A substance in honey bee larvae inhibits the growth of Paenibacillus larvae larvae
A substance in honey bee larvae inhibits the growth of Paenibacillus larvae larvae
rl CRAILSHEIM 0
0 Institut für Zoologie an der Karl-Franzens Universität Graz , Universitätsplatz 2, 8010 Graz , Austria
- Paenibacillus larvae larvae, a Gram-positive and spore-forming bacterium, is the cause of American foulbrood. We investigated the resistance of larvae of different ages from different colonies against P. larvae larvae. We prepared ethanol-water-extracts from two, three, four and five day old larvae and also larvae in two capped stages to test the ability of the homogenates to inhibit the growth of P. larvae larvae in vitro. There were age and colony dependent differences in the inhibiting potentials of larvae. The results suggest that the concentration of the inhibiting substance(s) in the extract might be responsible for the known different degree of resistance against P. larvae larvae in vivo. Our results further show that the extracts of the two-, three- and four-day-old larvae have the ability to inhibit the growth of P. larvae larvae very well, while starting around day five the inhibiting effect is gradually reduced.
Paenibacillus larvae subspecies larvae,
formerly Bacillus larvae
(Heyndrickx et al.,
, a Gram-positive, spore-forming
bacterium, is the causative agent of the American
foulbrood, one of the most serious and
destructive brood diseases of honeybees.
Different honey sample investigations
1984b; Grimm and Mossbeckhofer, 1993)
demonstrate that AFB is a problem for
One hypothesized mechanism of resistance
is built on genetically-based adult behavior.
Rothenbuhler and Thompson
Bamrick and Rothenbuhler (1961)
, 1967) investigated differences
between various genetic lines of honeybees.
They distinguished between resistant and
susceptible lines and were able to find
differences between the lines in their removal
of AFB-infected brood or hygienic behavior.
The resistant colony removed the dead brood
completely, while the susceptible colony
allowed some damaged brood to remain in the
Another hypothesized mechanism centers
on physiological features of the larvae.
also found differences in resistance among
individual larvae between the resistant and
susceptible colonies, in contrast to work by
, who reported that colony
resistance did not depend on larval resistance.
Rothenbuhler and Thompson
showed that the larvae of different lines
had different survival rates following
inoculation with spores of P. larvae larvae. After the
same treatment with spores,
determined that two
genetic lines differed in the age the larvae
became resistant against the infection. Larvae
of the resistant line became resistant after
about 36 hours of age, while the larvae of the
susceptible line did not become resistant until
about 48 hours.
A further hypothesis is that the ingredients
of the larval food (synthesized by nurse bee or
collected by foragers) or other substances have
an influence on the resistance of larvae against
P. larvae larvae.
Woodrow and Holst (1942)
that larvae older than two days were less
susceptible to an infection with AFB. Haydak
(1943) showed that the composition of the
larval food changed during larval development.
On the basis of these two facts, it was assumed
that there was a connection between larval
food and resistance.
showed that spores of P.
larvae larvae were not able to germinate at
high concentrations of reducing sugars. But
this result was disproved by
noticed a spore germination at even higher
reducing sugar concentrations than reported
Rinderer and Rothenbuhler (1974)
demonstrated the influence of pollen on the mortality
of spore-fed larvae. Pollen contains
microorganisms which act as antagonists of P. larvae
Brødsgaard et al., 1998
). The presence
of pollen in the intestine of the larvae may act
to inhibit P. larvae larvae
(Reiche et al.,
Bíliková et al. (2001)
royalisin, a peptide fraction of royal jelly (the
food of queen larvae) was found to inhibit the
growth of P. larvae larvae. Other investigated
substances include various saturated and
unsaturated free fatty acids
(Feldlaufer et al.,
, which were found to have antibiotic
The aim of the present work was to
elucidate the age related presence of P. larvae
larvae growth inhibiting substances in larvae,
especially in the larvae described as becoming
increasingly resistant at the age of about
2. MATERIALS AND METHODS
2.1. Paenibacillus larvae larvae
Cultivation and identification
of Paenibacillus larvae larvae
For our investigations we cultivated a fresh P.
larvae larvae culture from an infected colony in
Styria (Austria). We took material from a
contaminated brood comb and platted it on a
Columbia sheep blood agar (27.3 g Columbia agar
base (Oxoid®), 700 ml distilled water, 50 ml sheep
blood). Grown bacterial cultures were transferred
(after 3 days at 37 °C) from the plate to a liquid
medium (brain-heart-infusion (Oxoid®)) and
incubated for 48 hours at 37 °C, and then 1 ml
aliquots of the identified P. larvae larvae
suspension were frozen at – 70 °C until use.
The identification of P. larvae larvae was made
with the catalase-test
agar and with the “Plageman” test on Columbia
sheep blood slant agar
Starting a new experimental series, we
inoculated 40 ml autoclaved brain-heart-infusion
with 1ml defrosted bacteria suspension. After a
heat-shock at 77 °C for 10 min, we incubated the
suspension for 48 hours at 37 °C. After this time the
suspension reached an optical extinction of
0.22–0.23 measured at a wavelength of 546 nm.
The optical extinction was used as a measure for
the turbidity of the suspension and therefore as one
for the bacterial growth.
2.2. Colonies and samples
The samples for our investigations were taken
from six different full sized (two or three stories)
colonies, with approximately 35 000 bees, during
August 2000. The hives stood side-by-side in one
beeyard, so that we could exclude external
influences, e.g. different weather conditions.
Samples from the honey stores of each colony
were examined for spores of P. larvae larvae
(Hansen, 1984a, b; Hornitzky and Karlovskis, 1989;
von der Ohe and Dustmann, 1997)
. Our analysis did
not show any presence of P. larvae larvae spores.
2.2.1. Sampling the larvae
We sampled larvae of different weight classes.
In preliminary experiments, we caged queens to
determine the age and relative weights of larvae
(see Fig. 1a). We then compared our data with data
from the literature
(Stabe, 1930; Wang, 1965)
together with known morphological parameters,
e.g. size (Myser, 1954), we could estimate the age
of the larvae by its weight. For the present
experiment, we set up six weight classes consisting
of larvae with an average weight of 2.25 mg,
20.5 mg, 46.3 mg, 132.8 mg, 134.8 mg respectively
136.6 mg (see Fig. 1b). These six weight classes
correspond to six age classes (see Fig. 1b).
Hereafter, we refer to the age classes of the larvae,
based on their weights.
Larvae were removed from the brood combs and
washed with distilled water, then touched dry with
filter paper. For each sample (always about 130 mg
larval tissue) we either put several young larvae, or
only one old larva (five, six and seven day old) into
an Eppendorf vial, added 50 µL distilled water,
homogenized them ultrasonically and finally added
400 µL ethanol (96%). Then the samples were
frozen at –70 °C. Prior to use, they were thawed for
approximately 24 hours at 4 °C, centrifuged, and
the supernatants were lyophilized.
The lyophilized extracts were stored at 4 °C for
a possibly short period until use.
2.2.2. Larvae–weight and growth inhibition
From each colony used (n = 6), six larval age
groups were investigated . In each colony, one
agegroup comprised of six samples (n = 6; except the
two-day-old larvae of colony 4 in which n = 5; in
colony 6, n = 3; and in colony 7, n = 4). In case of
the five, six and seven day old larvae, single
individuals were used. Younger larvae had to be
pooled until a sample weight of about 130 mg was
obtained. Although intended, the weight of larvae
used for each sample was not exactly the same. To
exclude that such an inexactness might have caused
differences, we tested whether there was a
correlation between the weight of the larvae used to
generate a sample and the anti Paenibacillus larvae
larvae-activity of the pools. There was no such a
correlation, indicating our pooling method to be
2.3. Experimental setup
The method established by
was used to test the
antibacterial activity of the larval extract against P.
The lyophilized samples (see above) were
dissolved in 200 µL sterile water and different
concentrations of the solution were added to test
tubes filled with 1 mL liquid culture medium
(brainheart-infusion (Oxoid®)). The dilution series
consisted of five different concentrations of each
larval extract corresponding 32.5 mg to 2.03 mg
larval extract; the addition of the redissolved
extracts did not change the pH of the culture
medium (7.2). Sterile water was used as control for
normal growth of P. larvae larvae. For each
dilution steps two test tubes were used. Both were
filled with 1 ml brain-heart infusion and the same
concentration of larval extract. One of the two test
tubes was inoculated with 30 µL of P. larvae larvae
suspension with a defined optical extinction (E =
0.22–0.23 measured at a wavelength of 546 nm).
The second test tube was used as control for
unintended bacterial and fungus growth and also
served as a blank during later photometrical
measurements (wavelength: 546 nm).
All test tubes were incubated under aerobic
conditions at 37 °C (a temperature at which P. larvae
larvae grow best
) for 24 hours and
shaken before photometrical measurement.
For logistic reasons it was necessary to
investigate each colony in two steps. So we tested
the two-, four- and six-day-old larvae together in
one experimental series and three-, five- and
sevenday-old larvae in a different series.
Negative growth inhibition data were calculated
if the optical extinction (measured at a wavelength
of 546 nm after 24 hours of aerobically incubation
at 37 °C) was higher in larval extract samples
compared to the reference sample with sterile water.
Means and standard deviations are given. The
percent growth inhibition between all six colonies
was tested for differences with the Kruskal-Wallis
The percent growth inhibition of the different
colonies at certain larval ages (two, three and five
day old larvae) were tested for differences using a
Mann-Witney U-Test. The level of significance was
set at P < 0.05.
3.1. Different larval ages
The growth inhibiting effect was age
related; different larval ages are able to inhibit
the growth of the vegetative form of P. larvae
larvae to different extents (Fig. 2).
The equivalent of 32.5 mg larval extract
(the highest concentration in our experimental
set-up) of the two-, three- and four-day-old
larvae had the greatest potential to inhibit the
growth of P. larvae larvae. There were
variations in inhibition among identical age classes
taken from different colonies; the differences
ranged from 64% to 84% in the two day old
larvae, 73–86% in the three-day-old ones, and
68–85% in the four-day-old larvae. The ability
to inhibit the bacterial growth with extracts of
five-day-old larvae, the last uncapped larval
stage, decreased significantly, compared to the
two-day-old larvae (in all colonies except in
colony 4). Significant differences were found
in the growth inhibition of the five-day-old
larvae among the six investigated colonies.
While the larvae from colonies 1, 4 and 6 had
approximately the same inhibition potentials
as the three younger stages, the larval extract
of the colonies 3, 5 and 7 had nearly no
potential to suppress the growth of P. larvae larvae.
Differences are shown in Table Ia.
The six- and seven-day-old larvae – which
are already capped in their brood cells –
showed practically no inhibiting effect on
average (exception: colony 4).
Despite the differences between the
colonies at certain larval ages (see above), there
were no significant differences when we
compared the total age curves of the six colonies in
general (Kruskal-Wallis H-Test; Fig. 2).
3.2. Different growth inhibition of two different larval ages
We also tested whether there were
differences in the resistance against P. larvae larvae
within and between the investigated colonies
at two different young larval ages. Two- and
three-day-old larvae – being on the threshold
of resistance – were tested, and the percent
growth inhibition of an equivalent of 2.03 mg
larval extract was compared (2.03 mg is about
the weight of a two-day-old larva). There were
no differences between the extracts of the
twoand three-day-old larvae within each colony
Within the two-day-old larvae, significant
differences were found between colony 4 and
colony 5 as well as between colony 4 and
colony 7 (Tab. Ib).
Several significant differences among the
three-day-old larvae are shown in Table Ic.
Crailsheim and Riessberger-Gallé (2001)
found that larvae, and particularly adult
honeybees, had heat-resistant substances in their
midgut, which had the ability to inhibit the
growth of P. larvae larvae in vivo.
In the present work, we tested the time
course of resistance (the affectivity of
antibacterial substances) in the larvae. The three
youngest larval ages investigated, the two-,
three- and four-day-old larvae, inhibited the
growth of P. larvae larvae to the highest extent
(Fig. 2). Although we used larval extracts, we
can assume that larvae would have a good
resistance against an infection in vivo because
data from the literature
(Woodrow and Holst,
1942; Bailey and Lee, 1962; Bamrick, 1967)
show that young larvae (two-, three- and
fourday old) have increasing resistance with
increasing age. There are different explanations
found in the literature for why larvae are
resistant. Some authors
Rinderer and Rothenbuhler, 1974)
opinion, that the resistance of larvae against
AFB has something to do with the chemical
composition of larval food and its changes
during larval development
For example, Sturtevant found that the food of
the older larvae contained a higher percentage
of reducing sugar which was derived from
honey or nectar, and that a concentration of
reducing sugar of approximately 3 to 4 per
cent or more in the larval intestine was more
than sufficient to inhibit the growth of P.
larvae larvae spores.
On the other hand Tarr (1937b) showed,
that germination of the spores and
multiplication of the vegetative cells took place in the
presence of concentrations of reducing sugars
as high as 12.5%.
Rinderer and Rothenbuhler (1974)
that the resistance against AFB was associated
with the pollen consumption of the larvae.
With a certain amount of pollen fed together
with spores, they found decreasing mortality
of larvae. It is important to take into account
that the natural food of the worker larvae
during the first four days of their lives contains
only traces of pollen
Crailsheim and Riessberger-Gallé (2001)
found that there was a higher growth inhibition
effect of four day old larvae than could be
attributable to the amount of consumed pollen.
On the basis of their work, they assumed that
the inhibiting substances (or substance) were
produced by the honeybee larvae and by the
imagos, although they could not exclude an
involvement of food consumed as larvae in the
development of bacterial resistance. However
they could verify their assumption that the
substances at least in the adults were
“beemade” because even bees fed with artificial
food showed midgut-located resistance. The
midguts of foragers, that mainly feed on honey
and nectar and thus contain only traces of
pollen, have a great ability to inhibit the growth of
the vegetative form of P. larvae larvae.
and Crailsheim, 2001; Crailsheim et al., 1992)
Honey itself has no inhibiting effect at all if
(Riessberger-Gallé et al., 2001)
Another argument against the
“food-theory”, is the fact that worker jelly only has an
effect on the growth of the bacteria at high
Crailsheim and Riessberger-Gallé (2001)
showed that 2 mg of pure worker jelly had no
effect, while the equivalent extract from a
2.25 mg worker larva inhibited about 50%
The differences we found between the
inhibition potentials of the individual two day old
larvae (Fig. 3, Tab. 1b) were significant
between colonies 4 and 5, and colonies 4
and 7, but to determine whether the
differences were a function of genetic different lines
(Rothenbuhler and Thompson, 1956; Bamrick
and Rothenbuhler, 1961; Rothenbuhler, 1964)
more investigations would be necessary. The
lines Rothebuhler and Thompson used in their
investigations had been bred for resistance or
susceptibility to AFB. Further, Bamrick and
Rothenbuhler used lines that were bred over
several generations, while we just used a
variety of unselected colonies. To determine the
reasons for the significant differences between
the inhibition potentials of the equivalent
extracts of 2.03 mg of the three-day- old larvae
(Fig. 3), more investigations would have to be
performed. We think that our methods might
be a good way to discriminate between
resistant and susceptible lines, when the differences
in resistance could be attributed to genetic
The great variation between the growth
inhibition of the five-day-old larvae (Fig. 1)
may depend on the moment of taking the
samples, whether the larvae’s age was closer to the
four- or to the six-day-old larvae, and
therefore close to capped stage.
As we could detect different inhibiting
potentials of the larval extracts from different
colonies, one long-term aim of future
investigations will be the differentiation between
resistant and susceptible lines, followed by the
exclusive breeding of resistant colonies.
Another aim will be the identification of the
We thank Dr. Norbert Hrassnigg for his advise
and “keeping the bees” and we also thank Mag.
Ursula Platzer and Ross Little for linguistic
correction of the manuscript.
Résumé – Une substance dans les larves
d’abeilles (Apis mellifera) qui inhibe la
croissance de Paenibacillus larvae larvae.
Paenibacillus larvae larvae, bactérie gram+ et sporulante, est
l’agent de la loque américaine qui est l’une des
maladies les plus importantes du couvain de
l’Abeille domestique et qui cause le plus de dégâts.
Les larves d’abeilles peuvent être facilement
infectées par les spores des bactéries batonnets jusqu’à
l’âge de deux jours. Ensuite, en avançant en âge,
elles sont moins sensibles. Les abeilles adultes ne
peuvent plus du tout être infectées ; elles sont
devenues résistantes. Les spores, qui constituent le stade
infectieux, sont transférées aux larves par les
nourrices. Elles germent ensuite dans le tube digestif de
la larve et s’y multiplient rapidement, causant la
mort de la larve.
La résistance des larves à P. l. larvae a été étudiée
en fonction de leur âge. Nous avons utilisé pour
cette étude des larves âgées de deux, trois, quatre et
cinq jours, ainsi que deux stades operculés. Nous
avons produit un extrait des larves dans un mélange
eau-éthanol (le nombre de larves variait en fonction
de leur âge de façon à avoir toujours un échantillon
de 130 g), l’avons ajouté à un milieu de culture
liquide (infusion de cerveau-coeur [Oxoid] ) et
inoculé avec 30 µL d’une suspension de P. l. larvae
ayant une extinction optique précise (E = 0,22–
0,23). Après une période d’incubation de 24 h nous
avons mesuré l’extinction optique à la longueur
d’onde de 546 nm, comme mesure de la croissance
Nos résultats montrent que les extraits des larves de
deux, trois et quatre jours, sont capables d’inhiber la
croissance de P. l. larvae à près de 80 % par rapport
à la croissance normale. L’effet inhibiteur des
larves de cinq jours a varié entre 12 et 68 %. Nous
n’avons pas pu trouver d’effet inhibiteur des stades
operculés sur la croissance de la bactérie (Fig. 2).
La méthode décrite peut constituer un outil
utile pour discriminer des colonies plus ou moins
Apis mellifera carnica / Paenibacillus larvae
larvae / larve / résistance liée à l’âge
Zusammenfassung – Honigbienenlarven entha
lten eine Substanz die das Wachstum von
Paenibacillus larvae larvae hemmt. Paenibacillus
larvae larvae, ein gram-positives und sporenbildendes
Bakterium, ist der Erreger der Amerikanischen
Faulbrut. Diese ist eine der schwerwiegendsten und
destruktivsten Erkrankungen der Bienenbrut. Die
Honigbienenlarven können mittels der Sporen, des
stäbchenförmigen Bakteriums sehr leicht bis zu
einem Alter von zwei Tagen infiziert werden, aber
mit zunehmenden Alter sind sie weniger anfällig.
Adulte Honigbienen können nicht infiziert werden;
sie sind vollständig resistent.
Die Sporen, die das infektiöse Stadium darstellen,
werden durch die Ammenbienen auf die Larven
übertragen. Diese keimen dann im Darm der Larve
aus, vermehren sich sehr rasch und verursachen
somit den Tod der Larve.
Es wurde die Resistenz von unterschiedlichen
Altersklassen von Larven gegen P. larvae larvae
untersucht. Larven in einem Alter von zwei, drei,
vier und fünf Tagen und zwei verdeckelte
Larvenstadien wurden dazu verwendet. Wir stellten einen
Ethanol-Wasser-Extrakt aus den Larven
(unterschiedliche Anzahl von Larven, abhängig von
ihrem Alter; mit zunehmenden Gewicht benötigt
man weniger Larven um eine 130 mg schwere
Probe zu erhalten) her und fügten es einem
flüssigen Nährmedium (Herz-Hirn-Bouillon Oxoid ) bei
und beimpften es mit 30 µl P. larvae
larvae-Suspension mit einer definierten Extinktion (E = 0,22–
0,23). Nach einem Inkubationszeitraum von 24
Stunden, wurde die Extinktion bei einer
Wellenlänge von 546 nm, als Maß für das
Unsere Ergebnisse zeigen, daß der Extrakt aus den
zwei-, drei- und viertägigen Larven die Fähigkeit
besitzt, das Wachstum von P. larvae larvae im
Mittel beinahe zu 80 % des unbeeinflussten
Wachstums zu hemmen. Der Hemmeffekt der fünf Tage
alten Larven variiert, er reicht von 12 zu 68 %. Bei
den verdeckelten Stadien konnten wir beinahe
keine Hemmung auf das Bakterienwachstum
feststellen (Abb. 2). Die beschriebene Methode könnte
ein nützliches Werkzeug zur Diskriminierung von
mehr oder weniger resistenten Völkern sein.
Apis mellifera carnica / Paenibacillus larvae
larvae / Honigbienenlarven / altersabhängige
K. (2001) Free fatty acids
pollen and triolein in the
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