report DIAS

June 1999

Danmarks JordbrugsForskning

Forskningscenter Flakkebjerg 4200 Slagelse

DIAS report N o. 6 • H orticulture

Camilla J. Brødsgaard, Sten Erik Jensen, Carsten W. Hansen &

Henrik Hansen

Spring treatment with oxalic acid in honeybee colonies as varroa control

M inistry of Food, A griculture and Fish eries

Spring treatm ent w ith oxalic acid in honeybee colonies as varroa control

Camilla J. Brødsgaard, Sten Erik Jensen & Henrik Hansen Danish Institute o f A gricultural Sciences

Departm ent o f Crop Protection Research Centre Flakkebjerg DK-4200 Slagelse

Carsten W. Hansen

Danish Beekeeper's Association Møllevej 15

DK-4140 Borup

DIAS report H orticulture no. 6 • June 1999 • 2nd volume

Publisher: Danish Institute of Agricultural Sciences Tel. +45 89 99 19 00

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Cover ill. Oxalis acetosella L., Wood Sorrel (modified from Lindman 1974)

SUMMARY

In la te M a rc h 1 9 9 8 ,3 0 h o n e y b e e c o lo n ie s (A pis mellifera) in fo u r a p ia r ie s w e r e tre a te d fo r th e p a ra s itic m ite (Varroa jacobsoni) w ith e ith e r s p r a y in g o r tric k lin g o f oxalic ac id . F o u r c o lo n ie s w e r e n o t tr e a te d a n d s e rv e d as c o n tro ls. P rio r to th e tr e a tm e n t, e ig h t d a y s a fte r th e tr e a tm e n t, a n d a t th e firs t h o n e y h a r v e s t in J u n e o n e fo o d sa m p le w a s ta k e n in e a c h c o lo n y . O f th e s e s a m p le s five fro m s p r a y e d , fiv e fro m tric k le d , a n d th e fo u r f ro m c o n tro l c o lo n ie s w e r e c h o s e n a n d th e o x alic a c id r e s id u e le v el w a s d e te r m in e d b y m e a n s o f liq u id c h ro m a to g ra p h y . T h e re s u lts s h o w e d th a t th e m a x im u m r e s id u e le v e l w a s f o u n d e ig h t d a y s a fte r tr e a tm e n t in th e s p r a y e d g r o u p (x=0.0062% ) b u t a lso th a t th e re w a s n o sig n ific a n t d iffe re n c e in oxalic a c id c o n c e n tra ­ tio n b e tw e e n th e g r o u p s a t a n y o f th e s a m p lin g d a te s.

I n a n o th e r a p ia r y , th e g lu ta th io n e S -tra n s fe ra se (GST) a c tiv ity w a s m e a s u r e d in in d iv id u a l p u p a e a n d a d u lt b e e s fro m tric k le d a n d c o n tro l co lo n ies. T h e r e s u lt s h o w e d th a t 15 d a y s a fte r tr e a tm e n t th e G ST a c tiv ity in p u p a e a n d a d u l t b e e s fro m th e tric k le d c o lo n ie s w a s n o t d iffe re n t fro m th e G ST a c tiv ity f o u n d in n o n - tre a te d c o lo n ie s in d ic a tin g th a t tric k lin g tr e a tm e n t of c o lo n ie s w ith oxalic a c id d o e s n o t se em to h a v e a n effec t o n th e le v el o f G ST a c tiv ity in p u p a e o r n e w ly e m e rg e d a d u lt b ee s.

T h e v a r r o a m o r ta lity w a s r e c o rd e d a fte r th e s p rin g tr e a tm e n ts w ith oxalic acid tric k lin g a n d s p r a y in g a n d a g a in in th e a u tu m n a fte r a n oxalic a c id tric k lin g tr e a t­

m e n ts. F u rth e rm o re , th e b e e c o lo n y s tr e n g th a n d b r o o d a m o u n t w e r e r e c o rd e d p rio r to th e s p r in g tr e a tm e n t a n d a g a in a y e a r a fte r th e tr e a tm e n ts . A sig n ific a n t d iffe ren ce in v a r r o a m o r ta lity w a s s e e n a fte r th e s p r in g tr e a tm e n t b e tw e e n th e tre a te d co lo n ies a n d th e c o n tro ls. In th e tric k lin g g ro u p th e to ta l m ite d r o p - d o w n p e r c o lo n y w a s in a v e ra g e 61.53, in th e s p r a y e d g ro u p it w a s 145.47 a n d in th e c o n tro l g r o u p 1.50. A fter th e a u tu m n tr e a tm e n t, n o sig n ific a n t d iffe re n c e w a s f o u n d b e tw e e n th e th re e g ro u p s a n d th e m ite d r o p - d o w n r a n g e d b e tw e e n 936 a n d 1,400 m ite s. In 1998, th e m e a n b e e c o lo n y s tr e n g th w a s a p p ro x im a te ly 5.5 co m b g a te s b e fo re th e tre a tm e n t. A t th e sa m e tim e th e m e a n b r o o d a m o u n t r a n g e d fro m 1.77 to 3.25 d m 2. D u rin g th e 1998 se a so n , n o d iffe re n c e in c o lo n y d e v e lo p m e n t w a s o b s e rv e d a m o n g th e th re e tr ia l g ro u p s . O n e y e a r a fte r th e tre a tm e n ts th e m e a n c o lo n y s tr e n g th r a n g e d fro m 4.93 to 6.25 co m b g a te s. T h e b r o o d a m o u n t r a n g e d fro m 0.89 to 1.53 d m 2. T h e re w a s n o sig n ifi­

c a n t d iffe re n c e b e tw e e n th e tr e a te d g r o u p s a n d th e c o n tro l g r o u p a t a n y tim e.

INTRODUCTIO N

S ev era l s tu d ie s h a v e s h o w n th a t fo rm ic (F ries 1991) a n d lactic a c id (K o e n ig e r et al.

1983, K le p sc h et al. 1984, K ra u s 1991, B rø d s g a a r d et al. 1997) a re effec tiv e in c o n tro l­

lin g in fe s ta tio n w ith th e p a ra s itic m ite Varroa jacobsoni O u d e m a n s o n h o n e y b e e s (Apis mellifera, L.). H o w e v e r, in la b o ra to r y te s ts o xalic a c id w a s e v e n m o re p o is o n o u s th a n th e tw o fo rm e r o rg a n ic a c id s to v a r r o a m ite s (F u c h s, p e rs . co m m .). In fie ld te sts R a d e z k i (1994) f o u n d v e r y h ig h m ite m o r ta lity (97,3%) w h e n s p r a y in g 3% oxalic a c id o n a d u lt m ite in fe s te d bees.

T h o u g h o xalic a c id is f o u n d n a tu r a lly in v e ry lo w c o n c e n tra tio n s in e.g. s p in a c h a n d r h u b a r b (F a ssett 1973) it m a y b e h a r m f u l to h u m a n s e v e n in lo w c o n c e n tra tio n s . O ra l in g e s tio n o f o xalic a c id m a y b e d e a d ly , b y d e rm a l c o n ta c t it m a y c a u s e d a m a g e to th e s k in a n d tis s u e a n d b y in h a la tio n it m a y c a u se d a m a g e to th e m u c o u s m e m b r a ­ n e s. T h e re fo re , it is n e c e s s a ry th a t p r e c a u tio n s a r e ta k e n w h ile s p r a y in g oxalic acid ( a p p r o p r ia te u s e o f m a s k , g lo v e s, a n d g la sses) (R a d e z k i 1994).

T o a v o id o xalic ac id in a e ro so l fo rm , th e a p p lic a tio n o f oxalic a c id h a s b e e n fu rth e r d e v e lo p e d in Ita ly b y N a n e tti a n d S tra d i (1997). T h e y f o u n d th a t o n e a p p lic a tio n of oxalic a c id b y tric k lin g a n oxalic a c id s u g a r s o lu tio n o n to th e f ra m e s in th e c o lo n ies p r o v id e d efficacies r a n g in g fro m 89,6% to 96,8% d e p e n d in g u p o n th e o xalic ac id c o n c e n tra tio n .

H o w e v e r, b e s id e s b e in g h a r m f u l to th e v a r r o a m ite s , p h y s io lo g ic a l effects o n th e b e e s in th e c o lo n ie s tr e a te d w ith oxalic a c id b y th e tric k lin g m e th o d h a s b e e n s u g g e s te d (W . R itte r, p e rs . c o m m ., A . Im d o rf, p e rs . co m m .). T h e h y p o th e s is is th a t th e b e e s in g e s t so m e o f th e oxalic a c id -s u g a r s o lu tio n d u r in g th e tr e a tm e n t le a d in g to d a m a g e o n tis s u e s of th e d ig e stiv e sy ste m . If tis s u e s w ith g lu ta th io n e S -tra n s fe ra - se (GST) a c tiv ity a re d a m a g e d , a p o s s ib le effec t c o u ld b e a lo w e r in g o f th e le v e l of G ST a c tiv ity . It h a s b e e n s h o w n th a t se v e ra l d iffe r e n t tis s u e s , in c lu d in g g u t tis su e s h a v e G ST a c tiv ity in h o n e y b e e s a n d o th e r in s e c ts (e.g. C la rk 1989, E l-G h a re e b &

O m a r 1994). T h e G ST e n z y m e s a re a n im p o r ta n t g r o u p o f e n z y m e s fo r e lim in a tin g h a r m f u l s u b s ta n c e s th a t th e b e e s co m e in to c o n ta c t w ith (S m irle & W in s to n 1988, Y u et al. 1984). A lo w e r in g o f th e le v el o f G ST a c tiv ity c o u ld m a k e th e b e e s m o re v u ln e ra b le to toxic s u b s ta n c e s in th e e n v iro n m e n t. T h e re fo re a tr e a tm e n t o f b e e s th a t r e s u lts in a lo w e r in g of th e le v el o f G ST a c tiv ity m a y in flu e n c e th e ir o v e ra ll fitn e ss.

T h is s tu d y in c lu d e s p r e lim in a r y re s u lts o n th e u se o f G ST a c tiv ity a s a b io lo g ic a l m a rk e r fo r p o ssib le p h y s io lo g ic a l effects o n b e e s f ro m oxalic a c id tr e a te d colonies.

F u rth e rm o re , r e s id u e le v e ls in h o n e y h a v e o n ly b e e n s tu d ie d a fte r oxalic ac id tr e a tm e n t in a u tu m n a n d e a rly w in te r (R a d e z k i 1994, N a n e tti & S tra d i 1997) a n d in D e n m a r k th e n e e d fo r a n e ffic ie n t s p rin g tr e a tm e n t is p r o fo u n d . T h e re fo re , a fu rth e r aim o f th is s tu d y h a s b e e n to e x a m in e th e re s id u e le v e ls of o xalic a c id a fte r tr e a tm e n t of b e e c o lo n ie s in th e s p r in g tim e w ith e ith e r s p r a y in g o r tric k lin g o f o xalic a d d . To g e t a n im p r e s s io n o f d iffe re n c e s in th e tre a tm e n ts th e v a r r o a m o rta lity w a s m o n i­

to r e d s h o r tly a fte r th e s p r in g tr e a tm e n t a n d a g a in in th e a u t u m n a fte r a se c o n d sin g le tr e a tm e n t w ith oxalic a c id tric k lin g .

MATERIALS & M ETHODS

O xalic acid treatm en t

In s p r in g 1998 (23 M a rc h ), to ta lly 35 h o n e y b e e c o lo n ie s in fiv e a p ia rie s in D e n m a rk w e r e tr e a te d w ith oxalic ac id . T h e c o lo n ie s w e r e n a tu r a lly in fe s te d w ith v a r r o a m ite s. In b o th th e f irs t a n d th e se c o n d a p ia r y f o u r b e e c o lo n ie s w e r e s p r a y e d , fo u r w e r e tric k le d a n d o n e u n tr e a te d c o lo n y s e rv e d a s c o n tro l. In th e th ir d a p ia ry th re e b e e c o lo n ie s w e r e s p r a y e d , fo u r w e r e tric k le d , a n d o n e u n tr e a te d c o lo n y s e rv e d as c o n tro l. In th e f o u r th a p ia r y fo u r b e e c o lo n ie s w e r e s p r a y e d , th r e e w e re tric k le d , a n d o n e u n tr e a te d c o lo n y s e rv e d as c o n tro l. T he tr e a te d c o lo n ie s w e re s e le c te d ra n d o m ly w ith in th e a p ia rie s . T h e c o lo n ie s in th e la s t a p ia r y w e r e u s e d fo r a s s a y in g GST ac tiv ity in th e b e e s (see b e lo w ). In th is a p ia r y five ra n d o m ly s e le c te d b e e c o lo n ies w e r e tric k le d a n d fiv e u n tr e a te d c o lo n y s e rv e d a s co n tro ls.

F o r s p r a y tr e a tm e n t a n oxalic a c id s o lu tio n w a s m a d e o f 30 g oxalic a c id c ry sta ls (oxalic a c id d ih y d r a te ) d is s o lv e d in 11 d e m in e ra lis e d w a te r (R a d e tz k i 1994). 3-4 m l p e r co m b s id e w e r e s p r a y e d (Im d o rf et al. 1996) w ith a h a n d o p e r a te d a to m is e r (Ginge®, 0.51, n o . 20-05-00, a d ju s te d to th e fin e s t a to m isa tio n ).

T ric k lin g w ith oxalic a c id s u g a r s o lu tio n u s in g 1 p a r t (w e ig h t) oxalic a c id d ih y d r a te , 10 p a r t d e m in e r a lis e d w a te r , 10 p a r ts su c ro s e (N a n e tti & S tra d i 1997). 3 m l w e re tr ic k le d w ith a s y rin g e p e r f u lly o c c u p ie d co m b g ate. E.g. to ta lly , 30 m l w e r e u s e d

f o r a w e a k b e e c o lo n y , 40 m l fo r a m e d iu m s tro n g b e e c o lo n y a n d 50 m l fo r a stro n g c o lo n y (Im d o rf et al. 1998).

C lim a tic m e a su r e m e n ts

T h e o u td o o r te m p e r a tu re w a s a b o v e 5 °C a n d th e oxalic a c id s o lu tio n s w e r e a p p ro x i­

m a te ly 10°C b e fo re th e tre a tm e n t. O u td o o r te m p e r a tu re a n d re la tiv e a ir h u m id ity (R H ) w e r e r e c o r d e d in th e a p ia r ie s a t th e tim e o f th e tr e a tm e n t b y T in y Talk® d a ta lo g g e rs a t 17 m in s in te rv a l a n d c o n tin u e d fo r th r e e w e e k s.

V arroa m o r ta li ty

In th e c o lo n ie s in th e a p ia rie s 1-4 th e v a r r o a m o r ta lity w a s r e c o rd e d in sp e c ia lly d e s ig n e d w o o d e n in s e rts c o v e rin g th e e n tire b o tto m b o a r d e v e ry se c o n d d a y fo r fo u r w e e k s a fte r tre a tm e n ts . T h e in s e rts w e re e m p tie d e a c h s a m p lin g d a te . F u rth e rm o re , th e v a r r o a m o r ta lity w a s c a lc u la te d in all b e e c o lo n ie s a fte r o n e tric k lin g tr e a tm e n t w ith oxalic a c id in th e a u t u m n 1998. A t th e tim e o f th e a u t u m n tr e a tm e n t n o b r o o d w a s p r e s e n t in th e co lo n ies. T h e re s u lts w e re a n a ly s e d s ta tis tic a lly b y m e a n s o f K ru s k a l-W a llis M u ltip le C o m p a ris o n s (K-W ) (Siegel a n d C a s te lla n , 1988).

Bee c o lo n y s tr e n g th

In th e a p ia r ie s 1-4 th e a m o u n t of b r o o d w a s e s tim a te d in d m 2 b y v is u a lly d iv id in g th e c a p p e d b r o o d in to s q u a re s . T h e b e e c o lo n y s tre n g th w a s e s tim a te d as th e n u m b e r o f co m b g a te s o c c u p ie d b y th e b e e s w h e n lo o k in g in to th e c o lo n y w ith o u t re m o v in g th e co m b s. T h e e s tim a tio n s w e re c a rrie d o u t b e fo re th e tr e a tm e n t a n d a g a in o n e y e a r a fte r th e tre a tm e n t. T h e r e s u lts w e re a n a ly s e d s ta tis tic a lly b y m e a n s of K ru s k a l-W a llis M u ltip le C o m p a ris o n s (K-W ) (Siegel a n d C a s te lla n , 1988).

O x a lic a c id re sid u es

In th e a p ia r ie s 1-4 o n e fo o d sa m p le w a s ta k e n in e a c h o f th e c o lo n ie s b e fo re tr e a t­

m e n t. E ig h t d a y s a fte r th e tr e a tm e n t o n e sa m p le o f u n s e a le d fo o d o r h o n e y w e re ta k e n in e a c h o f th e co lo n ies. F u rth e rm o re , o n e s a m p le o f th e firs t h o n e y h a r v e s t (e a rly J u n e ) w e r e ta k e n in e a c h o f th e co lo n ies. T h e s a m p le s w e r e s to r e d in d a rk n e s s a t - 2 0 ° C u n til p ro c e s s in g . In th e a p ia rie s 1-4, fiv e s p r a y e d , fiv e tric k le d , a n d fo u r c o n tro l c o lo n ie s w e r e ra n d o m ly c h o s e n a n d a n a ly s e d fo r o xalic a c id r e s id u e s b y liq u id c h r o m a to g r a p h y a t I n s titu to Z o o p ro fila ttic o S p e rim e n ta le d e lle V en e zie , L e g n a ro , Italy . T h e r e s u lts w e re a n a ly s e d s ta tis tic a lly b y m e a n s o f K ru s k a l-W a llis M u ltip le C o m p a r is o n s (K-W ) (Siegel a n d C a s te lla n , 1988).

G lu ta th io n e S -tran sferase (G ST ) a ctiv ity

H o n eyb ees

In th e fifth a p ia r y , p u p a e a n d n e w ly e m e rg e d a d u lt w o r k e r h o n e y b e e s w e re co llec­

te d fro m th e co lo n ies. P u p a e w e re c o llec te d im m e d ia te ly b e fo re oxalic a c id tric k lin g tr e a tm e n t (as m e n tio n e d a b o v e ) in fiv e c o lo n ie s a n d a g a in 15 d a y s a fte r th e tre a t­

m e n t. N e w ly e m e r g e d a d u l t b e e s w e re c o lle c te d 15 d a y s a fte r th e oxalic ac id tre a tm e n t. A s c o n tro l, in d iv id u a ls w e r e c o llec te d s im u lta n e o u s ly fro m fiv e n o n ­ tre a te d c o lo n ie s a t th e sa m e lo cality . A fte r co lle c tio n th e in d iv i d u a l p u p a e a n d a d u lts w e r e s to r e d a t -80 °C u n til a s sa y e d .

C h em icals

R e d u c e d g lu ta th io n e (G SH ) a n d l-c h lo ro -2 ,4 -d in itro b e n z e n e (C D N B ) w e re p u r c h a ­ s e d fro m M e rc k ( D a rm s ta d t, G e rm a n y ), l- p h e n y l-2 - th io u r e a (PTU ) w a s p u r c h a s e d f ro m A ld ric h (M ilw a u k e e , W I) a n d e th y le n e d ia m in e te tra a c e tic a c id (ED TA ) w a s p u r c h a s e d f ro m S ig m a C h e m ic a l C o. (St. L o u is, M O ). T h e B ra d fo rd d y e r e a g e n t a n d b o v in e s e ru m a lb u m in (fra c tio n V) w e re p u r c h a s e d f ro m P ie rc e (R o ck fo rd , IL). A ll o th e r c h e m ic a ls w e r e o f a n a ly tic a l q u a lity a n d p u r c h a s e d f ro m c o m m e rc ia l s u p p li­

e rs.

H o m o g e n isa tio n o f p u p a e o r a d u lt bees f o r in vitro a n a ly s is

W h o le p u p a e o r w h o le a d u l t b e e s w e r e h o m o g e n is e d in d iv id u a lly in 0.1 M s o d iu m p h o s p h a te b u f fe r ( p H 6.5) c o n ta in in g 10 m M G S H , 1 m M P T U a n d 1 m M ED TA . T he in d iv id u a ls w e r e h o m o g e n is e d m a n u a lly in m ic ro tu b e s h e ld o n ice. T h e h o m o g e n a te w a s c e n trif u g e d a t 10,000 g fo r 20 m in a t 4 °C a n d th e s u p e r n a ta n t w a s u s e d in th e assay s.

G ST an d p r o te in a s s a y s

A ll m e a s u r e m e n ts in th e G ST a n d p r o te in a s s a y s w e re m a d e a t a n a m b ie n t te m p e r a ­ tu re (20-25°C ) a n d a b s o rb a n c e w a s re c o rd e d u s in g a M u ltis k a n M C C /3 4 0 m ic ro p la ­ te r e a d e r (L a b sy ste m s, F in la n d ).

G ST a c tiv ity w a s m e a s u r e d w ith C D N B as s u b s tra te as d e s c rib e d b y H a b ig et al.

(1974) a n d m o d ifie d for a m ic ro p la te . A ssa y s w e r e d o n e o n h o m o g e n a te d ilu te d w ith h o m o g e n is a tio n b u ffe r to a n a p p r o p r ia te c o n c e n tra tio n . D ilu te d h o m o g e n a te (20 ß1) w a s a d d e d p e r w e ll in a m ic ro p la te a n d 180 ß \ of G S H (10 m M fin a l c o n c e n tra tio n ) a n d C D N B (1.0 m M fin a l c o n c e n tra tio n ) in 0.1 M s o d iu m p h o s p h a te b u ffe r (p H 6.5) w a s a d d e d to e a c h w e ll. T h e m ic ro p la te w a s le ft 5 m in to e q u ilib r a te a n d th e n

a b s o rb a n c e a t 340 n m w a s re c o rd e d c o n tin u o u s ly . R e a c tio n r a te s w e r e c a lc u la te d b y lin e a r r e g r e s s io n a n a ly s is o n th e lin e a r p o r tio n o f th e c u rv e a fte r p lo ttin g a b s o rb a n c e a g a in s t tim e . D u p lic a te d e te r m in a tio n s w e r e m a d e o n e a c h in d iv id u a l.

P ro te in c o n te n t in h o m o g e n a te s w a s d e te r m in e d b y th e m e th o d o f B ra d fo rd (1976), as m o d if ie d in th e P ie rc e s ta n d a r d a s s a y fo r m ic ro p la te s , w ith b o v in e s e ru m a lb u m in as a s ta n d a r d .

T h e G ST a c tiv ity w a s m e a s u r e d in in d iv id u a ls fro m n o n - tr e a te d c o lo n ie s a n d in in d iv id u a ls f ro m c o lo n ie s tric k le d w ith oxalic ac id . E ig h t in d iv id u a ls w e r e a s sa y e d f ro m e a c h c o lo n y . G ST a c tiv ity w a s a s s a y e d 15 d a y s a fte r o x alic a c id tric k lin g tr e a tm e n t o f th e co lo n ies. C o n c e rn in g th e p u p a e , G ST a c tiv ity w a s also m e a s u r e d in in d iv id u a ls c o lle c te d im m e d ia te ly b e fo re tre a tm e n t.

RESULTS

C lim a tic m e a su re m e n ts

O n th e d a y o f th e oxalic a c id tr e a tm e n ts th e o u td o o r d a y te m p e r a tu r e w a s 17.0 °C.

In th e fo llo w in g f o u r w e e k s th e te m p e r a tu r e r a n g e d b e tw e e n - 0 .6 °C a t n ig h t a n d 26.3 °C in d a y tim e w ith a n a v e ra g e o f 7.0 °C. O n th e d a y o f th e tr e a tm e n ts th e re la tiv e h u m id i ty (RH ) w a s 28.6 %. In th e fo llo w in g 4 w e e k s th e R H r a n g e d b e tw e e n 21.9% a n d 100.0% w ith a n a v e ra g e o f 86.2%.

Varroa m orta lity

A fte r th e s p r in g tr e a tm e n t, th e m e a n v a r r o a m o r ta lity in c re a s e d in th e tre a te d g r o u p s re a c h in g a m a x im u m d a ily m ite d r o p - d o w n (±S.E.M .) o f 24.0±5.52 p e r c o lo n y in th e tric k le d g r o u p a n d o f 64.93±30.51 in th e s p r a y e d g r o u p 4 d a y s afte r tre a tm e n t. A t th a t tim e th e c o n tro l g r o u p h a d a m e a n d r o p - d o w n o f 0.25±0.25 v a rr o a m ite s. H e re a fte r, th e d a ily d r o p - d o w n in th e tr e a te d g r o u p s d e c re a s e d c o n tin u o u s ly to 0.47±0.24 in th e tric k le d g r o u p a n d 0.27±0.21 in th e s p r a y e d n e a r ly re a c h in g th e c o n tro l le v e l o f 0.00, fo u r w e e k s a fte r th e tr e a tm e n t (FIG U R E 1). T h e to ta l m e a n v a r r o a d r o p - d o w n (±S.E.M .) in th e tric k le d g r o u p in th e s p r in g s a m p lin g p e r io d w a s 61.53±15.42 m ite s , in th e s p r a y e d g r o u p it w a s 145.47±66.68 a n d in th e c o n tro l g r o u p 1.50 ±0.29. T h e r e s u lt o f th e c o n tro l d iffe re d sig n ific a n tly fro m th e tr e a te d c o lo n ie s (K -W , p< 0.05).

A fte r th e a u t u m n tric k lin g tre a tm e n t, th e g r o u p tr e a te d w ith tric k lin g in s p rin g h a d a m e a n v a r r o a d r o p - d o w n (±S.E.M .) o f 1,250.00±333.33. In th e s p r a y e d g r o u p th e m e a n d r o p - d o w n (±S.E.M .) w a s 935.80±391.53. T h e g r o u p s e rv in g a s c o n tro l in s p r in g h a d a m e a n v a r r o a d r o p - d o w n (±S.E.M .) o f 1,400.00±377.49 (FIG U R E 2). T he d iffe re n c e s in v a r r o a m o rta lity w e re n o t sig n ific a n t (K -W , p>0.05).

B ee c o lo n y stren g th

T h e m e a n b e e c o lo n ie s s tr e n g th (±S.E.M .) w a s 5.5±0.96 co m b g a te s in th e c o n tro l g r o u p , 5.4±0.30 co m b g a te s in th e s p ra y e d a n d 5.53±0.45 c o m b g a te s in th e tric k le d g r o u p b e fo re th e tr e a tm e n t. T h e re w a s n o sig n ific a n t d iffe re n c e b e tw e e n th e re s u lts (K -W , p>0.05). T h e m e a n b r o o d a m o u n t (±S.E.M .) in th e a b o v e m e n tio n e d g r o u p s w a s 3.25±0.75 d m 2,2 .0 7 ± 0 .3 0 d m 2 a n d 1.77±0.51 d m 2, re sp e c tiv e ly . T h e re s u lts d id n o t d iffe r s ig n ific a n tly (K -W , p> 0.05). D u rin g th e 1998 s e a so n , th e o xalic a c id tre a te d c o lo n ie s s e e m e d to d e v e lo p a s w e ll as th e c o n tro l co lo n ies. O n e y e a r a fte r th e tr e a tm e n t th e m e a n c o lo n y s tr e n g th (±S.E.M .) w a s 6.25±0.48 c o m b g a te s in th e c o n tro l g r o u p , 4.93±0.33 in th e s p r a y e d a n d 5.00±0.33 in th e g r o u p tric k le d in sp rin g 1998. T h e b r o o d a m o u n t in th e re sp e c tiv e g r o u p s a t th e sa m e tim e w a s 1.50±0.61 d m 2,1 .5 3 ± 0 .6 7 d m 2 a n d 0.89±0.24 d m 2. T h e re w a s n o s ig n ific a n t d iffe re n c e b e tw e e n th e tre a te d g r o u p s a n d th e c o n tro l (K -W , p>0.05).

O x a lic acid re sid u e s

J u s t b e fo re th e s p r in g tr e a tm e n ts th e m e a n n a tu r a l c o n c e n tra tio n o f oxalic acid (±S.E.M .) w a s m e a s u r e d to b e b e tw e e n 19.56±0.83 p p m a n d 35.85±5.96 p p m in th e th r e e e x p e r im e n ta l g r o u p s (FIG U R E 3). E ig h t d a y s a fte r th e tr e a tm e n t th e oxalic acid c o n c e n tra tio n w a s in c re a s e d in th e tr e a te d g r o u p s c o m p a r e d to b e fo re tr e a tm e n t b u t o n ly th e in c re a s e in th e s p r a y e d g ro u p to 62.84±15.88 w a s sig n ific a n t (K -W , p<0.05).

A t th e f irs t h o n e y h a r v e s t in Ju n e th e m e a n n a tu r a l c o n c e n tra tio n o f oxalic ac id (±S.E.M .) w a s m e a s u r e d to b e 37.78±5.55 p p m in th e s p r a y e d g r o u p , 41.56+8.54 in th e tric k le d g r o u p a n d 57.70±7.95 p p m in th e c o n tro l g ro u p . T h e re w a s n o sig n ific a n t d iffe re n c e in oxalic a c id c o n c e n tra tio n b e tw e e n th e g r o u p s a t a n y o f th e sa m p lin g d a te s (K -W , p>0.05).

G lu ta th io n e S -tran sferase a ctiv ity

T h e re s u lts fo r th e GST a c tiv ity in p u p a e are s u m m a r is e d in TA BLE 1 a n d th e re s u lts fo r th e n e w ly e m e r g e d a d u lts are s u m m a r is e d in TA BLE 2.

Sampling date 1998

F IG U R E 1. V a r r o a d r o p - d o w n o n w o o d e n in s e r ts a f te r tr ic k lin g o r s p r a y in g tr e a tm e n t w i t h o x a lic a c id . T h e tr e a tm e n t w a s c a rrie d o u t 23 M a rc h 1998.

. 1600- E 11200-

800-

400-

n---

Trickling, N=15 Spraying, N=15 Control, N=3

Treatment in spring 1998

F IG U R E 2. V a r ro a d r o p - d o w n o n w o o d e n i n s e r t s a f te r tr ic k lin g tr e a tm e n t w ith o x a lic a c id . T h e c o lo n ie s w e r e tr e a te d i n s p r in g '98 w i t h tr ic k lin g , s p r a y in g o r n o tr e a tm e n t, r e s p e c tiv e ly . T h e a u t u m n tr e a tm e n t w a s c a r rie d o u t i n S e p te m b e r.

80 - 7 0 - 60-

“ 40 - OX

i 30 - 20-

1 0-

Trickling, N=5

□ Spraying, N=5 I I Control, N=4

230398 310398

S a m p l i n g d a t e

080698

FIG URE 3. C o n cen tra tio n o f o x a lic acid r e sid u e s in h o n e y after sp r in g treatm en t o f b e e c o lo n ie s w it h o x a lic acid b y trick lin g . B lack arrow in d ic a te s treatm en t, w h ite arrow in d ic a te s h o n e y h arvest.

T h e d a t a fro m th e G ST a s s a y s o n th e p u p a e w e r e a n a ly z e d b y o n e w a y a n a ly s is o f v a ria n c e w ith m u ltip le c o m p a r is o n (T u k e y 's te s t, Z a r 1996). T h e o n ly g r o u p s th a t w e r e s ig n ific a n tly d iffe r e n t (P<0.05) w e r e p u p a e fro m n o n - tre a te d c o lo n ie s c o llec te d b e fo re tr e a tm e n t (184.4±5.8 n m o l m i n 1 m g p r o te in '1) a n d p u p a e fro m c o lo n ie s tr e a te d w ith oxalic a c id (207.0±4.2 n m o l m i n 1 m g p r o te in '1). T h u s , 15 d a y s a fte r tr e a tm e n t th e G ST a c tiv ity in p u p a e fro m th e tre a te d c o lo n ie s w a s n o t sig n ific a n tly d iffe re n t fro m th e G ST a c tiv ity in p u p a e fro m th e sa m e c o lo n ie s b e fo re tr e a tm e n t a n d n o t s ig n ific a n tly d if fe r e n t fro m th e G ST a c tiv ity in p u p a e f ro m n o n - tre a te d c o lo n ie s c o lle c te d a t th e s a m e tim e.

T h e d a t a f ro m th e G ST a s s a y s o n th e n e w ly e m e r g e d a d u l t b e e s w a s a n a ly s e d b y f te s t (Z a r 1996) a n d th e te s t s h o w e d th a t th e tw o g r o u p s w e r e n o t sig n ific a n tly d iffe re n t (P=0.14). T h u s, 15 d a y s a fte r tr e a tm e n t th e G ST a c tiv ity in th e a d u lts fro m th e oxalic a c id tric k le d c o lo n ie s w e re n o t d iffe re n t fro m th e G ST a c tiv ity fo u n d in a d u lts fro m n o n - tr e a te d co lo n ies.

TABLE 1. G lu ta th io n e S -tran sferase (G ST ) a c tiv ity in h o n e y b e e p u p a e co llec te d b e fo r e treatm en t an d 15 d a y s after tric k lin g treatm en t w ith o x a lic acid (O S).

C o lle c tio n o f p u p a e P u p ae for G S T a ssa y s

n G S T activity*

(n m o l m in 1 m g p r o te i n '1) Collection before treatm ent

P u p a e f ro m five n o n -tre a te d c o lo n ies

40 184.4±5.8

P u p a e fro m five co lo n i­

e s to be tric k le d w ith OS a fte r co llec tio n

40 194.1±5.5

Collection 15 days after treatm ent o f the colonies P u p a e fro m five n o n -tre a te d c o lo n ies

40 191.5+4.8

P u p a e f ro m fiv e co lo n i­

e s tric k le d w ith OS

40 207.0±4.2

“ Mean activity ± SEM.

TABLE 2. G lu ta th io n e S -tran sferase (G ST ) a ctiv ity in n e w ly em e r g e d a d u lt h o n e y b e e s c o lle c te d 15 d a y s after tric k lin g treatm en t w it h o x a lic acid (O S).

A d u lt b e e s n G S T activity"

(n m o l m in 1 m g p ro te in ’)

A d u lts fro m f o u r n o n - tr e a te d c o lo n ies 32 211.5±6.4

A d u lts fro m f o u r c o lo n ie s tr e a te d w ith OS tric k lin g

32 226.2±7.4

* Mean activity ± SEM.

D ISC U SSIO N

A t th e tim e o f th e oxalic a c id tre a tm e n ts th e b e e c o lo n ie s ' s tr e n g th a n d b r o o d a m o u n t w e re a v e ra g e fo r D a n is h c o n d itio n s. N e ith e r e ig h t d a y s a fte r th e M a rc h tr e a tm e n t, n o r a t th e f irs t h o n e y h a r v e s t in J u n e a s ig n ific a n t d iffe re n c e c o u ld b e d e te c te d in oxalic a c id c o n c e n tra tio n in fo o d o r h o n e y b e tw e e n th e tr e a te d g r o u p s a n d th e c o n tro l g ro u p . E ig h t d a y s a fte r th e tr e a tm e n t th e m a x im u m le v e l of oxalic a c id w a s f o u n d in th e s p r a y e d g ro u p w ith a m e a n c o n c e n tra tio n o f 0.0062% . F or c o m p a ris o n , th e n a tu r a l c o n c e n tra tio n o f oxalic a c id (o x ala tes) b a s e d o n fre s h w e ig h t in s p in a c h is 0.3-1.2% , in r h u b a r b 0.2-1.3% , in te a 0.3-2.0% a n d in c o c o a 0.5-0.9%

(F a sse tt 1973). S ince oxalic a c id is n o t fa t-so lu b le n o r e s id u e s w ill b u i l d u p in th e w a x in th e tr e a te d c o lo n ie s (Im d o rf et al. 1998). T h u s, re s id u e s in h o n e y a n d w a x afte r s p r in g tr e a tm e n t w ith oxalic a c id se e m s n o t to b e p ro b le m a tic .

If a d iffe re n c e in G ST a c tiv ity fro m tr e a te d vs. n o n - tre a te d c o lo n ie s w a s fo u n d it c o u ld b e a n in d ic a tio n o f a p h y s io lo g ic a l effect o n in d iv id u a ls in th e tr e a te d colonies.

In th is s tu d y tric k lin g tr e a tm e n t o f c o lo n ie s w ith oxalic a c id d o e s n o t se e m to h a v e a p r o lo n g e d n e g a tiv e effec t o n th e le v e l of G ST a c tiv ity in p u p a e o r n e w ly e m e rg e d a d u lts a s n o d iffe re n c e in G ST a c tiv ity in tr e a te d vs. n o n - tre a te d c o lo n ie s c o u ld be d e m o n s tr a te d . H o w e v e r, a la c k of effect o n th e le v el of G ST a c tiv ity d o e s n o t ru le o u t th a t in d iv id u a ls in oxalic a c id tre a te d c o lo n ie s w e r e p h y s io lo g ic a lly a ffe c te d b y th e tre a tm e n t.

D u rin g th e s e a s o n 1998, th e tr e a te d c o lo n ie s s e e m e d to d e v e lo p n o r m a lly c o m p a r e d to th e c o n tro l co lo n ies. O th e r D a n is h tria ls u s in g th e s a m e m e th o d s in s p rin g tim e c o n firm th is f in d in g (H a n s e n 1999). O n ly o n e of th e tr e a te d c o lo n ie s in th e p re s e n t s tu d y d id n o t s u rv iv e th e w in te r 9 8 /9 9 . In s p rin g 1999, th e c o lo n ie s in th e p re s e n t s tu d y h a d s tr e n g th r a n g in g fro m 4.93 to 6.25 co m b g a te s a n d a b r o o d a m o u n t o f 0.89 to 1.53 d m 2 w h ic h c o rr e s p o n d s w ith th e a v e ra g e in b e e c o lo n ie s a t th a t tim e th e c o u n try .

T h e re s u lts o f th is s tu d y d o n o t g iv e a d ire c t m e a s u r e o f th e effica cy o f th e tw o tre a tm e n ts . O x alic a c id s p ra y in g d o e s n o t h a v e a n effec t o n v a r r o a m ite s in th e s e a le d b r o o d (R a d e tz k i 1994). A lso tric k lin g is o n ly r e c o m m e n d e d in b r o o d le s s c o lo n ie s a s e x p e r im e n ts h a v e s u g g e s te d th a t th e efficacy o f o n e tr e a tm e n t in co lo n ies w ith b r o o d w a s to o p o o r a n d se v e ra l tr e a tm e n ts w e a k e n e d th e c o lo n ie s (Im d o rf et al.

1998). B ecau se o f th e b r o o d p r e s e n t in o u r c o lo n ies, w e a s s u m e th a t th e efficacy of

th e s e s p r in g tr e a tm e n t is lo w e r th a n th e a p p ro x im a te ly 98% f o u n d b y R a d e z k i (1994) a n d I m d o r f et al. (1998) fo r s p r a y in g a n d tric k lin g , re sp e c tiv e ly . In s p r in g in D e n ­ m a rk , it is n o t p o s s ib le to c u t o u t th e b r o o d to in c re a se th e efficacy a s it is d o n e in th e a u t u m n b y lactic a c id tr e a tm e n t ( B rø d s g a a rd et al. 1997). R e m o v in g th e b r o o d in s p r in g w o u ld m o s t lik e ly w e a k e n th e co lo n ies. A p r o p e r effica cy te s t s h o u ld of c o u rs e b e fo llo w e d b y a to ta l c o u n t o f m ite s in th e c o lo n ie s o r a tr e a tm e n t w ith a p e s tic id e w ith a w e ll-d o c u m e n te d effect. B u t sin c e th e re a re n o p e s tic id e s re g is te re d fo r u se in D e n m a r k th e h o n e y , w a x a n d e q u ip m e n t f ro m th e tre a te d c o lo n ie s w o u ld h a v e to b e d e s tro y e d a n d th is w a s n o t p o ssib le in th is p r e lim in a r y s tu d y . N e v e r th e ­ le ss, a s s u m in g th a t th e v a r r o a in fe s ta tio n w a s e v e n ly d is trib u te d b e tw e e n th e c o lo n ie s th e re se e m s to b e a te n d e n c y th a t th e s p r a y in g tr e a tm e n t w a s m o re efficien t th a n th e tric k lin g b a s e d o n th e o b s e rv e d v a r r o a d r o p -d o w n a lth o u g h th e d iffe re n c e w a s n o t sig n ific a n t (FIG U R E 1). T h e la ck o f d iffe re n c e in effica cy b e tw e e n tric k lin g a n d s p r a y in g tr e a tm e n t c o r r e s p o n d s w ith th e f in d in g s o f I m d o rf et al. (1998) w h o f o u n d n o d iffe re n c e s w h e n tre a tin g b r o o d le s s c o lo n ie s in a u t u m n w ith th e tw o m e th o d s . T h e v a r r o a d r o p - d o w n a fte r th e tric k lin g tr e a tm e n t in th e a u tu m n s h o w e d th a t th e v a r r o a p o p u la tio n in th e c o lo n ie s tr e a te d in s p r in g s e e m e d to h a v e d e v e lo ­ p e d as w e ll a s in th e c o n tro l. T h a t r e s u lt c o u ld b e e x p la in e d b y a p o o r efficacy o f th e s p r in g tr e a tm e n t, re in v a s io n fro m th e c o n tro l co lo n ie s to th e tr e a te d co lo n ies c o m b in e d w ith a v e r y s h o r t p e rs is te n c e o f o xalic a c id o r th e v e r y fe w c o n tro l co lo n ies.

N e ith e r th e r e s id u e s o f oxalic a c id in h o n e y , th e G ST a c tiv ity , n o r th e c o lo n y d e v e ­ lo p m e n t a fte r s p r in g tr e a tm e n t w ith e ith e r tric k lin g o r s p r a y in g w ith oxalic ac id s e e m to in d ic a te a n y p ro b le m s . H o w e v e r, b e fo re th e u s e o f o xalic a c id as s p rin g tr e a tm e n ts is r e c o m m e n d e d in D e n m a r k it is n e c e s s a ry to p u t m o re e ffo rt in to effica cy te s ts w ith a la rg e n u m b e r o f c o n tro l c o lo n ie s in fie ld tria ls.

ACKNOWLEDGEMENTS

W e th a n k b e e k e e p e r G. B org fo r c o o p e ra tio n , L. H a s m a r k a n d A . M a r th in for th e ir te c h n ic a l h e lp a n d I n s titu to Z o o p ro fila ttic o S p e rim e n ta le d e lle V e n e z ie , L e g n a ro , Ita ly fo r a n a ly s in g h o n e y s a m p le s fo r oxalic a c id re s id u e s .

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B ra d fo rd , M .M . (1976) A r a p id a n d se n s itiv e m e th o d fo r th e q u a n tita tio n o f m ic ro ­ g ra m q u a n titie s o f p r o te in u tiliz in g th e p rin c ip le o f p r o te in -d y e b in d in g . A n al.

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POSTB • BLfllS 7401 HRC 50866 686

Hannar k-s Jordbru9sf orskn i ns B i b i i o teke t ? M • B re n d s tru p F ia k k e b ie r9

4200 Sla9else

DIAS Foulum

P.O. Box 50, DK-8830 Tjele

Tel. +45 89 99 19 00. Fax +45 89 99 19 19 Management

Management Secretariat, Finance Secretariat Department of Animal Product Quality Department of Animal Breeding and Genetics Department of Animal Nutrition and Physiology Department of Animal Health and Welfare Department of Agricultural Systems Department of Crop Physiology and Soil Science Department of Farm Management and Experimental Stations

Department of Animal Research Facilities Central Laboratory

Public Relations

Computer Technical Services Library

International Unit

DIAS Aarslev

Kirstinebjergvej 10, DK-5792 Aarslev Tel. +45 63 90 43 43. Fax +45 65 99 17 22 Department of Ornamentals

Department of Fruit, Vegetable and Food Science Department of Plant Research Services

DIAS Flakkebjerg

Flakkebjerg, DK-4200 Slagelse

Tel. +45 58 11 33 00. Fax +45 58 11 33 01 Department of Plant Biology

Department of Crop Protection Department of Plant Research Services

DIAS Bygholm

P.O. Box 536, DK-8700 Horsens

Tel. +45 75 60 22 11. Fax +45 75 62 48 80 Department of Agricultural Engineering

Units at other locations

Department of Variety Testing

Teglværksvej 10, Tystofte, DK-4239 Skælskør Tel. +45 58 16 06 00. Fax +45 58 16 06 06 Askov Experimental Station

Vejenvej 55, DK-6600 Vejen

Tel. +45 75 36 02 77. Fax +45 75 36 62 77 Biotechnology Group (Dept. of. Plant biology) Thorvaldsensvej 4 0 ,1 .

DK-1871 Frederiksberg C

Tel. +45 35 28 25 88. Fax +45 35 28 25 89 Borris Experimental Station

Vestergade 46, DK-6900 Skjern Tel. +45 97 36 62 33. Fax +45 97 36 65 43 The Ecological Experimental Station

Rugballegaard

P.O. Box 536, DK-8700 Horsens Tel. +45 75 60 22 11. Fax +45 75 62 48 80

Foulumgaard, P.O. Box 50, DK-8830 Tjele Tel. +45 89 99 19 00. Fax +45 89 99 19 19 Jyndevad Experimental Station

Flensborgvej 22, 6360 Tinglev

Tel. +45 74 64 83 16. Fax +45 74 64 84 89 Rønhave Experimental Station

Hestehave 20, DK-6400 Sønderborg Tel. +45 74 42 38 97. Fax +45 74 42 38 94 Silstrup Experimental Station

Højmarken 12, DK-7700 Thisted Tel. +45 97 92 15 88. Fax+45 97 91 16 96 Tylstrup Experimental Station

Forsøgsvej 30, DK-9382 Tylstrup Tel. +45 98 26 13 99. Fax +45 98 26 02 11