T H E EFFECT OF TEMPERATURE ON NOSEMA APIS ZANDER (MICROSPORIDA,
NO S E M A T I D A E )INFECTION
IN HONEY BEES (APIS MELLIFERA)
WOYCIECHOWSKI M.* & CZEKOŃSKA K.*
S u m m a r y :
Newly emerged honey bee [Apis mellifera carnica L.) workers infected individually with Nosema apis Z . spores were divided into three groups and kept in incubators at 2 5 ° , 3 0 ° or 3 5 ° C . After 4 8 h all workers were kept at 30° C. The numbers of parasite spores in individual bees were counted in all groups on the 11th, 16th, 21st and 26th days of life. Generally higher numbers of spores were observed in workers infected at 25° C.
However, the numbers in workers infected at extreme temperatures (25° and 35°C) differed significantly. Choosing suitable temperature conditions might be employed by infected bees to reduce the parasite's fitness and prolong their life spans.
KEY WORDS : Apis mellifera, Nosema apis, nosema disease.
Resume : EFFET DE LA TEMPÉRATURE SUR L'INFECTION À NOSEMA APIS ZANDER (MICROSPORIDA, NOSEMATIDAE) CHEZ L'ABEILLE (APIS MELLIFERA) De jeunes abeilles ouvrières (Apis mellifera carnica L.)
individuellement infectées par des spores de Nosema apis ont été divisées en trois groupes et placées en incubateur à 25°, 30° ou 35° C. Après 48 heures, toutes les ouvrières ont été placées à 30° С. Le nombre de spores du parasite chez chaque abeille a été compté dans chaque groupe au 11e, 16e, 21e et 26e jour de vie. Globalement, un nombre plus élevé de spores a été observé chez les ouvrières infectées à 25°C. Cependant, le nombre d'ouvrières infectées aux températures extrêmes [25° et 35°C]
diffère de manière significative. Le choix de conditions thermiques adéquates pourrait être utilisé par les abeilles infectées pour réduire la charge parasitaire et prolonger leur durée de vie.
MOTS CLÉS : Apis mellifera, Nosema apis, microsporidiose à Nosema.
Nosema apis Z a n d e r (Microsporida, N o s e m a - tidae) d e v e l o p s within the epithelial cells o f t h e h o n e y b e e (APIS MELLIFERA L.) midgut. It is a c o m m o n parasite throughout t h e b e e k e e p i n g world, which not only limits the life spans o f workers but also d e e p l y influences their b e h a v i o r ( W a n g & Moeller, 1 9 7 0 ; M a l o n e ET AL, 1 9 9 5 ; W o y c i e c h o w s k i & Lomnicki, 1995; W o y c i e c h o w s k i & Kozlowski, 1 9 9 8 ) . T h e r e is m u c h e v i d e n c e for a significant relation b e t w e e n t h e parasite's d e v e l o p m e n t a n d t e m p e r a t u r e . K a r m o &
Morgenthaler ( 1 9 3 9 ) suggest that N. APIS finds optimal conditions for development b e t w e e n 3 0 ° and 34°C, that at 20-25° С their development is slower, and that b e l o w 10° С a n d a b o v e 3 7 ° С d e v e l o p m e n t is stopped. Lotmar ( 1 9 4 3 ) confirms t h e s e results a n d points to t h e adap
tation o f N. APIS to the narrow temperature interval ( 3 0 - 35° C ) prevailing in the b r o o d region o f t h e b e e nest ( B ü d e l , 1 9 6 0 ; Vogt & Heinrich, 1 9 8 5 ) . In s o m e parts o f the nest distant from the b r o o d the temperature m a y e v e n c o m e c l o s e to a m b i e n t temperatures. This might b e o n e r e a s o n w h y individuals have different levels o f infection. C h o o s i n g suitable temperature c o n d i t i o n s might reduce the parasite's fitness and prolong the lives o f infected b e e s .
T h e present p a p e r investigates the effects o f different temperatures only during infection o f h o n e y b e e wor
kers with N. APIS spores, while further parasite deve
l o p m e n t takes p l a c e at t h e s a m e temperature.
METHODS
T
h e e x p e r i m e n t w a s c o n d u c t e d in J u n e 1 9 9 7 . H o n e y b e e w o r k e r s (A. MELLIFERA CARNICA) w e r e derived from a single inseminated q u e e n . W o r kers emerging within 15 h in an incubator w e r e individually infected with t h e s a m e n u m b e r o f N. APIS
spores ( 4 8 6 x 1 03 spores p e r b e e ) dosed in 2 0 µL 50 % ( w / v ) sugar syrup. Each experimental group o f l6l b e e s w a s placed in a w o o d e n c a g e ( 1 5 x 1 4 x 6 c m ) with m e s h sides, provided with a small p i e c e o f b e e c o m b . T h e c a g e s w e r e kept in incubators, e a c h at o n e o f three temperatures: 2 5 ° , 30° a n d 35°C. After 4 8 h all c a g e s w e r e kept at 30°C. An additional fourth c a g e with l6l uninfected w o r k e r s ( c o n t r o l ) w a s kept at 30° С for the w h o l e time o f the e x p e r i m e n t . During the e x p e r i m e n t the cages w e r e c h e c k e d every day, the dead b e e s were r e m o v e d a n d food a n d water w e r e replenished in gra
vity feeders as necessary.
T o estimate the n u m b e r o f N. APIS spores in individual workers, o n the 11th, 16th, 21st a n d 26th days after infection 30 b e e s w e r e randomly c h o s e n from e a c h o f
* B e e Research Department, Agricultural University, Al. 29 Listo- pada 52, 31-425 Kraków, Poland.
Correspondence: Michal Woyciechowski.
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Note de recherche 185
Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/1999062185
W O Y C I E C H O W S K I M. & C Z E K O N S K A K.
the c a g e s after short treatment o f e a c h w o r k e r group with c a r b o n dioxide. Only o n the 26th day the n u m b e r o f remaining workers in the experimental groups was l o w e r than 3 0 individuals ( 2 5 ° С - 15, 3 0 ° С - 12, and 3 5 ° С - 22 individuals). Altogether 4 3 9 workers w e r e tested; their a b d o m e n s w e r e m a c e r a t e d in 1 mL dis
tilled water and the spores w e r e c o u n t e d in a h a e - m a c y t o m e t e r in 0.025 m m3 o f suspension. If less than 10 s p o r e s w e r e f o u n d in t h e s a m p l e t h e y w e r e r e c o u n t e d in 0.2 m m3 o f suspension. T h e results w e r e given as the n u m b e r o f spores per 1 mL, w h i c h w a s treated as the n u m b e r o f spores per b e e . T o assess the significance o f differences in spore numbers in infected workers at the various temperatures the Kruskal-Wallis test was used (Sokal & Rohlf, 1 9 8 1 ) .
RESULTS
T
here w e r e significant differences in the numbers of N. apis s p o r e s b e t w e e n the e x p e r i m e n t a l g r o u p o n the 1 1 , 21 and 26 days after infection (Fig. 1 ) . No such a difference w a s found only for 16- day-old w o r k e r s . T h e s e c o m p a r i s o n s e x c l u d e d the control group o f uninfected workers, in which the perc e n t a g e o f infected individuals in all samples w a s not high ( 1 5 . 8 % ) and the mean n u m b e r o f spores per indi
vidual did not markedly increase through time and equaled: 5,666, 2 , 3 3 3 , 3,000 and 3,333 spores o n the successive days. T h e biggest o f these values was 1,160 times smaller than the lowest in the e x p e r i m e n t a l groups (Fig. 1 ) .
Differences in the levels o f infection a m o n g the indi
viduals in the experimental groups (Fig. 1) w e r e due mainly to the different numbers o f spores in individuals infected at 2 5 ° and 3 5 ° C . O n three o f the four sam
pling days this difference w a s significantly higher in individuals infected in l o w e r temperatures (11th day:
H = 7 . 4 8 1 , η = 60, ρ < 0 . 0 1 ; l6th day: Η = 0 . 0 7 9 , η =
Fig. 1. - N. apis infection level in bees infected at various tempera
tures (11th day: Η = 6.367. η = 90, ρ < 0.05; l6th day: Η = 3.776, η = 90, ρ > 0.05; 21st day: Η = 9.027, η = 90, ρ < 0.05; 26th day:
Η = 10.108, η = 49; * ρ < 0.05,** ρ < 0.01, ns - ρ > 0.05).
1 8 6
60, ρ > 0.05; 21st day: Η = 8.311, η = 60, ρ < 0.01;
26th day: Η = 8.819, η = 37, ρ < 0.01).
Although on all the testing days the individuals infected at 35°C had the lowest numbers o f spores, they never differed significantly from those infected at 30° С ( 1 1 t h day: Η = 0.981, η = 60, ρ > 0.05; l 6 t h day; Η = 2.967, η = 60, ρ > 0.05; 21st day: Η = 1.598, η = 60, ρ > 0.05;
26th day: Η = 0.117, η = 34, ρ > 0.05). Individuals infected at 2 5 ° and 30° С differed significantly in the n u m b e r o f spores only o n the 26th day o f life ( 1 1 t h day: Η = 1.181, η = 60, ρ > 0.05; l ó t h day: Η = 2.597, η = 60, ρ > 0.05; 21st day; Η = 3-581, η = 60, ρ > 0.05;
26th day: Η = 5.952, η = 27, ρ < 0.05).
DISCUSSION
a n y a u t h o r s i n v e s t i g a t i n g t h e n u m b e r o f N. apis spores in the digestive system o f h o n e y b e e workers have o b s e r v e d great variation in the levels o f infection a m o n g individuals from the same c o l o n y (El-Shemy & Pickard, 1 9 8 9 ; W o y c i e c h o w s k i et al, 1996). Attention has b e e n given to different genetic- d e p e n d e n t levels o f resistance in individuals (Malone et al, 1 9 9 5 ) as well as to the significance o f their age (El-Shemy ík Pickard, 1 9 8 9 ) . T h e effects o f various tem
perature conditions have also b e e n observed, but only in c a g e e x p e r i m e n t s ( K a r m o & Morgenthaler, 1 9 3 9 ; Lotmar, 1 9 4 3 ) . Approximately the s a m e levels o f infec
tion have b e e n found in infected b e e s irrespective o f the s p o r e infection d o s e (Fries, 1 9 8 8 ) .
O u r e x p e r i m e n t points to another factor that s e e m s to have a significant influence o n the parasite d e v e l o p ment. It is the temperature o f the b e e s ' environment during infection. H e r e the results suggest that the optimal temperature during infection differs from the optimal temperature for multiplication o f the spores already present in the cells o f the worker's digestive system. T h e level o f infection in b e e s kept for 4 8 h at 25° С and fed N. apis s p o r e s w a s higher than in those kept at 3 5 ° C . This result is interesting b e c a u s e tem
peratures b e t w e e n 3 0 ° - 3 5 ° C are considered optimal for d e v e l o p m e n t o f N. apis ( K a r m o & Morgenthaler, 1939;
Lotmar, 1 9 4 3 ) . It a p p e a r e d that the results w e r e not influenced b y the l o w level o f uncontrolled infection n o t e d in the uninfected control g r o u p o f workers.
This conclusion confirms the fact that the biggest m e a n n u m b e r o f spores o b s e r v e d in control group w a s more than thousand times lower than the lowest value in any infected group o f workers.
Without m o r e e x p e r i m e n t s it is difficult to determine w h e t h e r the differences w e o b s e r v e d in the n u m b e r o f spores are the result o f the host's reaction (for e x a m p l e , turnover o f the epithelial cells o f the midgut, as suggested b y Lotmar in 1 9 4 3 ) o r the parasite's reac-
Parasite, 1999, 6, 185-187
N o t e d e r e c h e r c h e
EFFECT OF TEMPERATURE ON N. APIS INFECTION
tion (for e x a m p l e , different possibilities o f spore ger
mination d e p e n d i n g o n t e m p e r a t u r e c o n d i t i o n s o r spore dimorphism o b s e r v e d b y D e Graaf et al., 1994 during developmental c y c l e ) . Nor d o w e k n o w w h e ther b e e s c a n exploit temperature as a defense against parasite development, as d o Bombus terrestris parasi
tized with conopid fly larvae (Muller & Schmid-Hempel, 1 9 9 3 ) . Observations that N. apis infected b e e s tend to c o n g r e g a t e in the w a r m e r parts o f the hive (Moeller, 1 9 5 6 ) support this supposition.
ACKNOWLEDGEMENTS
W
e thank Magda Góral for technical assistance with this project, a n d Stefan Fuchs, Michael J a c o b s a n d an a n o n y m o u s referee for helpful c o m m e n t s o n the manuscript.
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Reçu le 4 janvier 1999 Accepté le 11 mars 1999
Note de recherche 187
