damages
the
brood
by
perforating
and
killing
the
pupa
within
the
brood
cell,
which
can
be
easily
performed
in
the
field
or
in
the
laboratory.
This
process
produces
a
small
hole
in
the
capped
cell
and
the
treated
brood
exudes
body
fluid.
Sometimes
this
fluid
is
visible
on
the
cell
cap.
The
presence
or
absence
of
this
fluid,
and
the
volume
of
such
fluid,
could
be
an
additional
non-standard
stimulus
for
hygienic
behavior.
We
were
concerned
that
this
would
affect
test
results.
The
objective
of
our
study
was
to
deter-
mine
the
effect
of
the
body
fluid
of
the
pupa,
which
appears
after
piercing
with
a
pin,
on
the
hygienic
behavior
of
worker
bees.
2.
MATERIALS
AND
METHODS
We
used
three
strong
colonies
of
Apis
mel-
lifera
carnica
Pollm.,
each
maintained
in
two
full-size
Langstroth
boxes,
at
the
Beekeeping
Laboratory
of
the
University
of Hohenheim
(Lan-
desanstalt
für
Bienenkunde),
in
Stuttgart,
Ger-
many.
A
comb
containing
sealed
pupae
aged
11-15
days
was
used
for
each
test,
with
four
rep-
etitions
for
each
treatment.
Each
comb
was
used
at
the
same
time
for
four
treatments
and
a
control.
Five
areas
identical
in
size
(3.5
cm
x
6
cm)
were
delimited
to
include
50
cells
of
the
sealed
worker
brood
(figure
I).
The
treatments
were
as
follows:
A
=
pin-killing
method
(worker
brood
cells
per-
forated
with
a
no.
1 insect
pin;
B
=
one
drop
(4
&mu;L)
of
pupal
body
fluid
placed
on
the
cap-
ping
of
an
otherwise
undisturbed
brood
cell;
C
=
pin-killing
method
and
introduction
of
4
&mu;L
of
body
fluid
inside
the
brood
cell
through
the
pin
hole;
D
=
control
(normal
undisturbed
brood);
E
= introduction
of
body
fluid
(4
&mu;L)
inside
the
capped
brood
cell.
In
treatment
E,
part
of
the
cell
capping
was
lifted
at
the
edge
with
fine-tipped
forceps
and
the
body
fluid
injected
through
the
hole, taking
care
to
avoid
touching
the
pupa,
and
then
resealing
the
cell
capping.
The
body
fluid
used
for
treatments
B and
E
was
obtained
by
cutting
worker
pupae
aged
11-15
days
with
a
glass
pipette,
and
letting
the
fluid
drip
into
small
sterilized
glass
flasks.
These
were
kept
on
ice
(0
°C)
for
a
few
minutes,
until
the
test
was
performed.
The
body
fluid
was
then
allowed
to
warm
to
room
temperature just
before
it
was
used.
For
the
pin-killing
of
the
brood
(treatments
A
and
C)
a
no.
1
insect
pin
was
used
to
pierce
the
sealed
brood
cells
through
the
center
of
the
cell
cap,
penetrating
the
body
of the
pupa
until the
pin
reached
the
base
of
the
cell.
The
sealed
worker
brood
comb
was
then
returned
to
the
respective
hive.
Twenty-four
hours
later
each
’treated’
comb
was
brought
to
the
laboratory
to
check
for
uncapped
cells
and
to
see
if the
brood
was
completely
removed
by
the
workers,
char-
acterizing
hygienic
or
cleaning
behavior.
Hygienic
behavior
was
calculated
according
to
the
following
formula:
where
EC
=
empty
cells,
CC
=
capped
cells
and
HB
=
hygienic
behavior
(in
%)
The
data
were
transformed
via
arcsine
of
the
square
root
of
the
proportion
removed,
for
com-
parisons
by
a
repeated
measures
analysis
of vari-
ance
and
the
Tukey
test.
3.
RESULTS
There
was
a
significant
difference
among
the
treatments
(table
I,
F
=
57.7,
DF
=
4,
P <
0.001).
Colony
(F =
2.10,
DF =
2,
P
=
0.204)
and
date
(F
= 0.515,
DF
=
3,
P
=
0.687)
had
no
significant
influence
on
the
results.
All
of
the
five
treatment
groups
gave
sig-
nificantly
different
hygienic
behavior
means
(Tukey
test,
P
<
0.05),
except
for
the
control
compared
to
the
body
fluid
placed
on
the
cell
capping.
The
sequence
of
results
was:
pin-killed
+
body
fluid >
pin-killed >
body
fluid
inside
capped
cells >
body
fluid
on
capped
cells
or
control
(table
I).
Treatment
A
(pin-killing
method)
and
treatment
C
(pin-
killing
method
plus
body
fluid
inside
the
capped
cells)
provoked
significantly
more
brood
removal
than
in
the
control
(table
I,
Tukey
test,
P
<
0.001).
Treatment
C
(pin-
killed
plus
body
fluid
injected
inside
the
cells)
provoked
a
18
%
higher
brood
removal
rate
than
treatment
A
(pin-killed
brood),
which
is
a
significant
difference
(Tukey
test,
P
<
0.01).
The
body
fluid
placed
on
the
capped
cells
had
no
influence
on
the
hygienic
behavior
of
the
worker
bees,
when
compared
to
the
con-
trol
(Tukey
test,
P
=
0.976).
Placement
of
body
fluid
inside
the
capped
brood
cells,
without
deliberately
piercing
the
pupa
(E),
provoked
about
25
%
removal
behavior
(table
I).
The
increase
over
the
con-
trol
was
significant
(Tukey
test,
P
<
0.01).
4.
DISCUSSION
AND
CONCLUSION
When
a
pupa
is
pierced,
a
whitish
fluid
is
exuded.
This
’body
fluid’
is
composed
of
hemolymph,
along
with
many
fat
body
cells
and
other
types
of
material
from
tissue
which
has
been
damaged.
In
a
recent
paper
[6]
’hemolymph’
was
extracted
from
live
and
dead
(frozen)
pupae
to
determine
the
effect
on
hygienic
behavior;
however,
this
was
actually
what
we
chose
to
call
body
fluid,
because
it
is
not
possible
by
normal
methods
to
obtain
clean
hemolymph
from
live
pupae.
We
found
that
pupal
body
fluid
placed
inside the
capped
cells
was
an
important
stimulus
for
hygienic
behavior.
Spivak
and
Downey
[6]
found
similar
effects
when
they
placed
pupal
body
fluid
(which
they
called
hemolymph)
on
pupae
in
an
artificial
comb,
though
when
this
material
was
taken
from
frozen
pupae
the
effect
was
significantly
stronger
than
when
fluid
was
taken
from
live
pupae.
Hemolymph
removed
from
dead
pupae
would
be
contaminated
with
burst
fat
body
and
hemolymph
cells,
which
may
have
been
the
reason
for
the
difference
in
reaction.
The
significant
effects
of
introducing
a
drop
of
pupal
body
fluid
inside
pin-killed
brood
in
our
experiment
show
that
this
fluid
has
an
important
effect
on
hygienic
behav-
ior
when
exposed
inside
the
brood
cell.
This
means
that
fluid
that
leaks
from
pin-killed
pupae
is
an
important
stimulus.
In
our
exper-
iment
the
addition
of
extra
pupal
body
fluid
increased
the
stimulus
for
the
bees
to
remove
the
brood,
whether
perforated
or
not.
We
were
concerned
with
preliminary
observations
that
a
drop
of
pupal
body
fluid
sometimes
appeared
on
the
capping
of
pin-
killed
brood
cells.
The
percentage
of
such
cells
with
a
visible
drop
of
fluid
varied
con-
siderably
(data
not
shown),
and
could
be
an
additional,
non-uniform
cue.
The
fact
that
the
worker
brood
cells
in
treatment
B
(body
fluid
placed
on
the
capped
brood
cells)
were
not
damaged
by
the
bees
indicates
that
the
deposition
of
body
fluid
on
top
of
the
capped
cells
does
not
provoke
hygienic
behavior.
Possibly
this
fluid
is
removed
by
the
bees
before
it
deteriorates
and
can
emanate
odors
which
stimulate
the
bees
to
take
further
action.
There
is
evidence
that
different
cohorts
of
bees
are
specialized
in
opening
suspicious
brood
cells
and
others
seal
the
brood
cell
again
or
remove
the
damaged
pupae
[1,
12].
This
is
also
confirmed
by
our
observations,
as
sometimes
damaged
brood
cells
were
uncapped
at
the
first
examination
and
capped
again
at
the
second
check.
We
found
that
when
the
capped
brood
cells
are
perfo-
rated
during
the
pin-killing
method,
with-
out
damaging
the
brood,
the
workers
nor-
mally
do
not
remove
these
brood
from
the
cells
(data
not
shown).
We
also
observed
that
when
the
capping
is
artificially
removed
without
disturbing
the
brood
inside
it,
the
workers
reconstruct
the
capping
without
removing
the
brood.
Pupal
body
fluid
placed
inside
brood
cells
increases adult
honey
bee
hygienic
behavior
directed
towards
normal,
otherwise
undam-
aged
pupae.
It
also
significantly
increases
the
hygienic
behavior
provoked
by
pin-killed
brood.
This
leads
us
to
conclude
that
the
fluid
that
leaks
from
pin-killed
pupae
inside
the
cell
is
an
important
stimulus
for
brood-
removal
(hygienic)
behavior.
ACKNOWLEDGMENTS
The
authors
are
grateful
for the
support
given
by
CAPES/MEC/DAAD,
Project
Probral
030/95
and
to
the
University
of
Hohenheim
(Lan-
desanstalt
für
Bienenkunde)
of
Stuttgart,
Ger-
many.
Our
special
thanks
to
Dr
Arcadio
de
los
Reyes
Borjas
of
the
Genetics
Department
of
the
University
of
São
Paulo
in
Ribeirão
Preto
for
help
with
the
statistical
analysis.
Résumé -
Influence
du
liquide
corporel
des
nymphes
d’abeilles
tuées
par
épingle
sur
le
comportement
hygiénique.
Diverses
techniques
ont
été
mises
au
point
pour
mesu-
rer le
comportement
hygiénique
des
abeilles.
Les
plus
courantes
consistent
soit
à
conge-
ler
le
couvain,
soit
à
le
tuer
avec
une
épingle,
mais
toutes
deux
détruisent
un
certain
nombre
de
cellules
de
couvain
d’ouvrières
operculé.
La
première
méthode
nécessite
de
découper
un
morceau
du
rayon
de
couvain
et
de
l’ôter
de
la
colonie
durant
24
h,
alors
que
la
seconde
méthode
peut
être
appliquée
en
quelques
minutes
sur
le
terrain
comme
au
laboratoire
sans
abîmer le
rayon.
Néanmoins
on
craint
que
la
méthode
de
l’épingle
ne
soit
moins
précise
et
moins
uniforme.
Au
cours
de
l’enfoncement
et
du
retrait
de
l’épingle
on
voit
souvent,
sur
l’épingle
et
sur l’opercule,
du
liquide
corporel
provenant
des
nymphes.
Cette
substance
pourrait
stimuler
une
réac-
tion
de
la
part
des
abeilles
nettoyeuses,
ce
qui influerait
sur
le
taux
de
désoperculation
et
d’élimination
du
couvain.
Nous
avons
testé
des
colonies
d’Apis
mellifera
carnica
pour
évaluer
cette
possibilité.
Un
rayon
de
couvain
d’ouvrières
operculé
(c.o.o.)
avec
des
nymphes
âgées
de
11
à
15 j
a
été
utilisé
pour
chaque
test
(rayon
expérimental).
Cinq
portions
de
rayon,
de
même
taille
(3,5
x
6
cm)
et
comprenant
50
cellules
de
c.o.o.,
ont
été
délimitées.
Après
avoir
tué
par
épingle
les
nymphes,
chaque
rayon
expéri-
mental
a
été
replacé
dans
sa
colonie.
24
h
plus
tard
chaque
rayon
expérimental
a
été
apporté
au
labo
et
les
cellules
désopercu-
lées,
ainsi
que
les
nymphes
totalement
éli-
minées
par
les
abeilles,
ont
été
dénombrées.
Dans
chacune
des
trois
colonies,
nous
avons
répété
quatre
fois
les
quatre
traitements
sui-
vants
(plus
le
témoin) :
a)
le
c.o.o.
est
percé
au
centre
avec
une
épingle
1
jusqu’à
atteindre
le
fond
de
la
cellule ;
b)
une
goutte
de
liquide
corporel
de
nymphe
est
déposée
sur
le
sommet
des
cellules
de
couvain,
sans
qu’elles
ne
soient
perforées,
c)
le
c.o.o.
est
percé
comme
précédemment
et
une
goutte
de
liquide
corporel
de
nymphe
est
déposée
à
l’intérieur
des
cellules,
d)
cellules
de
cou-
vain
operculé
(témoin),
e)
le
c.o.o.
n’est
pas
abîmé
et
une
goutte
de
liquide
corporel
est
introduite
à
l’intérieur
des
cellules.
Les
don-
nées
ont
été
traitées
par
analyse
de
variance
avec
mesures
répétées
et
test
de
Tukey,
après
transformation
Arcsin&radic;.
Le
comportement
hygiénique
chez
les
groupes
expérimentaux
n’était
pas
uniforme
(p
<
0,0001
). Il
se
clas-
sait
comme
suit
par
ordre
décroissant :
trai-
tement
c
>
traitement
a
>
traitement
e
>
trai-
tement
d
ou
b.
Ces
différences
sont
toutes
significatives
(p
>
0,05).
L’addition
de
liquide
corporel
s’écoulant
des
blessures
faites
aux
nymphes
à
l’intérieur
des
cellules
a
une
action
significative
sur
le
comporte-
ment
hygiénique
des
abeilles,
bien
qu’elle
n’en
ait
aucune
lorsque
les
gouttes
de
liquide
sont
déposées
sur
les
opercules.
&copy;
Inra/
DIB/AGIB/Elsevier,
Paris
Apis
mellifera
carnica
/
comportement
hygiénique
/
liquide
corporel
/
destruction
par
épingle
Zusammenfassung -
Einflu&szlig;
der
Kör-
perflüssigkeit
von
im
Nadeltest
getöteten
Bienenpuppen
auf
das
Ausräumverhal-
ten
(hygienisches
Verhalten).
Verschie-
dene
Methoden
wurden
entwickelt,
um
das
Ausräumverhalten
von
Honigbienen
zu
mes-
sen.
Am
häufigsten
wird
das
Einfrieren
von
Brut
und
der
Nadeltest
angewendet.
Eine
Standardanzahl
von
verdeckelten
Brutzel-
len
von
Arbeiterinnen
werden
durch
Ein-
frieren
oder
durch
Einstiche
mit
der
Nadel
verletzt.
Im
ersten
Fall
mu&szlig;
ein
Stück
Brut-
wabe
ausgeschnitten
und
24
Stunden
lang
aus
dem
Volk
entfernt
werden,
während
im
zweiten
Fall
der
Test
in
wenigen
Minuten
unter
Feld-
und
Laborbedingungen
durch-
geführt
werden
kann,
ohne
die
Wabe
zu
zer-
stören.
Trotzdem
gibt
es
Bedenken,
da&szlig;
der
Nadeltest
weniger
präzise
und
gleichmä&szlig;ig
ist.
Beim
Einstechen
und
herausziehen
der
Nadel
kann
man
oft
Körperflüssigkeit
an
der
Nadel
und
auf
dem
Zelldeckel
sehen.
Diese
Verunreinigung
könnte
eine
Reak-
tion
der
putzenden
Stockbienen
stimulie-
ren,
die die
Rate
des
Entdeckelns
der
Zellen
und
des Entfernens
der
Brut
beeinflussen
könnte.
Die
Stärke
eines
solchen
Effekts
testeten
wir
in
Völkern
von
Apis
mellifera
carnica.
Für
jeden
Test
wurde
eine
ver-
deckelte
Brutwabe
mit
Puppen
im
Alter
zwi-
schen
11
und
15
Tagen
benutzt.
Fünf
gleich
gro&szlig;e
Stücke
(3,5
cm
x
6
cm)
wurden
abge-
grenzt,
so
da&szlig;
sie
50
Zellen
verdeckelte
Arbeiterinnenbrut
enthielten.
Nach
Tötung
mit
der
Nadel
wurde
die
verdeckelte
Brut-
wabe
(Versuchswabe)
in
das
entsprechende
Volk
zurückgestellt.
Nach
24
Stunden
wurde
jede
Versuchswabe
ins
Labor
geholt,
um
die
Anzahl
sowohl
der
geöffneten
Zellen
als
auch
der
vollständig
entfernten
Puppen
zu
bestimmen.
Jede
der
4
folgenden
Ver-
suchsmethoden
und
die
Kontrolle
wurde
in
3
Völkern
4
mal
wiederholt:
a)
verdeckelte
Zellen
wurden
mit
nur
1
Nadel
bis
zum
Boden
durchstochen,
b)
ein
Tropfen
Kör-
perflüssigkeit
wurde
auf
den
Zelldeckel
gelegt,
ohne
die
Zelle
anzustechen;
c)
die
verdeckelte
Zelle
wurde
wie
oben
angesto-
chen
und
zusätzlich
ein
Tropfen
Körper-
flüssigkeit
in
die
Zelle
eingeführt;
d)
unbe-
handelte
verdeckelte
Zelle
(Kontrolle)
und
e)
unverletzte
Puppen
mit
einem
Tropfen
Körperflüssigkeit
in
der
Zelle.
Für
eine
Varianzanalyse
und
den
Tukey-Test
wurden
die
Quadratwurzeln
der
Daten
über
eine
Arcussinus
-Transformation
aufberei-
tet.
Das
Ausräumverhalten
war
in
den
Ver-
suchsgruppen
unterschiedlich
(P
<
0,0001).
Die
Reihenfolge
im
Ausräumverhalten
war:
Nadeltest
plus
Körperflüssigkeit
in
der
Brut-
zelle >
Nadeltest >
Körperflüssigkeit
in
der
Brutzelle
>
Kontrolle
oder
Körperflüssig-
keit
auf
dem
Zelldeckel.
Diese
Unterschiede
waren
alle
signifikant
(P
<
0,05).
Zusätz-
lich
hatte
die
Körperflüssigkeit,
die
aus
Ver-
letzungen
der
Puppe
in
der
Brutzelle
stamm-
ten,
einen
signifikanten
Einflu&szlig;
auf
das
Ausräumverhalten,
obwohl
es
keinen
Effekt
auf
dem
Zelldeckel
zeigte.
&copy;
Inra/DIB/
AGIB/Elsevier,
Paris
hygienisches
Verhalten
/
Körperflüssig-
keit
/
Tötung
durch
Nadeltest
/
Apis
mellifera
carnica
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D.C.,
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N.J.
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W.C.,
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M.,
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D.,
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M.,
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M.,
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M.,
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G.S.,
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M.,
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G.S.,
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M.,
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... For example, Spivak and Downey (1998) . Yet, this diameter may influence the quantity of leaking hemolymph, which is an important stimulus for the hygienic behavior (Gramacho, Gonçalves, Rosenkranz, & De Jong, 1999). The latter study showed, indeed, that the quantity of leaking hamolymph was reported to vary across the pierced cells, potentially affecting the strength of the stimulus. ...
... The higher stimulus creates a faster hygienic response, as recorded in all studies mentioned above. The higher intensity of this stimulus can be explained by the hole in the wax cap (Palacio et al., 1996) and the leaking hemolymph (Gramacho et al., 1999). The faster hygienic response for PKB compared to FKB might also potentially be explained by the set of hygienic tasks that workers have to perform. ...
Article
Full-text available
Individual immunity in honey bees (Apis mellifera L.) is complemented by highly evolved social behaviors. Among them, hygienic behavior has a key role involving the detection and removal of unhealthy or dead brood. Since the 1960s, several bioassays have been developed to quantify the hygienic behavior of honey bee colonies against chalkbrood, American foulbrood, and varroa infested brood. Here, we review the five main bioassays used since the late 1960s. We describe their advantages and disadvantages, including a special focus on their inherent biases. For each assay, we also discuss whether or not their use should be restricted to quantify the hygienic behavior against chalkbrood, or American foulbrood, or varroa infested brood. Overall, the bioassays involving the removal of freeze-killed brood are recommended over the bioassays relying on the removal of pin-killed brood but only for the quantification of hygienic behavior toward chalkbrood and American foulbrood. These bioassays are not recommended to quantify the hygienic behavior toward varroa infested brood, for which an accurate assessment should rely on assays based on the removal of brood artificially infested with varroa mites. Choosing an appropriate bioassay is crucial for an accurate assessment of the hygienic behavior against a defined pathogen, depending on the research question, or the goal of the breeding program. Bioensayos para cuantificar el comportamiento higiénico en las colonias de abejas melíferas (Apis mellifera L.): una revisión La inmunidad individual de las abejas melíferas (Apis mellifera L.) se complementa con comportamientos sociales altamente evolucionados. Entre ellos, el comportamiento higiénico tiene un papel clave en la detección y eliminación de crías enfermas o muertas. Desde la década de 1960, se han desarrollado varios bioensayos para cuantificar el comportamiento higiénico de las colonias de abejas melíferas frente a la cría infestada de Ascosfera, loque americana y varroa. Aquí revisamos los cinco principales bioensayos utilizados desde finales de la década de 1960. Describimos sus ventajas y desventajas, incluyendo un enfoque especial en sus sesgos inherentes. Para cada ensayo, también discutimos si su uso debe ser restringido o no para cuantificar el comportamiento higiénico contra la cría de loque americana o la cría infestada de varroa. En general, los bioensayos que involucran la eliminación de la cría muerta por congelación son más recomendables que los bioensayos que dependen de la eliminación de la cría muerta por pin, pero sólo para la cuantificación del comportamiento higiénico hacia la cría infestada de Ascosfera y la loque americana. Estos bioensayos no se recomiendan para cuantificar el comportamiento higiénico de la cría infestada de varroa, para lo cual una evaluación precisa debe basarse en ensayos basados en la extracción de la cría infestada artificialmente con ácaros de la varroa. La elección de un bioensayo apropiado es crucial para una evaluación precisa del comportamiento higiénico frente a un patógeno definido, dependiendo de la pregunta de investigación o del objetivo del programa de cría.
... According to Gramacho et al. [43], the pin-killed brood assay was used to detect which race showed more hygienic behaviors toward pin-killed brood between related or unrelated races. The combs were observed by using infrared photography video recording continuously for 3 days in each experiment [44] using a glass-walled observation unit ( Figure 1). ...
Article
Full-text available
The use of infra-red photography video recording is very useful for conducting behav-ioristic studies of honeybees against many brood diseases. The removal of dead or diseased brood from capped cells by honeybee workers is a heritable trait that confers colony-level resistance. This work aimed to compare the hygienic behavior of the native (Yemeni bees, A. mellifera jemenitica) and the exotic (Carniolan bees, A. m. carnica) honeybee races in Saudi Arabia using an infra-red photography video recording. In addition, hygienic behavior towards the related and non-related combs was examined. Therefore, it is possible to obtain honeybee colonies with greater disease resistance. The pin-killing method and infra-red photography video recording were used for the evaluation of hygienic behavior in colonies of the two races. Significant differences in hygienic behavior between the two races were detected at the beginning of the experiment. Under the environmental conditions of eastern Saudi Arabia, the Yemeni honeybee colonies showed a higher number of uncapped and cleaned cells containing dead brood in either the brood comb from the same colony, or the brood comb from the same race but a different colony, or brood comb from a different race. It was concluded that the honeybee's ability to detect and clean the dead brood from comb cells can be correlated with race and it is more efficient for the non-related individuals of the same race than from a related or another race. The outstanding performance of a few individuals in the expression of various traits indicates their usefulness in carrying out breeding programs for Varroa resistance.
... In the Ecuadoran highlands, the farmers select their hives somewhat arbitrarily and there is a lack of genetic crossbreeding program. Beehives with hygienic-behavior values ranging from 80-90% can be considered high HB [33]. Colonies with high HB removed more than 95% of the perforated offspring, albeit at 48 h [30]. ...
Article
Full-text available
The aim of this research was to analyze the relationship among hygienic behavior (HB), Varroa destructor infestation, and honey production in the central highlands of Ecuador. Overall, 75 honey bee colonies were evaluated before, during, and after production at three altitude levels (2600–2800, 2801–3000, and >3000 m.a.s.l.). The hygienic behavior percentage of the colonies was determined by the pin-killing method, and the colonies were classified into three groups: high HB (>85%), mid HB (60.1–85%), and low HB (≤60%). Varroa infestation was diagnosed as well, and honey production was evaluated only during production. HB was high and heterogeneous, averaging 80% ± 9.7%. Its highest expression was observed at lower altitudes. The infestation degree was low (3.47% ± 1.56%), although the mite was detected in all colonies upon sampling. A negative correlation was observed between HB and Varroa infestation in the first sampling (−0.49 **), suggesting that the high- and mid-altitude HB colonies underwent the lowest infestation rates, regardless of sampling. The correlations between HB and production were significant (0.26 *), indicating a positive effect of HB on production, meaning that colonies with high HB obtained the highest honey production (25.08 ± 4.82 kg/hive). The HB of bees showed an inverse relationship with altitude and it tended to reduce the effect of Varroa infestation, favoring honey production and, thus, suggesting the feasibility of selecting colonies with high HB.
... Although hygienic behavior occurs in response to damaged brood in a variety of contexts, the fundamental tasks of detection, uncapping, and removal of the brood generally occur in all contexts (Spivak and Danka 2020). Thus, several unspecific assays for studying hygienic behavior have been employed, among which the pin-killed and the freeze-killed brood (FKB) assays are most common (Gramacho et al. 1999). As suggested by their names, both assays rely on odors of dead brood for triggering hygienic behavior (McAfee et al. 2018) and thus may be only a crude surrogate for more subtle cues of Varroa-infested brood (Nazzi et al. 2004;Schöning et al. 2012;Mondet et al. 2016;Wagoner et al. 2020). ...
Article
Full-text available
Behavioral specialization and cooperation are fundamental in the organization and success of social groups. Honey bee workers display hygienic behavior, defined as the detection, uncapping, and removal of unhealthy brood. We present detailed analyses of behavioral specialization and task partitioning among hygienic worker bees, focusing on uncapping of brood cells and removal of the cells’ content in freeze-killed brood assays. We demonstrate specialization of hygienic workers on either uncapping or removal and task partitioning among multiple individuals. Speed of hygiene decreases with the number of behavioral instances, suggesting a time cost for cooperation of multiple individuals. Additional analyses of an individual agent-based simulation of hygienic removal of Varroa-mite-infested brood demonstrate that erroneous removal of healthy brood can be reduced by task partitioning due to collective decision-making. Combined, our results indicate a speed-accuracy trade-off in the collective performance of hygienic behavior: Hygienic behavior may take longer when many individuals contribute and specialize on different tasks, but this organization of work also can prevent costly mistakes. This trade-off may explain the observed combination of elite workers and numerous other workers that contribute only little. Significance statement Honey bees and other social animals form successful groups that are characterized by individual specialization on different tasks. Hygienic behavior of honey bees removes unhealthy brood from the nest and is important for the health of honey bee colonies. Here, we observed that individual worker bees specialize on either of the two main tasks involved in hygienic behavior: uncapping cells or removing brood. We also found that a few individuals perform the majority of the work and that the speed of hygienic behavior is higher when one individual works continuously instead of multiple individuals. Additionally, we show in computer simulations that the number of erroneous brood removals is decreased when separate workers contribute to the different aspects of hygienic behavior. Thus, our study indicates that a speed-accuracy trade-off might drive the evolution of task partitioning in hygienic behavior.
... European bees have behavioral defenses similar to those of Asian bees, such as grooming and hygienic behavior (LOCKE et al., 2015). However, this behavior may be less pronounced and efficient in European bees when compared to that in other species, such as African and Africanized bees (GRAMACHO, 1999;FRIES et al., 1996;LOCKE et al., 2015). ...
Article
Full-text available
Bees are very important insects for agriculture, fulfilling an important role in pollination and renewal of the ecosystem. However, in several countries significant losses of colonies and population decline of honeybees and native bees have been reported in recent years. Most researchers reported that premature losses are linked to several factors, including viruses that have a great impact on the colonies. This article reports the identification of new viruses, some transmission routes, the association of these parasites with the symptoms of the diseases that affect the health of honeybees, as well as viruses that have been described in Brazil.
Article
Hygienic behaviour is a natural mechanism of colony-level disease resistance to brood pathogens and has been reported in honey bees and stingless bees. A novel brood disease was recently confirmed in the Australian stingless bees Tetragonula carbonaria Smith and Tetragonula hockingsi Cockerell and there is a paucity of data available on hygienic behaviour in these species. To address this, we investigated hygienic behaviour in eight colonies of T. carbonaria and four colonies of T. hockingsi, using brood freeze-kill and pin-kill assays. Hygienic behaviour was present in both species and was rapidly expressed in both assays. In T. carbonaria, the mean time (± SE) for removal of freeze-killed and pin-killed brood was 9.1 ± 1.9 hours and 8.2 ± 0.9 hours, respectively (n = 8; one trial per assay). In T. hockingsi, removal of freeze-killed and pin-killed brood was 14.1 ± 5.1 hours and 10.4 (no SE) hours, respectively. There was no significant difference (α = 0.05) in time taken to complete the hygienic behaviour phases (detection, uncapping, removal or cell dismantling) between assay type or assay order in both species. However, intercolony variation was observed in both species in the assays, suggesting that like honey bees, hygienic behaviour may have a genetic component. Tetragonula carbonaria and T. hockingsi displayed significantly faster detection, uncapping, removal and cell dismantling times than any of the stingless bees or most honey bees studied previously. This may, in part, explain why stingless bees appear to suffer from relatively few brood diseases.
Article
Full-text available
Hygienic behavior is a social defense mechanism against parasites and pathogens in honeybees. We studied the genetic basis of hygienic behavior in African-derived Apis mellifera by performing RNA sequencing on brains of individual honeybee workers observed performing hygienic behavior, in order to identify expression changes linked with this behavior. We also used the transcriptome data to search for single nucleotide polymorphism (SNP) variation in genes previously associated with the trait in other A. mellifera populations. The analysis revealed 49 differentially expressed genes (DEG), most of them upregulated during hygienic acts. One DEG, Apidaecin, also showed SNP variation across samples, providing a promising candidate gene for both expression-based and heritable variation in hygienic behavior. We also identified 27 additional SNPs in the coding regions of five candidate genes previously linked with honeybee hygienic behavior. These results provide a useful starting place to analyze the genetic basis of hygienic behavior in African-derived honeybees.
Article
The two standardized assays for testing hygienic behavior in Apis mellifera in the field are the freeze-killed brood (FKB) and the pin-killed brood (PKB) assays. Correlation between the two tests is still in debate. It has been argued that the PKB assay was predictive of Varroa nonreproduction. We measured the agreement between the two methods in two apiaries with 36 and 59 colonies, respectively. The agreement between the two assays was very poor with a 95% limit of agreement greater than 100%. These assays and the SMR (Suppression of Mite Reproduction) trait were also measured in 21 single drone inseminated colonies infested with Varroa and with varying SMR efficacy. A PKB assay result of >46% at 24 h predicted an SMR trait >40% with a sensitivity and a specificity of 0.727 and 0.90, respectively, whereas the FKB assay did not exhibit any predictive value. In conclusion, the PKB and FKB assays are not correlated, but the PKB assay predicted SMR with a good accuracy.
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In Egypt, there was no considerable effort to incorporate the heritable trait, hygienic behavior (HB), into honey bee (Apis mellifera L.) stock due to the absence of accurate, fast and practical method that is acceptable by most of beekeepers specially queen producers. Present study was conducted to compare 3 designed assays with traditionally-used ones in ten honey bee colonies. Brood viability, presence of chalkbrood and American foulbrood, and Varroa mites on adult workers and within worker brood were evaluated. HB levels of local honey bee colonies from 5 districts at El-Beheira Governorate were investigated using a modified pin-killed brood assay. Colony strength, assay type and time of observation had significant effects on brood removal. Punctured cells (PC) stage was found to be highly significant and positively correlated with uncapped cells (UC) stage, and negatively correlated with cleaned cells (EC) stage, but it was nearly not correlated with partially removed cells (PRC) stage. HB was positively correlated with worker brood viability (WBV). But, it negatively correlated with other colony evaluation criteria. The negative correlation was significant in cases of chalkbrood mummies (CBM) and mites within brood (BI). Furthermore, there were significant effects of colony strength and assay type on the expression of HB at 6, 24 and 48 h. Also, their interactions at 6 and 48 h were significant. The modified pin-killed brood assay was found to be as accurate as freeze-killed brood assay but more practical in estimating HB. 02 J.Agric.&Env.Sci.Dam.Univ.,Egypt Vol.12 (1) 2013 Furthermore, using the potential new assay, there was a significant effect of apiary location and colonies of Ezbet Haggag apiary Ӏ had a significantly higher mean of slow HB (61.46 %) than that estimated in other apiaries, even those of the same location (Ezbet Haggag apiary ӀӀ), and none of evaluated apiaries had any hygienic colony.
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Beekeeping is a small livestock with significant contribution to the economy and environment. For the bees (Apis mellifera) the hygienic behavior is a mechanism of natural resistance against diseases whose variability and heritability qualify it as a basic trait in breeding programs. However, conventional evaluation tools require time waste which hinders their use. In this work, the evaluation of this mechanism is performed in a Breeding Center of Queen Bees by the punch method, using classical needle (instrument 1) and instrument 2, consisting of 100 needles inserted into an acrylic base, patented by the authors (DL68 Invention 07/10/2011). Fifteen Langstroth hives were worked during January-July, 2012. Two honey combs with brood 16-17 days (pink-eyed pupae) were taken from the center of the brood chamber, and an area of 100 cells/two honeyscombs/hive was selected with one of the instruments in each honeycombs. In the area selected, the cells to kill the pupae were punctured and the percentage of cleaned cells was determined at 24 hours. The apiary showed mean values of 90.3% hygienic behavior using instrument 1 and 92.5% with instrument 2, without significant differences. Saving time was 85 ± 5% with instrument 2 without the tedium performing 1500 punctures only against 15 actions.
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The method of using Infra-red photography was found very useful for conducting long-term behavioristic studies of honey bees against Varroa. The photographs show the defensive ability of this species and solved the earlier suspicion of direct damage. In total 30% and 26.6% artificially infested cells were uncapped and removed in two experiments. The correlations between uncapping and removal, uncapping and grooming and removal and grooming behavior were found to be r=0.37, p=0.001; 0.03, p=0.5146 and 0.01, p=0.8179, respectively. Large significant differences between colonies w.r.t. uncapping, removing and grooming and very similar results of three colonies in two experiments indicates a genetic basis for these traits. The correlations between percentages of damaged mites under field conditions and test performance of their individuals with regard to uncapping, removing and grooming were estimated to be 0.63 (p=0.07, n=9), 0.43 (p=0.25, n=9) and 0.72 (p=0.11, n=6), respectively. The outstanding performance of a few individuals in expression of various traits indicates their usefulness in carrying out breeding programs for Varroa tolerance.
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Honey bee, Apis mellifera L., hygienic behavior is a mechanism of disease resistance and a mode of defense against the parasitic mite Varroa jacobsoni Oudemans. Hygienic bees uncap and remove diseased and parasitized brood from the nest. The propagation of colonies that demonstrate resistance to chalkbrood and American foulbrood and that remove pupae infested by Varroa mites is becoming increasingly important in apiculture. This study evaluates 2 commonly used field assays used to screen colonies for hygienic behavior: the freeze-killed brood and the pierced brood assays. Both involve determining the time required for worker bees to remove dead capped brood from a section of comb. Colonies in the experiment displayed a wide range of removal rates and were grouped as hygienic, nonhygienic, or intermediate. The results of experiments 1 and 2 indicated that neither the age nor the source of the frozen brood had a significant effect on the removal rate by hygienic colonies (i.e., those colonies that consistently uncapped and removed freeze-killed brood within 48 h). In experiment 3, only a weak correlation was found between the removal of young freeze-killed and pierced pupae, but a significant correlation existed between the removal of pre-eclosion freeze-killed and pierced pupae. Experiment 4 examined cues that elicit removal behavior by hygienic and non hygienic colonies. When pupae were pierced with an insect pin through the base of the cell (without piercing the wax cell capping), there was no difference in the number of pupae removed by the hygienic and nonhygienic colonies. On average, 30% of all pierced pupae survived the treatment, which considerably diminished the accuracy and reproducibility of the test. When pupae were treated with hemolymph extracted from either a live or freeze-killed pupa, there was also no difference in the rate of removal by hygienic and nonhygienic colonies. These results indicate that bees from nonhygienic lines can be induced to express hygienic behavior only if a sufficiently strong stimulus is present. Both hygienic and nonhygienic colonies removed significantly more pupae treated with hemolymph from a dead pupa than hemolymph from a live pupa, indicating that the cue that stimulates removal behavior is stronger in dead pupae. It is concluded that the freeze-killed brood assay is the most conservative and reliable screening procedure for hygienic behavior. The following procedures are recommended: Randomly selected comb sections (5 by 6 cm each) of capped brood should be cut from 1 healthy colony, frozen, and introduced into the test colonies. The assay should be repeated at least twice. Only colonies that remove >95% of freeze-killed brood within 48 h in both tests should be considered hygienic. When developing hygienic breeder stock, the hygienic colonies should be challenged with the American foulbrood or chalkbrood pathogen to ensure resistance.
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Colonies with naturally mated queens from a hygienic line of Italian honey bees (Apis mellifera ligustica) were compared to colonies from a commercial line of Italian bees not selected for hygienic behavior. The following characteristics were compared: rate of removal of freeze-killed brood; amount of chalkbrood; incidence of American foulbrood; honey production; and the number of mites, Varroa jacobsoni, on adult bees. The hygienic colonies removed significantly more freeze-killed brood than the commercial colonies, had significantly less chalkbrood, had no American foulbrood, and produced significantly more honey than the commercial colonies. Estimates of the number of Varroa mites on adult bees indicated that the hygienic colonies had fewer mites than the commercial colonies in three of four apiaries. In previous studies on the relation between hygienic behavior and resistance to diseases and mites, the test colonies contained instrumentally inseminated queens. This is the first study to evaluate hygienic stock in large field colonies with naturally mated queens. © Inra/DIB/AGIB/Elsevier, Paris
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Eleven colonies of honey bees representing four inbred lines were tested to learn their behaviour toward American foulbrood-killed larvae and pupae in their broodnests. To set up the experiments, about 2,900 larvae were exposed to Bacillus larvae spores suspended in water, and 2,600 control larvae were treated with water alone. All brood cells were observed, daily in most cases, for about two weeks, to learn whether or not the adult bees had removed the brood contained therein. By the end of the experiment, two of the inbred lines, with a history of resistance to American foulbrood, had removed from their cells all but three individuals of the several hundred which did not survive. The two other inbred lines, with a history of susceptibility to American foulbrood, allowed hundreds of individuals dead of the disease to remain in the broodnest. Thus a striking difference in behaviour toward brood dead of American foulbrood was demonstrated.
Breeding honey bees for resistance to disease
  • S Iii Taber
  • M Gilliam
Taber S. III., Gilliam M., Breeding honey bees for resistance to disease, Korean J. Apic. 2 (1987) 15-20
Das Ausräumverhalten von Apis mellifera L. gegenüber Milben-infizierter Brut als ein Abwehrverhalten gegen die ektoparasitische Milbe Varroa jacobsoni Oud
  • O Boecking
Boecking O., Das Ausräumverhalten von Apis mellifera L. gegenüber Milben-infizierter Brut als ein Abwehrverhalten gegen die ektoparasitische Milbe Varroa jacobsoni Oud., Ph.D Thesis, Hohen Landwirtschaftliche Fakultät der Rheinischen Friedrich-Wilhelms-Universität, Bonn, Germany, 1994.
Estudo comparativo dos métodos de congelamento e perfuração dc crias para avaliação do comportamento higiênico em abelhas africanizadas, Anais do IV Congreso Iberolatinoamericano de Apicultura
  • K P Gramacho
  • L S Gonçalves
Gramacho K.P., Gonçalves L.S., Estudo comparativo dos métodos de congelamento e perfuração dc crias para avaliação do comportamento higiênico em abelhas africanizadas, Anais do IV Congreso Iberolatinoamericano de Apicultura, Rio Cuarto-Córdoba, Argentina, 1994, p. 45.